Development of rainbow trout hepatoma cell lines: effect of pro-IGF-I Ea4-peptide on morphological changes and anchorage-independent growth.
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 degrees 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.
Department of Molecular and Cell Biology, University of Connecticut, 91 North Eagleville Road, U-3125, Storrs, Connecticut 06269-3125, USA.
Maria J
MJ
Chen
P Peter
PP
Chiou
Bih-Ying
BY
Yang
Hung Chieh
HC
Lo
Jin-Ki
JK
Son
Jerry
J
Hendricks
George
G
Bailey
Thomas T
TT
Chen
D000818:Animals; D001580:Benzopyrenes / Q000633:toxicity; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D007334:Insulin-Like Growth Factor I / Q000627:therapeutic use*; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D017686:Oncorhynchus mykiss; D010446:Peptide Fragments / Q000627:therapeutic use*; D011498:Protein Precursors / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014410:Tumor Stem Cell Assay
D001580:Benzopyrenes; D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C119953:pro-insulin-like growth factor I; D007334:Insulin-Like Growth Factor I; C041517:dibenzo(a,l)pyrene