benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Osteosarcoma

Osteosarcoma is the predominant form of primary bone malignancy. Although the combinational application of neoadjuvant chemotherapy and surgical resection significantly increases the survival rate, the therapeutic outcome remains unsatisfactory. Deoxyelephantopin (DET), an active ingredient of Elephantopus scaber, has been reported to have an anti-tumor effect in recent publications. This study aimed to investigate whether DET has antineoplastic effects on osteosarcoma cells and its underlying mechanism.. Cell viability and morphological changes were assessed by MTT and Live/dead assays. Cell apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were detected utilizing Annexin V-FITC/PI double staining, DCFH-DA and JC-1 probes, respectively. Autophagy was detected by mRFP-GFP-LC3 adenovirus transfection and western blot.. DET dose-dependently reduced the viability of osteosarcoma cells following the increase in intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) reversed this effect. Furthermore, DET induced mitochondrial apoptosis. Depolarized cells were increased, and apoptosis-related proteins, such as Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3 and cleaved ploy ADP-ribose polymerase, were activated. Additionally, we found that DET could induce autophagy in osteosarcoma cells, but autophagy inhibition did not affect the decrease in cell viability.. DET induced apoptosis in osteosarcoma cells through ROS generation, mitochondrial dysfunction and caspase activation; in addition, autophagy was involved in the effects of DET on osteosarcoma cells.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Bone Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Humans; Lactones; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Reactive Oxygen Species; Sesquiterpenes

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Caspase Inhibitors; Cell Line, Tumor; Cyclosporine; Dose-Response Relationship, Drug; Humans; Imidazoles; Indoles; Microscopy, Electron, Transmission; Necrosis; Oligopeptides; Osteosarcoma; Time Factors

The mechanism of the cytotoxic effect exerted by parthenolide on tumor cells is not clearly defined today. This article shows that parthenolide stimulates in human osteosarcoma MG63 and melanoma SK-MEL-28 cells a mechanism of cell death, which is not prevented by z-VAD-fmk and other caspase inhibitors. In particular treatment with parthenolide rapidly stimulated (1-2 h) reactive oxygen species (ROS) generation by inducing activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, c-Jun N-terminal kinase (JNK) activation, cell detachment from the matrix, and cellular shrinkage. The increase of ROS generation together with the mitochondrial accumulation of Ca(2+) also favored dissipation of Δψm, which seemed primarily determined by permeability transition pore opening, since Δψm loss was partially prevented by the inhibitor cyclosporin A. Staining with Hoechst 33342 revealed in most cells, at 3-5 h of treatment, chromatin condensation, and fragmentation, while only few cells were propidium iodide (PI)-positive. In addition, at this stage apoptosis inducing factor (AIF) translocated to the nucleus and co-localized with areas of condensed chromatin. Prolonging the treatment (5-15 h) ATP content declined while PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by N-acetylcysteine, while caspase inhibitors were ineffective. We suggest that AIF exerts a crucial role in parthenolide action. In accordance, down-regulation of AIF markedly inhibited parthenolide effect on the production of cells with apoptotic or necrotic signs. Taken together our results demonstrate that parthenolide causes in the two cell lines a caspase-independent cell death, which is mediated by AIF.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis Inducing Factor; Caspase Inhibitors; Caspases; Cell Death; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Melanoma; NADPH Oxidases; NF-kappa B; Osteosarcoma; Reactive Oxygen Species; Sesquiterpenes

Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Chromatin; Cytidine; DNA; DNA Methylation; Dogs; Drug Screening Assays, Antitumor; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Osteosarcoma; Vorinostat

