Produced by dietary fiber, butyrate is a potential chemopreventive agent against colon cancer. It stimulates proliferation of normal colonic epithelial cells but induces growth inhibition, differentiation, apoptosis, or a combination of effects in colon carcinoma cells. In this study, we used cDNA membrane arrays and real-time reverse transcriptase– polymerase chain reaction to identify stress genes that were differentially regulated by sodium butyrate (NaB) in HT 29 human colon carcinoma cells. The results indicated that a group of heat shock protein (hsp) genes were upregulated by 3 mM NaB within the first 24 hours of exposure. Because the transcription of hsp genes is under the control of heat shock factors (HSFs), we measured the effects of overexpressed HSF-1 on the responses of HT 29 cells to NaB. Overexpression of HSF-1 inhibited NaB-induced differentiation as measured by alkaline phosphatase activity and carcinoembryonic antigen expression. These results suggest that increased expression of HSFs and Hsps might render colon carcinoma cells resistant to the chemopreventive effects of butyrate.

Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 ± 1.7 years) and middle-aged (mean age = 56.3 ± 4.7 years) subjects after 1 hour of heat shock at 42°C. Genotype-specific HSR was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible and inducible forms of Hsp70 decreased 1.3-fold (P < 0.001) and 1.4-fold (P < 0.001), respectively, in the monocytes with age. In the young subjects, a positive association was found between HSPA1L(T2437C) polymorphism and HSR. CC carriers had a significantly lower induction than TT carriers in both monocytes (P = 0.015) and lymphocytes (P = 0.044). This polymorphism, which is present in the coding region of HSPA1L gene, can affect the chaperoning function of Hsp70. These data consolidate our other observations that the CC genotype is unfavorable for human longevity and provide a functional explanation in terms of variations in HSR.

Hsp70s are a family of ATP-dependent chaperones of relative molecular mass around 70 kDa. Immunization of mice with Hsp70 isolated from tumor tissues has been proved to elicit specific protective immunity against the original tumor challenge. In this work, we investigated whether Hsp70 can be used as vehicle to elicit immune response to its covalence-accompanying antigen. A recombinant protein expression vector was constructed that permitted the production of recombinant protein fusing tumor-associated antigen (eg, Mela) to the C terminus of Hsp70. We found that the Hsp70–Mela fusion protein can elicit strong cellular immune responses against murine tumor B16, which expresses protein Mela. The Hsp70 peptide-binding domain deletion mutant of the fusion protein was sufficient for inducing Mela-specific cytotoxic T lymphocyte but was not sufficient for engendering potent anti-tumor immunity against B16. We also found that host natural killer (NK) cells were stimulated in vivo by C-terminal domain of Hsp70. We thus presume that Hsp70 fusion proteins suppress tumor growth via at least 2 distinct pathways: one is covalence-accompanying antigen dependent; another is antigen independent. The C-terminal domain of Hsp70 seemed to be the crucial part in eliciting antigen-independent responses, including NK cell stimulation, against tumor challenges. Furthermore, we found that immunization with multiple Hsp70 fusion proteins resulted in a better anti-tumor effect.

To address the question of a possible effect of magnetic fields (MF) at 50 Hz on living systems, gene expression analyses were performed on human primary vascular endothelial cells exposed to MF of various intensities compared to control cells. Exposure protocols included continuous exposure at a single intensity (10 and 700 μT), intermittent exposure at a single intensity (700 μT), and continuous exposure to a variable-intensity field (10–30 μT). The transcriptional response of the cells was investigated using oligonucleotide microarrays containing up to 30 000 unique features. Although in individual experiments genes were identified where the expression appeared to be affected by exposure to MF, none of these genes were regulated in the same manner in subsequent repetition experiments. This is the first report of a transcriptome-wide analysis of the effects of MF exposure on human cells. The lack of a reproducible effect of MF on the expression of any genes in our investigation adds further weight to the evidence that 50-Hz MF are not capable of interacting with biological systems and thus do not represent an endothelial stress factor.

