Is the DNA repair system involved in hepatitis-B-virus-mediated hepatocellular carcinogenesis?
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Carcinoma, Hepatocellular↗
Biomedical subjects
J S Butel
Publications and source records attributed to J S Butel.
Natural simian virus 40 strains are present in human choroid plexus and ependymoma tumors.
Simian virus 40 (SV40) sequences for large tumor antigen (T-ag) were recently detected in a significant fraction of certain human brain tumors of early childhood (Bergsagel et al., N. Engl. J. Med. 326, 988-993, 1992). In the current study, we sought to determine whether authentic SV40 was present in the choroid plexus and ependymoma tumors previously examined. Polymerase chain reaction and DNA sequence analysis revealed authentic SV40 regulatory region and major capsid (VP1) sequences in 14 of 17 tumors tested. Only one 72-basepair element was detected in the SV40 enhancer region of positive tumor samples, an arrangement designated as "archetypal." The C terminus of the T-ag gene was detected in the same 14 tumors and was sequenced from 5 tumors; some nucleotide changes were found that would result in amino acid changes in T-ag. Infectious SV40 was isolated from one sample after lipofection of tumor DNA into monkey kidney cells. Sequence analysis of the rescued virus SVCPC revealed (i) an archetypal regulatory region, (ii) nucleotide changes in the C terminus of the T-ag gene that distinguished it from SV40 laboratory strains 776 and SV40-B2 and from human isolate SVPML-1, and (iii) identity with previous human brain tumor isolate SVMEN in the three genomic regions sequenced. No human-isolate-specific distinguishing features were detected among the viral sequences analyzed. Thus, authentic SV40 is present in humans and associated with two tumor types known to be induced experimentally by the virus.
Amino Acid Sequence↗
Expression of the mouse mammary tumor virus long terminal repeat open reading frame promotes tumorigenic potential of hyperplastic mouse mammary epithelial cells.
The retrovirus mouse mammary tumor virus (MMTV) 3' long terminal repeat (LTR) contains an open reading frame (ORF) for a 36-kDa protein and encodes a superantigen activity [pORF(sag)]. We have tested the potential oncogenic activity of pORF(sag) in two immortalized mouse mammary epithelial cells. We subcloned MMTV LTR ORF DNA into the pRc/CMV mammalian expression vector in order to place LTR ORF transcription under the control of the constitutive CMV promoter. Mouse mammary epithelial cell lines TM3 and FSK7e4 were transfected and G418-resistant cell clones were isolated. Reverse transcription-polymerase chain reaction and Northern blot analyses revealed modest overexpression of LTR RNA in several transfected cell clones of each line. Individual cell clones were transplanted into cleared mammary gland fat pads of syngeneic BALB/c mice. The parental cell lines and FSK7e4-derived clones did not form tumors, whereas ORF-transfected clones derived from the TM3 cells formed tumors within 8 weeks in 100% of transplanted fat pads in multiple experiments. The tumor cells expressed exogenous LTR ORF RNA and were proven to be derivatives of TM3 cells based on a marker p53 mutation. Immunohistochemistry using a polyclonal antiserum raised against pORF(sag) expressed in insect cells revealed a cytoplasmic reaction in TM3-CMV-LTR tumor cells; a much weaker cytoplasmic reaction was detected in the transfected tissue culture cells. These observations suggest that MMTV pORF(sag) may act as an oncogene in certain mouse mammary epithelial cells and raise the possibility that pORF(sag) may have a role in mammary tumorigenesis. As the parental FSK7 cell line has produced only ductal outgrowths upon transplantation in vivo and the TM3 cell line produces a nontumorigenic hyperplasia, the results suggest further that pORF(sag) may influence the latter stages of mammary tumorigenesis, namely, the preneoplastic to neoplastic transformation.
Animals↗
Detection of retroviral superantigen and products of the envelope gene from endogenous mouse mammary tumor virus in B cells from BALB/c mice.
Minor lymphocyte-stimulating antigens and other superantigens have been shown to be encoded by the 3' long terminal repeat (LTR) open reading frame (ORF) of the endogenous and exogenous mammary tumor viruses. We have previously reported the presence of an antigen(s) related to mouse mammary tumor virus (MMTV) env products in splenic B cells of BALB/c mice. By Western blots an MMTV-related molecule of 68 kDa was detected in splenic preparations of B lymphocytes, but not in T cells. Antibodies against the MMTV envelope proteins gp52 and gp36, obtained by elution after binding to nitrocellulose in the presence of purified MMTV, reacted in Western blots with a 68-kDa protein present in B cells, indicating that this molecule is related to both MMTV envelope proteins. Using antibody against the MMTV 3' LTR ORF coding sequence, 10-15% of splenic B cells reacted by immunoperoxidase staining with this reagent, while no such staining was observed in splenic T cell preparations. Furthermore, in preparations of splenic B cells, but not T cells, two bands of 68 and 33 kDa, respectively, were detected by Western blots using the anti-ORF. These results demonstrate that the superantigen protein is present in B cells of BALB/c mice in two distinct forms, i.e., as a 68-kDa molecule, possibly associated with products of the env gene, and as a 33-kDa form.
Animals↗
Artificial modification of the viral regulatory region improves tissue culture growth of SV40 strain 776.
We describe here changes in the regulatory region of SV40 that influence its growth potential in cultured cells. Laboratory strains of papovaviruses BK and JC differ in the sequence of their regulatory regions from archetypes that have not been passaged in cell culture. These archetypes lack sequence repeats in the regulatory region; duplications that occur upon passage in cell culture confer selective growth advantage. Changes within the enhancer-promoter region of the well-characterized 776 strain of papovavirus SV40 that might affect its growth in tissue culture cells have not been documented. We measured the effect upon the growth of SV40 (776 strain) in CV-1 cells either of adding an additional 72-basepair (bp) enhancer element or of duplicating the entire 21-bp repeat region. SV40 growth in tissue culture was improved by reiteration of enhancer elements, whereas no growth advantage was conferred by tandem duplication of the 21-bp repeats. Viral DNA infectivity in CV-1 cells was directly proportional to the number of 72-bp elements but was unaffected by tandemly repeated 21-bp repeat elements. This study suggests that the 776 strain of SV40 is an evolutionary intermediate and that tissue-culture-adapted strains of SV40 do not accurately reflect the replication potential of natural isolates from primate hosts.
Animals↗
Tumor suppressor p53 mutations and breast cancer: a critical analysis.
Alterations in the tumor suppressor gene p53 are the most commonly identified changes in cancer, including neoplasia of the breast. The activity of p53 is regulated post-translationally. Phosphorylation state, subcellular localization, and interaction with any of a number of cellular proteins are likely to influence the function of p53. The exact effect of p53-mediated growth suppression seems to be cell-type specific but appears to be directly related to the ability of p53 to act as a specific transcriptional activator. The role that transcriptional repression plays in the function of WT p53 is less clear. It is also possible that p53 has a more direct activity in DNA replication and repair. Most documented p53 mutations result in single amino acid substitutions which may confer one or more of a spectrum of transforming abilities on the protein. Mutation may lead to nuclear accumulation of p53 protein; however, inactivation of p53 by nuclear exclusion and interaction with the mdm2 protein also appear to be important in tumorigenesis. Used in conjunction with other established factors, accumulation of cellular p53 may be a useful prognostic indicator in breast cancer. A syngeneic mouse model system yielded evidence that p53 mutations are important in the early, preneoplastic stages of mammary tumorigenesis. This murine system may provide the ability to investigate the functions of p53 in the early stages of breast cancer which are technically difficult to examine in the human system.
Base Sequence↗
Hepatitis B virus X protein interacts with a probable cellular DNA repair protein.
The mechanism of action of hepatitis B virus (HBV) X protein in transcriptional transactivation and in tumorigenesis remains obscure. We have used the yeast two-hybrid system to identify a cellular protein that can interact with HBV X protein. This protein, designated X-associated protein 1 (XAP-1), is a human homolog of the UV-damaged DNA-binding protein (UV-DDB) recovered from a monkey cell cDNA library. UV-DDB is presumed to be involved in DNA repair. The interaction between X protein and XAP-1 protein was verified by immunoprecipitation of yeast cell lysates expressing both proteins and by in vitro mixing with X protein expressed as a glutathione S-transferase fusion protein and XAP-1 protein either in HeLa cell extracts or synthesized by in vitro translation. We speculate that the interaction of X protein with a DNA repair protein may recruit cellular proteins to repair the partially double-stranded HBV genome or may modify cellular transcription processes. An effect on the cellular DNA repair system may explain a cofactor role for HBV in liver cancer development.
Amino Acid Sequence↗
Inhibition of HIV-1 by a double transdominant fusion gene.
A double transdominant fusion gene (trev) designed to inhibit two essential HIV functions simultaneously was constructed by linking tat and rev transdominant mutants. Trev independently inhibited both Tat and Rev functions, localized within the nucleus and cells transfected with trev showed a stable inhibition of HIV-1-mediated cytopathicity. A retroviral vector of trev was made and shown also to confer protection from HIV cytopathic effects. Simultaneous inhibition of two essential viral genes presents significant advantages for potential gene therapy treatment of HIV infection over conventional single effect molecules.
Amino Acid Sequence↗
Viral co-factors in liver cancer: lessons from hepatitis B virus.
Hepatitis B virus (HBV) is a co-factor in some hepatocellular carcinomas (HCC). Chronic infection with HBV is a risk factor for tumor development, suggesting the accumulation of cellular genetic changes. HBV DNA is frequently found integrated at random sites in HCC, with chromosomal deletions and rearrangements being common at the sites of viral integration. Tumor suppressor gene p53 is frequently altered in HCC. Environmental carcinogens are factors in HCC development in certain geographic locations. HBV encodes a protein (X) known to transactivate viral and cellular genes; the X gene is often retained in HCC. To learn more about X gene function. We employed the yeast two-hybrid genetic system to seek X-interactive proteins. A cellular protein, designated XAP-1, was recovered that interacts specifically with the X protein. XAP-1 is the human homologue of the monkey UV-damaged DNA-binding protein (UV-DDB); the UV-DDB protein functions in DNA repair and is defective in some xeroderma pigmentosum group E patients. The interaction between XAP-1 and HBV X protein was confirmed by several independent methods. This suggests that cellular DNA repair processes may be affected by HBV and that the resulting genetic instability may contribute to hepatocellular carcinogenesis. A unifying model of the molecular basis of HBV involvement in HCC development is presented. Fundamental components of the model are chronic infection by HBV and viral effects on cellular DNA repair. This model has implications for the possible role of HCV infection in the induction of HCV-associated HCC.
Carcinoma, Hepatocellular↗
Allelotype and loss of heterozygosity of p53 in primary and recurrent hepatocellular carcinomas. A study of 150 patients.
BACKGROUND: The allelotype and loss of heterozygosity (LOH) of the p53 gene in human hepatocellular carcinoma (HCC) were studied in 150 patients with resected primary HCC and 18 with recurrent HCC. METHODS: DNA samples of paired HCC and livers were cut with BanII enzyme for the study of p53 allelotype and allele loss. The medical records of the patients were carefully reviewed. RESULTS: Sixty-four (42.7%) patients were heterozygous for the p53 gene, 69 (46%) were homozygous for the 1.5/1.4 kb small (S) allele, and 17 (11.3%) were homozygous for the 2.9 kb large (L) allele. The frequencies of the minor L allele (0.323) and of the major S allele (0.677) in this population of Chinese patients differed from the frequencies previously reported for North American Caucasians (0.13 and 0.87, respectively). The heterozygous patients tended to have lower serum hepatitis B surface- and e antigens (HBsAg and HBeAg) and higher diabetes mellitus (DM) than did homozygous patients (SS and LL). Thirty-seven (57.8%) of the 64 heterozygous patients had a tumor-specific p53 allele LOH, being two times more common in HCC tumors larger than 8 cm than in HCC tumors 2 cm or smaller. The frequency of DM was four times higher in the heterozygous patients who had p53 LOH than in those who retained both alleles. LOH of p53 did not correlate with tumor invasiveness or differentiation, hepatitis B or C virus infection, or prognosis. CONCLUSION: The allelotype of p53 gene in HCC correlates with HBsAg and HBeAg seropositivities and DM. LOH of the p53 gene is a common event in HCC, correlates with DM, and occurs less often in familial HCC. LOH can identify the clonal origin of recurrent HCC but is not a critical prognostic factor.
