Molecular Biology of Plants Cancer

Molecular Aspects of Medicine 21 (2001) 167223 www.elsevier.
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Review
The molecular biology of cancer

John S. Bertram
*
Cancer Research Center of Hawaii, University of Hawaii at Manoa, 1236 Lauhala Street, Honolulu, HI 96813, USA
Abstract The process by which normal cells become progressively transformed to malignancy is now known to require the sequential acquisition of mutations which arise as a consequence of damage to the genome. This damage can be the result of endogenous processes such as errors in replication of DNA, the intrinsic chemical instability of certain DNA bases or from attack by free radicals generated during metabolism. DNA damage can also result from interactions with exogenous agents such as ionizing radiation, UV radiation and chemical carcinogens. Cells have evolved means to repair such damage, but for various reasons errors occur and permanent changes in the genome, mutations, are introduced. Some inactivating mutations occur in genes responsible for maintaining genomic integrity facilitating the acquisition of additional mutations. This review seeks rst to identify sources of mutational damage so as to identify the basic causes of human cancer. Through an understanding of cause, prevention may be possible. The evolution of the normal cell to a malignant one involves processes by which genes involved in normal homeostatic mechanisms that control proliferation and cell death suer mutational damage which results in the activation of genes stimulating proliferation or protection against cell death, the oncogenes, and the inactivation of genes which would normally inhibit proliferation, the tumor suppressor genes. Finally, having overcome normal controls on cell birth and cell death, an aspiring cancer cell faces two new challenges: it must overcome replicative senescence and become immortal and it must obtain adequate supplies of nutrients and oxygen to maintain this high rate of proliferation. This review examines the process of the sequential acquisition of mutations from the prospective of Darwinian evolution. Here, the ttest cell is one that survives to form a new population of genetically distinct cells, the tumor. This review does not attempt to be comprehensive but identies key genes directly involved in carcinogenesis and demonstrates how mutations in these genes allow cells to circumvent cellular controls. This detailed understanding of the process of carcinogenesis at the molecular level has only been possible because of the advent of modern molecular biology. This new discipline, by precisely identifying the molecular basis of the dierences between normal and malignant cells, has created novel opportunities and provided the means to specically target these modied genes. Whenever possible this review
Tel.: +1-808-586-2957; fax: +1-808-586-2970. E-mail address: john@crch.hawaii.edu (J.S. Bertram).
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J.S. Bertram / Molecular Aspects of Medicine 21 (2001) 167223
highlights these opportunities and the attempts being made to generate novel, molecular based therapies against cancer. Successful use of these new therapies will rely upon a detailed knowledge of the genetic defects in individual tumors. The review concludes with a discussion of how the use of high throughput molecular arrays will allow the molecular pathologist/ therapist to identify these defects and direct specic therapies to specic mutations. 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Cancer; Carcinogenesis; Mutations; DNA damage; DNA repair; Oncogenes; Tumor suppressor genes; Growth control; Angiogenesis; Apoptosis; Senescence
Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Carcinogenesis: the conversion of normal cells responsive to homeostatic feedback mechanisms to cells capable of autonomous growth and invasion 2.1. Mutations require proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. DNA is subject to chemical damage . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Induction of spontaneous DNA damage . . . . . . . . . . . . . . . . . . 2.3. Induction of DNA damage by exogenous agents . . . . . . . . . . . . . . . . 2.3.1. Chemical carcinogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Physical carcinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. Many cancer chemotherapeutic agents are carcinogenic . . . . . . . 2.4. Most DNA damage is repairable . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Defects in DNA repair are responsible for many familial cancer syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Cell-cycle checkpoints restrict replication of damaged DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 171 173 173 174 174 177 177 178
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Pathways to cancer: overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 Cancer cells are independent of external growth signals . . . . . . . . . . . . . . . . . . . 4.1. Inappropriate synthesis of growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Inappropriate expression of growth factor receptors. . . . . . . . . . . . . . . . . . . 4.2.1. Erb-B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Activation of downstream signal transduction pathways. . . . . . . . . . . . . . . . 4.3.1. c-abl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. ras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Inappropriate activation of nuclear transcription factors . . . . . . . . . . . . . . . 4.4.1. Inappropriate expression of c-myc, a transcription factor . . . . . . . . . . . 4.4.2. Mutation of a nuclear hormone receptor leads to blocked dierentiation Cancer cells become refractory to growth inhibitory signals: of tumor suppressor genes . . . . . . . . . . . . . . . . . . . . . . . . 5.1. The retinoblastoma gene RB . . . . . . . . . . . . . . . . . . . 5.1.1. RB functions to restrict entry into S-phase . . . . . 5.1.2. RB gene therapy . . . . . . . . . . . . . . . . . . . . . . . . 5.2. p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. p53 mutations . . . . . . . . . . . . . . . . . . . . . . . . . . the discovery .......... .......... .......... .......... .......... .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 183 183 183 184 184 185 188 188 189 190 191 192 193 193 193
5.
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5.2.2. p53 monitors genomic integrity. . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. p53 is a transcription factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4. Loss of p53 alters response to chemotherapeutic agents . . . . . . . . 5.2.5. p53 gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.6. Human papilloma virus (HPV) can inactivate both p53 and RB . . 5.3. Mutations in the APC gene link cell surface receptors with the nucleus . 5.3.1. The APC gene oers many targets for intervention. . . . . . . . . . . . 6. 7.
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195 195 197 197 198 198 199
Cancer cells are decient in intracellular communication mediated by gap junctions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Cancer cells evade apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Overexpression of bcl-2 protects lymphoma cells from apoptosis . . . . . . 7.2. Tumor cells evade apoptosis by modied FAS and FAS-L interactions . 7.3. The induction of apoptosis is an important target in cancer therapy . . . . . . . . . . . . . . . . . . . 202 202 203 204
8.
Cancer cells must avoid senescence and achieve immortality: role of telomeres . . . 205 8.1. Many cancer cells reactivate telomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 8.2. Telomerase oers an exciting novel target for cancer prevention and therapy . 206 Cancer cells require adequate supplies of nutrients and stimulate angiogenesis 9.1. Inhibitors of angiogenesis exert potent anti-tumor aects . . . . . . . . . . . . 9.2. Inhibitors of pro-angiogenic signals are eective anti-tumor agents . . . . . 9.3. Conventional chemotherapy can be targeted to endothelial cells . . . . . . . . . . . . . . . . . . . 207 208 209 210
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10. Putting it all together: prospects for molecular medicine in the 21st century . . . . 210 10.1. Use of genomic arrays in the molecular proling of cancers . . . . . . . . . . . . 211 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
1. Introduction The last two decades have seen enormous advances in our understanding of cancer at the molecular level. This understanding has revealed large numbers of exciting new targets for the development of eective therapies, some of which have already entered clinical practice. These new targets identify both early and late events in the carcinogenic process and thus oer opportunities for treatment and for prevention surely the most exciting goal in conquering this dreaded disease. By allowing the direct targeting of the genetic defects that are responsible for malignancy, it is a realistic expectation that increasing numbers of tumor-specic drugs will soon be available which will spare normal cells from the devastating eects of conventional cytotoxic therapeutic agents. To be eective, conventional agents must be used at dosages which are acutely life-threatening to the patient. Furthermore many currently available drugs also induce genetic damage which can itself be carcinogenic. New molecular therapies should allow the physician an unprecedented ability to treat the cancer without harming the patient. In order to fully exploit these new opportunities, it is becoming apparent that the wide diversity of genetic aberrations
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present in tumor cells will make it necessary to genotype individual tumors just as they are currently phenotyped by standard pathological procedures. High throughput screening tests which can simultaneously measure the expression pattern and presence of specic mutations in thousands of individual genes will make this possible. Their use is currently restricted to major research centers as research tools rather than diagnostic instruments; however, as new therapeutic agents become available whose use depends upon specic genetic information, it seems inevitable that this technology will be a necessary requirement for most cancer diagnoses. It is the purpose of this review to outline our current knowledge of cancer genetics and in so doing draw attention to the enormous possibilities for future research in the design of specic cancer therapeutic agents. 2. Carcinogenesis: the conversion of normal cells responsive to homeostatic feedback mechanisms to cells capable of autonomous growth and invasion The adult human is composed of approximately 1015 cells, many of which are required to divide and dierentiate in order to repopulate organs and tissues which require cell turnover. Obvious examples are cells in the basal layer of the skin which divide, dierentiate and are nally sloughed, cells composing the epithelial layer of the intestines which turnover and must be replaced approximately every 10 days, and cells in the bone marrow which divide and dierentiate to produce white and red cells whose life-time varies from 24 h in the case of some leukocytes to 112 days for mature red cells. Cells which have the capacity for division and replenishment are called stem cells. It can be calculated that there are approximately 1012 divisions per day in these stem cell compartments. Even in organs which normally exhibit low levels of cell division, the liver being the prime example, massive proliferation can be initiated by events such as trauma or infection. Yet in spite of this enormous production of new cells, the human body maintains a constant weight over many decades. Even obesity is not primarily the result of increased cell multiplicity but of increased volume and thus mass of adipocytes. This exquisite control over cell multiplicity is achieved by a network of overlapping molecular mechanisms which govern cell proliferation on one hand and cell death, termed apoptosis when the result of a programmed event, on the other. Any factor which alters this balance between birth and death, just as it would in an isolated species of individuals, has the potential if not corrected to alter the total number of cells in a particular organ or tissue. After many cell generations this increased cellular multiplicity would be clinically detectable as neoplasia, literally new growth. As will be described below, it is genes that alter the birth rate or the death rate of individual cells that have now been rmly implicated as causative in the carcinogenic process. Just as Darwinian evolution depends upon random mutations giving rise to a selective advantage to individuals, it now seems clear that random mutations in the genes which control proliferation or apoptosis are responsible for cancer. To take the analogy further, just as evolution allows the survival of the ttest individual, so too
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in the case of carcinogenesis, it is those mutations in individual genes which render cells most capable of evading normal homeostatic mechanisms that are the mutations detected in successful cancer cells. Success in this scenario must be viewed from the perspective of the individual cell, not from the perspective of the individual patient who harbors that cell. Clearly, this success is generally short-lived since if untreated it leads to the death of the host. Other cells have been more fortunate; HeLa cells, derived from a cervical carcinoma which killed their host in 1956, can be found in thousands of research institutes throughout the world. From an evolutionary perspective, in this particular environment, clearly HeLa cells have been highly successful. The vast majority of mutations that give rise to cancer are not inherited, but arise spontaneously as a consequence of chemical damage to DNA resulting in altered function of crucial genes. In a few specic cancers, the cervical cancer that gave rise to HeLa cells would be a prime example, genes encoded by the HPV virus directly interfere with gene action and perform the same function as mutations. However, as will be seen, mutations which inactivate these same genes in non-infected cells have the same carcinogenic consequences. Thus parallel evolution also occurs during the genesis of a cancer cell. In discussing mutations in the context of carcinogenesis we will be using the broadest denition: the change in the genome of a particular cell. This includes: point mutations which cause amino acid substitutions; frame-shift mutations or mutations to stop codons which either truncate the protein product or scramble its sequence; chromosomal imbalance or instability resulting in amplication, overexpression or inappropriate expression of a particular gene; loss of a gene or its fusion with another gene as a result of chromosomal breakage and rearrangement resulting in a chimeric protein with altered function; epigenetic modications to DNA of which the most important is the methylation of cytosine in CpG islands leading to gene silencing. Developing cancer cells select mutations having two basic functions: mutations which increase the activity of the proteins they code for; this class of genes are called oncogenes; or mutations which inactivate gene function in the case of genes classed as tumor suppressor genes. However, regardless of ultimate eect, the types of chemical damage causing these mutations are believed identical. A broad understanding of these chemical events are important for two reasons: rst since these initial events are causative of the whole process of carcinogenesis, their inhibition would be an eective preventive measure; secondly, several genetic diseases which predispose to cancer have as their origin mutations in genes whose purpose is to protect DNA from mutational events. Thus the understanding of these events has direct clinical relevance. 2.1. Mutations require proliferation It is important to note that chemical damage to DNA itself is not a mutagenic event. DNA replication and subsequent cell division is necessary to convert chemical damage to an inheritable change in DNA that we call a mutation. Thus, proliferation is a vital factor in the formation of mutations and in the expansion of clones of cells
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Fig. 1. Role of proliferation in the sequential acquisition of cancer-causing mutations. Because of the large number of normal stem cells there is a high probability of unrepaired DNA damage causing a single mutation in a critical gene leading to the formation of an initiated cell. Additional proliferation is necessary to produce a clone of at least 106 cells in order that a second, third, etc. mutation has a nite probability of occurring. As described in the text, each mutation results in a cell progressively better adapted to avoid normal controls on proliferation and apoptosis. Mutations in genes such as p53, and chromosome instability resulting from telomere erosion, will act to increase the mutation rate in cells progressing to neoplasia.
bearing these mutations. This is illustrated in Fig. 1 and has been eloquently discussed by Ames et al. (1993). Because of the multiple checks and balances that exist in stem cells to limit inappropriate proliferation, with few exceptions, malignant human cells must accumulate multiple mutations in crucial cellular genes that allow their autonomous replication and invasion. Yet mutation at a particular genetic locus is a relatively rare event. Even after deliberate chemical damage to a cell in a laboratory situation, the frequency of mutations at a particular allele is of the order of 106 , i.e., only one cell in one million is mutated. Mutation rates in human stem cells may be expected to be of the order of 1010 /cell division, a very low probability, yet because of the large number of proliferating stem cells it appears likely that initiation is a common event and all adults probably contain many mutated cells. Fortunately, a successful human cancer cell is required to have mutations in at least ve genes, as elegantly shown in the case of colon carcinoma, with each mutation creating a cell increasingly well adapted for autonomous growth in the host organism (Cahill et al., 1999; Cho and Vogelstein,
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1992). Because the probability of a single cell simultaneously acquiring these mutations is vanishingly small, this sequential process of acquisition of mutations can only be achieved if cells bearing the initial mutation, the so-called initiated cells, clonally expand until the population increases to many millions. In this population the probability of a second mutation at a critical locus in one of the cells again reaches or exceeds unity. This process of clonal expansion must then be repeated so that subsequent mutations can be amassed and cells become progressively better adapted to an independent life. The sequence of mutations is shown in Fig. 1. This process is observable clinically as disease progression characterized by an increased growth rate, acquisition of the ability to invade neighboring normal tissue and to metastasize and, after application of chemotherapeutic agents, to become progressively drug-resistant. 2.2. DNA is subject to chemical damage Although endowed with almost magical properties, DNA nevertheless is a molecule whose chemical bonds obey the same laws as other chemicals and which exists in an aqueous environment at 37C in the middle of a cell whose very existence depends upon making and breaking chemical bonds. Thus it is perhaps not surprising that DNA constantly suers chemical damage, some as a consequence of spontaneous thermal eects, some as a consequence of chemical attack by other reactive molecules. It has been estimated that approximately 70% of cancer in Western populations can be attributed to diet and lifestyle with exposure to tobacco products the major contributor at 30% (Doll and Peto, 1981). However, much of the remaining increased risk appears to be associated with deciencies in dietary factors, principally fruits and vegetables, which exert a protective role on cancer induction. When chemical damage occurs as a consequence of exposure to exogenous agents, either chemical or physical, these agents are generally carcinogenic and the type of damage and mutations they induce can act as a molecular ngerprint indicating exposure to these environmental carcinogens (Greenblatt et al., 1994; Multani et al., 2000). It is clear however that many human cancers occur in individuals without obvious exposure to environmental carcinogens and many human cancers occur in organs for which no environmental or genetic causes have yet been identied. It must be deduced then that spontaneous DNA damage does occur which gives rise to carcinogenic mutations. By understanding the causes underlying the genetic damage that results in cancer we are in a position to reduce its incidence. 20th-century medicine has made great strides in reducing the incidence of infectious diseases through eective vaccination programs, as for example with smallpox and polio, and creating eective public health programs providing for example, safe drinking water. It is hoped that 21st century medicine will place equal emphasis and have equivalent success in the reduction of cancer rates through focused preventive measures. 2.2.1. Induction of spontaneous DNA damage Spontaneous DNA mutations can occur directly as a consequence of errors in replication, or indirectly as a consequence of chemical damage to DNA leading to
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errors in the correct reading of the damaged DNA by DNA polymerase during the process of replication. Fortunately, cells have evolved highly ecient mechanisms for replicating their DNA which combine high-delity DNA polymerases with subsequent proofreading capabilities. As a consequence, the direct error rate during normal replication of DNA is of the order 1X3 1010 mutations/base pair/cell division in a human genome of approximately 2 109 base pairs. Thus, in a single stem cell, one miscoding error would be introduced every 10 divisions. Because approximately 97% of DNA is non-coding and because of the redundancy of codon recognition, many base changes do not give rise to amino acid substitutions. Thus, the functional mutation rate must be several orders of magnitude below the actual mutation rate. Nevertheless, with an estimated 1016 cell divisions occurring in an individual's life span, a total of 1015 base-pairs can accumulate, perhaps 103 base pair changes in each of the estimated 1012 cells capable of replication in an adult human. There thus seems a low probability that any one of these could eect the oncogenes or tumor suppressor genes known to be mutated in cancer. Other mechanisms therefore must exist that cause the observed mutations. Mutations as a result of chemical damage to DNA appear to be a major factor in initiating a cascade of events, one of which is an increased mutation rate, the so-called mutator phenotype, as a result of damage to genes whose function is to ensure the delity of DNA replication (Jackson and Loeb, 1998; Loeb, 1991) (see also Section 2.4.1). Spontaneous DNA damage is a frequent event as a result of the inherent instability of the DNA molecule: depurination from breakage of the N-glycosidic bond connecting purines to deoxyribose occurs at the rate of 104 events/cell/day (Lindahl and Nyberg, 1972); deamination of cytidine to uridine occurs about 20 times/cell/ day, while deamination of methylcytosine to form thymidine is probably the most frequent spontaneous chemical event with mutagenic potential (Jones et al., 1992). Mutations occur during replication because: apurinic sites can result in random base insertions; uridine when in DNA will base-pair with adenine leading to a G 3 T mutation, while deamination of methylcytosine will lead ultimately to a C 3 T transition, a mutation frequently observed in human cancers (Jones et al., 1992). In addition to these spontaneous changes, DNA damage occurs as the result of chemical attack, in large part by products of oxidative metabolism, and is probably the most frequent potentially mutagenic event. Although estimates vary, production of 8-hydroxydeoxyguanosine, perhaps the most dangerous of these mutagenic products (Cheng et al., 1992), occurs to the extent of 2 104 105 lesions/cell/day (Shigenaga et al., 1989). Fortunately, none of these lesions accumulate as evolution has developed a number of DNA-repair enzymes, which can rapidly restore the damaged sequence. 2.3. Induction of DNA damage by exogenous agents 2.3.1. Chemical carcinogens DNA is also subject to damage from exogenous agents both chemical and physical, most of which are now recognized as environmental carcinogens. For both types of agent the most frequent chemical reaction giving rise to DNA damage can
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be characterized as an electrophilic attack upon a tissue nucleophile (Miller and Miller, 1975). The most signicant of tissue nucleophiles to be damaged by chemical attack of this type is guanine, and the chemical changes induced are now known to interfere with base-pair recognition during replication. Perhaps the earliest example of environmental carcinogenesis was reported in 1775 and involved tumor induction in workers exposed to coal tar. This lead ultimately to the identication of the polycyclic aromatic hydrocarbon 3,4-benzpyrene and other polycyclic hydrocarbons in coal tar and the discovery of their action as skin carcinogens in laboratory animals. Similarly, the discovery of a high frequency of bladder carcinogenesis in workers in the rubber and chemical industries lead to the identication of 2-naphthylamine as a bladder carcinogen. With the growing realization that some human cancers have an environmental origin that could be linked directly to chemical exposure, the list of carcinogenic chemicals rapidly expanded (Doll and Peto, 1981). What was immediately apparent was the great chemical diversity of these structures, and for many of them such as the polycyclic aromatic hydrocarbons, their great chemical stability. How can we explain their similarity of actions in causing cancer and their ability to cause profound changes in cell behavior? Major insights to this question came from the work of the Millers in the 1960s and '70s with their discovery that these stable chemical carcinogens underwent a process of metabolic activation by enzymes normally involved in the detoxication of xenobiotic compounds, to yield highly reactive chemical species the electrophiles mentioned above (Miller and Miller, 1975). The sequence of events leading to DNA adduct formation and carcinogenesis can be best exemplied by reference to one of the simplest chemical carcinogens, dimethylnitrosamine. This compound was widely utilized as a chemical solvent and was investigated because of suspicions that it caused liver damage in exposed workers. This suspicion was conrmed when laboratory rats developed a similar pathology after exposure. Its carcinogenic potential was discovered serendipitously when animals surviving acute doses later were found to develop liver carcinomas (Magee, 1972). This accidental discovery had major repercussions: not only was a new industrial carcinogen discovered but this class of carcinogen, the N-nitrosamines, were found to be present in a large number of consumer items from beer, to tobacco smoke to cosmetics (Hecht, 1997). In addition, it was found that nitrosamines could be formed in the acid environment of the stomach after ingestion of primary and secondary amines, found in high levels in sh, and of sodium nitrite, also found in salted sh as a preserving agent. It is now believed that this endogenous production of nitrosamines explains the particularly high incidence of gastric cancer in Japan and Iceland where salt-preserved sh is a major dietary item (Mirvish, 1995). From the perspective of the cancer researcher striving to understand the nature of the interaction of chemical carcinogens with the cell, perhaps the greatest benet was the chemical simplicity of the electrophile a methylcarbonium ion CH3 generated by metabolic activation of dimethylnitrosamine. With the use of C-14-labeled carcinogens it was soon discovered that 06 methylguanine was a product of reaction of activated nitrosamines with DNA and that this base, if not repaired, could introduce point mutations which were potentially carcinogenic (Lawley, 1980; Lawley and
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Fig. 2. Metabolic activation of dimethylnitrosamine and reaction product with DNA. Metabolic activation by cytochrome P450 enzymes occurs mainly in the liver and results in the formation of the potentially mutagenic adduct 06 methylguanine. If unrepaired, this adduct can base-pair with adenine instead of cytidine during DNA replication to form a point mutation.
