Involvement of proteolytic activation of PKCdelta in cisplatin-induced apoptosis in human small cell lung cancer H69 cells.
Proteolytic activation of protein kinase C delta (PKCdelta) has been associated with apoptosis induced by the DNA damaging agent cisplatin. In cells undergoing apoptosis, caspase-3 cleaves PKCdelta at the site DMQD downward arrowN to generate a 40-kDa catalytic fragment. We have previously shown that the PKC signal transduction pathway regulates sensitivity of human small cell lung cancer H69 cells to cisplatin. In the present study, we have investigated if proteolytic activation of PKCdelta is essential for cisplatin-induced apoptosis in H69 cells. The caspase cleavage-resistant mutant PKCdelta (DMQA) was generated by mutating the aspartate residue at the site of proteolysis DMQD downward arrowN to alanine (D330A), and the wild-type and mutant PKCdelta were introduced into H69 cells. Cisplatin induced a substantial increase in PKCdelta catalytic fragment in H69 cells overexpressing PKCdelta (H69/delta, and the level of PKCdelta catalytic fragment in H69 cells expressing DMQA mutant (H69/DMQA) was equivalent to that in H69 cells. However, the cleavage of poly(ADP-ribose) polymerase (PARP), another substrate for caspase-3, was similar in cells overexpressing wild-type PKCdelta and DMQA mutant PKCdelta. The ability of cisplatin to induce mitochondrial depolarization and cell death was also equivalent among the cell lines tested. These results suggest that the proteolytic fragment of PKCdelta does not play a critical role in the induction of apoptosis in H69 cells.
Persaud
Shalini D
SD
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, 76107, USA.
Hoang
Van
V
Huang
Jie
J
Basu
Alakananda
A
D017209:Apoptosis; D018288:Carcinoma, Small Cell / Q000188:drug therapy* / Q000473:pathology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002945:Cisplatin / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology*; D008564:Membrane Potentials; D008928:Mitochondria / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051745:Protein Kinase C-delta; D017434:Protein Structure, Tertiary; D014162:Transfection
C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin