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Induction of apoptosis in plasma cells by B lymphocyte-induced maturation protein-1 knockdown.

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor that plays an


important role during plasmacytic differentiation and is expressed in normal and transformed plasma
cells. We here investigated the importance of continuous Blimp-1 expression. We found that
knockdown of Blimp-1 expression by lentiviral vector-delivered short hairpin RNA causes apoptosis in
multiple myeloma cell lines and plasmacytoma cells, indicating that continued expression of Blimp-1 is
required for cell survival.
We examined the mechanism underlying Blimp-1 knockdown-mediated apoptosis and found that the
Blimp-1 knockdown neither reversed the phenotypic markers of plasma cells nor caused cell cycle
arrest. Instead, our results show that knockdown of Blimp-1 induced the proapoptotic protein Bim,
reduced the antiapoptotic protein Mcl-1, and activated caspase-9 and caspase-3. We further link
apoptosis in transformed plasma cells mediated by proteasome inhibitors, the effective therapeutic
agent for multiple myeloma patients, with reduced expression of Blimp-1. Together, our data suggest
that Blimp-1 ensures the survival of transformed plasma cells.

Inhibition of Blimp-1 expression causes apoptosis in plasma cell lines.


Transduction of shRNAs for Blimp-1 (U6Blimp1-1004i or U6Blimp1-2254i),
but not a shRNA with a scrambled sequence (U6controli), causes cell death.
H929 or U266 cells were transduced with Blimp-1 shRNA-expressing
lentiviral vectors. The percentage of cell death was assayed by trypan blue
exclusion following various days of infection. 1

Cancer Res. 2007 Dec 15;67(24):11914-23.

Activation of caspases, induction of Bim and reduction of Mcl-1 during Blimp-1 RNAimediated apoptosis.
B. Caspase-9 and caspase-3 were activated by Blimp-1 RNAi in H929 cells. The
activities of caspase-8, caspase-9, and caspase-3 were determined from H929 cells
after 3 and 4 d of transduction with U6Blimp1-1004i or U6controli.
D. Immunoblot analysis of procaspase-4 and antiapoptotic Mcl-1, or proapoptotic Bim
& Bcl-2 family protein expression from H929 cell lysates after 4 d of transduction
with U6controli or U6Blimp1-1004i. Actin expression was analyzed for the loading
control.2

Cancer Res. 2007 Dec 15;67(24):11914-23.

Altered mRNA expression of Pax5 and Blimp-1 in B cells in multiple myeloma


Borson ND, Lacy MQ, Wettstein PJ. Dept of Immunology Mayo Clinic
Multiple myeloma (MM) is a plasma cell disorder that potentially initiates during an early stage of B-cell
development. The findings of the premature expression of Blimp-1, in conjunction with the potentially
disrupted transactivating domains in Pax5 and the frequent low levels of full-length Pax5 expression, suggest
that B-cell development in MM could be uncoupled from normal control by both of these transcription
factors. Blimp-1 is believed to induce either Ig secretion and terminal differentiation in B cells or growth
arrest and cell death, depending on the developmental stage of the cell, and it is up-regulated during the
transition of mature B cells to Ig-secreting plasma cells. Blimp-1 also regulates several target genes,
including transcription factor c-myc, antiapoptotic bcl-2 family member A1, syndecan-1, and J chain. We
also performed semiquantitative analyses of the mRNA expression levels of B lymphocyte-induced
maturation protein (Blimp-1), because expression levels of Pax5 and Blimp-1 have been shown to be
inversely correlated. We observed the expression of Blimp-1 in the B-cell populations in all 11 MM
patients but in none of 11 healthy subjects. We hypothesize that premature Blimp-1 expression coupled to
altered and deficient Pax5 expression causes some proliferating B cells to prematurely differentiate to plasma
cells in MM.
It has recently been found that Blimp-1 expression in germinal centers is associated with a subset of cells
with a phenotype intermediate between germinal center mature B cells and plasma cells and that these cells
are nonapoptotic, proliferating cells. It is conceivable that malignant plasma cells could develop if
proliferating B cells were pushed to differentiate prematurely. The expression of Blimp-1 in B cells of MM
patients may contribute to that paradox observed in MM: loss of polyclonality accompanied by proliferation
of a monoclonal cell type. We propose that the coupling of premature Blimp-1 expression with alterations and
deficits in Pax5 expression may cause some proliferating B cells to prematurely progress to a "plasma celllike" state while bypassing isotype switching events and control of proliferation.3
Identification of a functionally impaired positive regulatory domain I binding factor 1 transcription
repressor in myeloma cell lines.
Gyry I, Fejr G, Ghosh N, Seto E, Wright KL. H. Lee Moffitt Cancer Center and Research Institute,
Department of Interdisciplinary Oncology, University of South Florida, Tampa, FL
B cell differentiation into a plasma cell requires expression of the positive regulatory domain zinc finger
protein 1 gene (PRDM1) that encodes the positive regulatory domain I binding factor 1 (PRDI-BF1 or
Blimp-1) protein. It represses the transcription of specific target genes, including c-myc, the MHC class II
trans-activator, Pax-5, and CD23b.
Ectopic expression of Blimp-1 in the BCL1 mature B cell line induces many of the phenotypic changes
associated with plasma cell differentiation, including IgM secretion, J chain up-regulation, and Syndecan-1
expression. Expression of Blimp-1 in earlier stage B cell lines leads to increased apoptosis, consistent with a
central role for Blimp-1 or PRDI-BF1 in the cell fate choice of B cells.There are four identified target genes
that are directly repressed by PRDI-BF1 in the B lineage: c-myc, MHC class II trans-activator (CIITA),
CD23b, and Pax-5. The c-myc gene plays roles in growth control, apoptosis, and differentiation, and
repression of c-myc is important, but not sufficient, for plasma cell differentiation. The down-regulation of
CIITA by PRDI-BF1 results in silencing of MHC class II expression, another phenotypic feature of plasma
cells. CD23b is a type II integral membrane protein that is down-regulated in activated B cells and remains
3