Shah, 1973). The sequence of events from the activation of dimethylnitrosamine and production of the potentially mutagenic lesion 06 methylguanine is shown in Fig. 2. Shortly after this discovery, the metabolic conversion of polycyclic aromatic hydrocarbons to diol-epoxides and the subsequent reaction of the unstable epoxide group with the N-2 position of guanine was discovered, a lesion also with potential mutagenic properties (Jerey et al., 1977; Tucker et al., 1988). The signicance of these ndings cannot be understated as they represented the beginning of our molecular understanding of cancer. Prior to these discoveries the role of DNA damage in the process of carcinogenesis was unclear and many competing hypotheses existed. The development of rapid in vitro assays for the detection of environmental mutagens was an additional repercussion of the realization that carcinogens cause potentially mutagenic DNA adducts. Principal among these was the Ames test conducted in Salmonella bacteria which allowed the rapid and semi-quantitative assessment of the mutagenic ability of test chemicals in the presence or absence of mammalian metabolic activation (Ames, 1984). As a result of this and other tests there was a growing awareness of the presence of potential carcinogens in food and
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the environment (Ames, 1986). The frequent lack of correlation between the ability to induce mutations in bacteria and the positive carcinogenic potential of the same chemical when tested at high concentrations in experimental animals lead to erce debates regarding the validity of classication of mutagens as carcinogens. With the discovery of the genetic basis of cancer with an absolute requirement for mutation, this debate has largely subsided. Reasons for the lack of correlation between the positive long-term animal tests for carcinogenicity and the negative short-term bacterial tests have been persuasively explained by Ames as being due to the use of toxic concentrations of the test agent in animals leading to excessive regenerative proliferation with the consequences outlined in Fig. 1 (Ames and Gold, 1991). The need for adequate testing of chemicals for carcinogenic potential in humans remains a vital public health concern without as yet a totally satisfactory solution. Principal among the problems is that metabolic activation of carcinogens is species and tissue specic. Perhaps genetic engineering will allow the production of a humanized mouse in which the human pattern of metabolic activation of xenobiotics is faithfully replicated (Wolf and Henderson, 1998). 2.3.2. Physical carcinogens Here will briey be discussed the carcinogenic potential of ionizing radiation, both particulate and photon, and of ultraviolet radiation. Although the chemical reactions dier, both classes of physical carcinogens produce DNA damage which, as with the chemical carcinogens, can lead to mutations. Ionizing radiation can cause direct damage to DNA by causing single and double-strand breaks to the DNA helix, and can also induce indirect damage as a consequence of radiolysis of water to yield free radicals (Hall and Angele, 1999). It is of interest that the most biologically damaging radiation produces ionizations that are spaced approximately 2 nm apart the diameter of the DNA double helix (Hendry, 1991). Ultraviolet irradiation, though of insucient energy to produce ions, is absorbed by DNA bases and is suciently energetic to induce chemical reactions. The most relevant of these occurs between two adjacent thymidines in the DNA helix and results in covalent cross linking to form a cyclobutane-linked thymine dimer. This disrupts normal base pairing and presents a formidable obstacle to DNA polymerase, which if not repaired can give rise to mutations. It is no coincidence that approximately 90% of skin cancers arise in sun-exposed areas. A rare inherited disease, xeroderma pigmentosum, which results in acute sensitivity to ultraviolet rays and if not recognized early, an extremely high incidence of skin cancer, is a result in defects in the genes responsible for removal and repair of this DNA damage (Lehmann et al., 1977; van Steeg and Kraemer, 1999). The demonstration that ultraviolet causes DNA damage and that failure to repair this damage results in carcinogenesis, was the rst unequivocal evidence that damage to DNA was directly implicated in human cancer. 2.3.3. Many cancer chemotherapeutic agents are carcinogenic An unfortunate consequence of the DNA damage caused by many chemotherapeutic agents is that patients surviving therapy are at an increased risk of iatrogenic cancer (reviewed in Fraser and Tucker, 1989). Clearly, this concern also exists with
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radiotherapy, however the localized nature of the delivered dose, in comparison to the systemic therapy with chemotherapeutic agents, limits the overall risk of secondary cancer. The cancer chemotherapeutic agents of concern include alkylating agents such as cyclophosphamide, which chemically reacts with DNA in a manner similar to the carcinogens discussed above, and antibiotics such as Doxorubicin which interacts non-covalently with DNA and induces free-radical damage to the genome. As would be expected the risk is proportional to the cumulative dose, with younger patients being more susceptible. The most extensive data for increased risk has been accumulated in survivors of Hodgkin's disease in which the risk for developing any secondary cancer, excluding nonmelanoma skin cancer, was 17.6% vs 2.6% in the general population. It is of interest that the most rapidly developing tumors were leukemias, whose incidence peaked approximately eight years posttherapy, in contrast to solid tumors which rst appeared some ten years post-therapy and continued to increase in incidence with time (Tucker et al., 1988). This is consistent with evidence to be presented later that leukemias require fewer mutations than do solid tumors. It is to be hoped that the new opportunities presented by our increased understanding of the molecular biology of cancer will lead to specic therapies which do not themselves increase cancer risk. 2.4. Most DNA damage is repairable Although we have only recently become aware that man-made chemicals and ionizing radiation induce DNA damage, the genome has been constantly exposed to chemical damage, both endogenous and exogenous, since life began. In order to protect against the immediate and long-term eects of excessive mutational rates, genes, such as p53 discussed in detail below, have evolved whose sole purpose is to survey the genome for damage and/or to repair this damage. In addition genes exist whose function is to repair errors introduced during the replication process. Repair mechanisms dier according to the type of damage for example, the removal of thymine dimers formed as a consequence of UV radiation involves removal of a whole stretch of DNA followed by resynthesis using the opposing strand as template; alkylated bases such as 06 methylguanine can be directly removed without breaking the phosphate backbone; single-strand breaks in the DNA molecule formed as a consequence of damage from ionizing radiation can be directly repaired (reviewed in Frosina, 2000). Perhaps the only type of DNA damage, which is not repairable, consists of DNA double-strand breaks. Here, since both strands are damaged, the cell has no unmodied template that can provide the information necessary to reconstitute the damage strands. Depending upon the site of the doublestrand break this type of damage can lead to cell death, or, of signicance to the carcinogenic process, chromosome breakage and recombination with resulting activation or inactivation of crucial genes. 2.4.1. Defects in DNA repair are responsible for many familial cancer syndromes It is not proposed to exhaustively deal with these repair mechanisms in this section, however, the knowledge that repair capacity exists is vital to our understanding
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that defects in these repair processes play a vital role in carcinogenesis by increasing the rate of mutation and thus the rate of neoplastic progression. Several inherited diseases which predispose to cancer have as their genetic origin defects in DNA repair capacity. These include ataxia-telangiectasia, in which cells are sensitive to X-radiation (Lavin and Khanna, 1999) and the UV sensitivity disease xeroderma pigmentosum referred to above (van Steeg and Kraemer, 1999). The breast cancer susceptibility gene BRCA1 appears essential for repair in response to DNA damage and inactivation of BRCA1 in mouse cells results in increased cell sensitivity to DNA-damaging agents (Chen et al., 1999a). As discussed in more detail later, one of the most frequently mutated genes in human solid tumors is p53. p53, which has been called ``the guardian of the genome'' has among its functions the monitoring of the integrity of the genome and has the capacity to either delay replication until repair has been completed, or, if damage is too extensive, to induce a series of events leading to the programmed death of the cell by a process called apoptosis (Lakin and Jackson, 1999). Mutations in one allele of the p53 gene results in cancer susceptible individuals with the LiFraumani syndrome (Malkin et al., 1990). An additional syndrome, has been demonstrated to be responsible for the non-polyposis form of inherited colon cancer. Here, mutations in enzymes involved in mismatch repair, cause increased genomic instability, The role of mismatch repair deciencies has been recently reviewed (Lynch and de la Chapelle, 1999). 2.5. Cell-cycle checkpoints restrict replication of damaged DNA As discussed above, chemical damage to DNA is itself not a mutagenic event, but if unrepaired can be converted to a mutagenic event during the process of DNA replication. Because DNA synthesis itself is a tightly controlled, highly coordinated process, delays in progression through S-phase as a consequence of DNA damage or insucient availability of protein or DNA precursors frequently result in cell death, chromosomal abnormalities or mutations. Since these latter two events are intimately associated with carcinogenesis, it is not surprising that many of the genes found to be damaged in cancer cells have actions that relate to cell cycle checkpoint control. An overview of this G1/S checkpoint is shown in Fig. 3. To most eectively decrease the probability of mutations, the genome should be damage-free before the onset of replication. To achieve this, and to also ensure that a cell has all the nutritional support required for the synthesis of the new strands of DNA and the protein matrix to allow packaging of the newly synthesized DNA into chromatin, mammalian cells have devised elaborate checkpoints to prevent premature entry into the division cycle. The most signicant checkpoint occurs in late G1, approximately four hours prior to the cell's entry into S-phase. This restriction point, rst identied by Arthur Pardee (1974), represents the nal checkpoint after which the cell is irrevocably programmed to begin DNA synthesis. Activation of this checkpoint control in response to DNA damage, delays entry into S-phase and provides the cell the time necessary for repair. Many years ago, we demonstrated that mouse 10T1/2 cells are acutely sensitive to chemical carcinogenesis when damage occurs just as they exit this checkpoint and progress into S-phase (Bertram
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Fig. 3. Mammalian cell cycle checkpoints. Cells possess multiple mechanisms to prevent inappropriate passage from G1 into S-phase of the cell cycle were DNA synthesis occurs. Central to this is the phosphorylation of RB and RB family members such as p107 by cyclin dependent kinases (CDKs). Phosphorylation releases and activates the transcription factor E2F which in turn initiates the transcription of a number of genes required for S-phase entry and additional cyclins which maintain the phosphorylated state of RB making continued progression through S-phase a mitogen-independent event. Also shown are other cell cycle checkpoints which can be activated in G2 or M phase of cell cycle in response to DNA damage. (For additional discussion see Sherr, 1996, and Reed, 1997). (Figure courtesy of Biocarta.com, ``cyclins and cell cycle regulation'').
and Heidelberger, 1974). This suggests that the most dangerous DNA lesions occur in cells damaged in late G1 and early S-phase after the restriction point, pointing again to the role of proliferation in carcinogenesis. Central to the function of this restriction point is the interaction between the retinoblastoma protein RB and the E2F family of transcription factors. In its unphosphorylated form, RB tightly binds E2F to form a silencing complex restricting
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transcription of genes required for cell cycle entry (Weintraub et al., 1995). In response to mitogen stimulation, D-type cyclins are synthesized together with their associated kinases, the cyclin dependent kinases (CDKs) 4 and 6. Initially the activity of these kinases is inhibited by specic inhibitory factors, the so-called INK4 proteins. Sustained mitogenic stimulus results in the release of kinase inhibition and phosphorylation of RB. This alters its conformation so that it no longer binds E2F which is released and initiates transcription of two major classes of genes: genes such as thymidine kinase, dihydrofolate reductase, thymidylate synthase and DNA polymerase whose actions and products are essential to DNA synthesis, and genes such as cyclin E and CDK-2 whose actions are to maintain the phosphorylated state of RB and allow mitogen-independent passage through the remainder of S-phase cycle. Thus RB phosphorylation constitutes the molecular basis of the restriction point control (reviewed in Reed, 1997). The presence of DNA damage induces an independent block to passage through this restriction pathway. In response to damage the tumor suppressor gene p53 becomes a transcription factor and induces expression of a series of CDK inhibitors, p21, p27 and p57 which function to maintain RB in its unphosphorylated state even in the face of mitogenic stimulation (reviewed in Colman et al., 2000). This control is released once the cell has eectively repaired its damaged DNA. 3. Pathways to cancer: overview In the previous discussion I have attempted to present a brief overview of how mutations are introduced into the genome. The following chapters will present an overview with key examples of the consequences of these mutations to the development of a cancer cell. These examples were chosen to illustrate key genes whose involvement in human cancer has been most clearly demonstrated and, as an added criterion, I have chosen examples of genes that show great promise as targets for molecular intervention. This list of genes is by no means comprehensive but is intended to illustrate the many discrete pathways utilized by cancer cells in order to achieve unlimited replication. The incidence of most human cancers increases dramatically with age, and to briey repeat for emphasis what was discussed above, in these cancers which are predominantly tumors of epithelial origin, some 47 independent events must take place before such a cell can be considered malignant. From a functional perspective these mutations have two distinct consequences: they allow the inappropriate expression or activation of genes, or conversely, they result in the functional inactivation of the gene or its protein product. Genes which are activated by mutation are called oncogenes; those inactivated by mutation are called tumor suppressor genes. As may be deduced, oncogenes are involved in signaling pathways which stimulate proliferation, while most human suppressor genes code for proteins which normally act as checkpoints to cell proliferation or cell death. In discussing these genes and the mutations responsible for their altered function, it will be apparent that ``Murphy's Law'', which states: `ànything that can go wrong
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Fig. 4. Pathways to cancer. As a cell accumulates carcinogenic mutations, it progresses through preneoplastic stages characterized by the acquisition of properties, listed under the heading ``progression'' required for its survival. At each stage it must overcome control mechanisms, listed under ``protection'', which would act to eliminate the mutated cell from the host. The nal control of therapeutic intervention is becoming much more selective with the identication of crucial targets which distinguish cancer cells from their normal counterparts.
