Using computerized video time lapse for quantifying cell death of X-irradiated rat embryo cells transfected with c-myc or c-Ha-ras.

Rat embryo fibroblasts that had been transfected with the c-myc or c-Ha-ras oncogene were X-irradiated, after which individual cells and their progeny were followed in multiple fields for 5-6 days by computerized video time lapse microscopy to quantify the lethal events that resulted in loss of clonogenic survival. The loss of clonogenic survival of X-irradiated (9.5 or 2.5 Gy) REC:myc cells was attributed almost entirely to the cells dying by apoptosis, with almost all of the apoptosis occurring after the progeny had divided from one to four times. In contrast, the loss of clonogenic survival of X-irradiated REC:ras cells was attributed to two processes. After 9.5 Gy, approximately approximately 60% of the nonclonogenic cells died by apoptosis (with a very small amount of necrosis), and the other 40% underwent a senescent-type process in which some of the cells and their progeny stopped dividing but remained as viable cells throughout 140 h of observation. Both processes usually occurred after the cells had divided and continued to occur in the cells' progeny for up to five divisions after irradiation. Furthermore, the duration of the apoptotic process was shorter for REC:myc cells (0.5-1 h) than for REC:ras cells (4-5 h). By using computerized video time lapse to follow individual cells, we were able to determine the mode of cell death. This cannot be determined by conventional clonogenic survival experiments. Also, only by following the individual cells and their progeny can the true amount of apoptosis be determined. The cumulative percentage of apoptosis scored in whole populations, without distinguishing between the progeny of individually irradiated cells, does not reflect the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results because both clonogenic and nonclonogenic cells are dividing as nonclonogenic cells are apoptosing or senescing over a period of many days. For example, after 9.5 Gy, which causes reproductive cell death in 99% of both types of cells, the cumulative percentage of the cells scored as dead in the whole population at 60- 80 h after irradiation, when the maximum amount of cumulative apoptosis occurred, was approximately 60% for REC:myc cells, compared with only approximately 40% for REC:ras cells.

Unusual potentiation by vinca alkaloids of the cytostatic and cytocidal action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME) and its nonhydrolyzable ethanone analog (DIPE) on MDA-MB-231 human mammary cancer cells.

Drug interaction between DIME or DIPE ¿1-[3, 5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone¿ with vincristine and vinblastine on the growth rate of MDA-MB-231 human mammary cancer cells was determined by the median effect kinetic method. Mutually exclusive cellular binding sites were identified kinetically and isobologram analyses showed potentiation. The combind effect of 0.75 MICROM DIME and 2 nM vincristine demonstrated a nearly type of mutual activation. It was shown that the nonhydrolyzable DIME derivative DIPE is equivalent to DIME, but because of its biological stability is a preferred drug candidate. Vinblastine-DIME cooperative action is similar to that of vincristine-DIME (or DIPE). Activation of caspase 3 by both DIME and vincristine is greatly potentiated when both drugs are added simultaneously in a given proportion. We propose that following a primary binding of DIME and vinca alkaloids to microtubules, an as yet unrecognized mutual activation of caspase 3 apoptotic path is initiated, explaining DNA fragmentation and cell death. A subpopulation of cancer cells, capable of slow growth at 1.5 microM DIME was identified. This cell type was also killed by the DIME-vincristine drug combination.

Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome.

Pericentrin and gamma-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin-gamma-tubulin complex was distinct from the previously described gamma-tubulin ring complex (gamma-TuRC) as purified gamma-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and gamma-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.

Characterization of radiation-induced apoptosis in rodent cell lines.

For REC:myc(ch1), Rat1 and Rat1:myc(b) cells, we determined the events in the development of radiation-induced apoptosis to be in the following order: cell division followed by chromatin condensation, membrane blebbing, loss of adhesion and the uptake of vital dye. Experimental data which were obtained using 4He ions of well-defined energies and which compared the dependence of apoptosis and clonogenic survival on 4He range strongly suggested that in our cells both apoptosis and loss of clonogenic survival resulted from radiation damage to the cell nucleus. Corroboratory evidence was that BrdU incorporation sensitized these cells to radiation-induced apoptosis. Comparing the dose response for apoptosis and the clonogenic survival curves for Rat1 and Rat1:myc(b) cells, we concluded that radiation-induced apoptosis contributed to the overall radiation-induced cell inactivation as assayed by clonogenic survival, and that a modified linear-quadratic model, proposed previously, modeled such a contribution effectively. In the same context, the selective increase in radiation-induced apoptosis during late S and G2 phases reduced the relative radioresistance observed for clonogenic survival during late S and G2 phases.

Inhibition of migration of MDA-MB-231 cells by methyl-3,5-diiodo-4-(4'-methoxyphenoxy)benzoate (DIME).

The GTPase activity of purified dimeric tubulin (alpha+beta) at 5 mu M was insensitive to methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME), in contrast to nocodazole which activated GTPase. Cellular motility of MDA-MB-231 (human mammary cancer) cells migrating through 12-mu m pores was inhibited by DIME similar to nocodazole in a drug concentration-and DIME structure-dependent manner. An increase of cytoplasmic ATPase activity of DIME-treated cells without a decrease in ATP contents of intact cells suggests that DIME may also influence additional as yet unidentified ATP-dependent system(s) probably also involved in cell motility. These results show that DIME not only arrests cells in M phase but also inhibits cell motility in interphase. However the cellular mode of action of DIME is different from the action of other toxic tubulin-targeted drugs, despite the fact that DIME in a concentration-dependent manner disrupts microtubule structures in intact cells.

Structural specificity and tumoricidal action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME).

Seventeen homologs and analogs of methyl-3,5-diiodo-4(4'-methoxyphenoxy) benzoate (DIME), a hormonally inactive analog of thyroid hormones, have been synthesized and their antitumor activity scored by assaying their antitumorigenic effect in vivo following pretreatment of E-ras 20 cells with the drug. In vivo feeding of DIME in large doses had a similar antitumor effect on human tumor xenogafts in vivo without noticeable toxicity of DIME. Inhibition of clonogenicity with MDA-MB-231 cells by DIME yielded I-50 values similar to those found in tests measuring cell growth inhibition (median I-50 less than 1.0 mu M) The apparent ultimate signal for cytocidal action of DIME is the development of dose-dependent double strand cuts of DNA. All human tumor cells so far tested, with the exception of A-549 cells (lung cancer), show high sensitivity to DIME (I-50 1.0 mu M or below). The partially refractory behavior of A-549 cells to DIME is due to the presence of an esterase that cleaves the methyl ester group of DIME. Other tumor cells have negligible esterase activity, while such activity in homogenates of normal mouse tissues is high. Cells in culture take up DIME and the magnitude of uptake parallels drug sensitivity to DIME. Extrapolation from the correlations between drug efficacy, drug uptake, esterase activity and the absence of significant in vivo toxicity of DIME point to uptake of DIME by cells in culture but not by normal cells operating in intact organs in vivo. In contrast, tumor cells in vivo take up DIME and succumb to its cytocidal action just like cells in culture.

Cellular analysis of the mode of action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME) on tumor cells.

The hormonally inactive methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME) at 1-4 mu M concentration induces morphologic changes in E-ras 20 and MDA-MB-231 and other human cancer cells such as multinucleation and enlargement and arrests the cell cycle in the M phase without affecting interphase. Time-lapse videomicroscopy allowed us to follow individual cells. Cells exposed to DIME divided in an abnormal manner, leading to 20% cell fusion and multinucleation. Chromosome painting demonstrated a large accumulation of chromosomes after 5 days of treatment with DIME, consistent with the failure of cells to divide normally. Chromosome breakage was not observed. On the other hand, highly abnormal tubulin-containing structures ensue upon exposure to DIME (1 mu M for 18 h) treatment, indicating an early biochemical action of DIME on the spindle assembly system.

Heat shock causes protein aggregation and reduced protein solubility at the centrosome and other cytoplasmic locations.

