Induction of heat shock protein 27 by hydroxyurea and its relationship to experimental metastasis

Treatment of tumor cells with hydroxyurea (HU) has been shown to increase the experimental metastatic potential of these cells. We have previously described the induction of stress proteins (antioxidants) by in B16 murine melanoma cells and their relationship to the metastatic process. We have now investigated the induction by HU of another set of stress proteins, the heat shock proteins, and their role in experi-mental metastasis. HU markedly increased the cellular content of heat shock protein (hsp) 27 but not hsp 90, 72/73, or 60 as measured by immunoblotting. The induction of hsp27 protein was preceded specific increase in hsp27 mRNA. Furthermore, HU-treated cells were more thermotolerant. To investigate the functional role of hsp27, human hsp27 cDNA was constitutively overexpressed in B16 cells at seen in HU-treated cells. In separate experiments, we induced a global increase in hsps by heat shock. Neither the hsp27 transfectants nor the heat-shocked cells demonstrated an increase in their experimental metastatic capacity. We conclude that hsp27 protein is increased by HU by the specific induction of hsp27 mRNA in B16 melanoma cells but increased hsp27 protein is not responsible for the increase in experimental metastasis. Since high levels of hsp27 are associated with metastatic disease in breast and ovarian cancers, but not in our experimental system, the functional role of hsp27 in metastasis requires further study. © Rapid Science 1998     

Diathermy treatment increases heat shock protein expression in female,but not male skeletal muscle

Heat shock treatments have been used to appreciably increase heat shock protein 70 (Hsp70) content and the phosphorylation of heat shock protein 27 (Hsp27p). The purpose of this investigation was to determine whether diathermy can increase Hsp70 and Hsp27p content in skeletal muscle. Fourteen subjects (7 males and 7 females, 18–35 years) received a muscle biopsy from the v. lateralis and then underwent 20 min of diathermy followed by 20 min of hot pack heating on the contralateral leg. Twenty-four hours following treatment, a second muscle biopsy was performed on the treated leg. All samples were analyzed for Hsp70 and Hsp27p content using western immunoblotting. Images of the blots were obtained and analyzed via densitometry. A paired t-test was used to examine differences in heat shock protein content between the treated and untreated legs. Twenty-four hours following the heat treatment, female subjects significantly (P < 0.05) increased Hsp70 (+58%) and Hsp27p (+100%) content compared to the untreated leg. Male subjects had non-significant increases in Hsp70 (+35%) and Hsp27p (+32%) skeletal muscle content. These results implicate that diathermy can be an effective means to induce Hsp70 and Hsp27p in human skeletal muscle.     

Elevated levels of antibodies against 70 kDa heat shock proteins in the sera of patients with HIV infection

Heat shock proteins (Hsp), especially 70 kDa heat shock protein (Hsp70) play an important role in the life cycle of HIV-1 virus. Hsp70 is overexpressed in HIV-infected cells and this is the most abundant Hsp associated with HIV virions. The aim of our study was to investigate whether HIV infection increases the extent of specific humoral immune response against Hsp70. The serum concentration of anti-Hsp70 IgG antibodies was measured in 47 HIV-infected patients, and 62 healthy, HIV-seronegative persons. Nineteen patients on highly active anti-retroviral therapy (HAART) were followed for 24 months in a longitudinal study. Anti-Hsp70 antibodies were measured by ELISA, using recombinant human Hsp70. Levels of anti-Hsp70 antibodies were significantly (P < 0.0001) higher in the HIV-infected patients (median: 1409 (25th-75th percentile: 1031-2214) AU/ml) than in healthy control subjects (626 (429-970) AU/ml). In 19 HIV patients, serum levels of anti-Hsp70 antibodies significantly (P < 0.001) decreased during 24 (11-41) months HAART (1309 (887-2213) AU/ml before and 640 (386-959) AU/ml during HAART), accompanied by viral load reduction and CD4+ count elevation. It is concluded that HIV-infection induces a marked increase in the anti-Hsp70 antibody levels, which is consistent with the enhanced expression of Hsp70 on the surface of HIV-infected cells and/or incorporation of the protein into the membrane of HIV virions.     