does go wrong'', is applicable to the genesis of a cancer cell. It must also be remembered that the mutations which are available for analysis represent only those successful mutations allowing uncontrolled proliferation. This situation again mirrors that found in the Darwinian evolution where only benecial changes survive as new species. At the risk of over-simplication, ve major pathways must be activated or inactivated in the genesis of a cancer cell. These are presented in Fig. 4 and are listed below: development of independence in growth stimulatory signals; development of a refractory state to growth inhibitory signals; development of resistance to programmed cell death, i.e., apoptosis; development of an innite proliferative capacity, i.e., overcoming cellular senescence; development of angiogenic potential i.e. the capacity to form new blood vessels and capillaries. 4. Cancer cells are independent of external growth signals Normal cells proliferate in response to an array of external, mostly locally produced, growth factors produced by one cell type to activate a second. These factors
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include, epidermal growth factor (EGF), broblast growth factor (FGF), tumor growth factor alpha (TGF-a) and platelet derived growth factor (PDGF) produced by platelets at sites of wounding. These growth factors exert their proliferative action after binding to appropriate receptors and induce a cascade of responses most of which involve phosphorylation events. Tumor cells have found mechanisms to enable constant activation of these proliferative signals. These mechanisms dier from cancer to cancer depending upon cell type, and within a specic tumor type by pure chance, but the end result is continued mitogenic stimulation, centering as discussed above, on cyclin D. Examples are given below and are organized in terms of their position in the signal transduction pathway from the plasma membrane to the nucleus. 4.1. Inappropriate synthesis of growth factors A major growth factor in mesenchymal cells is PDGF. When malignantly transformed these cells give rise to sarcomas, meningiomas and gliomas and other connective tissue tumors. Inappropriate expression of PDGF can be demonstrated to induce neoplastic transformation in rodent cells. Various isoforms of PDGF are expressed in gliomas and in sarcomas whereas expression cannot be demonstrated in the normal cells giving rise to these tumors (Westermark et al., 1995). At present it is not clear if these forms of PDGF need to be secreted in order to activate the receptor for PDGF, or whether this receptor can be activated internally. In any event, autocrine stimulation by inappropriately expressed PDGF can be demonstrated to activate downstream signaling pathways leading to mitosis (Black et al., 1994). 4.2. Inappropriate expression of growth factor receptors 4.2.1. Erb-B There is currently considerable interest in the role of an overexpressed plasma membrane receptor for heregulin, a growth factor related to EGF. Erb-B is overexpressed in approximately 30% of breast carcinomas and is associated with a worse clinical outcome. In most cases analyzed, over-expression is a consequence of gene amplication, i.e., an increased copy number, usually as a result of end-to-end replication of this gene at the same chromosome location. Amplication can frequently be detected microscopically as homogeneously staining regions at the chromosomal location 17q12. The erb-B receptor when activated by its ligand heregulin, becomes an active Tyr kinase as do many cell surface receptors, and this Tyr phosphorylation stimulates downstream events resulting in mitotic activation (Neve et al., 2000). In addition to its mitogenic eects, overexpression of erb-B in breast carcinoma cells has been shown to lead to increased secretion of vascular endothelial growth factor (VEGF) which stimulates the angiogenesis necessary for progressive growth of the tumor (Yen et al., 2000). It is of interest that the protooncogene ras, (see below) which is very infrequently mutated in breast carcinomas becomes strongly activated in the presence of overexpressed erb-b, perhaps making this additional mutation unnecessary (von Lintig et al., 2000). It is at present unclear why overexpression alone should give rise to activation of this receptor, as no ac-
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tivating mutations have been described and there seems to be no concomitant increase in expression of its ligand. However, there is recent evidence that simultaneous secretion of prolactin occurs in erb-B over-expressing breast carcinoma cells and that this hormone may be responsible for receptor activation (Yamauchi et al., 2000). If substantiated, prolactin and its receptor would provide another exciting target for breast cancer therapy. Recently, a humanized monoclonal antibody directed against erb-B has been developed and has received FDA approval. Its use has been associated with dramatic tumor responses especially when combined with conventional chemotherapy (Pegram et al., 2000; Pegram and Slamon, 1999; Stebbing et al., 2000). This antibody, Herceptin, represents only the second monoclonal antibody approved for human use against cancer and represents an exciting prelude to the increasing numbers of therapeutically useful antibodies being developed against targets identied by molecular biology. Reports that erb-2 is expressed in other human tumors, including gastric carcinoma (Allgayer et al., 2000) and in 21% of several types of lung carcinoma (Scheurle et al., 2000), suggests that Herceptin may also be useful at these organ sites. 4.3. Activation of downstream signal transduction pathways 4.3.1. c-abl Chronic myelogenous leukemia (CML) is always associated with an abnormal chromosome, called the Philadelphia chromosome, formed by the fusion of a portion of chromosome 22 to chromosome 9. This chromosomal translocation occurs between a small 5.8 kb break point cluster region (BCR) within a gene called BCR on chromosome 22 and a region containing portions of the c-abl gene on chromosome 9. This fusion results in the transcription of a chimeric mRNA that is translated into a chimeric fusion protein BCR-ABL of 210 kb size. While the exact molecular consequences of this fusion protein are not yet clear, in murine models it is sucient to induce leukemia (Daley et al., 1990) and it may also be sucient in humans to induce the aberrant proliferation characteristic of CML. A similar translocation occurs in many cases of acute lymphocytic leukemia (ALL); here the break point in the BCR gene is in the rst exon leading to a shorter mRNA transcript size of 7.0 kb. However, the contribution of the c-abl gene is identical. c-abl, i.e., the normal cellular gene, is a Tyr kinase with structural similarities to many similar kinases involved in signal transduction. The BCR gene has serine kinase activity and it is believed that serine phosphorylation by BCR on the ABL portion of the chimeric protein results in constitutive activation of the Tyr kinase ability of this protein. However, it is at present unclear what is the substrate for this kinase and how subsequent events lead to aberrant proliferation. However, there is persuasive evidence that ABL signaling results in the inappropriate expression of many growth factors in CML cells. This topic has recently been reviewed (Sattler and Salgia, 1997). The presence of the BCR-ABL transcript is a sensitive indicator of disease state and the use of PCR to measure its expression in blood oers an at-
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tractive and rapid means of measuring disease progression or response to chemotherapy without the necessity for bone marrow aspiration (Branford et al., 1999). The apparent central role of the ABL kinase activity has led to eorts to synthesize specic inhibitors. One such inhibitor, STI 571 has been shown to be surprisingly specic in its inhibition of this kinase and not to be a general inhibitor of Tyr kinase. These kinases are used in many signal transduction processes, and if inhibited would be toxic to normal as well as neoplastic cells. Studies using X-ray crystallography have recently shown this specic activity is due to the ability of STI 571 to bind the catalytic site of the kinase only when the kinase is in its inactive state (Schindler et al., 2000). Pre-clinical studies in the mouse have shown that STI 571 can eradicate BCR-ABL positive human leukemia cells (le Coutre et al., 1999). Reports of a number of clinical trials of this compound have recently been reported in abstract form and the results look very encouraging: 96% of CML patients showed complete responses (Druker, 1999) with a 55% response rate in patients with ALL (Talpaz, 2000). These studies clearly indicate the crucial importance of an active BCR-ABL Tyr kinase to the continued proliferation of these leukemia cells and again indicate the power of rational drug design against a molecularly dened target. 4.3.2. ras This gene represents a family of signal transduction molecules which are plasmamembrane associated and which interact with a large series of downstream signal molecules with multiple functions including the stimulation of proliferation. The discovery of mutated ras was important for two reasons: (1) it represented the rst oncogene to be discovered in human cancer cells; (2) it represents the oncogene most widely activated in human cancers with incidence levels ranging from 90% in the pancreas, 50% in the colon, to 30% in the lung with comparable levels being found in most other solid tumors (Bos, 1989). Mutant ras was discovered by Weinberg's group (Parada and Weinberg, 1983) in mouse 10T1/2 cells which had been neoplastically transformed by Heidelberger's group a group in which this author was a member (Rezniko et al., 1973). Its presence was conrmed in human bladder carcinoma cells (Parada et al., 1982). In these studies fragments of DNA from the malignant cells were transfected into growth-controlled immortalized mouse broblasts. It was found that a small fraction of cells themselves became neoplastically transformed and that these transformants contained human DNA sequences encompassing the mutated ras gene. The importance of this nding cannot be overstated; just as the discovery of a DNA repair deciency for UVinduced lesions had identied DNA as a target for human carcinogens (Cleaver and Bootsma, 1975), now some 9 years later, a gene had been identied which could induce neoplastic transformation. This discovery, and the later discovery of the rst tumor suppressor gene RB, provided the groundwork for the enormous explosion in our understanding of human cancer that occurred from that date forward. ras is one of a large family of proteins that can bind GTP and act as a signal transduction molecule. The active state of ras is produced by the binding of GTP
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which produces a conformational change and allows interactions of ras with other downstream signaling molecules. The non-mutated, proto-oncogene form of ras binds then hydrolyses GTP to GDP which is then released and returns ras to its inactive state. Mutations in the proto-oncogene c-ras act to decrease the ability of this molecule to act as a GTPase. Since GTP is not released by mutated ras it now acts as a permanently activated signal transduction molecule. Studies of ras mutations have revealed mutational hotspots centered on codons 12, 13 and codon 61. At codon 12 the most frequent alteration is a G 3 T transversion causing a glycine 3 valine amino acid substitution (Minamoto et al., 2000). It is of interest that the induction of ras mutations appears to be an early event in the carcinogenic process. For example, in the lung approximately 39% of hyperplastic lesions, considered a carcinogenic precursor lesion, vs 42% of adenocarcinomas had codon 12 mutations. Furthermore, only infrequently was the same mutation found in geographically separate samples taken from the same patient indicating that independent events had given rise to these lesions (Westra et al., 1996) a result consistent with the eld cancerization theory of tobacco carcinogenesis. The presence of codon 12 mutations in the ras gene has been exploited recently as a sensitive indicator for the presence of pre-neoplastic cells in samples as diverse as feces, for the detection of early colon cancer, in bronchial washings for lung cancer, and duodenal samples for pancreatic cancer (Minamoto et al., 2000). The molecular basis for the hotspots on codons 12, 13 and 61 became clear when the ras protein was crystallized and its three-dimensional structure made apparent. As shown in Fig. 5, the binding pocket for GTP is dened at one edge by the amino acids coded by codons 12 and 13, while a crucial region of the protein involved in hydrolysis of GTP to GDP is encoded by codon 61. Thus both mutated portions of this protein are intimately involved in either binding or hydrolysis, thus explaining the biochemical observations that mutated ras lacks GTPase activity. In spite of intensive research, the full range of cellular and molecular consequences of activated ras are still not understood completely. Indeed, because of the plethora of signaling pathways that exist between plasma-membrane associated ras and the nucleus, the situation appears to be becoming more, rather than less complex (Campbell et al., 1998; Shields et al., 2000). ras is now known to associate with a second GTPase necessary for signal transduction activity called p120GAP, and its normal association with these proteins critically depends upon the amino acids coded by codons 12 and 61, again underlining the essential nature of these domains (Schezek et al., 1997). Recent studies have indicated that ras transformation is dependent upon the third protein Rho, which is a member of another large family of GTPases. These studies demonstrated that a dominant negative mutant of Rho was capable of blocking ras transformation in cell culture, while an activated form of Rho enhanced ras transformation (Prendergast et al., 1995). These Rho family members are known to be regulators of the actin cytoskeleton, to activate kinase cascades, and to regulate gene expression, thus making them important players in the overall regulation of cell homeostasis (McCormick, 1998; Zohn et al., 1998). There is also evidence that a third GTPase, RAC is also involved in ras signaling (Qiu et al., 1995). This signaling
J.S. Bertram / Molecular Aspects of Medicine 21 (2001) 167223 187
Fig. 5. Carcinogenic mutations in ras. As determined by X-ray crystallography, the mutagenic hotspots in ras are located in codons 12, 13 and 61, are localized to regions of the protein involved in interactions with GTP and the accessory protein p120. (Reproduced from Schezek, 1997, with permission.)
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cascade therefore interacts with two important regulatory pathways: (1) in organization of the cytoskeleton, a structure essential for maintaining normal morphology and perhaps also for nuclear functions, and (2) via activation of the Raf-1/mitogenactivated protein kinase pathway in causing activation of transcription factors such as C-jun by way of a series of signal transducing kinases cumulating in MAP kinase (Campbell et al., 1998). Because of the apparent central role of ras mutations in many human solid tumors, there have been many eorts to develop specic therapies directed against this oncogene. The most promising of these appears to be the development of drugs which inhibit the association of ras with the plasma membrane. This association is a result of the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase. Several inhibitors of this enzyme have been developed but unfortunately they appear to possess unacceptable side eects (Rowinsky et al., 1999). Some of these side eects may be related to inhibition of Rho function (Lebowitz and Prendergast, 1998). 4.4. Inappropriate activation of nuclear transcription factors The ultimate target of the oncogenes discussed above is to achieve activation of transcription factors such as cyclin D, primarily through protein phosphorylation. A more direct means would achieve direct activation of these transduction factors thus circumventing the complexity and feedback controls which exists in upstream signal transduction pathways. As suggested in the introductory section, Murphy's Law is obeyed in carcinogenesis and a number of tumors have evolved means to cause direct activation. This is achieved by overexpression of the transcription factor or the production of mutated proteins with altered functions. Important examples are given below. 4.4.1. Inappropriate expression of c-myc, a transcription factor C-myc is a transcription factor whose expression is tightly regulated in normal cells and is only expressed in S-phase of the cell cycle. In a large number of human tumor types this regulated expression is lost, and c-myc becomes inappropriately expressed and/or overexpressed throughout the cell cycle driving cells continuously towards proliferation. If this inappropriate expression occurs in epithelial cells and is the only genetic alteration then growth regulatory genes, in general the tumor suppressor genes to be discussed below, restrict this proliferation and in many cases will cause apoptosis. However, if these genes are themselves mutated inappropriate proliferation occurs. In contrast, hematopoietic cells appear to have fewer controls on their proliferation, perhaps explaining the early onset of many cancers aecting these cells, and c-myc expression can be oncogenic. Other family members of these transcription factors are N-myc, overexpressed in neuroblastoma, and L-myc, which is overexpressed in small cell lung cancer. One of the most interesting examples by which the regulation of c-myc expression is perturbed is in Burkitt's lymphoma. Here, a characteristic chromosomal translocation fuses the c-myc gene on chromosome 8q24 with either the heavy chain, j or k
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locus of the immunoglobulin genes on chromosomes 14q23, 2p12 and 22q11, respectively. This results in the removal of c-myc from normal cell cycle control and places it under the control of genes normally activated by infection. There is a strong association between prior infection with EpsteinBarr virus (EBV) and Burkitt's lymphoma. EBV gene products increase the translocation rate of c-myc (Li and Maizels, 1999) and viral infection itself could be expected to increase the transcription from immunoglobulin loci. There is also evidence that translocation may give rise to additional mutations in the c-myc gene by the antibody hypermutation mechanism. This has been demonstrated in a B cell line which mutates the c-myc allele that is translocated into the IgH locus whilst leaving untranslocated c-myc allele intact (Bemark and Neuberger, 2000). C-myc functions as a heterodimer with a second transcription factor, max, and while it seems clear these two function together to facilitate neoplastic transformation the exact sequence of events has yet to be discerned. A major problem is the complexity of cellular events modied by c-myc. It is now clear that this gene participates in many aspects of cellular function, including replication, growth, metabolism and dierentiation (Liao and Dickson, 2000). One central confusing feature of c-myc overexpression in many cells is that it induces apoptosis, apparently by increasing transcription of the cyclin-dependent kinase cdc25A, which can induce apoptosis in cells depleted of growth factors (Galaktionov et al., 1996). It would seem that in order to overcome apoptosis, tumor cells must also possess other mutated pathways (reviewed in Homan and Liebermann, 1998). Most breast cancers overexpress c-myc, and this overexpression acts to facilitate the ability of erb-B to cause proliferation (Neve et al., 2000). There is also suggestive evidence that the promoting eect of estrogen in estrogen receptor (ER) positive breast tumors may in part be due to the ability of the estrogen receptor to cause increased transcription of the c-myc gene and also of telomerase, an enzyme required for cell immortalization, a topic to be discussed below (Neve et al., 2000). 4.4.2. Mutation of a nuclear hormone receptor leads to blocked dierentiation A second example of how a chromosome translocation can give rise to a chimeric protein with altered function is the translocation between the nuclear receptor for all-trans retinoic acid (RAR-a ) and one of two other chromosomal locations. The result is a nuclear receptor with altered signaling properties manifested by strongly enhanced activity as a transcriptional repressor leading to arrested dierentiation. In the 1970s, there was intense interest in the development of retinoids, the natural and synthetic derivatives of the locally produced hormone, retinoic acid, as cancer chemopreventive agents. One test for retinoid function was the production of terminal dierentiation in a human promylocytic leukemia cell line, HL-60 (Strickland et al., 1983). The success of retinoids in model cell culture systems of dierentiation led Chinese physicians to access the ability of all-trans retinoic acid to induce remissions in human promylocytic leukemia, PML. This clinical trial was a dramatic success and was the rst example of dierentiation therapy in cancer (Huang et al., 1988). The studies were followed up by the French group led by
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Degos who, in 1990, conrmed these dramatic ndings in the majority of PML patients (Castaigne et al., 1990). Molecular and cytogenetic studies soon revealed the molecular basis for this disease; a chromosomal translocation which always involved the RAR-a locus (Castaigne et al., 1992; Chen et al., 1991). The translocation was found to involve one of two other chromosomes that gave rise to chimeric proteins: PML-RAR-a and PLZF-RAR-a. PML cells containing the former translocation were found to be sensitive to pharmacological doses of retinoic acid, whereas those expressing the PLZF-RAR-a fusion protein were found resistant. The molecular basis for both the block to dierentiation caused by the presence of these fusion proteins and the refractory nature of the PLZF-containing protein has now been resolved. Retinoic acid nuclear receptors are now known to act both as transcriptional silencers and transcriptional enhancers depending upon the binding of retinoic acid. In the absence of the ligand, RAR binds as a heterodimer with RXR, a closely related receptor, to retinoic acid responsive elements found in the promoter regions of retinoid responsive genes and silences transcription from these genes. This is achieved by the binding to the RAR/RXR complex of repressor proteins such as N-CoR, which possesses histone deacetylase activity. This enzyme removes acetyl groups from core histones and promotes the tight binding of DNA to histones thereby preventing access to transcriptional factors. Upon binding of retinoic acid to RAR-a a conformational change is induced which causes release of the repressor and its exchange for proteins with transcriptional activity. One of the actions of these transcription factors is to acetylate the core histones releasing DNA and allowing access to the transcription complex (Chen et al., 1999b; Wole, 1997). In studies of the PML fusion proteins it was found that PML-RAR-a would release the repressor in the presence of high doses of retinoic acid, whereas the PLZL-RAR-a fusion protein had an additional site of interaction which prevented release. In the presence of an inhibitor of histone deacetylase such as Trichostatin A, even PLZL-RAR-a containing PML cells regained sensitivity to retinoic acid and terminally dierentiated (Grignani et al., 1998; He et al., 1998). The studies very nicely illustrate successful transitional research from the laboratory to the clinic resulting in a novel, highly eective therapy for a previously refractory disease. 5. Cancer cells become refractory to growth inhibitory signals: the discovery of tumor suppressor genes The discovery of the ability of oncogenes to induce neoplastic transformation when transfected into immortalized mouse cell lines, initially seemed to answer many basic molecular questions about the molecular origins of cancer. However, it soon became clear that this was not the whole picture and that there existed other genes that could suppress transformation. For example, most of the studies demonstrating oncogene activity of ras and other oncogenes had been performed in a mouse NIH/3T3 cell line that was already immortal, easy to transfect and, from
