Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages.

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.     

Immortalization of murine primary spleen cells by v-myc, v-ras, and v-raf.

Growth factor-independent cell lines, including four lines characterized as macrophages, were isolated by infection of BALB/c mouse primary spleen cells with combinations of three retroviruses encoding v-myc, v-ras, and v-myc/v-raf. Proliferating cell lines were isolated only rarely, and after long crisis periods, following the introduction of myc and raf by infection with J2 virus, or of myc and ras by coinfection with myc309 and raszip6 viruses. However, sequential infections with all three viruses--myc plus ras cells reinfected with J2, or J2 followed by myc plus ras coinfection--resulted in rapid outgrowth of cell lines which grew at high growth rates to high densities. When cells were treated with anti-IgG F(ab')2/IL-4/IL-5 to specifically stimulate B cells, cell lines were isolated readily by infection with myc plus ras alone, J2 alone, or all three viruses. These cell lines arose after shorter crisis times and all grew at high growth rates and to high densities. Analysis of cell surface markers and immunoglobulin gene arrangement revealed no lymphoid characteristics in any of the lines. Four cell lines express all three macrophage markers analyzed (F4/80, Mac1, FcR), and many others are Mac1+ and/or FcR+. Out of 20 immortalized cell lines tested, 13 show clonal growth in soft agar, and 3/6 of these produced tumors in BALB/c mice, indicating that fully transformed cells may be isolated by these procedures. In at least one of the cell lines, integration of all three infecting viruses has occurred.     

Activity of an NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase in normal tissue, neoplastic cells, and oncogene-transformed cells

As an extension of the previously reported observation concerning the existence of NAD-dependent 5,10-methylenetrahydrofolate dehydrogenase in transformed cells a variety of tissues and cell lines have been assayed for this activity. This activity was found in all assayed transformed cells. Results with rat liver derived epithelial (RLE) cells transformed with a series of oncogenes (v-raf, v-raf/v-myc (J2), v-myc (J5), and v-Ha-ras (pRNR16)) indicated that expression of activity correlates with the extent of transformation and was independent of the oncogene used for transformation. Compared to previously reported values for normal tissue, surprisingly high levels of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase were found in the rat adrenal cortex. This activity was not seen in mouse or bovine adrenal. Enzymatic activity was also detected in mouse bone marrow and was strain dependent. The levels of activity in mouse bone marrow were lower than previously reported. The NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase activity in rat adrenal and RLE cells may represent tools for studying the regulation of expression of this activity.     

Activated v-myc and v-ras oncogenes do not transform normal human lymphocytes.

Activated v-myc (pSV v-myc) and v-Ha-ras (GT10) oncogenes were introduced into normal human lymphocytes, NIH 3T3 fibroblasts, B-lymphoblastoid cells, and human epithelial cells, using a reconstituted Sendai virus envelope-mediated gene transfer technique. Efficient transfer of the plasmid in each cell type was demonstrable within 1.5 h of transfection by Southern blotting of extrachromosomal DNA extracts, which unexpectedly revealed that v-myc plasmid DNA was unstable in normal lymphocytes but not in the other cell types. The v-myc plasmid was stabilized when cotransfected into lymphocytes together with v-Ha-ras. The transfected v-Ha-ras plasmid was stable in all the cell types tested. v-myc plasmid expression was clearly detectable by 5 h in all cell types except human lymphocytes. Lymphocytes expressed v-myc when transfected together with v-Ha-ras. Transfected ras oncogene was efficiently expressed in all the cell types tested. Expression of the transfected genes increased at 24 and 48 h after transfection. Even though plasmid stability and expression were achieved in myc-ras-cotransfected lymphocytes, no effects on cellular DNA synthesis or immortalization were observed, in contrast to efficient transformation of NIH 3T3 fibroblasts by the same procedure. Our data suggest that efficient expression of transfected myc and ras oncogenes in normal quiescent human lymphocytes is not sufficient for the induction of cell growth and immortalization.     

Generation of macrophage cell line from fresh bone marrow cells with a myc/raf recombinant retrovirus

