The naturally occurring mutants of DDB are impaired in stimulating nuclear import of the p125 subunit and E2F1-activated transcription.

The human UV-damaged-DNA binding protein DDB has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E), because a subset of XP-E patients lack the damaged-DNA binding function of DDB. Moreover, the microinjection of purified DDB complements the repair deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E mutations of DDB, 82TO and 2RO, have been characterized. They have single amino acid substitutions (K244E and R273H) within the WD motif of the p48 subunit of DDB, and the mutated proteins lack the damaged-DNA binding activity. In this report, we describe a new function of the p48 subunit of DDB, which reveals additional defects in the function of the XP-E mutants. We show that when the subunits of DDB were expressed individually, p48 localized in the nucleus and p125 localized in the cytoplasm. The coexpression of p125 with p48 resulted in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The mutant forms of p48, 2RO and 82TO, are deficient in stimulating the nuclear accumulation of the p125 subunit of DDB. In addition, the mutant 2RO fails to form a stable complex with the p125 subunit of DDB. Our previous studies indicated that DDB can associate with the transcription factor E2F1 and can function as a transcriptional partner of E2F1. Here we show that the two mutants, while they associate with E2F1 as efficiently as wild-type p48, are severely impaired in stimulating E2F1-activated transcription. This is consistent with our observation that both subunits of DDB are required to stimulate E2F1-activated transcription. The results provide insights into the functions of the subunits of DDB and suggest a possible link between the role of DDB in E2F1-activated transcription and the repair deficiency disease XP-E.

Mammary cancer stages in BALB/cV mice: mouse mammary tumor virus expression and virus-host interactions.

A unique subline of BALB/c mice, designated "BALB/cV," exhibits an intermediate mammary tumor incidence (47%) and harbors a distinct milk-transmitted mouse mammary tumor virus (MMTV). Virus expression and virus-host interactions were examined during the different stages of mammary tumorigenesis (normal, preneoplastic, and neoplastic) in the BALB/cV system. Protein immunoblot analyses established the presence of correctly processed (BALB/cV)MMTV structural proteins in all types of BALB/cV mammary tissues. Competition enzyme-linked immunosorbent assays demonstrated that cells from each biologic phenotype were capable of supporting high levels of (BALB/cV)MMTV protein expression. However, mammary epithelial cells that spontaneously underwent the inappropriate pathway of squamous metaplasia did not contain detectable levels of (BALB/cV)MMTV structural proteins. Iodination experiments revealed the presence of a 68K env-related protein on the surface of BALB/cV mammary cells. Nevertheless, sera from 40 mice bearing BALB/cV-positive mammary tissues did not contain detectable levels of anti-env antibodies. Metabolic labeling experiments showed that the half-life of transformation-related, host cell protein p53 (approximately equal to 60 min) in the distinct BALB/cV mammary cell populations was similar to that reported for normal mouse 3T3 cells. It appears that p53 is not stabilized by protein interactions involving any MMTV-encoded or MMTV-induced protein in mammary tumor cells. These characteristics of the BALB/cV system are compatible with the hypothesis that MMTV is only one of two or more cooperating factors required to mediate complete mammary transformation.

Frequent loss of chromosome 8p in hepatitis B virus-positive hepatocellular carcinomas from China.

