Characterization of the effects of cisplatin and carboplatin on cell cycle progression and DNA damage response activation in DNA polymerase eta-deficient human cells.

Abstract:

Translesion synthesis by DNA polymerase eta (poleta) is one mechanism by which cancer cells can tolerate DNA damage by platinum-based anti-cancer drugs. Cells lacking poleta are sensitive to these agents. To help define the consequences of poeta-deficiency, we characterized the effects of equitoxic doses of cisplatin and carboplatin on cell cycle progression and activation of DNA damage response pathways in a human cell line lacking poleta. We show that both cisplatin and carboplatin induce strong S-phase arrest in poleta-deficient XP30RO cells, associated with reduced expression of cyclin E and cyclin B. PIK kinase-mediated phosphorylation of Chk1, H2AX and RPA2 was strongly activated by both cisplatin and carboplatin, but phosphorylation of these proteins was induced earlier by cisplatin than by an equitoxic dose of carboplatin. Compared to Chk1 and H2AX phosphorylation, RPA2 hyperphosphorylation on serine4/serine8 is a late event in response to platinum-induced DNA damage. We directly demonstrate, using dual-labeling flow cytometry, that damage-induced phosphorylation of RPA2 on serine4/serine8 occurs primarily in the S and G(2) phases of the cell cycle, and show that the timing of RPA2 phosphorylation can be modulated by inhibition of the checkpoint kinase Chk1. Furthermore, Chk1 inhibition sensitizes poleta-deficient cells to the cytotoxic effects of carboplatin. Both hyperphosphorylated RPA2 and the homologous recombination protein Rad51 are present in nuclear foci after cisplatin treatment, but these are separable events in individual cells. These results provide insight into the relationship between cell cycle regulation and processing of platinum-induced DNA damage in human cells when poleta-mediated TLS is compromised.

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