Adult↗
Infrequent p53 mutations in 7,12-dimethylbenz[a]anthracene-induced mammary tumors in BALB/c and p53 hemizygous mice.
We conducted experiments to determine if p53 alterations, which are frequent in human breast cancers, were also common in murine mammary tumors. In 13 mammary tumors from 7,12-dimethylbenz[a]anthracene (DMBA)-treated BALB/c mice were immunohistochemically analyzed for overexpression of p53; p53 protein was not detectable. Three of the tumors were established as cell lines in vitro. p53 protein was rarely detected at passage 4 in these lines but was overexpressed by passage 8 in two of them. The p53 nucleotide sequence was shown to be wild type in one primary mammary tumor and in the two p53-overexpressing cell lines. One cell line that overexpressed p53 in vitro was implanted into BALB/c mice. The resulting tumors retained the wild-type p53 nucleotide sequence but no longer expressed detectable levels of p53 protein, suggesting that the overexpression of wild-type p53 was related to in vitro culture conditions. The effect of DMBA on mammary-tumor development was also tested in mice rendered hemizygous for p53. These mice and wild-type littermate controls had no differences in susceptibility to induction of mammary tumors by oral administration of DMBA. Furthermore, Southern blot hybridization detected no gross alterations in the wild-type p53 allele in mammary tumors from the p53-deficient mice. Point mutation of the wild-type p53 allele was also infrequent in the DMBA-induced mammary tumors from hemizygous p53 mice; it occurred in only one of seven tumors. Thus, the p53 gene is apparently not a primary target for genetic alterations in DMBA-induced mammary tumors. Next, we examined mammary tumors derived from D1 and D2 transplantable hyperplastic alveolar nodule (HAN) outgrowths, which rapidly form tumors containing Ha-ras mutations after DMBA treatment. As ras and p53 mutants can cooperate in transformation, we examined whether D1 and D2 HAN outgrowths have p53 mutations. Unlike in the DMBA-induced primary mammary tumors, nuclear p53 accumulation was observed frequently (10 of 14) in tumors that arose from D1 and D2 HAN outgrowths. Direct sequencing of the entire coding region of the p53 cDNA from six D1 and D2 tumors confirmed that the sequence was wild type. Although wild-type p53 was retained in both DMBA-induced mammary tumors and mammary tumors derived from D1 and D2 preneoplastic outgrowths, wild-type p53 overexpression was detected only in D1 and D2 tumors. Therefore, D1 and D2 tumors appear to arise by a pathway in which p53 expression is altered, whereas DMBA induction affects a different pathway that does not require such alteration.
9,10-Dimethyl-1,2-benzanthracene↗
p53 mutations in COMMA-D cells.
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Animals↗
Construction of SV40 deletion mutants and delimitation of the binding domain for heat shock protein to the amino terminus of large T-antigen.
SV40 large T-antigen (T-ag) mutants were generated using a cassette mutagenesis strategy and naturally occurring restriction sites. T-ag mutant constructs included internal in-frame deletions, frame-shift deletions that resulted in amino-terminal fragments, and internal initiation mutants that produced carboxy-terminal fragments; no foreign amino acids were introduced. The deletion mutants were stably expressed in BALB/c 3T3E cells and were analyzed for ability to bind heat shock cognate protein 70 using an ATP release assay of T-ag immunoprecipitates. Complex formation between heat shock protein and T-ag was independent of p53 involvement. The heat shock protein binding domain was narrowed to the amino-terminal 97 amino acids of T-ag, with the first 29 residues influencing the interaction. The amino-terminal domain of T-ag is important in both viral replication and cell transformation. We propose that the functional interactions of this highly interactive region of T-ag may be modulated by heat shock cognate protein 70.
