Biochemical analysis of heat-resistant mouse tumor cell strains: a new member of the HSP70 family.

A series of heat-resistant mutants selected from a murine tumor cell line, RIF-1, display a markedly increased and stable resistance to heat shock. The mutant cell lines were analyzed for differences that may explain their increased resistance. Membrane lipid analysis showed no change in cholesterol content but an increase in the proportion of saturated fatty acids in the phospholipid fraction. Two-dimensional gel analysis revealed a generally increased constitutive synthesis of several major heat shock proteins (HSP), including HSP90, 68, 60, and 28. In addition, a new protein in the 70-kilodalton region is present in the resistant lines. The new protein has a lower isoelectric point than the constitutive HSP70 does, is only weakly induced by heat shock, and is immunologically cross-reactive with other members of the HSP70 family. After heat shock, the mutants display increases in HSP similar to those seen in the wild-type cells and they develop further transient tolerance to heat. Analysis of these mutants may help in understanding the function of HSP, both in normal growth and after heat shock.

Isolation and initial characterization of thermoresistant RIF tumor cell strains.

Heat-resistant cell strains were obtained from RIF-1 mouse tumor cells by repeated heatings of cells derived from survivors of previous heating cycles (60 min; 45 degrees C). Twenty thermally resistant (TR) strains were derived from single cells that had survived 11 heating and regrowth cycles. These were then analyzed for appropriate characteristics in vitro and in vivo. In vitro we looked for: marked heat resistance; high plating efficiency; growth rate similar to that of RIF-1 cells; and no obvious morphological abnormalities. In syngeneic hosts, we looked for: ability of the cells to form tumors whose growth rates were similar to that of RIF-1 tumors; high cellular heat resistance; good plating efficiency of tumor-derived cells; and low immunogenicity. Five strains having these desired characteristics were analyzed for survival kinetics. The heat-resistant phenotype was found to be stable in vitro, although partial reversion in vivo was seen occasionally. The "break" in the Arrhenius plot was found to occur at 45 degrees C in TR strains versus 43 degrees C in RIF-1. All TR strains and the RIF-1 line developed similar levels of thermotolerance (as defined by slope ratios) when given isosurvival heat exposures. X-ray responses of TR and RIF-1 cells were indistinguishable both with respect to survival and to heat-induced radiosensitization. While the number of live cells required to give tumor takes in 50% of the recipients for TR strains was appreciably higher than that for RIF-1 cells, radiation-killed cells from none of the strains were able to immunize efficiently against subsequent challenges by live cells.

Modification of pH of normal and malignant mouse tissue by hydralazine and glucose, with and without breathing of 5% CO2 and 95% air.

We investigated the effects of hydralazine and glucose on the pH of several normal tissues and on RIF-1 radiation induced fibrosarcomas of mice that were breathing either air or a mixture of 5% CO2 and 95% air. Our goal was to investigate techniques to maximize the pH differences between normal tissue and tumors. Hydralazine (10 mg/kg) had only minor effects on pH of tumors and muscle; it lowered liver and kidney pH. In animals additionally breathing the air-gas mixture, pH was further lowered in liver and kidney. Glucose (0.6 mg/kg) by itself caused a major reduction in pH of liver, kidney, and adipose tissue. Only minor effects were seen in tumors and muscle. Causing the animals to breathe the gas-air mixture 4 h after glucose injection partially reversed the glucose effect in all normal tissues but caused additional reductions in tumors, particularly in small (less than 0.7 cm3) lesions. Thus, this last combination led to maximum differential between the pH of normal tissue and of tumor.

Tumor responses following multiple hyperthermia and X-ray treatments: role of thermotolerance at the cellular level.

