DEVELOPMENT OF RAINBOW TROUT HEPATOMA CELL LINES: EFFECT OF PRO-IGF-I EA4-PEPTIDE ON MORPHOLOGICAL CHANGES AND ANCHORAGE-INDEPENDENT GROWTH

• Published in: In vitro cellular & developmental biology. Animal Vol. 40; no. 3; pp. 118 - 128
• Main Authors: CHEN, MARIA J, CHIOU, P. PETER, YANG, BIH-YING, LO, HUNG CHIEH, SON, JIN-KI, HENDRICKS, JERRY, BAILEY, GEORGE, CHEN, THOMAS T
• Format: Journal Article
• Language: English
• Published: Germany Society for In Vitro Biology 01.03.2004
• Subjects: anchorage-independent cell growth; Animals; antitumor effect; Benzopyrenes - toxicity; Biomarkers, Tumor - genetics; Biomarkers, Tumor - metabolism; Cell Division - drug effects; Cell growth; CELL GROWTH/DIFFERENTIATION/APOPTOSIS; Cell Line, Tumor; Cell lines; Cells; Cultured cells; Gene Expression Regulation, Neoplastic - drug effects; Hepatocellular carcinoma; Hepatocytes; Insulin-Like Growth Factor I - therapeutic use; Liver; Liver Neoplasms, Experimental - drug therapy; Liver Neoplasms, Experimental - metabolism; Liver Neoplasms, Experimental - pathology; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Oncorhynchus mykiss; Peptide Fragments - therapeutic use; Protein Precursors - therapeutic use; Recombinant Proteins - therapeutic use; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA; RNA, Messenger - metabolism; single-cell clone; Trout; trout Ea4-peptide; trout hepatoma cells; Tumor cell line; Tumor Stem Cell Assay
• ISSN: 1071-2690; 1543-706X; 1543-706X
• DOI: 10.1290/1543-706X(2004)040<0118:DORTHC>2.0.CO;2

Our laboratory has shown previously that recombinant rainbow trout Ea4 (rtEa4)–peptide of pro–insulin-like growth factor-I (pro–IGF-I) exhibited antitumor activities against cancer cell lines derived from various human cancer tissues (Chen et al., 2002; Kuo and Chen, 2002). To confirm that rtEa4-peptide can exhibit the same spectrum of antitumor activities in fish tumor cells, we had developed permanent single-cell clones (RTH1B1A, RTH1B1D, RTH1B2A, and RTH1B2C) from a rainbow trout liver tumor induced by dibenzo[a,l]pyrene treatment. At 135 passages, the doubling time of these single-cell clones in CO2-independent medium at 20° C was 3.9, 3.5, 3.0, and 4.5 d, respectively. Reverse transcription–polymerase chain reaction analysis showed that the expression of liver signature genes (e.g., aldolase B, glucose-6-phosphatase [G-6-Pase], phosphoenolpyruvate carboxykinase [PEPCK], hepatic nuclear factor-1 [HNF-I], IGF-I, IGF-II, and growth hormone [GH] receptor-2 genes) and CYP1A1 and CYP1A3 genes was detected in these four single-cell clones. Furthermore, results of in vitro colony formation assay in a soft-agar medium showed different degrees of colony formation activities among them. These results confirmed that the single-cell clones were derived from the rainbow trout liver. Treatment of RTH1B1D with recombinant trout Ea4-peptide resulted in the induction of a dose-dependent morphological change and the suppression of colony formation in a soft-agar medium. In addition, both morphological change and reduction of colony formation were also observed in permanent transfectants of RTH1B1D cells carrying a trout Ea4-peptide gene or its human counterpart, hEb-peptide gene. These results confirm our earlier observations that trout pre–IGF-I Ea4-peptide and hEb possess activities counteracting malignant properties of cancer cells in vitro.