Grifolin, a natural biologically active substance isolated from the edible bodies of the mushroom Albatrellus confluens, has been shown to inhibit proliferation and induce apoptosis in several cancer cell lines. But the mechanisms remain poorly understood. In this study, we investigated the apoptosis-inducing effects and the mechanisms of grifolin on human osteosarcoma cells. Our results demonstrated that grifolin induced concentration- and time-dependent suppression of proliferation and induction of apoptosis in U2OS and MG63 osteosarcoma cell lines. Grifolin induced the release of cytochrome c accompanied by activation of caspase-9, caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, z-VAD-fmk, a universal inhibitor of caspases, prevented caspase-3 activation and PARP cleavage and inhibted grifolin-induced cell growth inhibition. Furthermore, grifolin treatment resulted in a reduction in level of phosphorylated AKT, FOXO transcription factor, and glycogen synthase kinase 3 (GSK3). Knockdown of GSK3 with siRNA inhibited the apoptotic effects of grifolin. On the other hand, grifolin treatment down-regulated the expression of the inhibitor of apoptosis protein(IAP) in both osteosarcoma cells. Taken together, our results suggested that grifolin is able to suppress the phosphorylation of Akt and its substrates FOXO transcription factor and GSK3 in osteosarcoma cells causing the suppression of proliferation and induction of mitochondria- and caspase-dependent apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Inhibitors; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; Mitochondria; Oncogene Protein v-akt; Osteosarcoma; Phosphatidylinositol 3-Kinases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; RNA, Small Interfering; Signal Transduction; Terpenes

Prostate cancer is one of the most common cancers in men, with more than 500,000 new worldwide cases reported annually, resulting in 200,000 deaths of mainly older men in developed countries. Existing treatments have not proved very effective in managing prostate cancer, and continuing efforts therefore are ongoing to explore novel targets and strategies for future therapies. LAPSER1 has been identified as a candidate tumor suppressor gene in prostate cancer, but its true functions remain unknown. We report here that LAPSER1 colocalizes to the centrosomes and midbodies in mitotic cells with gamma-tubulin, MKLP1, and p80 katanin, and is involved in cytokinesis. Moreover, RNAi-mediated disruption of LAPSER1, which is accompanied by the mislocalization of p80 katanin, results in malformation of the central spindle. Significantly, the enhanced expression of LAPSER1 induces binucleation and renders the cells resistant to oncogenic transformation. In cells transformed by the v-Fps oncogene, overexpressed LAPSER1 induces abortive cytokinesis, followed by mitotic catastrophe in a p80 katanin-dependent manner. Cells that are rescued from this apoptotic pathway with Z-VAD-fmk display karyokinesis. These results suggest that LAPSER1 participates in cytokinesis by interacting with p80 katanin, the disruption of which may potentially cause genetic instability and cancer.

Topics: Adenocarcinoma; Adenosine Triphosphatases; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bone Neoplasms; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Centrosome; CHO Cells; Cricetinae; Cricetulus; Cytokinesis; Fusion Proteins, gag-onc; Genes, Tumor Suppressor; Humans; Katanin; Leucine Zippers; Male; Membrane Proteins; Microtubule-Associated Proteins; Oncogene Protein p21(ras); Oncogene Proteins v-abl; Osteosarcoma; Polyploidy; Prostatic Neoplasms; Protein Subunits; Protein-Tyrosine Kinases; Rats; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering; Spindle Apparatus; Subcellular Fractions; Tubulin; Tumor Suppressor Proteins

The type of cell death occurring in the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as three-dimensional spheroids after exposure to 5 Gy of ionizing radiation was determined. Morphological analyses using the chromatin dye Hoechst 33258 demonstrated that spheroids showed the typical characteristics of apoptosis, while monolayer cells revealed those typical of mitotic catastrophe. In order to better characterize these two types of cell death, the role of caspases was examined in irradiated monolayer cells and spheroids using the broad spectrum caspase inhibitor zVAD-fmk. Death in monolayer cells was caspase-independent, whereas spheroid death was characterized by caspase dependence. Members of the Bcl-2 family of proteins and survivin involved in cell death processes were also studied by Western blot analysis. The pro-apoptotic protein Bax increased in spheroids, whereas this protein remained unchanged in monolayer cells after the same 5-Gy irradiation. The anti-apoptotic protein Bcl-2, on the other hand, remained unchanged in both monolayer cells and spheroids. Finally, survivin increased significantly after irradiation in both cells in monolayer and spheroids. The results presented suggest that three-dimensional cell organization leads to a different type of cell death after exposure to ionizing radiation. Thus, the use of spheroids, a cell model which mimics in vivo solid tumors more closely than cells grown in monolayer, is more appropriate when investigating the effects of antineoplastic treatments such as ionizing radiation.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Bisbenzimidazole; Blotting, Western; Bone Neoplasms; Caspase 3; Cell Line, Tumor; Cell Survival; Humans; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Mitosis; Neoplasm Proteins; Osteosarcoma; Proto-Oncogene Proteins c-bcl-2; Response Elements; Spheroids, Cellular; Survivin