We have investigated the effect of increasing numbers of hyperthermia fractions given at 7-day intervals, with or without fractionated radiotherapy, on tumor cure, tumor growth, and cell survival after in vivo or in vitro heat. The murine RIF tumor was treated by capacitive radiofrequency hyperthermia at 44.0 degrees C for 20 min for one to five treatments at weekly intervals (1-5 wk D1). Single treatments (1 wk D1) induced cure in 5% of tumors. Additional treatments (2-5 wk D1) induced similar rates of cure (0-16%, P greater than 0.05 for 1 wk versus 2, 3, 4 or 5 wk D1). 1 wk D1 resulted in marked growth delay compared to controls. Mean tumor diameter doubling times increased from 13.2 days to 27.5 days (P less than or equal to 0.01). 2-5 wk D1 induced little additional growth delay (doubling times, 27.8-32.3 days, P greater than 0.05 for 1 wk versus 2, 3, 4 or 5 wk D1). Fractionated radiotherapy of 3200 rads (400 rads given twice each week) significantly prolonged mean tumor doubling time to 26.2 days. The addition of one hyperthermia session to the fractionated radiotherapy (1 wk D1 + XRT) further increased doubling time to 34.2 days (P less than or equal to 0.01). Additional treatments (2-5 wk D1 + XRT) only modestly increased doubling times (36.0-39.5 days, P greater than 0.05 for 1 wk versus 2, 3, 4 or 5 wk D1). In vitro assay of cells dissociated from tumors 5, 10, or 15 days after 3 wk D1 showed increased survival to 44 degrees C compared to previously untreated controls, and this cellular thermoresistance proved to be transient and noninheritable (i.e., thermotolerance). These results indicate that tumors can develop a prolonged thermal resistance after multiple weekly treatments which significantly modifies the response to subsequent treatment and which is associated with cellular thermotolerance.

The significance of thermotolerance after 41 degrees C hyperthermia: in vivo and in vitro tumor and normal tissue investigations.

We have investigated the development of thermotolerance in both tumors and normal tissues after 41 degrees C for durations as brief as 15 minutes. The murine RIF tumor, treated by both local radiofrequency and systemic methods, was assayed for thermotolerance by both tumor growth and cell survival analyses. The murine leg and ear, treated by conductive methods, were assayed using pre-defined tissue damage scoring systems. All of these treatments quickly induced substantial levels of thermotolerance. In the tumor studies using local heating, RIF mean diameter doubling time decreased from 17.8 days to a minimum of 13.0 days with a 9 hr interval between 41.0 degrees C for 15 minutes and 44.0 degrees C for 30 minutes (9 hr D1-D2); cell survival increased from 1.2 X 10(-2) to 3.4 X 10(-1) (same interval). Both assays showed some degree of tolerance present immediately after 41.0 degrees C for 15, 30 or 60 minutes (0 hr D1-D2). In the tumor studies using systemic heating, the kinetic pattern of the induced tolerance was similar to that observed after local heating. In the leg studies, 41.0 degrees for 30 minutes increased the time at 45 degrees C necessary to induce a specified level of tissue damage (mean score of 7) by a maximum of 1.8 times (24 hr D1-D2). The kinetic pattern was similar to that for the tumors. In the ear studies, 41.0 degrees C for 30 minutes increased the time at 45 degrees C necessary to induce ear necrosis in 50% of animals by a maximum of 3.5 times (48 hr D1-D2). The peak tolerance level occurred later for the ears, which have a lower normal temperature of 28-30 degrees C, than for the tumors or legs. These results indicate that: thermotolerance can begin to appear in tumors during treatment if hyperthermia sessions involve initial low thermal exposures (near 41 degrees C) for 15 minutes or longer; thermotolerance can develop in tumors after systemic heating and occurs with a kinetic pattern similar to that following local heating; and normal tissues also can develop high levels of thermotolerance after similar thermal exposures.

Thermotolerance and heat shock protein induction by slow rates of heating.

The magnitude of thermotolerance and the level of heat shock protein (HSP) expression have been measured in Chinese hamster ovary cells after gradual temperature transients from 37 degrees or 39 degrees to 42 degrees or 43 degrees C. When the level of thermotolerance was measured by clonogenic survival after challenging temperatures between 42 degrees and 43 degrees, substantial thermotolerance was observed. However, when the challenging temperature was raised to 45 degrees C, proportionally less thermotolerance was apparent. Heat shock proteins were quantitated by scanning densitometry of radiographs and, in the case of HSP 70, by immunoassay. Scanning densitometry revealed that low levels of heat shock proteins were synthesized during the heating gradients, but less than after a heat shock at 45 degrees C that delivered an equivalent heat dose. The immunoassay of HSP 70 levels measures both pre-existing and newly synthesized protein, and showed that there was net increase in HSP 70 during two of the heating gradients tested, despite the increase in synthesis noted on the gels. Higher turnover of HSP 70 at the elevated temperatures possibly accounted for the failure to detect a net gain in total protein. In contrast, the total amount of HSP 70 doubled during the 6 hr following a heat shock of 45 degrees for 10 min, an equivalent heat dose to one of the gradients where no net increase in HSP 70 was measured by immunoassay. It appears, then, that tolerance to hyperthemia at 43 degrees C or below may occur under some conditions in the absence of elevated levels of HSP 70, but tolerance to higher temperatures is more closely correlated with increased levels of heat shock proteins. However, even at higher temperatures, our data show disparities between the levels of HSP measured and the thermotolerance expressed.

