Basal Levels Of Hsp70 Research Articles

Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70.

Aging impairs the expression of HSP70, an emergent player in vascular biology. However, it is unknown if age-related alterations in HSP70 are linked to a decline in arterial function. In this study, we test the hypothesis that the contributions of HSP70 to vascular contraction are diminished in middle-aged animals. We determined the basal levels of HSP70 in the aorta of young and middle-aged Sprague Dawley male rats using Western blotting. Functional studies were performed in a wire myograph system. Force development in response to phenylephrine was assessed in the presence or absence of extracellular calcium (Ca2+), and in aortic rings treated or non-treated with an HSP70 inhibitor. Fluorescent probes were used to evaluate vascular oxidative stress and nitric oxide levels. We report that middle-aged rats have significantly lower levels of HSP70. Blockade of HSP70 attenuated vascular phasic and tonic contraction in isolated aortas. It appears that a functional HSP70 is required for proper Ca2+ handling as inhibition of this protein led to reduced force–displacement in response to Ca2+ dynamics. Furthermore, middle-aged aortic rings exposed to the HSP70 inhibitor display higher reactive oxygen species levels without changes in nitric oxide. In summary, we show that middle-aged animals have lower levels of HSP70 in aortas, which associates with an age-related decline in vascular responses to α-1 adrenergic stimulation.

CHIP mutations affect the heat shock response differently in human fibroblasts and iPSC-derived neurons.

ABSTRACTC-terminus of HSC70-interacting protein (CHIP) encoded by the gene STUB1 is a co-chaperone and E3 ligase that acts as a key regulator of cellular protein homeostasis. Mutations in STUB1 cause autosomal recessive spinocerebellar ataxia type 16 (SCAR16) with widespread neurodegeneration manifesting as spastic-ataxic gait disorder, dementia and epilepsy. CHIP−/− mice display severe cerebellar atrophy, show high perinatal lethality and impaired heat stress tolerance. To decipher the pathomechanism underlying SCAR16, we investigated the heat shock response (HSR) in primary fibroblasts of three SCAR16 patients. We found impaired HSR induction and recovery compared to healthy controls. HSPA1A/B transcript levels (coding for HSP70) were reduced upon heat shock but HSP70 remained higher upon recovery in patient- compared to control-fibroblasts. As SCAR16 primarily affects the central nervous system we next investigated the HSR in cortical neurons (CNs) derived from induced pluripotent stem cells of SCAR16 patients. We found CNs of patients and controls to be surprisingly resistant to heat stress with high basal levels of HSP70 compared to fibroblasts. Although heat stress resulted in strong transcript level increases of many HSPs, this did not translate into higher HSP70 protein levels upon heat shock, independent of STUB1 mutations. Furthermore, STUB1(−/−) neurons generated by CRISPR/Cas9-mediated genome editing from an isogenic healthy control line showed a similar HSR to patients. Proteomic analysis of CNs showed dysfunctional protein (re)folding and higher basal oxidative stress levels in patients. Our results question the role of impaired HSR in SCAR16 neuropathology and highlight the need for careful selection of proper cell types for modeling human diseases.

Hsp70 basal levels, a tissue marker of the rate of aging and longevity in mice

Under stressful conditions, the expression of the chaperone Hsp70 is induced, which acts as a cellular defense mechanism. The impairment in this induction has been related to aging, whereas an increased expression has been related to longevity. Nevertheless, it is still not known if the basal levels of Hsp70 can be indicative of the aging rate of different tissues. The aim of this study was to quantify the basal levels of Hsp70 in tissues from female mice throughout their aging process including long-lived mice, as well as from prematurely aging mice (PAM). Adult, old and long-lived (6, 18 and 30months of age, respectively) female ICR-CD1 and Balb/C mice were used. Tissues with mainly mitotic (liver and renal medulla) or post-mitotic (heart, renal cortex, cerebral cortex, spleen) cells and peritoneal leukocytes from these animals as well as from adult PAM and non-prematurely aging mice (NPAM), were studied. Basal levels of Hsp70 were assessed using an ELISA method. The results showed that the aging-associated variation of the levels of Hsp70 followed a different pattern in post-mitotic and mitotic tissues, being lower or higher in old mice comparing to adults, respectively. In all the tissues analyzed the Hsp70 levels from long-lived mice were similar to those from adult animals. In addition, in adult PAM these Hsp70 levels were similar to those in chronologically old animals. In conclusion, Hsp70 basal levels show tissue-specific age-associated variations and are preserved in long-lived animals, demonstrating their role as markers of the rate of aging and longevity.

