scholarly journals Xenograft Models of Multiple Myeloma Reveal That PU.1 Serves As a Tumor Suppressor for Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2047-2047
Author(s):  
Nao Nishimura ◽  
Shinya Endo ◽  
Niina Ueno ◽  
Shikiko Ueno ◽  
Hiromichi Yuki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Suppressor Activity ◽  
Enhancer Region ◽  
Tumor Suppressor Activity

Abstract 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.

MiR-34a Replacement As a Novel Therapeutic Approach for Multiple Myeloma: Preclinical In Vitro and In Vivo Evidence

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2910-2910
Author(s):  
Maria Teresa Di Martino ◽  
Emanuela Leone ◽  
Nicola Amodio ◽  
Umberto Foresta ◽  
Marta Lionetti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Tumor Growth ◽  
Cell Lines ◽  
Complete Regression ◽  
Suppressor Activity ◽  
Empty Vector ◽  
Tumor Suppressor Activity

Abstract Abstract 2910 Multiple myeloma (MM) remains an incurable disease despite important therapeutic advances in the last few years. Small non-coding RNAs (miRNAs) synthetic mimics are a new class of biological agents which have recently demonstrated preclinical activity against a variety of human neoplasms. miRNA antitumor activity has been related to their capacity to interfere with mRNA stability and protein transducing activity. miR-34a has tumor suppressor activity and is transcriptionally regulated by p53. We investigated the in vitro and in vivo therapeutic potential of pre-miR-34a mimics against human MM cells. Transient expression of pre-miR-34a mimics, after electroporation of SKMM1 and RPMI-8226 MM cell lines which display low constitutive miR-34a levels, triggered antiproliferative effects, as demonstrated by MTT and long-term soft-agar colony assays. 48 hours after cell transfection, apoptotic events were detected in both cell lines exposed to miR-34a mimics. In parallel experiments, MM cells stably transduced with miR-34a gene cloned in a lentiviral vector showed significant growth reduction as compared to empty vector-transduced cell colonies, providing additional evidence of miR-34a tumor suppressor activity in MM cells. qRT-PCR analysis of treated MM cells showed that pre-miR-34a mimics induced down-regulation of mRNAs coding for Notch1 and the cell-cycle dependent kinase 6 (CDK6), validated miR-34a targets. Furthermore, decreased anti-apoptotic Bcl-2 and CDK6 proteins was detected after pre-miR-34a mimic expression, evidence by western blotting analysis. The anti-MM activity of pre-miR-34a mimics was also evaluated in vivo using xenografted SCID models of human MM. Intra-tumor delivery of pre-miR-34a was performed by a novel formulation with Neutral Lipid Emulsion (NLE). Following 4 injections (3 days apart) of pre-miR34a formulated in NLE particles, a highly significant inhibition of tumor growth was detected in SKMM1 xenografted SCID mice. At day 13 after the first treatment, tumors in mice treated with formulated pre-miR-34a were significantly smaller than tumors in mice treated with the formulated scrambled sequence (P=0.0002) or vehicle (P=0.0002) or PBS (P=0.0001). Interestingly, at day 21a three mice enrolled in the miR-34a treated group showed complete regression of tumors. Formulated synthetic pre-miR-34a also produced a significant increase of mice survival (P=0.01 versus formulated scrambled sequence). A similar in vivo tumor growth inhibition was observed in mice xenografted with SKMM1 MM cells stably transduced with a miR-34a lentiviral construct, as compared to cells transduced with the empty vector. We here provide the first proof-of-principle demonstrating that replacement of miR-34a produces therapeutic activity against MM cells with low constitutive miR-34a expression. Our findings provide a framework for development of miR-34a-based therapeutic strategies in MM. Supported by AIRC 5 per mille, Molecular Clinical Oncology Program No. 9980 Disclosures: Anderson: Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Identification of Tight Junction Protein (TJP)-1 As a Modulator of Sensitivity to Doxorubicin and Cisplatin in Models of Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3962-3962
Author(s):  
Xing-Ding Zhang ◽  
Robert Z. Orlowski ◽  
Lin Yang
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Hela Cells ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Cancer Center ◽  
In Vivo Models ◽  
Junction Protein ◽  
Rpmi 8226

