Impairment in S-phase entry of splenocytes of Parp-1 knockout mice

One immediate cellular response to DNA damage is the polyADP-ribosylation reaction by poly(ADP-ribose) polymerase-1 (Parp-1). The importance of Parp-1 has been established in many cellular processes, such as the maintenance of genomic stability, DNA repair and cell-death induction. Here, we established Parp-1(−/−) mice of C57BL/6J congenic strain and characterized the role of Parp-1 in cell-cycle progression. In this study, we also improved a method to observe G0/G1 to S-phase transition of splenocytes and bone marrow cells prepared from mice. The cells were cultured and stimulated with mitogens (50 μM ionomycin/1 μM phorbol 12, 13-dibutyrate). We found that addition of a commercially available growth supportive reagent, BM Condimed (R)H1, greatly enhanced the transition of G0/G1 to the S-phase, which was determined by bromodeoxyuridine (BrdU) incorporation to DNA. Using this method, G0/G1 to the S-phase entry was measured using splenocytes derived from Parp-1(−/−), Parp-1(+/−) and wild-type (Parp-1(+/+)) mice. DNA synthesis in Parp-1(+/+) and Parp-1(+/−) splenocytes started from day 1 after addition of mitogens, whereas that in Parp-1(−/−) cells started from day 2. The peak of the S-phase was at day 2 in all genotypes and notably DNA synthesis in Parp-1(−/−) cells was approximately halved compared to Parp-1(+/+) cells on day 2, 3 and 4. These results suggested that Parp-1 is involved in positive regulation of S-phase entry in quiescent mouse splenocytes.