A Drosophila heat shock response represents an exception rather than a rule amongst Diptera species.

Heat shock protein 70 (Hsp70) is the major player that underlies adaptive response to hyperthermia in all organisms studied to date. We investigated patterns of Hsp70 expression in larvae of dipteran species collected from natural populations of species belonging to four families from different evolutionary lineages of the order Diptera: Stratiomyidae, Tabanidae, Chironomidae and Ceratopogonidae. All investigated species showed a Hsp70 expression pattern that was different from the pattern in Drosophila. In contrast to Drosophila, all of the species in the families studied were characterized by high constitutive levels of Hsp70, which was more stable than that in Drosophila. When Stratiomyidae Hsp70 proteins were expressed in Drosophila cells, they became as short-lived as the endogenous Hsp70. Interestingly, three species of Ceratopogonidae and a cold-water species of Chironomidae exhibited high constitutive levels of Hsp70 mRNA and high basal levels of Hsp70. Furthermore, two species of Tabanidae were characterized by significant constitutive levels of Hsp70 and highly stable Hsp70 mRNA. In most cases, heat-resistant species were characterized by a higher basal level of Hsp70 than more thermosensitive species. These data suggest that different trends were realized during the evolution of the molecular mechanisms underlying the regulation of the responses of Hsp70 genes to temperature fluctuations in the studied families.

Epigallocatechin-3-gallate pretreatment attenuates doxorubicin-induced cardiotoxicity in rats: A mechanistic study

Doxorubicin (DOX) is a widely used chemotherapeutic agent however its clinical use is limited by cumulative cardiotoxicity. Epigallocatechin-3-gallate (EGCG), a main catechin in green tea, possesses a potent antioxidant, anti-apoptotic and anticancer properties. The current study aimed to investigate the potential protective effect of EGCG against DOX-induced cardiotoxicity. Firstly the potential cardioprotective dose of EGCG was screened at different doses (10, 20 and 40mg/kg/day) against a single dose of DOX (15mg/kg; i.p.). EGCG protected against DOX-induced ECG changes, leakage of cardiac enzymes (creatine kinase isoenzyme-MB, and lactate dehydrogenase) and histopathological changes. The dose of 40mg/kg EGCG was selected for further assessment to address the EGCG cardioprotective mechanisms. EGCG was given orally 3 times/week for 4 consecutive weeks and DOX (2.5mg/kg; i.p.) 3 times/week on the last 2 weeks. EGCG significantly ameliorated oxidative stress injury evoked by DOX as evidenced by inhibition of reduced glutathione depletion and lipid peroxidation as well as elevation of antioxidant enzyme activities. DOX caused down-regulation of ErbB2 expression while EGCG pretreatment significantly increased ErbB2 expression indicating its effect on pro-survival pathway. Furthermore, DOX provoked apoptotic responses evidenced by increasing the expression of nuclear factor kappa-B, tumor suppressor protein p53, calpain 2, caspases 3 and 12. Additionally basal level of Hsp70 was reduced in DOX-intoxicated group. EGCG pretreatment significantly ameliorated these apoptotic signals indicating its anti-inflammatory and anti-apoptotic actions. In conclusion, EGCG possesses cardioprotective action against DOX-induced cardiotoxicity by suppressing oxidative stress, inflammation and apoptotic signals as well as activation of pro-survival pathways.

