mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities.

The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v-mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes.     

Use of the mouse mammary tumor virus long terminal repeat to promote steroid-inducible expression of v-mos.

We used the mouse mammary tumor virus long terminal repeat to promote dexamethasone-regulated expression of the Moloney murine sarcoma virus (M-MSV) transforming gene, v-mos. A recombinant DNA vector containing the mouse mammary tumor virus long terminal repeat fused to the M-MSV 124 v-mos gene was cotransfected with a plasmid containing the herpes simplex virus thymidine kinase gene (tk) into 3T3TK- cells. Individual clones of cells which grew in hypoxanthine-aminopterin-thymidine medium were tested for dexamethasone-regulated expression of p37mos as well as several transformation-specific phenotypic parameters. In the absence of dexamethasone, the v-mos transfectants appeared morphologically similar to the control cells despite low basal levels of p37mos expression. Upon hormone treatment, the levels of p37mos increased 5- to 10-fold, coincident with morphological changes typical of M-MSV transformation of 3T3 cells. The ability to form foci in monolayers also correlated with p37mos induction. The extent of morphological changes varied in individual clones of cells with similar levels of induced p37mos. Although the induced levels of p37mos were comparable to those seen in stable M-MSV 124 virus-transformed NIH 3T3 cells, the transfectants were unable to grow in soft agar under conditions which support growth of the virus-transformed cells. Acute infection of the transfectants with M-MSV 124 virus, a situation which resulted in elevated levels of p37mos, allowed these cells to grow in soft agar. The results described in this paper suggest that different threshold levels of p37mos may be necessary for the expression of various parameters of the transformed phenotype and also that continued expression of p37mos is necessary for maintenance of the transformed state. However, it also appears that the sensitivity to given levels of p37mos varies among clonal cell lines.     

Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability.

Constitutive activation of mitogen-activated protein kinase (MAPK) is a property common to many oncoproteins, including Mos, Ras, and Raf, and is essential for their transforming activities. We have shown that high levels of expression of the Mos/MAPK pathway in Swiss 3T3 fibroblast cause cells in S phase to undergo apoptosis, while cells in G1 irreversibly growth arrest. Interestingly, cells in G2 and M phases also arrest at a G1-like checkpoint after proceeding through mitosis. These cells fail to undergo cytokinesis and are binucleated. Thus, constitutive overexpression of Mos and MAPK cannot be tolerated, and fibroblasts transformed by Mos express only low levels of the mos oncogene product. Here, we show that p53 plays a key role in preventing oncogene-mediated activation of MAPK. In the absence of p53 (p53-/-), the growth arrest normally observed in wild-type p53 (p53+/+) mouse embryo fibroblasts (MEFs) is markedly reduced. The mos transformation efficiency in p53-/- MEFs is two to three orders of magnitude higher than that in p53+/+ cells, and p53-/- cells tolerate > 10-fold higher levels of both Mos and activated MAPK. Moreover, we show that, like Mos, both v-ras and v-raf oncogene products induce apoptosis in p53+/+ MEFs. These oncogenes also display a high transforming activity in p53-/- MEFs, as does a gain-of-function MAPK kinase mutant (MEK*). Thus, the p53-dependent checkpoint pathway is responsive to oncogene-mediated MAPK activation in inducing irreversible G1 growth arrest and apoptosis. Moreover, we show that the chromosome instability induced by the loss of p53 is greatly enhanced by the constitutive activation of the Mos/MAPK pathway.     

Transformation of primary rat kidney cells by DNA fragments of weakly oncogenic adenoviruses.