The majority of hepatocellular carcinomas (HCCs) from hepatitis B virus (HBV)-endemic areas contain integrated viral sequences. To better understand the role of HBV DNA insertion in tumorigenesis, we examined the integration site of a HCC harboring a single insert. Cellular DNAs flanking the viral sequences were mapped to chromosomes 17 and 8, indicating a translocation had occurred at the site of viral integration. Regional mapping of chromosome 17 demonstrated that HBV had integrated in 17p12-pter, a region that harbors the p53 tumor suppressor gene. Many studies have shown that chromosome 17p allele loss occurs frequently in HCCs from certain geographical areas. To investigate the chromosome 8 allele status in Chinese HCCs, a panel of 37 matched normal and HCC DNAs from Qidong, China was analyzed for tumor-specific allele loss with RFLP probes from both arms of chromosome 8. Tumor-specific loss of heterozygosity was highest on the short arm with 71.4% (10/14) and 85.0% (17/20) of the informative patients missing an allele for 8p23 (YNM3) or 8p21 (NEFL), respectively. Allele loss from the long arm of chromosome 8 was also observed with 30.0% (6/20) and 33.3% (7/21) of the samples informative for 8q22 (CA2) and 8q24 (MCT128.2), respectively. The high allele loss on 8p correlates with recent studies of other human cancers and is interpreted to indicate that a tumor suppressor gene(s) whose loss is important for carcinogenesis lies within this region. These findings also support a model in which HBV insertions associated with gross chromosomal changes can identify genomic regions where alteration is important for development of some HCCs.

Hepatitis B virus integration event in human chromosome 17p near the p53 gene identifies the region of the chromosome commonly deleted in virus-positive hepatocellular carcinomas.

The development of hepatocellular carcinoma (HCC) presumably occurs in multiple steps and is influenced by numerous factors. Hepatitis B virus (HBV) is strongly associated with the development of HCC in people chronically infected with the virus, but the mechanism of viral involvement remains unclear. One possibility is that the gross chromosomal alterations frequently observed in HCC DNA at the site of HBV integration may alter the expression of important nearby cellular genes. We previously reported the cloning and characterization of a HBV insert from a Chinese HCC. The viral insert mapped to chromosome 17p11.2-12, and cellular sequences were duplicated at the site of viral integration. In the present study a DNA probe derived from cellular DNA sequences adjacent to the previously characterized HBV insert was used to analyze a set of 19 matched normal liver and HBV-positive hepatoma samples obtained from the same region of China, near Shanghai. Tumor-specific DNA changes were detected in two additional HCCs, suggesting that the small region of chromosome 17p defined by the flanking cell DNA probe is commonly altered in hepatomas. Restriction fragment length polymorphism studies demonstrated that the loss of one copy of portions of chromosome 17 occurred in 10 (53%) of the 19 patients. The loss of one allele of the p53 gene (located on chromosome 17p13) occurred in at least 6 (60%) of the 10 patients who were heterozygous at the p53 locus. As the p53 gene is known to possess tumor suppressor activity, the functional loss of this gene may be a significant step in the development of a subset of HCCs. High levels of allele loss also were detected for chromosomes 8q (4 of 9; 44%) and 16p (5 of 6; 83%) and may indicate the presence of additional cellular genes whose functional loss is important in the development of HCCs.

p53 mutations cluster at codon 249 in hepatitis B virus-positive hepatocellular carcinomas from China.

DNA samples from 36 hepatocellular carcinoma (HCC) patients from China were screened for a specific mutation affecting codon 249 of the p53 gene, recently identified as a hotspot mutation in some HCCs. We detected the tumor-specific p53 codon 249 mutation in 21 (58%) of 36 HCCs examined. Thirteen patients with the specific codon 249 mutation had lost the remaining allele of p53, whereas the remaining eight patients appeared to have retained both copies of the gene. These results suggest that alterations of p53 may be important events in the genesis of HCCs and that point mutation may precede allele loss.

Expression of mammary tumor virus proteins in preneoplastic outgrowth lines and mammary tumors of BALB/cV mice.