Basal and stress-induced Hsp70 are modulated by ataxin-3

Regulation of basal and induced levels of hsp70 is critical for cellular homeostasis. Ataxin-3 is a deubiquitinase with several cellular functions including transcriptional regulation and maintenance of protein homeostasis. While investigating potential roles of ataxin-3 in response to cellular stress, it appeared that ataxin-3 regulated hsp70. Basal levels of hsp70 were lower in ataxin-3 knockout (KO) mouse brain from 2 to 63 weeks of age and hsp70 was also lower in fibroblasts from ataxin-3 KO mice. Transfecting KO cells with ataxin-3 rescued basal levels of hsp70 protein. Western blots of representative chaperones including hsp110, hsp90, hsp70, hsc70, hsp60, hsp40/hdj2, and hsp25 indicated that only hsp70 was appreciably altered in KO fibroblasts and KO mouse brain. Turnover of hsp70 protein was similar in wild-type (WT) and KO cells; however, basal hsp70 promoter reporter activity was decreased in ataxin-3 KO cells. Transfecting ataxin-3 restored hsp70 basal promoter activity in KO fibroblasts to levels of promoter activity in WT cells; however, mutations that inactivated deubiquitinase activity or the ubiquitin interacting motifs did not restore full activity to hsp70 basal promoter activity. Hsp70 protein and promoter activity were higher in WT compared to KO cells exposed to heat shock and azetidine-2-carboxylic acid, but WT and KO cells had similar levels in response to cadmium. Heat shock factor-1 had decreased levels and increased turnover in ataxin-3 KO fibroblasts. Data in this study are consistent with ataxin-3 regulating basal level of hsp70 as well as modulating hsp70 in response to a subset of cellular stresses.

Hsp70 protein levels and thermotolerance in Drosophila subobscura: a reassessment of the thermal co‐adaptation hypothesis

Theory predicts that geographic variation in traits and genes associated with climatic adaptation may be initially driven by the correlated evolution of thermal preference and thermal sensitivity. This assumes that an organism's preferred body temperature corresponds with the thermal optimum in which performance is maximized; hence, shifts in thermal preferences affect the subsequent evolution of thermal-related traits. Drosophila subobscura evolved worldwide latitudinal clines in several traits including chromosome inversion frequencies, with some polymorphic inversions being apparently associated with thermal preference and thermal tolerance. Here we show that flies carrying the warm-climate chromosome arrangement O(3+4) have higher basal protein levels of Hsp70 than their cold-climate O(st) counterparts, but this difference disappears after heat hardening. O(3+4) carriers are also more heat tolerant, although it is difficult to conclude from our results that this is causally linked to their higher basal levels of Hsp70. The observed patterns are consistent with the thermal co-adaptation hypothesis and suggest that the interplay between behaviour and physiology underlies latitudinal and seasonal shifts in inversion frequencies.

Early Ischaemic Preconditioning of Spinal Cord Enhanced the Binding Profile of Heat Shock Protein 70 with Neurofilaments and Promoted its Nuclear Translocation after Thoraco-abdominal Aortic Occlusion in Pigs

Objective(s)Heat shock protein 70 (Hsp70) is detected in substantial amounts in normal neurons and this basal content may protect a cell against harmful conditions without the need for additional synthesis. Herein, we investigate the potential protective role of these basal levels of Hsp70, in an early ischaemic preconditioning (IPC) experimental model, suggesting a possible role of this protein as a first window of protection. Design, material and methodsForty-two pigs were used in an experimental thoraco-abdominal aortic occlusion model. Twelve animals (two groups) were used for neurological evaluation. The remaining 30 animals (five groups) were used for immunoprecipitation and immunohistochemical studies. These were performed to study the binding relationship of Hsp70/cytoskeleton elements and the cellular distribution of Hsp70, respectively. ResultsThe IPC + ischaemia-group showed significant better neurologic scores compared with those of the ischaemia group, indicating a protective role for IPC (P = 0.003). The immunoprecipitations demonstrated that early IPC increased significantly the binding profile of Hsp70/neurofilaments (P = 0.025). In addition, translocation of Hsp70 into the nucleus was observed, which was conserved until the sustained ischaemia. ConclusionsThese results indicate that Hsp70 may have an important role in early IPC of the spinal cord, by protecting neurofilaments and by ensuring the functionality and the integrity of the nucleus, at the time the intensive insult begins.

Cellular stress response pathways and ageing: intricate molecular relationships.

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, senescent decline and ageing is broadly influenced by genetic and extrinsic factors. Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Here, we survey the molecular mechanisms that link ageing to main stress response pathways, and mediate age-related changes in the effectiveness of the response to stress. We also discuss how each pathway contributes to modulate the ageing process. A better understanding of the dynamics and reciprocal interplay between stress responses and ageing is critical for the development of novel therapeutic strategies that exploit endogenous stress combat pathways against age-associated pathologies.

In vitro heat shock of human monocytes results in a proportional increase of inducible Hsp70 expression according to the basal content

Heat shock proteins play an important role as molecular chaperones of the cell. Inducible heat shock protein 70 is rapidly synthesised in response to numerous stressors and monocytes are sensitive to changes in core temperature resulting in a circadian variation of Hsp70 expression. Monocytes were isolated via density centrifugation from nine healthy male volunteers at 5 am, 1 pm and 9 pm, representing the nadir (5 am), peak (9 pm) and intermediate (1 pm) of Hsp70 expression in the 24-h cycle. Analysis of freshly isolated monocytes for Hsp70 expression confirmed Hsp70 levels at the three selected time points. Monocytes were subjected to in vitro heat shock at 40 degrees C (+/-0.1) for 90 min with a 90 min 37 degrees C (+/-0.1) exposure acting as a control. A significant increase in Hsp70 was observed at 5 am (p < 0.001) and 1 pm (p = 0.028) at 40 degrees C when compared to 37 degrees C but not at 9 pm (p = 0.19). A significant increase was also observed from the basal levels of Hsp70, measured on freshly isolated monocytes and the levels detected after heat shock at 40 degrees C at 5 am (p < 0.001) and 1 pm (p = 0.001), which was not observed at 9 pm (p = 0.15). Furthermore, a significant correlation was observed in the heat shock response at 40 degrees C and that obtained at 37 degrees C (p < 0.001). In conclusion, the heat shock response in monocytes is directly proportional to the amount of Hsp70 present in the cells and the stress response may be much higher at different times of the day.

Aging‐related differences in basal heat shock protein 70 levels in lymphocytes are linked to altered frequencies of lymphocyte subsets

Cell stress responses are ubiquitous in all organisms and are characterized by the induced synthesis of heat shock proteins (Hsp). Previous studies as well as recent reports by our group have consistently suggested that aging leads to an increase in the basal levels of Hsp70. Here we extend these studies by examining the differential Hsp70 response of peripheral blood lymphocyte (PBL) subsets. It is well established that with aging, one of the major changes in the T cell pool is an expansion of T cells with the memory phenotype as well as those deficient for the CD28 molecule. To determine if alterations in the frequency of T cell subsets might be responsible for the observations, we have carried out a more comprehensive flow cytometric analysis of the various phenotypes of PBL under unstimulated conditions. Cells were obtained from 10 young and 10 elderly normal subjects. The basal Hsp70 levels in the various PBL phenotypes were comparable between young and elderly subjects. However, different patterns of Hsp70 response were noticed among the PBL subtypes, which were similar in both young and elderly subjects. In particular, the memory cell phenotypes produced more Hsp70 than the naïve phenotypes. These results suggest that aging-related changes in basal Hsp70 levels in PBL are linked to the altered frequency of lymphocyte subsets and not to increases in aged lymphocytes per se. In addition, the increase in Hsp70 can be interpreted as the result of a tendency towards more pronounced cellular differentiation in aging.

