Zi-Zheng Hou

MECHANISM OF QUERCETIN-INDUCED SUPPRESSION AND DELAY OF HEAT SHOCK GENE EXPRESSION AND THERMOTOLERANCE DEVELOPMENT IN HT-29 CELLS

Previous studies have shown that a combination of low pH and quercetin (QCT) treatment following heat shock markedly suppresses and delays the expression of heat shock protein genes, particularly the HSP70 gene (Lee et al., Biochem. Biophys. Res. Commun., 186:1121–1128, 1992). The possible mechanism for alteration of gene expression by treatment with QCT at low pH was investigated in human colon carcinoma cells. Cells were heated at 45°C for 15 min and then incubated at 37°C for various times (0–12 h) with QCT (0.05–0.2 mM) at pH 7.4 or 6.5. Gel mobility-shift analysis of whole cell extracts from heated cells showed the formation of the heat shock transcription factor (HSF)-heat shock element (HSE) complex. Dissociation of HSF from the HSE of the human HSP70 promotor occurred within 4 h under both pH conditions. The kinetics of recovery were not affected by treatment with 0.1% dimethyl sulfoxide (DMSO). However, the dissociation of HSF-HSE complex was markedly delayed during treatment with a combination of low pH and QCT. In addition,in vitro transcription assays showed a suppression of initiation and elongation of HSP70 mRNA. These results may explain why the combination of low pH and QCT treatment suppresses and delays the HSP70 gene expression as well as thermotolerance development.

Effect of pH on quercetin-induced suppression of heat shock gene expression and thermotolerance development in HT-29 cells

When cells were heated for 15 min at 45 degrees C, they became thermotolerant to a second heat exposure at 45 degrees C. Thermotolerance developed rapidly, reached its maximum 6 hr after heat shock, and then gradually decayed. The development of thermotolerance was partially suppressed by treatment with various concentrations of quercetin (0.05-0.2 mM) at pH 7.4 after the initial heat treatment. In contrast, the drug markedly inhibited thermotolerance development at pH 6.5. Furthermore, a combination of low pH and quercetin treatment distinctively altered the expression of HSP70 gene compared with that of HSP28 or HSP90 gene. These results demonstrate a good correlation between the amount of HSP70 gene expression and development of thermotolerance.

Regulation of HSP70 and HSP28 gene expression: absence of compensatory interactions.

We have previously reported the lack of HSP28 gene expression during acute and chronic thermotolerance development in L929 cells (J Cell Physiol 152: 118–125, 1992; Cancer Res 52: 5787, 1992). In contrast to HSP28, an extremely high level of inducible HSP70 synthesis was observed. These results led us to investigate the possibility of compensatory interactions between HSP70 and HSP28. To test the hypothesis, L929 cells were transfected with the human HSP28 gene contained in plasmid pCMV27. Data from Western blot and two-dimensional gel electrophoresis of [3H] leucine and [32P] orthophosphate-labeled proteins showed the synthesis and phosphorylation of HSP28 in transfected cells after heating at 45°C for 10 min. However, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was not decreased after heat shock. These results suggest an independent regulation of HSP28 and HSP70 gene expression.

Comparison of heat shock gene expression in mild hyperthermia-sensitive human prostatic carcinoma cells and heat-resistant human breast carcinoma cells

Abstract 1. 1.|While human breast carcinoma (MCF-7) cells were resistant to heat shock, human prostatic carcinoma (DUT-145 and PC-3) cells were relatively sensitive to mild hyperthermia. For example, survival of both prostatic carcinoma cells or that of breast carcinoma cells was 10 or 93%, respectively, after heating at 41°C for 24h. 2. 2.|We investigated whether or not heat shock gene expression might be responsible for these differences in thermal sensitivity. 3. 3.|It was observed that transcriptional activation of heat shock genes was induced by heating at 41°C in all three cell lines. Studies from the gel mobility shift assay demonstrated the formation of heat shock factor and heat shock element (HSF-HSE) complex during heating at 41°C for 2 h in both MCF-7 and DUT-145 cell lines. 4. 4.|In addition, Northern blots and polyacrylamide gel electrophoresis demonstrated expression of heat shock genes, along with the synthesis of their proteins, particularly 68 kDa heat shock protein in all three cell lines. 5. 5.|These results demonstrate that differences in thermal sensitivity to mild hyperthermia are not due to differential heat shock gene expression.

