Abelson virus-infected cells can exhibit restricted in vitro growth and low oncogenic potential.

We have designed a method for growing bone marrow cells infected with Abelson murine leukemia virus which permits examination of target cell growth early after infection. This culture system increases the efficiency of target cell growth by favoring rapid growth of a mixed population of adherent cells in the primary culture. The nonadherent Abelson virus-infected cell populations expressed pre-B-cell differentiation markers characteristic of Abelson virus-transformed cells (mu-heavy chains of immunoglobulin M and terminal deoxynucleotidyltransferase). Early after infection, these cell populations exhibited restricted in vitro and in vivo growth properties which differed from those of an established Abelson virus-transformed cell line, 2M3. These included a marked dependency upon the adherent cell layer for growth and viability, a lower efficiency of agar colony formation, and a lower capacity for tumor production in syngeneic animals. Growth of the early populations could be maintained in the absence of the adherent cell layer by using conditioned medium from long-term adherent cell cultures established in the absence of viral infection. After passage of the populations for several weeks, the in vitro growth properties gradually shifted toward that of the 2M3 cell line. Twelve-week-old populations grew independently of the adherent cell layer and showed an increased efficiency of agar colony formation. These data indicate that many lymphoid target cells exhibit an intermediate transformed phenotype when infected with Abelson virus. Growth of these cells in culture is mediated via a synergistic interaction between intracellular expression of the viral transforming gene and an exogenous growth-promoting activity which can be provided by cultures of adherent bone marrow cells.     

In vitro transformation of murine pre-B lymphoid cells by Snyder-Theilen feline sarcoma virus

Snyder-Theilen feline sarcoma virus (ST-FeSV) codes for a protein kinase with specificity for tyrosine residues (Barbacid et al., Proc. Natl. Acad. Sci. U.S.A. 77:5158-5163, 1980), properties analogous to those of the transforming gene product of Abelson murine leukemia virus (Witte et al., Nature (London) 283:826-831, 1980). In the present report, ST-FeSV was demonstrated to transform murine hematopoietic cells under in vitro assay conditions which detect lymphoid cell transformation by Abelson murine leukemia virus. Bone marrow colony formation was shown to require ST-FeSV, follow single-hit kinetics, and require the presence of mercaptoethanol in the agar medium. ST-FeSV-induced colonies could be established in culture as continuous cell lines that demonstrated unrestricted self-renewal capacity and leukemogenicity in vivo. The hematopoietic blast cells transformed by ST-FeSV in culture appeared to be at an early stage of B cell differentiation. They possessed Lyb 2 surface antigens, were dependent on mercaptoethanol for growth, and contained only low levels of terminal deoxynucleotidyl transferase. Moreover, a large fraction of the lines synthesized immunoglobulin mu chain in the absence of light chains. Thus, the phenotype of ST-FeSV hematopoietic transformants was indistinguishable from that of the pre-B lymphoblast transformants induced by Abelson murine leukemia virus. These findings indicate that the in vitro functional similarities in the onc gene products of ST-FeSV and Abelson murine leukemia virus may reflect a common pathway by which they exert their oncogenic potential.     

Abelson murine leukemia virus mutants deficient in kinase activity and lymphoid cell transformation.

Abelson murine leukemia virus transforms both lymphoid cells and fibroblasts in vitro and induces a unique type of thymus-dependent lymphoma in vivo. Four fibroblast-transforming strains of Abelson murine leukemia virus were identified, based on the sizes of the Abelson murine leukemia virus-specific phosphoproteins produced by these isolates. Two of these strains, the standard P120- and the P160-producing viruses, transformed lymphoid cells efficiently in vitro and induced Abelson disease in vivo. Two other strains, which synthesized small Abelson murine leukemia virus-specific proteins with molecular weights of 90,000 (P90) and 100,000 (P100), transformed lymphoid cells very poorly both in vitro and in vivo. The reduced oncogenic potentials of these isolates were correlated with a high level of synthesis of fairly unstable P90 and P100. In addition, neither P90 nor P100 functional efficiently in protein kinase assays. The correlation of abnormal metabolism and deficient protein kinase activity with the reduced oncogenic potentials of these virus strains supported a direct role for these proteins and the kinase activity in transformation. Furthermore, these results suggested that the requirements for lymphoid cell transformation and fibroblast transformation are different.     

Conservation of function of Drosophila melanogaster abl and murine v-abl proteins in transformation of mammalian cells.