Primary cultures of baby rat kidney (BRK) cells were transformed by intact DNA and DNA fragments of weakly oncogenic human adenovirus types 3 and 7. The smallest fragment found to contain transforming activity was the left-terminal 4% endo R.HindIII fragment (for both adenovirus type 3 and 7 DNAs). The efficiency of transformation of this fragment was low, and no permanent cell line could be established. Left-terminal fragments ranging from 84 to 4,5% of the viral genome could all transform BRK cells with the same efficiency as intact viral DNA. A number of adenovirus type 7 DNA fragment-transformed lines were established and were found to contain persistent viral DNA sequences and adenovirus subgroup B-specific T antigen. Consequently, the transforming functions of adenovirus types 3 and 7 are located at the extreme left-hand end of the genome, and the minimum size for a DNA fragment with transforming activity is 1.0 X 10(6) daltons. These results do not rule out the possibility that viral genes located outside the transforming region may also influence transformation.     

The transforming protein of Moloney murine sarcoma virus is a soluble cytoplasmic protein

The transforming gene, v-mos, of Moloney murine sarcoma virus (M-MuSV) encodes a 37,000-dalton phosphoprotein, p37mos. Since the biochemical function of this protein is unknown, we have determined the subcellular location of p37mos in M-MuSV 124-transformed cells. Using two different methods of cell lysis and fractionation, we found that newly synthesized as well as mature p37mos is a soluble cytoplasmic protein. In agreement with these results, immunofluorescent staining of cells acutely infected with M-MuSV 124, using an antiserum directed against a synthetic v-mos peptide, produced a diffuse cytoplasmic pattern. Gel filtration experiments and glycerol gradient sedimentation analysis suggest that the bulk of p37mos exists as a monomer and is not involved in a specific association with other cellular proteins. These properties of p37mos are different from those of other characterized retroviral transforming proteins.     

Detection and preliminary characteristics of the transforming gene (oncogene) of Ha-ras type in human stomach cancer

High molecular weight DNA prepared from three undifferentiated human stomach carcinomas was assayed for transforming activity by transfection of mouse NIH 3T3 cells. One tumor DNA sample (stomach carcinoma CaVSt) induced (the transfection efficiency: 0.02 transformants/micrograms DNA X 10(-6) cells) transformation of NIH 3T3 recipient cells. Transforming gene of Ha-ras type was identified in transformants derived from this human carcinoma. The genetic lesion responsible for the activation of the CaVSt Ha-ras oncogene is not localized in the 12-th codon for p21c-Ha-ras protein.     

The effect of transforming growth factor beta on the intensity of cellular DNA synthesis in relation to the type and conditions of cultivation

A study was made of the influence of transforming growth factor beta (0.05-50.00 ng/ml) on the intensity of 3H-thymidine incorporation in the DNAs of pseudonormal mouse fibroblasts of NIH 3T3 line, of cells of NRK-49F line from normal rat kidney, and of cells of A-549 line from human lung adenocarcinoma. The experiments were carried out in the absence and in the presence of epidermal growth factor (5 ng/ml), and(or) insulin (1 micrograms/ml), as well as in the presence of different concentrations of fetal calf serum, and while using different time of incubation of cells with the above mentioned growth factors. It was shown that depending on the culture conditions the transforming growth factor beta exerted stimulatory, inhibitory or no action on the intensity of DNA synthesis in the cells of the same type. An attempt was made to analyse the reasons, which may significantly determine the direction of regulatory influence of the transforming growth factor beta on DNA replication in the cells.     

Sodium butyrate suppresses the transforming activity of an activated N-ras oncogene in human colon carcinoma cells

