High Bcl-xL Expression Research Articles

Effects of Trichostatin A and 5-aza-2’deoxycytidine on Nuclear Reprogramming in Pig Cloned Embryos

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2’deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

Loss of PDCD4 contributes to enhanced chemoresistance in Glioblastoma Multiforme through de-repression of Bcl-xL translation

Glioblastoma multiforme (GBM) is the most common and aggressive form of tumor of the central nervous system. Despite significant efforts to improve treatments, patient survival rarely exceeds 18 months largely due to the highly chemoresistant nature of these tumors. Importantly, misregulation of the apoptotic machinery plays a key role in the development of drug resistance. We previously demonstrated that Bcl-xL, an important anti-apoptotic protein, is regulated at the level of translation by the tumor suppressor programmed cell death 4 (PDCD4). We report here a strong correlation between low expression of PDCD4 and high expression of Bcl-xL in adult de novo GBM, GBM tumor initiating cells, and established GBM cell lines. Importantly, high Bcl-xL expression correlated significantly with poor progression and patient survival. We demonstrate that re-expression of PDCD4 in GBM cells down-regulated Bcl-xL expression and decreased cell viability. Finally, we show that direct inhibition of Bcl-xL by small molecule antagonist ABT-737 sensitizes GBM cells to doxorubicin. Our results identify Bcl-xL as a novel marker of GBM chemoresistance and advocate for the combined use of Bcl-xL antagonists and existing chemotherapeutics as a treatment option for this aggressive tumor.

Abstract 4607: Expression of Bcl-2 pro-survival family proteins predicts pharmacological responses to ABT-199, a novel and selective Bcl-2 antagonist, in multiple myeloma models.

Abstract A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues mediated, in part, by the the Bcl-2 family of pro-apoptotic and pro-survival proteins. ABT-199 is a potent BH3-only mimetic that selectively antagonizes the pro-survival function of Bcl-2. The work presented describes the efficacy of ABT-199 as a single agent and in combination with bortezomib in preclinical models of Multiple Myeloma (MM). Expression of Bcl-2 protein was detected in 95% of MM cell lines (n=21) evaluated and ABT-199 reduced cell viability in a sub-set of these lines (7/21) with EC50 values less than 0.5 μM. Expression of Bcl-xL and Mcl-1 at the mRNA and protein levels were also evaluated to determine predictors of drug sensitivity and resistance to ABT-199. MM lines that expressed higher levels of Bcl-2 relative to Bcl-xL and Mcl-1 were the most sensitive to ABT-199 treatment while those that were less sensitive expressed higher levels of Mcl-1 or Bcl-xL. Other predictors of sensitivity to ABT-199 included higher levels of Bcl-2/Bim complexes and t(11;14) status. Ex vivo, 75% of the MM bone marrow biopsies and aspirates (n=27) had high Bcl-2 levels whereas 50% had high Bcl-xL expression, demonstrating that a subset of patient samples (33%) have a favorable biomarker profile (Bcl-2-high/Bcl-xL-low) that may be predictive of ABT-199 activity. Efficacy of ABT-199 as a single agent and in combination with bortezomib was evaluated in vivo in MM xenograft models that expressed Bcl-2 (OPM-2, KMS-11, RPMI-8226, H929 and MM.1s). Bortezomib treatment alone at maximum tolerated doses resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at maximum tolerated doses was efficacious as a single agent in xenograft models that expressed higher protein levels of Bcl-2 but relatively lower levels of Bcl-xL. The combination of ABT-199 with bortezomib increased overall response rates, durability of anti-tumor activity and levels of apoptotic markers such as cleaved caspase 3 and PARP when compared to bortezomib alone even in MM xenograft models that also express relatively high levels of Mcl-1. Treatment with bortezomib in vivo increased levels of Noxa; a pro-apoptotic BH3-only protein that selectively antagonizes Mcl-1. Therefore, greater efficacy may be achieved when ABT-199 is combined with bortezomib due to inhibition of Bcl-2 by ABT-199 and neutralization of Mcl-1 by Noxa in tumors that express both pro-survival proteins. Our preclinical data supports the evaluation of ABT-199 as a single agent and in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Citation Format: Deepak Sampath, Elizabeth Punnoose, Erwin Boghaert, Lisa Belmont, Franklin Peale, Nguyen Tan, Jun Chen, Walter Darbonne, Peng Yue, Jason Oeh, Leslie Lee, Wayne J. Fairbrother, Andrew J. Souers, Steven W. Elmore, Joel D. Leverson. Expression of Bcl-2 pro-survival family proteins predicts pharmacological responses to ABT-199, a novel and selective Bcl-2 antagonist, in multiple myeloma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4607. doi:10.1158/1538-7445.AM2013-4607

Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines

MicroRNAs (miRNAs) have been found to be involved in cancer initiation, progression and metastasis and, as such, have been suggested as tools for cancer detection and therapy. In this work, a large-scale screening of the complete miRNA mimics library demonstrated that hsa-miR-15a-3p had a pro-apoptotic role in the following human cancer cells: HeLa, AsPc-1, MDA-MB-231, KB3, ME180, HCT-116 and A549. MiR-15a-3p is a novel member of the pro-apoptotic miRNA cluster, miR-15a/16, which was found to activate Caspase-3/7 and to cause viability loss in B/CMBA.Ov cells during preliminary screening. Subsequent microarrays and bioinformatics analyses identified the following four anti-apoptotic genes: bcl2l1, naip5, fgfr2 and mybl2 as possible targets for the mmu-miR-15a-3p in B/CMBA.Ov cells. Follow-up studies confirmed the pro-apoptotic role of hsa-miR-15a-3p in human cells by its ability to activate Caspase-3/7, to reduce cell viability and to inhibit the expression of bcl2l1 (bcl-xL) in HeLa and AsPc-1 cells. MiR-15-3p was also found to reduce viability in HEK293, MDA-MB-231, KB3, ME180, HCT-116 and A549 cell lines and, therefore, may be considered for apoptosis modulating therapies in cancers associated with high Bcl-xL expression (cervical, pancreatic, breast, lung and colorectal carcinomas). The capability of hsa-miR-15a-3p to induce apoptosis in these carcinomas may be dependent on the levels of Bcl-xL expression. The use of endogenous inhibitors of bcl-xL and other anti-apoptotic genes such as hsa-miR-15a-3p may provide improved options for apoptosis-modulating therapies in cancer treatment compared with the use of artificial antisense oligonucleotides.

Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

AbstractAbstract 5028Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM.A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity.ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures:Sampath:Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

Inhibition of Bcl-xL by ABT-737 enhances chemotherapy sensitivity in neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor cells

Neurofibromatosis type 1 (NF1) is one of the most commonly inherited autosomal dominant disorders. The malignant peripheral nerve sheath tumor (MPNST) is a major cause of mortality in patients with NF1. In this study, we found that overexpression of Bcl-xL in the established NF1-associated MPNST cell line and primary tissue cultured MPNST cells derived from an NF1 patient was closely associated with anticancer drug resistance of the NF1-associated MPNST cells. We demonstrated that high expression of Bcl-xL in the MPNST cells was caused by a decreased transcriptional expression of the NF1 gene. Downregulation of the NF1 gene by RNA interference (RNAi) induced an increase in Bcl-xL expression and a decrease in its sensitivity to apoptosis in the benign neurofibroma cell line and primary normal cells. These results suggest that an alteration of Bcl-xL expression levels by somatic expression changes in the NF1 locus may contribute to the malignant development of benign tumor tissues or normal tissues to MPNSTs. We further demonstrated that either depletion of Bcl-xL expression by RNAi or inactivation of Bcl-xL by ABT-737, a mimetic of the BH3-only protein BAD, was very effective in sensitizing the MPNST cells to apoptotic cell death by combined treatment with the tested anticancer drug doxorubicin. Notably, a low concentration of ABT-737 and doxorubicin could effectively induce synergistic cytotoxicity in the MPNST cells. These results suggest that pharmacological inhibition of Bcl-xL by ABT-737 in combination with doxorubicin can be a potential therapeutic strategy for the treatment of NF1-associated MPNSTs.

