Biomedical subjects
Publications and source records attributed to J S Butel.
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.
Simian virus 40 (SV40) is a small, DNA-containing tumour virus. One of its gene products, the large tumour antigen (T-ag), is essential for both viral replication and cell transformation. SV40 T-ag can be considered a dual oncogene protein; it is a composite transforming protein that provides distinct functions at different subcellular locations. In addition to its roles in virus replication, T-ag exerts numerous effects on host cells. Those cellular effects reflect viral stimulation of host cell entry into S phase. Numerous chemical modifications have been ascribed to T-ag. They might be involved in defining subpopulations of T-ag that are, in turn, responsible for mediating various T-ag biochemical functions. The T-ag polypeptide, 90,000-100,000 in molecular weight, appears to contain multiple, discrete functional domains; several biological activities have been assigned to relatively small defined regions of the molecule. The cellular progenitors of the T-ag biochemical activities are not obvious. A cellular protein, p53, thought to be involved in regulation of cell proliferation, becomes complexed with T-ag in transformed cells and is stabilized. The interaction of T-ag with this cellular substrate may play an important part in SV40 transformation. T-ag and T-ag/p53 complexes are localized in both the nucleus and plasma membrane of transformed cells. T-ag is transported to the nucleus because of a 7-residue nuclear transport signal contained within its primary sequence. Its migration to the membrane is by an unknown pathway. Only a minor fraction of the total cellular T-ag is present at the cell surface. Both amino and carboxy termini of the T-ag polypeptide are exposed on the extracellular face of the cell. Nuclear and membrane T-ag are structurally very similar, although a portion of membrane T-ag is acylated and nuclear T-ag is not. The nuclear and membrane forms of T-ag apparently provide separate and complementary functions necessary for cell transformation. Nuclear T-ag is important in immortalizing primary cells and membrane T-ag may mediate more pronounced morphological changes. A model is presented, postulating how the two forms of T-ag might cooperate to mediate phenotypic transformation.
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.
Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate. Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface. An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo. Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium. Induction medium containing lactogenic hormones was added at various times. The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads. Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins. No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen. Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths. The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis. Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively. While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species. These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation. It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression.
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.
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.
The SV40(cT)-3 mutant is defective in transport of SV40 large tumor antigen (T-ag) to the nucleus. Several properties of T-ag associated with SV40 lytic infection and attributed to its nuclear localization were examined to determine whether biologically significant levels of the mutant T-ag (cT-ag) that were immunologically undetectable were transported to the nucleus in SV40(cT)-3-infected TC-7 cells. SV40(cT)-3 was defective in regulation of T-ag synthesis and initiation of viral DNA synthesis. These defects were presumably due to the lack of nuclear transport of cT-ag, since cT-ag was capable of interacting with the SV40 origin of viral DNA synthesis in a solution binding assay. The level of fatty acid acylation, a modification specific for the cell surface associated T-ag, was not affected by the cT mutation. The cT mutation sufficiently suppressed the nuclear transport of wild-type (WT) T-ag in SV40(cT)-3-infected COS-1 cells to result in the cessation of WT-T-ag-stimulated SV40(cT)-3 viral DNA synthesis. These results are discussed with respect to the recent findings that SV40(cT)-3 is fully competent for the transformation of established cell lines and the induction of cellular DNA synthesis in quiescent cells.
The SV40-encoded transforming protein, large tumor antigen (T-ag), is multifunctional. Chemical modifications of the T-ag polypeptide may be important for its multifunctional capacity. T-ag is additionally modified by glycosylation. T-ag was metabolically labeled in SV40-infected cells with tritiated galactose or glucosamine, but not with mannose or fucose. The identity of glycosylated T-ag was established by immunoprecipitation with a variety of T-ag-specific antisera, including monoclonal antibodies. Incorporation of labeled sugar into T-ag was inhibited in the presence of excess unlabeled sugars, but not in the presence of excess unlabeled amino acids. Labeled monosaccharides could be preferentially removed from T-ag with a mixture of glycosidic enzymes. In addition, galactose was removed from purified T-ag by acid hydrolysis and identified as such by thin-layer chromatography. T-ag oligosaccharides were resistant to treatment with EndoH, and glycosylation was not inhibited by tunicamycin. Together, these data strongly suggest that T-ag is glycosylated. Several characteristics, including lack of mannose labeling, EndoH resistance, and tunicamycin resistance, suggest that T-ag is not an N-linked glycoprotein. Rather, these properties are more consistent with the identification of T-ag as an O-linked glycoprotein.
