calpain and Burkitt-Lymphoma

We have recently reported that the cytokine interferon-alpha (IFNalpha), commonly used in the treatment of cancer, induced a caspase-dependent apoptosis in tumor cell lines. The signaling mechanisms involved have not been defined. Here, we show that both proapoptotic Bcl-2 family members Bak and Bax were activated by IFNalpha, strictly in correlation with the induction of apoptosis. Using double stainings, we demonstrated that Bak was activated prior to cytochrome c (cyt c) release and caspase-3 activation, whereas activated Bax was only found in cells with released cyt c, mitochondrial depolarization, as well as activated caspase-3. Furthermore, IFNalpha-induced activation of Bak, and to a large extent also of Bax, was dependent on caspase activity. With the use of a panel of specific caspase inhibitors we found, however, that none of caspases-1 to -10 were responsible for this activation. Neither was the Ca(2+)-dependent protease calpain nor the stress-activated p38 SAPK pathway significantly involved. Overexpression of Bcl-2 blocked apoptosis induced by IFNalpha totally abolished Bak activation, as well as decreased the amount of activated Bax. We conclude that IFNalpha induces Bak and Bax activation via distinct mechanisms involving an unknown protease, and that their activation is regulated by Bcl-2.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Burkitt Lymphoma; Calpain; Carrier Proteins; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Inhibitors; Humans; Interferon-alpha; Membrane Potentials; Membrane Proteins; Mitochondria; Mitogen-Activated Protein Kinases; Multiple Myeloma; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

The molecular events involved in apoptosis induced by ionizing radiation remain unresolved. In this paper we show that the cleavage of fodrin to a 150 kDa fragment is an early proteolytic event in radiation-induced apoptosis in the Burkitts' Lymphoma cell line BL30A and requires 100 microM zVAD-fmk for inhibition. Caspases-1, -3, -6 and -7 were shown to cleave fodrin to the 150 kDa fragment in vitro and all were inhibited by 10 microM zVAD-fmk. We also show that the in vitro cleavage of fodrin by calpain is inhibited by 100 microM zVAD-fmk as was the calpain-mediated hydrolysis of casein. We demonstrate that calpain is activated within 15 min after radiation exposure, concomitant with the cleavage of fodrin to the 150 kDa fragment whereas caspase-3 is activated at 2 h correlating with the cleavage of fodrin to the 120 kDa fragment. These results support a role for calpain in the early phases of the radiation-induced apoptosis pathway, upstream of the caspases.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Apoptosis; Burkitt Lymphoma; Calpain; Carrier Proteins; Caspase 1; Caspase 3; Caspase 6; Caspase 7; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Activation; fas Receptor; Humans; Membrane Proteins; Microfilament Proteins; Molecular Sequence Data; Peptide Fragments; Tumor Cells, Cultured

The c-Fos and c-Jun transcription factors are rapidly turned over in vivo. One of the multiple pathways responsible for their breakdown is probably initiated by calpains, which are cytoplasmic calcium-dependent cysteine proteases. The c-fos gene has been transduced by two murine oncogenic retroviruses called Finkel-Biskis-Jenkins murine sarcoma virus (FBJ-MSV) and Finkel-Biskis-Reilly murine sarcoma virus (FBR-MSV); c-jun has been transduced by the chicken avian sarcoma virus 17 (ASV17) retrovirus. Using an in vitro degradation assay, we show that the mutated v-FosFBR, but not v-FosFBJ or v-JunASV17, is resistant to calpains. This property raises the interesting possibility that decreased sensitivity to calpains might contribute to the tumorigenic potential of FBR-MSV by allowing greater accumulation of the protein that it encodes in infected cells. It has also been demonstrated that resistance to cleavage by calpains does not result from mutations that have accumulated in the Fos moiety of the viral protein but rather from the addition of atypical peptide motifs at its both ends. This observation raises the interesting possibility that homologous regions in viral and cellular Fos either display slightly different conformations or are differentially accessible to interacting proteins.

Topics: Avian Sarcoma Viruses; Burkitt Lymphoma; Calcium; Calpain; Genes, fos; Genes, jun; Humans; Leukemia-Lymphoma, Adult T-Cell; Mutagenesis, Site-Directed; Neoplasm Proteins; Oncogene Protein p65(gag-jun); Oncogene Proteins v-fos; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Sarcoma Viruses, Murine; Sequence Deletion; Substrate Specificity; Tumor Cells, Cultured

In situ cytogenetic morphology and analysis showed that Epstein-Barr Virus (EBV)-infected Raji and EBV-producing P3HR-1 cells divide by meiosis and follow the life cycle of malignant cells in vitro. Meiosis was documented by the presence of condensed chromosomes, "o" chromosome, nuclear vlimata (NVs), NV invasion, extrusion of chromosomes, chromosomal transfer, metaphase fusion and aneuploidy. EBV-Raji, EBV-producing P3HR, HIV1-infected MOLT-4 cells (dividing by meiosis) and human spermatozoa (cellular products of meiosis) were highly sensitive to the endogenous inhibitor (CANP-I) of calcium-activated neutral proteinase (CANP). CANP-I-treated virally infected and virus producing cells showed necrosis and disappearance of immunofluorescent viral antigens, documenting the anti-viral action of CANP-I. CANP-I-treated spermatozoa exhibited arrested motility with subsequent necrosis documenting the in vitro spermicidal action of CANP-I. The testis of treated Wistar rats with 0.25 U CANP-I/gr body weight for six days, were devoid of spermatozoa, with pronounced toxicity and exfoliation of spermatocytes and spermatids, indicating the contraceptive action of CANP-I via spermatogenetic arrest. It is concluded that CANP-I is an anti-meiotic agent inhibiting CANP associated with meiosis. Hence CANP-I is a promising agent against various diseases involving meiosis.

Topics: Animals; Antigens, Viral; Antiviral Agents; Burkitt Lymphoma; Calpain; Cell Line; Chromosomes; Herpesvirus 4, Human; Humans; In Vitro Techniques; Male; Meiosis; Rats; Rats, Wistar; Spermatocidal Agents; Spermatogenesis; Spermatozoa; Testis