Tumors In Newborn Hamsters Research Articles

Enteric adenoviruses.

The 41 serotypes of human adenoviruses are classified into six subgenera (A-F) with different tropisms. Enteric infections are caused in children by serotypes Ad40 and Ad41 of subgenus F. Serotypes Ad40 and Ad41 transform embryonic cells but cannot induce tumours in newborn hamsters. They differ from all other (established) human adenoviruses by being unable to replicate in conventional cell cultures. Ad40 and Ad41 grow in 293 cells (human embryonic kidney cells immortalized by transfection with the E1A, E1B regions of Ad5). In spite of the difficulty of isolating Ad40 and Ad41 they can be directly identified in stools by enzyme-linked immunosorbent assay (ELISA) and solid-phase immuno-electron microscopy. The amount of viral DNA in stool preparations is sufficient for identification by DNA restriction or dot-blot analysis. Adenoviruses have been associated with 7-17% of cases of diarrhoea in children. Ad40 and Ad41 cause diarrhoea throughout the year. Clinical features are watery stools, vomiting and moderately elevated temperature; respiratory symptoms are infrequent. The diarrhoea is protracted (mean 8.6 and 12.2 days for Ad40 and Ad41 respectively). Children with rotavirus diarrhoea vomited more frequently and had a higher temperature and diarrhoea of shorter duration. The impact of enteric adenoviruses in the aetiology of diarrhoea world-wide is not known but is accessible to investigation.

Transformation by inorganic arsenic compounds of normal Syrian hamster embryo cells into a neoplastic state in which they become anchorage-independent and cause tumors in newborn hamsters.

Arsenic is a known human carcinogen, but little evidence exists for its carcinogenicity in animals. In order to investigate the ability of inorganic arsenics to transform normal cells into a neoplastic state, mass cultures of normal, diploid Syrian hamster embryo (SHE) cells exposed to various concentrations of sodium arsenite or sodium arsenate for 48 hr were continually passaged and tested for neoplastic transformation, as determined by anchorage-independent growth in semisolid agar and tumorigenicity in newborn hamsters. Twenty-one of 22 (96%) untreated, control cultures senesced by 20 passages. While 1 culture escaped senescence, it did not acquire the ability to either grow in semisolid agar or form tumors in animals. Ten of 14 (71%) cultures exposed to sodium arsenite or sodium arsenate escaped senescence. Nine of the 10 (90%) arsenic-treated immortal cultures acquired the anchorage-independent phenotype. Five of 5 anchorage-independent cultures examined were tumorigenic. Two of 3 morphologically transformed colonies induced by sodium arsenate also acquired the ability to grow in semisolid agar when isolated. Amplification of the c-myc or c-Ha-ras oncogene was detected in 3 of 5 and 4 of 5 tumorigenic cell lines, respectively. Both c-myc and c-Ha-ras were amplified even in a preneoplastic, anchorage-dependent cell line, but neither was amplified in 6 of 9 anchorage-independent cell lines. Overexpression of c-myc and c-Ha-ras mRNA was observed in most of the neoplastically transformed cell lines but not in the preneoplastic cell line. Experiments using the methylation-sensitive restriction endonuclease isoschizomers HpaII and MspI revealed hypomethylation of c-myc and c-Ha-ras in the 5'-CCGG sequence of arsenic-exposed cell lines but not in the parental SHE cells or a spontaneously transformed cell line. Thus, inorganic arsenics induce neoplastic transformation of normal, diploid mammalian cells. Overexpression of oncogenes by DNA hypomethylation may participate in the arsenic-induced neoplastic transformation of mammalian cells.

Tumorigenesis in transgenic mice by a nuclear transport-defective SV40 large T-antigen gene

The SV40(cT) mutant encodes a large tumor antigen (cT-ag) that is defective for transport from the cell cytoplasm into the nucleus. This mutant is able to transform established cell lines at near wild-type virus efficiencies, but has a markedly decreased ability to transform primary cells and to induce tumors in newborn hamsters ( R. E. Lanford, C. Wong, and J. S. Butel, 1985, Mol. Cell. Biol. 5, 1043–1050 ). To explore the biology of transport-defective T-ag in vivo, transgenic mice carrying the cT-ag gene were produced. Five of eight founder animals died early in life of choroid plexus tumors (mean age ± SE, 52 ± 11.0 days); renal and thymic lesions were also observed. Mice of an SV40(cT) transgenic line regularly succumb to brain tumors (mean age, 81 ± 1.2 days). SV40 T-ag is expressed in the tumor cells and is retained in the cytoplasm. The observation that SV40(cT) is equivalent to wild-type virus at tumor induction in transgenic mice emphasizes the probable importance of extranuclear forms of SV40 T-ag in brain tumor formation. This study also indicates that in vitro cell transformation assays may not always be accurate reflections of the oncogenic potential of a transforming gene in vivo, because of the different cell types involved.

Definition of a region required for transformation in E1a of adenovirus 12

In order to define functionally important regions of the E1a gene of adenovirus 12 (Ad12), a number of Ad12 mutants were studied. These mutants share an identical 69-bp deletion in the first exon of E1a as well as duplications of a long terminal repeat sequence at the end of the Ad12 genome. The mutants are fully competent for replication and growth in their normal hosts and have a host range extended to include the Vero cell line of African green monkey origin. The partially deleted E1a can stimulate the expression of all early adenoviral genes as well as the cellular heat shock gene, the transcription of which is stimulated by Ad5 E1a. However, plasmids containing the deleted E1a plus wild type E1b were unable to effect a transformation of either primary rat embryo fibroblasts or on an established cell line following transfection. Further, the mutant viruses were defective in generating tumors in newborn hamsters. We conclude that the deleted sequence of E1a described here is critical for transformation by E1a but is not necessary for multiplication of the virus or the transcriptional activating function of the gene.

Cellular targets for SV40 Large T-antigen

SV40 virus infection is able to induce tumours in newborn hamsters and to transform a wide range of eukaryotic cells in in vitro culture. This is achieved by integration of the viral DNA into the host cell DNA and expression of the virus-encoded Large T-antigen. The expression of Large T, a 708 amino acid phosphoprotein, is required both to induce and maintain the transformed state. The Large T protein initiates viral DNA synthesis and regulates viral transcription, apparently by binding in a specific manner to viral DNA sequences at and near the viral origin of replication. SV40 Large T also affects cellular DNA synthesis and transcription and this may account for its oncogenic activity. A novel immunochemical procedure has permitted the isolation of cellular DNA sequences occupied by SV40 Large T in the chromatin of SV40 transformed cells. Some of the cellular sequences contain high affinity binding sites for SV40 Large T, and hybridize to messenger RNAs expressed in SV40 transformed but not in normal cells. A second type of cellular target for Large T is the cell coded p53 protein that it binds to and stabilizes. A range of monoclonal antibodies to p53 has been isolated and characterized. They demonstrate that p53 is in the cytoplasm of normal cells but is located in the nucleus of transformed cells. One of the antibodies recognizes an epitope on p53 that is stabilized or induced by binding to Large T. Further studies on the T-p53 protein complex have been facilitated by constructing bacterial plasmids that direct the synthesis of substantial quantities of Large T-beta-galactosidase and p53-beta-galactosidase fusion proteins in bacteria. The results are discussed in the context of our current knowledge of oncogene action.

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.

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.

Transformation of rodent cells by DNA extracted from transformation-defective adenovirus mutants.

Complementation group II host range mutants of adenovirus type 5 which map in early region 1B (E1B, 4.5 to 11.0 map units) have been shown to be defective for the synthesis of the E1B 58,000-dalton (58K) antigen in infections of HeLa or KB cells (Lassam et al., Cell 18:781-791, 1979) and unable to transform cultured rodent cells (Graham et al., Virology 86:10-21, 1978). In this report we show that DNA extracted from group II mutants hr6 and hr50 can transform rat cells with the same efficiency as wild-type DNA. Furthermore, group II mutant-transformed hamster cells were shown to contain no detectable E1B 58K tumor antigen but were capable of inducing tumors in newborn hamsters. Hamster cell lines 1019-3 and 1019-C3, transformed by hr50 DNA, produced no detectable quantities of either the E1B 58K or 19K antigen but nonetheless exhibited a fully transformed oncogenic phenotype. Our results show that the E1B 58K antigen is not absolutely required for oncogenic transformation and suggest that even cells lacking the 19K protein can be oncogenic.

Information content of the adenovirus-2 genome.

Adenovirus 2 (Ad2) is a double-stranded DNA virus that is associated with infections of the human respiratory tract. It has been classified among the nononcogenic adenoviruses, since it fails to induce tumors in newborn hamsters. However, it will cause phenotypic transformation of rodent cells in culture, and such transformed cells can then lead to tumor formation when implanted into appropriate animals (Graham et al. 1975). Consequently, Ad2 and its close relative Ad5 have been used extensively as model systems with which to study cellular transformation and eukaryotic gene expression. It was in this system that RNA splicing was discovered (Berget et al. 1977; Chow et al. 1977a), and there has been widespread interest in the transcription, translation, and replication of adenoviruses (Tooze 1981).

