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.     

Phenotypically distinct target cells for murine sarcoma virus and murine leukemia virus marrow transformation in vitro.

An in vitro hematopoietic microenvironment was established from explained fragments of bone marrow from adult noninbred NIH Swiss mice with the use of corticosteroid-reconstituted horse serum. Infection with Kirsten murine sarcoma virus (Ki-MuSV) with either a Rauscher murine leukemia virus (R-MuLV) or Balb:virus-1 helper virus coat reduced proliferation of granulocytic and pluripotent hematopoietic stem cells and produced neoplastic transformation of both macrophages and preadipocytes in the adherent cell population within a 4-week period. Ki-MuSV-transformed, virus-releasing macrophages formed clusters of 4-49 cells in 0.8% methylcellulose-containing medium in the absence of added colony-stimulating factor (CSF), synthesized lysozyme, ASD-chloroacetate substrate-specific esterase-M, and CSF, and produced tumors following inoculation iv into adult NIH Swiss mice or ip into newborn NIH Swiss mice. In cultures infected with helper leukemia viruses R-MuLV or Balb:virus-1, gradual transformation of a distinct cell phenotype was observed over a 9-week period with generation of increasing numbers of atypical myeloblasts and promyelocytes which showed dyssynchronous nuclear-cytoplasmic maturation, basophilic granulation, cytoplasmic vacuolation, and formation of incompletely maturing CSF-dependent granulocyte-macrophage colonies in vitro and small spleen colonies in vivo. These data demonstrated that rapid biologic expression of the murine sarcoma virus genome in specific adherent "stromal" marrow cells prevents detection of a more subtle helper-virus-induced dysmyelopoiesis in a distinct nonadherent cell population.     

Focal infection and transformation in situ of thymus cell subclasses by a thymotropic murine leukemia virus.

Studies on the maturational lineages of thymic lymphocytes have revealed several subclasses which are distinguishable on the basis of cell size, topographic distribution within the thymus, DNA synthetic and mitotic activity, migratory behavior, and other properties. Strain C57BL/Ka mice were inoculated with radiation leukemia virus at different concentrations, and tissues were removed at defined intervals. Sequential sections were analyzed for virus-specific cytoplasmic antigen expression, for morphological evidence of neoplastic transformation, and for alkaline phosphatase activity. The first detectable sign of MuLV infection was the focal appearance of cytoplasmic viral antigens in cells of the outer thymic cortex, followed by coalescence of such foci and, several weeks later, by the appearance of morphologically transformed and alkaline phosphatase-positive cells, again often focally distributed in the outer thymic cortex. These observations strongly suggest that the large, mitotically active cells of the outer thymic cortex are the principal source of target cells for both productive infection and subsequent lymphoma induction by the virus.     

In vitro immune-receptor gene rearrangements in clonal thymic lymphomas induced by Abelson murine leukemia virus

The Abelson and Moloney murine leukemia virus complex (A-MuLV/M-MuLV) induces rapidly growing thymic lymphomas following direct injection into the thymus of newborn BALB/c and C57BL/6 mice. Southern blot analysis with a v-abl specific probe not only demonstrated that primary tumors are clonal, but also that the pattern of A-MuLV provirus integration is quite stable in primary tumor cells, as well as in their derived cell lines and clones. Most of the cell samples were able to rearrange the immunoglobulin heavy chain genes in culture, whereas in two cases the T cell receptor gamma chain genes also underwent rearrangement. Since the recombination mechanism is operative only in very immature lymphoid cells, these data provide indirect evidence for the lack of differentiation of A-MuLV cell targets in the thymus.     

Resistance of G mice to murine leukemia virus infection: apparent disparity in in vivo and in vitro resistances.

We observed a marked discordance between in vivo and in vitro sensitivities to Friend murine leukemia virus in G mice. In vivo resistance of G mice was more than 10(5)-fold relative to sensitive DDD mice, whereas in vitro resistance was only 50 to 100-fold. In vivo sensitivity to N- and NB-tropic Friend murine leukemia virus was recessive in (G X DDD)F1 mice, whereas in vitro sensitivity was dominant in the heterozygotes. The resistance of mouse fibroblasts was different from the resistance of fibroblasts of a certain NZB mouse strain.     