A subline of BALB/c mice, designated BALB/cV, has been segregated which exhibits an intermediate mammary tumor incidence and which harbors a unique milk-transmitted virus. Six stable hyperplastic alveolar nodule outgrowth lines were established from chemical carcinogen-treated and hormonally stimulated mice. Tumor incidences exhibited by the individual preneoplastic lines ranged from 22 to 95%; no differences in tumor-producing capability were observed when lines were transplanted in virus-negative (BALB/c) or virus-positive (BALB/cV) animals. Viral antigen expression was monitored using antisera prepared against C3H mouse mammary tumor virus (MMTV) proteins. The preneoplastic lines exhibited more virus antigenpositive cells in a peroxidase-antiperoxidase immunocytochemical assay than did primary tumors which arose from the hyperplastic alveolar nodule transplants. Analysis of BALB/cV preneoplastic and tumor tissue by metabolic labeling and by protein electroblotting methodologies revealed that viral precursor and structural proteins were expressed in both types of tissue; the polypeptides were similar in molecular weight to those encoded by exogenous MMTVs. These studies demonstrate that the coding capacity of the BALB/cV isolate of MMTV is similar to that of known MMTV isolates and that each of the BALB/cV structural polypeptides shares group-specific antigenic determinants with the analogous protein encoded by MMTV from the C3H mouse. The hyperplastic outgrowth lines established in the BALB/cV subline provide an additional system for the study of mammary tumorigenesis.

Development of a transgenic mouse system for the analysis of stages in liver carcinogenesis using tissue-specific expression of SV40 large T-antigen controlled by regulatory elements of the human alpha-1-antitrypsin gene.

alpha-1-Antitrypsin (AAT) is the major antiprotease in human plasma; it is synthesized primarily in hepatocytes and to a lesser extent in several nonhepatic tissues. Under the control of regulatory elements of the human AAT gene, expression of SV40-large tumor antigen (T-ag) in transgenic mice occurred in the liver, stomach, pancreas, and kidney. Among seven founder transgenic animals, six developed liver carcinoma, four showed gastric neoplasia, and one developed pancreatic carcinoma. In three animals the kidneys showed glomerular or tubular epithelial hyperplasia but no malignancy. A stable transgenic line, 1812, was established. Members of this line reproducibly develop liver tumors by 10 weeks of age but do not exhibit any phenotypic changes in other tissues. Histological changes leading to liver tumor formation occurred with predictable kinetics and could be classified into four distinct stages: (a) embryonal/fetal stage, no recognizable histological changes; (b) newborn to 2 weeks of age, hyperplastic hepatocytes with reduced amounts of cytoplasm but no nuclear alterations; (c) between 3 and 8 weeks of age, diffuse liver cell dysplasia without observable tumor nodules; and (d) 8 weeks of age and thereafter, hepatocellular carcinomas in a background of liver dysplasia. Embryonic and newborn liver tissue showed uniform, high level expression of T-ag in the majority of hepatocytes by immunohistochemistry, whereas the dysplastic and tumoral stages were characterized by considerable variation in both the intensity of T-ag staining and the proportion of T-ag-positive cells. Immunoprecipitation analyses showed that T-ag was complexed with cellular protein p53 in all tumor samples. This study showed that SV40 T-ag expression in the liver resulted in cellular hyperplasia and dysplasia; additional event(s) apparently were required for progression to neoplasia. Those cooperating events occurred with predictable kinetics. This transgenic mouse system displays several similarities with human liver disease and provides a practical model for the study of separate steps in hepatocarcinogenesis.

Dissociation of DDB1-binding and transactivation properties of the hepatitis B virus X protein.

The hepatitis B virus (HBV) X protein (HBx) is a transactivator encoded by mammalian hepadnaviruses, and is thought to stimulate transcription by interacting with one or more host cell factors. Numerous cellular proteins have been reported to interact with HBx including a component of the nucleotide excision repair complex called ultraviolet damaged DNA binding (UV-DDB, or DDB1) protein. Recent studies have identified a role for DDB1 in transcription, raising the possibility that HBx may acquire its broad transcriptional properties by interacting with DDB1. A panel of HBx mutant proteins, some of which no longer bind to DDB1, was used to test this hypothesis. Plasmid DNAs encoding HBx wildtype and mutant derivatives were transfected into HepG2 cells, and their ability to transactivate a cotransfected reporter plasmid tested. Results from the transactivation assays in HepG2 cells were then compared with data obtained from HBx-DDB1 binding studies performed in yeast. Several HBx mutant proteins unable to bind DDB1 remained competent for transactivation, indicating that HBx binding to DDB1 is not required for HBx transactivation of the ETS1 promoter. It remains possible that a subset of HBx transactivation function targets an as yet undefined DDB1-specific pathway.

