Enhanced 7-ethyl-10-hydroxycamptothecin (SN-38) lethality by methylselenocysteine is associated with Chk2 phosphorylation at threonine-68 and down-regulation of Cdc6 expression.

Methylselenocysteine (MSC) is an organic selenium compound in preventative clinical trials involving prostate, lung, and colon carcinoma. We found that methioninase-activated MSC potentiates 7-ethyl-10-hydroxycamptothecin (SN-38)-induced cell lethality in vitro in the p53-defective human head and neck carcinoma A253 cells. Activated MSC increases chk2 phosphorylation at threonine-68 induced by SN-38, with no significant effect on chk1 phosphorylation. Cell cycle arrest induced by SN-38, however, was not abrogated or potentiated by MSC. These results suggest that the enhanced cellular lethality of SN-38 by MSC was not associated with cell cycle regulation pathways. Because chk2, in addition to its role in cell cycle arrest, can induce apoptosis by phosphorylation/activation, we examined whether increased chk2 phosphorylation could induce preapoptotic DNA fragmentation. DNA damage analysis showed that megabase DNA fragmentation is decreased, accompanied by the increased 30 to 300 kilobase pairs of DNA fragmentation after exposure to SN-38 with MSC, compared with SN-38 alone. No significant changes in the amount of DNA fragments were observed in cells treated with SN-38 or MSC alone. Moreover, proteolytic destruction of DNA replication-associated proteins cdc6, MCM2, and cdc25A may induce a DNA damage checkpoint response. The observed down-regulation of DNA replication proteins cdc6, MCM2, and cdc25A after exposure to SN-38 with MSC further indicates a relationship between drug response and DNA damage. Exposure to SN-38 with MSC resulted in a significant increase of poly(ADP-ribose) polymerasecleavage and caspase 3 activation. All together, the data support the hypothesis that enhanced lethality of this combination is associated with increased chk2 phosphorylation at Thr68 and down-regulation of specific DNA replication-associated proteins, which result in poly(ADP-ribose) polymerase cleavage, caspase 3 activation, and the induction of 30 to 300 kilobase pairs of DNA fragmentation.