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Heat shock factor-1 (HSF1) is a transcriptional factor that binds to heat shock elements located on the promoter region of heat shock protein genes. The purpose of this study was to further investigate the regulation of the expression of the heat shock protein-70 (HSP-70) gene. The HSF1 gene was inserted into pCDNA3 plasmid and then transfected into human epidermoid A431 cells using the CaOP3 method. Control cells were transfected with vector alone. Expression of HSP-70, HSF1, and HSF2 genes and protein were determined. We found a significant increase in the expression of the HSF1 gene, but not HSP-70 and HSF2 genes, in the HSF1 gene-transfected cells. The amount of HSF1-heat shock element complex was significantly increased in both the nucleus and cytosol in HSF1 gene-transfected cells, indicating increased synthesis of HSF1. The amount of HSP-72 in these cells did not change. Therefore, overexpression of HSF1 protein failed to initiate transcription of the HSP-70 gene. Subsequently, we treated the cells with 1 microM PMA (a protein kinase C stimulator), and HSP-70 mRNA and protein were measured at 1 or 4 h of the treatment, respectively. The levels of both HSP-70 mRNA and HSP-72 protein were significantly increased in nontransfected and transfected cells; the levels of HSP-72 in HSF1 gene-transfected cells were greater than that found in the vector-transfected cells. The PMA-induced increase in HSP-72 protein peaked 8 h after treatment with PMA and returned to baseline levels at 72 h. This increase was blocked by a PKC inhibitor, staurosporine. After treatment with PMA, HSF1 translocated quickly from cytosol to nucleus. The results suggest that phosphorylation of newly synthesized HSF1 and possibly of other factors are necessary for the induction of HSP-72. Activation of PKC can cause phosphorylation of HSF1, which leads to an enhanced but transient increase in HSP-70 production.