Heterogeneity of heat response in murine, canine and human tumors: influence on predictive assays.

The heterogeneity of response to hyperthermia of cells taken from different regions of tumors was tested in a model tumor system (RIF-1) in the mouse and in specimens from spontaneous tumors taken from dogs and humans at the time of surgical resection. Cell survival was assayed by clonogenic survival in the murine tumor and by dansyl lysine staining in tumors from all three species. Using survival as an endpoint, it was found that the extent of heterogeneity depended on the temperature to which the tumor was heated and the duration of exposure. By increasing either of these factors, the coefficient of variation was increased. The large heterogeneity seen after in vivo heating could not be explained entirely by inhomogeneous heating within the tumor as evidenced by temperature mapping. It is concluded that other microenvironmental factors such as blood flow, pH, O2, and nutrient supply may cause variations in the heat response of the tumor cells in vivo. Little, if any, evidence of cellular heterogeneity was evident for all three species when comparisons were made between samples of 100-200 mg. The canine and human tumors were considerably more heat resistant when dansyl lysine was used as an endpoint. In the RIF-1 tumors, heterogeneity of heat response was greater after in vitro heating than after in vivo heating when small biopsy samples (10-20 mg) were taken, suggesting that some cellular heterogeneity was present.

Inhibition of the recovery from potentially lethal damage by lonidamine.

Lonidamine [1-(2,4-dichlorobenzyl)-1-H-indazol-3-carboxylic acid] is shown to inhibit recovery from potentially lethal damage after exposure of cells to X-rays, methyl methane sulfonate, or bleomycin and heat (43 degrees C, 1h). Inhibition is most effective when the drug is present before and after exposure of 10 to 25 mg l-1, a concentration readily achievable in vivo.

Investigation of adenylate energy charge, phosphorylation potential, and ATP concentration in cells stressed with starvation and heat.

We have attempted to determine the appropriate parameter of energy status associated with the survival of CHO fibroblasts under starvation conditions. Survival correlated well with adenylate energy charge (EC) but not so well with the phosphorylation potential or ATP concentration. Starved cells exhibited the capacity to resist (transiently) decreases in both EC and survival. A fall in EC was associated with decreased survival. Using this correlation, we subsequently investigated whether killing after thermal stress occurred by a mechanism analogous to starvation, perhaps due to inhibition of energy yielding pathways. This hypothesis proved to be false; over 99% of cells were killed before a decrease was observed in any of the parameters of energy status. Cells were, however, sensitized to heat under nutritionally deprived conditions, a finding which may be significant for tumor treatment by heat in vivo.

An immunoassay for heat shock protein 73/72: use of the assay to correlate HSP73/72 levels in mammalian cells with heat response.

An enzyme-linked immunosorbent assay (ELISA) for measurement of levels of heat shock proteins 73 and 72 (HSP73/72) in cultured cells and tissues is described. The assay involves detection of HSP73/72 in cell homogenates in 96-well plates using a specific monoclonal antibody. The assay has been used to explore the relationship between the amount of HSP73/72 in a cell and its response to heat shock, both before and after the development of thermotolerance. Six mammalian cell lines with differing responses to heat were characterized with respect to their response to heat treatments at 44 degrees C and concentrations of HSP73/72. Contrary to the widely expressed idea that the amount of HSP73/72 dictates the degree of heat resistance, no positive correlation between levels of HSP73/72 and heat resistance was found for the six lines tested here: if one particular line, a mutant selected for heat resistance, was excluded from the analysis, there was a negative correlation between HSP73/72 levels and heat resistance. A different result was, however, obtained when thermotolerant (transiently resistant) cells were compared to control cells. Here, we found a good correlation between the extent of thermotolerance and the amount of HSP73/72, suggesting that an increase in HSP73/72 level is important for the development of thermotolerance. The validity of the ELISA technique was checked using a second method for quantifying levels of HSP73/72. This involved uniform radiolabeling of cellular proteins, separation on two-dimensional gels and radioscanning to quantify radioactivity in each protein. The second technique is more powerful in that different isoforms of HSP73/72 can be distinguished, but it is more difficult to perform, is more labour intensive and requires an expensive device for gel scanning. The results using the second technique agreed well with those from the immunoassay and indicated that the level of the highly inducible HSP72 correlated best with the extent of thermotolerance.