To understand how the molecular chaperone Hsp90 participates in conformational maturation of the estrogen receptor (ER), we analyzed the interaction of immobilized purified avian Hsp90 with mammalian cytosolic ER. Hsp90 was either immunoadsorbed to BF4 antibody–Sepharose or GST-Hsp90 fusion protein (GST.90) was adsorbed to glutathione-Sepharose. GST.90 was able to retain specifically ER, similarly to immunoadsorbed Hsp90. When cells were treated with estradiol and the hormone treatment was maintained during cell homogenization, binding, and washing steps, GST.90 still interacted efficiently with ER, suggesting that ER may form complexes with Hsp90 even after its activation by hormone and salt extraction from nuclei. The GST.90-ER interaction was consistently reduced in the presence of increasing concentrations of potassium chloride or when cytosolic ER-Hsp90 complexes were previously stabilized by molybdate, indicating that GST.90-ER complexes behave like cytosolic Hsp90-ER complexes. A purified thioredoxin-ER fusion protein was also able to form complexes with GST.90, suggesting that the presence of other chaperones is not required. ER was retained only by GST.90 deletion mutants bearing an intact Hsp90 N-terminal region (1–224), the interaction being more efficient when the charged region A was present in the mutant (1–334). The N-terminal fragment 1–334, devoid of the dimeric GST moiety, was also able to interact with ER, pointing to the monomeric N-terminal adenosine triphosphate binding region of Hsp90 (1–224) as the region necessary and sufficient for interaction. These results contribute to understand the Hsp90-dependent process responsible for conformational competence of ER.

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41°C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39°C, and Hsp70 levels at 41°C were 10-fold higher than in the 37°C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39°C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type–specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.

Heat shock proteins (Hsps) are ubiquitous proteins that are induced following exposure to sublethal heat shock, are highly conserved during evolution, and protect cells from damage through their function as molecular chaperones. Some cancers demonstrate elevated levels of Hsp70, and their expression has been associated with cell proliferation, disease prognosis, and resistance to chemotherapy. In this study, we developed a tetracycline-regulated gene expression system to determine the specific effects of inducible Hsp70 on cell growth and protection against hyperthermia in MCF-7 breast cancer cells. MCF-7 cells expressing high levels of Hsp70 demonstrated a significantly faster doubling time (39 hours) compared with nonoverexpressing control cells (54 hours). The effect of elevated Hsp70 on cell proliferation was characterized further by 5-bromo-2′deoxyuridine labeling, which demonstrated a higher number of second and third division metaphases in cells at 42 and 69 hours, respectively. Estimates based on cell cycle analysis and mean doubling time indicated that Hsp70 may be exerting its growth-stimulating effect on MCF-7 cells primarily by shortening of the G₀/G₁ and S phases of the cell cycle. In addition to the effects on cell growth, we found that elevated levels of Hsp70 were sufficient to confer a significant level of protection against heat in MCF-7 cells. The results of this study support existing evidence linking Hsp70 expression with cell growth and cytoprotection in human cancer cells.

Mammalian cells respond to environmental stress by activating heat shock transcription factors (eg, Hsf1) that regulate increased synthesis of heat shock proteins (Hsps). Hsps prevent the disruption of normal cellular mitosis, meiosis, or differentiation by environmental stressors. To further characterize this stress response, transformed wild-type Hsf1 ^/ and mutant Hsf1^−/− mouse embryonic fibroblasts (MEFs) were exposed to (1) lethal heat (45°C, 60 minutes), (2) conditioning heat (43°C, 30 minutes), or (3) conditioning followed by lethal heat. Western blot analysis demonstrated that only Hsf1 ^/ MEFs expressed inducible Hsp70s and Hsp25 following conditioning or conditioning and lethal heat. Exposure of either Hsf1 ^/ or Hsf1^−/− MEFs to lethal heat resulted in cell death. However, if conditioning heat was applied 6 hours before lethal heat, more than 85% of Hsf1 ^/ MEFs survived, and cells in G₂/M transiently increased 3-fold. In contrast, conditioned Hsf1^−/− MEFs neither survived lethal heat nor exhibited this G₂/M accumulation. Coinfection with adenoviral Hsp70 and Hsp25 constructs did not fully recreate thermotolerance in either Hsf1 ^/ or Hsf1^−/− MEFs, indicating other Hsf1-mediated gene expression is required for complete thermotolerance. These results demonstrate the necessity of Hsf1-mediated gene expression for thermotolerance and the involvement of cell cycle regulation, particularly the G₂/M transition, in this thermotolerant response.