Stimulation of cellular proliferation by hepatitis B virus X protein.

Chronic infection with the hepatitis B virus (HBV) is a known risk factor in the development of human hepatocellular carcinoma (HCC). The HBV-encoded X protein, HBx, has been investigated for properties that may explain its cancer cofactor role in transgenic mouse lines. We discuss here recent data showing that HBx is able to induce hepatocellular proliferation in vitro and in vivo. This property of HBx is predicted to sensitize hepatocytes to other HCC cofactors, including exposure to carcinogens and to other hepatitis viruses. Cellular proliferation is intimately linked to the mechanism(s) by which most tumor-associated viruses transform virus-infected cells. The HBx alteration of the cell cycle provides an additional mechanism by which chronic HBV infection may contribute to HCC.

Hepatitis B virus-induced liver injury and altered expression of carcinogen metabolising enzymes: the role of the HBx protein.

Hepatitis B virus (HBV) and aflatoxins are major risk factors for hepatocellular carcinoma (HCC) exhibiting a synergistic interaction in the development of this disease. The molecular mechanisms of this interaction remain to be elucidated but an altered carcinogen metabolism in the presence of hepatitis-induced liver injury is one hypothesis. The availability of biomarkers of aflatoxin exposure and metabolism permits this hypothesis to be examined in human populations whilst animal models, such as HBV transgenic mice permit parallel studies in an experimental setting. The hepatitis B virus X protein (HBx) is suspected to play a role in the hepatocarcinogenic process by virtue of its capacity to transactivate oncogenes and several other cellular genes via cis-acting elements. In previous studies in HBV transgenic mice expressing the HB surface antigen and X genes we observed a marked induction of specific cytochrome P450s (CYP) (Kirby et al., 1994a). In the current study we investigated the status of CYP, glutathione S-transferases (GST) and antioxidant enzymes in mice carrying only the X gene under the control of the alpha-1 antitrypsin regulatory elements (ATX mice). Livers of ATX mice showed no major pathological alterations compared to age-matched non-transgenic control mice. Immunohistochemical staining for CYP1A, 2A5 and GST expression and determination of related enzymatic activities (7-ethoxyresorufin O-deethylation, 7-methoxyresorufin O-deethylation, coumarin 7-hydroxylation and GST activities) revealed no differences between control and ATX mice. In addition, no differences in antioxidant enzymes were observed. Overall, these results support the conclusion that HBx expression alone is insufficient to induce transactivation of CYP and GST genes or to alter the antioxidant system and that the induction in other HBV models is a result of inflammatory injury in the liver, a feature absent in ATX mice. These data are compared to biomarker studies of enzyme activities in aflatoxin-exposed human populations with and without HBV infection.

NF-kappaB signaling mediates the induction of MTA1 by hepatitis B virus transactivator protein HBx.

Metastasis-associated protein 1 (MTA1), a master chromatin modifier, has been shown to regulate cancer progression and is widely upregulated in human cancer, including hepatitis B virus-associated hepatocellular carcinomas (HCCs). Here we provide evidence that hepatitis B virus transactivator protein HBx stimulates the expression of MTA1 but not of MTA2 or MTA3. The underlying mechanism of HBx stimulation of MTA1 involves HBx targeting of transcription factor nuclear factor (NF)-kappaB and the recruitment of HBx/p65 complex to the NF-kappaB consensus motif on the relaxed MTA1 gene chromatin. We also discovered that MTA1 depletion in HBx-expressing cells severely impairs the ability of HBx to stimulate NF-kappaB signaling and the expression of target proinflammatory molecules. Furthermore, the presence of HBx in HBx-infected HCCs correlated well with increased MTA1 and NF-kappaB-p65. Collectively, these findings revealed a previously unrecognized integral role of MTA1 in HBx stimulation of NF-kappaB signaling and consequently, the expression of NF-kappaB targets gene products with functions in inflammation and tumorigenesis.