Chemical Genomics Identifies Small-Molecule MCL1 Repressors and BCL-xL as a Predictor of MCL1 Dependency

MCL1, which encodes the antiapoptotic protein MCL1, is among the most frequently amplified genes in human cancer. A chemical genomic screen identified compounds, including anthracyclines, that decreased MCL1 expression. Genomic profiling indicated that these compounds were global transcriptional repressors that preferentially affect MCL1 due to its short mRNA half-life. Transcriptional repressors and MCL1 shRNAs induced apoptosis in the same cancer cell lines and could be rescued by physiological levels of ectopic MCL1 expression. Repression of MCL1 released the proapoptotic protein BAK from MCL1, and Bak deficiency conferred resistance to transcriptional repressors. A computational model, validated invivo, indicated that high BCL-xL expression confers resistance to MCL1 repression, thereby identifying a patient-selection strategy for the clinical development of MCL1 inhibitors.

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

Identification of An Abnormal JAK2 WT CD34+ Cell Population Characterized by High Bcl-Xl Expression Levels Both in JAK2 Positive and Negative PV, ET and PMF Patients,

Abstract 3472 Background:The JAK2V617F gene mutation is a common feature of Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF); however the disease can occur without the JAK2 mutation suggesting that ET, PV and PMF share a common molecular mechanisms which is still unknown. In addition, in patients with the JAK2V617F mutation a substantial proportion of progenitor cells do not bear the JAK2V617F mutation and it is still unclear whether this population is normal or not. Methods: 64 patients affected by MPNs and 12 healthy subjects were included in the study. Twenty three patients were affected by ET, 25 by PV and 22 by PMF. We analyzed the expression, of the anti-apoptotic gene Bcl-xL, both at mRNA and protein level by qRT-PCR and immunofluorescence assay with specific antibodies. Erythroblasts, CD34+ and CD34+CD38-cell populations were selected and analyzed. Moreover, we designed specific PNA (Peptide Nucleic Acid) for JAK2V617F and for Bcl-xL isoform to identify their presence/expression at single CD34+ cell level. Results: Bcl-xL protein and mRNA were found progressively over-expressed in erythroblasts, in CD34+ and in CD34+ CD38- cells from ET, PV and PMF patients without any differences between JAK2V617F and wild type (WT) patients. The median protein level in erythroblasts increased from 22 Relative Units (RU) in ET, to 68,5 in PV and to 85 in PMF. Similarly, in CD34+ Bcl-xL increased from 30 RU in ET, to 89 RU in PV, and to 135 RU in PMF. Finally, CD34+CD38-increased from 17.4 RU in ET, to 45.5 RU in PV, to 101.7 RU in PMF. In addition, analysis by PNA identified distinct progenitor cells population. In patients bearing the JAK2 mutation, three CD34+ cell sub-populations were found: (a) one JAK2 positive and with high levels of Bcl-xL; (b) one JAK2 negative and with high levels of Bcl-xL. Thus, the cell population showing high levels of Bcl-xL is always greater than the JAK2 positive one; (c) one double negative population. In patients without JAK2 mutation, two CD34+ cell populations are identified: (a) one characterized by high levels of Bcl-xL and (b) one double negative. The distribution of these cell subpopulations is different in the distinct MPN. Conclusions: We have identified a previously undisclosed CD34+ cell population which is characterized by high level of Bcl-xL expression and represents the majority of CD34+ cells both in wild type and mutated samples. Interestingly, in mutated samples, this population encompasses the JAK2 mutated one. These findings suggest that Bcl-xL plays an important role in MPN, although it remains undisclosed whether this activation is dependent from an upstream oncogenetic signal. Finally, with this technique we can now identify a putative residual normal CD34+ cells in MPN which may be useful for monitoring patients undergoing targeted therapy with inhibitors. Altogether, these findings disclose unfolded aspects of pathophysiology of MPN and may contribute to a better understanding of the pathogenesis of MPN. Disclosures:Barosi:Novartis: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Paclitaxel sensitivity of breast cancer cells requires efficient mitotic arrest and disruption of Bcl-xL/Bak interaction