Development of chemotherapy resistance and evasion from apoptosis in osteosarcoma, a primary malignant bone tumor, is often correlated with constitutive nuclear factor-kappaB (NF-kappaB) activation. Here, we investigated the ability of a polyphenolic fraction of green tea (GTP) that has been shown to have antitumor effects on various malignant cell lines to inhibit growth and induce apoptosis in human osteosarcoma SAOS-2 cells. Treatment of SAOS-2 cells with GTP (20-60 microg/ml) resulted in reduced cell proliferation and induction of apoptosis, which correlated with decreased nuclear DNA binding of NF-kappaB/p65 and lowering of NF-kappaB/p65 and p50 levels in the cytoplasm and nucleus. GTP treatment of cells reduced IkappaB-alpha phosphorylation but had no effect on its protein expression. Furthermore, GTP treatment resulted in the inhibition of IKK-alpha and IKK-beta, the upstream kinases that phosphorylate IkappaB-alpha. The increase in apoptosis in SAOS-2 cells was accompanied with decrease in the protein expression of Bcl-2 and concomitant increase in the levels of Bax. GTP treatment of SAOS-2 cells also resulted in significant activation of caspases as was evident by increased levels of cleaved caspase-3 and caspase-8 in these cells. Treatment of SAOS-2 cells with a specific caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) and general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) rescued SAOS-2 cells from GTP-induced apoptosis. Taken together, these results indicate that GTP is a candidate therapeutic for osteosarcoma that mediates its antiproliferative and apoptotic effects via activation of caspases and inhibition of NF-kappaB.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Electrophoretic Mobility Shift Assay; Flavonoids; Humans; I-kappa B Kinase; I-kappa B Proteins; Immunoblotting; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Oligopeptides; Osteosarcoma; Phenols; Phosphorylation; Poly(ADP-ribose) Polymerases; Polyphenols; Tea; Transcription Factor RelA

The purpose of this study was to evaluate the anti-tumor effects of osteosarcoma (HOSM-1) cells via transfer of the Bax gene using a cationic liposome. We evaluated the levels of Bax, Bcl-xL, Bcl-2 and cytochrome c expression by Western blot analysis, and caspase-9 and -3 activities were determined in a colorimetric assay. Apoptosis was detected using a TUNEL assay, and cell growth inhibition was determined in an MTT assay. Following Bax gene transfer, release of cytochrome c to the cytosol was detected, the activities of caspase-9 and -3 increased, and TUNEL-positive cells (37.5%) were detected. Cell survival rate was 50.8% under these conditions. Induction of apoptosis was inhibited by a caspase inhibitor (zVAD-fmk), but only a slight increase in cell survival rate occurred. Hence, since not only apoptosis but also caspase-independent cell death is induced in HOSM-1 cells, we anticipate that Bax gene therapy with cationic liposomes will be useful for osteosarcoma.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cations; Cell Line, Tumor; Cell Survival; Humans; In Situ Nick-End Labeling; Liposomes; Osteosarcoma; Transfection

Paclitaxel has been found to exhibit cytotoxic and antitumor activity. There is little information regarding the mechanisms of apoptotic-inducing effect of paclitaxel on human osteogenic sarcoma U-2 OS cells. Several key regulatory proteins are involved in the initiation of apoptosis. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. We examined the effect of paclitaxel on the cell cycle arrest and apoptosis in U-2 OS cells using flow cytometric analysis and Western blotting. We also measured the inhibition of paclitaxel-induced apoptosis and the caspase-3 activity by the broad-spectrum caspase inhibitor z-VAD-fmk on U-2 OS cells. The increased levels of casapse-3 were also confirmed by cDNA microarray. Our observations were: (1) paclitaxel treatment resulted in G2/M-cycle arrest in U-2 OS cells; (2) time and dose dependent apoptosis of U-2 OS cells was induced by paclitaxel; (3) in U-2 OS cells, z-VAD-fmk blocked the paclitaxel-induced apoptosis and caspase-3 activation. These results suggest that paclitaxel-induced G2/M-cycle arrest of the G2/M phase and apoptosis via a caspase-3 pathway in U-2 OS cells.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Bone Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Osteosarcoma; Paclitaxel

