Modulation of in vivo HSP70 chaperone activity by Hip and Bag-1.

The chaperone activity of Hsp70 is influenced by the activities of both positive and negative regulatory proteins. In this study, we provide first time evidence for the stimulating effect of the Hsp70-interacting protein Hip on the chaperone activity in the mammalian cytosol. Overexpressing Hip enhances the refolding of the heat-inactivated reporter enzyme luciferase expressed in hamster lung fibroblasts. Also, it protects luciferase from irreversible denaturation under conditions of ATP depletion. We demonstrate that these stimulating actions depend on both the presence of the central Hsp70-binding site and the amino-terminal homo-oligomerization domain of Hip. The carboxyl terminus (amino acids 257-368) comprising the 7 GGMP repeats (Hsc70-like domain) and the Sti1p-like domain are dispensable for the Hip-mediated stimulation of the cellular chaperone activity. Bag-1, which inhibits the Hsp70 chaperone activity both in vitro and in vivo, was found to compete with the stimulatory action of Hip. In cells overexpressing both Hip and Bag-1, the inhibitory effects of Bag-1 were found to be dominant. Our results reveal that in vivo a complex level of regulation of the cellular chaperone activity exists that not only depends on the concentration of Hsp70 but also on the concentration, affinity, and intracellular localization of positive and negative coregulators. As the Hsp70 chaperone machine is also protective in the absence of ATP, our data also demonstrate that cycling between an ATP/ADP-bound state is not absolutely required for the Hsp70 chaperone machine to be active in vivo.

Cycloheximide- and puromycin-induced heat resistance: different effects on cytoplasmic and nuclear luciferases.

Inhibition of translation can result in cytoprotection against heat shock. The mechanism of this protection has remained elusive so far. Here, the thermoprotective effects of the translation inhibitor cycloheximide (CHX) and puromycin were investigated, using as reporter firefly luciferase localized either in the nucleus or in the cytoplasm. A short preincubation of O23 cells with either translation inhibitor was found to attenuate the heat inactivation of a luciferase directed into the cytoplasm, whereas the heat sensitivity of a nuclear-targeted luciferase remained unaffected. After a long-term CHX pretreatment, both luciferases were more heat resistant. Both the cytoplasmic and the nuclear luciferase are protected against heat-induced inactivation in thermotolerant cells and in cells overexpressing heat shock protein (Hsp)70. CHX incubations further attenuated cytoplasmic luciferase inactivation in thermotolerant and in Hsp70 overexpressing cells, even when Hsp70-mediated protection was saturated. It is concluded that protection by translation inhibition is unlikely due to an increase in the pool of free Hsps normally engaged in translation and released from the nascent polypeptide chains on the ribosomes. Rather, a decrease in nascent chains and thermolabile polypeptides may account for the heat resistance promoted by inhibitors of translation.

Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells.

Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-denatured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Ohtsuka, K., Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272, 33283-33289). We now provide further evidence for a functional interaction between Hsp70 and the J-domain of Hsp40 with denatured luciferase resulting in reactivation of heat-denatured luciferase within living mammalian cells. The stimulating effect of Hsp40 on the Hsp70-mediated refolding is lost when the proteins cannot interact as accomplished by their expression in different intracellular compartments. Likewise, the cooperation between Hsp40 and Hsp70 is lost by introduction of a point mutation in the conserved HPD motif of the Hsp40 J-domain or by deletion of the four C-terminal amino acids of Hsp70 (EEVD motif). Most strikingly, co-expression of a truncated protein restricted to the J-domain of Hsp40 had a dominant negative effect on Hsp70-facilitated luciferase reactivation. Taken together, these experiments indicate for the first time that the Hsp70/Hsp40 chaperones functionally interact with a heat-denatured protein within mammalian cells. The dominant negative effect of the Hsp40 J-domain on the activity of Hsp70 demonstrates the importance of J-domain-containing proteins in Hsp70-dependent processes.

Hsp70 and Hsp40 chaperone activities in the cytoplasm and the nucleus of mammalian cells.

