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 hepatitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatocarcinogenesis. 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 integrated WHV regulatory sequences responsible for N-myc2 promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the win locus in tumor 2260T1 and mapping approximately 175 kb 3' of N-myc2. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with We2 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 We1 element by itself was apparently devoid of activity. In contrast, the 2260T1 viral insert exhibited a potent enhancer capacity that depended both on multimerized We2 and on We1 sequences. In a survey of different woodchuck hepatomas, both elements were commonly found within integrated viral sequences involved in long-range N-myc2 activation.     

Hepatitis B virus and hepatocellular carcinoma

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.     

Insulin-like growth factor II blocks apoptosis of N-myc2-expressing woodchuck liver epithelial cells

N-myc2 and insulin-like growth factor II (IGF-II) are coordinately overexpressed in the great majority of altered hepatic foci, which are the earliest precancerous lesions observed in the liver of woodchuck hepatitis virus carrier woodchucks, and these genes continue to be overexpressed in hepatocellular carcinomas (HCCs). We have investigated the function of these genes in woodchuck hepatocarcinogenesis by using a woodchuck liver epithelial cell line (WC-3). WC-3 cells react positively with a monoclonal antibody (12.8.5) against woodchuck oval cells, suggesting a lineage relationship with oval cells. Overexpression of N-myc2 in three WC-3 cell lines caused their morphological transformation and increased their growth rate and saturation density in medium containing 10% serum. Removal of serum from the medium increased cell death of the N-myc2-expressing lines, whereas cell death in control lines was minimal. The death of N-myc2-expressing WC-3 cells was accompanied by nucleosomal fragmentation of cellular DNA, and DAPI (4',6-diamidino-2-phenylindole) staining revealed condensation and fragmentation of the nuclei, suggesting that N-myc2-expressing WC-3 cells undergo apoptosis in the absence of serum. In colony regression assays, conducted in the absence of serum, control colonies were stable, while N-myc2-expressing colonies regressed to various degrees. Addition of recombinant human IGF-II to the serum-free medium blocked both cell death and colony regression in all the N-myc2-expressing lines. Therefore, coordinate overexpression of N-myc2 and IGF-II in woodchuck altered hepatic foci may allow cells which otherwise might die to survive and progress to hepatocellular carcinoma.     

Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II

Induction of hepatocellular carcinoma in woodchucks by woodchuck hepatitis virus is associated with the activation of N-myc gene expression, usually by viral DNA integration in cis to the N-myc locus. We have examined the consequences of N-myc up-regulation in rodent hepatic cells in culture. Mouse alpha ML hepatocytes infected with a retroviral vector overexpressing the woodchuck N-myc2 gene display a higher proliferation rate than parental alpha ML cells but are morphologically unchanged and do not form colonies in soft agar. However, they display an increased propensity to undergo apoptosis, an effect that is markedly augmented by serum deprivation. Expression of the woodchuck hepatitis virus X gene in alpha ML cells does not alter the growth phenotype of the cells and has no effect upon N-myc-dependent apoptosis. However, apoptosis in N-myc2-expressing alpha ML cells is strongly inhibited by insulin-like growth factor II (IGF II). IGF II gene expression is also strongly up-regulated during hepatic carcinogenesis in vivo in virally infected animals and has been speculated to be part of an autocrine growth-stimulatory pathway. Our results suggest that IGF II may play another role in the development of virus-induced hepatoma: the prevention of programmed cell death triggered by deregulated N-myc expression.     

Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse. WE examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc-gene-family expression correlated with proliferative parameters. All tumours expressed at least one myc family member at the mRNA level. Exclusive c-myc mRNA expression was demonstrated in 8 tumours, L-myc in 7 and N-myc in I. Five tumours expressed both c-myc and L-myc, and 2 tumours expressed both c-myc and N-myc. In general, the level of expression of c-myc and N-myc was similar at the mRNA and the protein level. Expression of c-myc was positively correlated with the proliferative index (sum of S and G2+M phases) of cell lines, but not with the population doubling time. In general, L-myc-expressing cell lines had a low proliferative index. There was no systematic difference in myc expression between cell lines and xenografts of individual tumours.     

Absence of mutations in the p53 tumor suppressor gene in woodchuck hepatocellular carcinomas associated with hepadnavirus infection and intake of aflatoxin B1

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4-9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4-9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated a low rate of p53 mutations in HCCs of AFB1-treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development of p53 mutations less than in humans.     

Conservation and continuity of periodic bent DNA in genomic rearrangements between the c-myc and immunoglobulin heavy chain mu loci

Periodic bent DNA was mapped in the human c- myc and immunoglobulin heavy chain mu (Ig mu) loci. A total of 12 DNA bend sites in the c- myc gene and 11 sites in the Ig mu locus were aligned at average intervals of 694.2 +/- 281.4 and 654.5 +/- 222.7 bp respectively. Although some of the bend sites retained the distance of 700 bp, their periodicity was disturbed at several locations, including the exons of the c- myc gene and the enhancer element present in the Ig mu locus. Analysis of rearrangements that resulted in tumorigenesis of lymphocytes showed that the continuity of DNA bend sites was conserved in three lymphoma cell lines, Manca, BL22 and Ramos, suggesting that the genomic rearrangements gain stability by retaining their periodicity. This adds further evidence that the periodic bent DNA plays a crucial role in genomic structure.     

