Thermotolerance and heat shock proteins induced by hyperthermia in rat liver cells

Hepatic epithelial cells become thermotolerant when conditioned with a 30 minute heat-treatment at 43°C. The effect reaches a full amplitude after a 4–8 hour period at 37°C and lasts for more than one day at a level corresponding to a 50-fold increase in cellular thermoresistance. During the development period, electrophoretic patterns of proteins from cells incubated in presence of ³⁵S-methionine reveal an increased synthesis of a small set of proteins with molecular weights of 107, 89, 70, 68 and 27KD. The maximal synthesis of the induced proteins occurs concomitantly with the maximal increase of cell thermotolerance and has returned to normal when thermotolerance levels off. The induction of specific protein synthesis is also observed in other liver epithelial cells of normal and cancerous origins and in freshly isolated hepatocytes. It is suggested that the accumulation of these proteins in the cells plays a role in the process leading to a thermotolerant state.     

Investigation of the effects of heat shock and agents which induce a heat shock response on the induction of differentiation of HL-60 cells

A heat shock of 42.5-43.5 degrees C for 1 h applied to HL-60 promyelocytic leukemia cells induced the appearance of between 13 and 34% (n = 6) of cells which showed characteristics of mature metamyelocytes/granulocytes. This is the first time a physical agent has been shown to induce the differentiation of this leukemic cell line. The treatment of HL-60 cells with a variety of agents which have been documented to stress cells and induce thermotolerance or a heat shock-like response also induced granulocyte-like differentiation: continuous treatment for 4 days with ethanol (213 mM), sodium arsenite (6 microM), cadmium sulfate (60 microM), lidocaine (3 mM), and procaine (5 mM) induced 73, 54, 14, 54, and 55% of cells, respectively, to reduce the dye nitro blue tetrazolium. They were also capable of the phagocytosis of yeast particles. Examination of differentiated cells showed that those treated with ethanol, arsenite, lidocaine, and procaine also expressed nonspecific esterase activity, typical of monocytes, but did not adhere to plastic and had a cellular and nuclear morphology consistent with differentiation to metamyelocytes. Analysis of protein synthesis of HL-60 cells treated with 170 mM N-methylformamide, by the pulse labeling of cells for 2 h with [14C]leucine at various times, showed that the constitutive synthesis of both the Mr 90,000 and 70,000 heat shock proteins fell substantially after 2 h of exposure to N-methylformamide. When HL-60 cells were incubated with 1 M N-methylformamide, a toxic concentration of this agent, or were heat shocked, the synthesis of both the Mr 70,000 and Mr 90,000 proteins was induced. We propose that changes in heat shock protein synthesis may be an important element of the induction of differentiation of HL-60 cells, particularly as these proteins have recently been shown to regulate the stability of oncogene proteins, such as myc (Lüscher, B., and Eisenman, R. N., Mol. Cell Biol., 8: 2504-2512, 1988).     

How is the immune response affected by hyperthermia and heat shock proteins?

There is growing body of evidence linking the cellular response to heat stress with the response of the immune system to cancer. The anti-tumor immune response can be markedly enhanced by treatment with hyperthermia particularly in the fever range. In addition, the heat shock proteins (hsp) which are produced in abundant quantities in cells exposed to heat are potent immune modulators and can lead to stimulation of both the innate and adaptive immune responses to tumors. Immunostimulation by hyperthermia involves both direct effects of heat on the behavior of immune cells as well as indirect effects mediated through hsp release. In addition, the hsp can be deployed as components of antitumor vaccines in protocols that do not include hyperthermia. Understanding these process may permit the effective deployment of hyperthermia and hsp based vaccines in tumor treatment.     

用图像分析法研究银屑病表皮热休克蛋白的表达

目的 :了解热休克蛋白在银屑病表皮异常增生分化中的作用。方法 :采用免疫组化和图像定量分析技术研究银屑病皮损、非皮损表皮中热休克蛋白 (HSP2 7,HSP70 )含量的变化。结果 :正常人组和银屑病非皮损组表皮HSP2 7和HSP70的含量均明显高于银屑病皮损组 (P <0 0 1) ,而正常人组与银屑病非皮损组相比 ,两者含量无显著性差异 (P >0 0 5 )。结论 :热休克蛋白在银屑病发病中具有重要作用。而图像定量分析技术则大大提高了结果的可靠性和客观性。     

Effect of coumarins on HL-60 cell differentiation

Twenty-eight coumarins, including 7 furocoumarins, were examined for their activity of induction of terminal differentiation of human promyelocytic leukemia cells (HL-60) by nitro blue tetrazolium (NBT) reducing, nonspecific esterase, specific esterase and phagocytic activities. Esculetin, nordalbergin, 6,7-dihydroxy-4-methylcoumarin and imperatorin had strong activity among the coumarins examined. HL-60 cells treated with these coumarins differentiated into mature monocyte/macrophage. The structure-activity relationship established from the results revealed that 6,7-dihydroxy moiety had an important role in the induction of differentiation of HL-60.     

Down-regulation of a Mr 90,000 heat shock cognate protein during granulocytic differentiation in HL-60 human leukemia cells

Modulation of the synthesis of heat shock proteins in HL-60 human promyelocytic leukemia cells during differentiation was studied by metabolic labeling with [35S]methionine and Northern blot analysis. HL-60 cells were found to synthesize constitutively a high level of a Mr 90,000 protein (heat shock cognate protein, hsc90), which was very closely related to Mr 90,000 heat shock protein, hsp90, as far as molecular weight, isoelectric point, peptide maps, immunoreactivity, and encoded mRNA were concerned. Differentiation induction by dimethyl sulfoxide markedly decreased the level of the hsc90 expression, but preserved the ability to preferentially express hsp90 in response to heat stress. These results suggest distinct regulatory mechanisms of the synthesis between hsc90 and hsp90 although they are indistinguishable by conventional protein or mRNA analysis, and indicate that hsc90 has some role in cell growth and differentiation.     

黄芩苷作用HL-60细胞前后c-myc基因表达的变化

 目的 研究中药黄芩苷（baicalin）对人髓系白血病细胞株HL-60细胞增生、凋亡的影响及探讨c-myc基因在其中的作用。方法 培养人髓系白血病细胞株HL-60细胞,应用MTT法绘制细胞生长曲线观察黄芩苷对HL-60细胞增生的影响;DNA凝胶电泳观察细胞凋亡;RT-PCR检测黄芩苷作用前后c-myc基因mRNA表达水平的变化;Western blot检测黄芩苷作用前后c-myc蛋白表达水平的变化。结果　细胞生长曲线结果示黄芩苷能明显抑制HL-60细胞增生,半数抑制浓度（IC50）约为20μmol／L,DNA 片段化的检出表明黄芩苷能有效诱导HL-60细胞凋亡;黄芩苷作用后HL-60细胞c-myc基因和蛋白的表达水平均下降,并且随作用时间延长,表达下降更明显。结论 黄芩苷能有效抑制HL-60细胞增生,诱导其凋亡,c-myc基因可能参与了黄芩苷抑制HL-60细胞增生和诱导凋亡的过程。     

Effect of bioactive aldehydes on cell proliferation and c-myc expression in HL-60 human leukemic cells

Lipid peroxidation produces several toxic carbonyls, including biologically active aldehydes. In previous studies, we demonstrated that 4-hydroxynonenal (HNE), one of the major products of lipoperoxidation, inhibited growth and c-myc expression in K562 and HL-60 human leukemic cells. In this study, we compared the HNE effects with those of 4-hydroxyoctenal (HOE), 4-hydroxyundecenal (HUE; different lengths of the lipophilic tail), and the analogous aldehydes 2-trans-nonanal (lacking the OH group) and nonenal (lacking the OH group and the trans CC double bond), on HL-60 cell proliferation and c-myc expression. HUE and HOE inhibited growth and c-myc expression in a dose-dependent fashion, with an effectiveness comparable with that of HNE, whereas 2-nonenal and nonanal did not affect these parameters. Our results showed that different aldehydes produced from lipid peroxidation may contribute to growth inhibition by c-myc downregulation and that the molecular features involved seem to be the hydroxy group and the trans CC double bond.     

黄芪多糖对HL-60细胞端粒酶活性的作用

目的研究黄芪多糖对人早幼白血病HL-60细胞端粒酶活性的作用。方法不同浓度的黄芪多糖作用HL-60细胞后,MTT法测定细胞活性,计算细胞生长抑制率;TRAP-PCR-ELISA法测定端粒酶活性。结果黄芪多糖能抑制HL-60细胞的增殖,降低HL-60细胞的端粒酶活性,这两种作用均呈浓度和时间依赖性。结论黄芪多糖降低HL-60细胞端粒酶活性,可能是其诱导HL-60细胞凋亡的机制之一。     

Targeting heat shock proteins in cancer

Heat shock proteins (HSPs) HSP27, HSP70 and HSP90 are powerful chaperones. Their expression is induced in response to a wide variety of physiological and environmental insults including anti-cancer chemotherapy, thus allowing the cell to survive to lethal conditions. Different functions of HSPs have been described to account for their cytoprotective function, including their role as molecular chaperones as they play a central role in the correct folding of misfolded proteins, but also their anti-apoptotic properties. HSPs are often overexpressed in cancer cells and this constitutive expression is necessary for cancer cells’ survival. HSPs may have oncogene-like functions and likewise mediate “non-oncogene addiction” of stressed tumor cells that must adapt to a hostile microenvironment, thereby becoming dependent for their survival on HSPs. HSP-targeting drugs have therefore emerged as potential anti-cancer agents. This review describes the different molecules and approaches being used or proposed in cancer therapy based on the in inhibition of HSP90, HSP70 and HSP27.     