Analysis of the interaction between Rauscher murine leukemia virus and murine cell membrane receptor by in vitro binding assay

Binding of ¹²⁵I-labeled gp70 of Rauscher murine leukemia virus (R-MuLV) by 3 murine cell lines, $\text{BALB}\text{c}\text{-3T3}$, $\text{NIH}\text{3T3}$ and KA-31 (Kirsten murine sarcoma virus transformed clone A-31 of $\text{BALB}\text{c}\text{-3T3}$) cells was measured. The binding was a saturable process, dependent on the concentration of gp70 and on the number of cells. In no experiment could we demonstrate any quantitative utilization of gp70 in the medium. However, gp70 remaining in the spent medium could be bound to fresh cells in a subsequent incubation. $\text{BALB}\text{c}\text{-3T3}$, $\text{NIH}\text{3T3}$ and KA-31 cells showed similar association constants (1.2–2.5 × 10⁸ M^?1) for the binding. Moreover, all 3 cell lines had similar number of receptors (7.4–8.9 × 10⁵) per cell. Neither N- and B-tropism of the cells nor transformation by a sarcoma virus altered the number and type of the cell surface receptors.     

Further observations on in vitro cytopathic effects associated with murine leukemia virus infection

The previously reported cytopathic effects (CPE) in an established cell culture chronically infected with the Rauscher murine leukemia virus have been investigated in more detail. Initiation of CPE and morphologic transformation was observed in newly infected cultures. Suppression of CPE and transformation by NCTC 109 medium containing 25% human serum was also found to be reproducible in newly infected cultures. Adult human serum, human cord serum, fetal calf serum and fetuin had a suppressive effect on the CPE and transformation. Serological tests to detect non-leukemogenic mouse viruses in the transformed cells were negative. Infected, trans-formed cells were highly tumorigenic when inoculated subcutaneously into BALB/c mice. The resultant tumors were classified as myxofibrosarcomas. Approximately 50% of such tumor-bearing mice also developed leukemia. In contrast, control uninfected cells under similar conditions were less tumorigenic and produced spindle-cell sarcomas. None of the mice injected with control uninfected cells developed leukemia. The tumori-genicity of both the infected and uninfected cell cultures increased when the cells were cultured on 4 × BME medium containing 10% calf serum.     

Suppressor cells induced by total lymphoid irradiation affect proliferation and lymphokine production of murine T helper cell clones

A key response to antigen is the activation of helper T cells to release lymphokines which stimulate and effect the immune reaction. This T cell population can secrete many different factors with diverse, often multifunctional roles, such as amplifying T or B cell antigen responses or being effectors of cell mediated delayed type hypersensitivity. Among these lymphokines are gamma-interferon (gamma-IFN), interleukin-2 (IL-2), or T cell growth factor, and lymphotoxin (LT) which has cytotoxic activity against a variety of cells. Immune suppression in mice following total lymphoid irradiation (TLI) has been correlated with the presence in lympho-reticular tissues of an antigen non-specific, null suppressor cell. This study examined what effects radiation induced suppressor cells had upon the in vitro activation and lymphokine responses of the ovalbumin (OVA) specific T helper cell clone, 153E6, following antigen presentation. Splenocytes from TLI treated mice obtained early in the post-irradiation period exerted a pan-inhibitory effect upon OVA induced 153E6 proliferation and its concomitant release of gamma-IFN, LT, and IL-2. As the interval from irradiation increased, splenocytes from TLI treated mice showed persistent suppression of 153E6 proliferation and gamma-IFN release, but had rapidly diminishing effects on the T cell's capacity to produce LT and IL-2. These findings suggest that suppressor cells induced by TLI have a marked inhibitory effect in vivo upon T helper cell proliferative responses to antigen and the production of various T helper cell lymphokines necessary to mediate the immune response. Such processes could contribute to the immunosuppressive effects of extensive nodal irradiation.     

