Antisense downregulation of N-myc1 in woodchuck hepatoma cells reverses the malignant phenotype

Abstract

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.

FIG. 1

Characterizations of WHV DNA and N-myc and WHV X proteins in WH44KA cells. (A) Southern blot illustrating WHV DNA hybridization to a single 7-kb genomic DNA fragment of WH44KA DNA (KpnI digested); (B) Northern blot illustrating WHV DNA and N-myc1 cohybridization to the same 3.9-kb RNA plus WHV hybridization to lower-molecular-weight RNA species; (C) Western blot illustrating a high steady-state level of 63-kDa N-myc1 protein in WH44KA cells and its absence in WLC3 woodchuck liver epithelial cells.

FIG. 2

Nucleotide sequences of the left (A) and right (B) N-myc1-WHV junctions of the single WHV integration in the 3′ untranslated region of N-myc1 exon 3. A comparison of N-myc2, N-myc1, and WHV sequences with the left and right junction sequences is illustrated. Note the 2-bp homology between WHV and N-myc1 sequences at the integration site, which is a common feature of hepadnavirus integrations. Underlined sequences denote the inverted repeat of WHV sequences.

FIG. 3

(A) Map of the integrated WHV DNA in WH44KA cells determined by sequencing the entire integrated WHV DNA. The selected WHV open reading frames are illustrated in order to identify the sequences within the WHV map. The WHV nucleotide numbers at each WHV rearrangement point in the integration are noted below the map in small numbers (9). The N-myc1 nucleotide numbers at the integration junctions are noted in large numbers below the map (6). Ex3, exon 3; open boxes, WHV; shaded boxes, N-myc1; En1, WHV enhancer 1; X, WHx gene; C, woodchuck hepatoma C gene; P/S, entire Pre S-S open reading frame; Pre-S1, woodchuck hepatoma Pre S gene; a to e, oligonucleotides used for PCR amplification of the integration (see Materials and Methods); star, left junction sequence shown in panel B. (B) Nucleotide sequence map of the left N-myc1–WHV junction. The N-myc1 sequence is in lowercase letters, and WHV sequences are in uppercase letters and indicated by lines. The map illustrates the inverted repeat of WHV sequences at the N-myc junction. (C) Structures of the WHV and N-myc DNA sequences from the integration which were included in each antisense vector. Each of the sequences was inserted in the inverse orientation in the antisense vectors in order to synthesize antisense RNAs. The antisense vector was pCR3 (see Materials and Methods). The arrows in the figure denote the transcriptional direction to produce a sense-strand RNA. The constructs were inserted into the vector in the opposite direction to produce an antisense RNA.

FIG. 4

(A) Western blot illustrating the suppression of N-myc protein accumulation in WH44KA cells transfected with antisense vector 4 and not in cells transfected with antisense vector 1, 2, or 3 or in vector-only control cells. Lanes 1, 2, and 4, lysates from cell lines transfected with antisense vectors 1, 2, and 3, respectively; lanes 5 and 6, lysates of cell lines 4-5 and 4-11, respectively, transfected with antisense vector 4; lanes 3 and 9, lysates from cells transfected with pCR3 vector only; lane 7, lysate from WH44KA woodchuck hepatoma cells; lane 8, lysate from WLC-3 cells (a woodchuck liver epithelial cell line serving as a negative control). (B) Northern blot hybridized with an antisense N-myc probe illustrating the continued presence of sense N-myc1 RNA in antisense cell lines, along with positive and negative controls. Lane 1, positive control WH44KA cells; lane 2, positive control cells (WHK44A cells transfected with pCR3 vector); lane 3, antisense cell line 4-5 transfected with vector 4 (Fig. 3C); lane 4, antisense cell line 4-11 transfected with vector 4 (Fig. 3C); lane 5, negative control cells (WLC-3 cells). (C) Hybridization with a sense-strand N-myc probe to detect the antisense N-myc RNA produced by antisense vector 4 in cell lines 4-5 (lane 3) and 4-11 (lane 4).

FIG. 5

Growth curves of control and N-myc1 antisense cell lines. V--7, pCR3 vector-only control; 4--5 and 4--11, N-myc1 antisense cell lines in which the steady-state N-myc protein level was reduced 95 and 80%, respectively.

FIG. 6

(A) Morphology of vector control cell line V-7. (B) Morphological change in cell line 4-5, which shows flattened cells compared to those of the V-7 cell line. Phase contrast; magnification, X340.