Severe noise exposure can induce heat shock proteins (Hsps), and exposure to moderate noise has been reported to confer protection against noise-induced damage to hearing. Whether there is any association of genetic variation in both constitutive and inducible hsp70 genes with noise-induced hearing loss (NIHL) is presently unknown. Using polymerase chain reaction-restriction fragment length polymorphism, we genotyped 3 polymorphisms ( 190A/ B, 1267A/B, and 2437A/B) in the hsp70-1 (rs1043618), hsp70-2 (rs1061581), and hsp70-hom (rs2227956) genes, respectively, and investigated the associations of these polymorphisms with risk of developing NIHL in 194 automobile workers working in a similar noise environment as evaluated by audiological assessment. Multivariate logistic regression models were used to assess the associations with the risk genotypes, and Whap software was used to analyze their haplotypes. Our results showed that there was no statistically significant difference in the genotype and allele distributions of hsp70-1, hsp70-2, and hsp70-hom between the NIHL group and the normal group (P > 0.05) with and without adjustment for age, sex, smoking, history of explosive noise exposure, and cumulative noise exposure. However, haplotype analysis revealed that the Hap5 (ie, haplotype 190A/ 1267B/ 2437A) and Hap6 (ie, haplotype 190A/ 1267B/ 2437B) were significantly more frequent in the NIHL group than in the normal group (20/9, P = 0.022, and 7/0, P = 0.005, respectively). Compared with Hap1 (ie, 190A/ 1267A/ 2437A), Hap5 was associated with a nearly 3-fold increased risk of NIHL (adjusted odds ratio, 2.67; 95% confidence interval, 1.13–6.27). Seven of the NIHL patients had Hap6, but none of the controls had this haplotype. Our results suggest that some haplotypes of the hsp70 genes may be associated with a higher susceptibility to NIHL.

Base excision repair (BER) of DNA damage in irradiated THP1 human leukemic cells was stimulated by pretreating the cells with exogenous recombinant Hsp70. The treatment of THP1 cells with recombinant Hsp70 in cell culture promoted repair by reducing the frequency of apurinic, apyrimidinic (AP) sites in DNA before and after 1.3 Gy of radiation. However, by 30 minutes after 2.6 Gy, accelerated repair of abasic sites supervened, which may contribute to the loss of the very-low-dose cell hypersensitivity seen in clonogenic studies of other laboratories. After irradiation with 2.6 Gy, the crucial initial glycosylase step was markedly incomplete when cells had been transfected 24 hours before with a small interfering RNA (siRNA) designed to inhibit synthesis of Hsp70. In confirmation, lysates from irradiated siRNA-treated cells after 2.6 Gy were deficient in uracil glycosylase activity (UDG). Transfection with a scrambled RNA of the same size did not interfere with the glycosylase step, ie, the prompt conversion of damaged pyrimidine sites to abasic sites as well as the subsequent repair of those sites. BER measured by reduction of DNA AP sites before and after low-dose radiation was also deficient in THP1 cells that had been transfected with the siRNA designed to inhibit synthesis of Hsp70. These results implicate BER and the participation of Hsp70 in the repair of DNA in human leukemic cells with the doses of ionizing radiation used in clinical regimens.

The purpose of this study was to investigate (1) whether ischemia-reperfusion increased the content of heat shock protein 72 (Hsp72) transcripts and (2) whether myocardial content of Hsp72 is increased by ischemic preconditioning so that they can be considered as end effectors of preconditioning. Twelve male minipigs (8 protocol, 4 sham) were used, with the following ischemic preconditioning protocol: 3 ischemia and reperfusion 5-minute alternative cycles and last reperfusion cycle of 3 hours. Initial and final transmural biopsies (both in healthy and ischemic areas) were taken in all animals. Heat shock protein 72 messenger ribonucleic acid (mRNA) expression was measured by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method using complementary DNA normalized against the housekeeping gene cyclophilin. The identification of heat shock protein 72 was performed by immunoblot. In our “classic” preconditioning model, we found no changes in mRNA hsp72 levels or heat shock protein 72 content in the myocardium after 3 hours of reperfusion. Our experimental model is valid and the experimental techniques are appropriate, but the induction of heat shock proteins 72 as end effectors of cardioprotection in ischemic preconditioning does not occur in the first hours after ischemia, but probably at least 24 hours after it, in the so-called “second protection window.”

