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PubMed2003

c-Myc potentiates the mitochondrial pathway of apoptosis by acting upstream of apoptosis signal-regulating kinase 1 (Ask1) in the p38 signalling cascade.

Cell transformation by growth-promoting oncoproteins renders cells extremely sensitive to apoptosis through an unknown mechanism affecting the mitochondrial pathway of apoptosis. We have shown previously that sensitization to apoptosis also correlated with the activation of the stress-activated protein kinase p38. In the present study, we investigated the role of p38 in c-Myc-dependent apoptosis induced by the anticancer agent cisplatin. Cisplatin treatment of Rat1 cells with deregulated expression of c-Myc resulted in nuclear fragmentation that was accompanied in all cells by the activation of Bax and the translocation of cytochrome c from the mitochondria to the cytoplasm. None of these features of apoptosis was induced in control Rat-1 cells. p38 was also activated by cisplatin only in cells with deregulated expression of c-Myc, but, in contrast with all features of apoptosis, this activation was not affected by Bcl-2. Remarkably, overexpression of an interfering mutant of the p38alpha isoform, but not p38beta, blocked cisplatin-induced Bax activation or cytochrome c release and nuclear fragmentation. Analysis of the kinase cascade upstream of p38 revealed a c-Myc-dependent activation by cisplatin of mitogen-activated protein kinase kinase (MKK) 3/6 and apoptosis signal-regulating kinase 1 (Ask1). Inhibition of Ask1 blocked p38 activation by cisplatin and all features of apoptosis. Several of these data were confirmed using other DNA-damaging agents. The findings indicated that c-Myc potentiation of the mitochondrial pathway of apoptosis results, at least in part, from a sensitization of Ask1 activation, allowing DNA-damaging agents to induce in cascade Ask1, p38alpha and Bax.

Animals↗
PubMed2000

Role of c-jun expression increased by heat shock- and ceramide-activated caspase-3 in HL-60 cell apoptosis. Possible involvement of ceramide in heat shock-induced apoptosis.

Ceramide has emerged as a lipid mediator in apoptosis induced by a variety of stresses. As we previously showed that the activation of AP-1, a nuclear transcription factor was indispensable to ceramide-induced apoptosis in human leukemia HL-60 cells (Sawai, H., Okazaki, T., Yamamoto, H., Okano, H., Takeda, Y., Tashima, M., Sawada, H., Okuma, M., Ishikura, H., Umehara, H., and Domae, N. (1995) J. Biol. Chem. 270, 27326-27331), the role and mechanism of heat shock (HS)-increased c-jun expression in apoptosis was here investigated. HS increased morphological changes compatible with apoptosis in human leukemia HL-60 cells, and induced ceramide generation and sphingomyelin hydrolysis with an increase of neutral magnesium-dependent sphingomyelinase activity. When HS failed to induce apoptosis in HS-resistant HL-60 cells, ceramide generation was not detected, suggesting that ceramide was involved in downstream signals required for HS-induced apoptosis. Both HS and N-acetylsphingosine (C(2)-ceramide) increased the expression of c-jun/c-fos mRNAs with the peak 2 h after treatment. When we examined whether the inhibition of c-jun expression by its antisense oligodeoxynucleotides (AS) blocked HS- or C(2)-ceramide-induced apoptosis, AS of c-jun gene inhibited apoptotic morphological changes and DNA fragmentation whereas did not sense oligodeoxynucleotides. Moreover, a synthetic tetrapeptide, acetyl-Asp-Met-Gln-Asp-aldehyde (DMQD-CHO), which inhibited the formation of active form of caspase-3 more efficiently than those of caspase-4, -6, -7, and -8, blocked both caspase-3 like activity, c-jun expression and apoptosis induced by HS or C(2)-ceramide, although DMQD-CHO did not affect HS-induced ceramide generation. These results suggested that the ceramide was generated through sphingomyelin hydrolysis by HS-activated neutral, magnesium-dependent sphingomyelinase and that subsequent c-jun expression through activation of caspase-3 played a role in HS-induced HL-60 cell apoptosis.

Apoptosis↗
PubMed1997

The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis.

According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

Apoptosis↗
PubMed2003

Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates hydrogen peroxide-induced apoptosis in human pulmonary vascular endothelial cells.

