The major heat-shock protein hsp 68 is not induced by stress in mouse erythroleukemia cell lines

Most mammalian cells respond to brief incubation at elevated temperatures by enhanced or new synthesis of a set of heat-shock proteins (hsp). In mouse cells, as determined by SDS--one-dimensional gel electrophoresis, the most prominent hsps have molecular masses of approximately 89,000, 70,000, and 68,000 Da. When the heat-shock response of the mouse erythroleukemia cell line D1B, or two other DBA/2 cell lines (707C1 and 745C2), was examined by [35S]methionine labelling, following heat shocks of 10 min at 42 or 44 degrees C, or 1 h at 45 degrees C, no protein band corresponding to hsp 68 was observed. However, the synthesis of both hsp 89 and hsp 70 was enhanced. Northern blot analysis of cytoplasmic RNA extracted from control and stressed cells indicated that hsp 68 mRNA was absent, even after stresses of up to 1 h at 45 degrees C. Differentiation induced by dimethyl sulphoxide (DMSO) (monitored by the induction of globin synthesis) had no effect on hsp 68 expression in D1B cells; also, hsp 68 could not be induced at various stages of differentiation (0-72 h). Southern blot analysis showed that all three hsp-68 genes were present and not rearranged, and apparently did not carry any deletion in their 5' ends. To determine whether methylation could be involved in maintaining the genes in their silent state, we treated cells with 10 microM 5-azacytidine for 48 h. No hsp 68 expression was observed following such treatment in either undifferentiated or DMSO-induced differentiated D1B cells. Furthermore, Southern blot analysis of MspI/HpaII-digested genomic D1B DNA did not display any differences in methylation patterns around the promoter region of the probed gene compared with control cells, indicating that methylation is not involved in hsp-68 repression. When chimeric plasmids carrying the bacterial chloramphenicol acetyl transferase gene under regulation of the mouse hsp-68 or Drosophila hsp-70 promoters were transfected into D1B cells, minimal (2-fold) or no induction was observed, in contrast with the 60-fold induction seen in a control myeloma cell line. These results suggest a trans-acting mechanism of hsp-68 repression in erythroleukemia cells.     

The expression of Candida albicans enolase is not heat shock inducible

Abstract An isoprotein of enolase from the yeast Saccharomyces cerevisiae was reported to be a heat shock protein. The possible role of the C. albicans enolase as a heat shock protein was therefore investigated. The de novo synthesis of C. albicans enolase protein and mRNA did not increase during heat stress, but remained constitutively expressed. Amino acid similarity to the heat shock proteins suggests that although the C. albicans enolase is not a classical heat shock protein, it may be a memberof a group of constitutively expressed, structurally related proteins, the heat shock cognate proteins.     

Immunohistochemical detection of the inducible heat shock protein hsp70: a methodological study.

Stress-inducible Hsp70i and constitutively expressed Hsc70 are highly related heat shock proteins. Aberrant expression levels and intracellular localization of these proteins has been suggested as a potential marker in certain tumors. The aim of our study was to work out a reliable, immunohistochemical detection of the stress-inducible Hsp70i protein and enabling discrimination between Hsp70i and Hsc70 proteins in paraffin-embedded human tissues. We tested the effect of several fixative procedures and antigen retrieval on the effectiveness of the Hsp70i detection in murine cells cultured in vitro and in liver of rats subjected to heat shock. For cells grown in vitro, specific Hsp70i immunoreactivity was obtained with all fixatives used. However, samples fixed in 10% formalin and 4% paraformaldehyde required antigen retrieval. In liver tissue embedded in paraffin, regardless the fixative used, positive Hsp70i staining could be visible only if antigen retrieval was applied. We applied this procedure for detection of Hsp70i in routine sections of breast and lung cancers fixed with 10% formalin and found that the application of thermal antigen retrieval significantly enhanced the SPA810 immunoreactivity and reduced background staining. This procedure enabled also the differential detection of Hsp70i and Hsc70 in routine histopathological preparations.     

The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster.

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.     

