Diana A. Bates

Thermal adaptation in CHO cells at 40°C: the influence of growth conditions and the role of heat shock proteins

The kinetics of thermal adaptation at the nonlethal temperature of 40 degrees C was studied in CHO (Chinese hamster ovary) cells in vitro. Thermal resistance, demonstrated as an increase in mean 45 degrees C killing time or as an increase in the shoulder of the 45 degrees C survival curve, was fully developed by 2 h. Control cells in early logarithmic phase were more heat sensitive than those in stationary phase. Corresponding 45 degrees C killing time frequency distributions were unimodal with an increase in mean killing time from early logarithmic to stationary phase. Cells which were thermally adapted at 40 degrees C for 6 h had biphasic 45 degrees C killing time frequency distributions, and as cells progressed from early logarithmic to stationary phase the heat-sensitive subpopulation progressively declined. Exposure to 40 degrees C produced a 30% increase in total protein synthesis. Proteins with molecular weights 72, 89, and 109 kDa which correspond to those induced by lethal heat shock were synthesized at 40 degrees C, but there was no close temporal correlation between the development of heat resistance at 40 degrees C and synthesis of the heat shock proteins. Cycloheximide (100 micrograms/ml) reduced the mean 45 degrees C killing time but did not totally prevent the development of heat resistance at 40 degrees C.

The effect of hyperthermia on the sodium-potassium pump in Chinese hamster ovary cells.

The effect of hyperthermia on the Na+-K+ pump was determined by measuring influx and efflux of 86Rb+ in Chinese hamster ovary cells from 31 to 50 degrees C. The maximum initial rate of ouabain-sensitive influx increased with temperature between 31 and 45 degrees C although Km increased significantly above 37 degrees C, implying a diminished affinity of the transport protein for its substrate. The changes in the kinetics of influx at temperatures up to 45 degrees C were rapidly reversible on return to 37 degrees C. Above 45 degrees C an irreversible decrease in 86Rb+ uptake was observed. Efflux of 86Rb+ increased from 31 to 40 degrees C but above 43 degrees C showed a small but significant decrease. The study of 86Rb+ influx after varying times of exposure to elevated temperatures showed that the Na+-K+ pump remains functional in cells which are reproductively dead. We have shown that although the kinetics of K+ transport are sensitive to temperature changes in the range used in clinical hyperthermia, the inactivation of the Na+-K+ pump is not a primary event in cell killing.

The effect of hyperthermia on the uptake and cytotoxicity of melphalan in chinese hamster ovary cells

The effect of temperature on the cytotoxicity of melphalan in CHO cells was studied in an in vitro clonogenic assay. The cytotoxicity of melphalan was significantly increased at elevated temperatures with a 4 fold increase in cytotoxicity at 42 degrees C compared to 37 degrees C. The effect of temperature on membrane permeability to melphalan was studied to determine whether the increase in cytotoxicity was due to increased intracellular drug levels. Melphalan influx and efflux rates both increase with increasing temperature. There is, however, a small net increase in steady state intracellular drug levels with increasing temperature with a 20% increase in intracellular drug levels at 42 degrees C compared to 37 degrees C. The increase in drug uptake observed in insufficient to explain the much greater increase in cytotoxicity with increasing temperature.

Adriamycin uptake, intracellular binding and cytotoxicity in Chinese hamster ovary cells

The interaction between adriamycin influx, efflux and cytotoxicity has been studied in Chinese hamster ovary (CHO) cells. The rate of adriamycin uptake showed no evidence of saturation with increasing concentration suggesting that uptake occurred by passive diffusion. Plateau levels of adriamycin were reached within 20 min and more than 60% of this was firmly bound to the nucleus and did not efflux. Cytotoxicity studies did not correlate with uptake studies in that cell killing was an exponential function of drug exposure time well beyond the point at which intracellular concentrations had reached a maximum. This suggests that bound adriamycin is not primarily responsible for cytotoxicity.

