A. J. Moss

Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures

Cultured Chinese hamster V79 fibroblast cells at the transition from logarithmic to stationary growth have been shown to undergo apoptosis (programmed cell death) after cold shock [B. L. Soloff, W. A. Nagle, A. J. Moss, Jr., K. J. Henle, and J. T. Crawford, Biochem. Biophys. Res. Commun. 145, 876-883 (1987)]. In this report, we show that about 95% of the cell population was susceptible to cold-induced apoptosis, and the amount of cell killing was dependent on the duration of hypothermia. Cells treated for 0-90 min at 0 degrees C exhibited an exponential survival curve with a D0 of 32 min; thus, even short exposures to the cold (e.g., 5 min) produced measurable cell killing. The cold-induced injury was not produced by freezing, because similar results were observed at 6 degrees C, and cell killing was not influenced by the cryoprotective agent dimethyl sulfoxide. Cold-induced apoptosis was inhibited by rewarming at 23 degrees C, compared to 37 degrees C, by inhibitors of macromolecular synthesis, such as cycloheximide, and by 0.8 mM zinc sulfate. The results suggest that apoptosis represents a new manifestation of cell injury after brief exposure to 0-6 degrees C hypothermia.

Significance of protein-bound neuraminic acid levels in patients with prostatic and bladder carcinoma*

The role of plasma N-acetyl neuraminic acid (NANA) level in detection and staging prostatic and bladder carcinomas was evaluated. Multiple determinations were obtained in 73 male subjects, including normal controls, patients with prostatic or bladder carcinoma or with benign prostatic hyperplasia. Patients with prostatic or bladder carcinoma had significantly higher NANA levels than normal individuals. Further, in patients with prostatic carcinoma, NANA values correlated well with both the stage of the disease and the response to therapy.

Cellular ATP Content of Heated Chinese Hamster Ovary Cells

Hyperthermia at either 41.5 or 45 degrees C with variable heating times to reduce cell survival up to three orders of magnitude did not decrease significantly cellular ATP content when measured either immediately or up to 7 hr after a heat treatment. Similarly, cellular ATP content was not significantly reduced with step-down heating, precooling prior to hyperthermia, or thermotolerance induction. The data suggest that heat-induced depletion of intracellular ATP content is not a critical factor in the thermal death of cells heated under normal culture conditions.

Inhibitors of Poly(ADP-ribose) Synthetase Enhance the Cytotoxicity of 42°C and 45°C Hyperthermia in Cultured Chinese Hamster Cells

SummaryTwo inhibitors of poly(ADP-ribose) synthetase, 5-methylnicotinamide and m-methoxybenzamide, enhanced the cytotoxicity of 42°C and 45°C hyperthermia in cultured Chinese hamster V79 cells. The inhibitors showed minimal toxicity for cells treated at 37°C, and did not appreciably alter cellular ATP levels under any of the experimental conditions used. Enhanced cell killing occurred when the inhibitors were added after an acute (5–10 min) 45°C heat shock, and after 50 and 100 min exposures to 42°C. When present during heating at 42°C, the inhibitors reduced the shoulder of the 42°C survival curves but did not appreciably affect the slopes. The results suggest a possible role for poly(ADP-ribose) synthetase in the survival response of V79 cells to hyperthermia.

Sensitization of Cultured Chinese Hamster Cells to 42°C Hyperthermia by Pentalenolactone, an Inhibitor of Glycolytic ATP Synthesis

The antibiotic pentalenolactone, a specific inhibitor of glyceraldehydephosphate dehydrogenase, was used to investigate the effect of glycolytic adenosine triphosphate (ATP) synthesis on the survival response of aerobic and hypoxic Chinese hamster cells treated with 42 degrees C hyperthermia. Data obtained with aerobic cells, incubated in balanced salt solutions supplemented with different substrates for ATP production, showed that 50 microM pentalenolactone blocked ATP synthesis via glycolysis but not by oxidative phosphorylation. The glycolytic inhibition was reversed upon transfer of the cells to antibiotic-free medium, and minimal cytotoxicity (less than 20 per cent) was observed. Hypoxic cultures were obtained by incubating dense cell suspensions (2 X 10(6)/ml) to produce metabolic oxygen depletion. Concomitant with the development of hypoxia, pentalenolactone-treated cells became ATP-depleted; cellular ATP levels were reduced by about 70-fold as compared to hypoxic cells in the antibiotic-free medium. The ATP-depleted cells were more sensitive to killing by hyperthermia. Comparison of the 42 degrees C survival curves for control and the antibiotic-treated hypoxic cells yielded a dose-modifying factor of 4 (5 per cent survival level). The results indicate that inhibition of glycolytic ATP synthesis, for example by pentalenolactone, can selectively sensitize hypoxic cells to the lethal effects of mild hyperthermia.