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personal experience, was unstable and on the brink of spontaneous transformation. When oncogenic ras was instead transfected into hamster cells which had not undergone immortalization, transformation did not occur. However, after spontaneous immortalization in culture, ras was capably of inducing transformation indicating that ras did not act alone (Newbold, 1985; Newbold and Overell, 1983). These and other discoveries led to renewed interest in earlier observations by Harris that the fusion of normal cells with malignant cells frequently resulted in loss of tumorigenicity in the hybrids; results which strongly suggested that normal cells possessed genes which could suppress tumorigenicity an activity lost in tumor cells (Harris et al., 1969). The studies were taken further by Stanbridge's group in particular who developed techniques for transfer of single chromosomes and showed that some, but not all chromosomes derived from normal cells could achieve suppression of the neoplastic phenotype (Anderson and Stanbridge, 1993). The stage was thus set for the identication of these genes; a process which has led to completely new insights into cell control processes. This search is still continuing. In contrast to the mutagenic activation of oncogenes, where, because of the dominant nature of this activation step, mutation of a single allele is sucient to induce some aspects of the neoplastic phenotype, oncogenic mutations in tumor suppressor genes result in a lack of function. There are two important consequences of these dierences: rst, because in most cases the normal suppressor allele can function in the presence of the damaged allele, both copies must be inactivated before loss of function is manifested; second, again in contrast to oncogenes whose dominant eects would preclude normal embryonic development, loss of one allele of a suppressor gene is generally silent and allows germ-line inheritance of the damaged allele. Familial inheritance of mutated tumor suppressor genes has tragic results in leading to cancer-prone individuals, however, the study of such individuals has allowed signicant breakthroughs in identication of the genes responsible. 5.1. The retinoblastoma gene RB Retinoblastoma is a childhood cancer which is the most common malignant eye tumor and is responsible for 1% of cancer deaths in children. Approximately 40% of cases are familial and the remainder sporadic. In familial disease, retinoblastoma may be present neonatally or develops shortly after birth. It usually presents unilaterally in which multifocal tumors may be present. There is a high probability of the second orbit becoming involved within approximately four years. Survivors have an increased chance of secondary malignancies particularly osteosarcoma, brosarcoma and Wilm's tumor. In contrast, most of the sporadic cases have only a low incidence of involvement of the second orbit and a low incidence of secondary malignancies. This pattern of inherited disease led Knudson to hypothesize a two-hit theory of carcinogenesis. This hypothesized that in familial cases children are born with one damaged and one normal allele which itself becomes damaged shortly after birth. This explains the frequent occurrence of bilateral retinal tumors in the
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inherited form of this disease and the high frequency of tumors in other organs. Individuals with spontaneous disease, characterized by a low frequency of bilateral events and of distant tumors, were hypothesized to be born with two normal alleles resulting in a low probability that both alleles will be damaged in both retinas (Knudson et al., 1975; Knudson, 1978). This theory received support from the observation of frequent chromosomal abnormalities in retinal tumors involving lesions in both copies of chromosome 13q14 (Lemieux et al., 1989). 5.1.1. RB functions to restrict entry into S-phase The retinoblastoma gene was cloned and found to encode a nuclear protein, RB, which acts to control entry into the cell cycle. As discussed above in the section on cell cycle control, RB is normally not phosphorylated and associates with the transcription factor E2F. This combination acts as a silencing complex (Weintraub et al., 1995), whose mechanism has recently been elucidated. Just as described for the retinoic acid receptor, the complex maintains core histones in a non-acetylated form and restricts access to transcription factors (Brehm et al., 1998). After mitogen stimulation cyclin D/cdk4 phosphorylates RB in the C-terminal region of the protein which disrupts the binding region for E2F and causes its release. This altered conformation also appears to allow access of cdk2 during S-phase which produces additional phosphorylation further inhibiting E2F binding (Harbour et al., 1999). As described above, this disruption of RB/E2F allows transcription of crucial genes required for cell cycle entry. In view of the apparent central role of the RB proteins as a gatekeeper to S-phase entry, it is rather surprising that a wider spectrum of tumors is not observed in cases of familial retinoblastoma. There is evidence that only a sub-set of cells in the developing retina is susceptible to RB deletion and it has been suggested that these cells would be normally programmed for apoptosis during development, thus restricting disease to early childhood. Interruption of this program, perhaps through continued stimulation into the cell cycle as a consequence of RB deletion, may well be responsible for this tumor (Gallie et al., 1999). It is curious also that the human retina appears abnormally sensitive to this mutation. In mice, targeted heterozygous disruption of the RB locus allows the development of the embryo until about the day 14 when death occurs because of deciencies in blood-forming elements. Homozygous deletion, the genetic equivalent of human RB carriers, results primarily in pituitary adenomas, not retinoblastomas (reviewed in Vooijs and Berns, 1999). The ability of mouse embryos to live to day 14, and the lack of more extensive tumor development in both mouse and human heterozygotes suggests that other genes may well be able to substitute for RB. Two such genes have now been discovered, p107 and p130, and it seems likely that they have overlapping, but not always equivalent functions in the cell (Classon et al., 2000). Analysis of inactivating mutations in the RB gene indicate that most are the result of CT transitions at CpG dinucleotides (CpGs). Such recurrent CpG mutations, are likely the result of the deamination of 5-methylcytosine within these CpG islands. A major proportion of these mutations result in truncated proteins as the result of the premature termination of protein synthesis either through the introduction of chain-
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termination sequences or altered spice sites resulting in changes in the processing of mRNA (Lohmann, 1999; Mancini et al., 1997). These deletions in the RB protein primarily aects sites involved in nuclear localization and phosphorylation. They also disrupt the sites involved in binding by certain tumor viruses (Templeton et al., 1991), a topic to be discussed below. 5.1.2. RB gene therapy The realization that mutations in tumor suppressor genes result in a loss of function which is recessive and requires both copies of the gene to be damaged, opens up the possibility that gene therapy may be used to reintroduce one or more copies of the damaged gene. This has been successfully achieved in cell culture experiments utilizing RB negative human tumor lines. In one such experiment, reintroduction of RB into several human carcinoma cell lines led to a loss of invasion capacity but not necessarily loss of tumorigenicity in immunocompromised mice (Li et al., 1996). In a second experiment, reintroduction of RB function in human prostate carcinoma cells led to decreased tumorigenicity in mice but not to altered growth rates in cell culture (Bookstein et al., 1990). That other RB family members may also function as tumor suppressor genes has recently received conrmation in studies of human lung carcinoma lines which lack expression of functional p130; reintroduction of this gene strongly suppressed tumorigenicity in nude mice (Claudio et al., 2000). 5.2. p53 p53 is a tumor suppressor gene which monitors stress and directs the cell towards an appropriate response. The types of stress to which p53 is responsive include: anoxia; insuciency of nucleotides for DNA synthesis; the inappropriate activation of oncogenes; and DNA lesions as diverse as single-strand breaks and covalent adducts. There is also growing evidence that p53 monitors telomere length and thus is critically involved in cell senescence. Upon activation, p53 induces either cell cycle arrest or apoptosis. For these reasons, p53 has been called ``the guardian of the genome''. Its central role in eliminating the genomic damage so central to the successful genesis of the cancer cell is reected by the fact that over 70% of human cancers have defects in this gene, and virtually all have defects in genes upstream or downstream of p53 function (reviewed in Levine, 1997). As with RB, cancer-prone families have been identied which possess p53 mutations in one allele (the Li Fraumani syndrome). The importance of p53 function is indicated by the incidence of cancer in such individuals of approximately 100% (Malkin et al., 1990). 5.2.1. p53 mutations Analysis of p53 mutations revealed mutational hotspots localized in evolutionary conserved regions indicating that these regions were central to p53 function. When the crystal structure of p53 in a complex with a p53-specic DNA sequence was elucidated by X-ray methods, reasons for these mutational hot spots became apparent. Virtually all were localized in regions of the protein with close proximity to
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Fig. 6. Mutational hotspots in p53 are involved in protein/DNA interactions. In panel A is shown a histogram of the localization of mutations in p53 isolated from numerous tumors. These are localized to exons III, IV and V which code for regions of the protein involved in DNA binding. X-ray crystallography of wild-type p53 (shown as a green ribbon) associated with DNA (purple) reveals that these highly mutated regions (shown in white) are intimately involved in DNA binding. (Reproduced from Cho et al., 1994 with permission from the American Association for the Advancement of Science.)
DNA; these mutations act to either eliminate critical contacts with DNA or to destabilize key protein structures required for DNA binding (Cho et al., 1994). As discussed, the only mutations observed in successful cancers are those mutations that oer a selective advantage. It may be deduced therefore that this localization of mutations to the DNA binding domain identies regions critical to the function of p53 as a tumor suppressor gene. The mutational hotspots and the interaction with DNA are shown in Fig. 6. The high frequency with which p53 is mutated in human cancers has allowed its analysis in apparently identical tumors arising in dierent circumstances. Molecular evidence has been obtained for the involvement of dierent etiologic agents. This new discipline of molecular epidemiology has for instance revealed that hepatocellular carcinomas in China possess a dierent mutational spectrum than the same cancers in Japan or in Western countries perhaps reecting the role of aatoxin contamination of food in rural China (Aguilar et al., 1994); that lung
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cancers arising in nonsmokers living in regions with heavy air pollution contain mutations consistent with exposure to carcinogenic hydrocarbons (Rusin et al., 1999) and that the mutational spectrum in lung cancers arising in workers exposed to chromium, diered from the spectrum found in hydrocarbon exposed workers (Harty et al., 1996). The mutational spectra of p53 in human tumors are shown in Fig. 6. 5.2.2. p53 monitors genomic integrity p53 in solution aggregates as a tetramer and has a short half-life of approximately 20 min in non-stressed cells. This is due to its association with a protein, Mdm2 that targets it for degradation. In response to stress, p53 becomes post-translationally modied, initially by phosphorylation and later by acetylation. These modications cause release of Mdm-2, with resultant increase in half-life, and activates p53 as a transcription factor. Mutated p53 also has an increased half-life resulting in the positive immunostaining of p53 in cells containing mutated copies and in S-phase cells (Momand et al., 2000). While much remains to be learned about the mechanisms by which p53 senses damage there is evidence that the cell cycle checkpoint kinase, Chk2/hCds1, when in contact with damaged DNA becomes activated and phosphorylates p53 on serine 20. This results in dissociation of preformed complexes of p53 with Mdm2. The activated form of Chk2/hCds1, but not inactive mutants, causes G1 arrest in cells containing wild type but not mutant p53 (Chehab et al., 2000). These results are consistent with other reports that the inactivation of Chk2/hCds1 in knockout mice renders them unable to mount a p53 response after irradiation (Hirao et al., 2000), and that merely increasing the amount of p53 within the cell (which may be considered to be comparable to stabilization of the protein) was insucient to increase transcription of p53 activated genes. Transcriptional activation only occurs after post-translational modication induced by DNA damage (Xiao et al., 2000). At present it is not clear if the checkpoint kinase itself senses DNA damage or responds to unknown additional signals. There are several possible contenders: for example, individuals with the human genetic disorder ataxia-telangiectasia are unable to repair X-ray damage and have defects in the gene ATM. The protein product has been shown to bind to damaged DNA and to be capable of phosphorylating p53 (Lavin and Khanna, 1999). 5.2.3. p53 is a transcription factor Activation of p53 as a transcription factor results in increased transcription of several genes involved in DNA repair, G1 arrest and apoptosis. Events concerned with DNA repair and G1 arrest are the best understood. Transcription of Gadd45 by activated p53 produces a protein which can recognize an altered chromatin state and caused destabilization of histoneDNA interactions by interacting directly with the four core histones. The expected result is increased accessibility of damaged DNA to proteins involved in repair processes (Carrier et al., 1999). The role of GADD45 in DNA repair is further suggested from studies in mice in which this gene was deleted; these mice have increased genome instability and increased incidence of carcinogenesis (Hollander et al., 1999). Cells from these knockout animals were also
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Fig. 7. Pathways by which p53 causes cell cycle arrest or alternatively apoptosis. Shown is the activation of p53 in response to DNA damage and its role in causing transcription of genes such as GADD45 and p21 which facilitate DNA repair and inhibit S-phase entry or alternatively, the induction of a cascade event leading to apoptosis.
decient in ultraviolet repair (Smith et al., 2000). GADD45 also appears to have a second function in causing G2 arrest, probably by its known association with the Cdc2-cyclin B1 protein complex, causing its dissociation as a result of direct interaction between the Gadd45 and Cdc2 proteins (Jin et al., 2000). P53 also transcriptionally activates expression of p21, which as discussed above binds to a number of cyclin/Cdk kinases so as to inhibit their phosphorylation of RB and thus inhibit G1/S-phase transit (El Deiry et al., 1993; El Deiry et al., 1994). Thus activation of p53 induces genes which cause cell cycle arrest, thereby allowing time for DNA repair, and also modies the state of chromatin so as to allow access of repair proteins to DNA. These pathways are shown in Fig. 7. Mechanisms by which p53 stimulate apoptosis are poorly understood as are the conditions that determine whether p53 should signal cell cycle arrest, a reversible phenomena, or apoptosis which is clearly irreversible. One of the problems appears to be that dierent cells respond dierently to stressful stimuli making creation of an overall model dicult. Nevertheless, it is clear that in many situations but not all, p53 is required for apoptosis. For example, in p53 homozygous knockout mice, lymphocytes do not undergo apoptosis in response to X-irradiation, however they remain sensitive to glucocorticoid-induced apoptosis indicating that other pathways exist (Templeton et al., 1991). There is also evidence that p53 can trigger apoptosis by mechanisms requiring transcription and also by transcription-independent mechanisms. Apoptosis is triggered by changes in mitochondrial membrane potential leading to release of cytochrome C, calcium and other factors. Recent studies
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have shown that p53 localizes to mitochondria at the onset of p53-dependent apoptosis but not during p53-independent apoptosis or p53-mediated cell cycle arrest. Moreover targeting of p53 to mitochondria using peptide leader sequences is sucient to induce apoptosis strongly suggesting a central role for this translocation (Marchenko et al., 2000). In summary, p53 induces cell death by a multitude of molecular pathways involving activation of target genes and transcriptionally independent direct signaling and it is clear that much remains to be learned about the mechanisms of p53 induced apoptosis. 5.2.4. Loss of p53 alters response to chemotherapeutic agents The frequent loss of p53 function in human tumors may be expected to alter their response to chemotherapeutic agents which induce DNA damage (Lowe et al., 1993). This indeed has been reported with tumors containing mutated p53 being refractory to agents such as Doxorubicin (Aas et al., 1996) which damages DNA. However, this does not seem to be a universal occurrence as colon carcinoma cells genetically engineered to be p53 negative, were recently reported to be sensitized to killing by DNA damaging agents but be resistant to 5-FU, an antimetabolite (Bunz et al., 1999). 5.2.5. p53 gene therapy The loss of p53 in the majority of human cancers and the resulting resistance to apoptosis which would normally be triggered by genome instability or by many cancer chemotherapeutic agents, makes restoration of p53 an attractive target for gene therapy. In pre-clinical studies it has been shown that introduction of wild-type p53 into prostate and lung tumor cells containing mutated p53 results in ecient apoptosis, and that injection of p53 vectors into tumors growing in immunosuppressed nude mice results in decreased growth of these tumors (Eastham et al., 2000; Overholt et al., 1997). These very positive pre-clinical studies have fueled eorts to achieve the same results clinically. A number of clinical trials, primarily in head and neck cancer are currently underway (reviewed in Clayman, 2000a; Clayman, 2000b). Early results appear very encouraging. Direct injection of a replication-defective adenoviral expression vector encoding wild-type p53 has been evaluated in several studies in patients with lung cancer: in incurable non-small cell cancer one study reported a partial response in 8% and stabilization in 64% (Swisher et al., 1999); a second study found transient local disease control in 4/6 patients (Schuler et al., 1998); gene therapy was used as an adjuvant to surgery in historically incurable squamous cell carcinoma of the head and neck with 27% of patients reported disease-free after 18 months (Clayman et al., 1999). Use of a retroviral vector to deliver wild-type p53 has also been successful in patients with non-small cell lung cancer who had failed conventional therapy; 3/9 experienced tumor regression and 3/9 stabilization (Roth et al., 1996). Two major problems confront eective therapy: because p53 acts as a tetramer, the presence of mutated p53 can frequently act in a dominant manner and inhibit actions of the wild type protein. An innovative approach to overcome this limitation is the use of trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wild type p53 activity. In one report, sucient p53
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activity was generated to cause transcriptional activation of p53 responsive genes (Watanabe and Sullenger, 2000). A second problem common to other gene therapy protocols is the delivery of sucient copies of the molecular construct to target cells. An entirely dierent approach has been developed to exploit dierences between normal cells expressing wild type p53 and mutant tumor cells. A modied adenovirus, ONYX-015, has been engineered to selectively replicate in and lyse p53-decient cancer cells while sparing normal cells. Clinical success, again in head and neck cancer patients, has been achieved after direct injection of this construct in combination with conventional chemotherapy. A signicant number of complete responses were obtained with tumors showing evidence of necrosis (Khuri et al., 2000). It should be noted that tumors at this site have notoriously poor response rates to conventional therapy. 5.2.6. Human papilloma virus (HPV) can inactivate both p53 and RB In marked contrast to the slow acquisition of mutations in oncogenes and tumor suppressor genes that fortunately delay the development of most epithelial cancers until the later decades of life, cervical cancer frequently has a rapid early onset. Approximately 95% of cervical cancer in the West is associated with infection with high-risk HPV, and again in contrast to other epithelial tumors, these HPV associated cancers rarely show mutations in the p53 gene. This paradox is explicable by the presence in the HPV genome of genes whose protein products, E6 and E7, bind to and inhibit p53 and RB. Thus, by inactivating these two cell cycle regulators the HPV virus creates clones of epithelial cells with an enhanced proliferation rate which are resistant to apoptosis. Inhibition of both p53 and RB enhance the replicative capacity of HPV and the probability of its transmission. The carcinogenic consequence of HPV infection is probably an unfortunate side eect of this stratagy to enhance its replication. P53 and RB inhibition are necessary but not sucient for the malignant conversion of epithelial cells and it seems likely that the increased genomic instability as a consequence of p53 inactivation leads to rapid sequential acquisition of further changes which complete the process (reviewed in zur Hausen, 2000). 5.3. Mutations in the APC gene link cell surface receptors with the nucleus The detailed molecular analysis of color rectal carcinomas has revealed two classes of genes which when inactivated lead to colon cancer. As with p53 and RB, cancer-prone families have been identied with germ-line mutations in these genes. One syndrome, non-polyposis colorectal cancer involving mutations in enzymes involved in mismatch repair, thus increasing genomic instability, has been briey discussed above and has been recently reviewed (Lynch and de la Chapelle, 1999). A second mutation if inherited leads to the syndrome of familial adenomatous polyposis coli (FAP). Aected individuals generate literally thousands of colonic polyps at a young age. Some, perhaps all, of these polyps can progress to malignancy. Fortunately this is a rare disease aecting only approximately one in 10,000 individuals. The gene responsible, APC has all the features of a classical tumor
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suppressor and it, or proteins interacting with APC, are found to be mutated in the vast majority of sporadic human colon cancer. There is increasing evidence that mutations in this pathway are implicated in the etiology of many other cancers (reviewed in Morin, 1999). Although much remains to be discovered, the pathways by which APC mutations stimulate the malignant conversion of colonic epithelial cells is becoming clear. APC is now known to interact with beta-catenin, a protein with two apparently unrelated functions: it has a crucial role in cellcell adhesion by virtue of its interactions with E-cadherin, a plasma membrane adhesion molecule; in addition beta-catenin has a signaling role as a component of a transcriptional complex with transcription factors of the TCF/LEF family. This complex has been shown to directly activate transcription of the cyclin D1 gene (Shtutman et al., 1999) and there is some evidence that c-myc is also activated. As discussed previously, both genes are required for normal cell cycle transit and it may be that the role of E-cadherin signaling is to inform the intestinal stem cell when it is in contact with domains in the colonic crypt where proliferation is appropriate. Mutations in APC appear to be one of the earliest events occurring in colon carcinogenesis. The consequence of this mutation in producing polyps is exactly the proliferative stimulus leading to the accumulation of large populations of initiated cells which are required for the sequential acquisition of additional mutations described earlier in Fig. 1. Inactivating mutations of the APC tumor suppressor gene, or activating mutations of beta-catenin, result in the accumulation of beta-catenin and increased transcription of the cell cycle regulatory genes described above. Critical mutations in APC have been localized to the region of the protein involved in binding to beta-catenin. Transfection of plasmids coding for this binding domain have been shown to reverse aberrant proliferation in colon carcinoma cells (Shih et al., 2000). This suggests one avenue for selective gene therapy for this disease. There is evidence that APC acts as a chaperone protein to move beta-catenin from the cytoplasm to the nucleus (Henderson, 2000). Interestingly, the proliferative stimulus induced by APC mutation can be augmented by mutations in ras, again showing the cooperative nature of oncogenic mutations (Tetsu and McCormick, 1999). The complex role of APC in these signaling pathways has been subject to many recent reviews (Barker et al., 2000; Morin, 1999). 5.3.1. The APC gene oers many targets for intervention A mouse model for FAP has been developed, the mutant Min mouse. This model exhibits many of the abnormalities seen in patients with FAP. It has been utilized to probe the role of cyclooxygenase-2 (COX-2) in tumor development, studies which have been aided by the recent availability of specic inhibitors of this enzyme. Several drugs are currently in clinical use as anti-inammatory agents which lack many of the side eects of the conventional non-steroidal anti-inammatory drugs (NSAIDs). The selective COX-2 inhibitor celecoxib has been demonstrated to decrease tumor multiplicity to 29% and tumor size to 17% of controls in the Min mouse model (Jacoby et al., 2000). Several additional clinical trials with this drug are currently underway.