Blood. 2002 Dec 15;100(13):4629-39.

silenced during later stages of B cell development. Blimp-1 plays a role in maintaining low levels of CD23b
by interfering with activation of the CD23b promoter by IFN regulatory factor-4. The B cell lineage-specific
activator protein (BSAP/Pax-5) is indispensable for B cell lineage commitment and B cell development and
is silenced at the transcriptional level in terminally differentiated B cells by Blimp-1. PRDI-BF1 also
represses the IFN-beta promoter after viral induction in nonlymphoid cell lines.
In this study we demonstrate the presence of an alternative protein product of the PRDM1 gene. The new
protein, PRDI-BF1 beta, has a disrupted PR domain and lacks the amino-terminal 101 aa of the originally
described protein. PRDI-BF1 beta has a dramatic loss of repressive function on multiple target genes, but
maintains normal DNA-binding activity, nuclear localization, and association with histone deacetylases and
deacetylase activity.
Myeloma cell lines express the highest levels of PRDM1 beta mRNA relative to the full-length form,
while primary cells and several other cell lines have very low, but detectable, levels of PRDM1 beta.
RNA analysis and analysis of the PRDM1 promoters demonstrate that PRDI-BF1 beta is generated from the
same gene by alternative transcription initiation using an internal promoter. These newly described features
of the PRDM1 gene are highly analogous to the PRDM2 (RIZ) and PRDM3 (MDS1-EVI1) genes, in which
each express a truncated protein missing the PR domain. The expression of each of the truncated proteins is
elevated in cancerous cells and may play an important role in the disease.4
The expression of PRDI-BF1 beta isoform in multiple myeloma plasma cells.
The PRDM1 gene, a master regulator of plasma cells (PC), can generate two transcription factor isoforms:
PRDI-BF1alpha and PRDI-BF1beta. The present study shows that purified human normal PC have a
significantly lower levels of PRDI-BF1beta expression than that in tumoral PC isolated from multiple
myeloma (MM) (0.06+/-0.01 and 0.25+/-0.05, respectively; p<0.001)
Human positive regulatory domain I binding factor 1 (PRDI-BF1 or BLIMP-1) is a transcription factor that
has been demonstrated to act as a master regulator required and sufficient for the generation and for the
prolonged maintenance of plasma cells (PC). PRDI-BF1 essentially functions as a repressor, causing exit
from cell cycling and the extinction of the expression of several genes critical for B-cell development at
earlier stages. The PRDM1 gene, which codifies for PRDI-BF1, contains an alternative promoter capable of
generating a PRDI-BF1 deleted protein (called PRDI-BF1), which lacks 101 amino acids comprising most
of the regulatory domain.
PRDI-BF1 has been detected in relevant quantities in multiple myeloma (MM) cell lines. Since this
molecule contains the DNA-binding domain but bears a disrupted regulatory domain, PRDI- BF1 might
behave as an inhibitor of functional PRDI- BF1, called PRDI-BF1. We decided to compare, using real time
polymerase chain reaction (RT-PCR), the occurrence of PRDI-BF1 and PRDI-BF1 in human MM cell
lines, MM patients` tumoral PC (n=17) and normal human PC (n=11) purified from either bone marrow,
tonsil or colon lamina propria
The relatively high expression of the -isoform in all cell types tested could be expected bearing in mind that
its protein product, PRDI-BF1, is strictly required for commitment to the PC fate. When the expression
level of the PRDI- BF1 isoform was tested and compared in the same PC populations (Figure 2B),
significantly greater differences were observed. Interestingly, normal human PC had a markedly lower
transcript level for this factor (0.060.01) than did the other three MM PC populations MM cell lines, and
MM patients` PC. These data suggest that the level of PRDI- BF1 expression could be a feature
distinguishing between malignant and normal PC.