Differences in Thermotolerance Induced by Heat or Sodium Arsenite: Correlation between Redistribution of a 26-kDa Protein and Development of Protein Synthesis-Independent Thermotolerance in CHO Cells

In previous studies, we have demonstrated the differences in thermotolerance induced by heat and sodium arsenite (Lee et al., Radiat. Res. 121, 295-303, 1990). In this study, we investigated whether a 26-kDa protein might play an important role in evincing these differences. Chinese hamster ovary (CHO) cells treated for either 1 h with 100 microM sodium arsenite (ARS) or 10 min at 45.5 degrees C became thermotolerant to a test heat treatment at 43 degrees C administered 6 or 12 h later, respectively. After the test heating at 43 degrees C for 1.5 h, the level of 26-kDa protein in the nucleus was decreased by 92% in nonthermotolerant cells, 78% in ARS-induced thermotolerant cells, and 3% in heat-induced thermotolerant cells. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) after ARS treatment eliminated thermotolerance to 43 degrees C and delayed restoration of the 26-kDa protein in the nucleus. In contrast, CHM neither prevented the development of thermotolerance nor inhibited the restoration of the 26-kDa protein in heat-induced thermotolerant cells. However, when cells were exposed to cold (4 degrees C), immediately after initial heating, restoration of the 26-kDa protein and development of thermotolerance did not occur. These results demonstrate a good correlation between the restoration and/or the presence of this 26-kDa protein and the development of protein synthesis-independent thermotolerance.

Cycloheximide decreases nascent polypeptides level and affects up-regulation of HSP70 gene expression

Abstract 1. 1|Treatment with cycloheximide (CHM; 10 μg/ml) 2 h before and during heating produced a 13-fold increase in survival from 1 × 10 −2 to 1.3 × 10 −1 after 10h at 43°C in human colon carcinoma HT-29 cells. Little protection was observed if the drug was added only during heating. 2. 2|Pretreatment with 10 μg/m CHM which inhibited protein synthesis by 95%, decreased the level of nascent polypeptides and subsequently reduced the accumulation of polypeptides in the nucleus during heat shock. 3. 3|Adding CHM (10 μg/ml) 2h before and during heat facilitated the dissociation of heat shock transcription factor-heat shock element (HSF-HSE) complex and consequently terminated transcription activity earlier. 4. 4|These findings related to the literature suggest that the free pool of HSP70 is increased by inhibiting protein synthesis. An increase in the level of free HSP70 may more effectively protect or repair thermolabile targets and consequently affect the regulation of heat shock response.

Alteration of heat sensitivity by introduction of HSP70 or anti-HSP70 antibody in cho cells

Abstract 1. 1.An electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce HSP70 or anti-HSP70 antibody into Chinese hamster ovary cells. 2. 2.There was a 1.5- or 1.9-fold increase in the amount of intracellular HSP70 by electroporation in the presence of 0.75 or 1.5 mg/ml, respectively. 3. 3.Cells electroporated with HSP70 became resistant to hyperthermic killing; i.e. the survival increased 6–7-fold from 1.2 × 10 −2 to 7–8 × 10 −2 heating at 45.5°C for 20 min. 4. 4.In contrast, introduction of 1 mg/ml anti-HSP70 antibody sensitized cells to hyperthermic killing; i.e. the reciprocal of the survival slope was decreased from 1.68 to 1.25 min. 5. 5.Thus, our results support the hypothesis that HSP70 plays an important role in heat resistance.