The glucose-regulated protein grp94 is related to heat shock protein hsp90

We report the sequence of a cDNA clone that encodes the C-terminal half of the hamster 94 X 10(3) Mr glucose-regulated protein, grp94. The amino acid sequence of this protein is about 50% homologous to Drosophila hsp83 and yeast hsp90, suggesting that grp94 and hsp90 have similar functional properties. Unlike hsp90, grp94 is associated with the endoplasmic reticulum. It has the same C-terminal tetrapeptide as two other luminal endoplasmic reticulum proteins, grp78 and protein disulphide isomerase. We suggest that this sequence forms part of a signal for retention of proteins in the lumen of the endoplasmic reticulum.     

Mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast

Two main forms of immunoreactive insulin have been identified in cultures of foetal mouse brain using HPLC and gel filtration. The major component which resembled proinsulin was converted by trypsin to the minor form which was similar to authentic pancreatic insulin in chromatographic behaviour. Both components showed immunological properties comparable to insulin and proinsulin including sensitivity of the former to reduction and alkylation.     

Development and tissue-specific distribution of mouse small heat shock protein hsp25

We have investigated the developmental and tissue-specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13–20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related α, B-crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism. © 1993Wiley-Liss, Inc.     

The heat shock response in hydra: immunological relationship of hsp60, the major heat shock protein of Hydra vulgaris,to the ubiquitous hsp70 family

The major heat shock protein (hsp) of Hydra vulgaris has recently been found to be a 60 kDa protein. Since in all organisms studied so far, the major heat shock protein is a 70 kDa protein, we have analyzed the relationship of hydra hsp60 to the highly conserved 70 kDa heat shock protein family. Genes and proteins related to the 70 kDa class of stress proteins are present in hydra. However, antibodies known to cross-react with hsp70 proteins in several different organisms do not cross-react with hydra hsp60 suggesting that hsp60 is not related to the conserved hsp70 proteins.     

Role of inducible heat shock protein 70 in radiation-induced cell death

We previously demonstrated the protective effect of inducible heat shock protein 70 (Hsp70) against gamma radiation. Herein, we extend our studies on the possible role of Hsp70 to ionizing radiation-induced cell cycle regulation. The growth rate of inducible hsp70-transfected cells was 2-3 hours slower than that of control cells. Flow cytometric analysis of cells at G1 phase synchronized by serum starvation also showed the growth delay in the Hsp70-overexpressing cells. In addition, reduced cyclin D1 and Cdc2 levels and increased dephosphorylated phosphoretinoblastoma (pRb) were observed in inducible hsp70-transfected cells, which were probably responsible for the reduction of cell growth. To find out if inducible Hsp70-mediated growth delay affected radiation-induced cell cycle regulation, flow cytometric and molecular analyses of cell cycle regulatory proteins and their kinase were performed. The radiation-induced G2/M arrest was found to be inhibited by Hsp70 overexpression and reduced p21Waf induction and its kinase activity by radiation in the Hsp70-transfected cells. In addition, radiation-induced cyclin A or B1 expressions together with their kinase activities were also inhibited by inducible Hsp70, which represented reduced mitotic cell death. Indeed, hsp70 transfectants showed less induction of radiation-induced apoptosis. When treated with nocodazole, radiation-induced mitotic arrest was inhibited by inducible Hsp70. These results strongly suggested that inducible Hsp70 modified growth delay (increased G1 phase) and reduced G2/M phase arrest, subsequently resulting in inhibition of radiation-induced cell death.     

The structure and expression of maize genes encoding the major heat shock protein, hsp70

We have isolated and sequenced two maize genomic clones that are homologous to the Drosophila hsp70 gene. One of the maize hsp70 clones contains the entire hsp70 coding region and 81 nucleotides of the 5' nontranslated sequence. The predicted amino acid sequence for this maize protein is 68% homologous to the hsp70 of Drosophila. The second maize hsp70 clone contains only part of the coding sequence and 1.1 kb of the 5' flanking sequence. This 5' flanking sequence contains two sequences homologous to the consensus heat-shock-element sequence. Both maize genes are thermally inducible and each contains an intron in the same position as that of the heat-shock-cognate gene, hsc1, of Drosophila. The presence of an intron in the maize genes is a distinguishing feature in that no other thermally inducible hsp70 genes described to date contain an intron. We have constructed a hybrid hsp70 gene containing the entire hsp70 coding sequence with an intron, and 1.1 kb of the 5' flanking sequence. We demonstrate that this hybrid gene is thermally inducible in a transgenic petunia plant and that the gene is expressed from its own promoter.     