Role of heat shock proteins HSP70 and HSP32 in the protective effect of adaptation of cultured HT22 hippocampal cells to oxidative stress

Preadaptation of cultured HT22 mouse hippocampal neurons to oxidative stress prevented cell damage induced by severe oxidative stress. This protection manifested in a decrease in metabolic disturbances in neurons. Adaptation of neurons to oxidative stress was accompanied by accumulation of HSP32 and HSP70. HSP synthesis inhibitor quercetin abolished the protective effect of adaptation under conditions of oxidative stress. Activation of HSP70 synthesis in neurons is an important mechanism for adaptive protection of cells. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 138–142, August, 2007     

Expression of heat shock protein 47 is increased in remnant kidney and correlates with disease progression

Glomerulosclerosis is characterized by accumulation of the mesangial extracellular matrix, including type I and IV collagen. The processing for the collagens in the glomeruli may play a critical role for development of glomerulosclerosis. We examined the expression of heat shock protein 47 (HSP47), a collagen-binding molecular chaperone in the progresive glomerulosclerosis model. Subtotally nephrectomized rats, unlike sham-operated rats, developed focal and segmental glomerulosclerosis. Immunological staining demonstrated an increased expression of HSP47 which paralleled the expression of type I and IV collagen in the glomeruli of the nephrectomized rats as the glomerulosclerosis developed. The mRNA levels encoding type I and type IV collagen and HSP47 were increased 3.4 fold, 3.6 fold and 2.8 fold, respectively, at week 7 after nephrectomy. By in situ hybridization, the expression of HSP47 mRNA was determined to be localized to the glomeruli with segmental sclerosis. These results suggest that HSP47 may play a central role in the process of extracellular matrix accumulation during the development of glomerulosclerosis.     

热休克蛋白47和热休克蛋白60在小鼠胚胎器官形成过程中的表达

目的：研究小鼠胚胎器官形成过程中热休克蛋白47（Hsp47）和热休克蛋白60（Hsp60）的表达情况。方法：于GD11～GD18,取得小鼠胚胎脑、眼、心、肺、胃、肝、四肢,于GD13-GD18取得肾,利用RT-PCR方法半定量检测Hsp47和Hsp60在各器官的表达丰度。结果：Hsp47在GD11～GD12和GD18的脑、GD11～GD12和GD17～GD18的眼、GD11～GD12和GD16～GD18的肺、GD11-GD18的肝脏不表达,在其余时间和其余器官均有表达;Hsp60在CD11～GD18时段胚胎的脑、眼、心、肺、胃、肝、肾（GD13～GD18）、四肢中均有表达。结论：Hsp47和Hsp60在小鼠胚胎器官形成过程中有不同的表达丰度,其表达模式也不一致;Hsp47在小鼠胚胎发育过程中可能有重要功能,Hsp60则具有广泛表达的特点。     

结核杆菌热休克蛋白70基因的克隆与原核表达

目的：克隆结核杆菌热休克蛋白70(TBhsp70)基因，并在大肠杆菌中表达。方法：利用PCR技术从结核杆菌H37Rv中扩增Hsp70基因，并将其克隆到pUC19中，进行测序。将得到的Hsp70基因克隆到表达载体pGEX-4T-1中，构建重组表达质粒pGEX—TBhsp70，在大肠杆菌DH5α中进行表达。结果：成功地克隆了TBhsp70基因。DNA测序证实，与GenBank公布的序列一致。含pGEX-TBhsp70基因表达质粒的大肠杆菌经IPTG诱导后，能够表达相对分子质量(MR)约为96000的融合蛋白。结论：获得了TBhsp70基因，成功地构建了原核表达质粒pGEX-TBhsp70，并在大肠杆菌得到表达，为其相关研究奠定了一定的基础。     

