Lindali Curetty

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.

Heat-induced preferential synthesis and redistribution of HSP 70 and 28 families in Chinese hamster ovary cells.

We observed that members of two HSP families (70 and 28 kDa) preferentially redistributed into the nucleus after heating at 45.5 degrees C for 10 min. The rates of synthesis and redistribution of these proteins were different for each member of HSP families during incubation period at 37 degrees C after heat shock. The maximum rates of synthesis of HSP 70 and HSP 28 families, except HSP 28c, were 6-9 hr after heat shock, whereas the maximum rates of redistribution were 3-6 hr after heat shock. These results suggest that the rates of redistribution of these proteins may be dependent on the amount of intracellular proteins as well as the alteration of binding affinity of nucleoproteins following heat shock.

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.