The Drosophila melanogaster abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, we constructed Drosophila abl/v-abl-chimeric Abelson murine leukemia viruses. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-abl were replaced with the corresponding regions of D. melanogaster abl. The chimeric Abelson murine leukemia viruses were able to mediate morphological and oncogenic transformation of NIH 3T3 cells and were able to abrogate the interleukin-3 dependence of a lymphoid cell line. We also found that a virus that contained both TPK and carboxy-terminal Drosophila abl regions had no in vitro transforming activity for primary bone marrow cells and lacked the ability to induce tumors in susceptible mice. A virus that replaced only a portion of the v-abl TPK region with that of Drosophila abl had low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of abl TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-abl is a major determinant of its efficient lymphoid cell-transforming activity.     

Loss of viral gene expression and retention of tumorigenicity by Abelson lymphoma cells.

Lymphomas induced by the Abelson murine leukemia virus (A-MuLV) were examined for the expression of biochemical and biological markers associated with A-MuLV transformation before and after in vivo growth in genetically distinguishable host mice. Although all tumors and clonal lines derived from them initially expressed the A-MuLV-encoded gag fusion protein p160, they ceased synthesis of this molecule after several weeks of growth in vivo as ascites tumors. Transplanted clonal lines continued to express the alloantigenic marker H-2b and the isoenzyme marker Gpi-1b of the donor tumor cells, indicating that the cells were of donor and not host origin. Examination of cellular DNA obtained from p160-positive and derivative p160-negative lines indicated that p160-negative clones had lost A-MuLV-specific proviral sequences as detected by hybridization with several probes. Although the clonal lines no longer expressed p160, they retained their malignant phenotype and continued to express the Abelson antigen, a cell surface marker associated with A-MuLV lymphomagenesis. Continued expression of the A-MuLV genome was not required for maintenance of oncogenic potential under these conditions of in vivo tumor growth.     

Defective lymphopoiesis in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mutant mice. II. Description of a microenvironmental defect for the generation of terminal deoxynucleotidyltransferase-positive bone marrow cells in vitro

We have presented evidence in a previous paper that the development of prothymocytes, pre-B cells, and TdT+ lymphoid precursor cells in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mice is defective. In the present study, we have used a selective culture system that supports the generation of rat- and mouse-origin TdT+ bone marrow lymphoid cells in vitro to further investigate the early stages of lymphopoiesis in me/me and mev/mev mice. The results demonstrate that bone marrow stromal cell feeder layers derived from me/me and mev/mev mice do not support the growth of rat TdT+ cells in vitro, whereas stromal cell feeder layers from heterozygous (+/-) littermates and wild type (+/+) control mice do. Moreover, composite feeder layers formed by mixing as few as one part me/me and mev/mev bone marrow cells with 7 to 9 parts +/- littermate bone marrow cells also fail to effectively support the generation of TdT+ cells in vitro. In contrast to me/me and mev/mev mice, other mutant mouse models of autoimmune (NZB, NZB/W), immunodeficient (nu/nu), and hemopoietic (W/Wv, Sl/Sld) disorders form feeder layers that support normal to elevated levels of TdT+ cell growth in vitro. Thus, to date, only the me/me and mev/mev mutant mice have been found to lack the appropriate microenvironment for the generation of TdT+ bone marrow cells. Histologic analysis of the stromal cell feeder layers that are formed in our culture system shows that multilayered cellular patches, which normally are the most active sites of TdT+ cell development in vitro, are absent in feeder layers of me/me and mev/mev cells. Moreover, feeder layers from mev/mev mice contain a population of MAC 1+, basophilic, nonvacuolated, macrophage-like cells; whereas feeder layers from control mice contain MAC 1+, eosinophilic, vacuolated macrophage-like cells. Stromal cell feeder layers formed by mixtures of me/me or mev/mev and control mouse bone marrow cells contain numerous multilayered cellular patches and vacuolated mononuclear cells, but also contain large numbers of basophilic mononuclear cells. These composite feeder layers have a disproportionately reduced capacity to support the generation of TdT+ cells in vitro. Although the stromal microenvironment of me/me and mev/mev bone marrow does not support the growth of TdT+ cells in vivo or in vitro, the bone marrow from these mutant mice contains detectable numbers of pre-TdT+ cells. Thus, when cultured on normal mouse feeder layers, mutant mouse bone marrow rapidly generates TdT+ cells in vitro, albeit at significantly reduced levels as compared to +/- littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Proliferation and differentiation of Abelson virus-infected murine myeloid leukemia cell lines does not involve an autocrine mechanism