Heat shock markedly inhibited centrosome staining by antisera raised against the two centrosome-specific proteins, pericentrin and gamma tubulin. The inhibition of anti-pericentrin binding was measured by fluorescence imaging. Heat had the greatest effect on intact cells, followed in sensitivity by centrosomes attached to their companion nucleus, with purified centrosomes being least sensitive. The centrosomal content of pericentrin was measured by immunoprecipitation followed by western blotting. Heat caused the amount of pericentrin in the centrosomal fraction to increase, suggesting that pericentrin did not leave the centrosome during heat shock. Furthermore, the pericentrin of the centrosomal fraction became less soluble after heat shock, and could only be solubilized by the most denaturing condition of boiling in 0.1% SDS. Immunoelectron microscopy revealed a heat-induced increase in the electron-dense material comprising the pericentriolar material (PCM), consistent with protein aggregation. Lastly, in heated cells immunoelectron microscopy demonstrated an increase in the binding of heat shock protein 70 (HSP70) to numerous locations throughout the cytoplasm. These data suggest that heat shock reduces the solubility of centrosomal and other cytoplasmic proteins, most likely through protein aggregation.

Apoptosis induced by X-irradiation of rec-myc cells is postmitotic and not predicted by the time after irradiation or behavior of sister cells.

Rat embryo cells expressing the c-myc oncogene (rec-myc) were studied by time-lapse microscopy to determine whether radiation-induced apoptosis occurred before or after mitosis. Following X-irradiation with 9.5 Gy, cells were imaged every 3 min for 6 days. Episodes of apoptotic blebbing were very consistent from cell to cell, lasting 30-60 min, followed by cessation of movement and cell death. In contrast, the time of initiation of apoptotic blebbing was unpredictable. At least 96% of the apoptotic episodes were postmitotic, after one to four cell divisions and 2-97 h after a given division. Sister cells often behaved differently from one another, with apoptosis in one sister occurring many h or several divisions after apoptosis in the other. Thus, the onset of radiation-induced apoptosis in rec-myc cells is not strictly programmed but may result from the segregation of chromosome aberrations in the postirradiation generations.

p53-dependent G1 arrest and p53-independent apoptosis influence the radiobiologic response of glioblastoma.

PURPOSE: Loss of the p53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor response to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p53-mediated radiation-induced apoptosis and G1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells. METHODS AND MATERIALS: Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Isogenic derivatives of glioblastoma cells differing only in their p53 status were generated using a retroviral vector expressing a dominant negative mutant of p53. Radiation-induced apoptosis was assayed by Fluorescence-activated cell sorter (FACS) analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cells were synchronized in early G1 phase and mitotic and labeling indices were measured. RESULTS: Radiation-induced apoptosis of GM cells was independent of functional wild-type p53 (wt p53). Decreased susceptibility to radiation-induced apoptosis was associated with lower alpha values characterizing the shoulder of the clonogenic radiation survival curve. Using isogenic GM cells differing only in their p53 activity, we found that a p53-mediated function, radiation-induced G1 arrest, could also influence the value of alpha and clonogenic radiation resistance. Inactivation of wt p53 function by a dominant negative mutant of p53 resulted in a significantly diminished alpha value with no alteration in cellular susceptibility to radiation-induced apoptosis. The clonal derivative U87-LUX.8 expressing a functional wt p53 had an alpha (Gy-1) value of 0.609, whereas the isogenic clonal derivative U87-175.4 lacking wt p53 function had an alpha (Gy-1) value of 0.175. CONCLUSION: We conclude that two distinct cellular responses to radiation, p53-independent apoptosis and p53-dependent G1-arrest, influence radiobiological parameters that characterize the radiation response of glioblastoma cells. Further understanding of the molecular basis of GM radiation resistance will lead to improvement in existing therapeutic modalities and to the development of novel treatment approaches.

Cell cycle synchrony unmasks the influence of p53 function on radiosensitivity of human glioblastoma cells.

Although ionizing radiation causes DNA damage that can play a role in tumorigenesis, such irradiation is also an important modality of cancer therapy. We studied the radiation response of the U-87 MG human glioblastoma cell line and transfected derivatives in which p53 function had been inactivated. Although little effect of p53 on the radiation sensitivity of asynchronously growing cultures could be detected, inactivation of p53 resulted in a large increase in clonogenic survival when cells synchronized by mitotic selection were irradiated in early G1. The radiation dose sufficient to reduce cellular clonogenicity by 1 log in cells expressing functional p53 was 3.26 +/- 0.12 Gy, whereas a much higher dose (7.41 +/- 0.44 Gy) was required to achieve the same killing effect in cells in which p53 was inactivated. Apoptosis was excluded as a probable mechanism contributing to the radiosensitivity of these cells. Fluorescence-activated cell sorter analysis, continuous labeling with tritiated thymidine, and time-lapse videomicroscopy documented the first example of a prolonged p53-dependent G1 arrest induced by ionizing radiation during the first postirradiation cell cycle of tumor cells, suggesting a role for G1 arrest in determining the sensitivity of these cells to irradiation.

