No abstract

Measurement of fluorescein isothiocyanate (FITC) staining intensity of cultured lymphoblastoid cells following hyperthermia showed large increases without concomitant increases in nuclear protein. Similar measurements of cells following incubation with cytotoxic drugs showed fluorescent intensity increases that exceeded the increases in nuclear protein that were due to the cell cycle blocking action of the drug. The reverse, however, was true for cells following irradiation. In contrast, FITC staining intensity and nuclear protein measurements of cells proceeding through the cell cycle after removal of the cycle blocking agent showed nearly parallel changes, although there were reproducible minor differences, especially following blocking with hydroxyurea. These results suggest that FITC staining intensity is a function not only of nuclear protein content but also of stain access to the reaction sites of the protein constituents of the chromatin. Thus, it is possible that FITC staining may be used as a probe of changes in chromatin structure following experimental manipulation of cells in vitro or treatment of tumors in vivo.

Summary Sodium butyrate has been shown to affect cell proliferation, and, at concentrations above approximately 0.5 mM, to cause cell death in some tumour cell lines. When combined with cytotoxic drugs increase in chemosensitivity has been observed. We are presently carrying out a study of the combined effects of sodium butyrate and cytotoxic drugs on cultured cervix tumour cells. To provide a baseline for this study we Prasad, 1980;Kruh, 1982;Nordenberg et al., 1987). Possibly combined with the effect of n-butyrate on cell proliferation is its action in reversibly inducing what has been termed a 'better differentiated phenotype', of increased radiosensitivity and chemosensitivity (Spremuli & Dexter, 1984). Due to these properties n-butyrate has been the subject of clinical investigations in leukaemic patients (Novogrodsky et al., 1983;Miller et al., 1987). Its effects when combined with cytotoxic drugs (Wasserman et al., 1989) or irradiation (Arundel & Leith, 1987; Leith et al., 1986) have also been investigated.We are presently carrying out an investigation of the effect of n-butyrate, when combined with a range of cytotoxic drugs, on cervix tumour cell lines when cultured as multicell spheroids. As a necessary preliminary to this study we have carried out a systematic investigation of the effect of nbutyrate alone on the growth characteristics of cervix tumour cells cultured as multicell spheroids. The effect of n-butyrate concentrations in the range 0.005 to 3 mM has been investigated, and the culture of the cells as multicell spheroids has allowed measurements over periods of up to 24 days. The results of this study are presented in this report. Materials and methods Cell cultureThe cervix tumour cell lines employed in this study were established in primary culture from cervix biopsy tissue taken routinely during radiotherapy at Cookridge Hospital (Dyson et al., 1984a). Six cell lines were used: 754, 612, 995, 090, 329, 708. These were maintained as monolayer cultures from which multicellular aggregates were obtained as required by using the method of Sutherland and Durand (1976). Spheroid cultures were initiated with the same number of spheroids per flask to allow intercomparison of cell counts during the period of the experiments. Multicell spheroids were maintained in culture as previously described (Boothby et al., 1989). Excess spheroids were discarded at the time of media change to maintain cell numbers approximately constant.The necessary volumes of 50 mM or 500 mM sodium nbutyrate solution were added to the spheroid suspensions at the start of the experiment to adjust to the concentrations shown in the figures, with further additions at media changes to maintain these concentrations. The butyrate solution was prepared from n-butyric acid (BDH Limited, Poole, UK) in Hanks basic salt solution, adjusted to pH 7.2 with NaOH solution, then sterilised by filtration through an 0.2 gtm Acrodisc (Gelman Sciences Limited, Northampton, UK).Spheroid diameter This was measured by means of a laser diffracti...

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...