The transforming activity of DNA from a newly established undifferentiated human colon carcinoma cell line (MIP-101) was tested in the NIH-3T3 transfection assay. Southern blot analysis of the transfectant DNA revealed the presence of a human N-ras oncogene. Treatment of MIP-101 cells with the maturational agent sodium butyrate induced a more normal phenotype, including diminished growth rate, elimination of anchorage independent growth, and decreased tumorigenicity (R. Niles, S. Wilhelm, P. Thomas, and N. Zamcheck (1988) J. Cancer Invest. 6, 39). Here we report that there is a significant reduction in the transforming efficiency of the DNA from butyrate-treated MIP-101 cells. A nonspecific reduction in total DNA uptake as an explanation for these findings was eliminated by showing that there was similar uptake and expression of the thymidine kinase gene from the DNA of butyrate-treated and control MIP cells. Butyrate treatment had no detectable effect on the overall structure, methylation, and level of expression of the human N-ras gene from MIP-101 cells. An NIH-3T3 transformant ability after treatment with sodium butyrate. Although butyrate suppressed several transformed properties similar to MIP-101 cells, DNA from control and treated cultures had an identical level of transforming activity. The results suggest that the environment of the MIP cells may contain additional elements not present in the NIH-3T3 transformants which are required to observe the effect of butyrate on reduction of transforming activity.     

Chromosomal localization of the Moloney sarcoma virus mouse cellular (c-mos) sequence.

The Moloney sarcoma virus-specific onc gene, referred to as v-mos, was used as probe to hybridize to restricted DNAs from various mouse-Chinese hamster hybrid cell lines. These hybrid cells contain, in addition to all of the Chinese hamster chromosomes, various numbers (less than a full complement) of mouse chromosomes. Comparison of the presence or absence of the mouse cellular mos gene with the known karyotype in each of the hybrid cell lines allows us to conclude that the mos gene is on mouse chromosome 4.     

Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product.

Using an expression cDNA cloning approach, we examined human tumor cell lines for novel oncogenes that might evade detection by conventional techniques. We isolated a transforming sequence that was highly efficient in transforming NIH 3T3 mouse fibroblasts. DNA sequence analysis identified the gene as the human homolog of a recently cloned alpha subunit of mouse GTP-binding protein G alpha 12. NIH 3T3 cells transfected with G alpha 12 cDNA grew in soft agar and were tumorigenic in nude mice. There were no apparent mutations in the cloned cDNA in comparison with a G alpha 12 cDNA clone isolated from a normal human epithelial cell library, implying that overexpression alone was sufficient to cause NIH 3T3 cell transformation. The observed altered growth properties mediated by G alpha 12 showed a certain degree of dependency on serum factors, and its mitogenic potential was also potently inhibited by suramin treatment.     

Transforming activity of the lymphotoxin-beta receptor revealed by expression screening

Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies. To obtain insight into the molecular pathogenesis of PDC, we constructed a retroviral cDNA expression library with total RNA isolated from the PDC cell line MiaPaCa-2. Screening of this library with the use of a focus formation assay with NIH 3T3 mouse fibroblasts resulted in the identification of 13 independent genes with transforming activity. One of the cDNAs thus identified encodes an NH(2)-terminally truncated form of the lymphotoxin-beta receptor (LTBR). The transforming activity of this short-type LTBR in 3T3 cells was confirmed by both an in vitro assay of cell growth in soft agar and an in vivo assay of tumorigenicity in nude mice. The full-length (wild-type) LTBR protein was also found to manifest similar transforming activity. These observations suggest that LTBR, which belongs to the tumor necrosis factor receptor superfamily of proteins, may contribute to human carcinogenesis.     

Detection of a transforming gene product in cells transformed by Moloney murine sarcoma virus

We identified, in cells transformed by Moloney murine sarcoma virus (M-MuSV clone 124), a protein encoded by the M-MuSV transforming gene, v-mos. An antiserum against a synthetic peptide corresponding to the C terminus of a protein predicted from the v-mos nucleotide sequence specifically recognizes a protein doublet of approximately 37,000 daltons from 35S-methionine-labeled M-MuSV 124-transformed producer cells. By peptide mapping, this protein is almost identical to the 37 kd in vitro translation product from the M-MuSV v-mos gene. Immunoprecipitates from 32P-labeled cells contain a single v-mos-specific phosphoprotein, which has at least six sites of phosphorylation containing phosphoserine. Pulse-chase experiments show that the lower band in the 35S-methionine-labeled doublet is the primary translation product, which is modified, probably by phosphorylation, to yield the upper band. A similar mos protein is immunoprecipitated from HT1-MuSV-transformed cells, but not from uninfected NIH/3T3 cells. These mos proteins are present at very low levels in transformed cell lines. Cells acutely infected with M-MuSV 124, however, transiently contain much higher levels of the mos protein. These high levels coincide with extensive cell mortality.     

Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus.

BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.     

Expression in mouse L cells of mos-related ORAgp5 locus cloned from the human genome]

The recombinant locus ORAgp5 containing the regions homologous to protooncogene mos was previously cloned from the human genome. In order to monitor the expression of this human genome region, we have transfected the recombinant phage gp5 into mouse LMtk cells. One of these transfectants contained several copies of gp5. ORAgp5 sequences were found to be expressed in 1.5 kb RNA from this clone.     

Transformation of normal diploid cells by isolated metaphase chromosomes of virus-transformed or spontaneous tumor cells.

Normal diploid human cells with a limited life-span in culture, as well as primary or secondary cell cultures of mouse or rat embryos, can be transformed in vitro (i.e. grow in soft-agar or low-serum medium) after a single exposure to metaphase chromosomes from SV40-transformed human or rat cells, Ad5-transformed human cells and several spontaneous human or mouse tumor cells. Chromosomes from normal diploid cells do not show any such transforming activity. As judged from the number of colonies formed in selective medium, the efficiency of transformation is, with some exceptions, of the order of 10(-5)--10(-6) and is generally higher for homologous than for heterologous transfers. A fraction of the colonies demonstrate abortive transformation. Nevertheless, using chromosomes from all but one donor cell population, at least one transferent cell line expressing a stable transformed phenotype has been established. Our results demonstrate that transformation of normal diploid cells by a presumptive chromosome-mediated gene transfer can be obtained with a variety of donor and recipient cells.     

Mechanism of interferon action. Production and characterization of monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1

Monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1 were prepared using protein P1 purified from human amnion U cells as the immunogen. Rabbit antiserum to protein P1 recognized with comparable efficiency P1 both from human U cells and from mouse L929 cells. Immunoprecipitates that contained protein P1 also possessed a protein kinase activity that catalyzed the phosphorylation of protein P1 and the alpha subunit of initiation factor eIF-2. Three BALB/C mouse monoclonal antibodies efficiently recognized human protein P1, but either did not recognize or recognized very poorly P1 from mouse cells. A fourth monoclonal antibody against human P1 recognized mouse P1 with nearly equal efficiency. Immunoprecipitation of human P1 with different sequential combinations of the monoclonal antibodies suggest that two antigenic classes of protein P1 may exist.     

Molecular cloning and characterization of the tumor transforming gene (TUTR1): a novel gene in human tumorigenesis.

We cloned and sequenced the cDNA of a potent tumor transforming gene (TUTR1) from human testis and determined its primary structure. The TUTR1 cDNA is composed of 656 nucleotides and encodes a novel protein of 202 amino acids. The predicted TUTR1 protein is extremely hydrophilic and contains two proline-rich motifs at its C-terminus. Northern blot analysis of the mRNA from various human tissues and tumors revealed that TUTR1 mRNA is highly expressed in tumors of the pituitary gland, adrenal gland, ovary, endometrium, liver, uterus, and kidney as well as in cell lines derived from tumors of the pituitary, breast, endometrium, and ovary. With the exception of the testis, the levels of TUTR1 mRNA were either very low or undetectable in normal human tissues. Overexpression of TUTR1 in mouse fibroblasts (NIH 3T3) cells resulted in an increase in cell proliferation, induced cellular transformation in vitro, and promoted tumor formation in nude mice. These results suggest that TUTR1 is a novel and potent transforming gene, which may be involved in tumorigenesis in numerous different human tumors.