Taxanes are being used for the treatment of breast cancer. However, cancer cells frequently develop resistance to these drugs with the subsequent recurrence of the tumor. MDA-MB-231 and T-47D breast cancer cell lines were used to assess the effect of paclitaxel treatment on apoptosis and cell cycle, the possible mechanisms of paclitaxel resistance as well as the enhancement of paclitaxel-induced apoptosis based on its combination with phenylethyl isothiocyanate (PEITC). T-47D cells undergo apoptosis in response to paclitaxel treatment. The induction of apoptosis was associated with a robust mitotic arrest and the disruption of Bcl-xL/Bak interaction. By contrary, MDA-MB-231 cells were insensitive to paclitaxel-induced apoptosis and this was associated with a high percentage of cells that slip out of paclitaxel-imposed mitotic arrest and also with the maintenance of Bcl-xL/Bak interaction. The sequential treatment of MDA-MB-231 cells with PEITC followed by paclitaxel inhibited the slippage induced by paclitaxel and increased the apoptosis induction achieved with any of the drugs alone. In breast cancer tissues, high Bcl-xL expression was correlated with a shorter time of disease-free survival in patients treated with a chemotherapeutic regimen that contains paclitaxel, in a statistically significant way. Thus, resistance to paclitaxel in MDA-MB-231 cells is related to the inability to disrupt the Bcl-xL/Bak interaction and increased slippage. In this context, the combination of a drug that induces a strong mitotic arrest, such as paclitaxel, with another that inhibits slippage, such as PEITC, translates into increased apoptotic induction.

Trichostatin A Modulates Apoptotic-Related Gene Expression and Improves Embryo Viability in Cloned Bovine Embryos

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transferred nuclei into oocytes. Trichostatin A (TSA), a histone deacetylase inhibitor, has been used to enhance nuclear reprogramming following SCNT. However, the molecular mechanism of TSA for the improvement of the preimplantation embryo and fetal development following SCNT is not known. The present study investigates embryo viability and gene expression of cloned bovine preimplantation embryos in the presence and absence of TSA compared to embryos produced by in vitro fertilization or parthenogenetic activation. Our results indicated that TSA treatment significantly improved total and inner cell mass (ICM) cell number and ratio of ICM:trophectoderm (TE) and also decreased the apoptotic index including total, ICM, and ratio of ICM:TE. Four apoptotic-related genes, Bcl-xL, survivin, Bcl2-associated X protein (Bax), and caspase 3 (Casp3), and four pluripotency/differentiation related genes, Oct4, SRY (sex determining region Y)-box 2 (Sox2), Cdx2, and colony-stimulating factor 1 receptor (Csf1r), were measured by real-time RT-PCR. TSA treatment resulted in the high expression of antiapoptotic gene Bcl-xL and low expression of pro-apoptotic gene Bax compared to untreated NT embryos, fertilized embryos, or parthenotes. Furthermore, mRNA expression of Cdx2 was higher in NT-TSA embryos than in NT and in vitro fertilization (IVF) counterparts. Additionally, low expression of microRNA (mir)-21 in NT embryos was enhanced following TSA treatment. These results suggest that TSA positively regulates nuclear reprogramming, and TSA may increased resistance or reduced signal for induction of apoptosis.

