Phosphorylated histone H2AX in spheroids, tumors, and tissues of mice exposed to etoposide and 3-amino-1,2,4-benzotriazine-1,3-dioxide.

We reported recently that exposure of hamster V79 fibroblasts to 6 drugs that varied in their ability to produce DNA double-strand breaks stimulated formation of phosphorylated histone H2AX (serine 139 phosphorylated histone H2AX; gammaH2AX). Using flow cytometry to analyze gammaH2AX antibody-stained cells 1 h after a 30-min drug treatment, the fraction of cells that showed the control levels of gammaH2AX correlated well with the fraction of cells that survived to form colonies. This observation is now extended to V79 and SiHa human cervical carcinoma cells grown as multicell spheroids and SiHa xenografts and SCCVII tumors in mice. Animals were injected with etoposide, a topoisomerase-II inhibitor that targets proliferating cells or 3-amino-1,2,4-benzotriazine-1,3-dioxide (tirapazamine), a bioreductive cytotoxin that targets hypoxic cells. For spheroids, gammaH2AX intensity predicted clonogenic cell survival for cells recovered 90 min after drug injection, regardless of position of the cells within the spheroid. Similar results were obtained for etoposide in tumors; however, the gammaH2AX signal for tirapazamine was smaller than expected for the observed amount of cell killing. Frozen sections of tumors confirmed the greater intensity of gammaH2AX staining in cells close to blood vessels of tumors soon after treatment with etoposide and the opposite pattern for tumors exposed to tirapazamine. Analysis of cells or frozen sections from mouse spleen and kidney suggests that information can also be obtained on initial damage in normal tissues. These results support the possibility of using gammaH2AX antibody staining as a method to aid in prediction of tumor and normal tissue response to treatment.