siRNA对HL-60细胞c-myc蛋白表达的影响

 目的　研究小干扰RNA（siRNA）对白血病细胞c-myc蛋白表达的影响,探寻白血病基因治疗的新方法。方法　应用针对c-myc的siRNA转染HL-60细胞,分别收集转染后24、48和72 h细胞及对照组细胞,提取细胞总蛋白,用Western blotting方法检测c-myc蛋白的表达情况。结果　与对照组相比,转染c-myc siRNA后,细胞c-myc蛋白表达水平明显下降,且c-myc蛋白含量随作用时间的延长而逐渐降低,转染24 h后c-myc 蛋白含量下降约42 %,48 h下降66 %,72 h下降78 %;单因素方差分析显示与对照组相比,差异均有统计学意义（P < 0.05）。结论　c-myc siRNA能降低HL-60细胞c-myc蛋白的表达,其作用具有序列特异性和时间依赖性,有望成为白血病靶向基因治疗的新工具。     

THE STUDIES OF REVERSAL ON INHIBITING DNA SYNTHESIS AND CLONAL FORMATION OF HL－60 CELLS WITH HYPERTHERMIA

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Flow cytometric measurements of heat shock proteins

A fixation and immunofluorescence staining procedure for measurements of heat shock proteins (hsp) by flow cytometry is reported. Three fixatives were compared: 80% methanol at —20°C for 1 h, 70% ethanol at 0°C for 1 h, and 3% paraformaldehyde at 4°C for 1 h followed by 0·2% NP-40. Cells fixed with methanol showed strongest immunofluorescence and lowest nonspecific fluorescence. The level of hsp 70 as a function of time after heating followed the same kinetics as the development of thermotolerance reported by others. The level of hsp 70 increased with increasing heat dose up to a maximum heat dose, and above this heat dose a decrease in the level of hsp 70 was observed. Correlated measurements of the level of hsp 70 and DNA showed that hsp 70 was found in all phases of the cell cycle. The level of hsp 70 increased about two-fold in unheated cells throughout the cell cycle. The increase in G₂ + M cells compared with G₁ cells was lower in cells heated at 45°C for 20 min followed by incubation at 37°C for 24 h before fixation and staining, than in unheated cells. The results show that flow cytometry provides a rapid and quantitative technique for measuring hsp. Correlated measurements of hsp and other cellular parameters might also be obtained.     

淫羊藿甙对急性早幼粒白血病细胞端粒酶活性的影响

目的：探讨淫羊藿甙（ICA）对急性早幼粒白血病细胞端粒酶活性的影响。方法：采用ICA与全反式维甲酸（AT-RA）对比试验。观察ICA对白血病细胞端粒酶活性及增殖分化的影响。结果：ICA可明显抑制端粒酶活性，对白血病细胞均有较明显的诱导分化和抑制增殖作用。且与ATRA合用可产生明显的协同效应。结论：ICA具有抑制急性早幼粒白血病细胞端粒酶活性的作用。     

Effect of ara-A on differentiation and proliferation of HL-60 cells

The HL-60 human leukemic promyelocytic cell line can be induced to mature into terminally differentiated cells using certain chemotherapeutic agents. We have recently demonstrated that two inhibitors of DNA synthesis, cytosine arabinoside (ara-C) and aphidicolin, can induce HL-60 differentiation with the appearance of monocytic markers. These pyrimidine antimetabolites may have affected DNA methylation patterns and resulted in altered gene expression, or the differentiated phenotype may have occurred by inhibition of DNA replication. Consequently, we have extended these studies by using the purine analog, adenine arabinoside (ara-A), which also acts as an inhibitor of DNA synthesis. The results demonstrate that ara-A also induces HL-60 non-specific esterase activity and enhances expression of myeloid cell surface antigens, MY-4 and MO-1. The induction of a differentiated phenotype by ara-A occurs after partial inhibition of DNA synthesis, a finding similar to that observed with ara-C and aphidicolin. These observations indicate that purine, as well as pyrimidine analog inhibitors of DNA polymerization can induce differentiation of HL-60 cells along a monocytic lineage. These findings may be relevant to recent clinical trials that have employed low doses of ara-C in an attempt to induce differentiation of malignant hematopoietic cells.     

淫羊藿甙对急性早幼粒白血病细胞端粒酶活性的影响

NP方案有效治疗非小细胞肺癌屡有报道,但化疗的同时,往往伴有严重的骨髓及免疫抑制.为探讨有效治疗NSCLC的同时减轻化疗毒副作用的有效方法,我们用沙培林[国药准字(1998)鲁亚S-01号]配合NP方案治疗NSCLC,全部病例为确诊的NSCLC,共30例.均为初治,均无青霉素过敏.