Histologic and cell surface antigen studies of hematopoietic tumors induced by Cas-Br-M murine leukemia virus

Cas-Br-M, a cloned ecotropic murine leukemia virus (MuLV) of wild mouse origin that induces both neurogenic hindlimb paralysis and lymphomas, was injected into NFS/N inbred mice neonatally. Then the mice were observed for the development of neurologic disease and tumors. All mice manifested neurologic abnormalities by 6 months of age, and 58% of the animals died with hematopoietic neoplasms. The tumors included T- and B-cell lymphomas, lymphoblastic lymphoma, erythroleukemias, myelogenous leukemias, and a megakaryocytic leukemia. Cas-Br-M thus appeared to be unique among ecotropic MuLV in inducing a wide spectrum of hematopoietic tumors.     

Inhibition of in vitro Friend murine leukemia virus infection of lipopolysaccharide-activated B-cells with concanavalin A.

Stimulation with bacterial lipopolysaccharide (LPS) of splenic B-lymphocytes infected in vitro with Friend virus complex increased the number of cells with replicating murine leukemia virus (MuLV) [i.e., infectious centers (IC)] up to 100-fold. Concanavalin A (Con A) did not have such an effect. However, the addition of Con A to the LPS-stimulated cultures decreased the number of IC. The inhibitory concentration of Con A (2.5 microgram/ml) was eightfold less than that capable of neutralizing the in vitro infectivity of MuLV (20 microgram/ml). The effect of Con A was not mediated by T-cells; the inhibition of infection was comparable with use of whole spleen cell suspensions from normal BALB/c mice, with T-cell-depleted cell suspensions, or with spleen cells with congenitally athymic nude mice. However, specific removal of Con A from the surface of B-cells with alpha-methyl-D-mannopyranoside prior to the infection reversed the inhibitory effect entirely. It is suggested that the lectin interferes with MuLV on the membrane of B-cells.     

Lack of erythroid characteristics in Ia-positive leukemia cell lines induced by Friend murine leukemia virus: brief communication

A group of mouse leukemia cell lines induced by the Friend murine leukemia virus (F-MuLV) was examined for a cell membrane antigens (regulated by the I-region of the H-2 complex), as well as for erythroid characteristics. Erythroid traits tested were hemoglobin synthesis, incorporation of 59Fe into heme, and presence of globin mRNA. Of 19 lines, 13 were positive for erythroid characteristics. All of these 13 lines were a-negative. Of 19 lines, 6 were negative for erythroid characteristics, and 5 of the 6 were a-positive. The data suggested that F-MuLV-induced leukemogenesis may operate in more than 1 cell type. In addition to the primitive erythroid type of cell usually involved in leukemia induced by F-MuLV, nonerythroid Ia-positive cells may also be transformed. The exact origin of the Ia-positive leukemia cells is unknown.     

Redistribution and modulation of Gross murine leukemia virus antigens induced by specific antibodies.

Gross murine leukemia virus (G-MuLV)-induced rat leukemia cells in tissue culture replicate G-MuLV, express strong virus-associated membrane antigenicity, and are consistently killed by specific antibodies and complement in cytotoxicity tests. To explore the effect of specific antibodies, rat anti-G-MuLV antisera were added to the cultures of leukemia cells for variable periods of time. Redistribution of virus particles as well as of membrane virus antigens in the form of polar patches and caps was observed by electron microscopy, indirect immunofluorescence, and immunoelectron microscopy. Substantial decreases in cytotoxicity indexes accompanied these changes. The antigen modulation induced by anti-G-MuLV antibodies in vitro paralleled similar changes obtained in vivo by transplanttion of leukemia cells in rats with high anti-G-MuLV antibody titers. The importance of antigen modulation in this system resides in its direct relationship with the malignant potential of the leukemia cells.     

