Inhibitory guanine nucleotide regulatory protein activation of mitogen-activated protein kinase in experimental hepatocellular carcinoma in vitro

Objective Hepatocellular carcinoma (HCC) is associated with altered express ion and function of inhibitory guanine nucleotide regulatory proteins (Gi-p roteins), This study addresses the interaction between Gi-proteins and the extracellular regulated kinase (ERK) component of the mitogen activated pro tein kinase (MAPK) cascade in regulating mitogenesis in an experimental mod el of HCC. Design Pharmacological agents which selectively interact with specific targ et proteins involved in signal transduction through a Gi-MAPK pathway have recently become available. These agents in combination with scientific assa ys allow us to address the role of individual components of this cascade in the regulation of mitogenesis in HCC. Methods These studies were performed using rat hepatic tumorigenic cells (H 4IIE) and isolated cultured hepatocytes in vitro in conjunction with pharma cological agents which interact with Gi-protein or MAPK components of intra cellular signalling. Results Direct activation of Gi-proteins with mastoparan M7 (M7) significan tly increased nuclear thymidine incorporation in hepatic tumorigenic H4IIE cells in a dose-dependent manner (10-1000 nM, n = 4, P < 0.05), an effect t hat was abolished by treatment with either pertussis toxin (PTx) or the sel ective mitogen-activated ERK-regulated kinase (MEK) inhibitor PD098059, In contrast, M7 inhibited nuclear thymidine incorporation in serum-stimulated isolated hepatocytes, ERK2 activity was then determined as the ability of i mmunoprecipitated ERK2 proteins to phosphorylate the ERK substrate myelin b asic protein. These studies demonstrated a time- and dose-dependent increas e in ERK2 activity in H4IIE cells following Gi-protein activation with M7, a maximal response being attained at 20 min. In contrast, M7 failed to sign ificantly alter ERK2 activity in isolated cultured hepatocytes at any of th e doses or time points assayed (10-5000 nM, 10-120 min). Gi-stimulated ERK activation was completely blocked in tumorigenic cells following treatment with PTx, Conclusions These data demonstrate for the first time a Gi-linked MAPK casc ade in experimental HCC, activation of which stimulates cellular mitogenesi s. Eur J Gastroenterol Hepatol 11:761-768 (C) 1999 Lippincott Williams & Wi lkins.