Mechanism of synergistic effects of cytokine and hyperthermia on cytotoxicity in HT-29 and MCF-7 cells: Expression of MnSOD gene

Abstract 1. 1.A synergistic increase in the cytotoxic effects of recombinant human tumor necrosis factor-α (rhTNF-α) alone, recombinant human interferon-γ (rhIFN-γ) alone, or a combination of both drugs was observed when HT-29 and MCF-7 cells were heated at 42°C in the presence of the drug(s). 2. 2.We hypothesized that heat might alter the expression and synthesis of manganous superoxide dismutase (MnSOD) which is involved in the scavenging of superoxide radicals (O − 2 ). 3. 3.A 1.7–46-fold increase in two distinct species of MnSOD mRNA (1 and 4 kb) occurred by treatment with rhTNF-α (1000 U/ml), rhIFN-α (1000 U/ml), or a combination of both drugs in both cells. Particularly, the action of TNF and IFN on induction of 1 kb MnSOD mRNA was synergistic. 4. 4.Although the alteration of MnSOD gene expression was not observed by heat shock alone (42°C—4, 8, 12h), hyperthermic treatment suppressed the action of TNF, IFN or TNF + IFN on induction of MnSOD mRNA, especially 1 kb MnSOD mRNA in MCF-7. 5. 5.These results suggest that suppression of MnSOD gene expression is correlated with synergistic cytotoxic effects between cytokine and hyperthermia.

Alteration of heat sensitivity by treatment with nonpermissive temperature or cycloheximide in temperature-sensitive CHO mutant tsH1 cell

Abstract 1. 1.|The temperature-sensitive mutant CHO-tsH1 and wild type (CHO-SC) cells became thermal resistant when cells were treated for either 2 h at 39.5°C before heating at 43°C or 2 h with 10 μg/ml cycloheximide (CHM) before and during heating at 43°C. 2. 2.|There was a 2000-fold increase in survival after 2.5 h at 43°C by preincubation at 39.5°C in both cell types. There was also a 200- or 700-fold increase in survival after 2.5 h at 43°C by treatment with CHM in tsH1 or SC cell type respectively. 3. 3.|In contrast to the effects at 43°C, at 41.8°C these protective effects were not evident in tsH1 cells. In wild type, however, there was an 800- or 1800-fold increase in survival after 8 h at 41.8°C by preincubation at the temperature of 39.5°C or treatment with CHM, respectively. 4. 4.|Therefore, these results suggest that killing of tsH1 at low temperature hyperthermia (41.8°C) is probably due to denaturation of thermolabile leucyl-tRNA synthetase. 5. 5.|The denaturation of this enzyme may not be protected by inhibition of protein synthesis by preincubation at the nonpermissive temperature of 39.5°C or by CHM.

Comparison between tumour necrosis factor response and heat shock response in L929 cells : cellular and molecular aspects

1. 1.|When L929 cells were treated with 10 μg/ml cycloheximide, 100 μg/ml puromycin, or 50 mM histidinol 2 h before and during heating at 43°C, these drugs afforded heat protection, i.e. an 8-fold increase in survival from 6 × 10−2 to 4.5 × 10−1 after 1.5 h at 43°C by treatment with 10 μg/ml cycloheximide. 2. 2.|In contrast, when cells were treated with the drug 2 h before and during rhTNF-α treatment, the drugs enhanced rhTNF cytotoxicity, i.e. an 8 to 36-fold decrease in survival from 9 × 10−1 to 0.25–1.1 × 10−1 after 13 h with 100 U/ml rhTNF-α. 3. 3.|In addition, an increase in manganous superoxide dismutase (MnSOD) mRNA was induced by 100 U/ml rhTNF-α alone or in combination with 100 μg/ml puromycin, but not by heat shock. Moreover, heat shock protein synthesis was induced by heating at 45.5°C for 10 min, but not by TNF. 4. 4.|These results show that the TNF response appears to be distinct from the heat shock response in L929 cells.