Immunoglobulin synthesis by lymphoid cells transformed in vitro by Abelson murine leukemia virus.

The majority of cell lines derived by infection of murine bone marrow cells with Abelson murine leukemia virus (A-MuLV) synthesize a mu chain but no detectable light chain. Aside from this mu-only phenotype, lines that make only light chain, both chains or no immunoglobulin-related polypeptides have also been found. Two lines have been studied in detail: one that makes only mu chain and one that makes only kappa light chain. Synthesis of both polypeptides can be increased by modifying the culture conditions so as to decrease the growth rate of the cells. Although some kappa chain secretion was observed, neither secreted nor surface mu was detected. We suggest that the mu- only phenotype may be an early normal step in the pathway of B lymphocyte maturation.     

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.     

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.     

In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Synergism in the effect of gamma-radiation and quinoid radiotoxins on mice and cultured lymphoid cells

A considerable increase in the lethal effect of gamma-radiation on mice was noted after postirradiation administration of nontoxic quantities of quinoid radiotoxins (synergism). The same synergistic effect was observed on human lymphoid cells Raji in culture.     

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.     

Cytochemistry of dipeptidylaminopeptidase IV and II in normal and neoplastic lymphoid cells

Cytochemical methods were used to determine the distribution of dipeptidylaminopeptidase IV (DAP IV) and II (DAP II) in lymphoid cell populations from patients with lymphoproliferative diseases. Special attention was paid to unusual intracellular distribution patterns which might correlate with the presence of various membrane markers. In healthy patients, about 50% of the circulating lymphocytes were found to be positive to both reactions, the intracellular distribution patterns being variable. The DAP IV reaction was negative in all B-CLL cases. In 2 cases of T-CLL with phenotype E+ OKT3+T4-T8+ one was negative and one was weakly positive, while two cases of T-CLL with phenotype E+ OKT3+T4+T8- were both strongly positive. The other non-T lymphoproliferative diseases studied were negative for DAP IV, while one T-ALL and three T-lymphoma cases showed a strong granular or diffuse distribution. The DAP II reaction was strongly positive in all the T lymphoproliferative diseases studied, irrespective of their immunological phenotype. This reaction was also weakly positive in some cases of plasmocytoma and lymphoplasmacytoid lymphoma.     

Feline sarcoma virus induced in vitro progression from premalignant to neoplastic transformation of human diploid cells

Summary Human diploid cells morphologically transformed by feline sarcoma virus were serially propagated under selective cell culture conditions. When injected into nude mice prior to passage in soft agar (0.35%), morphologically transformed cells did not produce tumors. However, when propagated under selective cell culture conditions, transformed cells grew in soft agar and, when injected subcutaneously into the subcapsular region of the nμ/nμ mice, produced neoplastic nodules histopathologically interpreted as fibromas. Karyological examination of cell populations grown out from the tumors confirmed that the tumors were composed of human cells. Examination of electron micrographs of the excised tumor tissue revealed the presence of budding virus particles. Tumor cells isolated from nude mice and morphologicaly transformed cells both contained the feline concornativirus-associated cell membrane antigen. It was concluded that expression of feline oncornavirus-associated cell membrane antigen is associated with an early stage of feline rerovirus-induced carcinogenesis, namely focus formation. In addition, it was shown that FeLV-FeSV can induce morphological transformation in human cells in vitro and that there is a requirement for the cells to passage through soft agar before subsequent tumor formation (neoplastic transformation) can be demonstrated. This work was supported in part by NIH-NCI RO1-259007, NO1-CP-3571 and CPV08 103563, and Air Force F49620-77-C-110.     

Flow cytometric detection of the mitochondrial BCL-2 protein in normal and neoplastic human lymphoid cells.

The bcl-2 proto-oncogene, rearranged and deregulated in B-cell lymphomas bearing the t(14;18) translocation, encodes an inner mitochondrial membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single- and multicolor flow cytometric analysis of bcl-2 protein in relation to other markers and cell cycle, based on a fixation-permeation step of cells with paraformaldehyde and Triton X100 and the use of a bcl-2 specific monoclonal antibody (MoAb). As an application of this method, we have examined the expression of bcl-2 in normal and neoplastic lymphoid cells. We have found that greater than 80% of normal T-and B-cells are bcl-2 positive; following in vitro mitogen activation, the bcl-2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl-2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two-color analysis. On lymphoblastoid cell lines, the bcl-2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl-2 gene. Among fresh B-cell non-Hodgkin's lymphomas (B-NHL), most sporadic Burkitt's cases were bcl-2 negative. Of four centroblastic-centrocytic cases with rearrangements of the bcl-2 gene, only two presented elevated amounts of bcl-2 protein, indicating that the levels of bcl-2 are not diagnostic of the translocation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Feline sarcoma virus induced in vitro progression from premalignant to neoplastic transformation of human diploid cells