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J.S. Bertram / Molecular Aspects of Medicine 21 (2001) 167223 b
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Fig. 8. Proposed role of gap junctional communication in growth control. In panel Aa is shown the organization of connexins in the plasma membranes of opposing cells to form a water-lled pore. This pore is surrounded by 6 connexins from one cell tightly docked with 6 connexins from the opposing cell. This structural unit is called a connexon. In panel Ab, a connexin is shown integrated into the plasma membrane with four trans-membrane domains. The third domain contains hydrophilic amino acid residues and is believed to line the pore. Panel B depicts a model in which gap junction decient initiated cells have restricted communication with surrounding normal cells and can proliferate. By increasing expression of gap junction proteins, cancer preventive retinoids and carotenoids increase communication and thus limit aberrant proliferation. When human colon carcinoma cells, genetically engineered to express connexin 43 under the control of a tetracycline-inducible promoter were injected into immuno-compromised mice (panel C), treatment of the animals with doxycycline in the drinking water caused expression of connexin 43 in these cells and a major reduction in growth of subcutaneously growing tumors (..) in comparison with control animals ( ) (Panel A reproduced from Darnell et al. (Eds.) Molecular Cell Biology, Scientic American Books, W.H. Freeman, New York, with permission; panel D, from King et al., 2000, with permission).

Although cyclooxygenase inhibitors are generally considered to act by decreasing prostaglandin formation and subsequent inammatory actions, recent results have suggested that an alternative action of NSAIDs in colon tumor cells may be to cause a dramatic increase in arachidonic acid that in turn stimulates the conversion of sphingomyelin to ceramide, a known mediator of apoptosis (Chan et al., 1998). Other drugs, such as sulindac sulfone, originally also developed as cyclooxygenase inhibitors with the potential to act as chemopreventive agents in colon cancer, are now thought to induce apoptosis in colon cancer cells via yet a dierent mechanism. This involves increases in cGMP levels and cGMP-dependent protein kinase (PKG) induction (Thompson et al., 2000). The rate of new polyp formation in patients with FAP have been shown to be profoundly decreased after treatment with drugs of this type (these reports have so far only been as meeting abstracts). Clearly there is great potential in colon cancer for the development of preventive agents to interrupt disease progression. 6. Cancer cells are decient in intracellular communication mediated by gap junctions Virtually all cells in the body communicate with their neighbors through small, water-lled pores which directly link cytoplasm with cytoplasm. These pores are called gap junctions or connexons, which are created by the assembly of transmembrane proteins called connexins which assemble in a circle around the pore. The diameter of the pore is sucient to allow transfer of water-soluble nutrients, waste products, inter-cellular messengers such as cAMP and ions such as calcium, but small enough to exclude molecules such as mRNA and proteins above about 1000 daltons. The expression of gap junctions and their function is very frequently downregulated in cancer cells and there is much evidence that growth inhibitory signals pass through gap junctions during the normal homeostatic regulation of cell division (Mehta et al., 1986). For these reasons connexins are considered putative tumor suppressor genes (Lee et al., 1991). One function of retinoic acid, shown to be
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a cancer preventive agent in cervical and oral epithelium, is to upregulate gap junctional communication in epithelial cells (Guo et al., 1992). Downregulated communication occurs in cervical dysplasia and is thus an early event in cervical carcinogenesis. When gap junctional communication was restored in human cervical carcinoma cells which previously lacked junctional communication by the introduction of a tetracycline-inducible connexin gene, gene induction resulted in communication competent cells which had a reduced ability to grow as tumors in nude mice and to grow in an anchorage independent manner (King et al., 2000). The studies indicate that the loss of junctional communication may facilitate aberrant proliferation and that one function of cancer preventive agents may be to correct this deciency. A major unresolved question is the nature of the junctionally transmitted signal which mediates growth arrest. The role of gap junctions in cancer has recently been reviewed (Neveu and Bertram, 2000). The structure of gap junctions and their proposed role in limiting aberrant proliferation is shown in Fig. 8. 7. Cancer cells evade apoptosis The concept of programmed cell death, termed apoptosis, was introduced above in terms of the role of p53 in eliminating cells that have either acquired activating oncogenes or excessive genomic damage. Because apoptosis is such an eective mechanism of eliminating cells progressing to malignancy, it is not surprising that in addition to inhibiting p53 function by mutation, other p53 independent mechanisms are also utilized by many cancers as survival factors. Apoptosis is triggered by external plasma membrane associated receptors and internal sensors, principally involving the p53 gene. Both these external and internal mechanisms are perturbed in dierent cancers. Regardless of initial stimulus, pathways of apoptosis all appear to converge on the mitochondria causing loss of membrane integrity and release of cytochrome C. Cytochrome C is a potent mediator of apoptosis and appears to directly stimulate activity of a family of intracellular proteases, called caspases, whose function is to rapidly degrade cellular organelles and chromatin so as to induce cell destruction without inammatory responses that are typically seen after cell necrosis (Earnshaw et al., 1999). In addition to protecting the host against developing cancer cells, apoptosis is widely utilized during embryogenesis to eliminate populations of cells such as those between the digits or in the tail of human embryos and to eliminate self-recognizing immune cells. Apoptosis is also utilized to destroy viral genetic material and the infected cell after viral infection. As a consequence, many viruses have evolved mechanisms to evade apoptosis and several viral gene products interfere with mammalian proteins involved in apoptotic pathways (reviewed in Thompson, 1995). 7.1. Overexpression of bcl-2 protects lymphoma cells from apoptosis The rst indication that escape from apoptosis was an important factor in human cancer came from studies of follicular lymphoma. This disease bears great similarity
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to Burkitt's lymphoma as it also involves a chromosome translocation of the immunoglobulin heavy chain locus not to the c-myc locus as in Burkitt's, but to a chromosome 18 gene denoted bcl-2. When this gene was overexpressed in cells of B-cell lineage it allowed survival under conditions where normal cells died, through a process now identied as apoptosis (Vaux et al., 1988). Consistent with the central role of bcl-2 in follicular lymphoma, the use of an anti-sense construct against bcl-2 causes downregulated expression of this gene and massive apoptosis (Tormo et al., 1998). The similarities between these two diseases has recently been reviewed (Cory et al., 1999). 7.2. Tumor cells evade apoptosis by modied FAS and FAS-L interactions Major external signals triggering apoptosis are mediated by receptor/ligand interactions involving the FAS ligand ( FAS-L) binding to the FAS-receptor. While originally implicated as an important mediator of immune function, Fas/FasL signaling is now thought to play an important role in carcinogenesis, tumor outgrowth, and metastasis (reviewed in Owen-Schaub et al., 2000). A second family of membrane-associated death receptors which appear related to FAS receptors, is represented by the TNF-a, and related family members such as TRAIL, binding to their receptors (reviewed in Marsters et al., 1999). Recently a novel mechanism by which cancer cells avoid FAS- and TRAIL-mediated apoptosis has been discovered which involves the synthesis of decoy receptors to which the ligands bind without inducing apoptosis (Ashkenazi and Dixit, 1999; Marsters et al., 1999; Pitti et al., 1998). Apoptosis also results when epithelial cells are denied contact with basement membrane. Contact is signaled by integrin receptors which result in the formation of focal contacts which activate focal adhesion kinases (FAKs). The ability of epithelial cells to replicate in an anchorage-independent manner has long been used as an in vitro indicator of neoplastic potential. In the absence of focal contacts, normal cells undergo a programmed death that has been termed, anoikis, or loss of home. Apoptosis in this circumstance probably represents an additional fail-safe mechanism to prevent proliferation of epithelial stem cells which become detached from basement membrane. FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway. A hallmark of malignancy in epithelial cells is their ability to penetrate the basement membrane, invade surrounding tissue and ultimately metastasize. In order to achieve this they must bypass anoikis. In view of the previous discussion, it is not surprising that cancer cells have evolved another escape mechanism to avoid death control. Overexpression of FAK is frequently found in carcinoma cells. The importance of this overexpression has been demonstrated in studies where inhibition of FAK activity induces apoptosis in breast carcinoma cells (Cardone et al., 1997). Internal signals triggering apoptosis center around p53 which can directly regulate apoptosis by as yet undened mechanisms or alternatively can stimulate apoptosis
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via the FAS pathway. Both upstream and downstream signals of p53 mediated apoptosis and anoikis-mediated apoptosis converge on mitochondria and their release of cytochrome C (reviewed in Gottlieb, 2000; Kroemer, 1999). There is evidence that cells with wild-type p53 undergo the Fas-mediated killing in the presence of either FasL-expressing killer cells or activating Fas antibodies, whereas cells in which p53 was deleted or inactivated were protected from such killing (Maecker et al., 2000). At least in cell culture it has been shown that in p53 positive carcinomas, Fas and FasL expression and induction became transcriptionally repressed thus protecting cells from apoptosis (Maecker et al., 2000). In mammary carcinoma cells there is evidence that oncogenic ras overcomes anoikis by activation of a phosphoinositide 3-OH kinase (PI 3-kinase), which in turn activates a second protein kinase PKB/Akt (Khwaja et al., 1997; Rytomaa et al., 2000). As an additional twist to illustrate the diverse mechanisms by which tumor cells evade host surveillance mechanisms, esophageal carcinoma cells in vivo, and colon carcinoma cells in vitro, have been shown to express FAS-L which induces apoptotic destruction of invading lymphocytes and possibly also host surrounding tissue (O'Connell et al., 2000). Whereas bcl-2 acts to suppress apoptosis by stabilizing the mitochondrial membrane, a related family member bax, which forms heterodimers with bcl-2, stimulates apoptosis and is inducible by p53 activation. There is evidence that X-irradiation induction of apoptosis requires Bax expression, and may involve the DNA damage sensing kinase DNA-PKC, since apoptosis is signicantly suppressed in the thymocytes of DNA-PKC knock out mice (Wang et al., 2000a). There is limited evidence that in some tumors bax expression may be downregulated so as to evade apoptotic signals (Beerheide et al., 2000). 7.3. The induction of apoptosis is an important target in cancer therapy The induction of apoptosis by chemotherapy and radiotherapy is an important mechanism by which these modalities kill p53 positive tumor cells. Unfortunately, DNA damage in cycling normal cells also induces apoptosis and p53 signaling has been shown to be a major cause of chemotherapy induced hair loss or alopecia (Botchkarev et al., 2000) and is probably responsible for most other toxic responses to therapy. Many investigators are currently involved in research to exploit this cell killing pathway in tumor cells by developing molecular and chemical means to increase apoptosis in target cells. Drugs which selectively target mitochondria are being developed (Costantini et al., 2000), however at present it is not clear that tumor selectivity can be achieved by this means. The forced overexpression of BAX in tumor cells has been shown to increase their sensitivity to conventional chemotherapy (Guo et al., 2000), and to decrease their growth rate as xenografts in mice (Kagawa et al., 2000). Another approach has been to increase the degree of apoptosis induced by transfection of wild type p53 by the addition of a construct expressing FAS-L. This approach led to dramatically enhanced apoptosis in transfected glioma cells in culture (Shinoura et al., 2000). Yet another approach has been to inhibit the activity of bcl-2 by the use of drugs designed to bind to and activate its function. A candidate drug, HA14-1, induced apoptosis of human acute myeloid
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leukemia (HL-60) cells overexpressing Bcl-2 protein. Apoptosis was associated with a decrease in mitochondrial membrane potential and activation of caspases (Wang et al., 2000b). 8. Cancer cells must avoid senescence and achieve immortality: role of telomeres When normal human cells are placed in culture and serially passaged so as to progressively increase their cumulative generation number, it is found that after a total of from 50 to 100 generations cells cease to divide and enter what is called replicative senescence. This may be avoided for some time if their p53 and RB genes are inactivated, however after accumulation of further generations, cells exhibit massive chromosomal abnormalities and subsequently die. In contrast, when tumor cells are placed into the same in vitro environment most cells are capable of unlimited replication (Hayick, 2000). It is now known that the crucial event limiting the life span of normal cells is the progressive erosion of the ends of chromosomes which are normally capped by repetitive, six base pair sequences (TTAGGG in mammals) called telomeres which are approximately 612 kb long in humans. This erosion is an inescapable consequence of the unidirectional, 5H 3H nature of DNA replication. Due to the necessity for a small RNA primer to initiate DNA synthesis, the 5H end of the lagging strand of DNA cannot be replicated and becomes progressively shorter with each replicative cycle. In germ cells which must retain their replicative abilities, and in certain stem cells, particularly of the hematopoietic system, there is expression of an enzyme, telomerase, which can maintain normal telomere length (Yashima et al., 1998). However, telomerase is not expressed in most somatic cells in which the accumulation of replication cycles causes the progressive shortening of telomeres which are recognized by p53 causing cell-cycle arrest and the phenomenon of replicative senescence. Cloning of the enzyme telomerase which maintains telomere length resolved the question of how the 5H ends of chromosomes can be maintained. The enzyme is highly unusual as it consists of a reverse transcriptase, hTERT, together with an RNA sub-unit of 11 bases which is complementary to the telomere repetitive DNA (Counter et al., 1997). Sequence-specic proteins exist which recognize telomeres and serve to stabilize chromosomal ends and somehow signal telomere length (van Steensel et al., 1998). 8.1. Many cancer cells reactivate telomerase As discussed earlier, extensive proliferation of carcinogen-initiated cells is required for the progressive accumulation of mutations required for malignancy. Furthermore, after cytoreductive therapy of human tumors residual cells must proliferate massively in order to again threaten the life of the patient. Thus in the absence of telomere maintenance most candidate tumor cells would undergo senescence before creating a clinical problem, or would undergo senescence after initial therapy. To overcome this fatal aw in tumor evolution, most human malignancies
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can be shown to have reactivated telomerase in order to maintain telomere length. Moreover, the extent of activation seems to be positively correlated with the aggressiveness of the disease: 98% of small cell lung carcinomas but a much lower percent of less aggressive carcinoid tumors expressed telomerase (Sarvesvaran et al., 1999); 100% of invasive breast carcinomas but only 70% of ductal carcinoma in situ lesions expressed some activity (Umbricht et al., 1999); in melanoma (Ramirez et al., 1999) and in colon carcinoma (Tatsumoto et al., 2000) increased enzyme activities were found in more aggressive tumors. It is at present unclear whether all telomerase positive tumor cells have found mechanisms to reactivate this enzyme activity or whether in some situations tumors evolve from a self-renewing stem cell population which possesses constitutive activity. This may dier from organ to organ, for example normal breast is reported to be negative for expression, while activity can be demonstrated in the renewing population of the intestine. Nevertheless, much higher levels of telomerase were recorded in intestinal adenocarcinomas than in normal tissue (Bachor et al., 1999). Telomere length appears to be monitored by p53 which induces cell cycle arrest or alternatively apoptosis when a critical degree of shortening has occurred (Vaziri and Benchimol, 1999). Paradoxically, telomere shortening may present advantages to a developing cancer cell providing it can escape apoptosis due to p53 surveillance. As discussed above (van Steensel et al., 1998), telomeres are normally capped with proteins which limit degradation and prevent chromosomal instability. Since chromosomal instability favors rapid evolution due to activation of oncogenes or inactivation of tumor suppressor genes, tumor cells may benet in the short-term from chromosomal erosion, providing that telomerase becomes reactivated before massive chromosomal instability leads to cell death. This sequence of events appears to be illustrated in knockout mice which are decient either in telomerase or in telomerase and p53. Telomerase decient mice function normally and are fertile. However, after successive matings, animals in the fourth to sixth generations, which exhibit progressive telomere shortening, develop a high frequency of cancers, predominantly lymphomas originating from highly proliferative tissues. However mice which are doubly decient in telomerase and p53 exhibit a spectrum of cancers which are predominantly carcinomas with a spectrum similar to that seen in the human population. It may be speculated that the deciency in p53 allowed survival of cells which would otherwise undergo apoptosis and in these cells chromosomal aberrations could occur resulting the necessary additional mutations required for the genesis of the epithelial tumors (Artandi et al., 2000). 8.2. Telomerase oers an exciting novel target for cancer prevention and therapy The realization that the unlimited proliferative capacity of tumor cells is a consequence of their reexpression of telomerase has generated intense interest in inhibiting this activity and thus inducing replicative senescence in these cells. A number of approaches are currently being tested: these include use of antisense constructs to inhibit catalytic activity; transfection of mutant dominant negative TERT constructs and use of the hTERT promoter to target cells actively transcribing TERT. These
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studies have not yet reached clinical trial, however in vitro and in vivo experiments in nude mice look very encouraging. Transfection of a dominant negative mutant catalytic subunit of human telomerase into human tumor cells resulted in the complete inhibition of telomerase activity, reduction in telomere length, death of tumor cells in vitro and inhibition of tumorigenicity in vivo (Hahn et al., 1999). Antisense strategies have also been successful in three separate investigations involving three dierent tumor types. In these studies, a chimeric antisense construct was utilized involving short stretches of 2H ,5H oligoadenylates fused to a human antisense sequence directed against 19-mer RNA component of human telomerase hTR. The purpose of the 2H ,5H -oligoadenylates is to direct RNase L to an RNA target and cause its destruction. Administration of this construct to ovarian carcinoma cells which expressed hTERT cause rapid loss of telomeres within seven days and their apoptotic death. In contrast administration of the same constructs to normal, telomerase negative ovarian epithelial cells was without eect (Kushner et al., 2000). Use of a similar construct resulted within four days in a major reduction in viability in glioma cells and reduced their tumorigenicity as xenografts in the mouse. These constructs did not inuence the viability of cultured astrocytes (Mukai et al., 2000). Similarly, the viability of prostate carcinoma cells was reduced in vitro and their growth rate in nude mice strongly suppressed by treatment with this antisense strategy (Kondo et al., 2000). Using a dierent approach it was reasoned that tumor cells which strongly express TERT would drive a TERT promoter and if this were attached to a cytotoxic transgene, tumor-specic killing may be achieved. This was tested successfully by placing the pro-apoptotic Bax gene under control of this promoter. Induction of Bax elicited tumor-specic apoptosis in vitro and suppressed tumor growth in nude mice. Again, normal telomerase negative cells were not aected (Gu et al., 2000). Yet another approach aimed to decrease expression of the E6 and E7 proteins of HPV which actively prevent senescence from occurring in cervical carcinoma cells. To suppress expression of these proteins, cells were transfected with a construct coding for the E2 protein which normally suppresses E6 and E7 expression. Transfection caused a rapid decrease in telomerase activity and induction of apoptosis (Goodwin et al., 2000). The rapidity with which these therapies decreased telomerase activity and induced apoptosis suggests that this strategy could be utilized clinically without causing signicant toxicity to those normal host stem cells requiring telomerase activity for continued reproductive potential. 9. Cancer cells require adequate supplies of nutrients and stimulate angiogenesis During the evolution of a cancer cell it attained the capacity to proliferate, to avoid normal controls on proliferation, to avoid normal controls on cell death and nally it achieved immortality. It now faces one last challenge to ensure a satisfactory supply of nutrients necessary for continued growth and to eliminate products of metabolism which would potentially be lethal. This can only be achieved in two ways; by growing in suspension, either as ascites in the abdomen or the blood; or
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alternatively, by adopting a strategy utilized by normal cells, by creating a new network of vessels and capillaries to serve the needs of the tumor. This network needs to be extensive as it can be demonstrated that in order to survive cells need to be within approximately 0.1 mm of a capillary. Notable exceptions to this rule are cells forming the optically transparent cornea and lens; here gap junctions serve to transport nutrients and waste products. Angiogenesis occurs through the sprouting and invasion of endothelial cells from existing vessels in response to multiple extra-cellular signals such as vascular endothelial growth factor (VEGF) and broblast growth factors (FGF-1 and -2). This process is controlled and balanced by inhibitors of angiogenesis such as thrombospondin. Tumor cells have found means to enhance expression of pro-angiogenic factors as well as suppress negative regulators (Hahnfeldt et al., 1999). Interestingly, oncogenes and tumor suppressor genes seem intimately involved in both processes and the sequential acquisition of angiogenic properties can be demonstrated in the same cells. For example, expression of the inhibitor thrombospondin is positively regulated by p53 and in cells from patients with LiFraumani syndrome, which are heterozygous for mutant p53, loss of the second p53 allele causes major down regulation of thrombospondin expression. Subsequently, mutation of ras to its oncogenic form results in increased secretion of VEGF which further stimulated the migration and proliferation of endothelial cells. The net result is increased proliferation of both tumor cells and host endothelial cells which migrate into the growing tumor mass (Volpert et al., 1997). The complex interplay of positive and negative signals governing angiogenesis oers exciting opportunities to interfere in this process; opportunities that are currently being exploited (Hahnfeldt et al., 1999). 9.1. Inhibitors of angiogenesis exert potent anti-tumor aects Stimulated by very early observations that tumor cells rarely if ever invade collagen-rich tissues such as tendons, the group led by Folkman has for some 30 years been investigating the anti-angiogenic properties of collagen. Recently, with the identication of endostatin, a 20 kD fraction of collagen XVII, this eld has seen enormous advances. More than 30 dierent inhibitors of angiogenesis have now been described, several of which are currently in clinical trial (Table 1) (reviewed in Feldman and Libutti, 2000). One of the most attractive features of therapies aimed at inhibiting angiogenesis is that the target cells are normal host cells which are not subject to the extensive genomic instability found in cancer cells that gives rapid rise to drug-resistant phenotypes. However, a note to caution has recently been raised with the analysis of gene expression patterns in tumor-derived vs normal endothelial cells. Almost 80 dierent genes exhibited dierent patterns of expression although many of these were also dierently expressed during normal wound healing (St Croix et al., 2000). Endostatin has exhibited perhaps the most exciting activity to date and exemplies the potential for this novel type of therapy. In mice bearing Lewis lung carcinoma, T241 brosarcoma or B16F10 melanoma tumors, administration of
J.S. Bertram / Molecular Aspects of Medicine 21 (2001) 167223 Table 1 Angiogenesis inhibitors in clinical trialsa Drug EMD121974 PTK787/ZK2284 Endostatin BMS-275291 SU6668 Squalamine COL-3 CGS-27023A Vitaxin IL-12 Anti-VEGF Ab SU5416 Thalidomide Marimastat AG3340 Neovastat Interferon-a
a
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Phase I/II I I I/II/III I II II II II I/II I/II III/IV I/II/III III II III II/III
Mechanism Small molecule integrin antagonist Blocks VEGF-receptor signaling Induces endothelial cell apoptosis in vivo Synthetic MMP inhibitor Blocks VEGF-, FEG0 and PDGF-receptor signaling Inhibits Na /H exchanger Synthetic MMP inhibitor: tetracycline derivative Synthetic MMP inhibitor Fumagilin analogue; inhibits endothelial proliferation Induces interferon-c and IP-10 Monoclonal antibody to VEGF Blocks VEGF receptor signalling Unknown Synthetic MMP inhibitor Synthetic MMP inhibitor Natural MMP inhibitor Inhibition of bFGF and VEGF production
Data from the NCI clinical trials database November 2000.