Compared with normal cells, tumor cell lines exhibit an unusual plasma membrane localization of heat shock protein 70 (Hsp70). This tumor-selective Hsp70 membrane expression has been found to correlate with an increased sensitivity to lysis mediated by human natural killer (NK) cells that transiently adhere to plastic following cytokine stimulation. A human Hsp70-specific monoclonal antibody (mAb) detects membrane-bound Hsp70 on viable tumor cells and blocks the immune response of NK cells against Hsp70-expressing tumor cells. By peptide scanning (pepscan) analysis, the epitope of this mAb was mapped as the C-terminal–localized 8-mer NLLGRFEL (NLL, amino acids [aa] 454–461). Most interestingly, similar to full-length Hsp70 protein, the N-terminal–extended 14-mer peptide TKDNNLLGRFELSG (TKD, aa 450–463) was able to stimulate the cytolytic and proliferative activity of NK cells at concentrations equivalent to full-length Hsp70 protein. Blocking studies revealed that an excess of the 14-mer peptide TKDNNLLGRFELSG inhibits the cytolytic activity of NK cells similar to that of Hsp70 protein. In comparison, other TKD-related peptides, including the 8-mer antibody epitope NLLGRFEL (aa 454–461), the 12-mer TKDNNLLGRFEL (aa 450–461), the 13-mer C-terminal–extended peptide NLLGRFELSGIPP (aa 454–466), the 14-mer TKD-equivalent sequences of Hsp70hom TKDNNLLGRFELTG (aa 450–463), Hsc70 TKDNNLLGKFELTG (aa 450–463), and DnaK AADNKSLGQFNLDG (aa 447–460) failed to activate NK activity.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a hetero-oligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We recently reported that the expression level of CCT is closely correlated with growth rates of mammalian cultured cells. Here we examine the levels of CCT subunits and other molecular chaperones in tumor tissues of patients with hepatocelluar and colonic carcinoma, and compare them with nontumor tissues in the same patients. Expression levels of CCTβ in tumor tissues was significantly higher than in nontumor tissues in all patients with hepatocellular carcinoma (n = 15) and 83% of patients with colonic carcinoma (n = 17). The increased level of CCT expression in colonic cancer cells was confirmed by immunohistochemistry with anti-CCTβ antibody. The levels of CCTβ were highly correlated (r = 0.606) with those of the proliferating cell nuclear antigen (PCNA), which was used as an indicator of cell growth. CCTα gave similar results, although the correlation with PCNA levels was weaker. Other cytosolic and endoplasmic reticulum chaperones also showed higher expression in significant numbers of tumor tissues but less frequently than that observed with CCT. These results suggest that CCT is up-regulated in rapidly proliferating tumor cells in vivo to effectively produce proteins required for growth, and may serve as a useful tumor marker because it is widely distributed in the cytosol.

Metallothionein (MT), a cysteine-rich metal binding protein, is considered to play an essential role in the regulation of intracellular metals. Induction of MT in mammalian and nonmammalian tissues following heavy metal exposure may serve as a defense mechanism and a biomarker of environmental exposure to chemical stressors such as toxic metals. In this study, MT messenger RNA (mRNA) expression was characterized in male and female nonspawning and spawning killifish (Fundulus heteroclitus) following an 8-day exposure to specific sublethal stressors, which included temperature perturbation (26°C or 10°C) and/or 6 ppb of waterborne cadmium chloride (CdCl₂). Hepatic, gill, and intestinal MT mRNA, expressed as copy number per microgram of total RNA, was assessed by reverse transcriptase–polymerase chain reaction and electrochemiluminescence using winter flounder (Pleuronectes americanus) MT complementary DNA primers. Liver, gill, and intestine MT mRNA expression was significantly (P < 0.05) increased in nonspawning killifish exposed to 26°C compared with those exposed to 19°C (control). In addition, a significant (P < 0.05) increase in gill MT mRNA induction was observed in nonspawning killifish exposed to 6 ppb of waterborne CdCl₂ compared with controls. The results of this study demonstrate significant MT mRNA induction in nonspawning killifish following short-term exposure to physiological and chemical stressors. Thus, further research may be necessary before the use of killifish MT mRNA induction as a biomarker of environmental chemical stress exposure alone.

The small heat shock proteins Hsp12.2 and αB-crystallin differ in that the former occurs as tetramers, without chaperonelike activity, whereas the latter forms multimers and is a good chaperone. To investigate whether the lack of chaperone activity of Hsp12.2 is primarily due to its tetrameric structure or rather to intrinsic sequence features, we engineered chimeric proteins by swapping the N-terminal, C-terminal, and tail regions of Hsp12.2 and αB-crystallin, designated as n-c-t and N-C-T, respectively. Three of the chimeric sHsps, namely N-c-T, n-c-T, and N-C-t, showed nativelike secondary and quaternary structures as measured by circular dichroism and gel permeation chromatography. Combining the conserved α-crystallin domain of Hsp12.2 with the N-terminal and tail regions of αB-crystallin (N-c-T) resulted in multimeric complexes, but did not restore chaperonelike activity. Replacing the tail region of Hsp12.2 with that of αB-crystallin (n-c-T) did not alter the tetrameric structure and lack of chaperone activity. Similarly, providing αB-crystallin with the tail of Hsp12.2 (N-C-t) did not substantially influence the multimeric complex size, but it reduced the chaperoning ability, especially for small substrates. These results suggest that the conserved α-crystallin domain of Hsp12.2 is intrinsically unsuitable to confer chaperonelike activity and confirms that the tail region in αB-crystallin modulates chaperonelike capacity in a substrate-dependent manner.

Curcumin, a well-known dietary pigment derived from Curcuma longa, has been shown to be a potent anti-inflammatory, antioxidant, and anticarcinogenic compound. The present study was designed to investigate the cytotoxic potential of curcumin against a range of human tumor cell lines in an attempt to understand its mechanism of action, which may lead to its possible therapeutic applications. We have shown that different cancer cell lines differ in their sensitivity to curcumin. Cell lines established from malignancies like leukemia, breast, colon, hepatocellular, and ovarian carcinomas underwent apoptosis in the presence of curcumin, whereas cell lines from lung, kidney, prostate, cervix, CNS malignancies, and melanomas showed resistance to the cytotoxic effects of curcumin. Sensitivity of the cancer cell lines to curcumin correlated with the generation of superoxide radicals as determined by the reduction of ferricytochrome C. Curcumin-resistant tumor cell lines showed significantly higher production of Hsp70, thus mounting a stress response and protecting the cells from the apoptotic cell death. These observations yield clues toward understanding the regulation of the cell death machinery by the stress proteins. Interestingly, curcumin had no effect on nontransformed cell lines, which showed neither superoxide generation nor the induction of a stress response. These observations demonstrate that curcumin is an interesting molecule with varied actions, depending on the cell type.

