FACS analysis of a hyperthermia-induced alteration in Hoechst 33342 permeability and direct measurement of its relationship to cell survival.

Abstract

Heat-induced alterations in CHO-10B cell Hoechst 33342 (Ho342) permeability in vitro were analyzed by flow cytometry. Immediately after 45.5 degrees C heating, uptake was decreased in a dose-dependent manner with cytotoxicity. Kinetic analysis indicated that a treatment that reduced cell survival to approximately 10%, reduced the maximal velocity, Vmax, to 53% of control and increased the dissociation constant, Km, to 156% of control. Also, little change in Ho342 efflux was found to occur from control up to 90 min after heating. Upon incubation at 37 degrees C after the heat treatment from 1 to 24 hr (depending on the severity of the dose) diffuse heterogeneity of Ho342 staining developed which was not evident immediately after heating. The altered staining was not due to the presence of trypan blue staining cells. Membrane permeabilization and nuclei isolation studies indicated that the lesion responsible was most likely a plasma membrane event. Induction of the heterogenous staining was not inhibited by either actinomycin D or hydroxyurea but was inhibited by incubation at 4 degrees C. An inverse correlation existed between Ho342 permeability and clonogenicity, with nearly a 10-fold difference in survival between the high and low fluorescence intensity sorted cells. Also, larger fractions of heat-sensitive S and G2M-phase cells were found in the highly fluorescent sorted fractions. These results are discussed in terms of the putative molecular events that may be involved in hyperthermic modulation of Ho342 permeability.