Impaired Nuclear Accumulation and Shortened Phosphorylation of ERK After Growth Factor Stimulation in Cultured Hepatocytes From Rats Exposed to 2-Acetylaminofluorene

Abstract

The hepatic carcinogen 2‐acetylaminofluorene (AAF) exerts its effect as a tumor promoter by mitoinhibition of normal hepatocytes. Initiated cells proliferate selectively and develop into preneoplastic foci and subsequently into carcinomas. To study whether some of the mitoinhibitory effects of AAF could be attributed to an influence on intracellular signal transduction, growth factor signaling was studied in cultured hepatocytes from rats fed AAF for 7 d. Activation through the epidermal growth factor receptor (EGFR) was used to probe possible changes in downstream mitogenic signaling mechanisms. The proliferative response to epidermal growth factor (EGF), measured as proliferating cell nuclear antigen expression and thymidine incorporation, was almost completely inhibited in hepatocytes exposed to AAF. Neither EGFR protein levels nor EGF binding was notably altered in AAF‐exposed hepatocytes as opposed to normal hepatocytes. The initial tyrosine phosphorylation of EGFR and downstream activation of Sos, Raf‐1, and extracellular signal–regulated protein kinase (ERK) were similar in AAF‐treated and control hepatocytes. Even though ERK phosphorylation was unaffected, a remarkable (80%) reduction of ERK nuclear accumulation was observed in AAF‐exposed hepatocytes immediately after mitogen stimulation. EGFR tyrosine phosphorylation and downstream signaling lasted 6 h in control cells versus 2 h in AAF‐exposed hepatocytes. We previously demonstrated that AAF inhibits the growth factor–dependent induction of cyclin D1 and arrests hepatocyte cell‐cycle progression before the p21/CIP1‐controlled DNA‐damage check point. The present data indicate that the DNA‐damaging carcinogen AAF induces growth inhibition by a distinct inhibition of ERK nuclear accumulation after mitogen stimulation. Inhibition of intracellular signal transduction may represent a novel mechanism of growth arrest. Mol. Carcinog. 28:84–96, 2000. © 2000 Wiley‐Liss, Inc.

Cytoplasmic Retention of Peroxide-Activated ERK Provides Survival in Primary Cultures of Rat Hepatocytes *

“…Both the measurement of MAP kinase activities and the DNA synthesis were performed as previously described. 7 …”

“…Hepatocytes were isolated and seeded as previously described. 7 The cultures were kept in 95% air and 5% CO 2 at 37°C. Cell death was measured via trypan blue exclusion.…”

“…Western blot analysis was performed essentially as previously described. 7 Equal protein loading was controlled with an antibody to ␤-tubulin. For nuclear fractionation, hepatocytes were lysed in 10 mmol/L sodium phosphate buffer (pH 7.4) containing 10 mmol/L NaCl, 5 mmol/L MgCl 2 , 0.1% NP40, 0.2 mmol/L AEBSF, 20 mol/L leupeptin, 200 U/mL aprotinin, 65 mol/L sodium orthovanadate, and 10 mmol/L ␤-glycerophosphate.…”

“…Immunofluorescence staining was performed essentially as previously described. 7 The immunostained cells were examined with a Leica TCS SP confocal microscope (Leica, Heidelberg, Germany) equipped with TCS NT software and Ar (488 nm) and two He/Ne (543 and 633 nm) lasers. A Plan apochromat 100X 1.4 NA oil objective was used.…”

“…In hepatocytes, epidermal growth factor (EGF) binding to the EGF receptor initiates activation of ERK through the Raf/MEK/ERK pathway followed by nuclear accumulation of ERK. 7 In the nucleus, ERK phosphorylates substrates important for growth progression. 8 However, ERK also phosphorylates substrates in other cellular compartments, including membrane proteins and proteins in the cytoplasm.…”