endostatin caused dramatic decreases in tumor mass. Tumors regrew upon cessation of therapy, however successive cycles of therapy and regrowth resulted after four or ve cycles with the apparent permanent dormancy (cure?) of these tumors. This was achieved without evident toxicity to the host (Boehm et al., 1997). Similar results have been achieved in mice bearing human tumors with a second inhibitor, angiostastin, a 38 kb fragment of plasminogen (O'Reilly et al., 1996). These exciting results are not limited to implanted tumors as endostatin will also reduce growth rates in chemically induced rat mammary tumors (Perletti et al., 2000). Problems in the supply of these proteins have now been overcome with the successful generation of recombinant molecules in E. coli and insect cells (O'Reilly et al., 1997; Wu et al., 1997). As an alternative to delivery of the preformed protein, other investigators have shown that a single administration of a replication-defective adenoviral vector causing expression a secretable form of endostatin was capable of causing prolonged survival and apparent dormancy of 25% of human colon tumors in nude mice (Chen et al., 2000). 9.2. Inhibitors of pro-angiogenic signals are eective anti-tumor agents Most research has focused on inhibiting the action of VEGF either by direct means or by interfering with functioning of its receptor. Use of antisense constructs against VEGF has been shown to inhibit experimental angiogenesis in culture (Nakashima et al., 2000); while others have used monoclonal antibodies directed against VEGF endothelial receptors (Brekken et al., 2000). In yet another approach genetically engineered cells secreting a soluble form of the VEGF receptor have been shown capable of inhibiting angiogenesis at distant sites (Takayama et al., 2000). As
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with many trans-membrane receptors VEGF-R and PDGF-R activation stimulates Tyr kinases which modulate downstream events. A small molecule inhibitor of these two Tyr kinases, SU6668, has been shown to inhibit receptor tyrosine phosphorylation and to have potent anti-apoptotic aects in pre-clinical models (Laird et al., 2000). This molecule is currently undergoing clinical trial. Various interferons also are reported to inhibit angiogenesis and in one clinical report, interferon alfa-2a was administered to a 5-year-old girl with a large rapidly growing giant cell tumor of the mandible. Treatment resulted in dramatic decreases in urinary excretion of the angiogenic stimulator bFGF, and complete tumor regression over a three year treatment-free interval (Kaban et al., 1999). Progress in the area of anti-angiogenesis research has recently been reviewed (Feldman and Libutti, 2000). 9.3. Conventional chemotherapy can be targeted to endothelial cells Acting on the rationale that host endothelial cells in or around a successful tumor would be rapidly proliferating, and moreover would be highly sensitive to p53 mediated apoptosis resulting from DNA damage, Folkman's group devised a schedule of therapy targeted against endothelial cells. Proof of principal of this concept was demonstrated by the use of cyclophosphamide-resistant Lewis lung carcinoma and EMT-6 breast cancer implanted in nude mice and a chemotherapy schedule with cyclophosphamide designed to target endothelial cells. This antiangiogenic schedule suppressed tumor growth by induction of apoptosis far more eectively than a conventional schedule targeted at the tumor. When another angiogenesis inhibitor, TNP-470, to was added to this antiangiogenic protocol, drug-resistant Lewis lung carcinomas were eradicated (Browder et al., 2000). 10. Putting it all together: prospects for molecular medicine in the 21st century The preceding chapters have identied the plethora of mutations which are necessary for the genesis of a successful cancer cell. These mutations introduce defects in normal homeostatic control mechanisms that allow cancer cells to escape controls on cell birth, death, senescence and interactions with host cells such as those of the immune system and vasculature. I have attempted to illustrate how each of these mutations oer a window of opportunity for selective tumor-specic therapies. In the chapter on etiology of cancer was introduced the concept that, since mutations are in general random and that only the successful ones survive, each clone of successful cancer cells is probably unique in its sum total of acquired mutations. Furthermore, the genetic instability of cancer cells predicts that this uniqueness will continually evolve over time implying that a successful therapy must address some uniquely important attribute of a cancer cell (immortality, angiogenesis?) that cannot be circumvented by the acquisition of yet one more mutation. The therapeutic agents currently in development can be expected to be highly specic in their actions against cancer cells, but only those cancer cells utilizing a specic escape mechanism. Some therapies may require even greater specicity as they will be directed against specic
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mutations. For example, monoclonal antibodies recognize epitopes from 3 to 5 amino acid residues long, while antisense therapies may require an exact complementary mRNA sequence if they are to successfully inhibit translation. Thus it can be envisioned that the new generation of tumor-specic therapies must be specically targeted against only those tumors harboring the specic mutations to which the specic therapy is directed. The implication of this specicity is that while toxicity to the patient may be minimal, conventional pathology must be supplemented with molecular pathology capable of identifying specic mutations in an ever widening network of regulatory genes. Recent developments in high-throughput analysis of genomic and expressed sequences and the publication of the rst draft of the human genome oer a route to solving this problem. The automated fabrication of DNA arrays utilizing techniques borrowed from the computer chip industry coupled with PCR amplication now allow the simultaneous analysis of thousands of expressed genes or the presence of single nucleotide polymorphisms, SNPs. Moreover, these analyses can be performed on increasingly small numbers of cells isolated by the new technique of laser-assisted micro-dissection (Lockhart and Winzeler, 2000; Pappalardo et al., 1998). 10.1. Use of genomic arrays in the molecular proling of cancers Within the last year several publications have described the use of large-scale arrays to prole dierences in expression patterns between cancers and normal cells. These cancers include: lymphoma (Alizadeh et al., 2000), breast (Perou et al., 2000) and colon carcinoma (Alon et al., 1999). The results have conrmed the enormous variability in gene expression patterns that may be predicted from an extension of Murphy's Law to carcinogenesis `àny advantage will be exploited for tumor survival''. A distinctive feature discovered in all of these studies was that although cancers from dierent individuals diered extensively in their expression patterns, nevertheless clusters of gene expression patterns were detected which indicated the existence of sub-groups of cancers which diered in their cells of origin or state of dierentiation. In the case of B-cell lymphoma the results were particularly dramatic in that the disease, previously recognizable as a single entity by conventional pathology, could be divided into two separate categories by molecular pathology: one pattern characteristic of lymphocytes in germinal centers; a second characteristic of activated lymphocytes. Moreover these categories diered dramatically in their survival rates; patients with germinal center B-like lymphoma had a signicantly better overall survival than those with activated B-like lymphoma (Fig. 9). In the future it would appear that such knowledge will be required prior to therapeutic intervention so that more aggressive therapies be tailored to the more aggressive tumors. The ability to screen large numbers of genes simultaneously also allows for the rapid detection of dierentially expressed genes which may contribute to disease. As an example, comparisons of melanomas diering in their metastatic abilities revealed that a GTPase, RhoC, was strongly expressed in metastatic cells and is necessary for tumor invasion (Clark et al., 2000). High-throughput screening arrays will probably be integral to the molecular pathology that will be increasingly
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J.S. Bertram / Molecular Aspects of Medicine 21 (2001) 167223 b
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Fig. 9. Use of gene expression arrays to characterize lymphoma at the molecular level. mRNA was extracted from a series of diuse large B-cell lymphomas and the expression pattern of individual genes compared to the pattern seen in normal human tonsil and lymph nodes, and with a variety of lymphoma and leukemia cell lines. Expression of individual genes, identied at the right of the panel, greater than the mean expression in all cells analyzed is shown in red, while expression below the mean is shown in green. In panel A, left, the expression pattern from the lymphomas is shown; panel A, right, the expression pattern of normal lymphocytes is shown. While pathologically indistinguishable, it can be seen that the lymphoma expression proles can be distinguished into two groups as shown by the yellow, and blue groupings. The lymphomas organized on the left of this gure and identied by the yellow connections have a mRNA expression pattern which is similar to the expression pattern of normal germinal center B cells (GC B). Expression pattern of these cells is shown on the right side of panel A. In contrast, the second group of lymphomas had mRNA expression patterns similar to patterns seen in normal activated B lymphocytes. Shown in panel B is the probability of survival of patients with these dierent classes of lymphoma. It is striking that a comparison of all patients, (panel Ba) reveals that patients with GC B-like lymphomas had a signicantly better survival than those with the activated B-like lymphomas. Moreover, when patients were stratied according to low clinical risk and high clinical risk (panel Bb), even those patients classied as low risk contained both molecular types of lymphoma, and again the GC B-like lymphomas were signicantly less aggressive then the activated B-like lymphomas. (Reprinted by permission from Nature, Vol. 403, pp. 503511, 2000. Macmillan Magazines Ltd. Alizadeh et al., 2000).
necessary in order to fully exploit novel treatment modalities and are equally capable of detecting single mutations in genomic DNA (SNPs ) which may predict disease susceptibility or drug sensitivity (Lockhart and Winzeler, 2000). References
Aas, T., Borresen, A.L., Geisler, S., Smith-Sorensen, B., Johnsen, H., Varhaug, J.E., Akslen, L.A., Lonning, P.E., 1996. Specic p53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients. Nat. Med. 2, 811814. Aguilar, F., Harris, C.C., Sun, T., Hollstein, M., Cerutti, P., 1994. Geographic variation of p53 mutational prole in nonmalignant human liver. Science 264, 13171319. Alizadeh, A.A., Eisen, M.B., Davis, R.E., et al., 2000. Distinct types of diuse large B-cell lymphoma identied by gene expression proling. Nature 403, 503511. Allgayer, H., Babic, R., Gruetzner, K.U., Tarabichi, A., Schildberg, F.W., Heiss, M.M., 2000. c-erbB-2 is of independent prognostic relevance in gastric cancer and is associated with the expression of tumorassociated protease systems. J. Clin. Oncol. 18, 22012209. Alon, U., Barkai, N., Notterman, D.A., Gish, K., Ybarra, S., Mack, D., Levine, A.J., 1999. Broad patterns of gene expression revealed by clustering analysis of tumor and normal colon tissues probed by oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 96, 67456750. Ames, B.N., 1984. The detection of environmental mutagens and potential carcinogens. Cancer 53, 20342040. Ames, B.N., 1986. Food constituents as a source of mutagens, carcinogens, and anticarcinogens. Prog. Clin. Biol. Res. 206, 332. Ames, B.N., Gold, L.S., 1991. Mitogenesis, mutagenesis, and animal cancer tests. In: Chemically Induced Cell Proliferation: Implications for Risk Assessment. Wiley, New York, pp. 120. Ames, B.N., Shigenaga, M.K., Gold, L.S., 1993. DNA lesions, inducible DNA repair, and cell division: three key factors in mutagenesis and carcinogenesis. Environ. Health Perspect. 101 (Suppl. 5), 3544. Anderson, M.J., Stanbridge, E.J., 1993. Tumor suppressor genes studied by cell hybridization and chromosome transfer. FASEB J. 7, 826833.
214
Artandi, S.E., Chang, S., Lee, S.L., Alson, S., Gottlieb, G.J., Chin, L., DePinho, R.A., 2000. Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice. Nature 406, 641645. Ashkenazi, A., Dixit, V.M., 1999. Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol. 11, 255260. Bachor, C., Bachor, O.A., Boukamp, P., 1999. Telomerase is active in normal gastrointestinal mucosa and not up-regulated in precancerous lesions. J. Cancer Res. Clin. Oncol. 125, 453460. Barker, N., Morin, P.J., Clevers, H., 2000. The Yin-Yang of TCF/beta-catenin signaling. Adv. Cancer Res. 77, 124. Beerheide, W., Tan, Y.J., Teng, E., Ting, A.E., Jedpiyawongse, A., Srivatanakul, P., 2000. Downregulation of proapoptotic proteins Bax and Bcl-X(S) in p53 overexpressing hepatocellular carcinomas. Biochem. Biophys. Res. Commun. 273, 5461. Bemark, M., Neuberger, M.S., 2000. The c-MYC allele that is translocated into the IgH locus undergoes constitutive hypermutation in a Burkitt's lymphoma line. Oncogene 19, 34043410. Bertram, J.S., Heidelberger, C., 1974. Cell cycle dependency of oncogenic transformation induced by N-methyl-n'-nitro-N-nitrosoguanidine in culture. Cancer Res. 34, 526537. Black, P.M., Carroll, R., Glowacka, D., Riley, K., Dashner, K., 1994. Platelet-derived growth factor expression and stimulation in human meningiomas. J. Neurosurg. 81, 388393. Boehm, T., Folkman, J., Browder, T., O'Reilly, M.S., 1997. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, 404407. Bookstein, R., Shew, J.-Y., Chen, P.-L., Scully, P., Lee, W.-H., 1990. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science 247, 712715. Bos, J.L., 1989. ras Oncogenes in human cancer: a review. Cancer Res. 49, 46824689. Botchkarev, V.A., Komarova, E.A., Siebenhaar, F., Botchkareva, N.V., Komarov, P.G., Maurer, M., Gilchrest, B.A., Gudkov, A.V., 2000. p53 is essential for chemotherapy-induced hair loss. Cancer Res. 60, 50025006. Branford, S., Hughes, T.P., Rudzki, Z., 1999. Monitoring chronic myeloid leukaemia therapy by real-time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics. Br.J. Haematol. 107, 587599. Brehm, A., Miska, E.A., McCance, D.J., Reid, J.L., Bannister, A.J., Kouzarides, T., 1998. Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature 391, 597601. Brekken, R.A., Overholser, J.P., Stastny, V.A., Waltenberger, J., Minna, J.D., Thorpe, P.E., 2000. Selective inhibition of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk-1) activity by a monoclonal anti-VEGF antibody blocks tumor growth in mice. Cancer Res. 60, 51175124. Browder, T., Buttereld, C.E., Kraling, B.M., Shi, B., Marshall, B., O'Reilly, M.S., Folkman, J., 2000. Antiangiogenic scheduling of chemotherapy improves ecacy against experimental drug-resistant cancer. Cancer Res. 60, 18781886. Bunz, F., Hwang, P.M., Torrance, C., Waldman, T., Zhang, Y., Dillehay, L., Williams, J., Lengauer, C., Kinzler, K.W., Vogelstein, B., 1999. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J. Clin. Invest. 104, 263269. Cahill, D.P., Kinzler, K.W., Vogelstein, B., Lengauer, C., 1999. Genetic instability and darwinian selection in tumours. Trends Cell Biol. 9, M57M60. Campbell, S.L., Khosravi-Far, R., Rossman, K.L., Clark, G.J., Der, C.J., 1998. Increasing complexity of Ras signaling. Oncogene 17, 13951413. Cardone, M.H., Salvesen, G.S., Widmann, C., Johnson, G., Frisch, S.M., 1997. The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell 90, 315323. Carrier, F., Georgel, P.T., Pourquier, P., Blake, M., Kontny, H.U., Antinore, M.J., Gariboldi, M., Myers, T.G., Weinstein, J.N., Pommier, Y., Fornace Jr., A.J., 1999. Gadd45, a p53-responsive stress protein, modies DNA accessibility on damaged chromatin. Mol. Cell Biol. 19, 16731685. Castaigne, S., Balitrand, N., DeTh e, H., Dejean, A., Degos, L., Chomienne, C., 1992. A PML/retinoic acid receptor a fusion transcript is constantly detected by RNA-based polymerase chain reaction in acute promyelocytic leukemia. Blood 79, 31103115.