Exposure to Pretreatment Hypothermia as a Determinant of Heat Killing

When Chinese hamster ovary (CHO) cells were exposed to 22 degrees C for 2 hr prior to 42.4 degrees C hyperthermia, neither the shoulder region of the survival curve nor the characteristic development of thermotolerance after 3-4 hr of heating were observed. Absolute cell survival after 4 hr at 42.4 degrees C was decreased by a factor of between 10 and 100 (depending on the rate of heating of nonprecooled controls). Conditioning at 30 degrees C for 2 hr, 26 degrees C for 2 hr, or 22 degrees C for 20 min followed by heating to 42.4 degrees C over 30 min did not result in sensitization. Prolonged (16 hr) conditioning at 30 degrees C, however, increased the cytotoxicity of immediate exposure to 41.4 or 45 degrees C with maximum sensitization to 45 degrees C occurring after 6 hr at 30 degrees C. Both 3- and 18-hr pretreatments at 30 degrees C similarly increased the cytotoxicity of 45-41.5 degrees C step-down heating (D0 = 28 min in precooled versus 40 min in nonprecooled cells).

Heat protection by sugars and sugar analogues

We have examined the relative ability of 16 sugars and sugar analogues to reduce cell killing by hyperthermia of 40 min, 45 degrees C. In general, sugars were added to the culture medium 6 h prior to heating at a concentration of 100 mM (400 mosmol). The results show that D-hexoses, L-hexoses, methylated or thiolated sugars and disaccharides significantly protected cells against thermal damage, increasing survival by factors of 10 to 100. The degree of protection varied for specific sugars and could not be predicted on the basis of sugar conformation or the number of hydroxyl groups. Relative heat protection was partially dependent on the survival assay technique (pre- and post-plating); consistently lower cell survival was measured when cells were subcultured after hyperthermia, both in medium-control and sugar-protected cells. However, the time dependence of heat protection appeared independent of pre- and post-plating. Cell survival after heating was not increased by two sugars: (a) D-idose, and (b) 2-deoxy-D-galactose. The latter sugar, curiously, was also a heat protector but only when cells were trypsinized after hyperthermia. Both of these sugars were relatively more toxic at 37 degrees C under identical treatment conditions. The lack of protection by these two sugars is not understood. Another reported non-sugar heat protector, sodium butyrate, was included as an additional control. Heat protection by butyrate was not observed in CHO cells. The accumulation of intracellular free sugar was measured by gas chromatography after incubating cells for 6 h, 37 degrees C with talose, idose, L-galactose or 1-O-methyl-D-glucose. All of these sugars were found in high concentrations inside of cells. The data are consistent with the hypothesis that polyhydroxy compounds must accumulate intracellularly for cellular heat protection.

Interactions of BCNU, low pH, glucose and hyperthermia in cultured RIF cells

The interactions of BCNU (1,3-bis[2-chloroethyl]-1-nitrosourea) with low pH, glucose and hyperthermia were studied in cultured RIF tumor cells. The effect of a mild heat treatment of 43 degrees C, 1 h at pH 7.4 on cell killing [surviving fraction (S) = 0.27 +/- 0.05, standard error of the mean (S.E.)] was significantly enhanced by pH 6.5 (S = 0.11 +/- 0.02, S.E.) and 50 mM D-glucose (S = 0.14 +/- 0.01, S.E.). When heat (43 degrees C, 1 h) was added to BCNU, cytotoxicity was increased approximately 14-fold over BCNU alone. Moreover, pH 6.5 increased killing with BCNU and heat by an additional factor of 28. The presence of glucose at 37 degrees C at either pH 6.5 or 7.4 reduced BCNU toxicity in a dose dependent fashion. However, the presence of glucose did not reduce cell killing by BCNU at 43 degrees C. As a result BCNU cytotoxicity was enhanced by approximately 2 orders of magnitude when tumor cell acidification (glucose and low pH) was combined with BCNU and heat.

Initial studies with a line of radioresistant rat tumor cells.

This paper reports the results of studies with a line of cultured rat sarcoma cells (RT-2 cells). Survival curves indicate the cells to be highly radioresistant [

In situ repair by human malignant cells of radiation-induced DNA breaks.

D_{0}=240\ \text{rads}