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-.     

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).     

A murine recombinant retrovirus containing the src oncogene transforms erythroid precursor cells in vitro.

A murine retrovirus (MRSV) containing the src gene of Rous sarcoma virus has been shown to cause an erythroproliferative disease in mice (S. M. Anderson and E. M. Scolnick, J. Virol. 46:594-605, 1983). We now demonstrate that this same virus can transform erythroid progenitor cells in vitro. Infection of fetal liver cells or spleen and bone marrow cells from phenylhydrazine-treated adult mice gave rise to colonies of erythroid cells which grew in methylcellulose under conditions not favorable for the growth of normal erythroid cells. The presence of pp60src in the transformed erythroid cells was demonstrated by an immune complex protein kinase assay. The time course of appearance and subsequent differentiation of erythroid colonies indicated that the target cell for MRSV was a 6- to 8-day burst-forming unit. Differentiation of the erythroid progenitors was not blocked by the presence of pp60src, and the cells retained sensitivity to the hormone erythropoietin. In fact, the transformed cells exhibited increased hormone sensitivity since the number, the size, and the extent of hemoglobinization of the colonies were all increased by the addition of small amounts of erythropoietin. MRSV was not susceptible to restriction by the Fv-2 locus, as MRSV could transform hematopoietic cells from C57BL/6 mice. These results indicate that (i) the erythroid proliferation observed in vivo is caused by a direct effect of MRSV on erythroid progenitors and (ii) the transformed erythroid precursors acquire a growth advantage over uninfected cells without losing the ability to differentiate and respond to physiologic regulators.     

Immortalization of cloned mouse splenic macrophages with a retrovirus containing the v-raf/mil and v-myc oncogenes

The recombinant retrovirus J2, which contains the v-raf/mil and v-myc oncogenes, was used to immortalize mouse splenic macrophages that had been cloned in soft agar. When added to freshly harvested colonies, J2 failed to yield cell lines but it immortalized up to 30% of the clones if they had been maintained for at least 4 months in medium containing colony-stimulating factor 1 (CSF-1). All of the cell lines grew in agar in a CSF-1-independent manner, and they produced tumors in nude and syngeneic mice. The cell lines were judged to be macrophage based on morphological criteria and because they secreted lysozyme, were phagocytic for antibody-coated particles, and expressed both the Mac-1 antigen and the CSF-1 receptor. The cell lines could be divided into three groups based on their expression of Ia and their ability to present an antigen to a T-cell hybridoma. The majority of the lines did not constitutively express Ia or present antigen, but a lymphokine did induce Ia in all of the lines, with most of them also acquiring antigen-presenting activity. However, a small proportion of lymphokine-treated lines continued to lack antigen-presenting activity despite their ability to express Ia. The third and smallest group of cell lines constitutively expressed both Ia and antigen-presenting activity. These results show that the J2 recombinant retrovirus is a useful means of immortalizing functionally distinct populations of cloned splenic macrophages.     

Discontinuities in the DNA synthesized by an avian retrovirus

The unintegrated linear DNA synthesized in cells infected by Rous sarcoma virus is a predominantly double-stranded structure in which most of the minus-strand DNA, complementary to the viral RNA genome, is genome sized, whereas the plus-strand DNA is present as subgenomic fragments. We previously reported the application of benzoylated naphthoylated DEAE-cellulose chromatography to demonstrate that of the linear viral DNA species synthesized in quail embryo fibroblasts infected with Rous sarcoma virus greater than 99.5% contain single-stranded regions and these regions are predominantly composed of plus-strand DNA sequences (T. W. Hsu and J. M. Taylor, J. Virol. 44:47-53, 1982). We now present the following additional findings. (i) There were on the average 3.5 single-stranded regions per linear viral DNA, and these single-stranded regions could occur at many locations. (ii) With a probe to the long terminal repeat, we detected, in addition to a heterogeneous size distribution of subgenomic plus-strand DNA species, at least three prominent discrete size classes. Each of these discrete species had its own specific initiation site, but all had the same termination site. Such species were analogous to those reported by Kung et al. (J. Virol. 37: 127-138, 1981). (iii) These discrete size classes of plus-strand DNA were present not only on the major size class of linear DNA but also on a heterogeneous of slower-sedimenting species, which we have called immature linears. Our interpretation is that we have thus detected several additional sites for the initiation of plus-strand DNA. (iv) The 340-base plus-strand strong-stop DNA was only found associated with the immature linears. (v) From a size and hybridization comparison of these discrete size classes of plus-strand DNA with minus-strand DNA species, as synthesized in the endogenous reaction of melittin-disrupted virions, it was found that the putative additional initiation sites for plus-strand DNA synthesis corresponded to many of the pause sites in the synthesis of minus-strand DNA.     