Prognostic significance of Bcl-xL gene expression in human colorectal cancer

Bcl-xL is a pro-survival member of the Bcl-2 family that plays indispensable roles in regulating cell survival and apoptosis. It is overexpressed in many malignant tumors including colorectal cancer (CRC). However, it is still unclear if Bcl-xL can be used as an independent molecular marker for predicting the prognosis of CRC patients. In this study, reverse transcription-PCR assay was performed to detect the expression of Bcl-xL mRNA in CRC and corresponding non-tumor colon tissues. Immunohistochemistry was performed to detect the immunolocalization of Bcl-xL protein in sixty-eight primary CRC tissue samples. The association between Bcl-xL protein expression and clinicopathological factors of CRC patients was analyzed and the survival was assessed by the Kaplan–Meier method and proportional hazards model. The averaged level of Bcl-xL mRNA expression in CRC tissues (0.85 ± 0.13) was significantly higher than that in non-tumor colon tissues (0.08 ± 0.02). Immunohistochemical staining showed that the Bcl-xL protein was mainly located in the cytoplasm of tumor cells. The level of Bcl-xL protein expression was closely correlated with tumor differentiation ( P = 0.002), lymph node metastasis ( P = 0.010), venous permeation ( P = 0.004), and Duke's classification ( P = 0.021). Furthermore, patients with high Bcl-xL expression showed poorer overall survival than those with low Bcl-xL expression ( P = 0.016). Univariate and multivariate analysis indicated that the status of Bcl-xL protein expression might be an independent prognostic marker for CRC patients ( P = 0.032). Taken together, immunohistochemical assessment of status of Bcl-xL protein may offer a valuable approach for predicting survival after curative surgery for colorectal cancer.

Contributions of TRAIL-mediated megakaryocyte apoptosis to impaired megakaryocyte and platelet production in immune thrombocytopenia

AbstractRecent in vitro studies provide evidence for autoantibody-induced suppression of megakaryocytopoiesis and show a reduction in megakaryocyte production and maturation in the presence of immune thrombocytopenia (ITP) plasma. Here, we present CD34+ cells from healthy umbilical cord blood mononuclear cells cultured in medium containing thrombopoietin, stem cell factor, interleukin-3, and 10% plasma from either ITP patients or healthy subjects. The quantity, quality, and apoptosis of megakaryocytes were measured. We observed that most ITP plasma boosted megakaryocyte quantity but impaired quality, resulting in significantly less polyploidy cells (N ≥ 4) and platelet release. In these megakaryocytes, we found a lower percentage of cell apoptosis, a lower expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and a higher expression of Bcl-xL. Furthermore, there was a decrease of sTRAIL in ITP plasma and in cell culture supernatants of this group compared with the control group. Our findings suggest that decreased apoptosis of megakaryocytes also contributes to in vitro dysmegakaryocytopoiesis and reduced platelet production. The abnormal expression of sTRAIL in plasma and TRAIL and Bcl-xL in megakaryocytes may play a role in the pathogenesis of impaired megakaryocyte apoptosis in ITP.

Outcomes related to biomarkers in organ preservation for patients with advanced squamous cell carcinoma of the oral cavity (SCCOC).