OBJECTIVE: Reactive oxygen species initiate pulmonary vascular endothelial cell damage leading to an increase in endothelial permeability resulting in the production of pulmonary edema. Apoptosis signal-regulating kinase (ASK)-1 is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the MKK3/MKK6-p38 MAPK and the SEK1-c-Jun N-terminal kinase (JNK) signaling cascade. ASK1 has been implicated in cytokine- and stress-induced apoptosis. However, little is known about the role of ASK1 in apoptosis in hydrogen peroxide (H2O2)-stimulated pulmonary vascular endothelial cells and how ASK1-mediated apoptosis is executed. To clarify this issue, we examined the role of ASK1-p38 MAPK/JNK cascade in apoptosis and caspase-3 activation in H2O2-stimulated pulmonary vascular endothelial cells. DESIGN: Experimental laboratory study. SETTING: University laboratory. SUBJECTS: Normal human pulmonary artery endothelial cells. INTERVENTIONS: Western blot analysis and quantification of apoptosis in cells. MEASUREMENTS AND MAIN RESULTS: The results showed that H2O2 induced ASK1 phosphorylation and concomitantly p38 MAPK and JNK phosphorylation as well as induced caspase-3 activation in pulmonary vascular endothelial cells. To further characterize the role of ASK1 cascade in H2O2-induced apoptosis of pulmonary vascular endothelial cells, the dominant negative form of ASK1-stably transfected porcine artery endothelial cells was used. p38 MAPK and JNK phosphorylation, caspase-3 activation, and apoptosis in the dominant negative form of ASK1-stably transfected porcine artery endothelial cells were depressed compared with those in the parental porcine artery endothelial cells. CONCLUSION: ASK1-p38 MAPK/JNK cascade regulates apoptosis of H2O2-stimulated human pulmonary vascular endothelial cells.

Animals↗
PubMed2001

Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4.

The majority of oocytes present in fetal ovaries are depleted before birth, and only about 400 will ovulate during the normal fertile life span. Studies on animals have shown that apoptosis is the mechanism behind oocyte depletion and follicular atresia. In the present study, we investigated the extent and localization of apoptosis in human fetal (aged 13-40 weeks) and adult ovaries. Furthermore, the expression of apoptosis-regulating proteins, bcl-2 and bax, and the relationship of transcription factor GATA-4 were studied. Apoptosis was found in ovarian follicles throughout fetal and adult life. During fetal development, apoptosis was localized mainly to primary oocytes and was highest between weeks 14-28, decreasing thereafter toward term. Expression of bcl-2 was observed only in the youngest fetal ovaries (weeks 13-14), and bax was present in the ovaries throughout the entire fetal period. In adult ovaries, apoptosis was detected in granulosa cells of secondary and antral follicles, and Bcl-2 and bax were expressed from primary follicles onwards. During fetal ovarian development, GATA-4 messenger RNA and protein were localized to the granulosa cells, with expression being highest in the youngest ovaries and decreasing somewhat toward term. The expression pattern of GATA-4 suggests that it may be involved in the mechanisms protecting granulosa cells from apoptosis from fetal to adult life. The results indicate that depletion of ovarian follicles in the human fetus occurs through intrinsic mechanisms of apoptosis in oocytes, and later in adult life the survival of growing follicles may be primarily determined by granulosa cell apoptosis.

Aging↗
PubMedDate not supplied

Luteinizing hormone-releasing hormone (LHRH) inhibits apoptosis induced by cytotoxic agent and UV-light but not apoptosis mediated through CD95 in human ovarian and endometrial cancer cells.

