Here, we observed that this ther apy also induced the substantial activation of ATM and Chk2, which resulted in nuclear focus localisation of p ATM and p Chk2. Consistent with these results, treatment of MDA MB 231 cells with TMCG/ DIPY resulted in an increase in E2F1 phosphorylation SB203580 p38-MAPK at Ser31 and Ser364 .. Because TMCG/ DIPY treatment positively influenced E2F1 mediated cell death, we hypothesised that this combination might represent an attractive strategy to target over expressed E2F1 in 4OHT treated ER negative breast cancer cells. To test our hypothesis, MDA MB 231 cells were treated with the triple TMCG/DIPY/4OHT combination, and the effects of this combination on cell growth and apoptosis were compared to those of the double TMCG/DIPY combination treatment.

As observed in Figure 6A, addition of 4OHT to the double TMCG/DIPY combination significantly increased the number of apoptotic cells. This increase in apoptosis was also correlated with cellular damage, as indicated by an increase in the number of cells that were positive for phosphorylated histone H2AX and with an augmented ratio of H2AX per nucleus. The analysis of E2F1 phosphorylation at Serines 31 and 364 indicated that overexpressed E2F1 was fully phosphorylated in cells subjected to TMCG/DIPY/4OHT therapy, which is consistent with the pattern of Chk2 and ATM activation in these cells. Consistent with our hypothesis of TMCG/DIPY/4OHT combination inducing E2F1 mediated apoptosis, we observed a significant increase of p73 and Apaf1 mRNAs after treatment.

Conclusions As described in the introduction, the mutational status of the p53 gene and/or the levels of expression of ER determine the sensitivity or resistance of breast cancer cells to apoptosis. The p53 tumour suppressor protein is an essential component of the cell response induced by genotoxic stresses, but the p53 gene is inacti vated or mutated in the majority of human tumours. To overcome these obstacles, genes that can compensate or bypass cell death defects regardless of the p53 status are particularly useful. E2F1 and its proapoptotic genes represent such a group of molecules and therefore have direct implications as anti neoplastic therapeutics for cancers lacking p53 activity. Recently, our research group has generated novel antifolate drugs that have successfully been used in combined hypomethylating therapies against melanoma and breast cancer. Here, we present an experimental therapy that is effective in breast cancer cells independently of their p53 and ER status, and confirm the hypothesis Anacetrapib that the elevation of E2F1 in the presence of genotoxic stress could represent a valuable therapy against cancers.