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

We have examined the cell cycle specificity of 45.5$dG heat-induced toxicity and the induction and expression of thermal tolerance. Ultrapure populations of G1-, S-, and G2-M-phase cells were obtained through sequential centrifugal elutriatton and flow cytometric cell sorting of Hoechst 33342-stained cells. We found no interaction of Hoechst 33342 with hyperthermia under staining conditions that gave good cytometric resolution of DNA distributions. Single dose-response survival curves indicated that S phase was the most sensitive to 45.5$dG hyperthermia (D0 = 1.97,1.26, and 1.95 min for G$iG, S, and Gz-M, respectively). Both S and G2-M phases exhibited a decreased ability from d to accumulate sublethal neat lesions as evidenced by decreased heat survival curve shoulders (D.,) = 13.7,9.51, and 8.39 min for Gt, S, and G2-M, respectively). Thermal tolerance, as measured by the decreased inactivatton slope of the split-dose treatment, could be induced and expressed in G$iG, 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 G, 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 Gz-M indicated a near total inability to accumulate heat-induced sublethal damage. Simultaneous Divariate (90$dG light scatter and DNA content) progression analysis of heated replicates indicated that tolerance could probably be expressed in those cells which moved into other cycle compartments following the initial heat treatment. For instance, G,-phase cells preheated for 20 min began progression into normally heat-sensitive S phase between 24 and 28 hr after the heat treatment. This corresponded to approximately the time of maximal thermal tolerance expression. pHJThymidine suicide experiments also indicated that the ultimately donogenil cells began movement into S phase at or near the time of maximal tolerance. In this case then, tolerance expression appeared to supersede the S-phase acute heat sensitivity. Heated S-phase cells began progression into Gz-M between 4 and 12 hr, which corresponded temporally to large amounts of tolerance expression. Finally, a cohort of heated Gz-M-phase cells began division almost immediately after heating, although a large fraction was unable to divide even after 37 hr. A large number of irregular divisions occurred after heating Gz-M-phase cells, which were not evident in the heated G$iG- or S-phase fractions.