Antigenic relationship of SV40 early proteins to purified large T polypeptide.
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Antibodies, Viral↗
Biomedical subjects
J S Butel
Publications and source records attributed to J S Butel.
Mouse mammary tumor virus genome expression in chemical carcinogen-induced mammary tumors in low- and high-tumor-incidence mouse strains.
Involvement of mouse mammary tumor virus (MMTV) in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis was investigated in low- (BALB/c) and high- (BALB/cfC3H) mammary-tumor-incidence mouse strains. Both strains contain endogenous MMTV integrated into the cellular genome. Additionally, BALB/cfC3H mice are infected with exogenous MMTV-S which is responsible for a higher incidence of mammary tumors in breeding females. Administration of DMBA to virgin mice of both strains resulted in a moderate frequency of mammary tumors within 40 wk after treatment. No differences were found in DMBA-induced tumor incidences at 18 wk (6% and 7%) or at 38 wk (29% and 36%) after treatment of BALB/c and BALB/cfC3H mice, respectively. Expression of MMTV in these tumors was examined by assaying for the presence of MMTV RNA by hybridization using MMTV-specific cDNA and by immunohistochemical staining utilizing antibodies against MMTV 52,000-dalton glycoprotein, gp52, and 28,000-dalton internal protein, p28. Of 16 BALB/c tumors assayed, 11 did not contain detectable levels of MMTV RNA and the remaining 5 tumors contained only low levels (0.0005-0.0010%) of viral RNA. Importantly, MMTV RNA was not detected in 5 of 27 BALB/cfC3H tumors. The other BALB/cfC3H tumors contained quantities of MMTV RNA ranging from 0.0006 to 0.4170%. Most BALB/cfC3H tumors with detectable levels of MMTV RNA also synthesized viral proteins gp52 and p28. Thus, expression of the complete MMTV genome is not requisite for maintenance of the tumor phenotype in DMBA-induced mammary tumors in either BALB/c or BALB/cfC3H virgin mice under 1 year of age.
9,10-Dimethyl-1,2-benzanthracene↗
Subcellular Localization of simian virus 40 large tumor antigen.
The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [(35)S]-methionine- or (32)P(i)-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of approximately 96,000 was detected in both subcellular fractions. Minor components of approximately 68,000 and approximately 56,000 with anti-T reactivity which labeled with [(35)S]methionine were also detected in both fractions from H-50 cells, as were components of approximately 140,000 and approximately 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in (32)P(i)-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [(3)H]thymidine labeling, NADH-diaphorase activity, and Na(+)-K(+)-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.
Antigens, Neoplasm↗
Differential expression of poly(A)-adjacent sequences of mammary tumor virus RNA in murine mammary cells.
Two DNA probes representative of either the entire mouse mammary tumor virus (MMTV) genome or the poly(A)-adjacent sequences at the 3' end of MMTV RNA were synthesized with calf thymus DNA or oligo(dT) primers, respectively. These probes were used to study the expression of endogenous MMTV sequences in several BALB/c mammary tumor cell lines, in normal lactating BALB/c tissue, and in a cloned C3H tumor cell line. Both probes were characterized with respect to their rates of hybridization with template RNA, their size as determined by alkaline sucrose gradient centrifugation, and the thermal stability of the cDNA.MMTV RNA hybrids. In addition, the ability of the calf thymus oligodeoxy-nucleotide- or oligo(dT)-primed probes to protect (125)I-labeled MMTV RNA or (125)I-labeled poly(A)-adjacent MMTV RNA sequences from S1 nuclease digestion was determined. Hybridization analysis with these two probes indicated that (i) there were approximately 20-fold more oligo(dT)-primed sequences in BALB/c lactating tissue than there were sequences representing the entire genome; (ii) in BALB/c tumor cells, the oligo(dT):random oligonucleotide-primed cDNA sequence ratio was reduced to 4:1; and (iii) in virus-producer C3H tumor cells, there was only a 2-fold excess of oligo(dT)-primed sequences over that observed with a representative cDNA. These results are consistent with the presence of subgenomic viral mRNA species, integration of partial proviral copies, or altered mRNA processing.
Animals↗
Role of the simian virus 40 gene A product in regulation of DNA synthesis in transformed cells.
Cells transformed by tsA mutants of simian virus 40 (SV40) are temperature sensitive for the maintenance of the transformed phenotype. The kinetics of induction of DNA synthesis were determined for hamster cell transformants shifted to the permissive temperature after a 48-h serum arrest at the nonpermissive temperature. DNAsynthesis was initiated in the tsA transformants by 8 h after shiftdown was maximal by 12 h. The presence or absence of fetal bovine serum at the time of temperature shift had no effect on the kinetics of initiation of DNA synthesis. Analysis of TTP in tsA transformants revealed similar levels of incorporation of [3H]thymidine into TTP at both permissive and nonpermissive temperatures. Autoradiography revealed that by 12 h after a shift to the permissive temperature, approximately 50% of the cells exhibited labeled nuclei after a 60-min pulse with [3H]thymidine, indicating that a majority of the cells were actively synthesizing DNA. By 8 to 12 h after a shiftup of confluent tsA transformants to the nonpermissive temperature, the number of labeled nuclei was reduced to approximately 16%, regardless of serum concentration. These data indicate that the SV40 gene A product, either directly or indirectly, regulates cellular DNA synthesis in transformed cells.
Cell Line↗
Detection of mouse mammary tumor virus RNA in BALB/c tumor cell lines of nonviral etiologies.
A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a T(m) of 88.5 degrees C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (<three molecules per cell) by RNA excess hybridization. Two of the three "virus-negative" cell lines were derived from tumors induced by the chemical carcinogen 7,12-dimethylbenz(alpha)anthracene, whereas the third tumor occurred spontaneously. Two lines from tumors induced by either viral (mammary tumor virus) or hormonal (17-beta-estradiol) stimulus contained between three and nine molecules of MMTV RNA per cell by both RNA excess and cDNA excess hybridization. Clonal derivatives of these tumor lines had levels of viral RNA comparable to those of their parental lines. Therefore, it appears that the presence of detectable MMTV RNA sequences is not a necessary requirement for the maintenance of all murine mammary gland neoplasias.
Cell Line↗
Genetic evidence for a temperature-sensitive lesion in the adenovirus 7 region of the PARA genome.
It has previously been shown that the helper adenovirus (Ad) present in the defective Ad 7-SV40 hybrid population is temperature-sensitive (ts) for replication in human cells. This study has shown that the replication of the defective hybrid virus (PARA) in green monkey kidney cells is also restricted at 40.5 percent. Complementation tests between the parental or transcapsidant PARA populations and SV40 or various Ad serotypes revealed that the ts lesion is located in the Ad region of the hybrid genome. Wild-type (wt) Ad type 7 and Ad type 21 were able to complement the replication of PARA while Ad type 31 was unable to do so. Complementation was more efficient after the PARA genome was transcapsidated to the wt isolates of helper Ad than during multiple infections with the parental hybrid population. The SV40 function which complements the replication of human adenoviruses in simian cells is not expressed by PARA at the nonpermissive temperature. However, the ts lesion does not affect the expression of SV40 functions coded for by PARA under other conditions, i.e., complementation of the replication of human adenoviruses in transformed monkey cells, and synthesis of T antigen in lytically infected or transformed cells. Although the exact nature of the mutated Ad 7 gene product is unknown, heat inactivation data suggest that it may be a structural protein.
Adenoviridae↗
Temperature-sensitive mutants of simian virus 40. I. Isolation aand preliminary characterization of B/C gene mutants.
Sixteen temperature-sensitive mutants of simian virus 40 were isolated after treatment of wild-type virus with UV light, nitrous acid, or nitrosoguanidine. All of the mutants were assigned to the B complementation group on the basis of quantitative complementation analyses. Characterization of several representative mutants revealed that under restrictive conditions such mutants produced immunologically identifiable tumor and viral capsid antigens, induced the synthesis of cellular DNA, and replicated infectious viral DNA. In addition, mutant particles produced at 33 degrees were more heat-labile than wild-type virus. This collection of mutants will be useful in an analysis of SV40 particle morphogenesis.
Animals↗
Transformation by viruses: simian virus 40 as a model system.
Simian virus 40 (SV40), a DNA-containing tumor virus in the papovavirus group, represents an ideal model system for the analysis of the mechanism of viral-induced tumorigenesis because of the small size of its genome and its broad range of oncogenic potential. Viral genes persist and are expressed in SV40-transformed cells. Temperature-sensitive (ts) mutants of the virus have proved to be valuable tools for the identification and analysis of viral gene expression in transformed cells. Through the use of such mutants, it has been determined that a specific gene product (A-protein) is required to initiate cellular transformation. The role of virus genes in the maintenance of the transformed state was determined by transformation of the cells of mouse, hamster, and human origin by ts virus containing A-gene mutations. These cells were then examined under permissive and nonpermissive conditions for the presence of a variety of intracellular and surface alterations commonly associated with neoplastic transformation. From the results of such experiments, it has been concluded that an SV40-specific function is also necessary for the maintenance of at least some of the phenotypic properties of the transformed state. Indirect evidence, derived from a comparison of the biological and biochemical properties of the SV40-induced tumor (T) antigen and the gene A-protein, supports the idea that T-antigen is a product of the A-gene. One model devised to explain the mechanism by which the gene A-protein might function as an effector of transformation is presented.
Animals↗
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.
Animals↗
The role of SV40 gene A in the alteration of microfilaments in transformed cells.
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Animals↗
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.
Animals↗
Characterization of a temperature-sensitive mutant of human adenovirus type 7.
The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.
Adenoviruses, Human↗
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.
Animals↗
The solubility characteristics of SV 40 tumor antigen.
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Antigens, Neoplasm↗
Characterization of simian cells tranformed by temperature-sensitive mutants of simian virus 40.
Seven lines derived from primary African green monkey kidney cells, which had survived lytic infection by wild-type simian virus 40 (SV40) or temperature-sensitive mutants belonging to the A and B complementation groups, were established. These cultures synthesize SV40 tumor (T) antigen constitutively and have been passaged more than 60 times in vitro. The cells released small amounts of virus even at high passage levels but eventually became negative for the spontaneous release of virus. Virus rescued from such "nonproducer" cells by the transfection technique exhibited the growth properties of the original inoculum virus. Four of the cell lines were tested for the presence of altered growth patterns commonly associated with SV40-induced transformation. Although each of the cell lines was greater than 99% positive for T antigen, none of the cultures could be distinguished from primary or stable lines of normal simian cells on the basis of morphology, saturation density in high or low serum concentrations, colony formation on plastic or in soft agar, hexose transport, or concanavalin A agglutinability. However, the cells could be distinguished from the parental green monkey kidney cells by a prolonged life span, the presence of T antigen, a resistance to the replication of superinfecting SV40 virus or SV40 viral DNA, and, with three of the four lines, an ability to complement the growth of human adenovirus type 7. These properties were expressed independent of the temperature of incubation. These results indicate that the presence of an immunologically reactive SV40 T antigen is not sufficient to ensure induction of phenotypic transformation and suggest that a specific interaction between viral and cellular genes and/or gene products may be a necessary requirement.
Adenoviridae↗
Optimal conditions for uptake of exogenous DNA by Chinese hamster lung cells deficient in hypoxanthine-guanine phosphoribosyltransferase.
Conditions were characterized for maximizing the uptake of exogenous mammalian cell DNA by hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster lung cells. Recipient cell cultures in an exponential growth phase were found to be more competent in taking up DNA than stationary cultures. Polyornithine enhanced the uptake of exogenous DNA more reproducibly and to a greater extent than did any of the other facilitators tested (DEAE-dextran, CaCl2, latex spheres, spermine, polylysine and polyarginine). Maximal DNA incorporation occurred when polyornithine and DNA were mixed together prior to inoculation. About 25-30% of the DNA inoculum became deoxyribonuclease-resistant in a typical experiment utilizing polyornithine as the facilitator. Both homologous and heterologous exogenous DNAs rapidly became associated with recipient cell nuclei: approximately 95% of the deoxyribonuclease-resistant donor DNA was nuclear-associated 15 min after inoculation.
Animals↗
Demonstration of infectious DNA in transformed cells. II. Characterization of uptake of SV40-transformed mouse cell DNA by simian cells.
The initial steps in the DNA-transfer, or transfection, method of virus rescue were characterized using primary green monkey (GMK) cells exposed to SV40-transformed mouse (SV-3T3) cell DNA in the presence of 1 mg/ml DEAE-dextran. When large amounts (10-50mug) of high molecular weight DNA (greater than 10(7) daltons) were inoculated onto 10(6) GMK cells, usually less than 1 mug became cell-associated. DNA fragmented to a size of 1-3 X 10(6) daltons was bound more efficiently by the recipient cells, but generally only 5-10 per cent of the inoculum (representing 1-4 mug) was taken up. Approximately 50 per cent of the cell-associated DNA had penetrated to a DNase-resistant state by the end of the 30-minute incubation period. The effect of the size of thr transformed cell DNA molecule on the recovery of SV40 in transfection experiments was investigated. The trend appeared to be that rescue was more efficient with the larger molecular weight samples.
Animals↗