Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated β-galactosidase (SA-β-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.     

Expression levels of heat shock factors are not functionally coupled to the rate of expression of heat shock genes

The expression patterns of two mammalian heat shock factors (HSFs) were analysed in cell systems known to reflect an altered heat shock response. For being able to discriminate between the two closely related factors HSF 1 and HSF 2, specific cDNA sequences were cloned and used to generate antisense RNAs as hybridization probes. In general, in various cell lines expression of the two heat shock factors was clearly different. These expression patterns of the HSF genes were not influenced by retinoic acid-induced differentiation of human NT2 and mouse F9 teratocarcinoma cells. Generally, HSF 2 expression was extremely low, whereas the significantly higher expression of HSF 1 revealed cell specific differences. The highest expression rates of both HSFs were observed in 293 cells. To examine whether these high levels are involved in the constitutive expression of heat shock genes in these cells, we analysed the binding pattern of 293 cell proteins to the heat shock elements (HSEs). As with other cells, HSE-binding activity in 293 cells was only observed after heat shock treatment. This points to an HSE-independent way for high level expression of heat shock genes in these cells.     

Involvement of calcium in the differential induction of heat shock protein 70 by heat shock protein 90 inhibitors, geldanamycin and radicicol, in human non-small cell lung cancer H460 cells

Both geldanamycin (GA) and radicicol (RA) are HSP90 binding agents that possess antitumour activities. Although the in vitro data indicated that the inhibitory constant of RA is much bigger than that of GA, the in vivo data on drug efficacy might reveal different results. We have recently shown that treatment with GA induces a heat-shock response and that calcium mobilization may be involved in the process. By using induction of HSP70 as the endpoint assay, we found changes in upstream signaling mediators, including HSF1 and calcium mobilization, as well as possible involvement of protein kinase in human non-small cell lung cancer H460 cells treated with GA and RA. Our results demonstrated that calcium mobilization, a calcium dependent and H7-sensitive protein kinase, along with HSF1 activation by phosphorylation, are all involved in the HSP70 induction process triggered by the drugs. However, only GA, but not RA, can provoke a rapid calcium mobilization and thereby result in an instant induction of HSP70. Furthermore, the rapid calcium influx, followed by instant HSP induction, could be achieved in GA- or RA-treated cells placed in a medium containing excessive calcium while the response was completely abolished in cells depleted of calcium. Taken together, our findings suggest that differential calcium signaling may account for the differential induction of HSP and the action of GA and RA.     

转染人血红素加氧酶-1基因对大鼠血管平滑肌细胞抗氧化损伤的作用

本实验构建含人血红素加氧酶-1（hHO-1)基因的逆转录病毒载体XM-6/hHO-1,将其导入离体培养的大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC),观察外源性hHO-1基因在VSMC内的表达及其抗活性氧损伤作用,结果表明：（1）hHO-1基因可在靶细胞中明显表达,转染VSMC的HO-1蛋白表达和HO酶活性分别比非转染细胞高1.8倍和2.0倍;（2）转染hHO-1的VSMC可对抗大剂量H2O2对细胞的损伤作用,表现为细胞存活率增加和乳酸脱氢酶（LDH）漏出减少,上述保护作用可被HO特异性抑制剂锌原卟啉IX（Zinc-proto-porphyrinIX,ZnPP-IX)所阻断,研究结果提示,外源性HO-1的过量表达可增加VSMC对抗氧化损伤的能力。     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein （HSP） genes （hsp70, hsc70, hsp90） were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus （Homoptera： Delphacidae）, which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepidopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock （40 ℃） upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4℃ did not change the expression levels of any hsp in either species.     

Induction of heat shock gene expression in RAT1 primary fibroblast cells by ELF electric fields

Recent studies have demonstrated that the Ku70 gene fragment can be placed in the anti‐sense orientation under the control of a heat‐inducible heat shock protein 70 (HSP70) promoter and activated through heat shock exposure. This results in attenuation of the Ku70 protein expression, inhibiting cellular repair processes, and sensitizing the transfected cells to exposures such as the ionizing radiation exposures used clinically. However, achieving the tissue temperatures necessary to thermally induce the HSP70 response presents significant limitations to the clinical application of this strategy. Previous findings suggest an alternative approach to inducing a heat shock response, specifically through the use of extremely low frequency (ELF) electrical field stimulation. To further pursue this approach, we investigated HSP70 responses in transfected rat primary fibroblast (RAT1) cells exposed to 10 Hz electric fields at intensities of 20–500 V/m. We confirmed that low frequency electric fields can induce HSP70 heat shock expression, with peak responses obtained at 8 h following a 2 h field exposure. However, the approximate threefold increase in expression is substantially lower than that obtained using thermal stimulation, raising questions of the clinical utility of the response. Bioelectromagnetics 34:405–413, 2013. © 2012 Wiley Periodicals, Inc.