of Chk1 in a time and dose BMS-754807 dependent manner. It is interesting that Chk1 was similarly depleted in both parental and p53 null HCT116 cells, even though abrogation of the SN 38 induced G2 M checkpoint abrogation by 17AAG was seen only in the latter cell line. We therefore queried the basis for the selective abrogation of the G2 M checkpoint in cells that lack p53. We first studied the level of p53 and its downstream effector p21 during combination treatment. In parental cells, both p53 and p21 were up regulated by treatment with SN 38 alone, and their protein levels continue to increase in a time dependent fashion even upon removal of the drug.
After JNJ-26481585 sequential treatment with 17AAG, the up regulation of p53 was maintained, indicating that 17AAG treatment had no effect on the level of wild type p53 protein, which was consistent with reports in the literature showing that Hsp90 inhibition destabilized only mutated p53 proteins. The induction of p21 after sequential treatment with SN 38 and 17AAG seemed to be more robust than treatment with SN 38 followed by drug free medium. As expected, p21 was not induced in p53 null cells treated with SN 38 and 17AAG. To directly test the role of p21 in checkpoint maintenance in parental HCT116 cells after SN 38 and 17AAG treatment, we examined the checkpoint response of isogenic HCT116 p21 null cells treated by the combination. Sequential treatment with SN 38 followed by 17AAG resulted in a marked increase in mitosis that was not seen with SN 38 followed by drug free medium .
We have also confirmed that treatment with 17AAG resulted in down regulation of Chk1 in a dose dependent fashion in these cells similar to parental cells. To assess the effect of 17AAG treatment on Chk1 depedent signaling events, we examined the protein level of cdc25A, a dual specificity phosphatase that is known to be destabilized after phosphorylation by Chk1. Consistent with an interruption of Chk1 dependent signaling pathway, concurrent or sequential 17AAG treatment reversed the SN 38 induced down regulation of the cdc25A. Taken together, we conclude that the selective abrogation of the SN 38 induced G2 arrest by 17AAG in p53 null HCT116 cells was caused by a concomitant loss of two G2 M checkpoint pathways in these cells: loss of p21 as a result of p53 deletion and pharmacological disruption of the Chk1 mediated checkpoint by 17AAG.
Down Regulation of Wee1 by 17AAG Treatment. Because 17AAG treatment may lead to down regulation of a number of Hsp90 client proteins, we examined the effect of 17AAG treatment on other checkpoint targets, besides Chk1, that may be responsible for the G2 M checkpoint abrogation. The mitogen activated protein kinase MK2 pathway has been implicated recently in mediating the G2 M checkpoint induced by UV damage. However, treatment with 17AAG resulted in no appreciable change in the level of MK2 in either cell lines. Wee1 is a tyrosine kinase that prevents premature mitosis by phosphorylating cdc2 at Tyr15. We found that treatment with 17AAG caused a time and dose dependent depletion of Wee1 in both parental and p53 null HCT116 cells and in MKN 74 gastric cancer cells. It has been reported that Wee1 activity varies according to cell cycle phases, and its activity de