J Immunol. 2003 Mar 15;170(6):3125-33.

For this reason we decided to examine the ratio of PRDI-BF1/PRDI-BF1 transcript levels in the different
PC under study. As shown in Figure 2C, MM cell lines had very low ratios, a result that was due to the high
level of PRDI-BF1 expression. In contrast, normal PC had a ten- fold higher PRDI-BF1/PRDI-BF1
ratio (43.685.89). The ratios obtained in normal PC were more than three times higher than those of MM
patients` PC (16.371.04). Therefore, it is conceivable that the overexpression of PRDI-BF1 detected in
MM patients` PC could contribute to progression into the tumoral state.5
Coordination of upregulated XBP-1 and downregulated c-myc during myeloma cell differentiation
induced by 2-methoxyestradiol.
Our previous studies showed that 2-methoxyestradiol (2ME2) at low concentrations (0.1-0.5 micromol/l)
could induce differentiation of myeloma cells, and transcription factors Blimp-1 and XBP-1 repressed by
Pax-5 were both upregulated. The present study was aimed at elucidating the molecular mechanism
underlying 2ME2-induced myeloma cell differentiation. We demonstrated that cell differentiation required
not only Blimp-1 to upregulate XBP-1expression but also Blimp-1 to inhibit c-myc activity. These two signal
transduction pathways worked dependently of each other and synergistically to promote the differentiation
process of myeloma cells.6
MHC class II transactivator (CIITA) expression is upregulated in multiple myeloma cells by IFNgamma.
The MHC class II transactivator (CIITA) acts in the cell nucleus as the master regulator of MHC class II
(MHC II) gene expression. It is important to study CIITA regulation in multiple myeloma since MHC
expression is central to ability of myeloma cells to present antigen and to the ability of the immune system to
recognize and destroy this malignancy. PRDI-BF1/Blimp-1 diminishes the constitutive ability of these cells
to present antigen by limiting CIITA and MHC II expression.7
Regulation of c-myc expression by Blimp-1 during the differentiation of myeloma cells
OBJECTIVE: To investigate the repression of c-myc induced by Blimp-1 in 2-methoxyestradiol(2ME2)mediated differentiation of multiple myeloma cells. METHODS: CZ-1 and LP-1 myeloma cells lines were
exposed to 0.5 micromol/L of 2ME2 and 0.5 micromol/L of 2ME2 + antisense oligonucleotides (ASODN)
for 72 h. The effects on transcription of c-myc mRNA were studied by RT-PCR. The c-myc protein was
assayed with Western blot. The changes of the cell lines in morphology, expression of surface CD49e and
quantity of immunoglobin light chain secretion in the supernatant were studied. RESULTS: Incubation of the
cells with 0.5 micromol/L of 2ME2 could up-regulated Blimp-1 expression and increase the expression of cmyc gene. In contrast, c-myc expression was decreased with Blimp-1 expression down-regulated by
ASODN and the cell lines differentiation was arrested. CONCLUSION: Blimp-1 could directly repress
the expression of c-myc in 2ME2-mediated differentiation induction of multiple myeloma cells.8

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Haematologica. 2006 Nov;91(11):1579-80.


Leuk Res. 2007 Sep;31(9):1259-65.
Mol Immunol. 2007 Apr;44(11):2923-32.
Zhonghua Xue Ye Xue Za Zhi. 2007 May; 28(5):318-22.

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