The effects of dietary fat on 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumorigenesis were examined in BALB/c female mice. In the first experiment, BALB/c mice were treated with a total dose of 1, 3, or 6 mg DMBA to induce mammary tumors. One week after the last dose of DMBA, the mice were placed on a 5% (LF) or 20% (HF) corn oil semi-purified diet. The 20% corn oil diet resulted in a significantly decreased mean tumor latency period in each of the three groups of mice (p less than 0.05). However, the mammary tumor incidences at 11 months after DMBA treatment were the following: 6 mg DMBA (LF-14/32, HF-17/32); 3 mg DMBA (LF-13/50, HF-14/50); 1 mg (LF-2/50, HF-6/50). In the second experiment, prolonged hormonal stimulation by pituitary isografts, but not a HF diet, enhanced the incidence of mammary tumors in BALB/c mice given a threshold dose of DMBA (0.5 mg). In a further attempt to examine the mechanism of the enhancing effects of dietary fat, lipid peroxidation was measured in the microsomal-mitochondrial fractions of the mammary glands by two assays; measurements of conjugated dienes and TBA reactants (malonyldialdehyde). The high fat diet had no effect on the levels of conjugated dienes in mammary cell membrane preparations and decreased the levels of TBA reactants (malonyldialdehyde). The results indicated that a high fat diet did not lead to enhanced levels of lipid peroxidation in preparations of microsomal mitochondrial membranes from mouse mammary glands. The implications of these results are discussed with respect to the role of dietary fat as a promoter of mouse mammary tumorigenesis.
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.
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.
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.
Mouse mammary tumor virus-specific DNA sequences endogenous to the BALB/c mouse are shown to exhibit variable levels of methylation in a tissue-specific manner. In DNA from both lactating mammary gland and spleen, MMTV-specific sequences were hypomethylated at specific HpaII and HhaI sites. These variably methylated sites were found in the terminal repetitive sequences of the endogenous viral genomes. The specific hypomethylation of a HpaII site in Mtv-9 is associated with expression of a 1.6 kb transcript in the lactating mammary gland.
PARA(nT) is a defective SV40-adenovirus 7 hybrid virus which contains the entire early region of the SV40 genome and codes for the synthesis of SV40 large tumor antigen (T-ag). A transport-defective variant of this hybrid, PARA(cT), encodes T-ag that is not transported to the nucleus, but accumulates in the cytoplasm. The structures of T-ags extracted from wild-type (WT) SV40-, PARA(nT)-, and PARA(cT)-infected cells were compared by peptide mapping. All three types of T-ag underwent considerable degradation when extracted using Tris-buffered Nonidet P-40 at pH 8.0. The addition of 200 microM leupeptin to the extraction buffer significantly inhibited this degradation. Comparison of methionine-containing tryptic peptides revealed no differences among the T-ags, suggesting that their primary structures are similar or identical. Phosphopeptide mapping revealed no differences between SV40- and PARA(nT)-encoded T-ags. In contrast, PARA(cT)-encoded T-ag lacked a prominent phosphopeptide that was present in both of the others. The possible relevance of this difference in phosphorylation to the transport defect is discussed.
Primary and established murine mammary epithelial cells and wild-type SV40 were employed to study the phenomenon of epithelial cell transformation. Thirteen independent transformed cell lines were derived. All contained SV40 intranuclear T antigen. Eight transformed mammary cell lines were examined ultrastructurally and all were found to exhibit pronounced epithelial cell characteristics, including desmosomes and tight junctions. Growth studies revealed that while normal mammary cells were unable to grow in low serum (2% FBS), established Cl S1 mammary cells and SV40-transformed mammary epithelial cells replicated well. Cell densities achieved by the transformants were only slightly elevated in high serum (13% FBS) over normal cell values. All the transformants formed colonies on plastic and exhibited anchorage-independent growth in methylcellulose. Five of the transformed lines were tumorigenic in syngeneic animals, in marked contrast to the lack of transplantability usually observed with SV40-transformed mouse fibroblasts. Anchorage-independent growth was not a predictor of tumorigenic potential in this system. The transformants exhibited a spectrum of responsiveness to exogenous growth factors. This study establishes that the SV40-murine mammary cell system is a valid model for analyses of the process and consequences of epithelial cell transformation, in general, and mammary cell transformation in particular.
An SV40-adenovirus 7 hybrid virus, PARA(cT), has been described that is defective for the nuclear transport of SV40 large tumor antigen. An SV40(cT) mutant was constructed using SV40 early and late region DNA fragments derived from PARA(cT) and wild-type SV40 respectively. The SV40(cT)-3 construct is defective for viral replication, but can be propagated in COS-1 cells. T antigen induced by SV40(cT)-3 is localized in the cytoplasm of infected cells. The cT mutation also inhibits the transport of wild-type T antigen; COS-1 cells lose their constitutive expression of nuclear T antigen after infection with SV40(cT)-3. Sequence analysis revealed that the cT mutation results in the replacement of a positively charged lysine in wild-type T antigen with a neutral asparagine at amino acid number 128, demonstrating that the alteration of a single amino acid is sufficient to abolish nuclear transport. Implications of the cT mutation on possible mechanisms for the transport of proteins to the nucleus are discussed.
An epithelial cell line, designated COMMA-1D, was derived from mammary tissue of BALB/c mice in the middle of pregnancy. This line, in continuous cell culture for 12 months, exhibits several characteristics distinctive of normal mammary epithelial cells, including induction of casein synthesis in vitro and normal duct morphogenesis in the cleared mammary fat pads of syngeneic mice. The cells also form domes in high density culture and are positive for keratin intermediate filaments by indirect immunofluorescence. COMMA-1D cells have a near diploid number of chromosomes and do not grow in suspension culture or produce tumors in syngeneic hosts. This cell line should prove useful for studies examining the regulation of normal cellular differentiation of mammary cells as well as transformation of epithelial cells to the preneoplastic and neoplastic phenotypes.
A molecular complex of simian virus 40 large tumor antigen (T-Ag) and p53 cellular protein is present on the surface of simian virus 40-transformed mouse cells. The stability of the association of the two proteins with the cell surface was characterized. Cells were either surface iodinated by the lactoperoxidase technique or metabolically labeled with [35S]methionine, and surface antigens were detected by differential immunoprecipitation with specific antibodies immediately after labeling or after incubation at 37 degrees C. A rapid, concomitant disappearance of T-Ag and p53 from the cell surface was observed. The half-life of iodinated surface T-Ag was less than 30 min, whereas that of [35S]methionine-labeled surface T-Ag was 1 to 2 h. Although T-Ag and p53 were rapidly lost, both were also rapidly replaced on the cell surface, since newly exposed molecules could be detected when cells were reiodinated after a 2-h chase period. Control experiments established that the loss of the surface molecules was not induced by the iodination reaction. The appearance of surface T-Ag was prevented when cellular protein synthesis was inhibited with cycloheximide. The disappearance and replacement of T-Ag and p53 appeared to be energy-independent processes, as neither was inhibited by sodium azide or 2,4-dinitrophenol. Incubation of iodinated cells at 4 degrees C did block the loss of T-Ag and p53. These observations suggest that T-Ag and p53 are coordinately turned over in the plasma membrane. The nature of the association of the T-Ag-p53 complex with the cell surface can best be described as highly dynamic.