Effect of transient hypoxia on sensitivity to doxorubicin in human and murine cell lines

Overreplication of DNA associated with gene amplification and drug resistance has been reported to occur after transient hypoxia of rodent cells. Because oxygen levels fluctuate in solid tumors, clinical drug resistance might be stimulated by this mechanism. We have therefore studied the effect of transient hypoxia on sensitivity to doxorubicin in human and murine cell lines. Exposure to hypoxia led to a decreased rate of cell proliferation, and most of the observed changes in sensitivity to doxorubicin were consistent with cell cycle-dependent cytotoxicity of this drug. After transient hypoxia, about 10% of the murine cells (EMT6/Ro and KHT-LP1) contained greater than four times the haploid DNA content (greater than 4C DNA), but only 0%-5% of the human cells (MGH-U1, A549, and Hey) had greater than 4C DNA content. Murine cells that had been exposed to hypoxia and reoxygenation, and which had greater than 4C DNA content, were separated by flow cytometry. For KHT-LP1 cells, but not for EMT6/Ro cells, this subpopulation was found to be more resistant to doxorubicin than the subpopulation with less than 4C DNA content and the aerobic control. When resistant KHT-LP1 clones were expanded in the presence of doxorubicin, six of six clones showed amplification of the P-glycoprotein gene family. The ability and efficiency of hypoxia to induce DNA overreplication, gene amplification, and drug resistance appears to be cell-line dependent.