Mutagenesis, Vol. 15, No. 4, 361-366,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Induction of apoptosis and inhibition of signalling pathways by alkylated purines
1 IST, National Institute for Cancer Research, Genova, 2 CNR Institute of Mutagenesis and Differentiation, Pisa and 3 Department of Oncology, Biology and Genetics, University of Genova, Genova, Italy
Addition of growth factors such as EGF and insulin to serum-starved G0 Chinese hamster fibroblast cells results in activation of the phosphatidylinositol 3-kinase (PI3-K)/p70 S6 kinase (p70S6K) 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, O6-methylguanine (O6meG), O6-ethylguanine (O6etG) and 6-dimethylaminopurine (6DMAP) on two particular steps of these pathways, namely activation of p70S6K 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 G0/G1 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 p70S6K activity, while they had the opposite effect on MAPK. Rapamycin, a specific inhibitor of the p70S6K pathway, did not induce apoptosis at doses inhibiting p70S6K activity, suggesting that p70S6K is not directly involved in apoptosis. As expected, and in line with results reported by others, wortmannin, an upstream inhibitor of the p70S6K pathway, did induce apoptosis. We propose that activation of the MAPK pathway and simultaneous inhibition of the p70S6K pathway induce an apoptotic response in the cell.
4 To whom correspondence should be addressed at: IST, National Institute for Cancer Research, Largo R. Benzi 10, 16132 Genova, Italy. Fax: +39 010 560 0992; Email: abbondan{at}hp380.ist.unige.it
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