Luteinizing hormone-releasing hormone (LHRH) and its receptor are frequently expressed in human ovarian and endometrial cancers and are part of an autocrine mechanism of growth control. We have previously shown that the LHRH analog Triptorelin induces activation of nucleus factor kappa B (NFkappaB) and reduces apoptosis induced by doxorubicin in human ovarian cancer cells EFO-21 and EFO-27. The present study was performed to investigate the anti-apoptotic effects of LHRH analogs on apoptosis induced by doxorubicin, UV-light and ligation of CD95 in human endometrial and ovarian cancer cells. We further investigated the interaction of the LHRH system with the apoptotic pathway focusing on the effector-protease caspase 3. Doxorubicin (100 nM) induced apoptosis in the LHRH-receptor-positive human endometrial cancer cell line Ishikawa and in the human ovarian cancer cell lines EFO-21 and NIH:OVCAR-3. Pretreatment for 24 h with native LHRH, the LHRH agonist Triptorelin or the LHRH antagonist Cetrorelix (100 nM) significantly reduced apoptosis induced by doxorubicin in these cells. In EFO-21 cells pretreatment with 100 nM Triptorelin also reduced UV-light-induced apoptosis from 76% to 62.7% (p<0.01). EFO-21 cells express CD95. Cross-linking of CD95 with monoclonal antibody anti-APO-1 (500 ng/ml) increased apoptosis from spontaneous rate to 10.3% to 38.3% in EFO-21 cells (p<0.001). Pre-treatment with Triptorelin did not reduce CD95-mediated apoptosis in these cells. LHRH analogs protect human endometrial and ovarian cancer cells from DNA-replication-dependent cytotoxic agent and UV-light-induced apoptosis, but not from CD95-mediated apoptosis.

Antibiotics, Antineoplastic↗
PubMed2006

Signaling cascades in radiation-induced apoptosis: roles of protein kinase C in the apoptosis regulation.

Apoptosis is a biologic mechanism for eliminating damaged cells from the cell population. Apoptosis is known to be induced by irradiation and can prevent the development of disease states such as carcinogenesis or abnormal tissue formation. On the other hand, if the mechanism is properly controlled, radiotherapy can be used to kill cancer cells more efficiently. Radiation-induced apoptosis is regulated by the balance between cellular anti-apoptotic and (pro-)apoptotic signals. Many regulators of radiation-induced apoptosis have been identified and analyzed. Protein kinase C (PKC) is a family of serine/threonine kinases and one of the regulators in radiation-induced apoptosis. PKC has some subtypes, each of whose functions has been analyzed in radiation-induced signaling cascades. It has been demonstrated that each of PKC subtypes has distinct functions in radiation-induced apoptosis. Moreover, some participants in PKC-related signaling cascades have been identified in radiation-induced apoptosis. Interestingly, PKC-related signaling cascades have been found to be regulated in part by ATM (the gene that is mutated in the human genetic disorder ataxia telangiectasia). ATM is a protein related to cell-cycle checkpoints and cell radiosensitivity, and it also regulates radiation-induced apoptosis. This article reviews recent developments in the understanding of radiation-induced apoptosis, focusing on PKC functions, and the relationship with ATM.

Animals↗
PubMed1995

Analysis of apoptosis in lymph nodes of HIV-infected persons. Intensity of apoptosis correlates with the general state of activation of the lymphoid tissue and not with stage of disease or viral burden.

The occurrence of in vivo apoptosis was investigated in lymph node sections obtained from HIV-infected persons at different stages of disease. The degree of apoptosis in lymph nodes from HIV-infected individuals was compared with that observed in lymph nodes obtained from HIV-negative individuals. Apoptosis was readily detected in lymph nodes obtained from both HIV-negative and HIV-positive persons; however, the degree of apoptosis in lymph nodes obtained from HIV-positive persons was three to four times higher than that observed in the lymph nodes obtained from HIV-negative persons. In contrast to HIV-negative lymph nodes in which apoptosis was confined largely to germinal centers, in HIV-positive lymph nodes all functional compartments of the lymph node (i.e., cortex, paracortex, and sinuses) were extensively involved by this phenomenon. Furthermore, a significant correlation was observed between intensity of apoptosis and degree of activation of the lymphoid tissue associated with HIV infection. In contrast, intensity of apoptosis correlated neither with the clinical stage of HIV disease nor with the viral burden in the lymph node. Finally, apoptosis was not restricted only to CD4+ T cells; both B cells and CD8+ T cells were found to undergo apoptosis. Taken together, these results indicate that the increased intensity of the apoptotic phenomenon in HIV infection is caused by the general state of immune activation, and is independent of the progression of HIV disease and of the levels of viral load.

Acridine Orange↗
PubMed1996

p53-independent apoptosis during mammary tumor progression in C3(1)/SV40 large T antigen transgenic mice: suppression of apoptosis during the transition from preneoplasia to carcinoma.