Evidence for two tumour suppressor loci on chromosomal bands 1p35-36 involved in neuroblastoma: one probably imprinted, another associated with N-myc amplification

Previous reports on possible genomic imprinting of the neuroblastoma tumour suppressor gene on chromosome 1p36 have been conflicting. Here we report on the parental origin of 1p36 alleles lost in 47 neuroblastomas and on a detailed Southern blot analysis of the extent of the 1p deletions in 38 cases. The results are remarkably different for tumours with and without N-myc amplification. In the N-myc single copy tumours we show that the lost 1p36 alleles are of preferential maternal origin (16 of 17 cases) and that the commonly deleted region maps to 1p36.2-3. In contrast, all N-myc amplified neuroblastomas have larger 1p deletions, extending from the telomere to at least 1p35-36.1. These deletions are of random parental origin (18 of 30 maternal LOH). This strongly suggests that different suppressor genes on 1p are inactivated in these two types of neuroblastoma. Deletion of a more proximal suppressor gene is associated with N-myc amplification, while a distal, probably imprinted, suppressor can be deleted in N-myc single copy cases.     

Metabolic labeling of woodchuck hepatitis B virus X protein in naturally infected hepatocytes reveals a bimodal half-life and association with the nuclear framework

In order to identify potential sites of hepadnavirus X protein action, we have investigated the subcellular distribution and the stability of woodchuck hepatitis virus (WHV) X protein (WHx) in primary hepatocytes isolated from woodchucks with persistent WHV infection. In vivo cell labeling and cell fractionation studies showed that the majority of WHx is a soluble cytoplasmic protein while a minor part of newly synthesized WHx is associated with a nuclear framework fraction (20%) and with cytoskeletal components (5 to 10%). Pulse-chase experiments revealed that cytoplasmic WHx has a short half-life and decays with bimodal kinetics (approximately 20 min and 3 h). The rates of association and turnover of nucleus-associated WHx suggest that compartmentalization may be responsible for the bimodal turnover observed in the cytoplasm.     

Differential apoptotic behaviors of c-myc, N-myc, and L-myc oncoproteins

c-, N-, and L-myc are related nuclear oncoproteins that bind similar DNA sites and cooperate with activated ras oncogenes to transform primary fibroblasts. Although c-myc can also promote apoptosis in some cells after growth factor withdrawal or exposure to cytotoxic agents, roles for N- and L-myc in apoptosis remain undetermined. To address this, c-, N-, or L-myc were stably expressed in the interleukin 3 (IL-3)-dependent 32D hematopoietic cell line. The apoptotic response of each cell line was assessed after IL-3 withdrawal or treatment with four structurally unrelated cytotoxic agents. All three oncoproteins accelerated apoptosis after IL-3 withdrawal. In contrast, whereas c-myc overexpression generally sensitized cells to cytotoxic drugs, N-myc and L-myc overexpression produced resistance. myc expression tended to be associated with a more robust G2-M arrest after drug exposure, but this did not correlate with drug sensitivity or resistance. Bcl-2 and Bcl-X(L) protected control cells against apoptosis after either IL-3 withdrawal or drug exposure, although in some cases this effect could be overridden by myc oncoproteins, particularly N-myc and L-myc. Our results suggest that the apoptotic pathways activated upon IL-3 withdrawal and cytotoxic drug treatment are distinct and differentially affected by members of the myc and Bcl-2 families.     

Pharmacodynamics of (-)-beta-2',3'-dideoxy-3'-thiacytidine in chronically virus-infected woodchucks compared to its pharmacodynamics in humans

The pharmacodynamics of (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) was studied in chronically woodchuck hepatitis virus-infected woodchucks and compared to that in previous studies in hepatitis B virus (HBV)-infected humans. Net depletion rates of serum virus DNA in woodchucks receiving 3TC were modeled as a sum of an exponentially declining virus input and a first-order elimination. Preceding shoulders and pseudo-first-order virus half-lives in serum ranged from 1 to 7 days and were dose dependent. Higher plasma 3TC concentrations were needed in woodchucks for virus depletion similar to that attained in humans. Human HBV depletion curves from a previous clinical study with 3TC (>/=100 mg per day) were described by a biexponential relationship. The average half-life value in humans, normalized to fraction of area under the serum virus load-time curve, was similar to the average half-life value observed in woodchucks given the highest 3TC dose (2.4 and 2.0 days, respectively). On cessation of therapy, virus load rebounds in woodchucks were dose dependent and resembled posttherapy virus "flares" reported to occur in humans. The estimates of drug exposures that could lead to optimal antiviral effects presented indicate that 3TC should not be underdosed and compliance should be monitored. The study of chronically infected woodchucks may prove useful for optimizing drug regimens for hepadnavirus infections.