Abstract Abstract 3962 Background: Therapeutic advances in multiple myeloma have improved the outcomes of patients with this malignant plasma cell disorder, but the disease course is still strongly influenced by both innate, or primary, as well as acquired, or secondary mechanisms of drug resistance. Identification and validation of genes that may mediate these phenotypes is therefore of importance, since they could be useful prognostic markers, and also potential targets to overcome the emergence of resistance, or possibly preclude its emergence altogether. Methods: To identify non-redundant determinants of chemoresistance, we designed a robust, high-throughput RNA interference (RNAi) screen targeting 9610 human genes. The screen involved retroviral-mediated transduction first of HeLa cervical carcinoma cells with either the RNAi library, or with non-targeting retrovirus particles. After infection, cells were selected with puromycin, and treated with different concentrations of doxorubicin and cisplatin. Doxorubicin (Dox) treatment led to 33 surviving colonies from the cells transduced with the shRNA library, cisplatin (Cis) treatment led produced 22 surviving colonies, while non-targeting retrovirus-infected cells failed to form colonies after treatment. Screening was performed to identify the shRNA target gene(s) in each colony, and genes that were identified in both Dox- and Cis-treated HeLa cells, and that were expressed in myeloma cells, were selected for further study. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results: TJP1 (zona occludens (ZO)-1) was identified as one gene whose knockdown promoted survival in Dox- and Cis-treated HeLa cells, and which was expressed in myeloma cell lines and in primary plasma cells. To further examine its potential role in myeloma chemosensitivity, we performed mRNA and protein expression profiling in a panel of 11 cell lines and observed that TJP1 expression was silenced in 3 cell lines (ARP-1, INA-6, and MOLP-8), while it was moderately to highly expressed in 7 cell lines (including RPMI 8226, MM1.S, and U266). Comparing TJP1-positive MM1.S cells to TJP1-null MOLP-8 cells, the latter displayed a significantly higher median inhibitory concentration to Dox and Cis. Knockdown of TJP1 in RPMI 8226 and U266 cells, which produced a >75% target suppression, was sufficient to reduce the proportion of apoptotic cells in the sub-G1 fraction after treatment with Dox or Cis compared to control cells. Conversely, MOLP-8 cells transfected with human TJP1 cDNA exhibited an increase in the sub-G1 population in response to Dox and Cis treatment compared to vector controls. Conclusion: Taken together, these studies support the hypothesis that TJP1 expression mediates myeloma cell resistance to the DNA damaging agents doxorubicin and cisplatin. Further studies are underway to determine the mechanism by which TJP1 influences chemosensitivity, and to validate its impact using in vivo models. Disclosures: No relevant conflicts of interest to declare.

Anti-CD138-IFNα Fusion Proteins Are Effective in Treating Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 939-939
Author(s):  
Esther Yoo ◽  
Alex Vasuthasawat ◽  
Danh Tran ◽  
Alan Lichtenstein ◽  
Sherie Morrison
Keyword(s):  
Multiple Myeloma ◽  
Fusion Protein ◽  
Cell Lines ◽  
Dna Content ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Inhibition Of Proliferation ◽  
Discontinue Therapy