3T3 Cells↗
The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice.
Regulation of the cell cycle is a critical aspect of cellular proliferation, differentiation, and transformation. In many cell types, the differentiation process is accompanied by a loss of proliferative capability, so that terminally differentiated cells become postmitotic and no longer progress through the cell cycle. In the experiments described here, the ocular lens has been used as a system to examine the role of the retinoblastoma protein (pRb) family in regulation of the cell cycle during differentiation. The ocular lens is an ideal system for such studies, since it is composed of just two cell types: epithelial cells, which are capable of proliferation, and fiber cells, which are postmitotic. In order to inactivate pRb in viable mice, genes encoding either a truncated version of simian virus 40 large T antigen or the E7 protein of human papillomavirus were expressed in a lens-specific fashion in transgenic mice. Lens fiber cells in the transgenic mice were found to incorporate bromodeoxyuridine, implying inappropriate entry into the cell cycle. Surprisingly, the lens fiber cells did not proliferate as tumor cells but instead underwent programmed cell death, resulting in lens ablation and microphthalmia. Analogous lens alterations did not occur in mice expressing a modified version of the truncated T antigen that was mutated in the binding domain for the pRb family. These experimental results indicate that the retinoblastoma protein family plays a crucial role in blocking cell cycle progression and maintaining terminal differentiation in lens fiber cells. Apoptotic cell death ensues when fiber cells are induced to remain in or reenter the cell cycle.
Animals↗
Mutations in p53 are frequent in the preneoplastic stage of mouse mammary tumor development.
Preneoplastic lesions in the mammary gland represent a population of cells at increased risk of progression to tumors. Because p53 is the most commonly mutated gene in human breast cancer, we sought to determine whether mutations in p53 were present in preneoplastic lesions or were acquired during progression to overt tumors. In the mouse mammary gland, hyperplastic alveolar nodules (HAN) are the most common preneoplastic lesion. Analysis of the TM series of transplantable murine HAN outgrowths and tumors allowed the status of p53 to be determined at distinct stages of mammary tumorigenesis. Alterations in the p53 gene or in the pattern of p53 protein expression were observed in all five HAN outgrowth lines examined. Altered expression of p53 protein was detected in 3 of 5 TM HAN outgrowth lines as determined by immunohistochemistry. Overexpression of nuclear p53 was detected in only a fraction of the cells (10-50%) in TM3 and TM4 HAN outgrowths, whereas in tumors that arose from TM4 HAN outgrowths, the proportion of cells overexpressing p53 protein approached approximately 100%. Despite overexpression of p53 in TM3 HAN outgrowths, no tumors have developed in this line. The TM9 outgrowth line exhibited a different pattern of p53 expression by immunohistochemistry: p53 protein was overexpressed in the cytoplasm of virtually all cells in the HAN outgrowths but was localized to the nuclei of TM9 tumor cells. Direct sequencing of p53 transcripts from tumors and cell lines revealed various genetic changes: point mutations in exons 4 and 5 (TM2H, nonsense; TM4, missense); a deletion in exon 5 (TM4); and an insertion in exon 7 (TM3). Although p53 protein was overexpressed in TM9 tumors, it was shown to be wild-type both by immunoprecipitation and direct sequencing of the entire coding region of the cDNA. TM4 cells were homogeneous with respect to mutant p53 genotype and uniformly expressed p53 by immunohistochemical staining in vitro, but transplantation of TM4 cells to fat pads of BALB/c hosts resulted in HAN outgrowths in situ in which < 50% of the cells expressed the mutant p53 at detectable levels. In summary, mutation of the p53 gene and overexpression of p53 protein can occur in preneoplastic mammary epithelial cells, and those mutations are maintained in tumors that arise from the HAN. Conversely, expression of mutant p53 was decreased when cells were grown in situ, implicating the presence of cellular factors that can suppress p53 expression in vivo. These observations demonstrate that the p53 pathway may be a common target for mutation in murine mammary tumorigenesis.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
p53 mutations selected in vivo when mouse mammary epithelial cells form hyperplastic outgrowths are not necessary for establishment of mammary cell lines in vitro.
