Mcl-1 mRNA Expression Research Articles

Actinomycin D synergistically enhances the efficacy of the BH3 mimetic ABT-737 by downregulating Mcl-1 expression

Many types of cancer cells possess the ability to evade apoptosis, leading to their rapid and uncontrolled proliferation. As major regulators of apoptosis, Bcl-2 proteins serve as emerging targets for novel chemotherapeutic strategies. In this study, we examined the involvement of Bcl-2 proteins in apoptosis induced by the chemotherapeutic agent actinomycin D. A dramatic decrease in anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) mRNA and protein expression was detected upon actinomycin D treatment. Further, Mcl-l over-expression caused resistance to cell death upon treatment with actinomycin D, implicating a role for the down-regulation of Mcl-1 in actinomycin D-induced apoptosis. We also explored the therapeutic potential of actinomycin D in combination with ABT-737, an experimental agent that inhibits anti-apoptotic Bcl-2 proteins. Actinomycin D sensitized cells to ABT-737 treatment in a Bak- or Bax-dependent manner. Importantly, low concentrations of actinomycin D and ABT-737 were more effective in inducing cell death in transformed cells than their untransformed counterparts. A synergistic effect of actinomycin D and ABT-737 on cell death was observed in several human tumor cell lines. Like actinomycin D treatment, knocking down Mcl-1 expression greatly sensitized tumor cells to ABT-737, and Mcl-1 over-expression abrogated the cytotoxic effect induced by ABT-737 and actinomycin D. These results suggest that the down-regulation of Mcl-1 by actinomycin D is likely responsible for the observed synergistic effect between the two drugs. Overall, our studies provide compelling evidence that the combination of actinomycin D and ABT-737 may lead to an effective cancer treatment strategy.

Nucleoside analog ARC targets Mcl-1 to induce apoptosis in leukemia cells

Nucleoside analog ARC targets Mcl-1 to induce apoptosis in leukemia cells

Acoustic overstimulation modifies Mcl‐1 expression in cochlear sensory epithelial cells

Acoustic overstimulation causes apoptotic cell death in the cochlea. This death process is mediated, in part, by the mitochondrial signaling pathway involving Bcl-2 family proteins. Myeloid cell leukemia sequence 1 (Mcl-l) is an antiapoptotic member of the Bcl-2 family. Its involvement in noise-induced hair cell death has not been characterized. Here we report the endogenous expression and the noise-induced expression of Mcl-1 in Sprague Dawley rat cochleae. In the sensory epithelia of normal cochleae, there is strong constitutive expression of Mcl-1 mRNA, with an expression level higher than that of many other Bcl-2 family genes. The Mcl-1 protein is preferentially expressed in outer hair cells. After exposure to a high level of continuous noise at 115-dB sound pressure level for 1 hr, Mcl-1 expression displays a time-dependent alteration, with up-regulation of Mcl-1 mRNA at 4 hr postexposure and protein up-regulation at 1 day postexposure. Western blot analysis reveals the up-regulated Mcl-1 as the full-length form of Mcl-1. Immunolabeling of the Mcl-1 protein reveals the early increase in Mcl-1 immunoreactivity in the nuclear region of the hair cells displaying apoptotic phenotypes and a subsequent increase in survival hair cells. These results suggest that Mcl-1 is involved in the regulation of hair cell pathogenesis resulting from acoustic stress, possibly by influencing the nuclear events of apoptosis.

Adenovirus-mediated siRNA targeting Mcl-1 gene increases radiosensitivity of pancreatic carcinoma cells in vitro and in vivo

Myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic member of the B cell lymphoma/leukemia-2 (Bcl-2) family, has been shown to be involved in apoptosis and the cell cycle. Mcl-1 is overexpressed in many malignancies, including pancreatic cancer. The aim of this study was to investigate the effect of siRNA targeted against Mcl-1 on the radiosensitivity of human pancreatic carcinoma cells. In 3 pancreatic cancer cell lines, the expression of Mcl-1 mRNA was determined by RT-PCR assay, and the dose-dependent cytotoxicity of radiation was also assessed. Furthermore, the effects of adenovirus-mediated siRNA targeted against Mcl-1 on radiosensitivity of PANC-1 cells both in vitro and in vivo were evaluated. Pancreatic cancer cells showed various levels of Mcl-1 mRNA that were correlated with the radiosensitivity of tumor cells. AdU6/shMcl-1 significantly downregulated the expression of Mcl-1 gene in PANC-1 cells, the most radioresistant cell line. Furthermore, Mcl-1 downregulation could significantly enhance radiosensitivity of PANC-1 cells in vitro and in vivo. The mechanism might be correlated with apoptosis enhancement by activating celluar caspase-3. Mcl-1 might be a therapeutic target for radiosensitization of pancreatic carcinoma cells. Adenovirus-mediated siRNA targeting of Mcl-1 could enhance the radiosensitivity of pancreatic cancer cells both in vitro and in vivo, and thus might be a potential strategy for ameliorating cancer.