On the difficulties of fitting the double Michaelis-Menten equation to kinetic data

The double Michaelis-Menten equation describes the reaction kinetics of two independent, saturable uptake mechanisms. The use of this equation to describe drug uptake has been reported several times in the literature, and several methods have been published to fit the equation to data. So far, however, confidence intervals on the fitted kinetic parameters have not been provided. We present a grid-search method for fitting the double Michaelis-Menten equation to kinetic uptake data, and a Monte-Carlo procedure for estimating confidence intervals on the fitted parameters. We show that the fitting problem is extremely ill-conditioned, and that very accurate data are required before any confidence can be placed in the fitted parameters.

The effect of hyperthermia on the cytotoxicity of melphalan in Chinese hamster ovary cells.

Abstract The heat stability of melphalan during incubation at temperatures from 37°C to 45°C was determined by spectrophotometric and HPLC analyses and by direct measurement of melphalan cytotoxicity using a colonogenic assay Although OD 250 changed little during exposure to temperatures up to 45°C for periods of up to 1h, the melphalan HPLC peak decreased as function of incubation time and temperature. Loss of cytotoxicity following heating paralleled the decay of the melphalan HPLC peak. Despite the inactivation of melphalan by heat, the cytotoxic effects of melphalan were enhanced at elevated temperatures from 38°C to 42°C and synergism was observed at lethal temperatures above 42°C.

The effect of hyperthermia on intracellular K+ in Chinese hamster ovary cells.

The effect of hyperthermia on intracellular K+ concentrations was studied in Chinese hamster ovary (CHO) cells in vitro, using a flame photometer. Intracellular K+ concentrations decreased with increasing exposure time at temperatures from 40 degrees C to 45 degrees C. The decrease in K+ concentrations preceded any loss of reproductive capability at 43 degrees C and also occurred at the non-lethal temperature of 40 degrees C. Prolonged exposure to 45 degrees C resulted in an irreversible decrease in K+ concentrations. The decrease in K+ concentrations at elevated temperatures was not accounted for by changes in cell volume, loss of cells or failure of the Na+/K+ pump.

A Model-Free Way of Representing Hyperthermia Cell Survival Data

We present a method of fitting curves to cell survival data that is free from all model assumptions, requiring only that the fitted curves be decreasing and reasonably smooth, where the degree of smoothness is determined from considerations of experimental error. The fitted curves are then differentiated to yield frequency distributions of cell killing times, which may be of value in defining subpopulations with different sensitivities to the cytotoxic agent under study. In addition, confidence intervals on the fitted curves and frequency distributions are obtained by Monte Carlo simulation. The results allow the objective and model-free assessment of the effects of various experimental interventions on cell survival.

THE RELATIONSHIP BETWEEN MEMBRANE PERMEABILITY TO ADRIAMYCIN AND ADRIAMYCIN CYTOTOXICITY IN CHO CELLS AT ELEVATED TEMPERATURES

The cytotoxicity of adriamycin at elevated temperatures was studied in CHO cells in vitro using a clonogenic assay. There is a significant increase in the cytotoxicity of adriamycin at temperatures which are not lethal to mammalian cells (38 degrees C-42 degrees C). The uptake of [14C]adriamycin was studied in similar conditions to determine whether the thermal enhancement of adriamycin cytotoxicity was due to an increase in membrane permeability. Although measurable increases in adriamycin uptake occur in the elevated but non-lethal temperature range, they are insufficient to explain the observed increase in cytotoxicity.

The effect of hyperthermia on the cytotoxicity of melphalan in chinese hamster ovary cells in vitro

The heat stability of melphalan during incubation at temperatures from 37 degrees C to 45 degrees C was determined by spectrophotometric and HPLC analyses and by direct measurement of melphalan cytotoxicity using a colonogenic assay Although OD 250 changed little during exposure to temperatures up to 45 degrees C for periods of up to 1 h, the melphalan HPLC peak decreased as function of incubation time and temperature. Loss of cytotoxicity following heating paralleled the decay of the melphalan HPLC peak. Despite the inactivation of melphalan by heat, the cytotoxic effects of melphalan were enhanced at elevated temperatures from 38 degrees C to 42 degrees C and synergism was observed at lethal temperatures above 42 degrees C.