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.

Suppression of in vivo tumor formation induced by simian virus 40-transformed cells in mice receiving antiidiotypic antibodies.

This study characterizes four private idiotypes (Id) associated with monoclonal antibodies (mAb) to simian virus 40 (SV40) tumor antigen (T-Ag), and to a cellular protein, p53. Anti-Id recognized Id determinants associated with the antibody-combining site. BALB/c mice receiving a pool of anti-Id directed against mAb recognizing distinct amino and carboxyl terminal epitopes of T-Ag before receiving a tumorigenic dose of SV40-transformed cells showed suppression of tumor formation. Serum obtained from these mice before tumor challenge contained anti-anti-Id that failed to bind T-Ag. These data support the potential role of regulatory idiotopes in tumor immunity.

Replication of adenovirus type 7 in monkey cells: a new determinant and its transfer to adenovirus type 2.

A strain of human adenovirus type 7, adapted to replication in green-monkey kidney cells, requires the interaction of two particles to initiate plaque formation in the simian cells. One particle is a true adenovirion. The second, apparently defective, consists of a genome carrying amonkey-adapting component in an adenovirus capsid; this genome does not express known SV40 determinants. The addition of human adenovirus type 7 that is not adapted enhances the titer and changesconditions for plaque formation by the adapted virus to a one-particle requirement. Addition of nonadapted human adenovirus type 2 as helper virus results in the transfer of the monkey-adaptingcomponent from adenovirus type 7 to adenovirus type 2. The population containing the adenovirus 2 transcapsidant then has the ability to replicate in simian cells.

Surface T-antigen expression in simian virus 40-transformed mouse cells: correlation with cell growth rate.

Cell growth control appears to be drastically altered as a consequence of transformation. Because the cell surface appears to have a role in modulating cell growth and simian virus 40 (SV40)-transformed cells express large T antigen (T-Ag) in the plasma membrane, we investigated whether surface T-Ag expression varies according to cell growth rate. Different growth states were obtained by various combinations of seeding density, serum concentration, and temperature, and cell cycle distributions were determined by flow microcytofluorometry. Actively dividing SV40-transformed mouse cell cultures were consistently found to express higher levels of surface T-Ag and T-Ag/p53 complex than cultures in which cells were mostly resting. In addition, the T-Ag/p53 complex disappeared from the surface of tsA7-transformed cells cultured under restrictive conditions known to induce complete growth arrest (39.5 degrees C), although the surface complex did not disappear from other tsA transformants able to keep cycling at 39.5 degrees C. These results suggest that surface SV40 T-Ag or surface T-Ag/p53 complex, or both, are involved in determining the growth characteristics of SV40-transformed cells.

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- or proline-containing peptides, or by two-dimensional tryptic peptide mapping of methionine-containing peptides. The carboxy-terminal, methionine-containing tryptic peptide was identified in the two-dimensional maps and was shown to be identical in nT-ag and mT-ag. Thus, a structural basis for the recognition and differential localization of T-ags could not be demonstrated. The carboxy terminus of the T-ag encoded by mutant dlA2413 is derived from the alternate open reading frame of the simian virus 40 early region, in analogy with the theoretical early gene product, T*-ag. We used this mutant to identify peptides unique to T*-ag. None of these peptides were detected in maps of mT-ag; only wild-type T-ag-specific peptides were found. These findings suggest that T*-ag does not represent the membrane-associated form of T-ag, but that mT-ag is encoded within the same reading frame used for nT-ag.

Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen.

The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis.

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.

Mitogenic effects of the proto-oncogene and oncogene forms of c-H-ras DNA in human diploid fibroblasts.

Nuclear microinjection of c-H-ras DNA induced DNA synthesis in reversibly nonproliferating quiescent human cells. The proto-oncogene and oncogene forms were equally effective inducers. In contrast, c-H-ras DNA either alone or in combination with the adenovirus E1A gene did not cause terminally nondividing senescent cells to synthesize DNA.

Cell and molecular biology of simian virus 40: implications for human infections and disease.

