Antisense downregulation of N-myc1 in woodchuck hepatoma cells reverses the malignant phenotype.
Cell line WH44KA is a highly malignant woodchuck hepatoma cell line. WH44KA cells contain a single woodchuck hepatitis virus (WHV) DNA integration in the 3' untranslated region of exon 3 of the woodchuck N-myc1 gene. The highly rearranged WHV DNA contains WHV enhancers which activate the N-myc promoter, and a hybrid N-myc1-WHV mRNA is produced, which leads to a high steady-state level of N-myc1 protein. To investigate whether continuous N-myc1 expression is required to maintain the tumor phenotype, we knocked out N-myc expression using a WHV-N-myc1 antisense vector. We identified two WH44KA antisense cell lines, designated 4-5 and 4-11, in which steady-state N-mycl protein levels were reduced by 95 and 80%, respectively. The growth rates of both antisense cell lines were reduced in comparison to those of wild-type and vector controls. The phenotype of 4-5 and 4-11 cells changed to a flattened appearance, and the cells exhibited contact inhibition. Colony-forming ability in soft agar was reduced by 92% for 4-5 cells and by 88% for 4-11 cells. Cell line 4-11 formed only small, slow-growing tumors in nude mice, consistent with a low level of N-myc1 remaining in the cells. In contrast, 4-5 cells, in which N-myc protein was reduced by greater than 95%, failed to form tumors in nude mice. The integrated WHV DNA contained the complete WHV X gene (WHx) and its promoter; however, we did not detect any WHx protein in the cells by using a sensitive assay. These data demonstrate that N-myc overexpression is required to maintain the malignant phenotype of WH44KA woodchuck hepatoma cells and provide a direct function for integrated WHV DNA in hepatocarcinogenesis.
Wang
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HP
Marion Bessin Liver Research Center, Department of Medicine, Jack and Pearl Resnick Campus of the Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Zhang
L
L
Dandri
M
M
Rogler
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CE
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D004279:DNA, Viral; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D016259:Genes, myc; D005822:Genetic Vectors; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics*; D008392:Marmota; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D016372:RNA, Antisense; D012367:RNA, Viral; D014407:Tumor Cells, Cultured; D016662:Virus Integration
D004279:DNA, Viral; D016372:RNA, Antisense; D012367:RNA, Viral