Temperature-sensitive Rous Sarcoma Virus Research Articles

Specific increase in thymidine transport at a permissive temperature in the rat kidney cells infected with src ts-rous sarcoma virus

Thymidine transport was found to be increased two-fold at a permissive temperature in the cells of a normal rat kidney (NRK) line which was infected with a temperature-sensitive Rous sarcoma virus. This increase in thymidine transport was independent of cell density and coincided with the changes in cellular morphology that result from this temperature shift. A double reciprocal plot of the data demonstrated two saturable components (Km values of 60 microM and 250 microM at 39 degrees, non-permissive temperature) of the uptake of thymidine, and the drop of temperature (at 33 degrees, the permissive temperature for transformation) decreased Km to one half, but did not change Vmax. These results indicate that a qualitative alteration of thymidine transport took place in the presence of the active src gene product.

Calcium compartments and fluxes are affected by the src gene product of rat-1 cells transformed by temperature-sensitive rous sarcoma virus

Total cellular calcium content (determined by atomic absorption spectrometry) of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus decreases with cell density, but is found not significantly different at permissive and at non-permissive temperature. Kinetic analysis of 45Ca efflux from preloaded cells exhibits three separable pools of exchangeable calcium. The ratio of pool size of the fast-exchanging Ca-compartment (bound to cell surface) to pool size of the intermediate Ca-compartment (cytoplasmic) was found to decrease from 2.5 to 1.3 upon shift from non-permissive to permissive temperature. The slowly exchanging Ca-pool (presumably mitochondrial) did not change significantly upon temperature shift. These and further data demonstrate a close correlation between distribution of cellular Ca among different cellular compartments and characteristics of cellular proliferation, both attributable to the function(s) of a single oncogene.

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.

Monoclonal antibodies that prevent adhesion of B 16 melanoma cells and reduce metastases in mice: crossreaction with human tumor cells.

Monoclonal antibodies raised against B 16 melanoma cells in syngeneic mice were functionally screened for their ability to inhibit cell adhesion in tissue culture. Three of these antibodies (16/43, 16/77, 16/82), when preinjected into C57BL/6 mice, markedly reduced the number of experimental lung metastases produced by B 16 cells, possibly by interference with their adhesion to the lung endothelia. We now report that these monoclonal antibodies block in vitro attachment of the majority of human melanoma cell lines tested and also of carcinoma, neuroblastoma, and glioblastoma cells from both mice and humans but untransformed cell lines such as 3T3 mouse or MRC-5 human fibroblasts are not affected. The antibodies also react with mouse teratocarcinoma stem cells (F9, PCC4) but not with differentiated teratocarcinoma lines (PYS-2, 944). Furthermore, the antiadhesion activity of the antibodies could be quantitatively absorbed by intact human and mouse tumor cells but not by untransformed cells, suggesting that the corresponding antigens may represent tumor-associated cell surface components. Correspondingly, the antigens were found on simian virus 40-transformed 3T3 mouse fibroblasts and are expressed in a temperature-sensitive fashion in chicken fibroblasts transformed with a temperature-sensitive Rous sarcoma virus. On "immunoblots" of NaDodSO4-containing gels the three selected antibodies (16/43, 16/82, 19/1) were absorbed by antigens with molecular weights of 40,000 and 50,000.

Propagation of globin DNAase i-hypersensitive sites in absence of factors required for induction: A possible mechanism for determination

We tested whether DNAase I-hypersensitive sites, once induced, can be propagated to daughter cells in the absence of the original inducer. Chicken embryo fibroblasts infected with a temperature-sensitive Rous sarcoma virus at 41 °C and then shifted to 36°C become transformed and begin to transcribe globin RNA. DNAase I-hypersensitive sites appear in the α- and β-globin chromatin domains. Neither transcription nor hypersensitive sites are detected in cells infected and maintained at 41°C. Activation of the globin hypersensitive sites occurs within 30 min of a temperature shift to 36°C and does not require new protein synthesis. To test for the self-propagation of these hypersensitive structures, we inactivated the v-src gene product by a shift back up to 41°C, and allowed the cells to divide 20 times at 41°C. Although transcription of the globin genes was minimal after this treatment, the DNAase I-hypersensitive sites remained. The same sites can be induced by NaCI shock of cells. After the cells are returned to normal medium and allowed to grow for 20 doublings, the hypersensitive sites remain. This suggests that once formed, DNAase I-hypersensitive sites have the capacity to template their own structure independent of the initial “inductive” event. The single-stranded character of these DNAase I-hypersensitive sites could explain these results.