Identification and characterization of a mouse mammary tumor virus protein uniquely expressed on the surface of BALB/cV mammary tumor cells.

A unique subline of BALB/c mice, designated BALB/cV, exhibits an intermediate mammary tumor incidence (47%) and harbors a distinct milk-transmitted mouse mammary tumor virus (MMTV). The BALB/cV subline was used to study the molecular basis of potential virus-host interactions involving cell surface-expressed MMTV proteins. Cell surface iodination identified virus-specific proteins expressed on BALB/cV primary mammary tumor cells grown in culture. In contrast to (C3H)MMTV-producing cell lines which expressed MMTV gp52, BALB/cV tumor cells lacked gp52 and expressed instead a 68K, env-related protein. The 68Kenv protein was also detected on the surface of metabolically labeled BALB/cV tumor cells by an external immunoprecipitation technique. The expression of 68Kenv was restricted to mammary tissues of BALB/cV mice that also expressed other MMTV proteins. Biochemical analysis established that 68Kenv was not modified by N-linked glycosylation. 125I-labeled 68Kenv was rapidly released into the media of tumor cell cultures and was recovered both in the form of a soluble protein and in a 100,000 g pellet. The biologic function of this cell surface-expressed viral protein remains unknown.

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.

Expression of hepatitis B virus X protein does not alter the accumulation of spontaneous mutations in transgenic mice.

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens, although the mechanism for this cofactor role remains unknown. The ability of HBx to inhibit DNA repair in transiently transfected cell lines suggests one possible pathway. In the present study, primary hepatocytes isolated from transgenic mice that possess the HBV X gene under the control of the human alpha-1-antitrypsin regulatory region (ATX mice) were found to be deficient in their ability to conduct unscheduled DNA synthesis in response to UV-induced DNA damage. In order to measure the impact of HBx expression on DNA repair in vivo, double-transgenic mice that express HBx and possess a bacteriophage lambda transgene were sacrificed at 30, 90, and 240 days of age. Mutation frequency was determined for high-molecular-weight liver DNA of ATX and control mice by functional analysis of the lambda transgene. Expression of HBx did not significantly increase the accumulation of spontaneous mutations. These results are consistent with previous studies of HBx transgenic mice in which no effect of HBx on liver histology was apparent. This new animal model provides a powerful system in which to investigate the in vivo cooperation between HBx expression and environmental carcinogens.

Hepatitis B virus X protein acts as a tumor promoter in development of diethylnitrosamine-induced preneoplastic lesions.

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens. Although the mechanism for this cofactor role remains unknown, the ability of HBx to inhibit DNA repair and to influence cell cycle progression suggests two possible pathways. To investigate these possibilities in vivo, we treated double-transgenic mice that both express HBx (ATX mice) and possess a bacteriophage lambda transgene with the hepatocarcinogen diethylnitrosamine (DEN). Histological examination of liver tissue confirmed that DEN-treated ATX mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Treatment of mice with DEN resulted in a six- to eightfold increase in the mutation frequency (MF), as measured by a functional analysis of the lambda transgene. HBx expression was confirmed by immunoprecipitation and Western blotting and was associated with a modest 23% increase in the MF. Importantly, the extent of hepatocellular proliferation in 14-day-old mice, as measured by the detection of proliferating cell nuclear antigen and by the incorporation of 5-bromo-2'-deoxyuridine, was determined to be approximately twofold higher in ATX livers than in wild-type livers. These results are consistent with a model in which HBx expression contributes to the development of DEN-mediated carcinogenesis by promoting the proliferation of altered hepatocytes rather than by directly interfering with the repair of DNA lesions.

Deletion of the E-cadherin gene in hepatitis B virus-positive Chinese hepatocellular carcinomas.