Human diploid cells morphologically transformed by feline sarcoma virus were serially propagated under selective cell culture conditions. When injected into nude mice prior to passage in soft agar (0.35%), morphologically transformed cells did not produce tumors. However, when propagated under selective cell culture conditions, transformed cells grew in soft agar and, when injected subcutaneously into the subcapsular region of the n mu/n mu mice, produced neoplastic nodules histopathologically interpreted as fibromas. Karyological examination of cell populations grown out from the tumors confirmed that the tumors were composed of human cells. Examination of electron micrographs of the excised tumor tissue revealed the presence of budding virus particles. Tumor cells isolated from nude mice and morphologically transformed cells both contained the feline oncornavirus-associatied cell membrane antigen. It was concluded that expression of feline oncornavirus-associated cell membrane antigen is associated with an early stage of feline retrovirus-induced carcinogenesis, namely focus formation. In addition, it was shown that FeLV-FeSV can induce morphological transformation in human cells in vitro and that there is a requirement for the cells to passage through soft agar before subsequent tumor formation (neoplastic transformation) can be demonstrated.     

The subcellular distribution of adenylate and guanylate cyclases in murine lymphoid cells.

Membrane vesicles can be prepared from murine lymphoid cells by nitrogen cavitation and fractionated by sedimentation through nonlinear sucrose density gradients. Two subpopulations of membrane vesicles, PMI and PMII, can be distinguished on the basis of sedimentation rate. The subcellular distribution of adenylate and guanylate cyclases in these membrane subpopulations have been compared with the distribution of a number of marker enzymes. Approximately 20-30% of the total adenylate and guanylate cyclase activity is located at the top of the sucrose gradient (soluble enzyme), the remainder of the activity being distributed in the PMI and PMII fractions (membrane-bound enzyme). More than 90% of the 5'-nucleotidase and NADH oxidase activities detected in lymphoid cell homogenates are located in PMI and PMII fractions, whereas succinate cytochrome c reductase activity is detected only in the PMII fractions. In addition, beta-galactosidase activity is distributed in the soluble and PMII fractions of the sucrose density gradients. On the basis of the fractionation patterns of these various enzyme activities, it appears that PMI fractions contain vesicles of plasma membrane and endoplasmic reticulum, whereas PMII fractions contain mitochondria, lysomes, and plasma membrane vesicles. Approximately 30-40% of the adenylate and guanylate cyclase activities in PMII can be converted to a PMI-like form following dialysis and resedimentation through a second nonlinear sucrose gradient. Adenylate and guanulate cyclases can be distinguished on the basis of sensitivity to nonionic detergents.     

Transformation of mammalian fibroblasts and macrophages in vitro by a murine retrovirus encoding an avian v-myc oncogene

A murine retrovirus which expresses the avain v-myc OK10 oncogene was constructed. The virus, denoted MMCV, readily transforms fibroblasts of established lines, such as mouse NIH/3T3 and rat 208F cells, to anchorage-independent growth in agarose. The virus also transforms primary mouse cells: (i) virus-infected macrophages are induced to form large colonies in semi-solid media, and can easily be expanded into mass cultures; (ii) MMCV-infected fibroblastic cells from mouse limb buds undergo morphological transformation and grow in semi-solid medium. MMCV thus transforms both mouse fibroblastic cells and macrophages in vitro, in a fashion similar to the v-myc-containing avian viruses in chicken cells. The possibility of introducing a transforming myc gene into mammalian cells by virus infection provides a novel approach for studying the mechanism of myc transformation in cells from many lineages.     

Emperipolesis of lymphoid cells by human follicular dendritic cells in vitro.

Isolated follicular dendritic cells (FDCs) showed true and pseudoemperipolesis of fresh tonsillar lymphocytes, even after long-term (50-day) cultivation. Emperipolesis by FDCs was not restricted by allotype specificity, nor was it inhibited by the addition of antibodies against MHC-I & II antigens. Follicular dendritic cells predominantly engulfed B-cells; monocytes and macrophages were not found between FDC cytoplasmic extensions. When highly purified T-cell populations were added to FDC cultures emperipolesis of T-cells occurred, particularly those of the CD4-positive phenotype. Mitoses appeared within 6 h in the emperipolesed lymphocytes and, after an additional 18 h, some lymphocytes exhibited apoptosis.     

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-.     

Providing the TORC for cell cycle progression in neoplastic mast cells

Comment on: Smr? D, et al. Blood 2011; 118:6803–13