Apoptotic and clastogenic effects of methylating agents, alone or combined with poly(ADP-ribose) polymerase inhibitor, in peripheral blood lymphocytes

We recently demonstrated that mismatch repair deficient tumor cells, that are tolerant to O6-methyl guanine (O6meG) damage, are susceptible to MeOSO2(CH2)2-lexitropsin (Me-Lex) capable of inducing almost exclusively N3-methyl adenine (N3meA) adducts. Moreover, these cells can be rendered sensitive to O6methylating agents that generate low levels of N3meA (temozolomide, TZM), when used in combination with poly(ADP-ribose) polymerase (PARP) inhibitors. This is likely due to interruption of base excision repair (BER) process, that is involved in nucleotide replacement of N3meA. PARP inhibitors are also capable of enhancing the cytotoxic and clastogenic effects induced by Me-Lex. Purpose To evaluate the potential toxicity and clastogenicity in normal peripheral blood lymphocytes (PBL) of Me-Lex or TZM, as single agent or associated with PARP inhibitor. Methods and results PBL of healthy donors, untreated or activated with PHA for 3h, were exposed to graded concentrations of Me-Lex (1.5-25M) or TZM (62.5-250M), alone or combined with the PARP inhibitor 3-aminobenzamide (AB, 4mM). Cytotoxicity was evaluated at daily intervals during 72h of culture. Chromosome damage was assessed only in PHA-activated PBL at 72h. The results show that: a) when used alone, Me-Lex induced apoptosis in a dose-dependent fashion, either in non-stimulated or in PHA-activated PBL as early as 24h after treatment. Addition of AB significantly increased the percentage of apoptotic cells at all drug concentrations; b) in the case of TZM, at 24 h apoptosis was not observed either in resting or PHA-activated PBL. Only when the drug was combined with AB, a small percentage of apoptotic cells could be detected. In contrast, at 48 or 72h of culture, TZM induced apoptosis only in PHA activated PBL. This is likely the consequence of the toxicity derived from O6meG, that is known to occur after DNA replication. c) High concentrations of Me-Lex (12.5-25M) markedly reduced the mitotic index of PHA-activated cells and did not allow chromosome analysis. Me-Lex (1.5-3M) produced chromosome aberrations that were increased by addition of AB. TZM induced aberrations only at 250M and, when combined with AB, at all concentrations tested. d) MeLex (1-6M) induced sister chromatid exchanges (SCE) in a dose-dependent fashion and addition of AB significantly enhanced this effect. In the case of TZM, SCE were observed at all the concentrations tested. In the presence of AB, the number of SCE generated by TZM was significantly higher than that induced by the drug used alone or by Me-Lex ± AB. Conclusion High levels of N3meA or interruption of its repair through the use of PARP inhibitors might be toxic for effector cells of the immune system, including those mediating functions that do not require cell proliferation.