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Fluorescence-activated Cell Sorting Analysis of the Induction and Expression of Acute Thermal Tolerance within the Cell Cycle¹

Department of Radiation Oncology, University of California San Francisco, San Francisco, California 94143 [G. C. R., W. C. D.], and Lawrence Livermore National Laboratory, Biomedical Sciences Division, University of California, Livermore, California 94550 [J. W. G., P. N. D.]

We have examined the cell cycle specificity of 45.5° heat-induced toxicity and the induction and expression of thermal tolerance. Ultrapure populations of G₁-, S-, and G₂-M-phase cells were obtained through sequential centrifugal elutriation and flow cytometric cell sorting of Hoechst 33342-stained cells. We found no interaction of Hoechst 33342 with hyperthermia under staining conditions that gave good cytometric resolution of DNA distributions. Single dose-response survival curves indicated that S phase was the most sensitive to 45.5° hyperthermia (D₀ = 1.97, 1.26, and 1.95 min for G₁, S, and G₂-M, respectively). Both S and G₂-M phases exhibited a decreased ability from G₁ to accumulate sublethal heat lesions as evidenced by decreased heat survival curve shoulders (D_q) = 13.7, 9.51, and 8.39 min for G₁, S, and G₂-M, respectively). Thermal tolerance, as measured by the decreased inactivation slope of the split-dose treatment, could be induced and expressed in G₁, S, and G₂-M phases. However, both the magnitude and temporal expression of tolerance were dependent on the position of the cell within the cell cycle at the time of the initial heat treatment. S-phase cells exhibited slightly less thermal tolerance as compared to G₁ cells given isosurvival thermal induction doses as measured by the split-dose inactivation rate constants (heated/control = 8.37 and 5.62 for G₁ cells at 12 and 24 hr and 7.68 and 5.27 for S-phase cells at 12 and 28 hr). Also, split-dose survival curves for cells heated in G₂-M indicated a near total inability to accumulate heat-induced sublethal damage. Simultaneous bivariate (90° light scatter and DNA content) progression analysis of heated replicates indicated that tolerance could probably be expressed in those cells which moved into other cycle compartments following the initial heat treatment. For instance, G₁-phase cells preheated for 20 min began progression into normally heat-sensitive S phase between 24 and 28 hr after the heat treatment. This corresponded to approximately the time of maximal thermal tolerance expression. [³H]Thymidine suicide experiments also indicated that the ultimately clonogenic cells began movement into S phase at or near the time of maximal tolerance. In this case then, tolerance expression appeared to supersede the S-phase acute heat sensitivity. Heated S-phase cells began progression into G₂-M between 4 and 12 hr, which corresponded temporally to large amounts of tolerance expression. Finally, a cohort of heated G₂-M-phase cells began division almost immediately after heating, although a large fraction was unable to divide even after 37 hr. A large number of irregular divisions occurred after heating G₂-M-phase cells, which were not evident in the heated G₁- or S-phase fractions.

¹ Work performed under the auspices of the United States Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-ENG-48.

² Recipient of National Cancer Institute Grant CA 31813. Present address: Department of Radiology, Stanford University, Stanford, CA 94305.

³ Recipient of National Cancer Institute Grant CA 14533.

⁴ Recipient of National Cancer Institute Grant CA 31813. To whom requests for reprints should be addressed.

Received 10/11/83. Accepted 3/ 5/84.