Phosphorylation of NUMA occurs during nuclear breakdown and not mitotic spindle assembly.

NuMA, the nuclear mitotic apparatus protein, is a component of the nuclear matrix at interphase that redistributes to the spindle poles at mitosis. While the function of NuMA is not known, it has been implicated in spindle organization during mitosis and nuclear reformation. Phosphorylation is thought to play a regulatory role in NuMA function. In this study, NuMA phosphorylation was examined through the cell cycle using highly synchronized cells. In intact cells labeled with 32P-orthophosphate, NuMA appeared as a 250 kDa phosphoprotein in interphase that shifted to a higher apparent molecular mass in mitosis. The shift was due to phosphorylation as shown by reduction of the shifted band to interphase mobility by phosphatase treatment. This phosphorylation event occurred roughly at the G2/M transition at the time of NuMA's release from the nucleus and its redistribution to the mitotic spindle. However, mitotic phosphorylation did not require spindle formation since the phosphorylated species was detected in nocodazole-treated cells lacking microtubule spindles. Dephosphorylation of NuMA occurred in two distinct steps, after lamin B assembled into the nuclear lamina, in early G1 and at the end of G1. Based on the timing of the phosphorylation and dephosphorylation observed in this study, we propose that they may play a role in nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.

Thermotolerant cells possess an enhanced capacity to repair heat-induced alterations to centrosome structure and function.

To study the mechanisms of thermotolerance, the adaptive response by which cells become transiently resistant to killing by heat shock, we have focused on the centrosome, an organelle whose disorganization is closely correlated with thermal killing in Chinese hamster ovary (CHO) cells. Centrosome structure was studied by use of antisera directed against pericentrin, a 220 Kd protein of the pericentriolar material (PCM). Centrosome function was measured in intact cells by performing microtubule regrowth following exposure to the drug nocodazole. Immediately following heating at 45 degrees C for 4-18 min, centrosomal staining by antipericentrin decreased. Thereafter, staining gradually recovered, although abnormal configurations of staining appeared in heated cultures 10-20 h later. In contrast, abnormal patterns of staining rarely developed in thermotolerant cultures. Centriole number was not perturbed by heat, indicating that the heat effect was specific for the PCM. Heat also caused an immediate reduction in the number of microtubules nucleated by the PCM. As for staining by antipericentrin, microtubule nucleation recovered during 3-20 h at 37 degrees C after heating. The immediate, heat-induced decrease in antipericentrin staining or microtubule nucleation was similar in thermotolerant and nontolerant cells. In contrast, the inhibition for both endpoints recovered to control levels much more quickly in thermotolerant cells than in nontolerant cells. Furthermore, new protein synthesis was not required for the recovery of microtubule nucleation. These data show that thermotolerant cells have an enhanced capacity to repair thermal damage to centrosome structure and function, and suggest that a faster rate of recovery prevents disorganization of the PCM that is observed in nontolerant cells several hours after heating.

Plasma membrane activities retained after lethal heat shock.

Cultures of Chinese hamster ovary (CHO) cells were examined to determine if heat killing could be attributed to severe damage in the plasma membrane. Three independent transport activities of the plasma membrane were measured. Glucose transport into the cells (measured with the non-metabolizable analogue 3-O-methyl-D-glucose) was stimulated rather than inhibited by heat. Most of the stimulation was found after non-toxic heat doses. Although amino acid transport (measured with the non-metabolizable analogue 2-aminoisobutyric acid) was slightly inhibited by heat, heat-sterilized cells were able to accumulate high intracellular concentrations. Cellular uptake of the nucleoside uridine was unaffected for at least 4 h after heating. In contrast, its incorporation into RNA was immediately inhibited. To further study plasma membrane damage, cells were either heated or treated with drugs which localize to the plasma membrane, ionophore A23187 or amphotericin B. The mode of cell killing by heat was radically different from that of the two drugs: heat-sterilized cells retained a phase-bright morphology and excluded the viability dye trypan blue while drug-killed cells rapidly became phase-dark and absorbed the dye. These results add to a growing list of plasma membrane activities which are retained in heat-sterilized cells, and suggest that the initial thermal damage responsible for cell killing is at an alternate site(s).

