Embryonic lethalities and endothelial tumors in chimeric mice expressing polyoma virus middle T oncogene

The effect of the middle T oncogene of polyoma virus was studied in vivo using a replication-defective selectable retrovirus. Injection of virus into newborn and adult mice resulted in the rapid appearance of cavernous hemangiomas. Infection of embryos did not yield transgenic mice; therefore, embryonal stem (ES) cells were used as an alternative system. Several infected ES cell clones were established that constitutively expressed middle T and its associated tyrosine kinase activity. Chimeric embryos obtained by blastocyst injection of individual ES cell clones were specifically arrested at midgestation, when multiple hemangiomas disrupted blood vessel formation. From these tumors endothelial cell lines were established that retained expression of von Willebrand factor yet were tumorigenic in vivo. These results suggest that middle T acts in endothelial cells as a single-step oncogene and that ES cells provide a valuable system for the study of growth control during embryogenesis.     

Induction of clonal monocyte-macrophage tumors in vivo by a mouse c-myc retrovirus: rearrangement of the CSF-1 gene as a secondary transforming event.

A mouse retrovirus containing the c-myc oncogene was found to induce tumors of mononuclear phagocytic cells in vivo. All tumors expressed the c-myc retroviral gene but not the endogenous c-myc gene (with one exception), and virtually all tumors were clonal with a unique proviral integration. This observation, coupled with a lag time in tumor formation, suggests that a second event, in addition to c-myc proviral integration, is necessary for the generation of neoplastic cells in vivo. All of the tumor cells expressed high levels of mRNA for both the putative colony-stimulating factor 1 (CSF-1) receptor (c-fms proto-oncogene product), as well as the c-fos proto-oncogene. Although all of the tumor cells proliferated in culture without the addition of exogenous CSF-1, which is required for the proliferation of primary macrophages partially transformed by the same c-myc retrovirus, several phenotypes were observed with respect to the expression of CSF-1 and granulocyte-macrophage CSF and to their growth factor responsiveness. The proliferation of one tumor, which secreted high levels of CSF-1, was blocked by specific anti-CSF-1 serum. This tumor was found to express altered CSF-1 mRNA and to have a DNA rearrangement at the CSF-1 locus. In this particular case, the data indicate that a CSF-1 gene rearrangement was the secondary event in development of the tumor. The pleiotropy of phenotypes among the other tumors indicated that there are a variety of other mechanisms for such secondary events which can be investigated with this system.     

Clonal dominance and progression in Abelson murine leukemia virus lymphomagenesis.

We examined the clonality of tumors induced by an acutely transforming retrovirus which carries a single oncogene. Contrary to our expectation, tumors induced by the Abelson murine leukemia virus (A-MuLV) showed one to four major proviral integration events. To further investigate the process by which clonality was established, we analyzed the number of cells infected and transformed by A-MuLV at various times after in vivo infection. At the midpoint of tumor latency (14 days postinfection), we found that infection of total bone marrow cells by A-MuLV was efficient and polyclonal. However, only a minority of these infected cells were transformed as assayed in cell culture, and clonal dominance had already been established in this transformed cell population. Examination of the in vitro growth properties of transformed cells recovered from preleukemic and leukemic mice indicated that preleukemic cells had lower cloning efficiencies than primary tumor cells. Our results suggest that the rate-limiting step in this system of lymphomagenesis is the initial transformation of bone marrow target cells and that these cells undergo subsequent changes in cloning ability during the course of the disease that lead to an autonomous neoplastic state.     

Tumorigenicity of fibroblast lines expressing the adenovirus E1a, cellular p53, or normal c-myc genes.

Cellular and viral oncogenes have been linked to the transformation of established cell lines in vitro, to the induction of tumors in vivo, and to the partial transformation or immortalization of primary cells. Based on the ability to cooperate with mutated ras oncogenes in the transformation of primary cells, the adenovirus E1a and cellular p53 genes have been assigned an immortalizing activity. It is demonstrated in this paper that the adenovirus type 5 E1a gene and simian virus 40 promoter-linked p53 cDNA are able to transform previously immortalized cells to a tumorigenic phenotype without a significant change in cell morphology. It is also shown that, when linked to a constitutive promoter, the normal mouse and human c-myc genes have the same transforming activity. Cells transformed by each of these oncogenes have an increased capacity to grow in the absence of growth factors and a limited anchorage-independent growth capability.     

