Differentiation of Abelson murine leukemia virus-infected promonocytic leukemia cells

Virus-producing, tumorigenic, promonocytic leukemia cell lines were derived from Abelson murine leukemia virus-infected mice. This study shows that, of these 25 cloned lines, 22 were capable of extensive differentiation. It also shows that granulocyte-macrophage colony-stimulating activity increased the proportion of differentiating cells in 17/22 lines. Cells from agar colonies with a diffuse colony morphology had an increased expression of mature macrophage phenotypic characteristics, and a reduced proliferative capacity in vitro, compared to cells from agar colonies with a compact colony morphology. Cells from diffuse colonies also produced less Abelson virus, and were less tumorigenic in vivo than cells from compact colonies. Together, these results suggest some Abelson virus-producing leukemic cells are not blocked in their capacity to differentiate and are capable of reversing the transformed phenotype.     

Multistage progression of Abelson virus-infected murine pre-B-cells to the tumorigenic state

The tumorigenic potential of pre-B-cells at different stages of Abelson murine leukemia virus-induced transformation was determined. Cell lines with low growth potential in liquid culture were found (a) to have a dose-dependent growth requirement for conditioned medium obtained from bone marrow cultures, (b) to have low colony-forming ability in semisolid medium in the absence of conditioned medium, and (c) to be nontumorigenic when inoculated into syngeneic mice. Culture of the factor-dependent cells in vitro leads to the emergence of factor-independent variants, which eventually dominate the population by overgrowth. Cell lines that acquired a factor-independent phenotype were able to form colonies in semisolid medium and form tumors when inoculated into syngeneic mice. These results suggest that Abelson murine leukemia virus is sufficient to initiate transformation in the infected cell but that an additional genetic alteration is needed to confer tumorigenicity.     

Effect of Abelson murine leukemia virus on granulocytic differentiation and interleukin-3 dependence of a murine progenitor cell line

The murine diploid hematopoietic cell line 32D Cl3 strictly requires interleukin-3 (IL-3) for proliferation. When 32D Cl3 cells are transferred to IL-3-free medium which contains recombinant human granulocyte colony stimulating factor (rhG-CSF), the cell number increases four- to five-fold, and after 14 days the whole cell population is differentiated into morphologically normal and myeloperoxidase- and lactoferrin-positive metamyelocytes and granulocytes. Infection with Abelson murine leukemia virus (A-MuLV) of 32D Cl3 cells growing in the presence of IL-3 induces, within 2 weeks, the appearance of cells that are IL-3-independent for growth. The latter cells lack myeloid, T and B cell markers, and are unable to differentiate, even in the presence of very high doses of rhG-CSF. However, once the 32D Cl3 cells have been exposed to G-CSF, they become resistant to the transforming effects of A-MuLV as judged by the appearance of the IL-3-independent clones. These findings suggest that the ability of Abelson virus to transform immature progenitor cells is due to interference of the v-abl gene product with the mechanisms that control the commitment of the cells to differentiate.     

Increased sensitivity of murine leukemia virus-infected tumor cells to lymphocyte-mediated cytotoxicity

The cytotoxic sensitivity of murine leukemia virus (MuLV)-infected and noninfected fibrosarcoma cells in syngeneic inbred WKA/Hok rats was compared by in vitro cell-mediated 51Cr release cytotoxicity assay. A highly significant increase in cytotoxic sensitivity of target cells was observe in MuLV-infected tumor cells as compared with noninfected cells when spleen cells from syngeneic tumor-bearing hosts (TBH) were used as a source of effector lymphocytes. The cytotoxicity of spleen cells against MuLV-infected tumor cells was specifically directed to the tumor-associated antigen (TAA), but not to the virus-associated antigen. However, there was no quantitative difference in the amount of TAA on the cell membranes between virus-infected and noninfected tumor cells as measured by a quantitative absorption test of anti-TAA serum. The cytotoxic activity of spleen cells from TBH against MuLV-infected tumor cells was abrogated by the treatment of anti-T-serum plus complement and significantly decreased after trypsin treatment. Spleen cells from normal rats given injections of immune sera from TBH acquired the cytotoxic activity against MuLV-infected tumor cells.     