Immunological identification of cerebellar cell lines

Spleen cells from BALB/c mice, previously immunized with rat cerebellar tissue, were fused to the mouse myeloma cell line SP2/O-Ag14 and the cerebellar cell type specificity of the resultant hybridomas determined. In this report we describe the specificity of one hybridoma, C4/12. Monoclonal antibodies secreted by this hybridoma recognize granule cell neurons in adult cerebellar frozen sections, and in primary cultures started from 3 to 5-day-old newborn rats. In addition, C4/12 recognizes a subclass of astrocytes when screened on primary cultures but not adult cerebellar tissue. Two temperature sensitive Rous sarcoma virus transformed cerebellar cell lines, previously shown to be either neuronal or glial, were screened for the presence of the antigen. Both cell lines are positive at the temperature permissive for transformation, whereas the glial line but not the neuronal line exhibits the antigen at the nonpermissive temperature. These results are discussed in light of the cell lines being representative of precursor cells.

Intermediate-size filaments: changes in synthesis and distribution in cells of the myogenic and neurogenic lineages.

The number of intermediate-size filaments, their topographical distribution, and relationships with other cell organelles vary among cell types. These 10-nm filaments provide an unusual instance of an organelle that possesses a relatively constant morphology, although its constituent proteins are altered or replaced during differentiation. In this paper we focus on their qualitative and quantitative changes during myogenesis and neurogenesis. We stress the switch between the types of 10-nm filaments synthesized in precursor presumptive myoblasts and neuroblasts and those synthesized in their daughters, the postmitotic myoblasts and neuroblasts. Changes in the behavior of the 10-nm filaments are discussed in response to (1) arabinosyl-cytosine (Ara-C), (2) the cocarcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA), (3) transformation with temperature-sensitive Rous sarcoma virus (RSV), and (4) cell density. We also report that the effect of Colcemid in inducing aggregation of 10-nm filaments into immense cables is blocked by taxol, a microtubule-stabilizing drug.

Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture.

We have investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the src gene-associated protein kinase, the 34- to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response to TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation of other cellular phosphotyrosine-specific kinases. We have shown previously that temperature-sensitive RSV-infected cells at nonpermissive temperature demonstrate an increased sensitivity to TPA treatment [Bissell, M. J., Hatie, C. & Calvin, M. (1979) Proc. Natl. Acad. Sci. USA 76, 348-352]. Our present results indicate that this is not due to reactivation of the phosphorylating activity of the defective src gene product or to its leakiness, and they lend support to the notion of multistep viral carcinogenesis.

Transcription and site-specific hypomethylation of the delta-crystallin genes in the embryonic chicken lens.

Chicken delta-crystallin has at least two similar genes containing multiple intervening sequences. Hybridization experiments utilizing DNA probes derived from intervening sequences contained within cloned delta-crystallin gene fragments indicated that both of these two delta-crystallin genes are transcribed in the embryonic lens. No evidence for transcription of these genes was found in nuclei prepared from decapitated chicken embryos. Comparison of restriction enzyme digests of genomic DNA prepared from lens and non-lens tissues showed that the delta-crystallin gene sequences are not grossly rearranged in the lens during development. Msp I and Hpa II digests revealed that there are at lest three -CCGG- sites in the delta-crystallin genes that are hypomethylated in the embryonic lens DNA but are hypermethylated in sperm, red blood cell, or headless embryo DNAs. The delta-crystallin genes were not transcribed and these three sites were methylated in embryonic lens epithelial cells that were transformed with a temperature-sensitive Rous sarcoma virus. These results associate delta-crystallin gene transcription and site-specific hypomethylation of the delta-crystallin DNA.

Initial studies on cultured embryonic chick lens epithelial cells infected with a temperature-sensitive Rous sarcoma virus

Primary cultures of lens epithelial cells from 6-day-old chick embryos were infected with a temperature-sensitive Rous sarcoma virus (Schmidt-Ruppin strain, ts-68). After one month in culture, uninfected control cells had an epitheliod morphology and synthesized small amounts of δ-crystallin; by contrast, infected cells at the permissive temperature for src gene expression (and thus for transformation) had a fibroblastic appearance and showed no detectable synthesis of δ-crystallin. A hybridization test with δ-crystallin cDNA indicated that the transformed cells contained 10–100 times less δ-crystallin mRNA than the uninfected cells. The virally-infected cells adopted an epithelial-like appearance when cultured at the nonpermissive temperature for expression of the src gene, but did not initiate δ-crystallin synthesis or accumulate δ-crystallin mRNA.

Properties of two temperature-sensitive rous sarcoma virus transformed cerebellar cell lines

Cells from the cerebellum of 3-day-old BD-IX rats were obtained as permanent lines by transforming them with temperature-sensitive Rous sarcoma virus. The presence or absence of veratridine-stimulated Na +-uptake (voltage-dependent channels) was used to operationally classify them as neuronal or glial. When incubated at 34 °C, the permissive temperature for transformation, the cerebellar cells exhibit a transformed phenotype determined by anchorage independence, rounded morphology, high growth rate and absence of density-dependent inhibition of growth. In contrast, when the transformed cerebellar cell lines are kept at a temperature (38 °C) non-permissive for transformation, they exhibit a normal cellular phenotype with respect to the above properties. Moreover, changes toward neuronal morphology, increase in veratridine-stimulated Na +-uptake, decreased growth rate and the expression of the astrocyte specific protein, glial fibrillary acidic protein, suggest that a degree of differentiation is expressed at the non-permissive temperature.

The effects of temperature-sensitive Rous sarcoma virus and phorbol diester tumor promoters on cell lineages.

My colleagues and I, as well as many other investigators (Pierce 1967; Foulds 1969; Pierce and Wallace 1971; Berenblum 1974; Boutwell 1976; Potter 1978) have performed experiments based on the assumption that those intracellular controls which are involved in the transformation of normal cells into malignant cells are also involved in the generation of diversity among normal cells (Holtzer et al. 1972, 1974, 1975c). In addition, we have assumed that these intracellular controls are the very mechanisms which permit a “cloned” zygote, a “cloned” blastula cell, or a “cloned” limb bud precursor cell in early compartments of their respective lineages to generate diversified progeny (Abbott et al. 1974; Dienstman et al. 1974; Holtzer 1978). In all instances the fundamental question is: How does a single replicating “mother” cell, following a quantal cell cycle, yield daughter(s) with a predictably different, but equally limited, set of metabolic options? A replicating blastula cell, a replicating erythrogenic hematocytoblast or Friend erythroleukemic cell (Holtzer et al. 1972, 1975c; Lajtha and Schofield 1974; Weintraub 1975; Holtzer 1978), a replicating presumptive chondroblast (Holtzer 1978), or a replicating presumptive myoblast (Holtzer et al. 1975c; Holtzer 1978) has no option following a quantal cell cycle, but to yield specifically different kinds of gastrula cells, erythroblasts, chondroblasts, and myoblasts, respectively. Presumably, daughter cells, as a consequence of a quantal cell cycle, acquire the capacity to transcribe regions of the genome that could not be transcribed in their mother2. It is equally likely that the daughter cells lose the capacity to transcribe regions that were uniquely transcribed in their mother cells. Similarly, embryonal carcinoma cells (Martin 1975), chondrosarcoma cells, hepatoma cells, or lymphoma cells have each inherited a different set of metabolic options which (1) not only distinguish these different types of neoplastic cells one from the other, but (2) distinguish them from their normal mother cells.

Antiserum to murine leukemia virus recognizes novel cell surface molecules associated with growth control and transformation.

Antiserum directed against murine leukemia virus also reacts with several external proteins present in rat cells transformed by a temperature-sensitive Rous sarcoma virus. Reaction of iodinated cell extracts with anti-MLV (murine leukemia virus) serum revealed the presence of a 200,000 dalton iodinated component detectable also by metabolic labelling with glucosamine only in serum-starved cultures restricted in the expression of transformation. A similar assay with iodinated cells that express the transformed phenotype revealed the preferential recognition of two components with an approximate molecular weight of 100,00 daltons as well as an additional 65,000-dalton external component. Growth of the transformed non-producer NT3-KR cells in the presence of inducers of C-type viruses leads to an increased synthesis of a 100,000-dalton glycoprotein (gp100) recognized by the anti-MLV serum which is also recognized by the antiserum in NRK-MSV-MLV transformed producer cells, in addition to a virus-like glycoprotein of 71,000 dalton (gp71). Absorption of the anti-MLV serum with monolayers of NT3-KR cells eliminated the ability of the serum to recognize the gp100 but not the gp71 from NRK-MSV-MLV-transformed producer cells. The mediation of post-translational changes in growth control is suggested by the transformation-dependent alteration in the molecular weight of the non-virion surface proteins recognized by anti-MLV serum in the rat cells used in this study.

Immunochemical purification of probe-labeled plasma membrane proteins: an approach to the molecular anatomy of the cell surface.

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labeling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80--100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37 degrees C but not at 41 degrees C, the expression of this component is related to the expression of the transformed phenotype.

Effect of a tumour promoter on myogenesis.

EVIDENCE that viruses and chemical carcinogens alter the ongoing synthetic programme of cells has been presented1,2. Chondroblasts and melanoblasts transformed with a temperature-sensitive Rous sarcoma virus cease synthesising the chondroblast-unique sulphated proteoglycan and melanin, respectively3. Shifted to non-permissive temperature, these cells re-initiate the synthesis of the chondroblast-unique sulphated proteoglycan and melanin. Similar results have been obtained using chick myogenic cells4,5. Normal presumptive myoblasts undergo a limited number of replications, yielding postmitotic, mononucleated myoblasts. Postmitotic myoblasts initiate the synthesis of muscle-specific myosin heavy and light chains and fuse into myotubes6,7. In contrast, ts-viral transformed myogenic cells continue to replicate, do not initiate the synthesis of muscle-specific myosins and do not fuse. 24–48 h after shifting to non-permissive temperature, many transformed myogenic cells withdraw irreversibly from the cell cycle, initiate the synthesis of muscle-specific (unpublished data) myosins and fuse. Apparently when the transformation factor is expressed the cells do not withdraw from the cell cycle, and as a consequence of being forced to cycle, do not terminally differentiate. To probe the mechanisms that might be common to the generation of terminally differentiated cells from normal precursor cells, and to the generation of malignant cells from normal precursor cells, we studied the effects of phorbol-12-myristate-13-acetate (PMA) on myogenesis. Of the purified phorbol esters, PMA is the most potent as a tumour-promoting agent and as a mitogen8–12. We report here that PMA mimics some of the effects of the Rous sarcoma tumour agent on myogenesis.

Effects of Rous Sarcoma Virus on the synthetic programs of chondroblasts and retinal melanoblasts

Chondroblasts and retinal melanoblasts have been transformed with a temperature-sensitive Rous Sarcoma Virus. At permissive temperature the transformed cells do not display their unique phenotypic properties. When such transformed cells are shifted to non-permissive temperature they do display their unique phenotypic properties.

Reversion from transformed to normal phenotype by inhibition of protein synthesis in rat kidney cells infected with a temperature-sensitive mutant of Rous sarcoma virus.

By the use of a rat kidney cell line infected with a temperature-sensitive Rous sarcoma virus, we have shown that, at permissive temperatures where the cells are transformed, concanavalin A induces a clustering of its cell membrane receptors into patches, and the intracellular smooth muscle myosin-like protein is in a disordered state. By contrast, with infected cells grown at nonpermissive temperatures, the addition of concanavalin A does not alter the uniform distribution of its receptors, and the smooth muscle myosin-like protein is arranged in an ordered filamentous structure. These results are consistent with the hypothesis that the myosin protein is part of an intracellular aggregating-disaggregating complex. In the normal cell it is in its aggregated state and inhibits the lateral mobility of the concanavalin A receptors in the membrane; in the transformed cell the complex is relatively disaggregated and permits the concanavalin A receptors to be mobile. The addition of protein synthesis inhibitors to infected cells grown at the permissive temperature causes the cell to change from the transformed phenotype to the normal. Removal of the reversible inhibitors causes the cells to revert to the transformed phenotype. These results show that (i) protein synthesis, presumably of an unstable product of the transforming gene of the temperature-sensitive virus, is required to maintain the transformed state in these infected cells at the permissive temperature; and (ii) protein synthesis is not required for the intracellular myosin-containing complex to revert from its disordered transformed state to its ordered normal state. This suggests that the product of the transforming gene directly or indirectly causes the disaggregation of the myosin-containing complex in the process of transformation.