Effects of reoxygenation on cells from hypoxic regions of solid tumors: analysis of transplanted murine tumors for evidence of DNA overreplication.

Transient exposure of cultured cells to conditions of extreme hypoxia can induce DNA overreplication and the generation of cellular variants. This effect may be important for the development of tumor heterogeneity, since hypoxia may arise in solid tumors as a result of vascular insufficiency. We have investigated whether reoxygenation of cancer cells obtained from hypoxic regions of solid tumors is associated with DNA overreplication. Murine tumor cells were isolated from i.m. transplants as a function of their distance from the vasculature using a technique which involves in vivo staining of tumor tissues with the fluorochrome Hoechst 33342 (Chaplin et al., Br. J. Cancer, 51:569-572, 1985). Studies which determined the radiation sensitivity and cell cycle distribution of cells in the subpopulations indicated that cells were isolated from regions of the tumor which differed in oxygen levels. When KHT fibrosarcoma cells were isolated from hypoxic regions of tumors and introduced into culture (i.e., were reoxygenated), flow cytometric analysis revealed that they did not undergo any large scale DNA overreplication. These results indicate that hypoxic conditions which exist in transplanted tumors do not induce cells to undergo DNA overreplication to the same extent that is achieved after in vitro exposure of cells to hypoxia. We also found that at high concentrations (10 microM) the Hoechst dye itself induced DNA overreplication. These concentrations are frequently used to vitally stain cells for sorting according to DNA content, and this effect must be considered in the interpretation of such experiments.