Oncogenic transformation of murine lymphoid cells by in vitro infection with Abelson leukemia virus.

Spleen cell cultures stimulated to DNA synthesis by antigen or mitogen were infected with Abelson virus, a C-type RNA virus inducing nonthymic lymphomas in mice. After 3 days the cells were transferred to mice and caused 100 percent incidence of lymphomas in as few as 29 days. That a number of the tumors were of donor origin, as shown by female karyotypes in recipient male mice, indicated that cells infected by virus in vitro were transformed. The process depended upon both virus and stimulation of lymphocytes in culture. Lymphoid tumors did not develop in mice receiving cells from virus-infected cultures not exposed to antigen or mitogen.     

Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells

Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA). To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-induced cells by differential hybridization and isolated GTPase Ran as a down-regulated gene. We observed that Ran was expressed in a biphasic mode. Following a decrease in mRNA level during the initial hours of induction, Ran re-expressed at 24-48 h, and gradually declined again. To investigate the role of Ran during MEL differentiation we constructed MEL transfectants capable to express or block Ran mRNA production constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL cell differentiation resulting in a decrease of cell survival in the committed population.     

Regulation of c- and N-myc expression during induced differentiation of murine neuroblastoma cells

Using clones N1E-115 and N1A-103 from mouse neuroblastoma C1300, a comparative analysis of c- and N-myc gene expression was undertaken both in proliferating cells and in cultures exposed to conditions which induce differentiation. Under the latter conditions, while N1E-115 cells extend abundant neurites and express many biochemical features of mature neurons, clone N1A-103 stops dividing and expresses certain neurospecific markers but is unable to differentiate morphologically. In both clones, chemical agents, i.e. 1-methyl cyclohexane carboxylic acid (CCA) or dimethyl sulfoxide (DMSO), induce a decrease in c-myc expression. Similar results were found for N-myc gene in N1E-115 cells, but in contrast, in clone N1A-103, N-myc expression is increased with CCA and not modified with DMSO. Globally, this study favours the hypothesis that changes in c-myc expression would correspond to cell division blockade and differentiation, while modulations in N-myc are more closely related to an early phase of terminal differentiation.     

Abelson murine leukemia virus transforms preneoplastic Emu-myc transgene-carrying cells of the B-lymphocyte lineage into plasmablastic tumors

E mu-myc transgenic mice were back-crossed to BALB/c mice up to back-cross generation 3. The offspring that included transgene-carrying and -negative mice in approximately equal proportions were randomly divided into 2 groups. Thirty-four mice (group I) were treated with pristane, followed by A-MuLV, and 40 (group II) were injected with A-MuLV alone. Altogether, 16 lymphoid tumors developed in group I and 17 in group II. Nine of the tumors in group I and 4 in group II appeared as ascitic tumors. The ascites contained lymphoblasts and 10 to 45% plasmacytoid cells. These tumors were designated as plasmablastic lymphomas (PLs). All tumors except one were transgene-positive and did not carry translocations. An exceptional tumor in group I carried a variant 6;15 translocation but not the transgene. It obviously corresponds to the regular Abelson + pristane-induced plasmacytoma. Among 11 tested PLs, 10 had a single retroviral insertion site, while one tumor showed 3. Among 18 untreated transgenic descendants (group III), chosen randomly during serial back-crosses, 15 (83%) developed lymphomas, with no sign of plasmacytoid differentiation. The incidence was comparable in all 3 groups, assuming 50% of the mice in groups I and II to be transgenic. The time distribution of tumor development was also similar. Spleen cells from transgene-carrying mice with no clinical sign of lymphoma were infected in vitro with A-MuLV and transplanted i.p. into BALB/c recipients. PLs developed in 26 of 31 pristane-treated recipients, but in only one of 18 untreated recipients. One of 6 PLs tested was monoclonal, whereas the remaining 5 were oligoclonal. They all expressed v-abl. These results show that some of the preneoplastic B-cells that expressed constitutively active myc transgene turned into plasmablasts after infection with A-MuLV. Full development of their neoplastic potential was facilitated by the presence of pristane-granuloma.     