No heat shock protein synthesis is required for induced thermostabilization of translational machinery.

For Tetrahymena thermophila cells to survive at 43 degrees C, a normally lethal temperature, they require a pretreatment which either elicits the synthesis of heat shock proteins or one which brings about a change in the translational machinery of the cell such that is is not inactivated when transferred to 43 degrees C. In this report I present evidence showing that the latter modification can occur in the complete absence of protein synthesis, indicating that heat shock protein production is not required for the induced thermostabilization of the translational machinery.     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.     

The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila.

The heat shock protein Hsp90 has been shown to associate with various cellular signalling proteins such as steroid hormone receptors, src-like kinases and the serine/threonine kinase Raf. While the interaction between steroid hormone receptors and Hsp90 appears to be essential for ligand binding and activation of the receptors, the role of Hsp90 in Raf activation is less clear. We have identified mutations in the hsp83 gene, the Drosophila homologue of hsp90, in a search for dominant mutations that attenuate signalling from Raf in the developing eye. The mutations result in single amino acid substitutions in the Hsp83 protein and cause a dominant-negative effect on the function of the wild-type protein. We show that both wild-type and mutant forms of Hsp83 bind to the activated Drosophila Raf but the mutant Hsp83 protein causes a reduction in the kinase activity of Raf. Our results indicate that Hsp83 is essential for Raf function in vivo.     

Lactotransferrin is the major estrogen inducible protein of mouse uterine secretions

Antibody to an estrogen inducible mouse uterine protein (Teng, C. T., Walker, M. P., Bhattacharyya, S. N., Klapper, D. G., DiAugustine, R. P., and McLachlan, J. A. (1986) Biochem. J. 240, 413-422) has been used to isolate cDNA to the messenger RNA. Analysis of the deduced primary structure and additional biochemical characterization indicates that the protein is lactotransferrin. An increase in the level of lactotransferrin mRNA of at least 300-fold can be induced in the mouse uterus by estrogen. In contrast, the mRNA is virtually undetectable in rat uterine tissue following estrogen administration. The estrogenic stimulation in mouse uterus contrasts with the known prolactin dependence in mammary gland.     

Isolation of major heat shock protein (hsp70) gene-related sequences from rat genome

Rat genome was assayed for the presence of hsp70 gene-related sequences. Southern blots prepared from rat DNA digested with EcoRI or HindIII restriction endonucleases were hybridized with mouse, human and fruit fly hsp 70 gene probes at increasing stringencies. At the stringency which allows sequences divergent up to about 30% to form stable complexes all three probes detected 25–30 restriction fragments. Increased stringency of the hybridization reduced the number of detectable bands to a few and among them the DNA fragments hybridizing specifically either with mouse or human hsp70 gene probes were detected. Most of the genomic fragments containing hsp70 gene-related sequences were subsequently isolated by screening the rat genomic library with mouse hsp70 gene probe. 168 positive clones were plaque purified and on the basis of the restriction and hybridization pattern we deduced that inserts represented 20 different genomic regions. Partial restriction maps of all isolated genomic fragments were constructed and regions containing hsp70 gene related as well as highly repetitive DNA sequences were localized. A putative sequence rearrangement in the proximity of the hsp70 gene-related sequence was detected in one of the isolated genomic segments.     

Wild-type levels of abscisic Acid are not required for heat shock protein accumulation in tomato

Levels of endogenous abscisic acid (ABA) in wild type were not required for the synthesis of heat shock proteins in detached leaves of tomato (Lycopersicon esculentum Mill., cv Ailsa Craig). Heat-induced alterations in gene expression were the same in the ABA-deficient mutant of tomato, flacca, and the wild type. Heat tolerance of the mutant was marginally less that the wild type, and in contrast, ABA applications significantly reduced the heat tolerance of wild-type leaves. It was concluded that elevated levels of endogenous ABA are not involved in the tomato heat shock response.