Cultured cells subjected to oxygen deprivation have been shown to undergo anomalous DNA synthesis, which can result in DNA overreplication and the generation of cellular variants [Rice, G. C., Hoy, C. & Schimke, R. T. (1986) Proc. Natl. Arad. Sci. USA 83,[5978][5979][5980][5981][5982]. In the present study, murine tumor cells were exposed to severe hypoxia and then tested for their ability to form experimental metastases. Upon reoxygenation, cells transiently, yet dramatically, increased their metastatic potential. Flow cytometric analysis confirmed that hypoxia and reoxygenation induced cell cycle perturbations and DNA overreplication in these tumor cell lines. Fibrosarcoma cells with overreplicated DNA isolated by fluorescence-activated cell sorting proved to be highly metasiatic, although cells with 2-4 times the haploid DNA content in populations treated with hypoxia were also markedly

Tissue hypoxia in regions of solid tumors has been identified as a factor that may affect the behavior of cancer cells. In this study, cells were isolated from hypoxic regions of transplanted murine tumors and tested for sensitivity to anticancer drugs and ability to form experimental metastases. The tumors studied were KHT-C2-LP1 fibrosarcoma and SC-CVII squamous cell carcinoma in C3H mice and B16F10-A1 melanoma in C57BL mice. Our results indicate that the position of tumor cells relative to the vasculature, which determines the degree of tissue oxygenation, does not influence the in vitro sensitivity of cells to either doxorubicin or methotrexate. Conversely, 1-2 days after reoxygenation by introduction into culture, subpopulations of tumor cells demonstrated a transient increase in lung colonization ability. The most hypoxic cells exhibited a metastatic efficiency that was generally twice that of cells from well-oxygenated regions. This behavior is similar to behavior we observed in a previous study when tumor cells were exposed in vitro to conditions of extreme hypoxia. The findings in that study suggested that gene amplification associated with DNA over-replication is responsible for the enhanced metastatic potential, but we found no indication in the present study that gene amplification was involved in the effect observed with the hypoxic tumor subpopulations. These results provide additional evidence that reoxygenated cancer cells have a high colonization ability and that these cells may be important in the formation of distant metastases.

Recent studies have indicated that KHT fibrosarcoma or B16 melanoma cell variants capable of forming experimental metastases in the lungs of mice after i.v. injection are created stochastically at high rates (approximately 10(-5)/cell/generation). Expression of this phenotype is unstable and hence expanding populations of tumor cells establish a dynamic equilibrium between a small subpopulation of metastatic variants and a large compartment of nonmetastatic cells. In the present experiments, cell suspensions were prepared from the lungs of mice bearing 'experimental' metastases and the tumor cells contained in them were tested for their metastatic efficiency (ME) using the lung colony assay. The ME of the recovered tumor cell populations was found to be a function of the time of metastatic growth in the animal. Tumor cells isolated soon after the initial i.v. injection, i.e. derived from micrometastases, are highly metastatic while populations recovered from macroscopic nodules are similar to parental lines in their ability to colonize the lung. These results are consistent with the prediction of the above 'dynamic heterogeneity' model that nascent lung metastases should be composed largely of tumor cells expressing the variant metastatic phenotype, but that the proportion of such variants should decline during growth to the equilibrium (parental population) level. Mathematical analysis of the results indicates that the effective rate of reversion of the variant phenotype is approximately 10(-1)/cell/generation.

Summary The DNA-binding bisbenzimide fluorochrome Hoechst 33342 is being used routinely in radiobiological studies to assess cell kinetic parameters and tumour blood flow. However, there are reports in the literature which indicate that exposure to this compound can affect the radiation sensitivity of tumour cell populations. In this investigation, it was found that staining murine tumour cells in vitro with H33342 at concentrations >0.1 gLM before irradiation resulted in radioprotection. The protection factor calculated for fibrosarcoma cells stained with 10 IM H33342 was 1.7. Varying the time between radiation treatment and exposure to the fluorochrome demonstrated that the effect rapidly changed to radiosensitisation when staining was performed subsequent to irradiation. Cells in transplanted KHT tumours were stained in vivo by intravenous administration of H33342 to determine whether the radiation sensitivity of these populations might also be modified. Flow cytometric analysis of suspensions prepared from tumours stained in this manner revealed that recovered cells exhibited a >100-fold range in fluorescence intensities. These suspensions were irradiated in vitro and the cells were then fractionated according to fluorochrome content using cell sorting.Little evidence for a radioprotective effect was observed when these subpopulations were assessed for survival, even when tumour-bearing mice were given doses of H33342 which approached the LD50. Further analysis demonstrated that insufficient amounts of the fluorochrome were taken up by cells during in vivo staining to attain levels required for radioprotection. However, our results indicate that the amount of H33342 accumulated by cells may affect the radiation sensitivity of populations exposed to high concentrations of this fluorochrome, such as those required to achieve stoichiometric binding to DNA.