We have studied the effects of infection of fresh murine bone marrow (BM) cells by recombinant retroviruses carrying v-raf and v-myc oncogenes, either alone or in combination. Viruses containing v-raf or v-myc alone failed to induce BM proliferation in 24 out of 27 experiments performed so far, only the J2 virus containing both v-raf and v-myc oncogenes induced BM proliferation. Exogenous growth factors (GF) were not required to sustain the mitogenic effect of J2 virus. Infection with retroviruses carrying only v-raf or v-myc did not induce BM cell growth, indicating that co-expression of the two oncogenes was needed to provide the mitogenic signal(s) for BM proliferation. The kinetics of growth of the J2 virus-infected cells (J2 cells) were characteristically biphasic. The initial burst of proliferation was always followed by a quiescent phase culminating in cell death, which could not be reversed by addition of exogenous GF. In contrast, active proliferation of the quiescent monolayers could be restored by addition of dextran-based beads to the cultures, showing that the growth arrest of J2 cells was a reversible process. J2 cells actively growing in the presence of CT-beads could be expanded and cloned and subsequently grew continuously independent of the CT-beads. Eighteen clones obtained from different infections were all macrophages (M phi) by morphological criteria and all of them expressed the same membrane phenotype compatible with M phi, demonstrating that J2 virus infection leads to immortalization of the same BM-derived monocytic subpopulation. When injected in vivo, J2 cells produced histiocytic tumors in nude mice, but did not grow in immunocompetent syngeneic mice. The cells induced to proliferate in vitro in response to J2 virus infection appeared to be limited to the BM compartment, since spleen cells, thymocytes, peritoneal M phi and liver large granular lymphocytes did not grow in vitro in response to J2 virus. The immortalization of BM cells by J2 virus infection represents a novel reproducible experimental system to deliberately generate M phi lines, which proliferate in response to viral oncogenes and do not require exogenous GF to initiate or to sustain their continuous proliferation.     

Ras complements the carboxyl terminus of v-Abl protein in lymphoid transformation.

Abelson murine leukemia virus (Ab-MLV) mutants expressing v-Abl proteins lacking the carboxyl terminus are compromised in the ability to transform lymphoid but not NIH 3T3 cells. This feature correlates with the presence of low levels of phosphotyrosine in lymphoid cells infected with carboxyl-terminal truncation mutants. In contrast, high levels of phosphotyrosine are observed in NIH 3T3 cells infected with wild-type and mutant Ab-MLV. Two downstream targets affected in lymphoid transformants are the GTPase-activating protein and GTPase-activating protein-associated protein p62, molecules which are heavily tyrosine phosphorylated in lymphoid cells transformed by wild-type Ab-MLV but not carboxyl-terminal truncation mutants of Ab-MLV. This difference suggested that signaling mediated via the Ras pathway may be compromised in lymphoid cells expressing the carboxyl-terminal truncation mutants. Consistent with this idea, expression of v-Ha-ras complemented these mutants in primary bone marrow transformation assays and increased transformation frequencies obtained with the Ab-MLV mutants 8- to 20-fold. These data suggest that a biologically important link exists between the carboxyl terminus of v-Abl protein and the Ras pathway. Signals transmitted via this connection may enhance those mediated via other regions of the v-Abl protein and facilitate transformation of primary, nonimmortalized cells such as pre-B lymphocytes.     

Immortalization of macrophages from mouse bone marrow and fetal liver

Fresh bone marrow (BM)-derived cells infected with the J2 recombinant retrovirus (carrying v-myc and v-raf/mil oncogenes) grow as immortal cell lines belonging to the monocytic lineage. BM cells cultured for 24 h in conventional medium are no longer able to grow following infection with the J2 virus. We investigated whether specific growth factors affected the proliferative response of BM cells to the J2 virus. If the BM cells were cultured for 24 h in the presence of concanavalin A or CSF-1 and then infected with the J2 virus, immortalization of BM cells was observed. Under these conditions, the cell lines that we obtained were shown to belong to the monocytic lineage. We investigated whether target cells for the J2 virus existed in other hematopoietic organs. We observed J2-induced proliferation in fetal liver (FL) but not in spleen or thymus. The cells proliferating in the FL had macrophage characteristics during the early passages. However, some macrophage markers were lost upon extensive in vitro culture. We conclude that we have identified conditions in which J2 virus consistently and selectively stimulates the growth of macrophages from murine bone marrow and a wider range of hematopoietic cells from fetal liver.     

v-myc alters the response of a cloned mouse mammary epithelial cell line to lactogenic hormones

Several oncogenes have now been implicated in mammary carcinogenesis. We investigated the phenotypic effects of expressing three representative oncogenes in mammary epithelial cells. v-myc (coding for a nuclear protein), v-Ha-ras (a G-protein homologue) and v-fgr (a tyrosine kinase) genes were introduced into the nontumorigenic clone 14 of the mouse mammary epithelial cell line COMMA-1D. Their effects upon growth and differentiation were determined. Anchorage-independent growth was induced by all three oncogenes with low efficiency. v-Ha-ras and v-fgr induced tumorigenicity in nude mice. The effect of oncogenes upon parameters unique to mammary epithelial cells in vitro was assayed. Both v-myc and v-fgr abolished the ability of clone 14 to grow as three-dimensional branching structures in hydrated collagen gel. v-fgr completely and v-myc partially inhibited the expression of the epithelium specific cytokeratins. Clone 14 can be induced to produce the beta-casein milk protein by the combination of the lactogenic hormones, dexamethasone, insulin, and PRL. Introduction of v-myc into clone 14 cells resulted in an estimated 50-fold increased induction of beta-casein protein and at least a 60-fold increase in beta-casein mRNA. The number of cells stained with anti-beta casein antibodies also showed a 10-fold increase after v-myc introduction. This still required the synergistic action of all three lactogenic hormones. Thus v-myc can alter the normal response of mammary epithelial cells to lactogenic hormones.     

