ABSTRACT
The role of p53 in apoptosis and the contrasting p53 status in tumors prompted us to investigate the bleomycin-induced apoptosis in p53-null human leukemia HL-60 cells (bleomycin at 160 microM for 7.5 h). Cells with apoptotic phenotype increased from 0.87% in controls to 9.40% in bleomycin-treated cells. Both the enzymes, caspase-3 and -8, were activated. Furthermore, the apoptotic phenotypes totally disappeared with zVAD-fmk, a caspase inhibitor. Besides, cytochrome c release from mitochondria happened simultaneously to apoptotic phenotypes, shrinkage of mitochondria but being independent of the mitochondrial permeability transition, since cyclosporine A and bongkrekic acid were inefficient on induced apoptosis. On the other hand, incubations with bleomycin (BLM) did not result in detectable changes in the expression of Bcl-2- and Bax-mRNA neither Bcl-2- or Bax-proteins. In conclusion, we suggest that BLM can produce apoptosis independently of p53 through three mechanisms: i) at the nuclear level by its endonuclease activities; ii) at the cell membrane, by activating caspases; and iii) at the mitochondria by releasing cytochrome c. These results indicate that BLM-induced apoptosis in HL-60 cells results from the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.
Subject(s)
Apoptosis , Bleomycin/pharmacology , Genes, p53 , Leukemia/drug therapy , Leukemia/pathology , Amino Acid Chloromethyl Ketones/pharmacology , Antimetabolites, Antineoplastic/pharmacology , Blotting, Western , Bongkrekic Acid/pharmacology , Caspase 3 , Caspase 8 , Caspases/biosynthesis , Caspases/metabolism , Cell Line , Cyclosporine/pharmacology , Cytochromes c/metabolism , DNA Fragmentation , Enzyme Activation , HL-60 Cells , Humans , Microscopy, Electron , Mitochondria/pathology , Phenotype , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X ProteinABSTRACT
The subunit composition of nicotinic acetylcholine receptors involved in apoptosis is an ongoing question. HL-60 cells were used in order to investigate the implication of nicotinic acetylcholine receptors in bleomycin-induced apoptosis. We found that bleomycin-induced apoptosis was significantly enhanced by nicotine and was blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha 7 nicotinic receptor. Among the other agonists tested, 3-[2,4-dimethoxybenzylidene]anabaseine (GTS-21)-selective agonist for alpha 7-nicotinic acetylcholine receptor-, but not epibatidine or cytisine, enhanced bleomycin-induced apoptosis. In addition to these results, the detectable presence of alpha 7-mRNA supports a key role of alpha 7-nicotinic acetylcholine receptors in the modulation of the induced apoptosis by nicotine.
Subject(s)
Apoptosis/physiology , Receptors, Nicotinic/physiology , Apoptosis/drug effects , Dose-Response Relationship, Drug , HL-60 Cells , Humans , Nicotine/pharmacologyABSTRACT
The Bleomycin (BLM)-induced apoptosis in peripheral blood mononuclear cells (PBMC), from patients with advanced squamous cell carcinoma of the head and neck (SCCHN), cultured in vitro with PHA, was tested as a predictive indicator of the survival time. The rates of the PBMC BLM-induced apoptosis and of the cycling-PBMCs, measured respectively after Giemsa- and Feulgen-staining were determined in 25 patients before induction chemotherapy. By using the Cox model, the survival probability was significantly and independently increased for a high percentage of PBMC BLM-induced apoptosis and a high rate of cycling PBMCs. (Chi-2(2): 10; p=0.007). Six patients, in whom both PBMC variables were simultaneouly greater than the median values, showed a median survival time as long as 60 months versus 10 months for the other 19. Conclusively, the PBMC susceptibility to BLM-induced apoptosis as well as the PBMC capability for cycling may be regarded as independent pretherapeutic prognosis factors for patients with advanced SCCHN.
