WOODCHUCK HEPATITIS-VIRUS ENHANCER-I AND ENHANCER-II ARE BOTH INVOLVED IN N-MYC2 ACTIVATION IN WOODCHUCK LIVER-TUMORS

Direct activation of the N-myc2 oncogene by insertion of woodchuck hep atitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatoca rcinogenesis. We previously reported that WHV enhancer II (We2), which controls expression of the core/pregenome RNA, can also activate the N-myc2 promoter in hepatoma cell lines. To better define the integrate d WHV regulatory sequences responsible for N-myc2 promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer acti vity of a single viral integrant found at the win locus in tumor 2260T 1 and mapping approximately 175 kb 3' of N-myc2. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with W e2 sequences and 1 with WHV sequence homologous to that of hepatitis B virus enhancer I (We1), In transient transfection assays in hepatoma- derived cells, the We2 activator was found to be fully effective only when inserted in close proximity to the N-myc2 promoter whereas the We 1 element by itself was apparently devoid of activity. In contrast, th e 2260T1 viral insert exhibited a potent enhancer capacity that depend ed both on multimerized We2 and on We1 sequences. In a survey of diffe rent woodchuck hepatomas, both elements were commonly found within int egrated viral sequences involved in long-range N-myc2 activation.