The existence and function of a Hsp40-Hsp70 chaperone machinery in mammalian cells in vivo was investigated. The rate of heat inactivation of firefly luciferase transiently expressed in hamster O23 fibroblasts was analyzed in cells co-transfected with the gene encoding the human Hsp40 (Ohtsuka, K. (1993) Biochem. Biophys. Res. Commun. 197, 235-240), the human inducible Hsp70 (Hunt, C., and Morimoto, R. I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6455-6459), or a combination of both. Whereas the expression of human Hsp70 alone in hamster cells was sufficient for the protection of firefly luciferase during heat shock, expression of the human Hsp40 alone was not. Rather, this led to a small but significant increase in the heat sensitivity of luciferase. The expression of the human Hsp40 only led to heat protection when the human Hsp70 was expressed as well. Under such conditions the rate of luciferase reactivation from the heat-inactivated state was increased, but the rate of inactivation during heat shock was not affected. Using constructs that direct firefly luciferase either to the cytoplasm or to the nucleus (Michels, A. A., Nguyen, V.-T., Konings, A. W. T., Kampinga, H. H., and Bensaude, O. (1995) Eur. J. Biochem. 234, 382-389), it was demonstrated that these chaperone functions are found in both compartments. Our data provide the first evidence on how the Hsp40/Hsp70 chaperone complex acts as heat protector in mammalian cells in vivo.

Thermal radiosensitization in heat- and radiation-sensitive mutants of CHO cells.

Recently, it has been hypothesized (Iliakis and Seaner 1990) that DNA double-strand break (dsb) repair proficiency is a prerequisite for heat radiosensitization on the basis of the finding that the radiosensitive and dsb-repair-deficient mutant xrs-5 cell line shows no significant heat-induced radiosensitization (not even for severe heat doses), whereas their wildtype counterpart (CHO) did show such an effect. In the current study, the extent of hyperthermic radiosensitization in a new gamma-radiation-sensitive cell line, irs-20, recently isolated by Stackhouse and Bedford (1991) and a heat-sensitive mutant hs-36 (Harvey and Bedford 1988) was compared with the radiosensitization of their mutual parent CHO 10B12 cell line. The irs-20 and CHO 10B12 cells have comparable heat (43.5 degrees C) sensitivities, whereas hs-36 and CHO 10B12 show a similar sensitivity to gamma- and X-rays. Radiosensitization due to pre-exposure to 43.5 degrees C heating of plateau phase cultures was found for all three cell lines, even after relatively mild heat treatment killing < 20% of cells. Experiments using CHEF electrophoresis confirmed the dsb repair deficiency of the irs-20 cells (Stackhouse and Bedford 1992) and showed that heat inhibited dsb repair in all three cells lines. These data indicate that DNA repair deficiency (overall dsb repair) per se does not imply an absence of the ability for heat radiosensitization.

An image analysis technique for detection of radiation-induced DNA fragmentation after CHEF electrophoresis.

CHEF-electrophoresis was used as a technique to detect radiation-induced DNA breakage with special emphasis to biological relevant X-ray doses (0-10 Gy). Fluorescence detection of DNA-fragments using a sensitive image analysis system was directly compared with conventional scintillation counting of 3H-thymidine prelabelled DNA in HeLa S3 cells. It is shown that the image analysis-based fluorescence detection of fragmented DNA after ionizing radiation is as sensitive and reproducible as detection using radioactively prelabelled cells without the putative shortcomings of fluorescence detection methods described earlier (Blöcher and Kuhni 1990). Therefore, the image analysis-based detection of radiation-induced DNA fragmentation after CHEF electrophoresis seems to be the most reliable method for applications to non-cycling cells and biopsy material.

Heat-induced K+ loss, trypan blue uptake, and cell lysis in different cell lines: effect of serum.