Conjugated linoleic acid induces monocytic differentiation of murine myeloid leukemia cells

Conjugated linoleic acid (CLA) refers to a group of naturally occurring positional and geometrical conjugated dienoic isomers of linoleic acid (C18:2), of which the cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) isomers predominate. Accumulating evidence has demonstrated that CLA isomers are capable of inhibiting the growth of a variety of cancer cell lines in vitro; however, their modulatory effects on the proliferation and differentiation of myeloid leukemia cells remain poorly understood. In the present study, CLA was shown to inhibit the proliferation of murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. Morphological, flow cytometric and functional analyses revealed that CLA induced the differentiation of WEHI-3B JCS cells into matured macrophage-like cells, as indicated by increases in the cytoplasm:nucleus ratio and vacuolation, the expression of macrophage differentiation antigens (Mac-1 and F4/80) and the enhanced monocytic serine esterase activity of CLA-treated WEHI-3B JCS cells. RT-PCR analysis showed that CLA up-regulated the expression of TNF-alpha, IL-1beta and IFN-gamma genes in WEHI-3B JCS cells, which had previously been shown to play an important role in triggering the differentiation of myeloid leukemia cells. Moreover, CLA-treated WEHI-3B JCS cells had also shown reduced tumorigenicity in vivo. Collectively, our results indicate that CLA might exert its growth-inhibitory effects on myeloid leukemia cells by triggering their terminal differentiation, which is mediated, at least in part, by modulation of the cytokine gene expression in the leukemia cells.     

Selection and rejection of retrovirus-expressing tumor cells from a heterogeneous murine leukemia virus-infected cell population

We studied the basis of tumor recurrence at sites of rejection of retrovirus-infected guinea pig fibrosarcoma cells. Tumor recurrences, in contrast to the parent tumor, lacked retroviral antigens and did not release infectious virus. When reinjected into syngeneic animals, cell lines derived from tumor recurrences grew progressively. Tumor recurrences could be infected with the homologous retrovirus. Tumor rejection and recurrence were modulated by host immunity. In guinea pigs immunized to virus-infected cells, tumor recurrences occurred earlier and in a higher proportion of animals than in nonimmune guinea pigs. In some immunosuppressed guinea pigs, retrovirus-infected tumor cells grew progressively. Progressively growing tumors of immunosuppressed guinea pigs contained large amounts of infectious virus and expressed viral antigens. To identify the source of tumor recurrences, the parent virus-infected tumor was cloned. Clones were heterogeneous in virus expression; some clones released large quantities of infectious virus; others did not. Two clones formed tumors in syngeneic animals. Injection of a virus producer clone into virus-immune animals was not followed by tumor recurrence. The data suggest that the reappearance of tumors at sites of injection of retrovirus-infected fibrosarcoma cells represents immune selection and rejection of retrovirus-expressing cells. Cells with the potential to form tumor recurrences existed in the parent virus-infected tumor population.     

Involvement of the spleen in preleukemic development of a murine retrovirus-induced promonocytic leukemia.

An acute myeloid leukemia can result from the inoculation of Moloney murine leukemia virus into BALB/c mice undergoing a 2,6,10,14-tetramethylpentadecane-induced chronic inflammatory response in the peritoneal cavity. This leukemia is ultimately observed in the peritoneal cavity as an ascites with cells infiltrating the granulomatous tissue. It has been proposed, however, that hematopoietic organs such as the spleen and bone marrow are involved in preleukemic development of Moloney murine leukemia. Therefore, to determine if the spleen plays a role in this development, mice were splenectomized at various times relative to virus inoculation. When splenectomies were performed 3 days before and 2, 4, 6, and 8 weeks after virus inoculation there was, in all cases, a decreased death rate compared to sham-splenectomized controls. The greatest difference in death rate due to promonocytic leukemia was observed when mice were splenectomized at 4 weeks after virus inoculation. The decrease in disease incidence observed as a result of splenectomy was not caused by decreased virus spread in hematopoietic organs or an alteration in the profile of the cellular infiltrate in the granuloma. It was found, however, that the spleens of 2,6,10,14-tetramethylpentadecane-treated mice, relative to those of normal mice, have a significantly increased number of granulocyte-macrophage colony-forming cells and a slightly increased number of multipotential colony-forming cells. These observations suggest that a population of target cells for transformation, consisting of granulocyte-macrophage precursor cells, may reside in the spleen. Alternatively, partially transformed cells may reside temporarily in the spleen during the developmental stages of the disease process.     