Simian virus 40 (SV40), a polyomavirus of rhesus macaque origin, was discovered in 1960 as a contaminant of polio vaccines that were distributed to millions of people from 1955 through early 1963. SV40 is a potent DNA tumor virus that induces tumors in rodents and transforms many types of cells in culture, including those of human origin. This virus has been a favored laboratory model for mechanistic studies of molecular processes in eukaryotic cells and of cellular transformation. The viral replication protein, named large T antigen (T-ag), is also the viral oncoprotein. There is a single serotype of SV40, but multiple strains of virus exist that are distinguishable by nucleotide differences in the regulatory region of the viral genome and in the part of the T-ag gene that encodes the protein's carboxyl terminus. Natural infections in monkeys by SV40 are usually benign but may become pathogenic in immunocompromised animals, and multiple tissues can be infected. SV40 can replicate in certain types of simian and human cells. SV40-neutralizing antibodies have been detected in individuals not exposed to contaminated polio vaccines. SV40 DNA has been identified in some normal human tissues, and there are accumulating reports of detection of SV40 DNA and/or T-ag in a variety of human tumors. This review presents aspects of replication and cell transformation by SV40 and considers their implications for human infections and disease pathogenesis by the virus. Critical assessment of virologic and epidemiologic data suggests a probable causative role for SV40 in certain human cancers, but additional studies are necessary to prove etiology.

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.

Altered intracellular transport of exogenous DNA by murine mammary tumor cell lines.

The uptake of exogenous DNA by a series of murine mammary tumor cell lines was compared with DNA uptake by normal cells and other types of transformed cells. The mammary tumor cell lines all exhibited a decreased efficiency in the transport of DNase-resistant exogenous DNA into the nuclear fraction. None of the DNA facilitators tested were able to surmount this transport defect. Normal mouse mammary gland cells, mouse embryo cells, and normal kidney cells from various species efficiently transported exogenous DNA into the nucleus. Similarly, the transport defect was not exhibited by a series of transformed cell lines, including those of mouse origin and those transformed by C-type oncornaviruses. The only exception was a murine leukemia virus-producing mouse line that displayed decreased nuclear uptake of the DNA. The nature of the exogenous DNA was apparently not a factor, since identical results were obtained with simian virus 40 and mammalian cell DNA's. The inducing agents of the mammary tumor cells also did not appear to be a factor, since cell lines derived from tumors induced spontaneously or by viral, hormonal, or chemical carcinogens all behaved similarly.

Integrated state of subgenomic fragments of hepatitis B virus DNA in hepatocellular carcinoma from mainland China.

Hepatocellular carcinoma (HCC) samples from mainland China were examined for the presence and state of hepatitis B virus (HBV) DNA sequences. HBV DNA was detected by dot-blot hybridization in 13 of 17 cases of HCC from the Shanghai area and in three of six samples from Hangzhou. The HCC cases from Shanghai were then analyzed in more detail. Fifteen of the 17 patients had serologic evidence of past or present infection with HBV (with inadequate information available for the other two), and the 13 HCC samples positive for HBV DNA all came from serologically positive patients. Southern blot analysis showed that the HBV DNA sequences were always integrated in the HCC high-molecular-weight DNA; only one or two viral copies were present per tumor cell, and no common integration site was evident. Hybridization analyses using subgenomic probes of HBV DNA revealed that the tumors seldom retained an entire HBV genome. HBV S-region sequences were always present, X-region sequences were usually represented, and C-region sequences were rarely detectable in virus-positive tumors. A fragment within the HBV DNA X-region, between nucleotides 1441 and 1526, was found to hybridize nonspecifically with cellular DNA; reported sequence data indicated that this fragment would contain approximately 70% guanine + cytosine. Histologic sections were prepared from some of the frozen tissue specimens and stained by an indirect immunoperoxidase technique for hepatitis B surface antigen (HBsAg). Only 1 of 10 HBV DNA-positive samples contained HBsAg in the cytoplasm of tumor cells, although abundant HBsAg was present in adjacent normal cells in all 10 cases. There were no significant differences in histology between HCC that contained HBV DNA sequences and those that were virus negative. These data support the premise that HBV represents a major etiologic factor in the development of HCC in the Shanghai area of China, although the molecular basis of viral involvement remains obscure.

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.

Comparison of the growth properties in vitro and transplantability of continuous mouse mammary tumor cell lines and clonal derivatives.