Alterations in apoptosis and associated mechanisms during mammary tumor progression were investigated in transgenic mice expressing the SV40 large T antigen (T(AG)) driven by the rat prostatic steroid-binding protein C3(1) 5'-flanking region. Apoptosis levels, assessed by in situ end labeling, were low in normal mammary epithelial cells, highest in atypical hyperplasias (preneoplastic lesions), and less pronounced in adenocarcinomas. Preneoplastic cells maintain the ability to undergo apoptosis as a mechanism of tumor growth suppression, but this critical control of apoptosis is lost as these lesions progress to carcinomas. These alterations in apoptosis occur during mammary tumor progression in mice containing wild-type p53+/+ genotype as well as in mice with the p53-/- genotype. Thus, apoptosis in this tumor model occurs through a p53-independent mechanism. Because other studies have demonstrated p53-dependent apoptosis in T(AG)-induced choroid plexus tumors of transgenic mice, we propose that the role of p53 in apoptosis may be tissue-specific. In addition, bcl-2 protein was not expressed in any mammary lesions. SV40 T(AG) expression, which correlated with the nuclear p53 protein at all stages of tumor progression, was low in normal mammary epithelial cells, moderately high in atypical hyperplasias, and strongly expressed in adenocarcinomas. No p53 mutations were found at any stage of mammary adenocarcinoma development, suggesting that tumor progression does not require a dominantly acting p53 mutation in this transgenic model. p2l(Waf1/Cip1), a cyclin-dependent kinase inhibitor, was expressed in normal mammary tissue but was not detected in the mammary carcinomas, despite high nuclear accumulation of wild-type p53 protein, suggesting functional loss of p53 due to binding of SV40 T(AG), to p53. These findings suggest that suppression of apoptosis during the transition from atypical hyperplasia to adenocarcinoma appears to be a critical event for mammary cancer development in C3(1)/T(AG) transgenic mice and occurs by p53- and bcl-2-independent pathways.

Androgen-Binding Protein↗
PubMedDate not supplied

[Apoptosis and thymocyte development (epithelial cells as inducers of thymocyte apoptosis)].

Apoptosis, together with proliferation, is a main factor of selection of the clones of developing T-lymphocytes: the clones not supported by positive selection are subject to apoptosis and apoptosis accounts for discarding of potentially autoaggressive clones, i.e., for negative selection in the thymus and peripheral lymphoid tissue. Realization of apoptosis at different stages of the development of T-lymphocytes depends to a varying extent on Fas, Bcl-2, p53, and other regulators. The dendritic cells are the main cell type, the contact with determines apoptosis of T-lymphocytes. A possible role of the epithelial cells was shown in few models (on murine cells) and was not practically studied. We obtained a line of epithelial cells of the human thymus cells HTSC, cocultivation with which induces apoptosis of immature thymocytes and blood T-cells activated by mitogens. Development of apoptosis is suppressed by inhibitors of protein and RNA synthesis, chelators Ca2+, ions Zn2+, and factors destroying the cytoskeleton components. In this model, interaction of pairs of molecules CD4-HLA class II and LFA-1-ICAM-1. When in contact with the HTSC cells, the thymocytes of mice mutant for Fas-receptor (line MRL.lpr) are subject to apoptosis, but when this receptor is present, it affects the development of apoptosis.

Animals↗
PubMed2002

The involvement of oxidative stress in tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in HeLa cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves as an extracellular signal triggering apoptosis in tumor cells. However, the molecular mechanisms leading to the apoptosis are largely unknown. To characterize the molecular events involved in TRAIL-induced apoptosis, we examined the association of reactive oxygen species (ROS) in human adenocarcinoma HeLa cells. In this study, we show strong ROS accumulation upon TRAIL induction, with activation of caspases, followed by apoptosis. The pre-treatment with gamma-glutamylcysteinylglycine or estrogen, both effective antioxidants, significantly attenuated TRAIL-induced apoptosis through the reduction of ROS accumulation and diminished caspases activity. Furthermore, zVAD-fmk, an inhibitor of pan-caspase, effectively inhibited the activation of caspases and prevented apoptosis by TRAIL, although TRAIL-induced ROS generation was not attenuated. These data indicate that ROS may play a role as an upstream mediator of caspases. Taken together, our results suggest that oxidative stress mediates TRAIL-induced apoptosis in HeLa cells.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Identification of X-linked inhibitor of apoptosis-associated factor-1 as an interferon-stimulated gene that augments TRAIL Apo2L-induced apoptosis.

In the course of gene array studies aimed at identifying IFN-stimulated genes associated with interferon beta (IFN-beta)-induced apoptosis, we identified X-linked inhibitor of apoptosis-associated factor-1 (XAF1) as a novel IFN-stimulated gene. XAF1 mRNA was up-regulated by IFN-alpha and IFN-beta in all cells examined. However, IFNs induced high levels of XAF1 protein predominantly in cell lines sensitive to the proapoptotic effects of IFN-beta. In apoptosis-resistant cells including WM164 melanoma, WM35 melanoma, U937 pro-monocytic leukemia, and HT1080 fibrosarcoma cells, XAF1 mRNA was strongly up-regulated but XAF1 protein was up-regulated only weakly or not at all. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a critical mediator of IFN-beta-induced apoptosis, but most melanoma cell lines were resistant to recombinant TRAIL protein. For example, A375 melanoma cells were defective in TRAIL induction by IFN-beta and were resistant to TRAIL-induced apoptosis. However, IFN-beta pretreatment sensitized them to subsequent recombinant TRAIL-induced apoptosis. A375 cells expressing XAF1 constitutively were more sensitive to TRAIL-induced apoptosis compared with empty vector-transfected cells. The degree of sensitization by XAF1 was similar to that provided by IFN pretreatment and was correlated with the level of XAF1 expressed. Furthermore, the overexpression of the zinc-finger portion of XAF1 blocked IFN-dependent sensitization of A375 melanoma cells to the proapoptotic effects of TRAIL. These results suggested that IFN-dependent induction of XAF1 strongly influenced cellular sensitivity to the proapoptotic actions of TRAIL.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Upon drug-induced apoptosis in lymphoma cells X-linked inhibitor of apoptosis (XIAP) translocates from the cytosol to the nucleus.

The X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis-1 (cIAP-1) are emerging as versatile proteins in programmed cell death with a scope of possible functions reaching far beyond their well known inhibitory effects on caspases. We previously demonstrated that the ability of drugs to modify expression and cleavage of the IAPs are crucial for the synergistic effects achieved by the combinations of different cytotoxic drugs employed to treat malignant lymphomas. In order to more clearly assess the underlying molecular mechanisms, we here evaluated the consequences of drug-induced apoptosis on the localization and aggregation of XIAP and cIAP-1. The influence of drug-induced apoptosis on localization of IAPs was investigated using immunofluorescence microscopy as well as western blot analysis. Apoptosis was induced by chemotherapeutic drugs with different modes of action (bendamustine, cladribine, fludarabine, doxorubicin and mitoxantrone) and assessed by flow-cytometry using Annexin V. We demonstrate that XIAP and cIAP-1 are downregulated and/or cleaved in a dose-dependent manner upon treatment with a variety of anti-cancer drugs. Moreover we provide evidence that in the context of drug-induced apoptosis XIAP, its BIR3-RING cleavage product and cIAP-1 undergo an extensive change of subcellular localization. Immunofluorescence microscopy reveals that XIAP, in contrast to cIAP-1, is located in discrete cytosolic protein aggregates and-upon induction of apoptosis with cytotoxic drugs--redistributes into large nuclear inclusions. This translocation of XIAP and its BIR3-RING cleavage product from the cytosol into the nucleus is confirmed by cell fractionation and western blot analyses. Of note, in this experimental setting putative interaction partners of XIAP-such as Apaf-1, caspase-3 and -7--do not co-localize with XIAP. These results imply a new unknown function of XIAP and its BIR3-RING fragment in the nucleus in the context of drug-induced apoptosis. The localization of cIAP-1 in mitochondria and its liberation from these indicate a profoundly different function of this protein despite its similar modular structure to XIAP.

Antineoplastic Agents↗
PubMed2004

Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol.

Survivin is a member of the inhibitor of apoptosis proteins that is expressed at high levels in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Naturally occurring dietary compounds such as resveratrol have gained considerable attention as cancer chemopreventive agents. Here, we discovered a novel function of the chemopreventive agent resveratrol: resveratrol is a potent sensitizer of tumor cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through p53-independent induction of p21 and p21-mediated cell cycle arrest associated with survivin depletion. Concomitant analysis of cell cycle, survivin expression, and apoptosis revealed that resveratrol-induced G(1) arrest was associated with down-regulation of survivin expression and sensitization for TRAIL-induced apoptosis. Accordingly, G(1) arrest using the cell cycle inhibitor mimosine or induced by p21 overexpression reduced survivin expression and sensitized cells for TRAIL treatment. Likewise, resveratrol-mediated cell cycle arrest followed by survivin depletion and sensitization for TRAIL was impaired in p21- deficient cells. Also, down-regulation of survivin using survivin antisense oligonucleotides sensitized cells for TRAIL-induced apoptosis. Importantly, resveratrol sensitized various tumor cell lines, but not normal human fibroblasts, for apoptosis induced by death receptor ligation or anticancer drugs. Thus, this combined sensitizer (resveratrol)/inducer (e.g., TRAIL) strategy may be a novel approach to enhance the efficacy of TRAIL-based therapies in a variety of human cancers.

Anticarcinogenic Agents↗
PubMed2004

HPC1/RNASEL mediates apoptosis of prostate cancer cells treated with 2',5'-oligoadenylates, topoisomerase I inhibitors, and tumor necrosis factor-related apoptosis-inducing ligand.

The hereditary prostate cancer 1 (HPC1) allele maps to the RNASEL gene encoding a protein (RNase L) implicated in the antiviral activity of interferons. To investigate the possible role of RNase L in apoptosis of prostate cancer cells, we decreased levels of RNase L by severalfold in the DU145 human prostate cancer cell line through the stable expression of a small interfering RNA (siRNA). Control cells expressed siRNA with three mismatched nucleotides to the RNase L sequence. Cells deficient in RNase L, but not the control cells, were highly resistant to apoptosis by the RNase L activator, 2',5'-oligoadenylate (2-5A). Surprisingly, the RNase L-deficient cells were also highly resistant to apoptosis by combination treatments with a topoisomerase (Topo) I inhibitor (camptothecin, topotecan, or SN-38) and tumor necrosis factor-related apoptosis-inducing ligand [TRAIL (Apo2L)]. In contrast, cells expressing siRNA to the RNase L inhibitor RLI (HP68) showed enhanced apoptosis in response to Topo I inhibitor alone or in combination with TRAIL. An inhibitor of c-Jun NH(2)-terminal kinases reduced apoptosis induced by treatment with either 2-5A or the combination of camptothecin and TRAIL, thus implicating c-Jun NH(2)-terminal kinase in the apoptotic signaling pathway. Furthermore, prostate cancer cells were sensitive to apoptosis from the combination of 2-5A with either TRAIL or Topo I inhibitor, whereas normal prostate epithelial cells were partially resistant to apoptosis. These findings indicate that RNase L integrates and amplifies apoptotic signals generated during treatment of prostate cancer cells with 2-5A, Topo I inhibitors, and TRAIL.

Adenine Nucleotides↗
PubMed2005

Target cell-restricted apoptosis induction of acute leukemic T cells by a recombinant tumor necrosis factor-related apoptosis-inducing ligand fusion protein with specificity for human CD7.

Current treatment of human T-cell leukemia and lymphoma is predominantly limited to conventional cytotoxic therapy and is associated with limited therapeutic response and significant morbidity. Therefore, more potent and leukemia-specific therapies with favorable toxicity profiles are urgently needed. Here, we report on the construction of a novel therapeutic fusion protein, scFvCD7:sTRAIL, designed to induce target antigen-restricted apoptosis in human T-cell tumors. ScFvCD7:sTRAIL consists of the death-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to an scFv antibody fragment specific for the T-cell surface antigen CD7. Treatment with scFvCD7:sTRAIL induced potent CD7-restricted apoptosis in a series of malignant T-cell lines, whereas normal resting leukocytes, activated T cells, and vascular endothelial cells (human umbilical vein endothelial cells) showed no detectable apoptosis. The apoptosis-inducing activity of scFvCD7:sTRAIL was stronger than that of the immunotoxin scFvCD7:ETA. In mixed culture experiments with CD7-positive and CD7-negative tumor cells, scFvCD7:sTRAIL induced very potent bystander apoptosis of CD7-negative tumor cells. In vitro treatment of blood cells freshly derived from T-acute lymphoblastic leukemia patients resulted in marked apoptosis of the malignant T cells that was strongly augmented by vincristin. In conclusion, scFvCD7:sTRAIL is a novel recombinant protein causing restricted apoptosis in human leukemic T cells with low toxicity for normal human blood and endothelial cells.

