EPIDERMAL GROWTH-FACTOR ACTIVATES PROTEIN-KINASE-C IN THE HUMAN ENDOMETRIAL CANCER CELL-LINE HEC-1-A

Previous studies from this laboratory have shown that epidermal growth factor (EGF) and the tumor promoter, phorbol myristate acetate (PMA), are mitogenic in the endometrial cancer cell line HEC-1-A, Since the effects of EGF have been shown to be mediated by the protein kinase C (PKC) pathway transduction system, we examined the possibility that th e EGF-responsive signal in the endometrial cancer cell line HEC-1-A in volves protein kinase C activation, HEC-1-A cells were grown to conflu ency in 100-mm dishes and maintained in a serum-free medium for 24 hr prior to treatment, The cells were treated with EGF at varying time in tervals (0.25 to 60 min) and concentrations (0.1 to 200 ng/ml). The ce lls were then lysed, homogenized, and centrifuged at 105,000g for 1 hr at 4 degrees C. The supernatant was chromatographed on DEAE-Sephacel columns. The membranous pellet was resuspended in 5 mi of lysis buffer containing 1% Nonidet P-40 and also chromatographed on DEAE-Sephacel columns separately, The eluates were collected and assayed for protein kinase C activity by determining the amount of P-32 transferred from [gamma-P-32]ATP onto histones in the presence of the phospholipids, ph osphatidylserine, and diolein, Our results show that the cytoplasmic a nd membrane fraction of the HEC-1-A cell line contained phosphotransfe rase activity which displayed kinetic characteristics typical of the p rotein kinase C enzyme, The optimal incubation time for protein kinase C activation in the cytosol by EGF was 5 min (30-fold stimulation), T he protein kinase C activity was increased when the cell lines were in cubated with increasing concentrations of EGF, Enzyme saturation was s een at a concentration of 10 ng/ml of EGF (4.5-fold stimulation). West ern blot analysis confirmed the presence of the PRC enzyme in the cyto sol and membranes of our cancer cell line, These results suggest that EGF, at least partially, exerts its effects on the endometrial adenoca rcinoma cell line by activating protein kinase C through increased bre akdown of phosphatidyl inositol (PI), The PI cascade appears to be an important signal transduction system mediating the growth stimulatory effects of EGF on endometrial carcinoma. (C) 1997 Academic.