deltaNp63 is overexpressed in squamous carcinomas where it is associated with proliferation and is believed to enhance cell growth by blocking p53-mediated transactivation. In normal epithelium, deltaNp63alpha protein expression is abundant in basal cells and decreases with differentiation. To explore the biological consequences of deltaNp63alpha overexpression in relation to squamous carcinogenesis, we evaluated its effect on normal squamous differentiation and p53 transactivation function in keratinocytes. Forced overexpression of deltaNp63alpha in primary murine keratinocytes in vitro inhibits morphological differentiation induced by elevated extracellular [Ca(2+)], abrogates Ca(2)(+)-induced growth arrest, and blocks expression of maturation-specific proteins keratin 10 and filaggrin. This suggests that deltaNp63 overexpression in squamous carcinomas may serve to maintain the basal cell phenotype and promote cell survival. deltaNp63alpha blocks transactivation of p53 responsive reporter constructs mediated by endogenous or exogenous p53 at 17 h postinfection, as expected. However, at 41 h, when p53-mediated transactivation is diminished, deltaNp63alpha enhances transactivation of these reporter constructs by 2.2-12-fold over control. Maximal deltaNp63alpha-induced transactivation requires intact p53 responsive elements, but is independent of cellular p53 status. This positive transcriptional function of deltaNp63alpha appears to be cell-type specific, as it is not observed in primary dermal fibroblasts or Saos-2 cells. These findings support deltaNp63alpha as a master regulator of keratinocyte differentiation, and suggest a novel function of this protein in the maintenance of epithelial homeostasis.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Animals; Binding Sites; Bone Neoplasms; Calcium; Cell Differentiation; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Fibroblasts; Filaggrin Proteins; Genes, Reporter; Genes, Tumor Suppressor; Humans; Keratinocytes; Mice; Mice, Inbred C57BL; Organ Specificity; Osteosarcoma; Phosphoproteins; Protein Isoforms; Recombinant Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Leupeptins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Multienzyme Complexes; Osteosarcoma; Protease Inhibitors; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Retinoblastoma Protein; Time Factors; Transfection; Tumor Suppressor Protein p53

Three prevalent mitochondrial DNA pathogenic mutations at positions 11778, 3460, and 14484, which affect different subunits of Complex I, cause retinal ganglion cell death and optic nerve atrophy in Leber's hereditary optic neuropathy (LHON). The cell death is painless and without inflammation, consistent with an apoptotic mechanism. We have investigated the possibility that the LHON mutation confers a pro-apoptotic stimulus and have tested the sensitivity of osteosarcoma-derived cybrid cells carrying the most common and severe mutations (11778 and 3460) to cell death induced by Fas. We observed that LHON cybrids were sensitized to Fas-dependent death. Control cells that bear the same mitochondrial genetic background (the J haplogroup) without the pathogenic 11778 mutation are no more sensitive than other controls, indicating that increased Fas-dependent death in LHON cybrids was induced by the LHON pathogenic mutations. The type of death was apoptotic by several criteria, including induction by Fas, inhibition by the caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methyl ketone), activation of DEVDase activity (Asp-Glu-Val-Asp protease), specific cleavage of caspase-3, DNA fragmentation, and increased Annexin-V labeling. These data indicate that the most common and severe LHON pathogenic mutations 11778 and 3460 predispose cells to apoptosis, which may be relevant for the pathophysiology of cell death in LHON, and potential therapy.