Five continuous mouse mammary tumor cell lines and 12 clonal derivatives have been established from tumors arising in BALB/c or C3H mice either spontaneously or in response to viral (mammary tumor virus), hormonal (17 beta-estradiol), or chemical [7,12-dimethylbenz(alpha)anthracene] stimuli in vivo. The cell lines were examined for the following in vitro growth parameters:plating efficiency, saturation density, population-doubling times, colony formation on plastic surfaces, and anchorage-independent growth in methylcellulose. The majority of the mammary tumor cell lines were transplantable in syngeneic, immunocompetent mice and gave rise to tumors composed of epithelial cells. There was no growth parameter in vitro which invariably correlated with tumorigenicity in vivo. Most of the mammary tumor cell lines appeared to produce C-type virus. Only one, a C3H derivative, appeared to respond to stimulation by glucocorticoids with the enhanced production of B-type virus. Analysis of the growth properties exhibited in culture by transformed mammary epithelial cells revealed marked differences from those previously reported for transformed fibroblasts.

Correlation of amino acid fucoside labeling patterns with tumorigenicity of mouse mammary epithelial cells.

The amino acid fucosides of tumorigenic and nontumorigenic mouse mammary gland-derived cells were studied. The cells examined included tumorigenic cell lines derived from mammary carcinomas of the following etiologies: induction by hormone; virus; and chemical carcinogen. Also studied were cells derived from normal mammary glands and several clones of cells, which were derived from a mammary carcinoma but were not demonstrably tumorigenic at lower passage levels after cloning, while they were highly tumorigenic at higher passage levels. Cells were cultured in medium supplemented with radiolabeled fucose and extracted, and extracts were analyzed for the amino acid fucosides. Radiolabeled compounds which comigrated with the amino acid fucosides glucosylfucosylthreonine, fucosylthreonine, and fucosylserine were observed. There was a distinctive difference between the tumorigenic and nontumorigenic cells; the ratio of fucosylthreonine plus fucosylserine to glucosylfucosylthreonine was higher in all tumorigenic cells as compared to the ratio observed for the nontumorigenic cells.

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.

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.

Correlation of surface fucopeptide patterns with tumorigenicity of mouse mammary epithelial cells.

The plasma membrane fucopeptides of tumorigenic and nontumorigenic mouse mammary epithelial cells were studied. The types of cells analyzed included (a) cell lines derived from mouse mammary carcinomas of varying etiologies (viral, hormonal, chemical carcinogen), (b) a series of clonal cells lines which were nontumorigenic at lower passage levels and tumorigenic at higher passage levels, (c) normal primary cells derived from the mammary glands of pregnant or lactating animals, and (d) primary cells from tumors produced by s.c. injection of cultured mammary tumor cells into syngeneic animals. A distinctive difference was observed in the size distribution of the trypsin-sensitive surface fucopeptides from tumorigenic and nontumorigenic mammary cells; the tumorigenic cells were relatively enriched in the larger fucopeptides. The size distribution of the trypsin-sensitive surface fucopeptides was not markedly influenced by the physiological state of the cells or by cell population density. It appears that the trypsin-sensitive surface fucopeptide size pattern may be a distinguishing characteristic between tumorigenic and nontumorigenic mouse mammary epithelial cells.

Differential response of cultured mouse mammary cells of varying tumorigenicity to cytochalasin B.

Cultured BALB/c mouse mammary gland epithelial cells of varying oncogenic potential in vivo have been examined for their ability to multinucleate in the presence of cytochalasin B (CB). Highly tumorigenic cell lines derived from mammary tumors with hormonal, viral, or chemical carcinogen etiologies were extensively multinucleated when cultured in CB-supplemented medium. Normal mammary gland cells from either pregnant or lactating animals were predominantly mono- or binucleate under comparable conditions. At low passage levels after cloning, cell lines derived from a chemical carcinogen-induced mammary tumor were weakly oncogenic and remained largely mono-, or binucleated when cultured in CB-supplemented medium. At higher cell passage levels, both the ability to produce tumors in vivo and the degree of multinucleation in CB-supplemented medium increased dramatically with the clonal cell lines. Thus, the response of cultured mouse mammary gland epithelial cells to CB in vitro may be useful as a marker of the oncogenic potential of such cells.