Heat shock factor 2 (HSF2) is a member of the heat shock transcription factor family, which appears to be activated during differentiation and development rather than on cellular stress. Here we report the isolation and characterization of the human hsf2 gene and its 5′-flanking region. The transcription unit of the human hsf2 gene consists of 13 exons dispersed over 33 kbp of genomic DNA on chromosome 6. The hsf2 mRNA is transcribed from multiple start sites, and initiation from the major site results in a transcript of 2.45 kb. A functional promoter, as determined by the ability to direct expression of a transiently transfected luciferase reporter gene, resides in a 950-bp upstream region of the human hsf2 gene. Examination of the core promoter sequence revealed a high GC content and lack of a canonical TATA box. This feature seems to be common among various species, as comparison of the hsf2 proximal promoter sequences from human, mouse, and rat showed distinct conserved regions. Moreover, the overall architecture of the human hsf2 gene is similar to its mouse counterpart. A comparison between human hsf2 gene and other hsf genes showed striking similarities in exon size. However, the exons are assembled in an hsf-specific manner.

Recent evidence suggests that heat shock proteins (Hsps) may have an important systemic role as a signal to activate the immune system. Since acute exercise is known to induce Hsp72 (the inducible form of the 70-kDa family of Hsp) in a variety of tissues including contracting skeletal muscle, we hypothesized that such exercise would result in the release of Hsp72 from stressed cells into the blood. Six humans (5 males, 1 female) ran on a treadmill for 60 minutes at a workload corresponding to 70% of their peak oxygen consumption. Blood was sampled from a forearm vein at rest (R), 30 minutes during exercise, immediately postexercise (60 minutes), and 2, 8, and 24 hours after exercise. These samples were analyzed for serum Hsp72 protein. In addition, plasma creatine kinase (CK) was measured at these time points as a crude marker of muscle damage. With the exception of the sample collected at 30 minutes, muscle biopsies (n = 5 males) were also obtained from the vastus lateralis at the time of blood sampling and analyzed for Hsp72 gene and protein expression. Serum Hsp72 protein increased from rest, both during and after exercise (0.13 0.10 vs 0.87 ± 0.24 and 1.02 ± 0.41 ng/mL at rest, 30 and 60 minutes, respectively, P < 0.05, mean SE). In addition, plasma CK was elevated (P < 0.05) 8 hours postexercise. Skeletal muscle Hsp72 mRNA expression increased 6.5-fold (P < 0.05) from rest 2 hours postexercise, and although there was a tendency for Hsp72 protein expression to be elevated 2 and 8 hours following exercise compared with rest, results were not statistically significant. The increase in serum Hsp72 preceded any increase in Hsp72 gene or protein expression in contracting muscle, suggesting that Hsp72 was released from other tissues or organs. This study is the first to demonstrate that acute exercise can increase Hsp72 in the peripheral circulation, suggesting that during stress these proteins may indeed have a systemic role.

Autoantibodies against certain stress or heat shock proteins (Hsps) may play a role in the pathogenesis and/or prognosis of some diseases. Using immunoblotting with human recombinant Hsps and univariate and multivariate logistic regression models, we have investigated the presence of antibodies against Hsp70, the inducible member of the 70-kDa family of heat shock proteins, and analyzed its possible association with hypertension and working conditions. Plasma and serum were collected from 764 steel mill workers from 6 work sites exposed to (1) severe noise; (2) severe noise and dust; (3) noise, dust, and heat; (4) noise and heat; (5) severe noise and heat; and (6) office conditions (control). Workers with prolonged exposure to stresses such as noise, dust, and high temperature and a combination of these in the workplace had a high incidence (26.6% to 40.2%) of antibodies to Hsp70 compared to the lowest incidence (18.6%) of antibodies to Hsp70 in the control group of office workers. Moreover, there was a statistical association of antibodies against Hsp70 with hypertension. The statistical correlation between the presence of antibodies to Hsp70 and hypertension is higher in the group of workers with blood pressure of 160/95 mmHg than in the 140/90-mmHg group after excluding possible effects of the workplace stresses. These results suggest that harsh workplace conditions can increase the production of antibodies against Hsp70 and that the presence of antibodies to this stress protein may be associated with hypertension. The precise mechanism for the elevation of antibodies against Hsps by environmental and workplace stresses and their relation to hypertension remains to be established.