Arsenic trioxide induces apoptosis of glucocorticoid-resistant acute lymphoblastic leukemia CEM-C1 cells

To explore the effects of arsenic trioxide (ATO) on the apoptosis of glucocorticoid (GC)-resistant T-acute lymphoblastic leukemia (ALL) CEM-C1 cells and its possible mechanisms. Different concentrations of ATO (0.25 μmol/L-5 μmol/L) were used to induce the apoptosis of CEM-C1 cells. The inhibition rate of cell proliferation and apoptosis were detected by MTT test, Annexin V-FITC/PI flow cytometry and optical microscopy, respectively. RT-PCR was applied to semi-quantitatively analyze the mRNA expression of pro-apoptotic proteins (Bad and PDCD4) and anti-apoptotic proteins (XIAP and MCL-1) induced by different concentrations of ATO at different time points. ATO could inhibit proliferation and induce apoptosis of CEM-C1 cells at a concentration and time dependent manner. Low-dose ATO mildly inhibited the proliferation of CEM-C1 cells while higher concentrations (1 μmol/L and 5 μmol/L) had strong anti-tumor effect with the inhibiting rates of 40.07±7.98% and 88.67±2.88%, respectively. Annexin V-FITC/PI flow cytometry showed that the apoptotic rates of CEM-C1 cells were significantly increased after 48 hours treatment of different concentrations of ATO. RT-PCR demonstrated up-regulated mRNA expression of pro-apoptotic protein Bad and PDCD4 but down-regulated mRNA expression of anti-apoptotic protein XIAP when CEM-C1 cells were treated with different concentrations of ATO at different time points. The MCL-1 mRNA expression was down-regulated only after the treatment of 5 μmol/L ATO. ATO can inhibit cell proliferation and induce cell apoptosis in GC-resistant CEM-C1 cells. The molecular mechanisms might involve the increased mRNA expression of pro-apoptotic protein Bad and PDCD-4, and rapid down-regulation of XIAP mRNA expression.

Role of Nitric Oxide in the Pathogenesis of Encephalomyocarditis Virus-Induced Diabetes in Mice

The D variant of encephalomyocarditis virus (EMC-D virus) causes diabetes in mice by destroying pancreatic beta cells. In mice infected with a low dose of EMC-D virus, macrophages play an important role in beta-cell destruction by producing soluble mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). To investigate the role of NO and inducible NO synthase (iNOS) in the development of diabetes in EMC-D virus-infected mice, we infected iNOS-deficient DBA/2 mice with EMC-D virus (2 x 10(2) PFU/mouse). Mean blood glucose levels in EMC-D virus-infected iNOS-deficient mice and wild-type mice were 205.5 and 466.7 mg/dl, respectively. Insulitis and macrophage infiltration were reduced in islets of iNOS-deficient mice compared with wild-type mice at 3 days after EMC-D virus infection. Apoptosis of beta cells was decreased in iNOS-deficient mice, as evidenced by reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. There were no differences in mRNA expression of antiapoptotic molecules Bcl-2, Bcl-xL, Bcl-w, Mcl-1, cIAP-1, and cIAP-2 between wild-type and iNOS-deficient mice, whereas expression of proapoptotic Bax and Bak mRNAs was significantly decreased in iNOS-deficient mice. Expression of IL-1beta and TNF-alpha mRNAs was significantly decreased in both islets and macrophages of iNOS-deficient mice compared with wild-type mice after EMC-D virus infection. Nuclear factor kappaB was less activated in macrophages of iNOS-deficient mice after virus infection. We conclude that NO plays an important role in the activation of macrophages and apoptosis of pancreatic beta cells in EMC-D virus-infected mice and that deficient iNOS gene expression inhibits macrophage activation and beta-cell apoptosis, contributing to prevention of EMC-D virus-induced diabetes.

Bcl-xL and Myeloid cell leukaemia-1 contribute to apoptosis resistance of colorectal cancer cells

To explore the role of Bcl-x(L) and Myeloid cell leukaemia (Mcl)-1 for the apoptosis resistance of colorectal carcinoma (CRC) cells towards current treatment modalities. Bcl-x(L) and Mcl-1 mRNA and protein expression were analyzed in CRC cell lines as well as human CRC tissue by Western blot, quantitative PCR and immunohistochemistry. Bcl-x(L) and Mcl-1 protein expression was knocked down or increased in CRC cell lines by applying specific siRNAs or expression plasmids, respectively. After modulation of protein expression, CRC cells were treated with chemotherapeutic agents, an antagonistic epidermal growth factor receptor (EGFR1) antibody, an EGFR1 tyrosine kinase inhibitor, or with the death receptor ligand TRAIL. Apoptosis induction and cell viability were analyzed. Here we show that in human CRC tissue and various CRC cell lines both Bcl-x(L) and Mcl-1 are expressed. Bcl-x(L) expression was higher in CRC tissue than in surrounding non-malignant tissue, both on protein and mRNA level. Mcl-1 mRNA expression was significantly lower in malignant tissues. However, protein expression was slightly higher. Viability rates of CRC cells were significantly decreased after knock down of Bcl-x(L) expression, and, to a lower extent, after knock down of Mcl-1 expression. Furthermore, cells with reduced Bcl-x(L) or Mcl-1 expression was more sensitive towards oxaliplatin- and irinotecan-induced apoptosis, and in the case of Bcl-x(L) also towards 5-FU-induced apoptosis. On the other hand, upregulation of Bcl-x(L) by transfection of an expression plasmid decreased chemotherapeutic drug-induced apoptosis. EGF treatment clearly induced Bcl-x(L) and Mcl-1 expression in CRC cells. Apoptosis induction upon EGFR1 blockage by cetuximab or PD168393 was increased by inhibiting Mcl-1 and Bcl-x(L) expression. More strikingly, CD95- and TRAIL-induced apoptosis was increased by Bcl-x(L) knock down. Our data suggest that Bcl-x(L) and, to a lower extent, Mcl-1, are important anti-apoptotic factors in CRC. Specific downregulation of Bcl-x(L) is a promising approach to sensitize CRC cells towards chemotherapy and targeted therapy.

Galiellalactone is a novel therapeutic candidate against hormone‐refractory prostate cancer expressing activated Stat3

Signal transducer and activator of transcription 3 (Stat3) is constitutively active (phosphorylated) in several forms of cancer, including prostate cancer (PCa). Stat3 signaling may be an interesting target for cancer therapy since inhibition of this pathway mediates growth inhibition and apoptosis of these cells. In this study we investigated the in vitro and in vivo effects of the fungal metabolite galiellalactone, a direct inhibitor of Stat3, on PCa cells. The human PCa cell lines DU145, PC-3, and LNCaP were used. Nude mice with subcutaneous PCa cell xenografts were subjected to daily intraperitoneal injections of galiellalactone for 3 weeks. The effect of galiellalactone on the induction of apoptosis of cultured PCa cells was investigated by Western blot analysis, immunocytochemistry, and annexin V staining. Effects of galiellalactone on Stat3 signaling were investigated by a luciferase reporter gene assay. Expression of Stat3 associated proteins and mRNA was investigated by Western blot and real-time quantitative PCR analysis. Galiellalactone induced apoptosis of p-Stat3 positive PCa cells (androgen-insensitive DU145 and PC-3) but not in cells lacking p-Stat3 (androgen-sensitive LNCaP). Galiellalactone inhibited Stat3-mediated luciferase activity (IC(50) approximately 5 microM) and reduced the expression of Bcl-2, Bcl-x(L), c-myc, and cyclin D1. Furthermore, galiellalactone significantly suppressed DU145 xenograft growth in vivo (42% growth reduction; P<0.002) and reduced the relative mRNA expression of Bcl-x(L) and Mcl-1. Galiellalactone induced growth inhibition and apoptosis in androgen-insensitive PCa cells expressing p-Stat3. We suggest that galiellalactone is a potential anti-tumor lead against hormone-refractory PCa with constitutively active Stat3.

In Vivo Evaluation of AT-101 (R-(—)-Gossypol Acetic Acid) in Androgen-Independent Growth of VCaP Prostate Cancer Cells in Combination with Surgical Castration

PURPOSE: Upregulation of Bcl-2 family members is a well-established mechanism in the development of androgen-independent prostate cancer. Inhibition of the antiapoptotic proteins Bcl-2 and Mcl-1 may delay the transition to androgen-independent growth. EXPERIMENTAL DESIGN: We have established a prostate cancer model with VCaP prostate cancer cells in vivo to study the transition to androgen independence. Here, we investigated the efficacy of AT-101 (R-(—)-gossypol acetic acid; a pan small molecule inhibitor of Bcl-2, BCI-xL, Mcl-1) in combination with surgical castration to delay the onset of androgen-independent growth in vivo. RESULTS: AT-101 (15 mg/kg, per os (p.o.) 5 days/week) in combination with surgical castration delayed the onset of androgen-independent prostate cancer growth in vivo. In addition, we demonstrate the induction of caspase-9- and caspase-3-dependent induction of apoptosis following AT-101 treatment in vitro which was accompanied by an AT-101-induced downregulation of Bcl-2 and Mcl-1 mRNA and protein expression. CONCLUSIONS: We conclude that AT-101 in combination with surgical castration delays the onset of androgen-independent prostate cancer in vivo by disrupting the antiapoptotic activity of BCl-2 upregulation during the transition to androgen independence. Further studies are needed to define the mechanism of action by which AT-101 attenuates the expression of Bcl-2 and Mcl-1 and to characterize the potential for AT-101 in combination with hormone therapy.

Low MCL-1 mRNA expression correlates with prolonged survival in B-cell chronic lymphocytic leukemia

Low MCL-1 mRNA expression correlates with prolonged survival in B-cell chronic lymphocytic leukemia

Mcl-1 Depletion in Apoptosis Elicited by Ionizing Radiation in Peritoneal Resident Macrophages of C3H Mice

Remarkably, apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice, to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins, Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1, but not Bcl-2, Bcl-X(L), Bax, A1, or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand, Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis.

Cyclic AMP delays neutrophil apoptosis via stabilization of Mcl-1

Human neutrophils underwent spontaneous apoptosis, which was accompanied by degradation of Mcl-1, but not other anti-apoptotic molecules (cIAP1, cIAP2, A1, survivin and Bcl-2). Spontaneous neutrophil apoptosis and Mcl-1 degradation were prevented by cyclic AMP (cAMP) agonists (dibutyryl cAMP and prostaglandin E 1), and the effects of cAMP agonists on neutrophils were highly resistant to cycloheximide, a protein synthesis inhibitor, although slight increase in Mcl-1 mRNA expression was induced by cAMP agonists. Proteasome inhibitors (epoxomicin and lactacystin) also prevented spontaneous neutrophil apoptosis and Mcl-1 degradation to the same extent as cAMP agonists, and no additive effect was obtained by combination of cAMP agonists and proteasome inhibitors. These findings suggest that cAMP agonists, like proteasome inhibitors, delay neutrophil apoptosis primarily via stabilization of Mcl-1.

Inducer-and cell type-specific regulation of antiapoptotic MCL1 in myeloid leukemia and multiple myeloma cells exposed to differentiation-inducing or microtubule-disrupting agents

The antiapoptotic BCL2 family member MCL1 is rapidly upregulated upon exposure of ML-1 myeloid leukemia cells to either differentiation-inducing phorbol 12'-myristate 13'-acetate (PMA) or chemotherapeutic microtubule disrupting agents (MTDAs). This report examined how signaling for MCL1 upregulation is coupled to these two different phenotypic changes, and tested for upregulation in other hematopoietic cancers. With PMA, ERK stimulated MCL1 mRNA expression and ML-1 cell differentiation, and ERK additionally stabilized expression of the MCL1 protein. However, with MTDAs, transient ERK and ensuing JNK activation contributed to initial MCL1 upregulation and viability-retention, but sustained JNK activation eventually resulted in cell death. MCL1 was upregulated by PMA in THP-1 and U937 myeloid leukemia cells, but by MTDAs only in THP-1 cells. MCL1 expression was constitutively elevated in multiple myeloma cell lines, and was not affected by PMA/ERK or MTDAs. Thus, MCL1 expression level and sensitivity to regulation are important considerations in selecting approaches for targeting this antiapoptotic gene product to kill cancer cells.

M. leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression

BackgroundVirulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG.ResultsM. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1–4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA.ConclusionThis study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.

Over-expression of interleukin-6 enhances cell survival and transformed cell growth in human malignant cholangiocytes

Over-expression of IL-6 has been implicated in cholangiocarcinoma growth but the cellular mechanisms involved are unknown. Our aims were to assess the mechanisms by which over-expression of IL-6 promotes transformed cell growth in malignant cholangiocytes. Stably transfected cell lines over-expressing IL-6 were derived from malignant human cholangiocytes. Transformed cell growth was assessed by anchorage independent growth in vitro and by xenograft growth in nude mice. Expression of the anti-apoptotic protein Mcl-1 was quantitated by immunoblot analysis and by real-time PCR. Gene silencing was performed using siRNA. Dominant negative upstream kinase activators and isoform-specific constructs were used to evaluate the involvement of p38 MAP kinase signaling pathways. Over-expression of IL-6 increased xenograft growth, anchorage independent growth and cell survival but did not significantly alter cell proliferation. The basal expression of Mcl-1 was increased in IL-6 over-expressing cells. Selective knockdown of Mcl-1 by siRNA increased gemcitabine-induced cytotoxicity. Moreover, IL-6 increased Mcl-1 mRNA and protein expression via a p38 MAPK dependent mechanism. These data demonstrate a major role of survival signaling pathways in mediating the effects of IL-6 over-expression in cholangiocarcinoma growth. Mcl-1 is identified as a mediator of IL-6-induced tumor cell survival and shown to be transcriptionally regulated by IL-6 via a p38 MAPK dependent pathway. We conclude that modulation of IL-6 mediated survival signaling pathways involving the p38 MAPK or downstream targets such as Mcl-1 may prove useful therapeutic strategies for human cholangiocarcinoma.

HIF‐1α has an anti‐apoptotic effect in human airway epithelium that is mediated via Mcl‐1 gene expression

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.

High expression of Mcl-1 in ALK positive and negative anaplastic large cell lymphoma

Aim: To gain more insight into the genes involved in the aetiology and pathogenesis of anaplastic large cell lymphoma (ALCL). Methods: Serial analysis of gene expression (SAGE) was undertaken on...

Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides

AbstractAntiapoptotic members of the bcl-2 family have recently been implicated in the pathogenesis of chronic myeloid leukemia (CML), a hematopoietic neoplasm associated with the BCR/ABL oncogene. We have examined expression of MCL-1 in primary CML cells and BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed myeloid cell leukemia-1 (mcl-1) mRNA and the MCL-1 protein in a constitutive manner. The BCR/ABL inhibitor imatinib (=STI571) decreased the expression of MCL-1 in these cells. Correspondingly, BCR/ABL enhanced mcl-1 promoter activity, mcl-1 mRNA expression, and the MCL-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of MCL-1 in Ba/F3 cells was counteracted by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Identical results were obtained for constitutive expression of MCL-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of MCL-1 as a survival-related target in CML cells, mcl-1 siRNA and mcl-1 antisense oligonucleotides (ASOs) were applied. The resulting down-regulation of MCL-1 was found to be associated with a substantial decrease in viability of K562 cells. Moreover, the mcl-1 ASO was found to synergize with imatinib in producing growth inhibition in these cells. Together, our data identify MCL-1 as a BCR/ABL-dependent survival factor and interesting target in CML. (Blood. 2005;105:3303-3311)

Hypoxia-inducible Factor-1-dependent Overexpression of Myeloid Cell Factor-1 Protects Hypoxic Cells against tert-Butyl Hydroperoxide-induced Apoptosis

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1alpha short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tert-butyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein.

Sensitization of cervical cancer cell lines to low-dose radiation by retinoic acid does not require functional p53

Objective. Current therapy for cervical cancer includes radiation therapy. Retinoic acid (RA) can increase the sensitivity of cervical cancer cell lines to radiation. The mechanism of this sensitization may not involve the p53 protein because the human papillomavirus (HPV) E6 protein, which is present in the majority of cervical cancers, promotes p53 degradation. The objective of this study was to determine if p53 is involved in the mechanism of RA radiosensitization. Methods. The effects of radiation on cervical (SiHa, CC-1, and C33a) and vulvar (SW962) cancer cell lines under various experimental conditions were evaluated using clonogenic, Coulter Counter, electrophoretic mobility shift (EMSA) and a multi-probe RNase protection assay of p53-inducible genes. Results. RA (5 μM 9- cis-RA) radiosensitized the SiHa and CC-1 cell lines that contain HPV-degraded p53, but did not radiosensitize the SW962 cell line, which is HPV negative and contains wild-type p53, nor the C33a cell line, which contains mutant p53 (R273C). Expression of mutant p53 (R273H) in SiHa cells increased the growth rate, but did not prevent RA-induced differentiation or radiosensitization at clinically relevant doses. Inhibition of p53 transactivation with pifithirin α did not prevent RA radiosensitization of SiHa at 5 Gy. RA repressed c- fos mRNA expression in control and irradiated SiHa cultures, but did not repress bcl-x L, p53, GADD45, p21, bax, bcl-2, or mcl-1 mRNA expression. Conclusions. The mechanism of RA radiosensitization does not require functional p53 and may involve c- fos in cervical cancer cell lines.