Breast cancer is a consequence of multiple alterations occurring over a long period of time. Genetic changes in early stages of tumorigenesis have not been defined. A recently developed murine system permits the study of mammary preneoplastic cells in vivo and in vitro (F. S. Kittrell et al., Cancer Res., 52: 1924-1932, 1992). To assess the potential role of p53 mutations in early stages of breast cancer, the status of p53 was determined in a series of mouse mammary epithelial cell lines which give rise to preneoplastic outgrowths (hyperplastic alveolar nodules) when transplanted into cleared mammary fat pads of syngeneic mice. Protein stability and conformation were analyzed using immunoprecipitations and immunochemical assays; p53 transcripts were sequenced using a polymerase chain reaction approach. The parental cell lines (FSK lines) showed no evidence of p53 alterations at either the protein or the nucleic acid level, indicating that p53 mutations are not essential for the establishment of mammary epithelial cell lines in vitro. In contrast, cell lines (TM lines) derived from hyperplastic alveolar nodule outgrowths induced by FSK cells expressed only mutant p53 genes. The mutation in one outgrowth cell line (TM-2H) resulted in the loss of p53 protein synthesis, whereas two other outgrowth lines (TM-3, TM-4) overexpressed mutant p53 protein. Mutation of p53 appears to correlate with preneoplastic growth in vivo. Although it is not clear if the mutations occur before or after transplantation of cells in vivo, there appears to be a pronounced growth advantage in the mammary gland for cells expressing mutant p53.
Animals↗
Phylogenetic and structural analyses of MMTV LTR ORF sequences of exogenous and endogenous origins.
The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) harbors an open reading frame (ORF) that encodes a glycoprotein and is present in all exogenous and endogenous MMTV proviruses. The ORF protein has been reported to interact with the immune system of mice to cause deletion of specific V beta-bearing subsets of T cells. Twenty-two MMTV LTR ORF sequences were analyzed. Although highly conserved, the MMTV ORF sequences are not identical, with approximately 35% of the total variation clustered at the carboxy terminus. Statistical analysis revealed the presence of two conserved regions in the protein, one of which contained a transmembrane-like domain (residues 45-63). Two potential nuclear localization signals were recognized. Many ORF sequences shared polymorphisms. To analyze relationships, phylogenetic trees were constructed on the basis of alignments of LTR ORF sequences. A tree generated from the carboxy-terminal 35 residues clustered the sequences into three divergent families. The topology of the tree based on the amino-terminal 288 residues differed significantly, with some MMTV sequences rearranged relative to their carboxy-terminal families. A continuum of exogenous-like to endogenous-like character was suggested by the amino-terminal tree. The discordance between the topologies of the two trees suggests that some type of genetic exchange has occurred in the MMTV LTR gene. Mechanisms and implications of such genetic exchange are discussed.
Amino Acid Sequence↗
Spontaneous and carcinogen-induced tumorigenesis in p53-deficient mice.
Using gene targeting techniques, mice that have been generated with two germ-line p53 null alleles (homozygotes) develop normally but are highly susceptible to early onset spontaneous tumours. Here, we show that mice with a single null p53 allele (heterozygotes) produced in the same way are also susceptible to spontaneous tumours, but with a delayed onset compared to homozygotes. The most frequent tumour type in homozygotes was malignant lymphoma; in heterozygotes, osteosarcomas and soft tissue sarcomas predominated. Heterozygous mice treated with a liver carcinogen, dimethylnitrosamine, showed a decreased survival time in comparison to treated wild type mice, suggesting that the p53-deficient mice may be useful for some in vivo carcinogenesis assays.
Animals↗