Heat shock alters centrosome organization leading to mitotic dysfunction and cell death.

To identify the cellular target(s) responsible for thermal killing in the G1 phase of the cell cycle, synchronous cultures of Chinese hamster ovary cells (CHO) were heat shocked and studied for one cell cycle by time-lapse videomicroscopy and immunocytochemistry. At the first mitosis post-heating, the fraction of cells giving rise to multinucleated progeny approximately equaled the nonclonogenic fraction. In addition, the cells yielding multinucleated progeny were delayed in prophase-metaphase relative to the cells yielding two uninucleated progeny (clonogenic cells). To study the basis for the delay in prophase-metaphase and subsequent formation of multinucleated cells, cells in mitosis were examined by immunofluorescence for spindle abnormalities. Multipolar mitotic spindles and chromosome misalignment were induced by heat. All multiple spindle poles induced by heat stained for pericentriolar material (PCM), the microtubule nucleating material of centrosomes. Heated cells in mitosis also contained additional foci of PCM which were not associated with the spindle. Cells made thermotolerant by a nonlethal heat shock were resistant to both thermal killing and the induction of multiple foci of PCM. Quantitative analysis revealed a good correlation between the fraction of cells with multipolar spindles, the fraction with more than two foci of PCM, and the nonclonogenic fraction. These data indicate that heat-induced alterations to the PCM of centrosomes resulted in multipolar mitotic spindles, delay in prophase-metaphase, and formation of multinucleated cells which were nonclonogenic. These results identify the centrosome as a G1 target for cell killing.

Similar heat-induced cell cycle delays in the clonogenic and nonclonogenic fractions of a thermotolerant population.

We have tested the theory that a faster rate of recovery from thermal damage in thermotolerant cells relative to nontolerant cells is the critical factor which confers heat resistance. We measured the rate of recovery from the heat-induced cell cycle delay in Chinese hamster ovary cells, since this delay is shortened in thermotolerant cells (i.e., exhibits thermotolerance) and can be measured in individual cells. Individual thermotolerant cells were followed by discontinuous microscopic observation from shortly after heating, through the first mitosis after heating, until a colony formed or failed to form by 8 days. The heat-induced delay in the clonogenic and nonclonogenic fractions was the same. This shows that the rate of recovery from the cell cycle delay was not the determining factor as to whether or not a cell survived to form a colony. Either additional factors are involved or the rate of recovery from the cell cycle delay plays no role in cell survival. These data show the importance of determining whether a faster rate of repair of thermal damage is specific for the clonogenic fraction, since most if not all types of thermal damage are likely to be repaired more quickly when thermotolerant and nontolerant cells are compared at isodose.

Maintenance of intracellular free Ca2+ homeostasis following lethal heat shock.

We have manipulated the extracellular Ca2+ concentration (1.8 mM in normal Dulbecco's modified Eagle's medium) to test whether the resulting effect on intracellular free Ca2+ homeostasis was similar in heat-sterilized and nonheated mouse NIH-3T3 cells. The responsiveness of the intracellular free Ca2+ concentration to changes in the extracellular Ca2+ concentration was not affected by prior treatment of the cells with trypsin, or by the extracellular Ca2+ concentration during dye loading (indo-1, AM). Rather, the intracellular free Ca2+ concentration was dependent upon the ambient Ca2+ concentration during analysis by flow cytometry. When the extracellular Ca2+ concentration was decreased to 0.017 mM, either before or after a lethal heat shock, the intracellular free Ca2+ concentration (approximately 300 nM) decreased to a similar extent in both heated and control cells (to approximately 30-100 nM). Similarly, when the extracellular Ca2+ concentration was increased to 15.0 mM, either before or after a lethal heat shock, the intracellular free Ca2+ concentration exhibited a quantitatively similar increase in both heated and nonheated cells (to approximately 400-1000 nM). These data indicate that a lethal heat dose does not inhibit the intact cell's ability to maintain intracellular free Ca2+ homeostasis.

Division-associated and division-independent hyperthermic cell death: comparison with other cytotoxic agents.