Abstract Abstract 939 Although IFNα has shown some efficacy in the treatment of multiple myeloma (MM), this efficacy has been limited in large part because systemic toxicity makes it difficult if not impossible to reach therapeutically effective doses at the site of the tumor. The short half-life of IFN also makes it difficult to sustain high levels during treatment, and because of the side effects, the patients often discontinue therapy. To address these issues, we have genetically fused IFNα2 to a chimeric IgG1 antibody specific for the antigen CD138 expressed on the surface of MM cells, yielding anti-CD138-IFNα. We have also produced a fusion protein (anti-CD138-mutIFNα) using a mutant IFNα that binds the IFN receptor (IFNAR) more tightly. The fusion proteins continued to bind CD138 and retained IFN activity and showed anti-proliferative activity against a broad panel of myeloma cell lines (HMCL) representing MM with different characteristic. To investigate the events responsible for the inhibition of proliferation, 8226/S, ANBL-6, MM1-144, H929, OCI-My5 and U266 cells were incubated with 500 pM anti-CD138-IFNα for 72 h and their DNA content analyzed by FLOW cytometry following permeabilization and staining with PI. The different cell lines exhibited different responses. All of the cell lines except OCI-My5 underwent apoptosis. For 8226/S, OCI-My5 and U266 there was little change in DNA content following treatment. ANBL-6 showed a slight increase in the number of cells in S. However, MM1-144 and H929 showed a marked accumulation in G2 with H929 also showing accumulation of cells with sub-G0content of DNA. Therefore, there is heterogeneity in the response of different HMCL to treatment with targeted IFNα2. For many but not all of the cell lines, anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inhibiting proliferation and causing DNA fragmentation. Anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inducing senescence-associated β-galactosidase and STAT1 activation in OCI-My5 cells. Treatment with anti-CD138-IFNα or anti-CD138-mutIFNα resulted in a decrease in the amount of IRF4 present in U266, suggesting that this may be responsible for the efficacy of the fusion proteins in this cell line. Treatment of the other cell lines did not alter the level of IRF4 present, but anti-CD138-IFNα and anti-CD138-mutIFNα treatment caused a decrease in the amount of ppRB present in 8226/S, OCI-My5 and MM1-144, and to a lesser extent in H929. To determine the in vivo efficacy of fusion protein treatment, SCID mice were injected subcutaneously with OCI-My5 cells and treated intravenously on days 14, 16 and 18 with 100 μg of the indicated proteins and monitored for tumor growth (Figure 1). Mice were sacrificed when tumors exceeded 1.5 cm in diameter. Treatment with anti-CD138-IFNα provided some protection (p ≤ 0.0001 compared to PBS). However, treatment with anti-CD138-mutIFNα was even more effective (p = 0.0004 compared to anti-CD138-IFNα). Anti-CD138-mutIFNα was also found to be more effective than anti-CD138-IFNα against primary MM cells. Patients with active myeloma were biopsied while off therapy and the marrow cells isolated by a negative antibody selection to >95% purity. After 72 h incubation with 25 nM of protein, anti-CD138 was found to have little effect. In contrast treatment with anti-CD138-IFNα caused a decrease in viability with anti-CD138-mutIFNα treatment leading to an even greater decrease in cell viability. Following 72 h of treatment, 25 nM of anti-CD138-mutIFNα was found to have more potent cytoreductive effects than 100 nM of anti-CD138-IFNα. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

scholarly journals Interleukin-6 promotes multiple myeloma cell growth via phosphorylation of retinoblastoma protein

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 2219-2227 ◽  
Author(s):  
M Urashima ◽  
A Ogata ◽  
D Chauhan ◽  
MB Vidriales ◽  
G Teoh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
Cell Growth ◽  
Tumor Cell ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Growth Arrest ◽  
Retinoblastoma Protein ◽  
Myeloma Cell ◽  
Phosphorylated Form

Interleukin-6 (IL-6) mediates autocrine and paracrine growth of multiple myeloma (MM) cells and inhibits tumor cell apoptosis. Abnormalities of retinoblastoma protein (pRB) and mutations of RB gene have been reported in up to 70% of MM patients and 80% of MM-derived cell lines. Because dephosphorylated (activated) pRB blocks transition from G1 to S phase of the cell cycle whereas phosphorylated (inactivated) pRB releases this growth arrest, we characterized the role of pRB in IL-6-mediated MM cell growth. Both phosphorylated and dephosphorylated pRB were expressed in all serum-starved MM patient cells and MM-derived cell lines, but pRB was predominantly in its phosphorylated form. In MM cells that proliferated in response to IL-6, exogenous IL-6 downregulated dephosphorylated pRB and decreased dephosphorylated pRB-E2F complexes. Importantly, culture of MM cells with RB antisense, but not RB sense, oligonucleotide (ODN) triggered IL- 6 secretion and proliferation in MM cells; however, proliferation was only partially inhibited by neutralizing anti-IL-6 monoclonal antibody (MoAb). In contrast to MM cells, normal splenic B cells express dephosphorylated pRB. Although CD40 ligand (CD40L) triggers a shift from dephosphorylated to phosphorylated pRB and proliferation of B cells, the addition of exogenous IL-6 to CD40L-treated B cells does not alter either pRB or proliferation, as observed in MM cells. These results suggest that phosphorylated pRB is constitutively expressed in MM cells and that IL-6 further shifts pRB from its dephosphorylated to its phosphorylated form, thereby promoting MM cell growth via two mechanisms; by decreasing the amount of E2F bound by dephosphorylated pRB due to reduced dephosphorylated pRB, thereby releasing growth arrest; and by upregulating IL-6 secretion by MM cells and related IL-6- mediated autocrine tumor cell growth.

scholarly journals Mortaparib, a novel dual inhibitor of mortalin and PARP1, is a potential drug candidate for ovarian and cervical cancers