The effects of heat shock protein 70 (Hsp70), a molecular chaperone, on the degradation and functional alterations of a mutant large T antigen induced by a nonpermissive temperature were examined. In this study, mouse tracheal epithelial TM02-3 cells harboring temperature-sensitive simian virus 40 large T antigen and stable TM02-3 cells overexpressing human Hsp70 and/or Hsp40 were used. Although the temperature shift from 33°C (permissive temperature) to 39°C (nonpermissive temperature) induced increases in the endogenous chaperones including Hsp70 and Hsp40, degradation of the T antigen, activation of the p53-p21^waf1 pathway, and an arrest of cell growth were observed in the mock cells. In contrast, these changes induced by the temperature shift were partially but significantly prevented in stable cells overexpressing human Hsp70 and/or Hsp40. A combination of Hsp70 and Hsp40 was the most effective, suggesting that Hsp40 may cooperate with Hsp70. Moreover, immunocytochemical observation indicated that human Hsp70 was expressed in the cytoplasm at 33°C, but it colocalized with T antigen in the nucleus at 39°C. These results suggest that overexpressed Hsp70 translocates from the cytoplasm to nucleus, and significantly restores the structural stability and functional defects of mutant large T antigen in the cells.

Enhanced survival of both individual cells and whole organisms following a heat stress is termed thermotolerance. In organisms, the maintenance of tissue function rather than the survival of individual cells ultimately determines outcome following thermal challenge. We used MDCK kidney epithelial cells to compare alterations in chaperone activity (as a measure of cellular tolerance) and epithelial barrier function (as a measure of physiological tolerance) after thermal challenge. Quercetin, an inhibitor of heat shock factor–dependent transcriptional activity, both potentiated the effects of heat on naive monolayers and blocked conditioning of monolayers following moderate heat shock, suggesting a central role of heat shock protein (HSP) family members in the maintenance of epithelial integrity. We used MDCK cells that constitutively overexpressed HSP70 to demonstrate 2 functionally distinct components of the response of monolayers to thermal stress. The maintenance of epithelial barrier function during exposure to elevated temperatures is regulated by a complex network of processes that involve the actions of HSP70 but that are independent of alterations in chaperone activity as reflected by changes in the thermal inactivation/refolding of luciferase. In contrast, the restoration of barrier function following a heat stress is directly modulated by HSP70 in a manner that can be fully accounted for by changes in chaperone activity. This study demonstrates an important, albeit complex, protective role for heat shock proteins in the modulation of MDCK epithelial barrier function following a thermal stress.

Mammalian responses to bacterial lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria can lead to an uncontrolled inflammatory reaction that can be deadly for the host. We checked whether heat shock protein 70 (Hsp70) protein is able to protect animals from the deleterious effects of bacterial LPS by monitoring the effect of exogenous Hsp70 injections before and after LPS administration. Our research with rats demonstrates for the first time that administration of exogeneous Hsp70 before and after LPS challenges can reduce mortality rates and modify several parameters of hemostasis and hemodynamics. Hsp70 isolated from bovine muscles showed significant protective effects against the impaired coagulation and fibrinolytic systems caused by LPS, and reduced the mortality caused by Escherichia coli and Salmonella typhimurium LPS injections significantly. Characteristically, Hsp70 preparations used in the experiments result in different effects when administered before and after an LPS challenge, and the effects of Hsp70 injections also differ significantly depending on the origin of the LPS (E coli vs S typhimurium). Based on our data, mammalian Hsp70 appears to be an attractive target in therapeutic strategies designed to stimulate endogenous protective mechanisms against many deleterious consequences of septic shock by accelerating the functional recovery of susceptible organs in humans.