Comparison of promoter suppression in avian and murine retrovirus vectors.

Previously, we described "promoter suppression" in infectious retrovirus vectors with two genes and an internal promoter. Here, we examined several parameters of promoter suppression and found that the amount of suppression in an integrated retrovirus vector was dependent both on whether the vector was derived from spleen necrosis virus or murine leukemia virus and on which internal promoter was present in the vector. Murine leukemia virus vectors showed less suppression than analogous spleen necrosis virus vectors. Furthermore, the amount of suppression was not dependent on either the relative strengths of the promoters nor the distance between the promoters. Moreover, we found that in vectors in which one promoter was suppressed, there was an inverse correlation between the DNaseI sensitivity of the chromatin surrounding a promoter and the suppression of its expression.     

Sequences in human cytomegalovirus which hybridize with the avian retrovirus oncogene v-myc are G + C rich and do not hybridize with the human c-myc gene.

The degree of relatedness between previously identified cross-hybridizing regions within human cytomegalovirus strain AD169 and the avian retrovirus oncogene v-myc were investigated by nucleotide sequence comparison. We found that the homologous regions between the human cytomegalovirus genome and v-myc are limited to short G + C-rich regions in each genome and that the human cytomegalovirus genome shares little or no homology with the human c-myc gene.     

Apoptosis of murine peritoneal macrophages induced by an avian pathogenic strain of Escherichia coli

Ehrlichiae are obligatory intracellular, Gram-negative bacteria which belong to the alpha subclass of the phylum Proteobacteria and are responsible for infectious diseases of humans. Little is known about genetics and genomic organization of Ehrlichia spp. The genome sizes of four representatives of the genus Ehrlichia were determined for the first time by pulsed field gel electrophoresis. The sizes for E. sennetsu, E. risticii, E. chaffeensis (strain Arkansas and strain 91HE17), and the HGE agent were 878.5 kb, 880.3 kb, 1225.8 kb, 1262.3 kb and 1494 kb respectively.     

Transformation of quail embryo fibroblasts by a retrovirus carrying a normal human c-myc gene.

We have constructed avian retroviruses expressing the human c-myc oncogene. These viruses morphologically transformed primary quail embryo fibroblasts upon transfection and infection. Transformed cells produced viruses harboring a spliced c-myc gene and contained high levels of p64-67c-myc protein. One of these infectious viruses, vSX-AHM, was molecularly cloned and the nucleotide sequence of the spliced c-myc insert determined. No mutation was found within the c-myc coding sequence of this transforming clone when compared to the normal genomic progenitor. Thus, we concluded that no mutation within the human c-myc gene is required to induce primary avian embryo fibroblast transformation.     

Efficient autointegration of avian retrovirus DNA in vitro.

We have developed a cell-free system for an avian retrovirus that promotes autointegration, one-long-terminal-repeat (LTR) circle formation, and correct integration into exogenous target DNA. In this system, autointegration and one-LTR circle formation occurred far more frequently than integration into exogenous target DNA. Autointegration had the same characteristics of normal integration into target DNA except in its selection of target. Highly efficient autointegration as well as one-LTR circle formation in vitro suggest that there may be a mechanism to prevent these processes in vivo.     

Molecular cloning of the intronless EJ ras oncogene using a murine retrovirus shuttle vector

We have inserted a genomic clone of the human EJ bladder oncogene into a murine retrovirus shuttle vector. Cotransfection of this shuttle vector DNA containing the activated ras oncogene with molecularly cloned Moloney murine leukemia virus into NIH/3T3 cells was able to rescue a replicating and transforming retrovirus. The viral DNA from infected cells was excised by fusion to mouse COP-5 cells and recloned into Escherichia coli as plasmid DNA. Analysis of the recloned plasmids by size, restriction enzyme mapping, and DNA sequence indicated that approximately 5% of the recloned plasmids contained the intronless EJ ras oncogene.     

