ALBERT

Description: PU.1 is an Ets family transcription factor, which is important for differentiation of granulocytes, monocytes/macrophages, and B cells. In the Friend leukemia model, it is reported that the failure of PU.1 down-regulation in erythroblasts reportedly results in differentiation arrest that leads to erythroleukemia. In conditional knockout mice of the 3.5 kb length of enhancer region located in14 kb 5′ of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells down to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like disease. In addition, a deletion of the 3.5 kb enhancer region, which also contains the suppressor region for PU.1 in T cells, results in ectopic expression of PU.1 in T cells, which leads to T cell lymphoma in those mice. Taken together, the failure of up-regulation or down-regulation of PU.1 in certain differentiation stages for each lineage appears to cause differentiation arrest and hematological malignancies. We recently reported that PU.1 is down-regulated in a majority of myeloma cell lines through the methylation of the promoter and enhancer region located in17 kb 5′ of human PU.1 gene which is homologous to that in14 kb 5′ of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of those PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that down-regulation of PU.1 is necessary for myeloma cell growth. In addition, we reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of some myeloma patients. Myeloma patients with low-to-negative PU.1 expression (lower 25th percentile of PU.1 expression level distribution among 30 patients we examined) may have poor prognosis compared to those with high PU.1 expression, although more patient samples have to be examined to define the significance of the relationship of PU.1 expression levels and prognosis. To elucidate the mechanisms of PU.1 induced cell growth arrest and apoptosis of myeloma cells, we next performed DNA microarray analysis to compare gene expression levels before and after PU.1 induction. We utilized Illumina Sentrix® Human-6 Expression BeadChip. Of 47296 genes, 479 genes were up-regulated (〉2fold) and 1697 genes down-regulated (

Description: PU.1 is an Ets family transcription factor, which is necessary for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream regulatory element (URE) located in 14 kb 5' of the PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Since the URE contains a suppressor region for PU.1 expression in T cells, such mice express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages in certain cell lineages appears to result in hematological malignancies. We previously reported that PU.1 is down-regulated in various myeloma cell lines. In addition, PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In those myeloma cell lines, the promoter and URE of the PU.1 gene are highly methylated. A demethylation agent, 5-aza-2'-deoxycytidine, induced PU.1 up-regulation, growth arrest, and apoptosis in myeloma cell lines, KMS12PE and KHM11. In addition, conditionally expressed PU.1 induced cell growth arrest and apoptosis in PU.1-low-negative myeloma cell lines, U266 and KMS12PE, suggesting that PU.1 is a tumor suppressor for myeloma cells. To elucidate the mechanisms of the cell growth arrest and apoptosis in myeloma cells induced by PU.1, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, while among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. With further investigation, we concluded that PU.1 directly transactivated the TRAIL gene in myeloma cells, leading to apoptosis. Based on the DNA microarray data generated, we found that IRF4 is downregulated in U266 myeloma cells after PU.1 induction. It has been reported that knockdown of IRF4 induces apoptosis in myeloma cell lines. Therefore, we examined whether IRF4 was down-regulated in three myeloma cell lines, U266, KMS12PE, and KHM11 following PU.1 induction. Conditional expression of PU.1 by tet-off system induced IRF4 down-regulation in U266and KMS12PE cells. With lentiviral transduction method, ectopic expression of PU.1 also induced IRF4 down-regulation, cell-cycle arrest, and apoptosis in KHM11 cells. To investigate the role of IRF4 in PU.1-expressing U266 cells, we stably expressed IRF4, partially rescuing U266 cells from apoptosis. IRF4 is known to directly bind to the IRF7 promoter and down-regulate IRF7 expression in activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma cells. Therefore, we examined whether IRF4 bound to the IRF7 promoter in KMS12PEand U266cells using chromatin immunoprecipitation assays. We found that IRF4 directly bound to the IRF7 promoter in both myeloma cell lines. When we overexpressed PU.1, IRF4 levels were decreased and the IRF4 binding to the IRF7 promoter was significantly reduced in those cell lines. Moreover, knockdown of IRF7 significantly rescued PU.1-expressing U266cells from apoptosis. These data strongly suggest that PU.1-induced apoptosis is associated with IRF4 down-regulation and subsequent IRF7 up-regulation in myeloma cells. Since IRF4 is essential transcription factor for myeloma cell survival, up-regulation of PU.1 by demethylation agents, including 5-aza-2'-deoxycytidine may serve as a promising therapeutic modality of multiple myeloma by inducing down-regulation of IRF4. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2337 The nucleosome remodeling and deacetylase (NuRD) complex is involved in gene repression and normal hematopoiesis. In embryonic stem (ES) cells, NuRD complex directly maintains the self-renewal from lineage commitment. Murine models with defects in NuRD complex present with increased hematopoietic stem/progenitor cells (HSPCs). Several transcriptional factors (TFs), such as SALL4, FOG1, BCL11A, BCL11B, ZNF521 use a conservative 12 amino acid domain to recruit NuRD components for targeted gene repression. In this study, we plan to test the hypothesis that by blocking the interaction between NuRD and its TFs, we can phenocopy its knockdown effects on human HSPC. Human cord blood (CB) CD34+ cells were purified, and treated with peptide with the NuRD interacting sequences (wt-pep) or its controls mutant peptide (mut-pep) or scramble peptide (scr-pep). In vitro and in vivo assays were performed to evaluate whether wt-pep treatment can maintain or expand human cord blood HSPCs, which were further characterized as CD34+CD90+ and CD34+CD38-CD90+ sub-population with known repopulating potential after ex vivo culture. First we observed wt-pep treated CB CD34+ (1×104) cultures with cytokines (SCF, Flt3 ligand, IL-3 and IL-6) yielded 10.6 times greater numbers of CD34+CD90+ cells (Fig 1) and 19.3 times greater numbers of CD34+CD38-CD90+ as compared to the cultures containing cytokines with scr-pep after 6 days of culture (p

Description: Abstract 2844 Poster Board II-820 Introduction: Skeletal complications including bone fracture, bone pain and hypercalcemia are major clinical events in patients of multiple myeloma (MM). Osteoclastgenesis is known to be induced by free receptor activator of nuclear factor kappa β ligand (RANKL) and inhibited by dimerization of RANKL and osteoprotegerin (OPG). OPG is also known as a soluble inhibitor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); therefore, a possible role of TRAIL as an osteoclast inducer is suggested, although the association of TRAIL with bone lesions in MM is a matter of debate. We thus investigated the expression of TRAIL mRNA in purified MM cells and analyzed its association with skeletal-related events. Patients and Methods: MM cells were purified from bone marrow samples from 40 MM patients by CD138-immunomagnetic beads (Miltenyi Biotech, Paris, France). TRAIL mRNA expression in purified MM cells was analyzed by real time PCR(ABI PRISM 7700 Sequence Detector, Applied Biosystems). Simultaneous analysis of serum TRAIL concentrations, analyzed by ELISA (Diaclone, Cedex, France), and TRAIL mRNA-expression levels was also performed in 23 cases. Each of these patients was given a score called skeletal-related event score (SRE score) according to the skeletal complications (pathological fracture, bone-associated plasmacytoma, 〉12mg/dL hypercalcemia, and receiving an pathological fracture-related operation or radiation therapy). Results: Significant association (p=0.0006) was seen between TRAIL mRNA expression levels and the SRE score (Fig.1). Serum calcium levels also had significant association to TRAIL mRNA expression levels (p=0.0050). On the other hand, no association of TRAIL mRNA with hemoglobin (p=0.3970) and platelets (p=0.9401) was seen. Serum TRAIL concentrations in MM cases, which were equivalent to those in healthy individuals, did not correlate to TRAIL mRNA expression levels in purified MM cells (p=0.4094). Conclusions: The data suggest that MM patients with high TRAIL expression in MM cells tend to have more skeletal complications, which may be mediated by increased osteoclastgenesis. Since serum TRAIL concentrations did not correlate with TRAIL mRNA levels in MM cells, increased TRAIL expression in bone marrow microenvironment could be important. Despite of previous reports suggesting TRAIL-induced apoptosis of hematopoietic cells, the observed high TRAIL expression did not correlate with anemia or thrombocytopenia in our cases. Although mechanisms regulating TRAIL expression in MM cells and protection from TRAIL-induced apoptosis remain to be determined, our findings may introduce a new strategy targeting TRAIL to reduce skeletal events in MM. Disclosures: No relevant conflicts of interest to declare.

