Induction of apoptosis and inhibition of signalling pathways by alkylated purines.
Addition of growth factors such as EGF and insulin to serum-starved G(0) Chinese hamster fibroblast cells results in activation of the phosphatidylinositol 3-kinase (PI3-K)/p70 S6 kinase (p70(S6K)) pathway and the ras-raf mitogen-activated kinase (MAPK) pathway. Activation of these pathways is usually associated with protection of cells from apoptosis. We have studied the effect of three alkylpurines, O(6)-methylguanine (O6meG), O(6)-ethylguanine (O6etG) and 6-dimethylaminopurine (6DMAP) on two particular steps of these pathways, namely activation of p70(S6K) and of MAPK. Under the same experimental conditions we studied the ability of these alkylpurines to induce apoptosis. Our results show that the three alkylpurines induced apoptosis with increasing efficiency from O6meG to 6DMAP to O6etG. The induction of apoptosis was phase specific, with the G(0)/G(1) phase being most sensitive. A reduced apoptotic response was observed in cells with abnormal nuclear accumulation of mutant or wild-type p53, suggesting that functional p53 was required for the induction of apoptosis. At concentrations inducing apoptosis the three alkylpurines inhibited p70(S6K) activity, while they had the opposite effect on MAPK. Rapamycin, a specific inhibitor of the p70(S6K) pathway, did not induce apoptosis at doses inhibiting p70(S6K) activity, suggesting that p70(S6K) is not directly involved in apoptosis. As expected, and in line with results reported by others, wortmannin, an upstream inhibitor of the p70(S6K) pathway, did induce apoptosis. We propose that activation of the MAPK pathway and simultaneous inhibition of the p70(S6K) pathway induce an apoptotic response in the cell.
Bonatti
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IST, National Institute for Cancer Research, Genova, CNR Institute of Mutagenesis and Differentiation, Pisa, Italy.
Pigullo
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Simili
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Abbondandolo
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D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000478:Alkylation; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D006224:Cricetinae; D053938:DNA Fragmentation / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects; D016193:G1 Phase / Q000187:drug effects; D016158:Genes, p53 / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011687:Purines; D016192:Resting Phase, Cell Cycle / Q000187:drug effects; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D020123:Sirolimus / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D000903:Antibiotics, Antineoplastic; D004791:Enzyme Inhibitors; D011687:Purines; C007673:6-ethylguanine; D006147:Guanine; C001020:N(6),N(6)-dimethyladenine; C008449:O-(6)-methylguanine; D019893:Ribosomal Protein S6 Kinases; D000225:Adenine; D020123:Sirolimus; D000077191:Wortmannin