热休克对大鼠再灌注性心律失常及心肌动作电位的影响

55只SD大鼠随机分为热休克组（H组,n=29)和对照组（Co组,n=26)。H组予以热休克预处理,Co组则否。两组先各取16只大鼠行Langendorff离体心脏灌注。以心电图记录模拟缺血后复灌时心律失常情况,并测定复灌时心脏流出液CK的活性,同时检测两组心脏组织中HSP70的含量和抗氧化酶的活性。接着对其余H组13只和Co且10只大鼠进行离体心脏乳头肌动作电位的测定。结果显示H组再灌注性心律失常明显减轻,心肌CK的释放量显著减少（P＜0．001）。同时H组心脏组织HSP70表达量显著增多,抗氧化酶活性也明显增强。H组台氏液灌流时心肌动作电位Vmax显著降低（P＜0．01）。以台氏液复灌后,心肌动作电位恢复时间也明显以H组为短。结果提示热休克预处理可以减轻大鼠心肌再灌注性损伤和再灌注性心律失常,此作用与心脏组织中HSP70含量的增加和抗氧化酶性的增强有关,同时还可能与热休克对心肌快Na^ 通道的抑制作用有关。     

热休克蛋白在子宫内膜异位症中的表达及意义

目的 探讨热休克蛋白27（HSP27）和HSP70在子宫内膜异位症发病中的作用。方法 采用免疫组化链霉菌抗生素蛋白－过氧化物酶染色法（SP法），检测子宫内膜异位症中56例异位内膜与在位内膜（30例）中HSP27和HSP70的表达。采用原位杂交，检测HSP70mRNA的水平，并与正常子宫内膜相比较。结果 免疫组化结果显示，异位内膜组织中HSP27与HSP70均呈高表达，与正常内膜组的表达差异显著（P＜0．01），而且失去其在正常内膜组织中表达的周期性变化。卵巢子宫内膜异位症组（OEM）与子宫腺肌症组（AM）组中，HSP27和HSP70的表达无显著差异（P＞0．05）。原位杂交结果显示，HSP70 mRNA（分别为P＜0．01和P＜0．05）。HSP27与HSP70的表达水平，同OEM的严重性无关。结论 异位内膜组织中的HSP70基因被激活，转录增加。HSP27和HSP70呈高表达，可能在EM的发病中起重要作用。     

Responses of rat myocardial antioxidant defences and heat shock protein HSP72 induced by 12 and 24‐week treadmill training

Aim: The purpose of this study was to determine the effects of both a short (12 weeks) and a long‐term (24 weeks) endurance treadmill‐training programme on the levels of oxidative stress markers, the activity of the enzymatic antioxidants, and the content of the 72 kDa heat shock protein (HSP72) in rat myocardium. Methods: Thirty male Wistar rats were randomly assigned to exercise trained (n = 16) and sedentary (n = 14) groups. After 12 week of training, eight rats were killed while the remaining eight continued the training programme until 24 week. Results: Seven sedentary controls were killed together with each trained group. Levels of thiobarbituric acid‐reactive substances (TBARS), protein carbonyls, and total and oxidized glutathione (tGSH and GSSG) in myocardial homogenates were unchanged by training irrespective of the protocol duration. However, an increased content of the oxidative stress biomarkers was detected in hearts from both the 24‐week trained rats and their sedentary controls when compared with their corresponding 12‐week groups. The antioxidant enzymatic activities total and mitochondrial superoxide dismutase (tSOD and mtSOD, respectively), glutathione peroxidase (GPX) and glutathione reductase (GR), remained unchanged after the 12‐week training period whereas a significant increase in tSOD and mtSOD activities (18%, P < 0.05) was observed in heart homogenates of 24‐week trained animals as compared with their sedentary controls. HSP72 expression levels were not significantly modified after 12 week of training but a threefold increase was detected after 24 week (P < 0.05). Conclusion: These results demonstrate that a long‐term endurance training (24 weeks) induced discrete increases in antioxidant enzyme activities in rat myocardium and elicited a marked enhancement in HSP72 expression levels. However, a shorter training programme (12 weeks), was not effective in increasing heart antioxidant defences.     

