DNA damage checkpoint responses in the S phase of synchronized diploid human fibroblasts.

We investigated the hypothesis that the strength of the activation of the intra-S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m(-2) UVC in early, mid- and late-S phase. The endpoints measured were the following: (1) radio-resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC-induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10-fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra-S checkpoint activated by UVC-induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.

Effects and mechanism of suberoylanilide hydroxamic acid on the proliferation and apoptosis of human hepatoma cell line Bel-7402.

PURPOSE: To investigate the effects and mechanism of suberoylanilide hydroxamic acid (SAHA) on the proliferation and apoptosis of human hepatoma cell line Bel-7402. METHODS: SAHA treatment and control groups were designed in this study. To observe the morphological characteristics and the inhibition of cell proliferation, we conducted confocal microscopy and methyl thiazolyl tetrazolium (MTT) assay, respectively. Changes in cell apoptosis and cell cycle were then determined by flow cytometry. Real-time polymerase chain reaction (RT-PCR) was also conducted to detect the mRNA expressions of p53, bcl-2 and bax genes. Caspase-3 protein activity was determined by spectrophotometry. RESULTS: Cell proliferation in the SAHA treatment group could be inhibited in a time- and dose-dependent manner. FCM analysis showed that the early apoptosis rate in the SAHA treatment group increased significantly. Furthermore, cell cycle was arrested at the S phase. RT-PCR assay confirmed that SAHA could upregulate the mRNA expressions of p53 and bax genes. By comparison, SAHA could downregulate the mRNA expression of bcl-2. SAHA induced apoptosis by activating the caspase-3 pathway. CONCLUSION: SAHA inhibited cell proliferation and promoted human hepatoma Bel-7402 cell apoptosis by affecting caspase-3 protein activity and mRNA expressions of p53, bcl-2 and bax genes.