Regulation of the synthesis of heat-shock proteins in heat-resistant variants of Chinese hamster fibroblasts

The synthesis of the major heat-shock proteins (hsp) was compared in normal and heat-resistant Chinese hamster fibroblasts which express higher levels of the 70 kDa heat-shock protein (hsp70). Following exposure to a variety of experimental conditions that induce the elevated synthesis of the hsp, higher relative levels of hsp70 and lower relative levels of hsp89 and hsp110 were found in the heat-resistant variants. This effect was observed with all inducers tested. The relatively greater synthesis of hsp70 and relatively lower synthesis of hsp89 occurred at all temperatures tested and was found to be independent of cell culture conditions. The relatively greater increase in the levels of hsp70 in the heat-resistant variants after a mild heat shock was found to be a reflection of elevated levels of messenger RNA coding for this polypeptide. These results indicate that the heat-shock response in mammalian cells displays coordinate regulatory features and that the alteration of the expression of one of the hsp may affect the expression of the others.     

Effect of heat shock on the intracellular distribution of proteins

Exposure of cells to heat induces thermotolerance, a transient resistance to subsequent heat challenges. It has been shown that thermotolerance is correlated in time with the enhanced synthesis of heat shock proteins. In this study, the association of induced heat shock proteins with various cellular fractions was investigated and the heat-induced changes in skeletal protein composition in thermotolerant and control cells was compared. All three major heat shock proteins induced in Chinese hamster fibroblasts after a 46 degrees C, 4-min heat treatment (70, 87, and 110 kDa) were purified with the cytoplasmic fraction, whereas only the 70-kDa protein was also found in other cell fractions, including that containing the cellular skeleton. Immediately after a second heat treatment at 45 degrees C for 45 min, the 110-kDa protein from thermotolerant cells also purified extensively with the cellular skeletal fraction. In this regard, the 110-kDa protein behaved similarly to many other cellular proteins, since we observed an overall temperature-dependent increase in the total labeled protein content of the high-salt-resistant cellular skeletal fraction after heat shock. Pulse-chase studies demonstrated that this increased protein content gradually returned to normal levels after a 3-hr incubation at 37 degrees C. The alteration or recovery kinetics of the total labeled protein content of the cellular skeletal fraction after heat shock did not correlate with the dramatic increase in survival observed in thermotolerant cells. The relationship between heat shock proteins and thermotolerance, therefore, does not correlate directly with changes in the heat-induced cellular alterations leading to differences in protein fractionation.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Use of surface-enhanced laser desorption ionization-time-of-flight to identify heat shock protein 70 isoforms in closely related species of the virilis group of Drosophila

The 70-kDa heat shock protein (Hsp) family in all Drosophila species includes 2 environmentally inducible family members, Hsp70 and Hsp68. Two-dimensional gel electrophoresis revealed an unusual pattern of heat shock-inducible proteins in the species of the virilis group. Trypsin fingerprinting and microsequencing of tryptic peptides using ProteinChip Array technology identified the major isoelectric variants of Hsp70 family, including Hsp68 isoforms that differ in both molecular mass and isoelectric point from those in Drosophila melanogaster. The peculiar electrophoretic mobility is consistent with the deduced amino acid sequence of corresponding hsp genes from the species of the virilis group.     