Culture in agar of cloned promonocytic leukemia cell lines derived from Abelson virus-infected mice produced colonies of both a compact and diffuse morphology. Diffuse colonies contained fewer cells capable of forming colonies when recultured in agar than did compact colonies. Serial subcloning of cells from diffuse, but not compact, colonies ultimately led to the complete loss of colony-forming cells, i.e., to clonal extinction. The production of both compact and diffuse agar colonies was independent of the cell density of either the static liquid culture from which cells were taken for culture in agar, or the number of cells per agar culture. Furthermore, bioassays of culture supernatants indicated the leukemia cells did not secrete hemopoietic growth factors active on normal hemopoietic cells, transforming growth factors active on adherent cell lines, or factors that influenced the growth of the leukemic cells themselves. Collectively, these data suggest neither growth-factor independent replication nor the spontaneous differentiation of Abelson virus-infected myeloid cells involves autocrine secretion of growth regulators.     

不同饲养层对鸡胚胎干细胞培养效果的影响

目的:为探索鸡胚胎干细胞培养的优化条件,比较不同饲养层对鸡胚胎干细胞离体培养的效果。方法:用传至第2代的鸡胚成纤维细胞与鸭胚成纤维细胞,经丝裂霉素处理后制作饲养层,比较这2种饲养层以及不用饲养层对鸡胚胎干细胞离体培养效果的影响。结果:在以鸡胚成纤维细胞和鸭胚成纤维细胞作为饲养层的培养体系中,鸡胚胎干细胞均可保持良好的生长状态,而且2种饲养层对鸡胚胎干细胞克隆形成的影响差异不显著(P0.05)。结论:鸡胚成纤维细胞和鸭胚成纤维细胞均可作为较好的饲养层细胞用于鸡胚胎干细胞的离体培养。     

Cloned murine non-malignant, spontaneously transformed and chemical tumour-derived cell lines related to the type 2 pneumocyte

NAL1A is a murine type 2 pneumocyte-related cell line cultured from normal BALB/c adult mouse lung. In vitro spontaneous transformation of 3 out of 7 clones of NAL1A has led to the isolation and establishment in continuous cell culture of sibling-related non-neoplastic (NAL1A) and spontaneously arising neoplastic (NAL1As) cell strains. NAL1As cells exhibited a similar phenotype to cloned NUL1 cells cultured from urethane-induced mouse lung adenomas. All NAL1As and NUL1 clones grew vigorously in 0.3% agar and formed invasive, poorly differentiated carcinomas following subcutaneous inoculation into immunesuppressed mice. Several subcutaneous nodules metastasised preferentially to the lung. All spontaneous and chemically-derived malignant clones were less differentiated than the non-malignant clones as assessed by staining with a type 2 pneumocyte-specific polyclonal antiserum. The clones described in this report form a useful model in the study of spontaneous and chemically-induced neoplastic transformation in mouse epithelial lung cells.     

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells.

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.     

The extreme carboxyl terminus of v-Abl is required for lymphoid cell transformation by Abelson virus

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells in vivo and in vitro and can transform immortalized fibroblast cell lines in vitro. Although the kinase activity of the protein is required for these events, most previously studied mutants encoding truncated v-Abl proteins that lack the extreme carboxyl terminus retain high transforming capacity in NIH 3T3 cells but transform lymphocytes poorly. To understand the mechanisms responsible for poor lymphoid transformation, mutants expressing a v-Abl protein lacking portions of the COOH terminus were compared for their ability to transform pre-B cells. Although all mutants lacking sequences within the COOH terminus were compromised for lymphoid transformation, loss of amino acids in the central region of the COOH terminus, including those implicated in JAK interaction and DNA binding, decreased transformation twofold or less. In contrast, loss of the extreme COOH terminus rendered the protein unstable and led to rapid proteosome-mediated degradation, a feature that was more prominent when the protein was expressed in Ab-MLV-transformed lymphoid cells. These data indicate that the central portion of the COOH terminus is not essential for lymphoid transformation and reveal that one important function of the COOH terminus is to stabilize the v-Abl protein in lymphoid cells.     