e16009 Background: A previous trial at the University of Michigan was conducted in SCCOC pts using 1 cycle of induction chemotherapy (IC) to select pts for organ preservation. Here we report outcomes related to biomarkers. Methods: Pts with advanced SCCOC received one cycle of cisplatin and 5-fluorouracil. Those who achieved 50% reduction of their tumor received concurrent chemoradiation (CRT); those with less than 50% response underwent salvage surgery. CRT consisted of 70 Gy (2 Gy fraction daily × 7 weeks) with concurrent cisplatin 100 mg/m2 every 3 weeks × 3 cycles. After CRT, pts with a complete histologic response received 2 cycles of paclitaxel 175 mg/m2; those with residual tumor had salvage surgery. Biopsies were obtained and assessed for HPV, p16, p53 mutation, Bcl-xL, and epidermal growth factor receptor (EGFR). Results: Between 7/24/2000 and 3/29/2004, 19 pts were enrolled (11 men; 8 women); 10 were responders to IC, but only 5/10 responded to CRT. 7/19 (37%) were alive at 3 yrs and 4/19 (21%) were alive at ≥ 6 yrs. Three of thirteen tested positive for HPV/p16 and 2/3 were long term survivors. High Bcl-xL expression was associated with local recurrence or a second primary SCC head and neck (p=0.0397). The association between low Bcl-xL expression and disease-specific survival (DSS) approaches significance (p=0.067; HR = 3.768; CI [0.911, 15.582]). Using a Cox regression model to adjust for the effects of smoking, low Bcl-xL expression held prognostic value for better DSS (p =0.0207). Conclusions: In this small sample of pts with SCCOC, no single marker predicted survival or response to therapy. Low Bcl-xL expression may predict better disease-specific survival for organ preservation for pts with SCCOC. Further investigation is warranted. No significant financial relationships to disclose.

An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats

BackgroundCystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence.MethodsWe compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.ResultsThe proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins.ConclusionThese results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.

EGFR, p16, HPV Titer, Bcl-xL and p53, Sex, and Smoking As Indicators of Response to Therapy and Survival in Oropharyngeal Cancer

To prospectively identify markers of response to therapy and outcome in an organ-sparing trial for advanced oropharyngeal cancer. Pretreatment biopsies were examined for expression of epidermal growth factor receptor (EGFR), p16, Bcl-xL, and p53 as well as for p53 mutation. These markers were assessed for association with high-risk human papillomavirus (HPV), response to therapy, and survival. Patient variables included smoking history, sex, age, primary site, tumor stage, and nodal status. EGFR expression was inversely associated with response to induction chemotherapy (IC) (P = .01), chemotherapy/radiotherapy (CRT; P = .055), overall survival (OS; P = .001), and disease-specific survival (DSS; P = .002) and was directly associated with current smoking (P = .04), female sex (P = .053), and lower HPV titer (P = .03). HPV titer was significantly associated with p16 expression (P < .0001); p16 was significantly associated with response to IC (P = .008), CRT (P = .009), OS (P = .001), and DSS (P = .003). As combined markers, lower HPV titer and high EGFR expression were associated with worse OS (rho(EGFR) = 0.008; rho(HPV) = 0.03) and DSS (rho(EGFR) = 0.01; rho(HPV) = 0.016). In 36 of 42 biopsies, p53 was wild-type, and only one HPV-positive tumor had mutant p53. The combination of low p53 and high Bcl-xL expression was associated with poor OS (P = .005) and DSS (P = .002). Low EGFR and high p16 (or higher HPV titer) expression are markers of good response to organ-sparing therapy and outcome, whereas high EGFR expression, combined low p53/high Bcl-xL expression, female sex, and smoking are associated with a poor outcome. Smoking cessation and strategies to target EGFR and Bcl-xL are important adjuncts to the treatment of oropharyngeal cancer.

Expression of p53 and Bcl-xL as Predictive Markers for Larynx Preservation in Advanced Laryngeal Cancer