The relationship between hsp 70 localization and heat resistance

Using indirect immunofluorescence we have investigated the kinetics of nuclear accumulation and removal of hsp 70 in HA-1 Chinese hamster fibroblasts exposed to elevated temperatures. The kinetics of accumulation of hsp 70 in the nuclei were found to be time/temperature dependent at all temperatures tested (42-45 degrees C). At a given temperature, the fraction of cells manifesting nuclear localization of hsp 70 increased with exposure time. For a given duration of heating, the fraction of cells manifesting nuclear localization of hsp 70 increased with the temperature. The kinetics of the nuclear accumulation of hsp 70 were similar for normal HA-1 cells, their heat-resistant variants, and transiently thermotolerant cells (triggered by prior exposure to a brief heat shock or to sodium arsenite). Upon return to 37 degrees C after heat shock, the kinetics of removal of the hsp 70 associated with the nucleus was dependent on the severity of the initial heat challenge. However, for a given heat dose, the decay of nuclear localization of hsp 70 was more rapid in thermotolerant and heat-resistant cells than in their normal counterparts. These results suggest that the increased levels of hsp 70 associated with the transient or permanently heat-resistant state may play a direct role in restoring and/or repairing heat-induced nuclear and nucleolar alterations associated with heat-induced cell killing. Furthermore, they also suggest that the heat-resistant state may involve ameliorated repair of heat-induced cellular alterations.     

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.     

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.     

Molecular cloning of sequences encoding the human heat-shock proteins and their expression during hyperthermia

Plasmids containing cDNA copies of mRNAs induced in HeLa cells by heat shock have been isolated and characterized. In vitro translation of RNAs selected by hybridization to plasmid DNAs identified sequences representing the three major classes (89, 70 and 27-kDa) of heat-shock proteins (hsp) and a 60-kDa minor hsp. Plasmids with inserts specific for the 27, 60, and 70-kDa hsp each hybridize with a single discrete size class of heat-inducible mRNA. Plasmids specific for the 89-kDa protein, however, hybridize with either a 2.7- or 2.95-kb mRNA species. Both mRNAs are coordinately induced during heat shock. We show that the characteristic pattern of induction and repression of each class of hsp during sustained hyperthermia is the result of changes in the steady state level of each mRNA.     

Amino acid analogs while inducing heat shock proteins sensitize CHO cells to thermal damage

Amino acid analogs have been shown to induce heat shock proteins (HSPs). We have examined the effect of these analogs on the thermal sensitivity of Chinese hamster fibroblasts (HA-1) and their stable heat-resistant variants. We found that exposure of HA-1 cells and their heat-resistant variants to canavanine or L-azetidine-2-carboxylic acid cause enhanced synthesis of the three major mammalian HSPs (molecular weight 70,000, 87,000, and 110,000 kd). Although the synthesis of HSPs was increased, the analogs did not induce thermotolerance, a transient ability to protect cells from thermal damage. On the contrary, the analog treatment increased the thermal sensitivity of HA-1 cells, but not of the heat-resistant strains, when these cells were exposed subsequently to elevated temperatures. Our tentative explanation for these findings is that the incorporation of amino acid analogs into HSPs or other cellular proteins sensitizes HA-1 cells to heat. The heat-resistant strains contain higher levels of constitutive HSPs. The additional functional HSPs in the heat-resistant variants may protect these cells from thermal stress. The presence of some newly synthesized analog-substituted, perhaps nonfunctional, HSPs need not affect this thermal protection.     

Heat shock response in mycoplasmas, genome-limited organisms.

We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JA1 and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 heat shock proteins in A. laidlawii JA1, and 7 heat shock proteins in M. capricolum was observed by electrophoretic analysis of proteins from heat-shocked cells in sodium dodecyl sulfate-polyacrylamide gels. In all three strains, major heat shock proteins (66 to 68 and 26 to 29 kilodaltons [kDa]) were found. The 66- to 68-kDa protein cross-reacted with antibody to Escherichia coli DnaK protein, suggesting that this heat shock protein has been conserved in spite of major reductions in genetic complexity during mycoplasma evolution. A. laidlawii also contained a 60-kDa protein that cross-reacted with eubacterial GroEL protein and a 40-kDa protein that cross-reacted with E. coli RecA protein. Unlike with coliphages, the mycoplasma virus L2 progeny yield was not increased when virus was plated on heat-shocked A. laidlawii host cells. However, UV-irradiated L2 virus could be host cell reactivated by both A. laidlawii SOS repair and heat shock systems.     