The effect of 15 consecutive days of heat–exercise acclimation on heat shock protein 70

The purpose of this study was to investigate the alterations in serum heat shock protein (Hsp) 70 levels during a 15-consecutive-day intermittent heat-exercise protocol in a 29-year-old male ultra marathon runner. Heat acclimation, for the purpose of physical activities in elevated ambient temperatures, has numerous physiological benefits including mechanisms such as improved cardiac output, increased plasma volume and a decreased core temperature (T (c)). In addition to the central adaptations, the role of Hsp during heat acclimation has received an increasing amount of attention. The acclimation protocol applied was designed to correspond with the athlete's tapering period for the 2007 Marathon Des Sables. The subject (VO(2)max = 50.7 ml.kg(-1).min(-1), peak power output [PPO] = 376 W) cycled daily for 90 min at a workload corresponding to 50% of VO(2)max in a temperature-controlled room (average WBGT = 31.9 +/- 0.9 degrees C). Venous blood was sampled before and after each session for measurement of serum osmolality and serum Hsp70. In addition, T (c), heart rate (HR) and power output (PO) was measured throughout the 90 min to ensure that heat acclimation was achieved during the 15-day period. The results show that the subject was successfully heat acclimated as seen by the lowered HR at rest and during exercise, decreased resting and exercising T (c) and an increased PO. The heat exercise resulted in an initial increase in Hsp70 concentrations, known as thermotolerance, and the increase in Hsp70 after exercise was inversely correlated to the resting values of Hsp70 (Spearman's rank correlation = -0.81, p < 0.01). Furthermore, the 15-day heat-exercise protocol also increased the basal levels of Hsp70, a response different from that of thermotolerance. This is, as far as we are aware, the first report showing Hsp70 levels during consecutive days of intermittent heat exposure giving rise to heat acclimation. In conclusion, a relatively longer heat acclimation protocol is suggested to obtain maximum benefit of heat acclimation inclusive of both cellular and systemic adaptations.

Characterization of the heat shock response in mature zebrafish ( Danio rerio)

Heat shock proteins (Hsps) are involved in many physiological and pathological processes and are diminished with age in a variety of species. As zebrafish embryos have proven to be excellent models for studying Hsp response during development, we sought to characterize the response in mature zebrafish to demonstrate the utility of the zebrafish model in studying late-life diseases and the biology of aging. Accordingly, mature zebrafish were exposed to a 37 °C heat stress and mRNA was isolated from various tissues and subjected to analysis by RT-PCR. We found that Hsp70 was upregulated in brain, liver, and muscle, while Hsp47 was upregulated in brain, but not liver or muscle. Hsp90alpha, Hsp90beta, and heat shock factor 1a (Hsf1a) were expressed in all three tissues, but were not upregulated in response to heat stress. A comparison of Hsp expression in young versus mature zebrafish revealed decreased basal levels of Hsp70 and increased levels of Hsf1a in mature fish. These results indicate that the heat shock response is detectable in mature zebrafish and that there are age differences in their heat shock response, suggesting that mature zebrafish may be a useful model for studying Hsp response during the aging process.

Basal and inducible levels of Hsp70 in patients with acute heat illness induced during training.

Heat shock proteins (Hsps) or stress proteins, and, in particular, the inducible, cytosolic Hsp70, represent a highly conserved response to heat exposure and to a variety of noxious stimuli. Many investigations have shown correlations between the aberrant expression of Hsps and disease states. Whether the basal and inducible levels of Hsp70 are of any biological significance in patients with heat-induced diseases remains unknown. In the present study, we compared the basal and inducible levels of Hsp70 by flow cytometry in lymphocytes of patients with heat-induced diseases and after recovery from this disease, and in matched controls. Both groups comprised individuals who exercised by running in the same hot environment. The level of inducible Hsp70 was also measured after a heat treatment of lymphocytes in vitro. The results show that there is variation of basal and inducible Hsp70 levels among individuals. However, the group of patients suffering from heat-induced illnesses in May shows a significantly higher basal (P = 0.02) level of Hsp70 than does the control group. Individuals who have an increased level of Hsp70 may be more sensitive to heat or may respond differently. The level of Hsp70 may represent a biomarker to evaluate whether they are more susceptible to stresses than other individuals. Interestingly, the basal level of Hsp70 is higher in both the patient group and the control group in November than in May. In fact, the basal levels of Hsp70 in the patient and control groups are essentially the same in November, perhaps reflecting the successful stress conditioning of both groups.