The effect of the rate of heat storage on serum heat shock protein 72 in humans

Hsp72 concentration has been shown to be higher in the serum (eHsp72) of runners with symptoms of heat illness than in non-ill runners. Recently, it has been suggested that the rate of heat storage during exercise in the heat may be an important factor in the development of heat stroke. Therefore, we compared the effect of two rates of heat storage on eHsp72 concentration during exercise in which subjects reached the same final core temperature. We hypothesized that with a lower rate of heat storage the increase in eHsp72 would be attenuated compared to a higher rate of heat storage. Nine heat acclimated subjects performed two exercise trials in a counterbalanced order in the heat (42°C, 30% relative humidity). The trials consisted of walking on a treadmill (~50% VO ₂ peak) dressed in military summer fatigues until rectal temperature reached 38.5°C. A high rate of heat storage (HS, 1.04 ± 0.10 W m⁻² min⁻¹, mean ± SE) occurred when subjects walked without cooling. To produce a lower rate of heat storage (LS, 0.54 ± 0.09 W m⁻² min⁻¹) subjects walked while wearing a water-perfused cooling vest underneath clothing. eHsp72 increased pre- to post-exercise (P < 0.05) but there was no difference (P > 0.05) in eHSP between the two rates of heat storage (LS 1.25 ± 0.73 to 2.23 ± 0.70 ng ml⁻¹, HS 1.04 ± 0.57 to 2.02 ± 0.60 ng ml⁻¹). This result suggests that eHsp72 is a function of the core temperature attained rather than the rate of heat storage.     

热休克蛋白70对荷瘤鼠的治疗作用

目的研究肿瘤来源的热休克蛋白70(heat shock protein 70,HSP70)对荷瘤小鼠的治疗作用,为其应用提供参考依据.方法细胞培养、蛋白质提纯技术、western-blot法、毛细管电泳技术和动物实验等.结果应用5μgHSP70能延长小鼠的生存期限,平均生存(28.7±4.5)d,与对照组(18.5±3.9)d相比差异显著(P＜0.05);而10μg治疗组的小鼠生存期为＞(62.3±29.1)d,40%获长期生存(＞90d),所接种肿瘤完全消退.与其他组相比,差异具显著性(P＜0.01).结论肿瘤来源的HSP70具有明确的治疗作用,本研究对于研究应用肿瘤来源的热休克蛋白70治疗人类恶性肿瘤具有较重要的参考意义.     

Successful primate immunization with peptides conjugated to purified protein derivative or mycobacterial heat shock proteins in the absence of adjuvants

We have previously shown in mice that antibodies can be induced to synthetic malaria peptides conjugated to mycobacterial antigens, such as purified protein derivative (PPD) or heat shock proteins (hsp), and given in the absence of adjuvants after a previous priming with bacille Calmette—Guérin (BCG). In the present study we investigated this model of immunization in the non-human primates, Saimiri sciureus monkeys. Monkeys primed with BCG subcutaneously and then immunized subcutaneously with the Plasmodium falciparum sporozoite (NANP)₄₀ synthetic peptide conjugated to PPD or mycobacterial hsp of 65 or 70 kD, in the absence of adjuvants, produced anti-peptide and anti-sporozoite IgG antibodies. Interestingly, the carrier effect of the hsp of 70 kD for the induction of anti-(NANP)₄₀ antibodies was also observed in the absence of a previous priming with BCG. These data suggest that such a vaccination strategy may be applied to humans.     

Lack of cytotoxic activity against Mycobacterium leprae 65-kD heat shock protein (hsp) in multibacillary leprosy patients.

Cytotoxic T cells play an important role in host defence mechanisms, as well as in the immunopathology of leprosy. In this study, we evaluated whether Mycobacterium leprae hsp18, hsp65 and Myco. tuberculosis hsp71 could induce cytotoxic T cell activity against autologous macrophages pulsed with these hsp. Paucibacillary (PB) patients and normal controls generated more effector cells than multibacillary (MB) patients with all three hsp tested. There was no cross-reactivity between any of the hsp tested. Mycobacterium leprae hsp65 induced cytotoxic responses only in those MB patients undergoing an erythema nodosum leprosum (ENL) episode. Although hsp65 and hsp18 induced similar proliferation in MB patients, a high proportion of these patients did not generate cytotoxic effector cells in response to hsp65. Hence, those T cells reacting to hsp65 may play an important role in the control of Myco. leprae infection.