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Cell Cycle Distribution of Chronically Hypoxic Cells and Determination of the Clonogenic Potential of Cells Accumulated in G₂ + M Phases after Irradiation of a Solid Tumor in Vivo¹

The Ontario Cancer Institute Incorporating the Princess Margaret Hospital, Toronto, Ontario, M4X 1K9, Canada

Information obtained from flow cytometric analysis of cell cycle perturbations has been limited by the problem of distinguishing clonogenic from nonclonogenic cells in a DNA distribution. Hoechst 33342 is a DNA-specific stain reported to have minimal effects on cell viability. It was used in these studies to investigate the clonogenicity of unirradiated and irradiated solid tumor cells sorted according to their DNA content. The clonogenic potential of unirradiated KHT sarcoma cells was determined with an in vitro assay and was not significantly different for cells in G₁, S, and G₂ + M phases. The KHT tumor cells are tetraploid, and a "normal diploid population" was detected in the tumor. The plating efficiency of the diploid population was minimal. After a large dose of radiation (1700 rads) to the in situ tumor, 85% of the surviving hypoxic cells were found in G₁ phase, and 9.6 and 5.6% were in S and G₂ + M phases, respectively.

The temporal course of radiation-induced cell cycle perturbations in the KHT tumor was documented by flow cytometric analysis of mithramycin-stained cells. A 2.5-fold increase in the percentage of cells in G₂ + M was observed 10 hr after radiation. Significant perturbations still existed at 48 hr. Similar results were obtained with Hoechst 33342, and the clonogenic potential of the cells comprising the DNA distribution was determined 10 hr after radiation. Approximately 77% of the surviving clonogenic cells were in G₁ phase, and only 23% were found in G₂ + M in spite of the large increase in the total number of tumor cells in these phases. These data demonstrate applications of flow cytometric analysis and sorting of cells obtained from solid tumors to the investigations of radiobiological phenomena.

¹ This work was supported in part by the National Cancer Institute of Canada.

² To whom requests for reprints should be addressed, at Biomedical Sciences Division, Lawrence Livermore Laboratory, P. O. Box 5507, Livermore, Calif. 94550.

Received 10/16/78. Accepted 2/21/79.