Introducing Professor Masataka Mori, Asia-Australian Regional Editor.

Introducing Professor Masataka Mori, Asia-Australian Regional Editor.

Heat shock protein Hsp70 expression and DNA damage in Baikalian sponges exposed to model pollutants and wastewater from Baikalsk Pulp and Paper Plant

Lake Baikal, a unique habitat for a great number of endemic species, is the largest freshwater reservoir in the world which is still largely unaffected by anthropogenic pollution, except for some shore regions with industrial activity. The expressions of a biomarker of exposure (heat shock protein Hsp70) and a biomarker of effect (DNA single-strand breaks) were measured for the first time in endemic Baikalian sponge species ( Baikalospongia intermedia, Lubomirskia fusifera, and Lubomirskia abietina). Tissue cubes of B. intermedia and dissociated cells of L. fusifera and L. abietina reacted to temperature stress (10–16 °C above ambient temperature) with a time-dependent increase in expression of Hsp70. In B. intermedia, the effects of model pollutants (lead, copper, and zinc, and the organochlorines tetrachloroguaiacol, TCG, and pentachlorophenol, PCP) and of the wastewater from the final refinement and aeration reservoirs of the Baikalsk Pulp and Paper Plant (BPPP), located at the shore of the southern basin of Lake Baikal, on the expression of Hsp70 and the extent of DNA damage were investigated. It was found that lead and zinc but not copper cause a strong induction of Hsp70 in this sponge, while the frequency of DNA single-strand breaks increased after exposure to all these heavy metals tested. Induction of DNA single-strand breaks was also observed after exposure to TCG and PCP, but these compounds did not (consistently) enhance Hsp70 expression. Wastewater taken from the final water aeration pond of BPPP caused a concentration-dependent increase in Hsp70 expression in B. intermedia. However, there was no difference in the basal levels of Hsp70 between sponges collected in the shallow water at an unpolluted site near Baikalsk City and at a polluted site where the wastewaters of BPPP are discharged into the lake. There was also no clear difference in the wastewater concentration-dependent induction of Hsp70 expression between sponges collected at these sites, indicating no adaptation to continuous stress exposure.

Reduced levels of oxidized and glycoxidized proteins in human fibroblasts exposed to repeated mild heat shock during serial passaging in vitro

Repeated mild heat shock (RMHS) has beneficial hormesis-like effects on various characteristics of human skin fibroblasts undergoing replicative senescence in vitro. We have tested whether RMHS could reduce the accumulation of oxidized and glycoxidized proteins, which is a major age-related change. Levels of carbonylated proteins, furosine, Nε-carboxymethyl-lysine-rich proteins and advanced glycation end products increased during serial passaging of fibroblasts in culture. However, the extent of accumulation of oxidized and glycoxidized proteins was significantly reduced in RMHS cells. The basal concentration of reduced glutathione was higher and that of oxidized glutathione was lower in RMHS cells. Whereas the basal level of heat shock protein HSP27 decreased in both RMHS and control cells during serial passaging, the increase of the basal level of HSP70 with increasing passage level was significantly higher in RMHS cells. These results show that the slower accumulation of damaged proteins in fibroblasts exposed to RMHS results partly from the increased ability of these cells to cope with oxidative stress, and to synthesize HSP responsible for protein capping and refolding.

Increase in basal level of Hsp70, consisting chiefly of constitutively expressed Hsp70 (Hsc70) in aged rat brain.