215
Castaigne, S., Chomienne, C., Daniel, M.T., Ballerini, P., Berger, R., Fenaux, P., Degos, L., 1990. Alltrans retinoic acid as a dierentiation therapy for acute promyelocytic leukemia. I. Clinical results. Blood 76, 17041709. Chan, T.A., Morin, P.J., Vogelstein, B., Kinzler, K.W., 1998. Mechanisms underlying nonsteroidal antiinammatory drug-mediated apoptosis. Proc. Natl. Acad. Sci. USA 95, 681686. Chehab, N.H., Malikzay, A., Appel, M., Halazonetis, T.D., 2000. Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev. 14, 278288. Chen, C.T., Lin, J., Li, Q., Phipps, S.S., Jakubczak, J.L., Stewart, D.A., Skripchenko, Y., ForrySchaudies, S., Wood, J., Schnell, C., Hallenbeck, P.L., 2000. Antiangiogenic gene therapy for cancer via systemic administration of adenoviral vectors expressing secretable endostatin. Hum. Gene Ther. 11, 19831996. Chen, H.W., Lin, R.J., Xie, W., Wilpitz, D., Evans, R.M., 1999b. Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acetylase. Cell 98, 675686. Chen, S.-J., Zhu, Y.-J., Tong, J.-H., Dong, S., Huang, W., Chen, Y., Xiang, W.-M., Zhang, L., Li, X.-S., Qian, G.-Q., Wang, Z.-Y., Chen, Z., Larsen, C.-J., Berger, R., 1991. Rearrangements in the second intron of the RARA gene are present in a large majority of patients with acute promyelocytic leukemia and are used as molecular marker for retinoic acid-induced leukemic cell dierentiation. Blood 78, 26962701. Chen, Y., Lee, W.H., H.K, 1999a. Chew. Emerging roles of BRCA1 in transcriptional regulation and DNA repair. J. Cell Physiol. 181, 385392. Cheng, K.C., Cahill, D.S., Kasai, H., Nishimura, S., Loeb, L.A., 1992. 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes GT and AC substitutions. J. Biol. Chem. 267, 166172. Cho, K.R., Vogelstein, B., 1992. Genetic alterations in the adenomacarcinoma sequence. Cancer 70, 17271731. Cho, Y., Gorina, S., Jerey, P.D., Pavletich, N.P., 1994. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265, 346355. Clark, E.A., Golub, T.R., Lander, E.S., Hynes, R.O., 2000. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 406, 532535. Classon, M., Salama, S., Gorka, C., Mulloy, R., Braun, P., Harlow, E., 2000. Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control. Proc. Natl. Acad. Sci. USA 97, 1082010825. Claudio, P.P., Howard, C.M., Pacilio, C., Cinti, C., Romano, G., Minimo, C., Maraldi, N.M., Minna, J.D., Gelbert, L., Leoncini, L., Tosi, G.M., Hicheli, P., Caputi, M., Giordano, G.G., Giordano, A., 2000. Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer. Cancer Res. 60, 372382. Clayman, G.L., 2000b. The current status of gene therapy. Semin. Oncol. 27, 3943. Clayman, G.L., 2000a. The current status of gene therapy. Semin. Oncol. 27, 3943. Clayman, G.L., Frank, D.K., Bruso, P.A., Goepfert, H., 1999. Adenovirus-mediated wild-type p53 gene transfer as a surgical adjuvant in advanced head and neck cancers. Clin. Cancer Res. 5, 17151722. Cleaver, J.E., Bootsma, D., 1975. Xeroderma pigmentosum: biochemical and genetic characteristics. Annu. Rev. Genet. 9, 1938. M.S, Colman, Afshari, C.A., Barrett, J.C., 2000. Regulation of p53 stability and activity in response to genotoxic stress. Mutat. Res. 462, 179188. Cory, S., Vaux, D.L., Strasser, A., Harris, A.W., Adams, J.M., 1999. Insights from Bcl-2 and Myc: malignancy involves abrogation of apoptosis as well as sustained proliferation. Cancer Res. 59, 1685s1692s. Costantini, P., Jacotot, E., Decaudin, D., Kroemer, G., 2000. Mitochondrion as a novel target of anticancer chemotherapy. J. Natl. Cancer Inst. 92, 10421053. Counter, C.M., Meyerson, M., Eaton, E.N., Weinberg, R.A., 1997. The catalytic subunit of yeast telomerase. Proc. Natl. Acad. Sci. USA 94, 92029207. Daley, G.Q., Van Etten, R.A., Baltimore, D., 1990. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 247, 824830.
216
Doll, R., Peto, 1981. R. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J. Natl. Cancer Inst. 66, 11911308. Druker, B.J., 1999. Clinical ecacy and safety of an Abl specic tyrosine kinase inhibitor as targeted therapy for chronic myelogenous leukemia. Am. Soc. Hematol. Abstract. Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383424. Eastham, J.A., Grafton, W., Martin, C.M., Williams, B.J., 2000. Suppression of primary tumor growth and the progression to metastasis with p53 adenovirus in human prostate cancer. J. Urol. 164, 814819. El Deiry, W.S., Harper, J.W., O'Connor, P.M., Velculescu, V.E., Canman, C.E., Jackman, J., Pietenpol, J.A., Burrell, M., Hill, D.E., Wang, Y., 1994. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 54, 11691174. W.S, El Deiry, Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., Vogelstein, B., 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817825. Feldman, A.L., Libutti, S.K., 2000. Progress in antiangiogenic gene therapy of cancer. Cancer 89, 11811194. Fraser, M.C., Tucker, M.A., 1989. Second malignancies following cancer therapy. Semin. Oncol. Nurs. 5, 4355. Frosina, G., 2000. Overexpression of enzymes that repair endogenous damage to DNA. Eur. J. Biochem. 267, 21352149. Galaktionov, K., Chen, X., Beach, D., 1996. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 382, 511517. Gallie, B.L., Campbell, C., Devlin, H., Duckett, A., Squire, J.A., 1999. Developmental basis of retinalspecic induction of cancer by RB mutation. Cancer Res. 59, 1731s1735s. Goodwin, E.C., Yang, E., Lee, C.J., Lee, H.W., DiMaio, D., Hwang, E.S., 2000. Rapid induction of senescence in human cervical carcinoma cells [In Process Citation]. Proc. Natl. Acad. Sci. USA 97, 1097810983. Gottlieb, R.A., 2000. Mitochondria: execution central. FEBS Lett. 482, 612. Greenblatt, M.S., Bennett, W.P., Hollstein, M., Harris, C.C., 1994. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 48554878. Grignani, F., DeMatteis, S., Nervi, C., Tomassoni, L., Gelmetti, V., Cioce, M., Fanelli, M., Ruthardt, M., Ferrara, F.F., Zamir, I., Seiser, C., Lazar, M.A., Minucci, S., Pelicci, P.G., 1998. Fusion proteins of the retinoic acid receptor-a recruit histone deacetylase in promyelocytic leukaemia. Nature 391, 815818. Gu, J., Kagawa, S., Takakura, M., Kyo, S., Inoue, M., Roth, J.A., Fang, B., 2000. Tumor-specic transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic eects of the Bax gene to cancers. Cancer Res. 60, 53595364. Guo, B., Cao, S., Toth, K., Azrak, R.G., Rustum, Y.M., 2000. Overexpression of Bax enhances antitumor activity of chemotherapeutic agents in human head and neck squamous cell carcinoma. Clin. Cancer Res. 6, 718724. Guo, H., Acevedo, P., Parsa, D.F., Bertram, J.S., 1992. The gap-junctional protein connexin 43 is expressed in dermis and epidermis of human skin: dierential modulation by retinoids. J. Invest. Dermatol. 99, 460467. Hahn, W.C., Stewart, S.A., Brooks, M.W., York, S.G., Eaton, E., Kurachi, A., Beijersbergen, R.L., Knoll, J.H., Meyerson, M., Weinberg, R.A., 1999. Inhibition of telomerase limits the growth of human cancer cells. Nat. Med. 5, 11641170. Hahnfeldt, P., Panigrahy, D., Folkman, J., Hlatky, L., 1999. Tumor development under angiogenic signaling: a dynamical theory of tumor growth, treatment response, and postvascular dormancy. Cancer Res. 59, 47704775. Hall, J., Angele, S., 1999. Radiation, DNA damage and cancer. Mol. Med. Today 5, 157164. Harbour, J.W., Luo, R.X., Dei, S.A., Postigo, A.A., Dean, D.C., 1999. Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell 98, 859869.
217
Harris, H., Miller, O.J., Klein, G., Worst, P., Tachibana, T., 1969. Suppression of malignancy by cell fusion. Nature 223, 363368. Harty, L.C., Guinee Jr., D.G., Travis, W.D., Bennett, W.P., Jett, J., Colby, T.V., Tazelaar, H., Trastek, V., Pairolero, P., Liotta, L.A., Harris, C.C., Caporaso, N.E., 1996. p53 mutations and occupational exposures in a surgical series of lung cancers. Cancer Epidemiol. Biomarkers Prev. 5, 9971003. Hayick, L., 2000. The illusion of cell immortality. Br. J. Cancer 83, 841846. He, L.Z., Guidez, F., Tribioli, C., Peruzzi, D., Ruthardt, M., Zelent, A., Pandol, P.P., 1998. Distinct interactions of PML-RAR a and PLZF-RAR a with co-repressors determine dierential responses to RA in APL. Nature Genet. 18, 126135. Hecht, S.S., 1997. Approaches to cancer prevention based on an understanding of N-nitrosamine carcinogenesis. Proc. Soc. Exp. Biol. Med. 216, 181191. Henderson, B.R., 2000. Nuclear-cytoplasmic shuttling of APC regulates beta-catenin subcellular localization and turnover. Nat. Cell Biol. 2, 653660. Hendry, J.H., 1991. The slower cellular recovery after higher-LET irradiations, including neutrons, focuses on the quality of DNA breaks. Radiat. Res. 128, S111S113. Hirao, A., Kong, Y.Y., Matsuoka, S., Wakeham, A., Ruland, J., Yoshida, H., Liu, D., Elledge, S.J., Mak, T.W., 2000. DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287, 18241827. Homan, B., Liebermann, D.A., 1998. The proto-oncogene c-myc and apoptosis. Oncogene 17, 33513357. Hollander, M.C., Sheikh, M.S., Bulavin, D.V., Lundgren, K., Augeri-Henmueller, L., Shehee, R., Molinaro, T.A., Kim, K.E., Tolosa, E., Ashwell, J.D., Rosenberg, M.P., Zhan, Q., FernandezSalguero, P.M., Morgan, W.F., Deng, C.X., Fornace Jr., A.J., 1999. Genomic instability in Gadd45adecient mice. Nat. Genet. 23, 176184. Huang, M.-E., Ye, Y.-C., Chen, S.-R., Chai, J.-R., Lu, J.-X., Zhoa, L., Gu, L.-J., Wang, Z.-Y., 1988. Use of all- trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72, 567572. Jackson, A.L., Loeb, L.A., 1998. The mutation rate and cancer. Genetics 148, 14831490. Jacoby, R.F., Seibert, K., Cole, C.E., Kello, G., Lubet, R.A., 2000. The cyclooxygenase-2 inhibitor celecoxib is a potent preventive and therapeutic agent in the min mouse model of adenomatous polyposis. Cancer Res. 60, 50405044. Jerey, A.M., Weinstein, I.B., Jennette, K.W., Grzeskowiak, K., Nakanishi, K., Harvey, R.G., Autrup, H., Harris, C., 1977. Structures of benzoapyrenenucleic acid adducts formed in human and bovine bronchial explants. Nature 269, 348350. Jin, S., Antinore, M.J., Lung, F.D., Dong, X., Zhao, H., Fan, F., Colchagie, A.B., Blanck, P., Roller, P.P., Fornace Jr., A.J., Zhan, Q., 2000. The GADD45 inhibition of Cdc2 kinase correlates with GADD45mediated growth suppression. J. Biol. Chem. 275, 1660216608. Jones, P.A., Rideout III, W.M., Shen, J.C., Spruck, C.H., Tsai, Y.C., 1992. Methylation, mutation and cancer. Bioessays 14, 3336. Kaban, L.B., Mulliken, J.B., Ezekowitz, R.A., Ebb, D., Smith, P.S., Folkman, J., 1999. Antiangiogenic therapy of a recurrent giant cell tumor of the mandible with interferon alfa-2a. Pediatrics 103, 11451149. Kagawa, S., Gu, J., Swisher, S.G., Ji, L., Roth, J.A., Lai, D., Stephens, L.C., Fang, B., 2000. Antitumor eect of adenovirus-mediated Bax gene transfer on p53-sensitive and p53-resistant cancer lines. Cancer Res. 60, 11571161. Khuri, F.R., Nemunaitis, J., Ganly, I., Arseneau, J., Tannock, I.F., Romel, L., Gore, M., Ironside, J., MacDougall, R.H., Heise, C., Randlev, B., Gillenwater, A.M., Bruso, P., Kaye, S.B., Hong, W.K., Kirn, D.H., 2000. a controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-uorouracil in patients with recurrent head and neck cancer. Nat. Med. 6, 879885. Khwaja, A., Rodriguez-Viciana, P., Wennstrom, S., Warne, P.H., Downward, J., 1997. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J. 16, 27832793.
218
King, T.J., Fukushima, L.H., Hieber, A.D., Shimabukuro, K.A., Sakr, W.A., Bertram, J.S., 2000. Reduced levels of connexin43 in cervical dysplasia: inducible expression in a cervical carcinoma cell line decreases neoplastic potential with implications for tumor progression [In Process Citation]. Carcinogenesis 21, 10971109. Knudson, A.G., Jr., 1978. Retinoblastoma: a prototypic hereditary neoplasm. Semin. Oncol. 5, 5760. Knudson Jr., A.G., Hethcote, H.W., Brown, B.W., 1975. Mutation and childhood cancer: a probabilistic model for the incidence of retinoblastoma. Proc. Natl. Acad. Sci. USA 72, 51165120. Kondo, Y., Koga, S., Komata, T., Kondo, S., 2000. Treatment of prostate cancer in vitro and in vivo with 2-5A-anti-telomerase RNA component. Oncogene 19, 22052211. Kroemer, G., 1999. Mitochondrial control of apoptosis: an overview. Biochem. Soc. Symp. 66, 115. Kushner, D.M., Paranjape, J.M., Bandyopadhyay, B., Cramer, H., Leaman, D.W., Kennedy, A.W., Silverman, R.H., Cowell, J.K., 2000. 2-5A antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells. Gynecol. Oncol. 76, 183192. Laird, A.D., Vajkoczy, P., Shawver, L.K., Thurnher, A., Liang, C., Mohammadi, M., Schlessinger, J., Ullrich, A., Hubbard, S.R., Blake, R.A., Fong, T.A., Strawn, L.M., Sun, L., Tang, C., Hawtin, R., Tang, F., Shenoy, N., Hirth, K.P., McMahon, G., Cherrington, G., 2000. SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. Cancer Res. 60, 41524160. Lakin, N.D., Jackson, S.P., 1999. Regulation of p53 in response to DNA damage. Oncogene 18, 76447655. Lavin, M.F., Khanna, K.K., 1999. ATM: the protein encoded by the gene mutated in the radiosensitive syndrome ataxia-telangiectasia. Int. J. Radiat. Biol. 75, 12011214. Lawley, P.D., 1980. DNA as a target of alkylating carcinogens. Br. Med. Bull. 36, 1924. Lawley, P.D., Shah, S.A., 1973. Methylation of DNA by 3H-14C-methyl-labelled N-methyl-Nnitrosoureaevidence for transfer of the intact methyl group. Chem. Biol. Interact. 7, 115120. le Coutre, P., Mologni, L., Cleris, L., Marchesi, E., Buchdunger, E., Giardini, R., Formelli, F., Gambacorti-Passerini, C., 1999. In vivo eradication of human BCR/ABL-positive leukemia cells with an ABL kinase inhibitor. J. Natl. Cancer Inst. 91, 163168. Lebowitz, P.F., Prendergast, G.C., 1998. Non-Ras targets of farnesyltransferase inhibitors: focus on Rho. Oncogene 17, 14391445. Lee, S.W., Tomasetto, C., Sager, R., 1991. Positive selection of candidate tumor-suppressor genes by subtractive hybridization. Proc. Natl. Acad. Sci. USA. 88, 28252829. Lehmann, A.R., Kirk-Bell, S., Arlett, C.F., Harcourt, S.A., Weerd-Kastelein, E.A., Keijzer, W., HallSmith, P., 1977. Repair of ultraviolet light damage in a variety of human broblast cell strains. Cancer Res. 37, 904910. Lemieux, N., Milot, J., Barsoum-Homsy, M., Michaud, J., Leung, T.K., Richer, C.L., 1989. First cytogenetic evidence of homozygosity for the retinoblastoma deletion in chromosome 13. Cancer Genet. Cytogenet. 43, 7378. Levine, A.J., 1997. p53, the cellular gatekeeper for growth and division. Cell 88, 323331. Li, J., Hu, S.X., Perng, G.S., Zhou, Y.L., Xu, K., Zhang, C.Y., Seigne, J., Benedict, W.F., Xu, H.J., 1996. Expression of the retinoblastoma (RB) tumor suppressor gene inhibits tumor cell invasion in vitro. Oncogene 13, 23792386. Li, M.J., Maizels, N., 1999. Activation and targeting of immunoglobulin switch recombination by activities induced by EBV infection. J. Immunol. 163, 66596664. Liao, D.J., Dickson, R.B., 2000. c-Myc in breast cancer. Endocr. Relat Cancer 7, 143164. Lindahl, T., Nyberg, B., 1972. Rate of depurination of native deoxyribonucleic acid. Biochemistry 11, 36103618. Lockhart, D.J., Winzeler, E.A., 2000. Genomics, gene expression and DNA arrays. Nature 405, 827836. Loeb, L.A., 1991. Mutator phenotype may be required for multistage carcinogenesis. Cancer Res. 51, 30753079. Lohmann, D.R., 1999. RB1 gene mutations in retinoblastoma. Hum. Mutat. 14, 283288. Lowe, S.W., Ruley, H.E., Jacks, T., Housman, D.E., 1993. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74, 957967.