Experiments were performed with three different cell lines, mouse fibroblast LM cells, HeLa S3 cells, and Ehrlich Ascites Tumor (EAT) cells, to establish the possible importance of hyperthermic-induced alterations in cellular K+ content in the mechanism of cell killing by heat. At different time points after the hyperthermic treatment, the K+ content in the cells, the uptake of the dye Trypan Blue (TB), and cell lysis were assayed. Heat-induced K+ loss preceded TB uptake which was followed by the heat-induced cell lysis. Lysis was assayed as disappearance of cells by counting the cells at different time points in a hematocytometer. The presence of serum during and after the heat treatment was of considerable importance with respect to K+ loss and TB uptake. K+ loss and TB uptake after the heat treatment were less when serum was present during and after hyperthermia. To protect against cell lysis, however, the serum had to be present during a preincubation period of 24 h. Clonogenic ability was not affected by the presence of serum. It is concluded that the intracellular K+ level of hyperthermic-treated cells is not a direct cause for cell killing and that heat-induced alterations in the cell leading to cell lysis are different from the processes decreasing cellular K+ content and permeabilizing the plasma membrane for trypan blue.

Na+/K+ ATPase activity in mouse lung fibroblasts and HeLa S3 cells during and after hyperthermia.

The ouabain-sensitive ATP-hydrolysing activity, representing the Na+/K+ ATPase capacity, of isolated membranes and whole cells during and after hyperthermia treatments was investigated. In isolated membranes no heat damage after treatments up to 46 degrees C during 45 min or up to 6 h at 44 degrees C could be detected. The ATP hydrolysing activity of Na+/K+ ATPase seems not to be impaired by direct heat attack in the range of commonly used hyperthermic temperatures (39-46 degrees C). Heat effects on the ATP hydrolysing activity of Na+/K+ ATPase of whole mouse fibroblasts could only be detected after heat doses (greater than 40 min at 44 degrees C) necessary to yield over 99 per cent dead cells. Potassium influx, measured with 86RB+ as the K+ tracer, was initially enhanced during incubation at 44 degrees C proportionally with the enhancement of the ATP-hydrolysing activity after raising the temperature. Replacement of non-lethally (10 min at 44 degrees C) and lethally (40 min at 44 degrees C) treated mouse fibroblasts to 37 degrees C showed complete reversibility of the enhanced activity at 44 degrees C to the control level at 37 degrees C. For comparison, the ATP-hydrolysing activity of Na+/K+ ATPase of HeLa S3 cells growing as monolayer was also tested. The activity after heat treatments up to 60 min at 44 degrees C was also found to be unchanged in these experiments. No indication of irreversible damage to the ATP-hydrolysing capacity of mouse fibroblasts and HeLa S3 cells, or K+ pumping activity of mouse fibroblasts by heat treatments up to 40 min at 44 degrees C was found.

Correlation of colony forming ability of mammalian cells with potassium content after hyperthermia under different experimental conditions.

When the potassium content of LM mouse fibroblasts was determined 16 hr after a heat treatment, a correlation was found between the concentration of potassium in the cell population and the level of cellular survival. When the cells were made thermotolerant, the degree of acquired heat resistance corresponded to a higher level of residual potassium. Also the degree of heat protection by erythritol corresponded to a higher level of residual potassium in the cells, while heat sensitization by procaine resulted in a lower level of potassium. It is concluded that the amount of residual potassium in these cells measured 16 hr after heat treatment may be considered as a suitable index for cell survival after hyperthermia treatment under different conditions.

Correlation between cellular survival and potassium loss in mouse fibroblasts after hyperthermia alone and after a combined treatment with X rays.

Mouse fibroblast LM cells have been heated at 44 degrees C for different periods. Potassium content of the cells was measured at certain intervals during the postheating period at 37 degrees C for up to 24 hr. The level of K+ decreased gradually in time starting within some hours after the heat treatment. The rate of K+ loss as well as the ultimate level reached was heat-dose dependent. When the potassium content of the cell population was determined 16 hr after the heat treatment, a correlation was observed between the concentration of potassium and the level of cell survival. When X irradiation was applied immediately after hyperthermia, radiosensitization on the level of cell survival was obtained as expected, the extent being dependent on the severity of heat treatments. No added K+ loss was observed, however, when hyperthermia was combined with radiation. It is suggested that plasma membrane related functions are disturbed by the heat treatment. This points to membranes as possible candidates for primary targets in the case of cell inactivation by heat alone, and not with respect to the radiosensitization by hyperthermia.