Differentiation of cloned populations of immature B cells after transformation with Abelson murine leukemia virus

The nature of the target cell for Abelson virus transformation and the effect of transformation on B cell differentiation were studied with six cloned lines of nontransformed immature B lymphocytes. Three clones were at the pre-B cell stage of maturation prior to A-MuLV infection; two were at the B cell stage, and one appeared to represent a stage prior to rearrangement of the mu heavy chain gene. All six cloned lines could be transformed by Abelson virus. Many of the transformants of the pre-B cell clones underwent kappa light chain gene rearrangement and expression following viral infection. Distinct light chain gene rearrangements were segregated by further subcloning these transformed lines. Abelson virus infection of one cloned cell line believed to represent a stage of maturation prior to the pre-B cell stage produced pre-B cell transformants with a variety of heavy chain gene rearrangements. Thus B lymphoid target cells for Abelson virus are not restricted to a single developmental stage, and some transformed subclones can undergo extensive immunoglobulin gene rearrangements shortly after viral infection.     

Functional macrophage cell lines transformed by Abelson leukemia virus.

Three cloned cell lines have been established from murine tumors induced with Abelson leukemia virus which express properties of macrophages. Two of the three original tumors in addition yielded lymphocyte cell lines, one typical of the Abelson virus disease and the other a thymic lymphoma. Two of the macrophage lines are tumorigenic when placed in syngeneic mice. All of the macrophage lines pinocytose neutral red, phagocytose zymosan and latex beads, mediate antibody-dependent killing and phagocytosis of sheep erythrocyte targets, and secrete high levels of lysozyme. None of these properties was exhibited by the lymphocyte lines. Of the two macrophage cell lines tested, neither was capable of replacing the adherent cell population required for the induction of in vitro immune responses. An agent that activates normal macrophages, bacterial lipopolysaccharide, specifically inhibits the growth of the transformed macrophages in culture. Secretion of infectious Abelson leukemia virus by two of the macrophage lines, RAW 309Cr and WR 19M, provides conclusive evidence that the Abelson virus is capable of productively infecting the macrophage cell type. The other macrophage line, RAW 264, fails to secrete detectable virus particles and is negative in the XC plaque formation assay, as well as the fibroblast transformation assay for Abelson virus, but becomes positive for Abelson virus production after rescue by Moloney leukemia virus.     

Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus.

The single protein encoded by Abelson murine leukemia virus is a fusion of sequence from the retroviral gag genes with the v-abl sequence. Deletion of most of the gag region from the transforming protein results in a virus capable of transforming fibroblasts but no longer capable of transforming lymphoid cells. Smaller deletions in gag reveal that p15 gag sequences are responsible for this effect, whereas deletion of p12 sequences had no effect on lymphoid transformation. In transformed fibroblasts, p15-deleted and normal proteins had similar activities and subcellular localization. When the p15-deleted genome was introduced into previously transformed lymphoid lines, its protein product exhibited a marked instability. The tyrosine-specific autophosphorylation activity per cell was less than 1/20th that of the nondeleted protein. Although pulse-Ia-beling showed that the p15-deleted protein was synthesized efficiently, immunoblotting demonstrated that its steady-state level was less than 1/10th that of the nondeleted Abelson protein. The specific instability of the p15-deleted protein in lymphoid cells explains the requirement of these sequences for lymphoid but not fibroblast transformation.     

Abelson virus potentiates long-term growth of mature B lymphocytes.

Abelson murine leukemia virus (A-MuLV) infection of mouse bone marrow cells usually leads to transformation of pre-B cells. However, when the environment is modified by the continuous presence of lipopolysaccharide (LPS), two novel types of membrane immunoglobulin (mIg)-positive B cell lines are generated. Because the cells which give rise to these cell lines copurify with mIg-positive bone marrow cells, the cell lines arise as a result of A-MuLV interaction with a new type of in vitro target cell. The cell lines generated fall into two groups which differ in several phenotypic characteristics. Group 1 cells are more differentiated than the typical pre-B cell transformant in that they synthesize mIgM and appear to resemble virgin B cells. The group 1 cells do not secrete immunoglobulin and are independent of LPS for growth. In addition, these cell lines synthesize the Abelson P160 protein, contain integrated abl proviral DNA, and are highly tumorigenic in syngeneic animals. The group 2 cell lines differ markedly from both the group 1 cells and from typical, pre-B cell A-MuLV transformants. These cells are mIgG positive and secrete large amounts of immunoglobulin into the culture medium. The cell lines are comprised of both adherent and nonadherent cells and do not synthesize P160 or contain integrated v-abl sequences. The group 2 cells are nontumorigenic in syngeneic animals and require LPS for growth and viability. Both types of cells have remained in culture for over 2 years with no changes in their phenotypic characteristics. This A-MuLV infection system and the novel mIg-positive cell lines may serve as useful models for studying biochemical and molecular properties of mature B cells.