To assess tumor markers in advanced laryngeal cancer. Marker expression and clinical outcome. Pretreatment tumor biopsy specimens were analyzed from patients enrolled in the Department of Veterans Affairs Laryngeal Cancer Study. Expression of p53 (OMIM TP53) and Bcl-xL (OMIM 600039) in pretreatment biopsy specimens was assessed for correlation with chemotherapy response, laryngeal preservation, and survival. Higher rates of larynx preservation were observed in patients whose tumors expressed p53 vs those that did not (80% [36 of 45 patients] vs 59% [24 of 41 patients], P =.03). Higher rates of larynx preservation were also observed in patients whose tumors expressed low levels of Bcl-xL vs high levels of Bcl-xL (90% [18 of 20 patients] vs 60% [30 of 50 patients], P =.02). Patients were categorized into 3 risk groups (low, intermediate, and high) based on their tumor p53 and Bcl-xL expression status. Patients whose tumors had the high-risk biomarker profile (low p53 expression and high Bcl-xL expression) were less likely to preserve their larynx than patients whose tumors had the intermediate-risk biomarker profile (high p53 expression and low or high Bcl-xL expression) or the low-risk biomarker profile (low p53 expression and low Bcl-xL expression). The larynx preservation rates were 100% (10 of 10 patients), 77% (26 of 34 patients), and 54% (7 of 13 patients) for the low-risk, intermediate-risk, and high-risk groups, respectively (P =.04, Fisher exact test). Tumor expression of p53 and Bcl-xL is a strong predictor of successful larynx preservation in patients treated with induction chemotherapy and followed by radiation therapy in responding tumors.

Clinicopathological correlations of Bcl‐xL and Bax expression in differentiated thyroid carcinoma

The Bcl-2 family proteins are essential mediators in the apoptotic process. Our aim was to investigate whether anti-apoptotic Bcl-xL and pro-apoptotic Bax were over-expressed in a large series of differentiated thyroid carcinomas (DTC) and to study their association with tumour presentation at diagnosis and prognosis. We examined the immunohistochemical expression of Bcl-xL and Bax in benign nodular thyroid disease (BNTD) and DTC and their association with clinicopathological parameters. Thyroid tissue samples were collected from an unselected series of patients undergoing surgical resection for DTC (n = 74) or BNTD (n = 15). Among DTC cases, expression of Bcl-xL was found to be high in 43.2% and low or absent in 56.8%. Expression of Bax was high in 75.7% and low or absent in 24.3%. Non-neoplastic thyroid tissue was largely unstained for both proteins. Among BNTD cases, expression of Bcl-xL was high in 13.3% and low or absent in 86.6%. Expression of Bax was high in 14.3% and low or absent in 86.6%. A significant association was found between Bcl-xL expression and the presence of high-risk histological subtype (P < 0.05), and regional lymph node (P < 0.01) and distant metastases (P < 0.01). The association between high Bcl-xL expression levels and a longer time of persistent disease after radioiodine ablation was also significant (P < 0.01). Bcl-xL expression was confirmed as an independent prognostic factor for persistent disease in DTC (relative risk, 2.5; 95% confidence interval, 1.1-5.9; P < 0.05). Immunohistochemical expression of Bcl-xL might be a valuable tool in the prediction of tumour aggressiveness in DTC.

Modulation of the Mitochondrial (Type 2) Apoptotic Pathway Can Enhance Apo2L/TRAIL - Mediated Killing of Human Multiple Myeloma (MM) Cells.

