Fluorescein isothiocyanate staining intensity as a probe of hyperthermia‐induced changes in chromatin conformation

Abstract

In a previous report we presented evidence for large increases in fluorescein isothiocyanate (FITC) fluorescent intensity caused by hyperthermia which were not associated with synthesis of heat-shock proteins. We have now refined and considerably extended the measurements of increases in FITC fluorescent intensity caused by hyperthermia within the range 41.0"C to 5O.O0C, and associated these with the extent of cell death caused by the hyperthermia. It appears that cell death ensues when the FITC fluorescent intensity has not returned to its baseline value within the time of one cell cycle. If thermotolerance is induced, there is a concomitant reduction in the increase in FITC staining intensity and the extent of cell death. When hyperthermia is followed by acid extraction, an additional increase in FITC staining intensity (above that due to hyperthermia alone) is observed, indicating separate sites of action on basic nuclear proteins. Hyperthermia and acid extraction have related effects on the relationship between FITC and propidium iodide staining. Hyperthermia-induced increases in FITC staining intensity are almost completely reversed by 6.7 mM formaldehyde with a marginal effect on the control FITC staining at this formaldehyde concentration. We suggest that hyperthermia causes extensive dissociation of basic protein-protein binding within nuclear chromatin, and that this may be a contributory cause of hyperthermia-induced cell death.Key terms: Flow cytometry, fluorescein isothiocyanate, hyperthermia, cell deathThe fluorescent intensity resulting from reaction of the DNA of a cell with a DNA-specific dye (assuming RNase treatment where necessary), under conditions of equil.ibrium with the dye, is influenced by other factors, in addition to the total DNA content of the cell, due to steric hindrance preventing complete reaction of dye with DNA. Thus ionic strength (3), M?' concentration (2,20) metabolic state (3), cell-cycle phase or compartment (3), and degree of differentiation (4,20) have subtle effects on the fluorescent intensity because of their influence on chromatin conformation, which depends on the dye used, on the type of cell, and the type of complex formed between the dye and DNA. Extensive increases are also observed with many DNA-specific dyes following dissociation of basic chromatin proteins by acid, due to unmasking of further dye binding sites on the DNA (4). In contrast to DNA, however, little attention has been paid to the effect of chromatin conformation on the extent of the reaction between chromatin proteins and fluorochromes reactive with protein binding sites.In a previous report from this laboratory (9) we presented measurements demonstrating considerable increases in fluorescein isothiocyanate (FITC) staining intensity of nuclear proteins during and following hyperthermia, and following irradiation or chemotherapy of lymphoid cells in vitro and irradiation of human tumours in vivo. These increases were over and above those due to unbalanced protein synthesis due to cellcycle block...

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A flow cytometric study of the effect of heat on the kinetics of cell proliferation of Chinese hamster V‐79 cells

“…When cells are heated, there is an increase in the stainability of the nuclei with FITC. This is caused by an increase in nuclear protein (Blair et al 1979, Roti Roti et al 1986) and also a concomitant change in the conformation of the chromatin proteins (Dyson et al ,1989. The phenonemon observed is dependent on the experimental protocol.…”