219
Lynch, H.T., de la, C.A., 1999. Genetic susceptibility to non-polyposis colorectal cancer. J. Med. Genet. 36, 801818. Maecker, H.L., Koumenis, C., Giaccia, A.J., 2000. p53 promotes selection for Fas-mediated apoptotic resistance. Cancer Res. 60, 46384644. Magee, P.N., 1972. Possibilities of hazard from nitrosamines in industry. Ann. Occup. Hyg. 15, 1923. Malkin, D., Li, F.P., Strong, L.C., Fraumeni Jr., J.F., Nelson, C.E., Kim, D.H., Kassel, J., Gryka, M.A., Bischo, F.Z., Tainsky, , Friend, S.H., 1990. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Sci. 250, 12331238. Mancini, D., Singh, S., Ainsworth, P., Rodenhiser, D., 1997. Constitutively methylated CpG dinucleotides as mutation hot spots in the retinoblastoma gene (RB1). Am J. Hum. Genet. 61, 8087. Marchenko, N.D., Zaika, A., Moll, U.M., 2000. Death signal-induced localization of p53 protein to mitochondria. A potential role in apoptotic signaling. J. Biol. Chem. 275, 1620216212. Marsters, S.A., Pitti, R.A., Sheridan, J.P., Ashkenazi, A., 1999. Control of apoptosis signaling by Apo2 ligand. Recent Prog. Horm. Res. 54, 225234. McCormick, F., 1998. Signal transduction. Why Ras needs Rho. Nature 394, 220221. Mehta, P.P., Bertram, J.S., Loewenstein, W.R., 1986. Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell 44, 187196. Miller, J.A., Miller, E.C., 1975. Metabolic activation and reactivity of chemical carcinogens. Mutat. Res. 33, 2526. Minamoto, T., Mai, M., Ronai, Z., 2000. K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancersa review. Cancer Detect. Prev. 24, 112. Mirvish, S.S., 1995. Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC [published erratum appears in Cancer Lett. 1995 Nov 6;97(2), 271]. Cancer Lett. 93, 1748. Momand, J., Wu, H.H., Dasgupta, G., 2000. MDM2master regulator of the p53 tumor suppressor protein. Gene 242, 1529. Morin, P.J., 1999. beta-catenin signaling and cancer. Bioessays 21, 10211030. Mukai, S., Kondo, Y., Koga, S., Komata, T., Barna, B.P., Kondo, S., 2000. 2-5A antisense telomerase RNA therapy for intracranial malignant gliomas. Cancer Res. 60, 44614467. Multani, A.S., Ozen, M., Narayan, S., Kumar, V., Chandra, J., McConkey, D.J., Newman, R.A., Pathak, S., 2000. Caspase-dependent apoptosis induced by telomere cleavage and TRF2 loss. Neoplasia 2, 339345. Nakashima, T., Hudson, J.M., Clayman, G.L., 2000. Antisense inhibition of vascular endothelial growth factor in human head and neck squamous cell carcinoma. Head Neck 22, 483488. Neve, R.M., Sutterluty, H., Pullen, N., Lane, H.A., Daly, J.M., Krek, W., Hynes, N.E., 2000. Eects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells. Oncogene 19, 16471656. Neveu, M., Bertram, J.S., 2000. Gap junctions and neoplasia. In: Hertzberg, E.L., Bittar, E.E. (Eds.), Gap Junctions. JAI Press, Greenwich, CT, pp. 221262. Newbold, R.F., 1985. Multistep malignant transformation of mammalian cells by carcinogens: induction of immortality as a key event. Carcinog. Comput. Surv. 9, 1728. Newbold, R.F., Overell, R.W., 1983. Fibroblast immortality is a prerequisite for transformation by EJcHa-ras oncogene. Nature 304, 648651. O'Connell, J., Bennett, M.W., Nally, K., Houston, A., O'Sullivan, G.C., Shanahan, F., 2000. Altered mechanisms of apoptosis in colon cancer: Fas resistance and counterattack in the tumor-immune conict. Ann. NY Acad. Sci. 910, 178192. O'Reilly, M.S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W.S., Flynn, E., Birkhead, J.R., Olsen, B.R., Folkman, J., 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277285. O'Reilly, M.S., Holmgren, L., Chen, C., Folkman, J., 1996. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat. Med. 2, 689692.
220
Overholt, S.M., Liu, T.J., Taylor, D.L., Wang, M., ElNaggar, A.K., Shillitoe, E., Adler-Storthz, K., John, L.S., Zhang, W.W., Roth, J.A., Clayman, G.L., 1997. Head and neck squamous cell growth suppression using adenovirus-p53-FLAG: a potential marker for gene therapy trials. Clin. Cancer Res. 3, 185191. Owen-Schaub, L., Chan, H., Cusack, J.C., Roth, J., Hill, L.L., 2000. Fas and Fas ligand interactions in malignant disease. Int. J. Oncol. 17, 512. Pappalardo, P.A., Bonner, R., Krizman, D.B., Emmert-Buck, M.R., Liotta, L.A., 1998. Microdissection, microchip arrays, and molecular analysis of tumor cells (primary and metastases). Semin. Radiat. Oncol. 8, 217223. Parada, L.F., Tabin, C.J., Shih, C., Weinberg, R.A., 1982. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature 297, 474478. Parada, L.F., Weinberg, R.A., 1983. Presence of a kirsten murine sarcoma virus ras oncogene in cells transformed by 3-methylcholanthrene. Mol. Cell. Biol. 3, 22982301. Pardee, A.B., 1974. A restriction point for control of normal animal cell proliferation. Proc. Natl. Acad. Sci. USA 71, 12861290. Pegram, M.D., Konecny, G., Slamon, D.J., 2000. The molecular and cellular biology of HER2/neu gene amplication/overexpression and the clinical development of herceptin (trastuzumab) therapy for breast cancer. Cancer Treat. Res. 103, 5775. Pegram, M.D., Slamon, D.J., 1999. Combination therapy with trastuzumab (Herceptin) and cisplatin for chemoresistant metastatic breast cancer: evidence for receptor-enhanced chemosensitivity. Semin. Oncol. 26, 8995. Perletti, G., Concari, P., Giardini, R., Marras, E., Piccinini, F., Folkman, J., Chen, L., 2000. Antitumor activity of endostatin against carcinogen-induced rat primary mammary tumors. Cancer Res. 60, 17931796. Perou, C.M., Sorlie, T., Eisen, M.B., van de, R.M., Jerey, S.S., Rees, C.A., Pollack, J.R., Ross, D.T., Johnsen, H., Akslen, L.A., Fluge, O., Pergamenschikov, A., Williams, C., Zhu, S.X., Lonning, P.E., Borresen-Dale, A.L., Brown, P.O., Botstein, D., 2000. Molecular portraits of human breast tumours. Nature 406, 747752. Pitti, R.M., Marsters, S.A., Lawrence, D.A., Roy, M., Kischkel, F.C., Dowd, P., Huang, A., Donahue, C.J., Sherwood, S.W., Baldwin, D.T., Godowski, P.J., Wood, W.I., Gurney, A.L., Hillan, K.J., Cohen, R.L., Goddard, A.D., Botstein, D., Ashkenazi, A., 1998. Genomic amplication of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396, 699703. Prendergast, G.C., Khosravi-Far, R., Solski, P.A., Kurzawa, H., Lebowitz, P.F., Der, C.J., 1995. Critical role of Rho in cell transformation by oncogenic Ras. Oncogene 10, 22892296. Qiu, R.G., Chen, J., McCormick, F., Symons, M., 1995. A role for Rho in Ras transformation. Proc. Natl. Acad. Sci. USA 92, 1178111785. Ramirez, R.D., D'Atri, S., Pagani, E., Faraggiana, T., Lacal, P.M., Taylor, R.S., Shay, J.W., 1999. Progressive increase in telomerase activity from benign melanocytic conditions to malignant melanoma. Neoplasia 1, 4249. Reed, S.I., 1997. Control of the G1/S transition. Cancer Surv. 29, 723. Rezniko, C.A., Bertram, J.S., Brankow, D.W., Heidelberger, C., 1973. Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconuence inhibition of cell division. Cancer Res. 33, 23392349. Roth, J.A., Nguyen, D., Lawrence, D.D., Kemp, B.L., Carrasco, C.H., Ferson, D.Z., Hong, W.K., Komaki, R., Lee, J.J., Nesbitt, J.C., Pisters, K.M., Putnam, J.B., Schea, R., Shin, D.M., Walsh, G.L., Dolormente, M.M., Han, C.I., Martin, F.D., Yen, N., Xu, K., Stephens, L.C., McDonnell, T.J., Mukhopadhyay, T., Cai, D., 1996. Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat. Med. 2, 985991. Rowinsky, E.K., Windle, J.J., Ho, D.D., 1999. Ras protein farnesyltransferase: a strategic target for anticancer therapeutic development. J. Clin. Oncol. 17, 36313652. Rusin, M., Butkiewicz, D., Malusecka, E., Zborek, A., Harasim, J., Czyzewski, K., Bennett, W.P., Shields, P.G., Weston, A., Welsh, J.A., Krzyzowska-Gruca, S., Chorazy, M., Harris, C.C., 1999. Molecular epidemiological study of non-small-cell lung cancer from an environmentally polluted region of Poland. Br. J. Cancer 80, 14451452.
221
Rytomaa, M., Lehmann, K., Downward, J., 2000. Matrix detachment induces caspase-dependent cytochrome c release from mitochondria: inhibition by PKB/Akt but not Raf signalling. Oncogene 19, 44614468. Sarvesvaran, J., Going, J.J., Milroy, R., Kaye, S.B., Keith, W.N., 1999. Is small cell lung cancer the perfect target for anti-telomerase treatment?. Carcinogenesis 20, 16491651. Sattler, M., Salgia, R., 1997. Activation of hematopoietic growth factor signal transduction pathways by the human oncogene BCR/ABL. Cytokine Growth Factor Rev. 8, 6379. Schezek, K., Ahmadian, M.R., Kabsch, W., Wiesmuller, L., Lautwein, A., Schmitz, F., Wittinghofer, A., 1997. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333338. Scheurle, D., Jahanzeb, M., Aronsohn, R.S., Watzek, L., Narayanan, R., 2000. HER-2/neu expression in archival non-small cell lung carcinomas using FDA-approved Hercep test. Anticancer Res. 20, 20912096. Schindler, T., Bornmann, W., Pellicena, P., Miller, W.T., Clarkson, B., Kuriyan, J., 2000. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 289, 19381942. Schuler, M., Rochlitz, C., Horowitz, J.A., Schlegel, J., Perruchoud, A.P., Kommoss, F., Bolliger, C.T., Kauczor, H.U., Dalquen, P., Fritz, M.A., Swanson, S., Herrmann, R., Huber, C., 1998. A phase I study of adenovirus-mediated wild-type p53 gene transfer in patients with advanced non-small cell lung cancer. Hum. Gene Ther. 9, 20752082. Sherr, C.J., 1996. Cancer cell cycles. Science 274, 16721677. Shields, J.M., Pruitt, K., McFall, A., Shaub, A., Der, C.J., 2000. Understanding Ras: ìt ain't over 'til it's over'. Trends Cell Biol. 10, 147154. Shigenaga, M.K., Gimeno, C.J., Ames, B.N., 1989. Urinary -8-hydroxy-2-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc. Natl. Acad. Sci. USA 86, 96979701. Shih, I.M., Yu, J., He, T.C., Vogelstein, B., Kinzler, K.W., 2000. The beta-catenin binding domain of adenomatous polyposis coli is sucient for tumor suppression. Cancer Res. 60, 16711676. Shinoura, N., Yoshida, Y., Asai, A., Kirino, T., Hamada, H., 2000. Adenovirus-mediated transfer of p53 and Fas ligand drastically enhances apoptosis in gliomas. Cancer Gene Ther. 7, 732738. Shtutman, M., Zhurinsky, J., Simcha, I., Albanese, C., D'Amico, M., Pestell, R., BenZe'ev, A., 1999. The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc. Natl. Acad. Sci. USA 96, 55225527. Smith, M.L., Ford, J.M., Hollander, M.C., Bortnick, R.A., Amundson, S.A., Seo, Y.R., Deng, C.X., Hanawalt, P.C., Fornace Jr., A.J., 2000. p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21, and/or gadd45 genes. Mol. Cell Biol. 20, 37053714. Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K.E., Montgomery, E., Lal, A., Riggins, G.J., Lengauer, C., Vogelstein, B., Kinzler, K.W., 2000. Genes expressed in human tumor endothelium. Science 289, 11971202. Stebbing, J., Copson, E., O'Reilly, S., 2000. Herceptin (trastuzamab) in advanced breast cancer. Cancer Treat. Rev. 26, 287290. Strickland, S., Breitman, T.R., Frickel, F., Nurrenbach, A., Hadicke, E., Sporn, M.B., 1983. Structureactivity relationships of a new series of retinoidal benzoic acid derivatives as measured by induction of dierentiation of murine F9 teratocarcinoma cells and human HL-60 promyelocytic leukemia cells. Cancer Res. 43, 52685272. Swisher, S.G., Roth, J.A., Nemunaitis, J., Lawrence, D.D., Kemp, B.L., Carrasco, C.H., Connors, D.G., Naggar, A.K., Fossella, F., Glisson, B.S., Hong, W.K., Khuri, F.R., Kurie, J.M., Lee, J.J., Lee, J.S., Mack, M., Merritt, J.A., Nguyen, D.M., Nesbitt, J.C., Perez-Soler, R., Pisters, K.M., El Putnam Jr., J.B., Richli, W.R., Savin, M., Waugh, M.K., 1999. Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer. J. Natl. Cancer Inst. 91, 763771. Takayama, K., Ueno, H., Nakanishi, Y., Sakamoto, T., Inoue, K., Shimizu, K., Oohashi, H., Hara, N., 2000. Suppression of tumor angiogenesis and growth by gene transfer of a soluble form of vascular endothelial growth factor receptor into a remote organ. Cancer Res. 60, 21692177.
222
Talpaz, C.L., 2000. Activity of an Abl specic tyrosine kinase inhibitor in patients with BcrAbl positive acute leukemias, including chronic myelogenous leukemia in blast crisis. Am. Soc. Clin Oncology. (Abstract). Tatsumoto, N., Hiyama, E., Murakami, Y., Imamura, Y., Shay, J.W., Matsuura, Y., Yokoyama, T., 2000. High telomerase activity is an independent prognostic indicator of poor outcome in colorectal cancer. Clin. Cancer Res. 6, 26962701. Templeton, D.J., Park, S.H., Lanier, L., Weinberg, R.A., 1991. Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering. Proc. Natl. Acad. Sci. USA 88, 30333037. Tetsu, O., McCormick, F., 1999. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422426. Thompson, C.B., 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267, 14561462. Thompson, W.J., Piazza, G.A., Li, H., Liu, L., Fetter, J., Zhu, B., Sperl, G., Ahnen, D., Pamukcu, R., 2000. Exisulind induction of apoptosis involves guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition, protein kinase G activation, and attenuated beta-catenin. Cancer Res. 60, 33383342. Tormo, M., Tari, A.M., McDonnell, T.J., Cabanillas, F., Garcia-Conde, J., Lopez-Berestein, G., 1998. Apoptotic induction in transformed follicular lymphoma cells by Bcl-2 downregulation. Leuk. Lymphoma 30, 367379. Tucker, M.A., Coleman, C.N., Cox, R.S., Varghese, A., Rosenberg, S.A., 1988. Risk of second cancers after treatment for Hodgkin's disease. N. Engl. J. Med. 318, 7681. Umbricht, C.B., Sherman, M.E., Dome, J., Carey, L.A., Marks, J., Kim, N., Sukumar, S., 1999. Telomerase activity in ductal carcinoma in situ and invasive breast cancer. Oncogene 18, 34073414. van Steeg, H., Kraemer, K.H., 1999. Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer. Mol. Med. Today 5, 8694. van Steensel, B., Smogorzewska, A., de Lange, T., 1998. TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401413. Vaux, D.L., Cory, S., Adams, J.M., 1988. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335, 440442. Vaziri, H., Benchimol, S., 1999. Alternative pathways for the extension of cellular life span: inactivation of p53/pRb and expression of telomerase. Oncogene 18, 76767680. Volpert, O.V., Dameron, K.M., Bouck, N., 1997. Sequential development of an angiogenic phenotype by human broblasts progressing to tumorigenicity. Oncogene 14, 14951502. vonLintig, F.C., Dreilinger, A.D., Varki, N.M., Wallace, A.M., Casteel, D.E., Boss, G.R., 2000. Ras activation in human breast cancer. Breast Cancer Res. Treat. 62, 5162. Vooijs, M., Berns, A., 1999. Developmental defects and tumor predisposition in Rb mutant mice. Oncogene 18, 52935303. Wang, J.L., Liu, D., Zhang, Z.J., Shan, S., Han, X., Srinivasula, S.M., Croce, C.M., Alnemri, E.S., Huang, Z., 2000b. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc. Natl. Acad. Sci. USA 97, 71247129. Wang, S., Guo, M., Ouyang, H., Li, X., Cordon-Cardo, C., Kurimasa, A., Chen, D.J., Fuks, Z., Ling, C.C., G.C, 2000a. Li. The catalytic subunit of DNA-dependent protein kinase selectively regulates p53dependent apoptosis but not cell-cycle arrest.. Proc. Natl. Acad. Sci. USA 97, 15841588. Watanabe, T., Sullenger, B.A., 2000. Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts. Proc. Natl. Acad. Sci. USA 97, 84908494. Weintraub, S.J., B.Chow, K.N., Luo, R.X., Zhang, S.H., He, S., Dean, D.C., 1995. Mechanism of active transcriptional repression by the retinoblastoma protein. Nature 375, 812815. Westermark, B., Heldin, C.H., Nister, M., 1995. Platelet-derived growth factor in human glioma. Glia 15, 257263. Westra, W.H., Baas, I.O., Hruban, R.H., Askin, F.B., Wilson, K., Oerhaus, G.J., Slebos, R.J., 1996. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res. 56, 22242228. Wolf, C.R., Henderson, C.J., 1998. Use of transgenic animals in understanding molecular mechanisms of toxicity. J. Pharm. Pharmacol. 50, 567574.
223
Wole, A.P., 1997. Sinful repression. Nature 387, 1617. Wu, Z., O'Reilly, M.S., Folkman, J., Shing, Y., 1997. Suppression of tumor growth with recombinant murine angiostatin. Biochem. Biophys. Res. Commun. 236, 651654. Xiao, G., Chicas, A., Olivier, M., Taya, Y., Tyagi, S., Kramer, F.R., Bargonetti, J., 2000. A DNA damage signal is required for p53 to activate gadd45. Cancer Res. 60, 17111719. Yamauchi, T., Yamauchi, N., Ueki, K., Sugiyama, T., Waki, H., Miki, H., Tobe, K., Matsuda, S., Tsushima, T., Yamamoto, T., Fujita, T., Taketani, Y., Fukayama, M., Kimura, S., Yazaki, Y., Nagai, R., Kadowaki, T., 2000. Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer. J. Biol. Chem. 275, 3393733944. Yashima, K., Maitra, A., Rogers, B.B., Timmons, C.F., Rathi, A., Pinar, H., Wright, W.E., Shay, J.W., Gazdar, A.F., 1998. Expression of the RNA component of telomerase during human development and dierentiation. Cell Growth Dier. 9, 805813. Yen, L., You, X.L., AlMoustafa, A.E., Batist, G., Hynes, N.E., Mader, S., Meloche, S., Alaoui-Jamali, M.A., 2000. Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis. Oncogene 19, 34603469. Zohn, I.M., Campbell, S.L., Khosravi-Far, R., Rossman, K.L., Der, C.J., 1998. Rho family proteins and Ras transformation: the RHOad less traveled gets congested. Oncogene 17, 14151438. zur Hausen, H., 2000. Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis.J. Natl. Cancer Inst. 92, 690698.

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The molecular biology of cancer

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Data from the NCI clinical trials database November 2000.

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J.S. Bertram / Molecular Aspects of Medicine 21 (2001) 167223

J.S. Bertram / Molecular Aspects of Medicine 21 (2001) 167223

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