Replication of endogenous avian retrovirus in permissive and nonpermissive chicken embryo fibroblasts.

Clones of chicken embryo fibroblasts exogenously infected with the endogenous avian retrovirus were analyzed to examine the replication of this virus in permissive (Gr+) and nonpermissive (Gr-) cells. The results demonstrate that the endogenous virus was capable of infecting both Gr+ and Gr- cells with equal efficiency. Infected clones of Gr+ and Gr- cells differed, however, in two significant ways. At the time of their initial characterization, the Gr+ clones produced 100- to 1,000-fold more virus than the Gr- clones. Further, the amount of virus produced by Gr+ clones did not change significantly during serial passage of the cells. In contrast, continued passage of the infected Gr- clones resulted in a gradual increase in the amount of virus produced. Individual clones of infected Gr- cells produced infectious virus at rates that, initially, differed by a factor of more than 10(4). The large differences in the production of virus by these clones could not be explained by equally large differences in the number of infected cells within the clonal populations. Greater than 80% of the clonal populations examined ultimately produced virus at rates that were not significantly different from the rates observed in infected Gr+ cells. Virus produced by these infected Gr- cells exhibited the same restricted replication upon establishing a new infection in nonpermissive cells. Analysis of the appearance of free and integrated viral DNA sequences during endogenous virus infection of Gr+ and Gr- cells demonstrated that, after an initial delay in the synthesis of free viral DNA in Gr- cells, the nonpermissive cells ultimately acquired as many integrated viral DNA sequences as were found in infected Gr+ cells. These results indicate that a majority of the infectious particles of the endogenous virus are capable of establishing infection in a Gr- cell and, ultimately, of producing virus at a rate that is not significantly different from that produced by infected Gr+ cells. The virus produced from the Gr- cells is not a stable genetic variant of the original endogenous virus that is capable of unrestricted replication in nonpermissive cells. The reduced efficiency with which the endogenous virus initially replicates in nonpermissive cells and the increased length of time required for infected Gr- cells to produce maximal virus titers suggest that the endogenous virus may utilize a different mechanism of replication in Gr+ and Gr- fibroblasts.     

Studies on the recognition of xenogeneic cells by nonimmune macrophages. I. Destruction of chicken fibroblasts in vitro by murine macrophages.

Human cord blood lymphocytes were compared with adult lymphocytes with regard to proportions of cells with surface markers for surface immunoglobulin (Ig), receptors for C′3 and the Fc-portion of IgG, as well as two types of erythrocyte rosettes (rapid and late E-rosettes). A significant decrease (P < 0.02 ? 0.05) in both early and late E-rosettes was noted when cord cells were compared to adult lymphocytes. After 20 hr of incubation at 37 °C, proportions of cells bearing Fc receptors in cord blood samples showed striking increments (P < 0.001) when compared with adult lymphocytes. T cell enrichment studies and sequential depletion of cells bearing Fc receptors as well as E-rosette forming cells indicated that the precursors of cells generating Fc receptors in vitro did not arise from cells with Fc receptors or T cell markers.     

Temperature-sensitive mutants of MH2 avian leukemia virus that map in the v-mil and the v-myc oncogene respectively.

MH2 is an avian retrovirus that contains the v-mil and v-myc oncogenes. In vitro it transforms chick macrophages that are capable of proliferation in the absence of growth factor. Earlier work showed that v-myc induces macrophage transformation and that v-mil induces the production of chicken myelomonocytic growth factor (cMGF), thus generating an autocrine system. We describe the isolation of temperature-sensitive (ts) mutants of MH2 virus. As suggested by marker rescue experiments, one mutant bears a ts lesion in v-mil, whereas the other carries a mutation in v-myc. Ts v-mil MH2-transformed macrophages become factor-dependent at the non-permissive temperature (42 degrees C), while ts-v-myc MH2-transformed macrophages cease growing and acquire a more normal macrophage phenotype at 42 degrees C irrespective of the presence of cMGF. Both phenotypes can be reversed by backshift to the permissive temperature. These results suggest that the gene products of v-mil and v-myc function independently of each other and that v-mil is necessary for the maintenance of autocrine growth, whereas v-myc is required to maintain the transformed phenotype.     

