TW-37 were harvested at serial time points

TW-37 After transfection, cells were harvested at serial time points for immunoblot and immunofluorescence analysis after labeling with MPM 2 and staining with DAPI. Results Abrogation of the G2 M Checkpoint by Treatment with 17AAG Occurs Selectively in p53 Null HCT116 Cells. It has been shown that treatment with 17AAG resulted in specific depletion of Chk1 in several tumor cell lines. Down regulation of Chk1 resulted in an abrogation of the S phase checkpoint induced by gemcitabine. We have shown previously that sequential treatment of HCT116 colon cancer cells with SN 38, the active metabolite of irinotecan, followed by UCN 01 resulted in abrogation of the G2 arrest induced by SN 38. This checkpoint inhibitory effect is more selective in cells that lack p53 .
We therefore examined the effect of Hsp90 inhibition on regulation of the G2 M DNA damage checkpoint after treatment with SN 38 and 17AAG in both parental and p53 null HCT116 cells. Parental HCT116 cells bearing wild type p53 underwent a late S G2 arrest with loss of mitotic cells after a 24 h treatment with 20 nM SN 38, which was expected, based on our previous results. ITF2357 Treatment with 500 nM 17AAG alone resulted in an accumulation of cells in both G1 and G2, also consistent with the reported effect of this drug . Parental cells treated with a combination of SN 38 and 17AAG either concurrently or sequentially underwent a G2 arrest without mitosis, suggesting that 17AAG was unable to abrogate the G2 M checkpoint induced by SN 38 in this cell line.
It is interesting that we consistently found that in addition to the G1 and G2 arrest seen with 17AAG treatment in parental cells, treatment of p53 null HCT116 cells with this drug resulted in an increase in mitosis. Examination of the nuclear morphology of these mitotic cells by fluorescent microscopy after DAPI staining revealed an increase in apparently normal metaphases compared with untreated HCT116 p53 null cells . After SN 38 treatment, p53 null cells underwent a late S G2 arrest in a way similar to parental HCT116 cells. However, upon removal of SN 38, approximately 14 of p53 null cells had escaped the G2 M checkpoint and entered mitosis, consistent with an intrinsic defect in maintaining the G2 M checkpoint in these cells. This checkpoint defect was markedly enhanced by sequential treatment with 17AAG, resulting in an increase in mitotic index up to 74.
8 . Concurrent treatment with SN 38 and 17AAG also resulted in a higher level of mitotic entry than with either agent alone. When cells were followed for an additional 24 h after drug washout, p53 wild type cells remained arrested in G2, whereas p53 null cells had begun to exit mitosis as evidenced by a decrease in MPM 2 positive cells from 74.8 to 35.8 . Cells that had exited mitosis contained 4 N rather 2 N DNA, indicating a failure of cytokinesis in these cells, an observation consistent with results obtained with compounds that directly inhibit Chk1. Finally, abrogation of the SN 38 indued G2 M checkpoint by 17AAG is schedule dependent because the reverse sequence did not result in any increase in mitotic cells in both cell lines. Treatment with 17AAG Depletes Cellular Chk1 in Both Parental and p53 Null HCT116 Cells. In accord with results published previously, we found that treatme