We have heated Chinese hamster ovary (CHO) cells in G1 phase, and followed them by microscopic observation for 8 days. Non-clonogenic cells were monitored with regard to when they died, and whether death was preceded by cell division. Such cells underwent either a division-independent or division-associated mode of death. At high survival levels most cells died a division-associated death, while at low survival levels division-independent death predominated. Mouse 10T1/2 cells died primarily according to the division-independent mode, regardless of survival level. These data are consistent with two distinct targets for the thermal killing of G1 cells, one of which involves the cell's ability to divide properly. The heat-induced division delay was similar for both clonogenic and non-clonogenic cells, suggesting that cell death was not due to lengthened cell cycle times. Hydrogen peroxide caused more division-independent death than heat, at isosurvival. The mode of death following cis-platinum treatment was largely division-associated, regardless of survival level. The combination of heat and cis-platinum caused synergistic killing that was due to a potentiation of cis-platinum killing by heat, since the pattern of death was clearly similar to that caused by cis-platinum alone.

Noninvolvement of the heat-induced increase in the concentration of intracellular free Ca2+ in killing by heat and induction of thermotolerance.

Mouse C3H 10T1/2 cells exhibited a two- to threefold increase in the concentration of free Ca2+ during heating at 45 degrees C. The increase was maximal for a heat dose which was still in the shoulder region of the survival curve. The increase was fully reversible in heat-sterilized cells. By changing the concentration of extracellular Ca2+, it was possible to modulate the concentration of intracellular free Ca2+ in heated cells. Lowering the extracellular concentration to 0.03 mM reduced the baseline concentration of intracellular free Ca2+, and prevented it from increasing in heated cells to a level exceeding that of nonheated cells incubated in medium containing 2.0 or 5.0 mM Ca2+. Raising the concentration of extracellular Ca2+ to 15.0 mM raised the baseline, and resulted in a heat-induced increase in free Ca2+ which was twofold higher than that of cells heated in medium containing 2.0 or 5.0 mM Ca2+. An elevated concentration of intracellular free Ca2+ during and after heating did not potentiate thermal killing, nor did a reduced concentration during and after heating mitigate killing. Furthermore, the data argue against a heat-induced increase in free Ca2+ to some threshold level, which potentiates cell killing by some other parameter. In addition, cells heat-shocked in either 0.03 or 5.0 mM extracellular Ca2+, and then incubated in the same concentration for 12 h at 37 degrees C, developed quantitatively similar amounts of tolerance to a second heating. The data suggest that the concentration of intracellular free Ca2+ does not play a critical role in thermal killing or the induction and development of thermotolerance.

Rapidly reversible enzyme inhibition in a temperature-sensitive mammalian cell mutant lacks thermotolerance.

The temperature-sensitive (ts) Chinese hamster ovary (CHO) cell mutant tsH1 contains a thermolabile leucyl-tRNA synthetase. Upon incubation at the nonpermissive temperature of 39.5 degrees C, the enzyme became reversibly inhibited over a period of minutes, and the cells lost viability over a period of many hours. However, killing of tsH1 by acute heating at 45 degrees C was identical to that of wild-type (SC) cells. In addition, the heat-induced inhibition of protein synthesis was similar for both cell types, as measured after acute heating at 45 degrees C. Furthermore, both killing and inhibition of protein synthesis showed thermotolerance in both cell types. In contrast to the effects at 45 degrees C, at 39.5 degrees C, neither the inhibition of leucyl-tRNA synthetase activity nor the killing of tsH1 expressed thermotolerance. Also, treatment of tsH1 at 39.5 degrees C did not induce thermotolerance to killing at 45 degrees C. The inhibition of leucyl-tRNA synthetase activity in tsH1 at 39.5 degrees C was further distinguished from the 45 degrees C-induced inhibition of protein synthesis in SC cells by a much more rapid reversal of the inhibition of leucyl-tRNA synthetase activity. Also, the rate of reversal of the inhibition of protein synthesis by 45 degrees C in SC cells was decreased by increased heat dose. Such was not true for the 39.5 degrees C inhibition of leucyl-tRNA synthetase activity in tsH1. The data indicate that there exist two distinct types of thermal inhibition--one slowly reversible type which was observed during and after heating at 45 degrees C and both induced and expressed thermotolerance, and a second, rapidly reversible type, which was evident only during heating of tsH1 at 39.5 degrees C and neither induced nor expressed thermotolerance.