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes.

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.     

Oncogene cooperation in lymphocyte transformation: malignant conversion of E mu-myc transgenic pre-B cells in vitro is enhanced by v-H-ras or v-raf but not v-abl.

Although transgenic mice bearing a c-myc gene controlled by the immunoglobulin heavy-chain enhancer (E mu) eventually develop B-lymphoid tumors, B-lineage cells from preneoplastic bone marrow express the transgene but do not grow autonomously or produce tumors in mice. To determine whether other oncogenes can cooperate with myc to transform B-lineage cells, we compared the in vitro growth and tumorigenicity of normal and E mu-myc bone marrow cells infected with retroviruses bearing the v-H-ras, v-raf, or v-abl oncogene. The v-H-ras and v-raf viruses both generated a rapid polyclonal expansion of E mu-myc pre-B bone marrow cells in liquid culture and 10- to 100-fold more pre-B lymphoid colonies than normal in soft agar. The infected transgenic cells were autonomous, cloned efficiently in agar, and grew as tumors in nude mice. While many pre-B cells from normal marrow could also be induced to proliferate by the v-raf virus, these cells required a stromal feeder layer, did not clone in agar, and were not malignant. Most normal cells stimulated to grow by v-H-ras also cloned poorly in agar, and only rare cells were tumorigenic. With the v-abl virus, no more cells were transformed from E mu-myc than normal marrow and the proportion of tumorigenic pre-B clones was not elevated. These results suggest that both v-H-ras and v-raf, but apparently not v-abl, collaborate with constitutive myc expression to promote autonomous proliferation and tumorigenicity of pre-B lymphoid cells.     

Coinfection with viruses carrying the v-Ha-ras and v-myc oncogenes leads to growth factor independence by an indirect mechanism.

The concomitant expression of certain oncogenes can transform normal diploid rodent cells into transplantable tumorigenic cells. The mechanism by which these oncogenes collaborate is unclear. Recent findings (M. Oshimura, T. M. Gilmer, and J. C. Barrett, Nature [London] 316:636-639, 1985) raise the possibility that karyotypic changes, including monosomy for chromosome 15, are required to induce tumorigenicity in Syrian hamster embryo cells transfected in vitro with v-Ha-ras and v-myc DNAs. We studied the effect of the oncogenes v-Ha-ras and v-myc, introduced by viral infection, on murine hematopoietic cells. The induction of growth factor independence by the two oncogenes was used as an in vitro correlate of tumorigenicity. After a period of reduced growth rate reminiscent of the growth rate of cells in crisis, the doubly infected cells became growth factor independent. These cells showed a great variability in their karyotypes.     

Expression of functional human EGF receptor on murine bone marrow cells.

The human epidermal growth factor-receptor (EGF-R) was introduced into primary mouse bone marrow cells (BMC), utilizing retrovirus mediated gene transfer. Cultivation of infected BMC in the presence of interleukin-3 (IL-3) led to the outgrowth of IL-3 dependent myeloid cells, which efficiently expressed functional EGF-R, exhibiting its two characteristic affinity states. EGF acts on these cells synergistically with IL-3 in stimulating DNA synthesis and cell proliferation even under IL-3 saturation conditions. However, EGF was not sufficient to replace the requirement for IL-3. In contrast, EGF was able to maintain proliferation of a factor-dependent hemopoietic cell line (FDC-P1) infected with the EGF-R retrovirus in the absence of IL-3, but these cells did not respond to EGF in the presence of IL-3. No influence of EGF on IL-3 induced mast cell differentiation of BMC expressing the EGF-R could be observed by histological criteria. These data show that the expression of EGF-R alone is not sufficient to induce or maintain cell proliferation in IL-3 dependent bone marrow derived cells, although it can do so in established hemopoietic cell lines.     

Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.     

Retroviral-mediated gene transfer into mammalian cells

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.     

Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice.

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo.     

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.     

Growth of measles virus in various human lymphoid cell lines.

Neurovirulent TYCSA strain and attenuated Schwarz strain of measles virus and Halle strain of subacute sclerosing panencephalitis (SSPE) virus replicated in cultures of human lymphoid cell lines of the T-cell type, MOLT-3, MOLT-4 and CCRF-CEM. TYCSA and Halle strains grew rapidly, but Schwarz strain grew slowly in these cell lines. Furthermore, these three strains established persistent infection in CCRF-CEM cells but not in the other cell lines. In these persistently infected cultures an almost entire population of cells were shown to be infected and infectious virus was produced constantly for over 100 days. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titered infectious virus, whereas nucleocapsid structures were observed only in the cytoplasm of cells persistently infected with either TYCSA or Halle strain and the titers of infectious virus produced from these cells were high.