Description: PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream enhancer region (URE) located in 14 kb 5’ of the murine PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Therefore, down-regulation of PU.1 in myeloid and B cell lineages results in hematological malignancies. We previously reported that PU.1 is down-regulated in 5 out of 7 myeloma cell lines as well as primary myeloma cells from a subset of myeloma patients; that the promoter and the URE located in 17 kb 5’ of the human PU.1 gene that is homologous to that in 14 kb 5’ of murine PU.1 gene are highly methylated in these cell lines; and that conditionally expressed PU.1 with tet-off system induces cell growth arrest and apoptosis in myeloma cell lines, U266 and KMS12PE, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. Here, to evaluate tumor suppressor activity of PU.1 in mature B and plasma cells in vivo, we generated Cγ1-Cre PU.1 knockout mice by crossing Cγ1-Cre and PU.1-loxP mice. We confirmed that PU.1 alleles were both conditionally deleted in the maturation stages of B cells from post germinal center B to plasma cells. By 18-24 months of age, about 77.7% (10 of 13) of the knockout mice had developed serum M proteins. To induce B cell differentiation to plasma cells, those mice were immunized with NP-CGG and 76.9% (20 of 26) of the mice developed serum M protein. ELISA of sera from those mice revealed that IgG was not elevated compared to those from the PU.1-loxP mice, which was thought because Cγ1-Cre locus fails to produce IgG1. Instead, a small number (5 of 20) of the mice showed relatively large amounts of IgM and/or IgA. When 11 such mice were sacrificed, 7 had developed splenomegaly and/or intestinal B cell lymphoma. Immunostaining revealed that B220+ cells had infiltrated into the tumors and various organs including the spleen, liver, and bone marrow. Those cells were monoclonal for κ chain and partly CD138 positive. When we transplanted those tumor cells into Rag2-/- Jak3-/- immunedeficient mice, all the mice died within 3 weeks. Thus, PU.1 apparently functions as a tumor suppressor in mature B cells and its deletion in late B cell maturation stages produces B cell lymphoma with M proteinemia. The remaining 4 mice developed high titer IgM and/or IgA levels and flow cytometry of bone marrow cells and splenocytes revealed that those cells were monoclonal for κ chain and positive for B220 and IgM and/or IgA, suggesting that those mice suffered from multiple myeloma or monoclonal gammopathy with undetermined sighnificance (MGUS). These data strongly suggest that conditional knockout of PU.1 in post germinal center B and plasma cells results in B cell lymphoma and plasma cell neoplasms related to multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Description: PU.1 is an essential transcription factor for hematopoiesis and important for differentiation of both myeloid and lymphoid lineages. In mice conditionally knocked-out of 3.4 kb length of the enhancer region located in14 kb 5’ upstream of the PU.1 gene (URE), PU.1 is down-regulated in myeloid cells and B cells by 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. These data strongly suggest that PU.1 has tumor suppressor activity in hematopoietic cells. We previously reported that human PU.1 is down-regulated in the majority of myeloma cell lines through the methylation of the promoter and the 17 kb upstream enhancer region (URE) of the PU.1 gene that is homologous to that in 14 kb 5’ upstream of the murine PU.1 gene. Conditionally expressed PU.1 with tet-off system induced cell growth arrest and apoptosis in two myeloma cell lines, KMS12PE and U266, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and in contrast, PU.1 is down-regulated in primary myeloma cells from a subset of myeloma patients, who appear to have poor prognosis. In the present study, to test whether PU.1 has tumor suppressor activity in vivo, we generated xenograft mouse models. 