Oncogenicity of heat-inactivated simian adenovirus SA7.

Simian adenovirus SA7 heated for 30 minutes at 70 degrees C retained part of its ability to induce tumors in newborn hamsters. Tumors were also induced by DNA extracted from the heated virus. However, neither residual replicating, i.e. cytopathogenic, nor transforming and T-antigen-inducing activities could be detected in vitro in CV-1 and hamster embryo cells inoculated with the heated virus. The results are discussed in terms of a possible release of viral nucleic acid at the high temperature. They also show that current tests for effective virus inactivation based exclusively on the recognition of replicating virus should be reevaluated.

A cloned polyoma DNA fragment representing the 5' half of the early gene region is oncogenic.

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.

Extensive homology of nuclear ribonucleic acid and polysomal poly(adenylic acid) messenger ribonucleic acid between normal and neoplastically transformed cells

A cell line, designated BP6T, derived from Syrian hamster embryo (SHE) cells following treatment with benzo[a]pyrene is capable of producing tumors in newborn hamsters following the injection of as few as 1-10 cells. Polysomal poly(A) mRNA and total nuclear RNA obtained from this highly tumorigenic cell line were compared to RNAs obtained from the nonneoplastic parental embryo cells by a variety of techniques. RNA excess hybridizations to normal cell radiolabeled single-copy DNA or to a single-copy DNA tracer enriched for sequences transcribed in neoplastically transformed cells were unable to detect any significant differences in RNA sequence complexity between normal SHE cells and neoplastic BP6T cells. This finding of extensive homology of polysomal poly(A) mRNA and total nuclear RNA between normal and neoplastic cells, together with our previous finding of extensive homology of the major 35S-labeled nuclear or cytoplasmic polypeptides observable on two-dimensional gels [Leavitt, J. C., & Moyzis, R. K. (1978) J. Biol. Chem. 253, 2497-2500], demonstrates that the phenotypic changes associated with neoplastic transformation by chemical carcinogens are accompanied by relatively few changes in the qualitative pattern of gene expression in cells cultured in vitro.

Structure and function of adenovirus type 12 defective virions

Purified human adenovirus type 12 preparations contain defective virions with a lighter density. These defective virions were isolated, and their biological functions and DNA were characterized. They can induce early and late antigens in infected cells and tumors in newborn hamsters with similar efficiency as complete virions. The majority of the DNA molecules from light virions contain deletions mapping near 16% from the left-hand end of the genome. Mechanisms for the generation of these molecules are discussed.

8-Methoxypsoralen Induced Alterations of Mammalian Cells

In vitro and in vivo assays demonstrate that 8-methoxypsoralen, in the presence of ultraviolet light (8MOP-UV), induces phenotypic alterations in certain mammalian cells. Transformation for certain growth characteristics occurred in high percentages of 8MOP-UV treated primary mouse, rat, and hamster embryo cultures, and in continuous cell lines (BHK 21 /Cl 13 , NIH/3T3, and Balb/c 3T3). Morphological alterations were observed at both macroscopic (focus formation) and microscopic (random growth patterns, multilayering, etc.,) levels of examination. Treated cells which demonstrated altered phenotypes were tested for growth in soft agarose. Those cultures with altered morphology and whose cells grew in soft agarose. Those cultures with altered morphology and whose cells grew in soft agarose, were, in addition, tested for murine leukemia virus (MuLV) production by the XC cytopathogenicity test. 8MOP-UV transformed mouse, rat, and hamster (except BHK 21 /Clone 13 ) cells produced positive XC results. Florescent antibody tests of those cells which produced giant cell cytopathogenicity were positive. Cytoplasmic fluorescence was observed when these cells were tested with antisera to oncornavirus group specific (gs-3) antigen. Control nontreated cells were negative in all experiments. Hamster embryo fibroblasts transformed with 8MOP-UV, acquired apparent unlimited in vitro growth characteristics (> 150 population density levels); they exhibited mixoploidy; small numbers of these cells caused spindel cell sarcomas in hamster cheek pouches, and produced tumors in new born hamsters; and their generation times (x = 22.5 hr) were characteristic of transformed cells. Expression of altered growth characteristics by BHK 21 /CI 13 cells may be related to selection for cytotoxic resistant cells rather than being caused by 8MOP-UV induced transformation.

Immunological identification of two adenovirus 2-induced early proteins possibly involved in cell transformation.

HUMAN adenovirus 2(Ad2), a DNA tumour virus that replicates in the nucleus of permissive human cells1,2, transforms non-permissive or semipermissive cultured rodent cells, but does not induce tumours in newborn hamsters (oncogenic group C). The constant functioning of protein product(s) from Ad2 ‘transforming gene(s)’ is probably responsible for the transformed phenotype, although this has not been proved. Identification and functional characterisation of ‘transforming protein(s)’ are important for the understanding of cell transformation and growth control. We describe here immunoprecipitation studies made with antisera against presumptive Ad2 ‘transforming protein(s)’, that identify two candidate transforming proteins of 53,000 (53k) and 15kdaltons.

Retention of tumour-inducing capacity by adenovirus DNA after cleavage by restriction endonucleases.

WE have previously reported that fragments of Simian adenovirus SA7 DNA produced by physical breakage are oncogenic1. These fragments had an average molecular weight corresponding to half-molecules (11.4× 106). After separation of the heavy and light molecular halves by density gradient centrifugation, we demonstrated that both half-molecule preparations retain the ability to initiate tumours in newborn hamsters. We suggested that this may be caused by the presence of one or more “oncogenic regions” in both molecular halves, or by random breaks near the centre of the DNA which could result in a single small oncogenic region, residing near the centre and occurring with equal probability in either the heavy or light half-molecule preparation. To clarify these results we have studied the effect of several restriction endonucleases on the oncogenic activity of SA7 DNA. These enzymes produce defined fragments of the SA7 genome rather than the heterogeneous populations produced by physical shear.

Nonproductive infection and induction of cellular deoxyribonucleic acid synthesis by bovine adenovirus type 3 in a contact-inhibited mouse cell line.

Bovine adenovirus type 3 (BAV-3), which has been reported to produce tumors in newborn hamsters, induced cellular deoxyribonucleic acid (DNA) synthesis in a contact-inhibited mouse kidney cell line (C3H2K). In this system, the virus did not multiply, whereas virus-specific tumor antigen (T antigen) was detected in nearly all cells. Replication of viral DNA could not be detected by DNA-DNA hybridization on membrane filters. The cellular DNA synthesis induced by BAV-3 did occur in the absence of added serum. Extent of induction of cellular DNA synthesis was closely correlated with the multiplicity of infection. Cells activated to synthesize DNA in the serum-free medium by the virus infection progressed to cell division without noticeable cell killing.

Tumor induction in hamster by para(3cT)-adenovirus 7 mutant. Morphology and antigenic profiles of neoplastic cells.

AbstractThe variant 3ct of PARA‐adenovirus 7, which was able to synthesize SV40‐T antigen in cytoplasm, induced tumors in newborn hamsters. The neoplasms were either undifferentiated adenovirus tumors or pleomorphic sarcomas. Several cell lines were obtained from the tumors: some had the cytomorphology typical of adenovirus or SV40‐transformed cells, while others showed ribbon‐cell formation.Only the cells derived from the “early” tumors displayed adenovirus T antigen (s); in these cells the SV40 T antigen had a diffuse, granular pattern and was mainly localized on the perinuclear region. In the other cell lines no adenovirus T antigen was shown by the indirect immunofluorescence test; the SV40 T antigen was present in the nuclei, either with the regular pattern of SV40‐transformed cells or in the form of faint and sparse fluorescent spots.

Tumour induction by simian adenovirus SA7 DNA fragments.

DNA extracted from simian adenovirus SA7 can induce tumours in hamsters and is infectious in tissue culture1,2. We now have evidence that fragments of the SA7 genome can initiate tumours in newborn hamsters.

Chromosome, transplantation and tumour antigen studies of three virus-induced tumour cell lines.

The transplantability to adult hamsters, the CF titers of tumour antigen and the chromosome content was determined at a single passage level for two established adenovirus 12-induced hamster tumour cell lines and an SV40-virus tumour cell line. The cell lines had been passaged more than 200 timesin vitro, and immunofluorescent tests showed that all the cells contained specific virusinduced tumour antigen. The SV40 cell line and one of the adenovirus 12 tumour cell lines (TAd III cells) were transplantable to adult hamsters. The second adenovirus 12 tumour cell line (H212 cells) did not produce tumours in adult hamsters, but produced tumours in new-born hamsters. TAd III cells contain relatively a low CF titre of tumour antigen while H212 cells contain high CF titres of antigen. Cytogenetic analysis showed that all tumour cell lines were markedly different from normal hamster cells. The incidence of marker chromosomes was greatest for SV40 tumour cells, and smallest for H212 cells. The results of cytogenetic and antigen studies are discussed in relation to the transplantability of the tumour cell lines.