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Jayarani F. Putri ◽  
Priyanshu Bhargava ◽  
Jaspreet Kaur Dhanjal ◽  
Tomoko Yaguchi ◽  
Durai Sundar ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Growth Arrest ◽  
Suppressor Activity ◽  
Dual Inhibitor ◽  
Tumor Suppressor Activity ◽  
And Migration

Abstract Background Mortalin is enriched in a large variety of cancers and has been shown to contribute to proliferation and migration of cancer cells in multiple ways. It has been shown to bind to p53 protein in cell cytoplasm and nucleus causing inactivation of its tumor suppressor activity in cancer cells. Several other activities of mortalin including mitochondrial biogenesis, ATP production, chaperoning, anti-apoptosis contribute to pro-proliferative and migration characteristics of cancer cells. Mortalin-compromised cancer cells have been shown to undergo apoptosis in in vitro and in vivo implying that it could be a potential target for cancer therapy. Methods We implemented a screening of a chemical library for compounds with potential to abrogate cancer cell specific mortalin-p53 interactions, and identified a new compound (named it as Mortaparib) that caused nuclear enrichment of p53 and shift in mortalin from perinuclear (typical of cancer cells) to pancytoplasmic (typical of normal cells). Biochemical and molecular assays were used to demonstrate the effect of Mortaparib on mortalin, p53 and PARP1 activities. Results Molecular homology search revealed that Mortaparib is a novel compound that showed strong cytotoxicity to ovarian, cervical and breast cancer cells. Bioinformatics analysis revealed that although Mortaparib could interact with mortalin, its binding with p53 interaction site was not stable. Instead, it caused transcriptional repression of mortalin leading to activation of p53 and growth arrest/apoptosis of cancer cells. By extensive computational and experimental analyses, we demonstrate that Mortaparib is a dual inhibitor of mortalin and PARP1. It targets mortalin, PARP1 and mortalin-PARP1 interactions leading to inactivation of PARP1 that triggers growth arrest/apoptosis signaling. Consistent with the role of mortalin and PARP1 in cancer cell migration, metastasis and angiogenesis, Mortaparib-treated cells showed inhibition of these phenotypes. In vivo tumor suppression assays showed that Mortaparib is a potent tumor suppressor small molecule and awaits clinical trials. Conclusion These findings report (i) the discovery of Mortaparib as a first dual inhibitor of mortalin and PARP1 (both frequently enriched in cancers), (ii) its molecular mechanism of action, and (iii) in vitro and in vivo tumor suppressor activity that emphasize its potential as an anticancer drug.

scholarly journals Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 11-13 ◽  
Author(s):  
XG Zhang ◽  
B Klein ◽  
R Bataille
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
S Phase ◽  
Myeloma Cells ◽  
Severity Of The Disease

Abstract It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

scholarly journals Synthetic miR-145 mimic inhibits multiple myeloma cell growth in vitro and in vivo

2014 ◽  
Vol 33 (1) ◽  
pp. 448-456 ◽  
Author(s):  
QI ZHANG ◽  
WEIQUN YAN ◽  
YANG BAI ◽  
HAO XU ◽  
CHANGHAO FU ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell

PU.1 Is Downregulated in a Subset of Multiple Myeloma by the Methylation of Its -17 kb Upstream Regulatory Region and the Promoter.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3417-3417
Author(s):  
Yutaka Okuno ◽  
Hiro Tatetsu ◽  
Shikiko Ueno ◽  
Hiroyuki Hata ◽  
Yasuhiro Yamada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Promoter Region ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Upstream Region ◽  
Cell Growth Arrest ◽  
Myeloma Cells ◽  
Expression Levels

Abstract 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.

Microarray Analysis of PU.1-Induced Growth Arrest and Apoptosis of Myeloma Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2499-2499
Author(s):  
Shikiko Ueno ◽  
Hiro Tatetsu ◽  
Naoko Harada ◽  
Hiroyuki Hata ◽  
Tadafumi Iino ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cell Growth ◽  
Cell Lines ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Down Regulation ◽  
Cell Growth Arrest ◽  
Myeloma Cells ◽  
Enhancer Region

Abstract 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 (<0.5 fold) either day 1 or 3 after PU.1 induction in U266 cells. Among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. Stably expressed siRNA for TRAIL partially inhibited apoptosis of U266 cells expressing PU.1, suggesting that TRAIL is related to PU.1 induced cell death of U266 cells. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, which was confirmed with protein levels. We are now examining the roles of the observed up-regulated genes in both U266 and KMS12PE myeloma cell lines.

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