Transformation of diploid human fibroblasts by DNA transfection with the v-sis oncogene

The simian sarcoma virus (SSV) oncogene (v-sis) has a high degree of homology to the cellular gene coding for the B peptide of human platelet-derived growth factor (PDGF), a potent fibroblast mitogen. The cellular homolog of v-sis is activated in some mesenchymal human tumors and cell lines derived from them. To determine the phenotype produced by v-sis in diploid human fibroblasts, we constructed plasmids containing the SSV provirus and drug-resistance markers and transfected them into early-passage human cells. Fibroblasts that had integrated the plasmid were selected for drug resistance and shown to contain and express the v-sis oncogene by DNA and RNA hybridization. The v-sis-expressing cells grew to higher saturation densities than control cells transfected with the vector plasmid alone and formed large, well defined foci. This allowed selection of transfectants directly for focus formation. The v-sis transformed cells continued to grow well in the absence of serum, whereas age-matched, vector-transfected control cells ceased replicating under these conditions so that the final difference in density between the two populations was tenfold. Incorporation of thymidine in serum-free medium by the v-sis-transformed cells was independent of exogenous PDGF. In contrast, PDGF increased thymidine incorporation in such medium by the control cells to the level found in the v-sis-transformed cells with or without added PDGF. These results suggest that expression of the v-sis oncogene in diploid human fibroblasts causes sufficient endogenous synthesis of the B chain of PDGF to allow transformants to grow to abnormally high cell densities. When individual v-sis-transformed cells were grown on a background of normal cells, this higher cell density at confluence could be visualized as a focus.     

Transformation of chicken myelomonocytic cells by a retrovirus expressing the v-myb oncogene from the long terminal repeats of avian myeloblastosis virus but not Rous sarcoma virus.

To test the effect of long terminal repeat (LTR) regulatory sequences on the transforming capability of the v-myb oncogene from avian myeloblastosis virus (AMV), we have constructed replication-competent avian retroviral vectors with nearly identical structural genes that express v-myb from either AMV or Rous sarcoma virus (RSV) LTRs. After transfection into chicken embryo fibroblasts, virus-containing cell supernatants were used to infect chicken myelomonocytic target cells from preparations of 16-day-old embryonic spleen cells. Both wild-type AMV and the virus expressing v-myb from AMV LTRs (RCAMV-v-myb) were able to transform the splenocyte cultures into a population of immature myelomonocytic cells. The transformed cells expressed the p48v-Myb oncoprotein and formed compact foci when grown in soft agar. In contrast, the virus expressing v-myb from RSV LTRs (RCAS-v-myb) was repeatedly unable to transform the same splenocyte cells, despite being able to infect fibroblasts with high efficiency. This difference in the transforming activities of v-myb-expressing viruses with different LTRs most likely results from the presence of a factor (or factors) within the appropriate myelomonocytic target cell that promotes specific expression from the AMV but not from the RSV LTR.     

Polyhormonal regulation of avian and mammalian corticosteroidogenesis in vitro

1. The combined actions of ACTH, corticosterone and prolactin (PRL) in the acute regulation of corticosteroidogenesis were investigated using isolated adrenocortical cells from intact and hypophysectomized (hypox) rats (Rattus norvegicus) and from intact male domestic fowl (Gallus gallus domesticus). 2. Exogenous corticosterone suppressed to about 50% ACTH-induced corticosterone production of cells from either species. This suppression, in part, was due to corticosterone degradation. 3. oPRL, in the presence or absence of ACTH, raised corticosterone production of hypox rat cells, but not intact rat and domestic fowl cells. 4. In addition, oPRL counteracted the corticosterone-induced suppression of net ACTH-stimulated corticosterone production of hypox rat and intact domestic fowl cells, but not intact rat cells. 5. The potency of oPRL with domestic fowl cells was 4 times that with hypox rat cells. 6. Furthermore, in domestic fowl cells, the effect of oPRL was Ca2+-dependent.