Topics: Amino Acid Chloromethyl Ketones; Antibodies; Antigens, CD; Apoptosis; Cell Culture Techniques; Cell Death; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; DNA, Mitochondrial; fas Receptor; Humans; Kinetics; Mutation; Optic Atrophy, Hereditary, Leber; Osteosarcoma; Protein Subunits; Retinal Ganglion Cells; Tumor Cells, Cultured

Insulin-like growth factor-I exerts potent mitogenic effects through the type I IGF receptor, a member of the insulin receptor family, and exhibits at the same time some insulin-like metabolic activities. We have questioned whether IGF-I presents moreover a modulatory effect upon programmed cell death (PCD)(apoptosis) in serum-deprived human osteosarcoma U-2 OS cells, a cell line synthesizing IGF-II and exhibiting an increased DNA synthesis following treatment with IGF-I. U-2 OS cells were cultured in a medium containing 0.8% FCS and growth arrest was induced by transfer to serum-free growth conditions. PCD was measured using a commercially available DNA degradation ELISA while viable cell numbers were counted microscopically after trypan exclusion to estimate net proliferative activity. Following serum withdrawal for 24 hrs., the level of PCD in U-2 OS cells was increased six-fold while cell number was reduced by approximately 35% compared to cells grown in the presence of 15% serum. Incubation with recombinant human IGF-I for 24 hrs. caused a dose-dependent inhibition of the level of programmed cell death. Co-incubation with an IGF-I receptor monoclonal antibody (alphaIR3) dose-dependently blocked the effects of 10 ng/ml IGF-I on PCD, with an ED50 of 1-10 ng/ml of alphaIR3 immunoglobulin. Conversely IGF-1 provoked a significant cell number increase, an effect blocked by addition of alphaIR3. The addition of an inhibitor of caspase 1 (ICE) had little effect on PCD but resulted in a net increase in the number of viable cells. In summary, IGF-I treatment of U-2 OS cells at the same time inhibits the induced programmed cell death and increases the cell number, effects which are blocked by addition of IGF-I receptor antibodies. These data support the hypothesis that IGF-I affects cells in a dual way, both by enhancing proliferative responses and by suppressing programmed cell death. The differential response between PCD and cell number to ICE inhibitors suggests the existence of independent control systems for these processes although the role of IGF-I in this study has yet to be determined.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Cell Count; Culture Media, Serum-Free; Enzyme Inhibitors; Humans; Insulin-Like Growth Factor I; Osteosarcoma; Receptor, IGF Type 1; Tumor Cells, Cultured

Cell death processes are progressively inactivated during malignant development, in part by loss of tumor suppressors that can promote cell death. The Bin1 gene encodes a nucleocytosolic adaptor protein with tumor suppressor properties, initially identified through its ability to interact with and inhibit malignant transformation by c-Myc and other oncogenes. Bin1 is frequently missing or functionally inactivated in breast and prostate cancers and in melanoma. In this study, we show that Bin1 engages a caspase-independent cell death process similar to type II apoptosis, characterized by cell shrinkage, substratum detachment, vacuolated cytoplasm, and DNA degradation. Cell death induction was relieved by mutation of the BAR domain, a putative effector domain, or by a missplicing event that occurs in melanoma and inactivates suppressor activity. Cells in all phases of the cell cycle were susceptible to death and p53 and Rb were dispensable. Notably, Bin1 did not activate caspases and the broad spectrum caspase inhibitor ZVAD.fmk did not block cell death. Consistent with the lack of caspase involvement, dying cells lacked nucleosomal DNA cleavage and nuclear lamina degradation. Moreover, neither Bcl-2 or dominant inhibition of the Fas pathway had any effect. In previous work, we showed that Bin1 could not suppress cell transformation by SV40 large T antigen. Consistent with this finding, we observed that T antigen suppressed the death program engaged by Bin1. This observation was interesting in light of emerging evidence that T antigen has roles in cell immortalization and human cell transformation beyond Rb and p53 inactivation. In support of a link to c-Myc-induced death processes, AEBSF, a serine protease inhibitor that inhibits apoptosis by c-Myc, potently suppressed DNA degradation by Bin1. Our findings suggest that the tumor suppressor activity of Bin1 reflects engagement of a unique cell death program. We propose that loss of Bin1 may promote malignancy by blunting death penalties associated with oncogene activation.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Antigens, Polyomavirus Transforming; Apoptosis; Bone Neoplasms; Carcinoma, Hepatocellular; Carrier Proteins; Caspases; Cell Adhesion; Cell Size; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; DNA Fragmentation; Enzyme Activation; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mitochondria; Nuclear Proteins; Osteosarcoma; Protein Structure, Tertiary; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Recombinant Fusion Proteins; Retinoblastoma Protein; Serine Proteinase Inhibitors; Sulfones; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins