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

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

Influence of mammary cell differentiation on the expression of proteins encoded by endogenous BALB/c mouse mammary tumor virus genes.

The interactions between differentiation-associated cellular events in the intact mammary gland or in cultured mammary cells and the post-transcriptional activity of the endogenous mouse mammary tumor virus (MMTV) loci were investigated. The transcriptional activities of the endogenous MMTV proviruses of the BALB/c mouse strain (Mtv-6, Mtv-8 and Mtv-9) appear to be regulated differentially during pregnancy-induced mammary gland development (J.E. Knepper, D. Medina and J.S. Butel, J. Virol. 59, 518-521, 1986). Analysis of MMTV-specific proteins at various stages of mammary gland development (virgin, midpregnant, lactating, regressing) established the presence of steady-state levels of a 67,000-Mr env precursor-type polypeptide at all physiological stages. However, processing to lower-molecular-weight env-specific proteins, including a predominant 50,000-Mr species, was detected only with the transition to the functional mammary gland phenotype. The contributions of cell proliferation, cell-matrix interactions, and modulation of functional activity to the pattern of endogenous MMTV protein expression were investigated using a 3-dimensional collagen type I culture system. Growth and cell-matrix interactions (cell polarization, lumen formation) leading to formation of 3-dimensional duct-like structures were permissive for the synthesis and processing of MMTV-specific proteins; accumulation of high levels of the 50,000-Mr env-specific polypeptide was associated with the onset of the fully functional mammary cell phenotype. Expression of MMTV-specific proteins was not due to amplification of a specific cell subpopulation. The potential of the full-length Mtv-8 and Mtv-9 proviruses to be transcribed, as indicated by their methylation status, was not dramatically different between differentiated and undifferentiated mammary cells in culture. This study indicates that MMTV transcriptional activity is reflected at the protein level in mammary tissue of BALB/c mice and that viral protein synthesis and processing may serve as important markers of different physiological stages of mammary epithelial cells. These observations also suggest a general approach to the examination of potential modulatory effects of cellular interactions (cell-cell, cell-matrix or both) known to be important in various differentiated epithelial cell systems for the expression of viral genes.

Disappearance of specific proteins from the membranes of colicin-treated cells.

Two colicins that affect energy metabolism in Escherichia coli (colicins K and E1) are shown to cause loss of specific membrane proteins from treated cells. Disappearance of these proteins after treatment with colicin K occurs at low multiplicities and is independent of ATPase (EC 3.6.1.4) and phospholipase A (EC 3.1.1.4) activities. The uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol do not alter the pattern of membrane proteins.

Cloning and characterization of BK virus-related DNA sequences from normal and neoplastic human tissues.

DNA homologous to that of the known papovarius BK was cloned from the high molecular weight DNA of two human tissues: a normal liver and a kidney carcinoma. The clone isolated from human liver consisted of DNA indistinguishable from prototype BK by restriction enzyme analysis that used ten different enzymes. The DNA cloned from the human carcinoma of the kidney was subject to rearrangement in recombination-deficient bacteria, and exhibited a deletion of a small segment of DNA localized to the BK late region. Restriction fragments representing the BK origin and promoter regions are overrepresented in the tumor-derived clone. The possible significance of retrieval of defective viral genomes from tumor tissues is discussed.

Differential expression of endogenous mouse mammary tumor virus genes during development of the BALB/c mammary gland.

Expression of endogenous mouse mammary tumor virus sequences varied over the course of development of the mammary gland during primary pregnancy and lactation in virus-free BALB/c mice. Although RNA from all regions of the genome was detected, both the level and temporal regulation of expression were different for long terminal repeat-, env-, and gag-pol-specific RNAs. Analysis of the methylation status of proviral DNA indicated differential accessibility of the three endogenous units during development. The results demonstrated noncoordinate regulation of mouse mammary tumor virus expression with respect to provirus template utilized and specific transcripts accumulated.

Sequence similarity between the long terminal repeat coding regions of mammary-tumorigenic BALB/cV and renal-tumorigenic C3H-K strains of mouse mammary tumor virus.

The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) encodes a protein which functions as a superantigen. The BALB/cV strain differs from other exogenous MMTVs antigenically, biochemically, on the basis of restriction fragment analysis, and by the specificity of its superantigen for V beta 2+ T cells. In order to elucidate the origin of the BALB/cV virus and to better understand the interaction of its superantigen with the T cell receptor, we have determined the nucleotide sequence of the BALB/cV LTR open reading frame, including 93 bases downstream of the translation termination site. The encoded protein's C-terminal portion, thought to control superantigenic specificity, is identical to the C3H-K strain of MMTV, isolated from a rare kidney adenocarcinoma. The remainder of the coding sequence is highly related to many MMTV strains. Like other MMTV strains, the BALB/cV LTR maintains intact an 18 base pair sequence, located downstream of the translational termination site, which is lacking in the C3H-K LTR. Sequence comparison between the BALB/cV LTR and other MMTV strains indicates that the most likely origin for the BALB/cV open reading frame sequence is a recombination event involving the endogenous provirus mtv-6.

Activation of endogenous MMTV proviruses in murine mammary cancer induced by chemical carcinogen.

A study was undertaken to determine whether activation of expression of silent endogenous mouse mammary tumor virus (MMTV) proviruses may occur during tumor induction by a chemical carcinogen. A series of transplantable mammary tumors induced in BALB/c mice by treatment with dimethylbenz(alpha)anthracene (DMBA), pituitary isograft, or both was examined. The results obtained suggest that chemical carcinogens may induce mammary tumors through more than one pathway. Two of 9 tumor lines produced virus-specific products at levels above those observed during the course of normal mammary gland development. One tumor contained high levels of MMTV-specific envelope [3.8 kilobase (kb)] and genomic length (8.9 kb) RNAs. This tumor expressed core- and envelope-related proteins detectable by immunoblotting (including p28, gp52, and gp36), displayed an acquired provirus with a restriction map different from those of described exogenous MMTV strains, and contained abundant virus particles. The other tumor that expressed high levels of MMTV gene products contained envelope-specific (3.8 kb) and long-terminal-repeat-specific (1.6 kb) messages but no full-length RNA. It exhibited an aberrant 39 kDa, envelope-related protein, but no virus particles. Methylation data implicated the usually silent endogenous Mtv-8 provirus as the source of the abnormal envelope protein. None of the tumors expressed RNA from the putative mammary oncogenes, int-1 or int-2. We propose that chemical carcinogens may activate different cellular genes by mutation and that, in a subset of DMBA-induced mammary tumors, the target genes include endogenous MMTV proviruses that are normally not expressed. The effect on provirus expression varies from tumor to tumor, but is stable over passage of a given tumor. MMTV may be of etiological importance in the genesis of those DMBA-induced tumors which contain high levels of MMTV-specific products, but its action in the BALB/c system is not mediated through enhanced expression of the int-1 or int-2 preferred integration regions.

Minor histocompatibility antigen-dependent rejection of Pam 212 epidermoid carcinoma by DBA/2 mice.

Since Pam 212 cells express low levels of class I major histocompatibility (MHC) antigens, we tested their ability to present alloantigens or minor histocompatibility (mH)/minor lymphocyte stimulatory (mls) antigens in disparate hosts. After subcutaneous injection, Pam 212 cells grew progressive tumors in normal BALB/c mice but were rejected rapidly by naive C3H mice (3 weeks) and slowly by DBA/2 mice (8 weeks). Pam 212 cells (high or low class I MHC expression) induced a strong primary MLR in DBA/2 T cells, but a weak BALB/c T-cell response. In contrast, splenic APC (BALB/c) did not induce an MLR, suggesting that Pam 212 cells represented mH antigens to naive DBA/2 T cells. This MLR was blocked by anti-TCR alpha/beta, anti-class II, and anti-CD4 monoclonal antibodies, but was independent of ICAM-1 and B7. Repeated immunization using IFN-gamma-treated Pam 212 cells induced anti-Pam 212 CTL in DBA/2 mice but not in BALB/c mice. DBA/2 T-cell responses did not appear to be mls (MMTV superantigen)-specific, because Pam 212 cells did not express MMTV mRNA detectable by RT-PCR. Pam 212 cells presented non-lymphoid-associated mH antigens that served as potent stimuli for tumor rejection in mH/mls-disparate hosts, which is similar to tumor rejection mediated by MHC alloantigens.

Association of a V beta 2-specific superantigen with a tumorigenic milk-borne mouse mammary tumor virus.

A number of endogenous mouse mammary tumor virus (MMTV) proviruses encode superantigens that have the property of stimulating mature T lymphocytes in a TCR V beta-specific fashion and of mediating V beta-specific clonal deletion in developing T cells. The tumorigenic milk-borne MMTV carried by C3H and GR mice also have superantigen properties in vivo, and it has been proposed that this superantigenic function is critical to the infectivity and/or tumorigenicity of the virus. To test the requirement for superantigen properties in tumorigenic MMTV, a highly tumorigenic strain of MMTV isolated from BALB/c mice (BALB/cV virus) was analyzed for its effect on TCR V beta expression. It was found that exposure of newborn mice to milk-borne virus results in marked deletion of V beta 2-expressing CD4+ peripheral T cells. This deletion is detected in mature TCRhigh thymocytes as well as in peripheral T cells from virus-exposed mice. Deletion is dependent on expression of a permissive MHC type in mice exposed to virus. Subcutaneous injection of adult mice with virus-containing milk induces a substantial increase in V beta 2+ CD4+ cells in draining lymph nodes within 4 days. Thus, tumorigenic BALB/cV is associated with V beta 2-specific superantigen activity capable of mediating both T cell expansion and clonal deletion in vivo. These findings are consistent with a critical role of superantigen-mediated T cell activation in MMTV infection and tumorigenesis.

Spontaneous progression of hyperplastic outgrowths of the D1 lineage to mammary tumors: expression of mouse mammary tumor virus and cellular proto-oncogenes.

Mammary cancer in mice is characterized by progression through defined stages of preneoplasia, with the most common preneoplastic stage being the hyperplastic alveolar nodule (HAN). We determined the relative levels of RNA expression of various cellular proto-oncogenes and endogenous mouse mammary tumor virus genes in outgrowths and tumors of three sublines of the transplantable D1 HAN preneoplastic outgrowth line. The three sublines differed in relative tumor-producing capabilities. Subline D1B produced a high incidence of tumors with short latency periods, whereas sublines D1C and D1D produced low incidences of tumors with long latency periods. No consistent alteration in proto-oncogene expression correlated with relative tumorigenicity, although tumors frequently contained higher levels of one or more proto-oncogene transcripts as compared with preneoplastic tissue. Slightly elevated (2- to 6-fold) levels of different oncogene transcripts were detected in 13 of 17 tumors as compared with outgrowth tissue, including abl (2 tumors), fps (5 tumors), Ha-ras (6 tumors), and Ki-ras (8 tumors). One tumor contained 45 times more Ki-ras-specific RNA than outgrowth tissue because of a comparable amplification of Ki-ras DNA sequences. Elevated levels of Ha-ras occurred more frequently in tumors of a high-incidence subline than in a less-aggressive subline (5/10 vs 1/7), but this difference was not statistically significant. However, consistent changes in MMTV expression accompanied progression from preneoplastic tissues to mammary tumors. All 17 tumors displayed reduced levels of the MMTV-specific long terminal repeat (LTR) transcript (1.6 kb) as compared with HAN tissue; tumors with moderate levels of LTR transcript expressed the 3.8-kb envelope message as well, one not detected in HANs. Expression of the LTR transcript is apparently influenced by factors in addition to the methylation status of endogenous mouse mammary tumor virus genes, which was similar in outgrowths and tumors. As the survey of representative proto-oncogenes failed to identify a uniform change between HAN and tumors, it is likely that other genes are involved in tumor progression in the mammary gland.

Clonal populations of the mouse mammary cell line, COMMA-D, which retain capability of morphogenesis in vivo.

Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland.