Characterization of lymphoid tumors induced by a recombinant murine retrovirus carrying the avian v-myc oncogene. Identification of novel (B-lymphoid) tumors in the thymus

Lymphoid tumors induced by a recombinant murine retrovirus carrying the v-myc oncogene of avian MC29 virus were characterized. The Moloney murine leukemia virus myc oncogene (M-MuLV (myc], carried by an amphotropic MuLV helper, induced tumors in NIH Swiss and NFS/N mice after a relatively long latency (8 to 24 wk). Tumor masses appeared in the thymus, spleen, and lymph nodes. Flow cytometry of the tumor cells indicated that approximately 50% were positive for Thy 1.2. Most of these tumors also expressed one or more other cell surface markers of thymocytes and mature T cells (CD4, CD8). Southern blot hybridization revealed genomic rearrangements for the TCR beta genes. The TCR beta analysis suggested that the M-MuLV(myc)-induced Thy 1.2+ tumors were derived from somewhat less mature cells than tumors induced by M-MuLV, which is a classical non-acute retrovirus lacking an oncogene. The remainder of the M-MuLV(myc)-induced tumors were Thy 1.2-, but they were positive for Ly-5 (B220) and also for MAC-2. The Thy 1.2- tumors were characteristically located in the thymus. However, they were negative for TCR beta gene rearrangements. Some, but not all, of the Thy 1.2- tumors contained rearrangements for Ig genes. Additionally, they typically expressed mRNA specific for B but not for T cells. Thus, these thymic tumors had characteristics of the B cell lineage. Tumor transplantation experiments demonstrated that the Thy 1.2- tumor cells could reestablish in the thymus and spleen of irradiated hosts, and low level expression of the Thy 1 molecule was observed in the thymus but not the spleen on the first passage. After serial passage, one Thy 1- tumor altered its cell surface phenotype to Thy 1low B220-.     

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.     

Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

Coordinate regulation of myelomonocytic phenotype by v-myb and v-myc.

Both avian myeloblastosis virus (by the action of v-myb) and avian myelocytomatosis virus MC29 (by the action of v-myc) transform cells of the myelomonocytic lineage. Whereas avian myeloblastosis virus elicits a relatively immature phenotype, cells transformed by MC29 resemble mature macrophages. When cells previously transformed by v-myb were superinfected with MC29, their phenotype was rapidly altered to that of a more mature cell. These superinfected cells expressed both v-myb (at a level similar to that found before superinfection) and v-myc. It therefore appears that the expression of v-myc can elicit certain properties of a more differentiated phenotype. In addition, unlike cells transformed by v-myb alone, the cells expressing both v-myb and v-myc could not be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to differentiate to fully mature macrophages. Cells with a morphology similar to that of the superinfected cells were elicited by simultaneously infecting yolk sac macrophages with avian myeloblastosis virus and MC29. Such cells expressed both v-myb and v-myc. These results indicate that expression of v-myb and v-myc in infected cells coordinately regulates myelomonocytic phenotype and that the two viral oncogenes vary in their ability to interfere with tumor promoter-induced differentiation. Our findings also sustain previous suggestions that the oncogenes v-myb and v-myc may not transform target cells by simply blocking differentiation.     

Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.     

Molecular analysis of myc gene mutants

We describe the generation and characterization of a series of deletion mutants of the avian acute leukaemia virus MC29 which allow the study of the function of the myc in transformation of quail embryo fibroblasts in vitro and tumour induction in vivo. These mutants, which are deleted in the 3' portion of the myc gene, fail to transform macrophages in vitro or induce tumours in vivo but are still able to transform morphologically fibroblasts. From one of these mutants a 'recovered' MC29 virus was generated which, like wild type MC29, transformed fibroblasts and macrophages in vitro. When tested in vivo this virus induced lymphomas of T and B cells rather that the endotheliomas induced by wild type MC29. This system allows us to investigate another question which is the mechanism by which the virus (or oncogene it contains) preferentially transforms one cell type.     

Distinct and different effects of the oncogenes v-myc and v-src on avian sympathetic neurons: retroviral transfer of v-myc stimulates neuronal proliferation whereas v-src transfer enhances neuronal differentiation

Immature avian sympathetic neurons are able to proliferate in culture for a limited number of divisions albeit expressing several neuron- specific properties. The effect of avian retroviral transfer of oncogenes on proliferation and differentiation of sympathetic neurons was investigated. Primary cultures of 6-d-old quail sympathetic ganglia, consisting of 90% neuronal cells, were infected by Myelocytomatosis virus (MC29), which contains the oncogene v-myc, and by the v-src-containing Rous sarcoma virus (RSV). RSV infection, in contrast to findings in other cellular systems, resulted in a reduction of neuronal proliferation as determined by 3H-thymidine incorporation (50% of control 4 d after infection) and in increased morphological differentiation. This is reflected by increased neurite production, cell size, and expression of neurofilament protein. In addition, RSV- infected neurons, unlike uninfected cells, are able to survive in culture for time periods up to 14 d in the absence of added neurotrophic factors. In contrast, retroviral transfer of v-myc stimulated the proliferation of immature sympathetic neurons preserving many properties of uninfected cells. The neuron-specific cell surface antigen Q211 and the adrenergic marker enzyme tyrosine hydroxylase were maintained in MC29-infected cells and in the presence of chick embryo extract the cells could be propagated over several weeks and five passages. Within 7 d after infection, the number of Q211-positive neurons increased approximately 100-fold. These data demonstrate distinct and different effects of v-src and v-myc-containing retroviruses on proliferation and differentiation of sympathetic neurons: v-src transfer results in increased differentiation, whereas v- myc transfer maintains an immature status reflected by proliferation, immature morphology, and complex growth requirements. The possibility of expanding immature neuronal populations by transfer of v-myc will be of considerable importance for the molecular analysis of neuronal proliferation and differentiation.     

Generation of a recombinant Moloney murine leukemia virus carrying the v-src gene of avian sarcoma virus: transformation in vitro and pathogenesis in vivo.

A Moloney murine leukemia virus (M-MuLV) recombinant carrying the v-src gene of avian sarcoma virus was generated by the introduction of a cloned portion of v-src from Schmidt-Ruppin A avian sarcoma virus into a molecular clone of M-MuLV provirus at the recombinant DNA level. The v-src sequences (lacking a portion of the 5' end of v-src) were inserted into the p30 region of the M-MulV gag gene so that M-MuLV gag and v-src were in the same reading frame. Transfection of this chimeric clone, pMLV(src), into NIH 3T3 cells which were constitutively producing M-MuLV gag and pol protein resulted in the formation of foci of transformed cells. Infectious and transforming virus could be recovered from the transformed cells. This virus was designated M-MuLV(src). M-MuLV(src)-transformed cells contained two novel proteins of 78 and 90 kilodaltons. The 78-kilodalton protein, p78gag-src, contained both gag and src determinants, exhibited kinase activity in an immune kinase assay, and is probably a fusion of Pr65gag and src. The 90-kilodalton protein, which is of the appropriate size to be the gPr80gag fused to src, contained gag determinants as well as a V8 protease cleavage fragment typical of the carboxy terminus of avian sarcoma virus pp60src. However, it could not be immunoprecipitated with an anti-v-src serum. M-MuLV(src)-transformed cells showed elevated levels of intracellular phosphotyrosine in proteins, although the elevation was intermediate compared with cells transformed with wild-type v-src. M-MuLV and amphotropic murine leukemia virus pseudotypes of M-MuLV(src) were inoculated into newborn NIH Swiss mice. Inoculated mice developed solid tumors at the site of inoculation after 3 to 6 weeks, with most animals dying by 14 weeks. Histopathological analysis indicated that the solid tumors were mesenchymally derived fibrosarcomas that were both invasive and metastatic.     

Transforming potential of a myc-containing variant of feline leukemia virus in vitro in early-passage feline cells.

We studied a naturally occurring variant of feline leukemia virus (FeLV) in which the oncogene myc has substituted for a portion of the viral structural genes (myc-FeLV). myc-FeLV was rescued by replication in the presence of FeLV as helper, and its biological activity was examined in early-passage feline cells in vitro. Infection of leukocytes from peripheral blood, spleen, or thymus, or of kitten fibroblasts did not immortalize these cells or alter them morphologically. Northern blot (RNA blot) analysis of virion RNA prepared from the supernatant of infected cells demonstrated the 8.2-kilobase genome of FeLV, but did not demonstrate the 5.0-kilobase genome of myc-FeLV. Apparently, the myc-FeLV genome was lost in the absence of the selective pressure of transformation. In contrast, infection of embryonic fibroblasts with myc-FeLV(FeLV) rendered these cells capable of greatly increased, if not infinite, proliferative potential. The cells were morphologically altered compared with controls and were only loosely adherent to the substrate. The cells failed to proliferate in semisolid medium and did not form tumors when inoculated subcutaneously into athymic mice. Blot analyses demonstrated the presence and expression of integrated proviral DNAs of both FeLV and myc-FeLV in these cells. They appear, then, to represent cells partially transformed by infection with myc-FeLV(FeLV). The action of feline v-myc in early-passage cells in vitro was compared to that of avian v-myc.     

Nuclear location of the putative transforming protein of avian myelocytomatosis virus

The putative transforming protein of avian myelocytomatosis virus MC29 is a 110,000 dalton (P110gag-myc) polyprotein comprised of sequences derived from both the gag region and the MC29-specific myc region. Two approaches have been taken to determine the location of the MC29 gag-related proteins in transformed cells: subcellular fractionation and immunofluorescence. Analysis of subcellular fractions of MC29-transformed cells by immunoprecipitation indicates that the majority of the gag-myc polyprotein is found in the nuclear fractions of Q8 cells (a nonproducer line of MC29-transformed quail embryo fibroblasts) and nonproducer cells derived from a liver tumor of MC20-infected quail. This is in contrast to the distribution of gag-related helper virus proteins lacking myc, which are found only in nonnuclear fractions of superinfected Q8 cells. The purity of unlabeled nuclei was assessed by electron microscopy and enzyme assays, revealing little contaminating material from other subcellular fractions. Immunofluorescence experiments using monospecific anti-gag serum showed specific, intense immunofluorescence in the nuclei of fixed Q8 cells. In contrast, the majority of P75gag-erb, a candidate transforming protein produced by avian erythroblastosis virus (AEV), is absent from the nuclei of nonproducer AEV-transformed chick embryo fibroblasts. The nuclear association of the MC29 transforming protein may be related to some of the unique properties of MC29-transformed cells.     

FH3, a v-myc avian retrovirus with limited transforming ability.

We have isolated a new acute avian transforming virus which contains the oncogene myc. This virus, designated FH3, was isolated after injection of a 10-day-old chick embryo with avian leukosis virus. While FH3 shares many properties with other v-myc-containing avian retroviruses, it also has several unique properties. The primary target for transformation in vitro is chicken macrophages; infection of chicken fibroblasts does not lead to complete morphological transformation. FH3 also exhibits a limited host range, in that Japanese quail macrophages and fibroblasts are infected but are not completely transformed. FH3 induces in vivo a limited tumor type if injected into 10-day-old chick embryos; only a cranial myelocytoma, which does not appear to be metastatic, can be detected. The v-myc gene of FH3 is expressed predominantly as a P145 Gag-Myc protein which is encoded by a ca. 8-kilobase genomic RNA. This FH3-encoded polyprotein is localized in the nucleus of all infected cells, whether or not they are transformed.     

Activation of ras and myc proto-oncogenes in human breast carcinoma and neuroblastoma

DNA from human breast carcinoma (SK-BR-3) and neuroblastoma (LA-N-1) cell lines are capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. The blot hybridization analysis of DNA from primary and secondary NIH 3T3 transformants identified additional sequences homologous to the c-Ha-ras 1 oncogene, and revealed amplification of nucleotide sequences homologous to the v-myc oncogene. Restriction fragments of the amplified myc-related sequences correspond to c-myc (SK-BR-3) and N-myc (LA-N-1) loci of the human genome. The results show that active Ha-ras oncogenes can coexist with altered myc oncogenes in breast carcinomas and neuroblastomas. This suggests that a multi-step mechanism involves both ras and myc genes and their cooperation in the development of these tumors.     

Differential induction of cytolytic susceptibility by E1A, myc, and ras oncogenes in immortalized cells.

The E1A oncogene of adenovirus serotypes 2 and 5 induces susceptibility to the cytolytic effects of natural killer lymphocytes and activated macrophages when expressed in infected and transformed mammalian cells (cytolysis-susceptible phenotype). E1A and the oncogenes v-myc, long-terminal-repeat-promoted c-myc, and activated c-ras share the ability to immortalize transfected low-passage rodent cells. The cytolytic phenotypes of well-characterized rodent cell lines immortalized by these three oncogenes were defined. In contrast to target cells expressing the intact E1A gene, myc- and ras-expressing, immortalized primary transfectants were resistant to lysis by both types of killer cell populations. The same patterns of susceptibility (E1A) and resistance (myc and ras) to cytolysis were observed in oncogene-transfected continuous rat (REF52) and mouse (NIH 3T3) cell lines, indicating that differences in the cytolytic phenotypes associated with expression of these oncogenes are not due to cell selection during immortalization. The results suggest that the E1A oncogene may possess a functional domain that is different from those of other oncogenes, such as myc and ras, and that the activity linked to this postulated domain is dissociable from the process of immortalization.     

Myeloblasts transformed by the avian acute leukemia virus E26 are hormone-dependent for growth and for the expression of a putative myb-containing protein, p135 E26.

Avian leukemia virus E26 contains the myb oncogene and transforms erythroid and myeloid hematopoietic cells in vivo and in vitro. E26-transformed nonproducer myeloblasts but not avian erythroleukemia virus (AEV)-transformed erythroblasts nor MC29-transformed macrophages were shown to be dependent for growth on factor(s) present in supernatants from Concanavalin A-stimulated chicken spleen cells. The same factor enhanced the synthesis of p135 E26, the candidate transforming protein of E26, but did not induce the synthesis of the transforming proteins of AEV and MC29 viruses nor that of helper virus-derived structural proteins. P135 E26 was shown to contain sequences related to the viral gag gene as well as sequences which may be related to the myb gene product. P135 E26 might constitute the first example of a viral onc protein whose synthesis is regulated directly or indirectly by an exogenous hematopoietic growth factor.     

Transformation by myc prevents fusion but not biochemical differentiation of C2C12 myoblasts: mechanisms of phenotypic correction in mixed culture with normal cells

To study the effects of myc oncogene on muscle differentiation, we infected the murine skeletal muscle cell line C2C12 with retroviral vectors encoding various forms of avian c- or v-myc oncogene. myc expression induced cell transformation but, unlike many other oncogenes, prevented neither biochemical differentiation, nor commitment (irreversible withdrawal from the cell cycle). Yet, myotube formation by fusion of differentiated cells was strongly inhibited. Comparison of uninfected C2C12 myotubes with differentiated myc- expressing C2C12 did not reveal consistent differences in the expression of several muscle regulatory or structural genes. The present results lead us to conclude that transformation by myc is compatible with differentiation in C2C12 cells. myc expression induced cell death under growth restricting conditions. Differentiated cells escaped cell death despite continuing expression of myc, suggesting that the muscle differentiation programme interferes with the mechanism of myc-induced cell death. Cocultivation of v-myc-transformed C2C12 cells with normal fibroblasts or myoblasts restored fusion competence and revealed two distinguishable mechanisms that lead to correction of the fusion defect.     

Genome structure of HBI, a variant of acute leukemia virus MC29 with unique oncogenic properties.

We have analyzed the viral RNA of a variant of avian acute leukemia virus MC29, termed HBI. This virus was isolated during in vitro passage of a partially transformation-defective (td) mutant of MC29 (td10H-MC29) in chicken macrophages. While td10H-MC29 has a reduced ability to transform macrophages in vitro or to induce tumors in vivo, HBI-MC29 transforms macrophages efficiently and induces in vivo a high incidence of lymphoid tumors. Electrophoretic analysis of HBI-MC29 genomic RNA revealed that it has a complexity of 5.7 kilobases, like the RNA of wild-type (wt) MC29, and that it is 0.6 kilobases longer than the 5.1-kilobase RNA of the deletion mutant td10H-MC29. Analysis of the viral RNAs of two clonal isolates of HBI-MC29 by T1 oligonucleotide fingerprinting showed that sequences from the viral transformation-specific region, v-myc, which are deleted in td10H RNA, are present in HBI RNA. Moreover, hybridization of HBI RNA to molecularly cloned subgenomic fragments of wtMC29 proviral DNA, followed by fingerprint analysis of hybridized RNA, showed that the entire v-myc-specific RNA sequences defined previously are present. Hybridization to cloned DNA of the normal chicken locus c-myc shows a close relationship between HBI v-myc RNA and c-myc DNA, especially in the sequences which were deleted from td10H-MC29. T1 oligonucleotide maps of HBI and td10H RNAs were prepared and compared. Total conservation of the oligonucleotide pattern is observed in the overlapping v-myc regions, while the partial structural genes gag and env show some variations, most of which can be directly proven to be due to point mutations or recombination with helper viral RNAs that were analyzed in parallel. Recombination of td10H-MC29 with c-myc, followed by recombinational and mutational changes in the structural genes during passage with helper virus, could be a possible explanation for the origin of HBI.     

Quail embryo fibroblasts transformed by four v-myc-containing virus isolates show enhanced proliferation but are non tumorigenic.

Quail embryo fibroblasts infected with any of the four natural avian myc gene-containing virus strains (MC29, CMII, OK10 and MH2) or with the myb, ets-containing E26 acute leukemia virus, were examined for their expression of several transformation-associated parameters. All myc-containing viruses, but not E26 or Rous sarcoma virus (used as a control) induced a dramatic stimulation of cell proliferation. In addition, the myc virus-transformed cells exhibited prominent nucleoli, possibly as a consequence of their increased proliferation. Cells transformed by MC29, OK10, MH2 and E26 were capable of growing in semi-solid medium and showed a loss of actin cables and, in most cases, of an ordered fibronectin distribution. All of the myc virus-transformed fibroblasts, as well as the E26-transformed cells, were unable to form tumors in nude mice, indicating that the myc gene (and the myb/ets genes) are not sufficient for the induction of a fully malignant phenotype in avian fibroblasts.     

Isolation and characterization of the human cellular myc gene product

Antibodies against the product of the human cellular myc gene (c-myc) were prepared against a bacterially expressed human c-myc protein by inserting the ClaI/BclI fragment of the human c-myc DNA clone in an expression vector derived from pPLc24. These antibodies cross-react with viral-coded myc (v-myc) proteins from MC29 and OK10 viruses. Furthermore, IgGs specific for synthetic peptides, corresponding to the 12 carboxy-terminal amino acids of the human c-myc gene and 16 internal amino acids, were isolated. By use of the various myc-specific antisera or IgGs, a protein of Mr 64 000 was detected in several human tumor cell lines including Colo320, small cell cancer of the lung (417d), HL60, Raji, and HeLa. This protein is larger than the corresponding v-myc or chicken c-myc proteins from avian virus transformed cells or avian bursa lymphoma cells (RP9), both of which are proteins of Mr 55 000. The human c-myc protein is located in the nucleus of Colo320 cells, exhibits a half-life of about 15 min, and is expressed at significantly lower levels than the viral protein. The human c-myc protein was enriched about 3000-fold from Colo320 cells using c-myc-specific IgG coupled to Sepharose beads. The protein binds to double-stranded DNA in vitro, a reaction that can be inhibited to more than 90% by c-myc specific IgG.