Heat shock proteins within the mammalian cell cycle: relationship to thermal sensitivity, thermal tolerance, and cell cycle progression

We have measured endogenous and induced rates of 70-kD, 89-kD, and 110-kD heat shock proteins in highly pure G1-, S-, or G2-M phase fractions of Chinese hamster fibroblasts (CHO) separated by fluorescence-activated cell sorting (FACS). Relative rates of synthesis of all three polypeptides as measured by two-dimensional gel electrophoresis were similar throughout the cell cycle, and therefore, endogenous levels were unlikely to explain the thermal sensitivity of S-phase cells. Distinct heterogeneity in induced rates of these polypeptides was noted in all phase fractions. Enhanced rates of 70-kD polypeptide were measured in S and G2-M as compared to G1 following heat shock. Little increase in either the 89-kD or 110k-kD heat shock proteins was observed in heated G1 cells. This heterogeneity in induced rates of synthesis was in contrast to the similarity in thermal tolerance expression kinetics between each phase. Finally, enhanced synthesis of these polypeptides appeared unrelated to regulation of either heat-induced cell cycle delay or to the resumption of phase-specific progression after heat shock as measured by simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content.     

Acquired thermotolerance and heat shock proteins in thermophiles from the three phylogenetic domains.

Thermophilic organisms from each of the three phylogenetic domains (Bacteria, Archaea, and Eucarya) acquired thermotolerance after heat shock. Bacillus caldolyticus grown at 60 degrees C and heat shocked at 69 degrees C for 10 min showed thermotolerance at 74 degrees C, Sulfolobus shibatae grown at 70 degrees C and heat shocked at 88 degrees C for 60 min showed thermotolerance at 95 degrees C, and Thermomyces lanuginosus grown at 50 degrees C and heat shocked at 55 degrees C for 60 min showed thermotolerance at 58 degrees C. Determinations of protein synthesis during heat shock revealed differences in the dominant heat shock proteins for each species. For B. caldolyticus, a 70-kDa protein dominated while for S. shibatae, a 55-kDa protein dominated and for T. lanuginosus, 31- to 33-kDa proteins dominated. Reagents that disrupted normal protein synthesis during heat shock prevented the enhanced thermotolerance.     

Effects of heat shock on neuroblastoma (N1E 115) cell proliferation and differentiation.

Heat shock (44 degrees C) applied for only 15 min induced the development of neurites in neuroblastoma cells 3-6 days later. During the first day after heat shock a transient increase in the rate of cytokinesis together with a synchronizing effect was observed, which led to waves of cytokinesis 14.5 h apart. Individual cell cycles were determined and showed a lengthening in the minimal cell cycle duration and a decrease in the cell cycle variance after shock. Two to 3 days after heat shock the proliferation rate decreased and then recovered. During the 6 days after heat shock, total protein synthesis was lower compared to the untreated cultures. The synthesis of heat shock proteins (100, 90, 84, 70, 68 kDa and some of lower MW) reached a maximum 6 h after heat shock. Parallel changes in the phosphorylation state of proteins were observed in an in vitro assay. Four proteins (100, 89, 67, and 15 kDa) increased and two proteins (97, 73 kDa) decreased their phosphorylation state significantly. Six days after heat shock two proteins (89, 55 kDa) increased their phosphorylation state; the 55-kDa phosphoprotein was identified as tubulin. The effect of heat shock on the intracellular calcium level was determined by measuring Fura 2 fluorescence. Six hours after shock, the Ca2+ level increased to a maximum (about three times the control value) and then dropped during the following days below the control values. We conclude from these results that a decrease in the calcium level may be causally involved in the differentiation process. The calcium effect is probably mediated by changes in the activity of different kinases. This assumption is compatible with the results of experiments with cyclic nucleotides when 10(-5) M cAMP and cGMP were added to in vitro assays of protein phosphorylation. They had different stimulating effects in heat-shocked, differentiating, and growing (control) cells.     

Drug sensitivity of heat-resistant mouse B16 melanoma variants

Induction of transient thermotolerance by heat or other cytotoxic stressors has been reported to confer a moderate degree of drug resistance to tumor cells in vitro. In this study, a genetically stable, heat-resistant mouse B16 melanoma variant (W-H75) was tested for its sensitivity to various cytotoxic and antiproliferative agents. The heat-resistant W-H75 cells displayed a moderate two- to threefold resistance to doxorubicin, VP-16, VM-26, colchicine, cis-dichlorodiammineplatinum(II), HgCl2, and CdCl2. Marginal resistance to 4'(9-acridinylamino)methanesulfon-m-anisidide vinblastine, 1,3-bis(2-chloroethyl)-1-nitro-sourea, and NaAsO2 was observed, while no difference in sensitivity to the anticancer drugs, actinomycin D and camptothecin, was observed. Although W-H75 cells were generally more resistant than the parental cells to most of the agents that were tested, they were collaterally sensitive to the antimetabolites methotrexate and 6-mercaptopurine. Resistance of the W-H75 cells to epipodophyllotoxins and anthracyclines was not due to differences in steady-state drug accumulation. For the epipodophyllotoxin VP-16, resistance may be related to a relative decrease in the number of drug-induced DNA strand breaks in W-H75 cells. However, no difference in DNA strand breakage was observed between W-H75 and parental cells which were treated with doxorubicin, suggesting that resistance to this drug occurred by a different mechanism. The possible involvement of glutathione and glutathione S-transferase in resistance was also investigated. The glutathione content in W-H75 cells was 35% higher than that in the parental line. However, glutathione S-transferase activity appeared to be identical in both cell lines. Two other heat-resistant B16 melanoma variants, B-H103 and R-H92, were also tested for sensitivity to doxorubicin and VP-16. In contrast to the W-H75 cells, these two heat-resistant variants were hypersensitive to doxorubicin. The B-H103 cells were also hypersensitive to VP-16. This study suggests that selection for cellular resistance to heat may result in cells that have an altered sensitivity to drugs.     

Analysis of the expression of the two major proteins of the 70 kilodalton mammalian heat shock family

This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.     

Cell-specific expression of heat shock proteins in chicken reticulocytes and lymphocytes

We have found that chicken reticulocytes respond to elevated temperatures by the induction of only one heat shock protein, HSP70, whereas lymphocytes induce the synthesis of all four heat shock proteins (89,000 mol wt, HSP89; 70,000 mol wt, HSP70; 23,000 mol wt, HSP23; and 22,000 mol wt, HSP22). The synthesis of HSP70 in lymphocytes was rapidly induced by small increases in temperature (2 degrees-3 degrees C) and blocked by preincubation with actinomycin D. Proteins normally translated at control temperatures in reticulocytes or lymphocytes were not efficiently translated after incubation at elevated temperatures. The preferential translation of mRNAs that encode the heat shock proteins paralleled a block in the translation of other cellular proteins. This effect was most prominently observed in reticulocytes where heat shock almost completely repressed alpha- and beta-globin synthesis. HSP70 is one of the major nonglobin proteins in chicken reticulocytes, present in the non-heat-shocked cell at approximately 3 X 10(6) molecules per cell. We compared HSP70 from normal and heat-shocked reticulocytes by two-dimensional gel electrophoresis and by digestion with Staphylococcus aureus V8 protease and found no detectable differences to suggest that the P70 in the normal cell is different from the heat shock-induced protein, HSP70. P70 separated by isoelectric focusing gel electrophoresis into two major protein spots, an acidic P70A (apparent pl = 5.95) and a basic P70B (apparent pl = 6.2). We observed a tissue-specific expression of P70A and P70B in lymphocytes and reticulocytes. In lymphocytes, P70A is the major 70,000-mol-wt protein synthesized at normal temperatures whereas only P70B is synthesized at normal temperatures in reticulocytes. Following incubation at elevated temperatures, the synthesis of both HSP70A and HSP70B was rapidly induced in lymphocytes, but synthesis of only HSP70B was induced in reticulocytes.     

Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP.

Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.