LMP1 of Epstein-Barr virus induces proliferation of primary mouse embryonic fibroblasts and cooperatively transforms the cells with a p16-insensitive CDK4 oncogene

The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkin's lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recombinant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the proliferation of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4(R24C) oncogene in transforming MEF cells. Our results provide the first evidence of the abilities of the LMP1 gene, acting alone, to effectively induce the proliferation of primary MEF cells and of its cooperativity with another cellular oncogene in transforming primary cells.     

Transformation of mammalian fibroblasts and macrophages in vitro by a murine retrovirus encoding an avian v-myc oncogene

A murine retrovirus which expresses the avain v-myc OK10 oncogene was constructed. The virus, denoted MMCV, readily transforms fibroblasts of established lines, such as mouse NIH/3T3 and rat 208F cells, to anchorage-independent growth in agarose. The virus also transforms primary mouse cells: (i) virus-infected macrophages are induced to form large colonies in semi-solid media, and can easily be expanded into mass cultures; (ii) MMCV-infected fibroblastic cells from mouse limb buds undergo morphological transformation and grow in semi-solid medium. MMCV thus transforms both mouse fibroblastic cells and macrophages in vitro, in a fashion similar to the v-myc-containing avian viruses in chicken cells. The possibility of introducing a transforming myc gene into mammalian cells by virus infection provides a novel approach for studying the mechanism of myc transformation in cells from many lineages.     

Immortalization by c-myc, H-ras, and Ela oncogenes induces differential cellular gene expression and growth factor responses.

Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a "competence" function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus, transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell.     

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts.

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.     

Altered regulation of c-myc expression in adenovirus-transformed cells

Expression of the oncogenes c-myc, c-raski, and p53 is studied in normal primary mouse cultures and in two adenovirus-transformed mouse cell lines. In all cases oncogene expression is measured in cells arrested in G1 (or G0 for primary cells) by serum starvation and at different times after cell cycle traverse is stimulated by addition of high serum. For primary mouse cells, c-myc mRNA levels are found to increase four- to six-fold within 1 h of serum addition and then decline by 4 h to nearly the level observed in serum-starved cells. This level is maintained throughout the remainder of the cell cycle. The early induction of c-myc is dependent on serum concentration and is independent of cell density. These results confirm and extend previous observations for primary cells. By contrast, expression of c-raski does not vary at all through the cell cycle and p53 increases with time after mitogenic stimulation. In the adenovirus-transformed cell lines, the regulation of expression of c-myc with respect to the cell cycle is altered. There is an increase in c-myc in S phase cells which is dependent on cell density and the early induction in response to serum addition as seen in primary cells is absent. Expressions of c-raski and p53 are found to show similar profiles to those observed for primary cells.     

Establishment of murine cell lines by constitutive and conditional immortalization

Mouse cell lines were immortalized by introduction of specific immortalizing genes. Embryonic and adult animals and an embryonal stem cell line were used as a source of primary cells. The immortalizing genes were either introduced by DNA transfection or by ecotropic retrovirus transduction. Fibroblasts were obtained by expression of SV40 virus large T antigen (TAg). The properties of the resulting fibroblast cell lines were reproducible, independent of the donor mouse strains employed and the cells showed no transformed properties in vitro and did not form tumors in vivo. Endothelial cell lines were generated by Polyoma virus middle T antigen expression in primary embryonal cells. These cell lines consistently expressed relevant endothelial cell surface markers. Since the expression of the immortalizing genes was expected to strongly influence the cellular characteristics fibroblastoid cells were reversibly immortalized by using a vector that allows conditional expression of the TAg. Under inducing conditions, these cells exhibited properties that were highly similar to the properties of constitutively immortalized cells. In the absence of TAg expression, cell proliferation stops. Cell growth is resumed when TAg expression is restored. Gene expression profiling indicates that TAg influences the expression levels of more than 1000 genes that are involved in diverse cellular processes. The data show that conditionally immortalized cell lines have several advantageous properties over constitutively immortalized cells.     

Generation and characterization of a recombinant Moloney murine leukemia virus containing the v-myc oncogene of avian MC29 virus: in vitro transformation and in vivo pathogenesis.

A new retrovirus consisting of the v-myc oncogene sequences of avian MC29 virus inserted into the genome of Moloney murine leukemia virus (M-MuLV) was generated. This was accomplished by constructing a recombinant DNA clone containing the desired organization, introducing the recombinant DNA into mouse NIH 3T3 cells, and superinfecting the cells with replication-competent M-MuLV. The construction was designed so that an M-MuLV gag-myc fusion protein would be produced. The resulting virus, M-MuLV(myc), morphologically transformed uninfected NIH 3T3 cells. Stocks of M-MuLV(myc)-M-MuLV were infected into secondary mouse embryo cultures. M-MuLV(myc) induced striking growth and proliferation of hematopoietic cells. These cells were of the myeloid lineage by morphology, phagocytic properties, and surface staining with Mac-1 and Mac-2 monoclonal antibodies. They resembled mature macrophages, although they displayed minor properties of immaturity. The myeloid cells were transformed in comparison with uninfected myeloid cells since they were less adherent and had unlimited proliferative capacity and reduced growth factor requirements. The transformed myeloid cells with proliferative potential were actually myeloid progenitors which apparently underwent terminal differentiation to macrophages. It was possible to derive a permanent line of factor-independent macrophages from M-MuLV(myc)-transformed myeloid cells. M-MuLV(myc) also immortalized and morphologically transformed mouse embryo fibroblasts. These in vitro properties closely resembled the biological activity of MC29 virus in avian cells and suggested that the nature of the v-myc oncogene was an important determinant in transformation specificity. Neonatal NIH Swiss mice inoculated intraperitoneally with M-MuLV(myc)-M-MuLV only developed lymphoblastic lymphoma characteristic of the M-MuLV helper alone, and no acute fibrosarcomas or myeloid tumors resulted. In light of the strong myeloid transformation observed in vitro, the absence of acute in vivo myeloid disease was noteworthy. Interestingly, when a derivative of M-MuLV(myc) carried by a nonpathogenic amphotropic MuLV helper was inoculated, T lymphomas developed with long latency. Molecular hybridization confirmed that these tumors contained M-MuLV(myc).     

Production of replication-defective retrovirus by transient transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRalpha, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80 degrees C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.     

Cell properties after repeated transplantation of spontaneously and of SV40 virus transformed mouse cell lines. I. Growth in culture.

"Spontaneously" or SV40 virus transformed AL/N mouse cell lines were passed repeatedly through syngeneic mice. Cell lines were re-established in culture from minced pieces of tumors in the presence of concentrated fetal calf serum or from tumor cells dispersed by trypsin. The aim of this study was to compare the two cell lines in regard to the selection processes which operate during such procedures by characterization of the resulting cell lines. Measurements of growth in tissue culture on substratum showed no significant difference between any of the transformed cell lines. The SV40 transformed cells and its derivative cells had a low anchorage requirement for growth. The greatest anchorage requirement for growth was in the normal untransformed cells and in the derivative cells from the "spontaneously" transformed cells which were established from minced tumors. The spontaneously transformed cells and all derivative cells had high tumorigenicity (TD50 is less than 10-2). The SV40 transformed cells had no observable tumorigenicity (TD50 is greater than 10-8), except when injected into irradiated mice (TD50 = 1-5 X 10-5 in the immunocompetent mice, 5 X 10-4 in the irradiated mice). The SV40 transformed derivative cells maintained their SV40 specific T antigen and their susceptibility to lysis by specific antiserum.     

Isolation of sarcoma virus from noninbred rats

The data are presented on the activation of endogenous retrovirus in vaccinia virus--malignant transformed cells of rat tissue culture. Infection of the cells by Mazurenko mouse leukemia virus induced rat sarcoma virus. The latter was formed as a result of recombination of sarcoma virus-specific sequences received from the rat cells malignantly transformed by vaccinia virus and virus-helper (Mazurenko mouse leukemia virus).     

An E mu-v-abl transgene elicits plasmacytomas in concert with an activated myc gene.

To clarify how the v-abl oncogene of Abelson murine leukemia virus contributes to lymphoid tumorigenesis, we introduced the gene linked to an immunoglobulin heavy chain enhancer (E mu) into the mouse germline. Although lymphoid development was not detectably affected in young E mu-v-abl mice, three transgenic lines shared a high predisposition to develop clonal plasmacytomas that secreted IgA or IgG. The unexpected absence of pre-B lymphomas suggests that Abelson virus generates such tumors by infecting an early lymphoid progenitor cell that has not yet activated the heavy chain enhancer. Most plasmacytomas bore a rearranged c-myc gene, apparently as a result of spontaneous translocation to the Igh locus. Moreover, progeny of a cross with analogous E mu-myc mice rapidly developed oligoclonal plasmacytomas. Thus, the collusion of v-abl with c-myc is stage specific, efficiently transforming plasma cells but not pre-B cells or B cells.     

