Aim-To assess the effect of insulin-like growth factor II (IGF-II) on proliferation of hepatocellular carcinoma (HCC) cells. Methods-Expression of IGF-II mRNA and protein was detected in 10 archival HCC specimens using in situ hybridisation and inumunohistochemistry, respectively. Expression of the Ki-67 antigen, a proliferation marker, was determined immunohistochemically on the same sec-
MethodsTen archival HCCs complicating liver cirrhosis were studied. All 10 patients had died of their disease. Seven tumours were associated with hepatitis C virus (HCV) and three with hepatitis B virus (HBV) infection. All liver samples had been fixed in 10% buffered formalin and embedded in paraffin wax. Serial, 5 gm thick, sections were cut and mounted on 2% 3-aminopropyltriethoxysilane coated slides. Sections from all tumours were stained with haematoxylin and eosin for histopathological examination.
IN SITU HYBRIDISATIONThe in situ IGF-II mRNA hybridisation protocols used were as described by Yun et al.3 Briefly, the hybridisation solution contained 50% formamide, 2 x SSC, 0.15 M NaCl, 0.2 mg/ml Escherichia coli tRNA, 1 mg/ml degraded herring sperm DNA, 0.1 mg/ml bovine serum albumin, 10% polyethylene glycol, 0.2 M dithiothreitol, and [35S] CTP labelled antisense or sense RNA probe (40 000 cpm/pl) and was kept at 50°C overnight. The slides were washed prior to RNase treatment. For autoradiography, slides were dipped in nuclear emulsion and exposed for two weeks at 4°C. After development, the slides were counterstained with haematoxylin and eosin.
IMMUNOHISTOCHEMISTRYFor IGF-II, dewaxed sections were rehydrated through an alcohol series and overlaid with a 10% non-fat milk solution to block nonspecific protein binding. Mouse monoclonal antibody directed against rat IGF-II (Amano Pharmaceutical Co, Osaka, Japan) was used at a final dilution of 1 in 400 and incubated with the sections overnight at 4°C. Sections were incubated with biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, California, USA) and alkaline phosphatase avidin D (Vector Laboratories). Sections were then treated with biotinylated goat anti-avidin D (Vector Laboratories) and again with alkaline phosphatase avidin D. Nitroblue tetrazolium with 5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP: BluGENE kit, BRL, Gaithersburg, Maryland, USA) was used as the immunoreactive alkaline phophatase substrate.Cellular proliferation was assessed in serial sections using the MIB 1 antibody (Immunotech S.A., Marseille, France) directed against the Ki-67 antigen by means of the streptavidin-biotin complex method. Initially, dewaxed sections were autoclaved for 10 minutes at 120°C in a 0.01 M citrate buffer (pH 6.0) to improve antigen retrieval. MIBI was used at a final dilution of 1 in 100. Naphthol-