We and others have previously demonstrated that Apo2/TRAIL effectively induces apoptosis of human myeloma cell lines (HMCL) but exhibits only modest activity against primary MM cells derived from patients with advanced MM. A commonly acquired drug-resistance mechanism found in these patients is the over-expression of the anti-apoptotic protein Bcl-xl. Based on these observations and the potential of Apo2/TRAIL as a specific anti-MM therapeutic agent we have investigated further the mechanism(s) of apoptosis induction in MM and whether inhibition of bcl-xl expression enhances the efficacy of Apo2L/TRAIL induced killing of MM cells. Four HMCL selected on the basis of high (U266 and RPMI 8226) or low (LP-1 and NCI H929) Bcl-xl expression, as determined by both Western blotting and ELISA, were chosen for study. We have previously demonstrated no correlation between the absolute levels of caspase 8 and cFLIP or the caspase 8:cFLIP ratio of these 4 HMCL and their sensitivity to Apo2/TRAIL. HMCL were treated with 1μg/ml of recombinant human trimeric TRAIL (LZ-TRAIL) (Amgen) and the level of apoptosis was measured by AnnexinV expression at 2 hours - U266 24%, RPMI 8226 53%, LP-1 57% and NCI H929 26%. The experiment was then repeated and both whole cell lysates and cytosolic fractions were prepared for immunoblot analysis of caspase 8 cleavage and cytochrome C/SMAC protein levels, respectively, at various time-points from 0 to 120 minutes following treatment. Two patterns of apoptosis induction were observed. All 4 HMCL had demonstrable increases in cytosolic cytochrome c and SMAC by 15 minutes post LZ-TRAIL. Similarly both RPMI and LP-1 demonstrated contemporaneous cleavage of caspase 8. In contrast, no evidence of caspase 8 cleavage was seen in U266 or NCI H929 until 30 – 60 minutes post LZ-TRAIL. The impact of pre-treatment with a caspase 9 inhibitor, (Z-LEHD-FMK) prior to LZ-TRAIL correlated with the immunoblot findings: RPMI 8226 and LP-1 (7% and 20%, respectively) showing less relative reduction in apoptosis inhibition than U266 and NCI H929 (64% and 49%, respectively). Finally, treatment with TRAIL alone, the bcl-xl inhibitor 2MAME (10, 15 or 20μg/ml) alone or both (2MAME 4 hours prior to TRAIL) demonstrated synergistic killing of both 'high' bcl-xl expressing HMCL (RPMI 8226 synergism quotients = 1.1, 1.3 and 1.3 and U266 synergism quotients = 1.6, 1.4 and 1.4 for 2MAME 10, 15 and 20μg/ml, respectively) but not the 'low' bcl-xl expressing HMCL (NCI H929 and LP-1). From these data we conclude that human MM cells exhibit variable apoptotic responses to Apo2L/TRAIL that involve both the Type 1 and Type 2 apoptotic pathways and that the targeting of known inhibitors of the Type 2 pathway can enhance Apo2L/TRAIL-induced apoptosis.

Expression of bcl2 family proteins and active caspase 3 in classical Hodgkin's lymphomas

The expression of various bcl2 family proteins has been reported in Hodgkin and Reed-Sternberg cells, but the proteins bad, bid, and bim have not been analyzed in classical Hodgkin's lymphomas (HLs). This study aimed to investigate the expression of the proteins bcl2, bcl-xl, mcl1, bax, bak, bad, bid, bim, and active caspase 3, and the TUNEL (terminal deoxynucleotidyl transferase-mediated in situ labeling) index to gain further insight into the apoptosis profile of classical HLs. A high expression of the proteins bcl2, bcl-xl, mcl1, bax, bak, bad, bid, and bim in HRS cells was found in 27 of 101 (27%), 95 of 101 (94%), 27 of 97 (29%), 73 of 95 (77%), 37 of 102 (36%), 85 of 94 (90%), 19 of 109 (17%), and 43 of 91 (47%) cases, respectively. The high expression of bcl-xl, bax, and bad in HRS cells in most classical HLs indicates that these proteins may play predominant roles in the regulation of apoptosis in classical HLs. Active caspase 3-positive and TUNEL-positive Reed-Sternberg cells were detected in 47 of 70 (67%; range, 0%-12%) and 60 of 71 (85%; range, 0%-19%) cases, respectively. Significant positive correlations were found between bax/bcl2 (P = .002), bad/bcl2 (P = .020), bad/bcl-xl (P = .003), and bim/mcl1 (P = .036). Based on these findings, it could be hypothesized that the antiapoptotic proteins bcl2, bcl-xl, and mcl1 may counteract the expression of the proapoptotic proteins bax, bad, and bim, thereby contributing to the survival of Reed-Sternberg cells.