Restoration of anchorage regulation in transformed cells by retinoic acid (RA) is independent of the presence of cytoplasmic RA-binding proteins

In an attempt to analyse the cause-effect relationship between anchorage-independent growth (a property which correlates best with in vivo tumorigenicity) and a set of other common transformation-related properties, the effect of retinoic acid (RA) treatment on six unrelated transformed cell lines (including DNA tumor virus, retrovirus, and spontaneously transformed cells) were studied. The data show that the changes in morphology and cellular orientation in culture, loss of cell surface fibronectin, disruption of actin microfilaments, increased hexose uptake, loss of density-dependent growth, and decreased binding of EGF, properties which are often associated with oncogenic transformation of cells, are dissociable from one another and from anchorage-independent growth. RA appears to interfere with anchorage-independent growth of all the retrovirus and spontaneously transformed cell lines (responsive cells) that we examined; however, such treatment failed to inhibit anchorage-independent growth in both of the DNA tumor virus-transformed cell lines (non-responsive cells) that we used in the present study. The presence of RA-binding proteins in both responsive and non-responsive cells suggests that the mechanism of RA action for the inhibition of anchorage-independent growth in transformed cells may be independent of the presence of such cytoplasmic proteins. Finally, the present study clearly indicates that the use of RA treatment, like partial transformation mutants of oncogenic viruses, can be a novel approach in analysing the general mechanism by which transformed cells grow without anchorage.     

Induction of tumorigenesis and chromosomal abnormalities in human amniocytes infected with simian virus 40 and kirstein sarcoma virus

Summary Cell cultures of epithelial-like human amniocytes were infected with simian virus 40 (SV40) and Kirsten sarcoma virus (KSV) in various sequential orders, and tested for agar growth, chromosome abnormalities, and tumorigenesis in the nude mouse assay. We observed that regardless of the order in which the viruses were introduced, the doubly infected cells always exhibited the typical SV40 premalignantly transformed phenotype before changing to the malignant phenotype. All doubly transformed cells from different cell donors produced tumors in adult and suckling nude athymic mice, classified as poorly differentiated sarcomas. Infection with SV40 alone conferred extended life span and accelerated growth without, the malignant capability of tumor production. Kirsten sarcoma virus alone produced only focal cell alteration with no change in cell longevity or tumorigenesis. Chromosome studies of the premalignant and malignant cells from one cell donor did not reveal any significant clonal development for marker chromosomes in either cell line. Chromosome 12, which carries the homologous cellular oncogene to KSV, had no increase in aberrations in the malignant cells. Chromosome 8 was most often involved in aberrations, and the most frequent aberration for both series was dicentric chromosomes due to telomere fusion. For other translocations the breakpoints were almost exclusively in the centromere regions. The vulnerability of telomere and centromere regions to the free virus present in these precrisis cells is discussed, and similarities in regard to types of aberrations in transfection experiments are noted.     

Addition of constitutive c-myc expression to Abelson murine leukemia virus changes the phenotype of the cells transformed by the virus from pre-B-cell lymphomas to plasmacytomas.

Abelson murine leukemia virus (A-MuLV), a retrovirus that expresses the v-abl oncogene, characteristically induces pre-B-cell lymphomas following in vivo infection of BALB/c mice or in vitro infection of suspensions of fetal liver or bone marrow cells. ABL-MYC, a retrovirus that expresses both v-abl and c-myc, induces solely plasmacytomas in BALB/c mice. To investigate how the addition of overexpression of c-myc to that of v-abl accomplishes this dramatic change in the phenotype of the cells transformed by these closely related retroviruses, we utilized helper-free A-MuLV (psi 2) and ABL-MYC (psi 2) in vitro to infect suspensions of cells from different lymphoid tissues and purified immature and purified mature B cells. As expected, A-MuLV(psi 2) induced only pre-B-cell lymphomas in vivo and in vitro when immature B cells were present. ABL-MYC(psi 2), on the other hand, produced only plasmacytomas, even when purified immature B lymphocytes were infected in vitro. Although the A-MuLV(psi 2)-induced pre-B-cell lymphomas express easily detectable levels of c-myc mRNA, maturation into more-mature forms of B lymphocytes is blocked. The constitutively overexpressed c-myc in the ABL-MYC retrovirus abrogates this block, permits maturation of infected immature B cells, and yields transformed plasma cells.