FLUORESCENCE-ACTIVATED CELL SORTING ANALYSIS OF THE INDUCTION AND EXPRESSION OF ACUTE THERMAL TOLERANCE WITHIN THE CELL-CYCLE

  • 1 January 1984
    • journal article
    • research article
    • Vol. 44  (6) , 2368-2376
Abstract
The cell [CHO; Chinese hamster ovary] cycle specificity of 45.5.degree. heat-induced toxicity and the induction and expression of thermal tolerance were examined. 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. No interaction of Hoechst 33342 with hyperthermia was found 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.degree. hyperthermia (Do = 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 (Dq = 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 h). 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.degree. 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 h 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 h, which corresponded temporally to large amounts of tolerance expression. 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 h. 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. [Clinical tumor therapy applications is implied.].