Abelson murine leukemia virus: Effect of helper virus from regressing friend virus on leukemia development

Replication defective Abelson murine leukemia virus (A-MuLV) induces a non-thymic lymphoma in vivo and transforms both hematopoietic and fibroblastic cells in vitro. In vivo leukemogenicity and the efficiency of in vitro transformation of hematopoietic cells by A-MuLV are known to be affected by the replication competent helper virus present in A-MuLV stocks. The helper virus isolated from the regressing strain of Friend virus (RF-MuLV) is responsible for the spontaneous regression of erythroleukemia induced by replication defective spleen-focus forming virus and itself induces a lymphocytic leukemia which spontaneously regresses. The diseases produced by A-MuLV stocks containing either RF-MuLV or Moloney leukemia virus, the helper virus associated with the original isolate of A-MuLV, were compared to determine if RF-MuLV can influence the disease produced by a replication defective virus with a discrete transforming gene. Both virus stocks induced leukemias with similar efficiency and gross pathology. Spontaneous regression was not observed when RF-MuLV was used as the helper virus. Examination of the leukemic cells and cell lines derived from leukemic tissues indicated that the target cell for A-MuLV transformation was not affected by the helper virus. Both transformed lymphoid and monocytic cells were cultured from leukemic tissues and established as cell lines. The lymphoid cells were phenotypically similar to pre-B cells or null cells, while the monocytic cell lines resemble promonocytes. The frequency with which promonocytic cell lines were isolated from leukemic mice suggests that A-MuLV leukemogenesis may often involve transformation of monocytic series cells as well as lymphoid cells. Thus, RF-MuLV can serve as an efficient helper virus for A-MuLV and does not appear to alter the in vivo target cell for transformation. It is unable, however, to alter the progressive course of Abelson virus induced disease.     

Bone marrow-transforming activity of linker insertion mutants of Abelson murine leukemia virus.

Two sets of mutants of the Abelson murine leukemia virus, generated by linker insertion mutagenesis of a cloned proviral DNA, were tested for their ability to transform bone marrow cultures in vitro. All the viruses retained an intact tyrosine kinase domain and were competent for transformation of NIH3T3 fibroblasts in culture. One series contained 12-bp linker insertions in the regions flanking the kinase domain, and the other contained frameshift mutations that truncated the gene product downstream of the kinase domain. The majority of the 12-bp insertion mutants retained full bone marrow-transforming activity; only one insertion in the SH2 domain showed reduced activity. This mutant suggests that some aspect of the SH2 domain may be more important in transformation of lymphocytes than fibroblasts. In contrast to the first set of mutants, the bone marrow-transforming activity of the majority of the truncation mutants was significantly reduced or completely lost. We conclude that there is a broad requirement for an intact C-terminal domain of the v-abl protein for the transformation of pre-B cells, but that no single part of this domain is critical.     

Abelson murine leukemia virus-induced thymic lymphomas: transformation of a primitive lymphoid precursor

For the investigation of whether Abelson murine leukemia virus (A-MuLV) is able to transform in vivo lymphocytes other than those of the B-cell lineage, newborn BALB/c and C57BL/6 mice were given an injection of A-MuLV directly into the thymus. Thymic lymphomas appeared with a short latent period of 4-5 weeks in BALB/c mice and 8 weeks in C57BL/6 mice. Cell lines derived from some thymic lymphomas presented a very immature phenotype and did not express cellular markers of either T-cells (Thy 1.2, Lyt 1.2, and Lyt 2.2) or B-cells (cytoplasmic IgM) even after treatment with several differentiation inducers. Molecular analysis showed that T-cell receptor (TCR) beta chain genes were never rearranged; in one case only, rearrangement of TCR gamma chain genes could be demonstrated, confirming the immaturity of the presumptive T-cell lines studied. Furthermore, the cell lines consistently carried diversity (D)-joining (J) but not variable (V)-D-J rearrangements of the immunoglobulin heavy chain genes. On the whole, these findings suggest that following intrathymic A-MuLV injection neoplastic transformation does involve lymphocytes possibly of T-cell lineage, at a very early stage of differentiation.