Abstract 2972: In vitro quantification and modeling of DNA damage by histone H2A after exposure to gemcitabine and a Chk1 inhibitor

Background: Gemcitabine is a nucleoside analog used as an anti-cancer chemotherapeutic drug. To enhance its disruption of DNA replication and repair, Chk1 inhibitors are being developed for use in combination with Gemcitabine and other cytotoxic agents. Evaluation of these therapies with mechanistic biomarkers can be an important part of the drug development process. One such biomarker is the phosphorylation of histone H2A (γH2AX), which is a DNA damage response biomarker. An ELISA based assay has been developed to quantify γH2AX. This quantitative assay provides data that is incorporated into a mathematical model relating drug exposure to DNA damage and cell viability. Experiment Design: MiaPaCa-2 cells, a human pancreatic tumor cell line, were subjected to Gemcitabine alone (0.1 to 5uM for 2 hours), a Chk1 inhibitor alone (0.1-5uM for 2 – 48 hours), or the Chk1 inhibitor in combination with 0.5 and 5uM Gemcitabine. Cellular concentrations of Gemcitabine and the Chk1 inhibitor were detected with LC/MS. DNA damage was assessed by the γH2AX ELISA at 2, 6, 24 and 48 hours after the start of incubation with drug. In addition to γH2AX, cell viability was measured by cell number at 48 hours. A mathematical model was developed to quantify the modulation of γH2AX when exposed to each drug separately and in combination. Result: Transient exposure of the cells to Gemcitabine resulted in an increase in γH2AX at later times (24 and 48 hours). The Chk1 inhibitor resulted in a dose dependent increase in γH2AX, with a greater effect being observed at later times of exposure. The combination therapy showed a greater response than Gemcitabine or Chk1 alone. However it was observed that higher doses of Gemcitabine and Chk1 in combination did not always yield the largest γH2AX response or the greatest effect on cell viability. This may be due to cellular resistance and is still being investigated. Conclusion: The ELISA based assay for γH2AX provides a quantitative assessment of the level of DNA damage. This information can then be utilized in developing mathematical models of drug action alone and in combination with other drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2972. doi:10.1158/1538-7445.AM2011-2972