ATM/ATR-independent inhibition of cyclin B accumulation in response to hydroxyurea in nontransformed cell lines is altered in tumour cell lines

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Abstract
The DNA replication checkpoint is an inhibitory pathway ensuring that mitosis occurs only after completion of DNA synthesis. Its function may be relevant to the stability of the genome. The essential elements of this checkpoint are ATM/ATR kinases that indirectly lead to the phosphorylation and inhibition of the mitosis-promoting factor (Cdc2/cyclin B1). The function of this checkpoint was analysed in diverse nontransformed and tumour-derived cell lines. All cell lines tested arrested mitosis entry when DNA synthesis was inhibited by hydroxyurea (HU) treatment. But, unlike what has been described in yeast and Xenopus, in normal rat kidney (NRK) cells and NIH 3T3 fibroblasts, the arrest induced by HU treatment was not abrogated by caffeine, an ATM and ATR inhibitor. This indicated the presence of an ATM/ATR-independent response to DNA synthesis inhibition in these nontransformed mammalian cell lines. Interestingly, the behaviour of different tumour cell lines after caffeine treatment varied. While SW480, NP29, NP18 and HeLa cells did not enter mitosis in the presence of caffeine after HU treatment, in CaCo2, DLD1, HCT116 and HT29 caffeine abrogated the checkpoint response. In nontransformed cell lines, lack of cyclin B1 accumulation was observed when DNA synthesis was inhibited. This response was not abrogated by caffeine. In the tumour cell lines, a good correlation between the ability to arrest cell cycle when DNA synthesis was inhibited in the presence of caffeine and the lack of cyclin B1 accumulation was observed. Thus, there is an ATM/ATR-independent checkpoint response that leads to a decrease in cyclin B1 accumulation. However, this response is not functional in some tumour cell lines. Using inhibitors of p38 and , Mek1, 2 and p53-/- knocked-out fibroblasts, we showed that these proteins were also not involved in this particular checkpoint response. Lack of cyclin B1 accumulation after DNA synthesis inhibition in NRK cells was not due to increased degradation of the protein, but correlated with a decrease in mRNA accumulation.