Alteration of proteins in the function and higher structure has been observed in aged organisms. Stress proteins, which have a role to protect proteins from denaturation, may respond to the protein denaturation with aging. We found that the basal level of Hsp70, consisting chiefly of constitutively expressed Hsp70 (Hsc70), in 24-month-old Wistar rats was significantly higher in some parts of the brain than that in 6-month-old rats. On the other hand, the basal level of Hsp70 was significantly lower in 24-month-old rats after restricted feeding, which is known to suppress aging, than those fed the diet ad libitum. In the pons, medulla, striatum, and thalamus of 24-month-old rat brain, the level of Hsp70 increased and the denaturation of the cytosol proteins was suppressed. These results suggest that the expression of Hsp70, mostly Hsc70, increases with aging and may have a role to suppress protein denaturation.

Expression of 70-kDa heat shock protein in oral lesions: marker of biological stress or pathogenicity.

We showed differential expression of HSP70 during oral tumorigenesis. The precise functional role of HSP70 overexpression in the pathogenesis of betel and tobacco related oral cancer remains to be determined. To evaluate the utility of HSP70 as an indicator of the biological stress experienced by tumour cells or the malignant potential of oral epithelial lesions and predicting clinical outcome, its expression was assessed in different stages of oral carcinogenesis by immunohistochemical analysis and correlated with clinicopathological parameters. Overexpression of HSP70 protein was observed in 38 of 64 (59%) dysplastic lesions and 92 of 125 (74%) oral squamous cell carcinomas (SCCs) which included 76 of 105 cases (72%) of primary oral SCCs and 16 of 20 (80%) of recurrent oral SCCs. A significant correlation of HSP70 expression was observed with severity of dysplasia (P = 0.0006767), poor histological differentiation of primary tumours (P = 0.0184348), increase primary tumour size (P = 0.0221103) and consumption of betel and tobacco (P < 0.01). Follow-up studies showed that in patients with premalignant lesions the median transition time (premalignancy to malignancy) was significantly shorter in HSP70 overexpressing cases than those showing basal level of HSP70 (P = 0.012). Oral cancer patients with elevated levels of HSP70 showed decreased median disease-free survival time (no recurrence/metastasis) than those showing basal HSP70 immunoreactivity (P = 0.0246). The results suggest that HSP70 expression may not be a mere marker of biological stress but may also be implicated in the pathogenesis of oral cancer.

Characterization of Human Proximal Tubular Cells after Hypoxic Preconditioning: Constitutive and Hypoxia-Induced Expression of Heat Shock Proteins HSP70 (A, B, and C), HSC70, and HSP90

In animal models of cardiac or cerebral ischemic preconditioning, induction of heat shock proteins (HSPs), especially HSP70, correlates with protection from subsequent injury. The extent of HSP70 induction after stress correlates inversely with initial HSP70 levels. Primate cells, unlike nonprimate cells, express high basal levels of HSP70; thus, primate cells may respond differently to preconditioning than nonprimate cells. We have demonstrated that exposing cultured human proximal tubular epithelial cells (PTEC) to 12 h of hypoxia followed by a 24-h recovery period (hypoxic preconditioning) induces resistance to subsequent hypoxic injury. Herein, we characterize the expression of HSP70, HSP90, and heat shock cognate-70 (HSC70) in PTEC under basal conditions and after hypoxic preconditioning. By Northern blot analysis, we demonstrate that hypoxic preconditioning of PTEC increases mRNA for HSP70 > HSP90 > HSC70. With reverse transcription and polymerase chain reaction, mRNA transcripts from three different HSP70 genes (HSP70 A, B, and C) were detected in unstressed PTEC. Transcripts from these genes were also detected in freshly isolated human renal cortex, indicating that all three genes are expressedin vivo.By Western blot analysis, we demonstrate that PTEC express high basal levels of HSP70, HSC70, and HSP90. Hypoxic preconditioning did not lead to a significant increase in protein content of any of these HSPs, despite increased mRNA levels. This suggests that HSP accumulation cannot account for the development of cytoresistance after hypoxic preconditioning in PTEC. However, high basal expression of HSP70 in human PTEC may contribute to their innate resistance for hypoxia.