benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Cell-Transformation--Viral

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

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase Inhibitors; Caspases; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytochromes c; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Etoposide; Fibroblasts; Kinetics; Mitochondria; Nucleic Acid Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction; Staurosporine; Temperature; Tumor Suppressor Protein p53

The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation.

Topics: 3C Viral Proteases; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Transformation, Viral; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; HeLa Cells; Humans; Microscopy, Electron; Poliovirus; Viral Proteins

Epstein-Barr virus (EBV) transforms B lymphocytes into lymphoblastoid cell lines usurping the Notch and tumor necrosis factor receptor pathways to effect transcription including NF-kappaB activation. To determine whether NF-kappaB activity is essential in the growth and survival of EBV-transformed lymphoblastoid cell lines, a nondegradable IkappaBalpha mutant was expressed under tetracycline regulation. Despite continued Bcl-2 and Bcl-x/L expression, NF-kappaB inhibition induced apoptosis as evidenced by poly(ADP-ribose) polymerase cleavage, nuclear condensation and fragmentation, and hypodiploid DNA content. Both caspase 3 and 8 activation and loss of mitochondrial membrane potential were observed in apoptotic cells. However, caspase inhibition failed to block apoptosis. These experiments indicate that NF-kappaB inhibitors may be useful in the therapy of EBV-induced cellular proliferation.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; B-Lymphocytes; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Transformed; Cell Transformation, Viral; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Drug Design; Flow Cytometry; Gene Expression Regulation; Herpesvirus 4, Human; Humans; I-kappa B Proteins; Intracellular Membranes; Lymphoproliferative Disorders; Membrane Potentials; Microscopy, Confocal; Minor Histocompatibility Antigens; Mitochondria; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Transfection