Animals↗
PubMed2006

Human melanoma cells selected for resistance to apoptosis by prolonged exposure to tumor necrosis factor-related apoptosis-inducing ligand are more vulnerable to necrotic cell death induced by cisplatin.

PURPOSE: Heterogeneous sensitivity of melanoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis may lead to outgrowth of TRAIL-resistant cells and limit successful treatment by TRAIL. The present study aims to better understand the biological characteristics of melanoma cells resistant to TRAIL-induced apoptosis. EXPERIMENTAL DESIGN: We generated TRAIL-resistant melanoma cells by prolonged exposure to TRAIL and characterized the cells in terms of their sensitivity to killing induced by a panel of cytotoxic agents using biological and biochemical methods. RESULTS: TRAIL-resistant melanoma cells are cross-resistant to apoptosis induced by another death ligand FasL, the DNA-damaging agent cisplatin, the histone deacetylase inhibitor suberic bishydroxamate, and the antimicrotubule Vinca alkaloid, vincristine. The apoptotic signaling seemed to be inhibited upstream of mitochondrial apoptotic events and was associated with decreased expression of multiple apoptotic mediators, including pro-caspase-8, Fas-associated death domain, Bid, Bim, p53, and the products of its proapoptotic target genes. Despite being resistant to apoptosis, TRAIL-resistant melanoma cells were more vulnerable to cisplatin-induced nonapoptotic cell death. This was characterized by lack of DNA fragmentation, delayed externalization of phosphatidylserine, caspase and p53 independence, and severe mitochondrial disruption, and was preceded by poly(ADP)ribose polymerase (PARP) activation and depletion of intracellular ATP, indicative of necrotic cell death. Inhibition of PARP activity partially converted the mode of cell death from necrosis to apoptosis. CONCLUSIONS: TRAIL-resistant melanoma cells are cross-resistant to apoptosis induced by various apoptotic stimuli but are more sensitive to nonapoptotic cell death induced by cisplatin. Exploration of chemotherapy-induced nonapoptotic cell death may provide an alternative strategy in overcoming resistance of melanoma cells to TRAIL-induced apoptosis.

Antineoplastic Agents↗
PubMed2006

Sequential combination of flavopiridol and docetaxel reduces the levels of X-linked inhibitor of apoptosis and AKT proteins and stimulates apoptosis in human LNCaP prostate cancer cells.

Clinical trials have shown that chemotherapy with docetaxel combined with prednisone can improve survival of patients with androgen-independent prostate cancer. It is likely that the combination of docetaxel with other novel chemotherapeutic agents would also improve the survival of androgen-independent prostate cancer patients. We investigated whether the combination of docetaxel and flavopiridol, a broad cyclin-dependent kinase inhibitor, can increase apoptotic cell death in prostate cancer cells. Treatment of DU 145 prostate cancer cells with 500 nmol/L flavopiridol and 10 nmol/L docetaxel inhibited apoptosis probably because of their opposing effects on cyclin B1-dependent kinase activity. In contrast, when LNCaP prostate cancer cells were treated with flavopiridol for 24 hours followed by docetaxel for another 24 hours (FD), there was a maximal induction of apoptosis. However, there was greater induction of apoptosis in DU 145 cells when docetaxel was followed by flavopiridol or docetaxel. These findings indicate a heterogeneous response depending on the type of prostate cancer cell. Substantial decreases in X-linked inhibitor of apoptosis (XIAP) protein but not survivin, both being members of the IAP family, were required for FD enhanced apoptosis in LNCaP cells. Androgen ablation in androgen-independent LNCaP cells increased activated AKT and chemoresistance to apoptosis after treatment with FD. The proteasome inhibitor MG-132 blocked FD-mediated reduction of XIAP and AKT and antagonized apoptosis, suggesting that the activation of the proteasome pathway is one of the mechanisms involved. Overall, our data suggest that the docetaxel and flavopiridol combination requires a maximal effect on cyclin B1-dependent kinase activity and a reduction of XIAP and AKT prosurvival proteins for augmentation of apoptosis in LNCaP cells.

Antineoplastic Combined Chemotherapy Protocols↗