Continuous expos ure to either drug individually for as long as 48 hours was unable to robustly induce H2AX staining, appearing in only 10% or selleck chemicals Brefeldin A less of treated cells. When combined, however, the same concentrations of MK 1775 and MK 8776 demonstrated synergistic induction of H2AX in as many as 45% to 75% of treated cells, which was maximally induced by 24 hours. In addition to their effects on DNA metabolism, both WEE1 and CHK1 inhibitors are known to disrupt the G2 M cell cycle checkpoint and accelerate mitotic entry. There fore, to determine whether premature mitosis could con tribute to the synergism of MK 1775 and MK 8776, the mitotic index of treated cells was scored using the mitotic marker phosphorylated serine 28 of histone H3.

In both HT 29 and, to a lesser extent, A2058 cells, we observed a more than additive increase of pHH3 positive cells in the combination treated population, indicating that accelerated or premature mitosis can indeed result from combination treatment. Interest ingly, however, no increase in pHH3 positive cells was observed when LoVo cells were treated with the combin ation despite equally robust inhibition of cell proliferation and induction of DNA damage as in the A2058 and HT 29 cells. This suggests that DNA damage is the primary mech anism underlying the cytotoxic synergy of WEE1 and CHK1 inhibitors, whereas premature mitosis may or may not contribute as a secondary mechanism of action. To better determine whether DNA damage is associated with the anti proliferative effect of the drug combination, we analyzed three less responsive cell lines for induction of H2AX or pHH3.

We treated KPL 1, NCI H460, and T47D cells each with 150 nM MK 1775, 300 nM MK 8776, or both. These drug concentrations are equal to or in ex cess of those used for the three sensitive cell lines. As expected, only minimal effects were observed on cell via bility. In KPL 1 cells we observed induction of H2AX, though unlike all three sensitive lines, the DNA damage in the combination treated sample was not maximally induced by 24 hours, did not exceed 32%, and was not ob viously supra additive. This cell line did not show an in crease in pHH3 positive cells when treated with the combination. Neither NCI H460 nor T47D cells showed any appreciable evidence of DNA damage or premature mitosis, supporting the notion that MK 8776 and MK 1775 synergize to inhibit cell proliferation by inducing DNA damage in sensitive cell lines.

DNA damage is present in the S phase population of cells and is CDK dependent Both siRNA knockdown and pharmacologic inhibition of WEE1 are known to result in damaged DNA specifically in S phase cells. Cell cycle analysis based Cilengitide on DNA content of the three sensitive cell lines above demonstrated that DNA damage caused by the MK 1775 and MK 8776 combination is detected in S phase.