PCR and found no difference in Wee1 mRNA levels between the two conditions. Thus, our results indicate that Wee1 interacts with Hsp90 in vivo, and inhibition of Hsp90 by 17AAG results in accelerated degradation of Wee1, which at least partially depends on the 26S INO-1001 proteasome. Taken together, these data strongly suggest that Wee1 is an Hsp90 client protein in mammalian cells. Gene Knockdown of Chk1 and Wee1 by siRNA Phenocopied the Pharmacological Effect of 17AAG. To confirm that the down regulation of Chk1 and Wee1 upon 17AAG treatment caused the abrogation of the G2 M checkpoint rather than being part of a pleiotropic effect caused by Hsp90 inhibition, we knocked down the expression of these two checkpoint kinases by siRNA and determined the effect of their individual or combined depletion on the G2 M checkpoint.
To mimic the schedule of sequential treatment with SN 38 and 17AAG, HCT116 p53 null cells were pretreated with SN 38 for 24 h to induce a G2 checkpoint arrest before siRNA transfection. As shown in Fig. 5A, transfection with siRNA oligonucleotides specific KRN 633 for Chk1 or Wee1, but not control siRNA, resulted in a substantial down regulation of their respective protein targets. It is noteworthy that we consistently observed a slight decrease in Wee1 protein level in cells transfected with Chk1 siRNA. We postulated that this reduction in Wee1 level was caused by mitotic entry induced by Chk1 knockdown rather than an off target effect of the Chk1 directed siRNA oligonucleotide used, because the decline in Wee1 could be reproduced with a different Chk1 specific siRNA duplex.
We next examined the effect of gene knockdown on the G2 M DNA damage checkpoint in these cells by monitoring the percentage of mitotic cells 8, 12, 16, 20, and 24 h after siRNA transfection. Compared with SN 38 treated cells transfected with control siRNA, cells transfected with siRNA specific for Chk1 or Wee1 showed a progressive increase in mitotic index . The kinetics of mitotic entry were somewhat faster in cells transfected with both Chk1 and Wee1 siRNA than in those transfected with each individual oligonucleotide. However, the extent of checkpoint escape seen in cells transfected with the pooled oligonucleotides was lower than what one would have expected if the combined effect of down regulating each kinase was additive, suggesting that Chk1 and Wee1 may function along the same signaling pathway in controlling the G2 M checkpoint.
Together, gene knockdown of Chk1 and Wee1 recapitulated in part the pharmacological effects of 17AAG in causing abrogation of the G2 M checkpoint. Treatment with SN 38 and 17AAG Is Selectively More Cytotoxic in HCT116 Cells Lacking p53. Finally, we explored the therapeutic potential of combining SN 38 and 17AAG to target p53 defective cells. Apoptosis was measured in parental and p53 null HCT116 after combined treatment with SN 38 and 17AAG in various schedules. As shown in Fig. 6A, single agent treatment with 20 nM SN 38 or 500 nM 17AAG resulted in minimal apoptosis in both cell lines. The combination of SN 38 and 17AAG was ineffective in causing apoptosis in the parental cells, regardless of the sequence of drug treatment. This result is in agreement with the flow cytometry data, which showed