Effects of insulin, glucagon and triiodothyronine on DNA synthesis in rat hepatocyte primary cultures induced by liver tumour promoters and EGF

Abstract

Enhanced cell proliferation may favour DNA-damaging processes (tumour initiation) as well as the expansion of clones of altered cells (tumour promotion). The latter process appears to be responsible for carcinogenesis by non-genotoxic compounds. We previously have established rat hepatocyte primary cultures under serum-free conditions and studied the effect of the rat liver tumour promoter cyproterone acetate (CPA) on DNA synthesis. It was found that glucocorticoid concentration of 100 n m dexamethasone was necessary for the induction of DNA synthesis. In the present study the growth supporting effect of insulin, glucagon and triiodothyronine (T3) were examined. Pretested concentrations of CPA, pregnenolone-16α-carbonitrile (PCN), α-hexachlorocyclohexane (HCH), nafenopin (NAF) and phenobarbital (PB) were added for stimulation of DNA synthesis. EGF served as a reference stimulator. Beginning with standard medium concentrations of insulin (7 nM), glucagon (0.4 nM) and triiodothyronine (100 nM) dose-response studies were obtained by leaving all but one of the hormone concentrations constant. DNA synthesis was measured by incorporation of [ 3H]thymidine into DNA. In solvent-treated dimethyl sulfoxide (DMSO) cultures insulin at 6 nM and higher induced a 2.5-fold induction of DNA synthesis. Insulin also enhanced DNA synthesis induced by CPA, HCH, NAF, PCN and EGF but not by phenobarbital (EC 50, 2–3 n m). The effect of glucagon (0.016n m–16n m) appeared to be much less prominent and was observed only at superphysiological concentrations with CPA, PCN, HCH and EGF. DNA synthesis induced by NAF was slightly suppressed at 1.6n m glucagon. T3 did not induce DNA synthesis by itself, nor was there modulation of DNA synthesis induced by rat liver tumour promoters. Thus the so-called hepatotrophic hormones differentially modulate the induction of DNA synthesis by rat liver tumour promoters in primary rat hepatocyte cultures.