Frequent allele loss from chromosome 16q was recently described for human tumors of the breast, prostate gland and liver, indicating the possible presence of a tumor-suppressor gene on that chromosome arm. In this study, the chromosome 16 allele status of 38 hepatocellular carcinomas in Chinese patients was determined with restriction-fragment-length polymorphism analysis. Tumor-specific allele loss was detected in 14 (74%) of 19 patients informative for 16p markers and in 22 (85%) of 26 patients informative for 16q markers. Quantitative densitometric analysis revealed reduction to hemizygosity of the E-cadherin cell adhesion gene (localized to 16q22.1) in 18 (64%) of the 28 patients for whom quantitative data were available. Reduced expression of E-cadherin has been associated with invasion and metastasis in several human cell lines and primary tumors, and our results suggest that one mechanism of reduced E-cadherin expression is the loss of one copy of the E-cadherin gene.

Identification of a conserved sequence in the non-coding regions of many human genes.

We have analyzed a sequence of approximately 70 base pairs (bp) that shows a high degree of similarity to sequences present in the non-coding regions of a number of human and other mammalian genes. The sequence was discovered in a fragment of human genomic DNA adjacent to an integrated hepatitis B virus genome in cells derived from human hepatocellular carcinoma tissue. When one of the viral flanking sequences was compared to nucleotide sequences in GenBank, more than thirty human genes were identified that contained a similar sequence in their non-coding regions. The sequence element was usually found once or twice in a gene, either in an intron or in the 5' or 3' flanking regions. It did not share any similarities with known short interspersed nucleotide elements (SINEs) or presently known gene regulatory elements. This element was highly conserved at the same position within the corresponding human and mouse genes for myoglobin and N-myc, indicating evolutionary conservation and possible functional importance. Preliminary DNase I footprinting data suggested that the element or its adjacent sequences may bind nuclear factors to generate specific DNase I hypersensitive sites. The size, structure, and evolutionary conservation of this sequence indicates that it is distinct from other types of short interspersed repetitive elements. It is possible that the element may have a cis-acting functional role in the genome.

Activation of the HTLV-I long terminal repeat by the hepatitis B virus X protein.

The human T-cell leukemia virus type I (HTLV-I) Tax protein and the hepatitis B virus (HBV) X protein have each been shown to activate transcription of their respective viral promoters as well as a subset of cellular gene promoters. Here we show that the HTLV-I long terminal repeat (LTR) is responsive to HBV X transactivation. Maximum levels of X-mediated transactivation of the LTR were 8-fold. An X-responsive-region (XRR) of the LTR is located between nucleotides -355 and -276 and contains an AP-2 binding site, a previously recognized X-responsive element. We demonstrated that Tax and X synergize to activate transcription from the HTLV-I LTR, although the AP-2 binding site was not required for this synergy. These results raise the possibility that the HBV X protein may affect the level of HTLV-I gene expression in co-infected individuals.

Molecular basis of altered mouse mammary tumor virus expression in the D-2 hyperplastic alveolar nodule line of BALB/c mice.

The preneoplastic D-2 hyperplastic outgrowth line, which was derived from a hormone-induced hyperplastic alveolar nodule (HAN) of a BALB/c mouse, was used for a detailed analysis of mouse mammary tumor virus (MMTV) expression. The D-2 HAN line has previously been shown to express viral RNA representative of the entire genome, although viral particles have been noted only rarely. The MMTV-specific mRNA, protein, and DNA content of the D-2 tissues was defined in an effort to better understand the molecular basis of the aberrant virus expression. Northern blotting techniques demonstrated the presence of properly processed 8.9 kb (genomic) and 3.6 kb (envelope) mRNA. Protein electroblotting procedures established the presence of properly processed viral core protein p28. In contrast, the envelope precursor polyprotein was not processed into detectable levels of gp52. Analysis of MMTV proviral content by Southern blot methodology revealed the presence of a newly acquired provirus which serves as a marker for the clonal nature of the D-2 line. The origin of the new provirus is unknown. Methylation studies established that the new proviral insert is hypomethylated and, therefore, is likely serving as the template for the MMTV expression observed in the D-2 HAN line. These characteristics of the D-2 line make it an excellent system in which to study the role, if any, of MMTV in the progression of D-2 preneoplastic tissues to the tumor phenotype.