0.6 - 1 x 107 KMS12PE cells were subcutaneously injected in 16 immunodeficient mice (Rag2-/- Jak3-/- bulb/c). The mice were then administered doxycycline through drinking water. Half of the mice (N=8) stopped taking doxycycline when the tumor sizes reached 1 cm in diameter, whereas the other half (N=8) kept taking doxycycline. Although the tumors in the mice taking doxycycline continued to grow, the tumor growth in the mice not taking doxycycline significantly slowed down. Flow cytometry analysis of the tumors in the mice that stopped taking doxycycline revealed that the cells from the tumor had completely lost PU.1 expression. Moreover, when U266 cells conditionally expressing PU.1 were subcutaneously injected to another 10 mice and the same experiment was conducted, although the tumors in the mice taking doxycycline (N=5) kept growing, the tumors in the mice not taking doxycycline (N=5), did not grow any further. The present data suggest that PU.1 serves as a tumor a suppressor in the multiple myeloma cell lines as examined in vivo. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2428 SALL4 is a zinc-finger transcriptional factor and a member of the SALL gene family. It plays an essential role in the maintenance of ESC pluripotent and self-renewal properties by interacting with other two key regulators in ESCs, Nanog and Oct4. We previously have shown that stem cell factor SALL4 is aberrantly expressed in 75% of acute B-cell lymphoblastic leukemia (B-ALL). We have also shown that SALL4 is aberrantly expressed in AML, and down-regulation of SALL4 in AML leads to significant cell death. In this study, we focused on investigating the functional role of SALL4 in human B-ALL leukemogenesis. We first assessed the SALL4 mRNA level in four B-ALL cell lines (REH, Nalm6, 697, Blin-1) and five primary patient samples by qRT-PCR. We observed SALL4 mRNA in these four cell lines increased when compared to normal human CD34 negative BM cells. Moreover 4 of 5 primary samples showed high level expression of SALL4, suggesting that SALL4 might play a role in B-ALL pathogenesis. Then, we selected a SALL4 expressing B-ALL cell line (REH and Nalm6) and attenuated SALL4 expression through GFP-labeled shRNA approach in these cell lines. We monitored the growth of SALL4 knockdown and control REH and Nalm6 cells through MTS assay. SALL4 knockdown cells had a decreased growth rate compared to that of the control cells. We also stained SALL4 knockdown and control cells with Annexin V and 7-AAD by flow cytometric quantitation of apoptotic cells. The percentages of apopotic cells in SALL4 knockdown cells were much higher than these in controls. These data demonstrated that inhibition of SALL4 in REH cells and Nalm6 cells led to reduced proliferation and increased apoptosis. We then examined the oncogenesis ability of SALL4 knockdown REH cells in a mouse xenotransplantation model. SALL4 knockdown or control REH cells were injected intravenously into immunodeficient mice. All the recipients succumbed to fatal leukemia within 4 to 6 weeks post transplantation. In both BM and spleen of SALL4 knockdown recipients the engrafted proportion of GFP+ cells was significantly decreased compared to the initial donor cells. Whereas, in both BM and spleen control recipients the percentage of GFP+ REH cells engrafted was similar to that of initial donor cells. This suggests that down-regulation of SALL4 is essential for B-ALL engraftment. To rule out the observed engraftment defect was due to homing, we next performed homing assay. SALL4 knockdown or control cells were injected intravenously into immunodeficient mice as well. Three hours of the injection, mice were sacrificed and analyzed the percentage of GFP+ cells in BM and spleen by flow cytometry. There was no difference among SALL4 knockdown and the control. Furthermore, we performed gene expression profiling on apoptosis-related genes in SALL4 knockdown and control REH cells. The result showed that in SALL4-knockdown REH, TNF mediated cell apoptosis pathways was up-regulated as well as multiple caspase members. The expression of Caspase 3, Caspase 8, FADD was up-regulated in both SALL4 knockdown REH and Nalm6 when compared to these controls, and was verified by real time RT-PCR. This suggests that SALL4 could repress apoptosis through the TNF signal pathway. In summary, we report a novel SALL4/TNF pathway in maintaining cell survival in B-ALL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2628 PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth. In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo. We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: PU.1 is an Ets family transcription factor, which is important for differentiation of both myeloid and lymphoid lineages. In the Friend leukemia model, the failure of PU.1 down-regulation in erythroblasts reportedly results in differentiation arrest, leading to erythroleukemia. In mice conditionally knocked-out of the 3.5 kb length of enhancer region located in14 kb 5′ upstream of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. Since the 3.5 kb enhancer region contains a suppressor region for PU.1 expression in T cells, such mice ectopically express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages for certain cell lineages appears to result in hematological malignancies. We recently reported that human PU.1 is down-regulated in a majority of myeloma cell lines through the methylation of the promoter and enhancer region located in17 kb 5′ upstream of the PU.1 gene which is homologous to that in14 kb 5′ upstream of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of two PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that the down-regulation of PU.1 is essential for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In the present study, to elucidate the mechanisms of the cell growth arrest and apoptosis in PU.1-conditionally expressing myeloma cells, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression, utilizing Illumina Sentrix® Human-6 Expression BeadChip. Of 47,296 genes, 479 genes were up-regulated (〉2fold) and 1,697 genes down-regulated (

Description: It has been reported that disruption of transcription factors critical for hematopoiesis, such as C/EBPa and AML1, is involved in leukemogenesis. PU.1 is a transcription factor important for both myeloid and lymphoid development. We reported that mice in which the levels of PU.1 were 20% of that of wild-type developed acute myeloid leukemia, T cell lymphoma, and a CLL-like disease. These findings strongly suggest that PU.1 has tumor suppressive activity in multiple hematopoietic lineages. Last year, we reported that PU.1 is downregulated in a majority of multiple myeloma cell lines and and freshly isolated CD138 positive myeloma cells from certain number of myeloma patients, and that tet-off inducible exogenous expression of PU.1 in PU.1 negative myeloma cell lines induced cell growth arrest and apoptosis. Based on their PU.1 expression levels, we divided the myeloma patients into two groups, namely PU.1 high and PU.1 low-to-negative, (cutoff index of 25th percentile of the PU.1 expression level distribution among all patients). The PU.1 low-to-negative patients had a significantly poorer prognosis than the PU.1 high patients. To elucidate the mechanisms of downregulation of PU.1, we performed sequence and epigenetic analysis of the promoter region and the -17 kb upstream region that is conserved among mammalians and important for proper expression of PU.1. There are no mutations in these regions of all five myeloma cell lines. In contrast, the -17 kb upstream region was highly methylated in 3 of 4 PU.1 negative myeloma cell lines, while the promoter region was also methylated to various levels in all five myeloma cell lines including one PU.1 positive cell line. These data suggested that the downregulation of PU.1 in myeloma cell lines might be dependent on the methylation of both regulatory regions of PU.1 gene, especially the -17 kb upstream region. We also evaluated the mechanisms of cell growth arrest and apoptosis of myeloma cell lines induced by PU.1. Among apoptosis-related genes, we identified that TRAIL was upregulated after PU.1 induction. To evaluate the effect of upregulation of TRAIL, we stably introduced siRNA for TRAIL into myeloma cell lines expressing PU.1, and we found that apoptosis of these cells was partially suppressed by siRNA for TRAIL, suggesting that apoptosis of myeloma cells induced by PU.1 might be at least partially due to TRAIL upregulation. We are currently performing DNA microarray analysis to compare the expression levels of genes between before and after PU.1 induction, in order to further elucidate the mechanisms of cell growth arrest and apoptosis.