Fig. 3

Rhamm^−/− tumor cells are resistant to DNA damage. A DNA fragmentation was quantified by comet assays as a measure of DNA damage using frozen samples of isolated, unstimulated Wildtype and Rhamm^−/− primary tumor cells. Quantification of % DNA in tail and tail moment is not affected by Rhamm-loss consistent with similar mutation burdens in the two genotypes identified by the genotyping array. Values are the Mean and S.E.M of n = 3 cell samples/genotype and n = 21 wells/genotype. B The amount of ROS-induced DNA damage in Wildtype and Rhamm^−/− tumors was quantified using ELISA as described in Methods. Results show that DNA from lung metastases of both genotypes carry more ROS-induced DNA damage that can cause point mutations and CNVs than primary tumors, which is consistent with the higher levels of ROS in lungs versus mammary fat pads. Values are the Mean and S.E.M. N = 4 biological replicates and 3 technical replicates. *p < 0.05, ***p < 0.001. C, D Cultures of Wildtype and Rhamm^−/− primary tumor cells were exposed to H₂O₂ (C) and cisplatin (D) to induce DNA damage. DNA damage was detected by gamma-H2AX staining, and cell death was quantified by Apoptag reactivity as described in Methods. Rhamm^−/− tumor cells survive with more DNA damage than Wildtype comparators. Values are the Mean and S.E.M. n = 3 biological replicates *p < 0.05, **p < 0.01