Leukemogenesis in vitro induced by thymus epithelial reticulum cells transmitting murine leukemia viruses.

The role of the thymus in induction of leukemia was studied in vitro. Curltivation of normal thymus cells on thymus epithelial reticulum cell monolayers that had been grown from radiation leukemia virus-induced leukemic thymuses rendered the thymocytes leukemic. C57BL/6 thymocytes were cultivated for 3 days on leukemic thymus reticulum monolayers, and 106 thymocytes were injected i.p. into young adult C57BL/6 mice. After 3 to 4 weeks all mice died of disseminated lymphatic leukemia. Mice given thymocytes that had been cultivated on thymus epithelial reticulum monolayers from normal mice did not develop lymphomas. The leukemic thymus epithelial reticulum cells were shown to produce thymotropic as well as ecotropic and xenotropic radiation leukemia virus. (Thymotropic virus has affinity for thymus lymphocytes but noes not infect fibroblasts.) The cells were brightly positive for murine leukemia virus group-specific antigen in immunofluorescence tests. Leukemic thymus epithelial reticulum cells produced ample infectious exotropic virus in the culture supernatant, although the cells were negative in the XC syncytia test. Upon infection of mouse fibroblasts with ecotropic virus produced by the leukemic reticulum cells, XC syncytia were readily obtained. Thymocytes that were cultivated on leukemic thymus reticulum cells became positive for murine leukemia virus group-specific antigen and produced syncytia in the XC test. Thus, in vitro lymphomagenesis of the thymocytes that were cultured on leukemic thymus reticulum cells was associated with their infection with thymotropic and ecotropic radiation leukemia virus.     

Pathogenesis and virus content of lymphomas induced by pure ecotropic Graffi murine leukemia virus

Tumors induced by wild type Graffi murine leukemia virus (Gi-MuLV) contained high titers of MuLV consisting of a predominant ecotropic (e)-MuLV and a scarcer titer recombinant (RM) MuLV component. Each of these was purified by biological cloning and examined for its envelope properties and leukemogenicity. Both the e- and the RM-MuLV's were single isolates and unique in terms of their neutralization profiles and peptide maps. The cloned e-Gi-MuLV was highly leukemogenic in C57Bl mice, inducing a very rapid lethal thymic lymphoma but no myeloid leukemia. e-Gi-MuLV also accelerated thymic lymphoma in AKR mice. The purified RM-MuLV did not induce any tumors. Infectious cell center (ICC) experiments of organs of mice inoculated with e-Gi-MuLV showed that virus replicated very rapidly and reached maximal titers in about one week in C57Bl mice. There was a highly preferential replication in the thymus of the animal so that this e-Gi-MuLV can be considered as thymotropic. Within two weeks after infection of mice, infected cells of the thymus also began to release low levels of a non-ecotropic MuLV. The rapid induction of lymphoma is compared to that induced by other e-MuLV's and their RM-MuLV's, and to the natural AKR-MuLV-associated disease. These findings are discussed in the context of prevailing theories on envelope gene rearrangements in the virus and in the proviral sequences in resulting tumors.     

Enhancement of erythroid target cells for Friend murine leukemia virus by intravenous pyran treatment.

Male BALB/c mice that received prophylactic iv treatment with pyran had significantly enhanced splenomegaly, an increased number of splenic foci induced by the spleen focus forming virus (SFFV) in the Friend murine leukemia virus (F-MuLV) complex, and a slightly decreased mean survival time as compared with untreated controls infected with F-MuLV. A corresponding increase in the lymphatic leukemia virus component of the F-MuLV complex was not observed, which suggests that the enhancement of the disease was due primarily to a selective increase in the SFFV component of the F-MuLV complex. That the enhancement was related to an increased number of target cells for SFFV was substantiated by data concerning erythropoiesis in iv pyran-treated animals. Increases in splenic hematocrits and in uptake of 59Fe in the spleens of animals treated iv with pyran provided quantitative evidence for the histologic finding of increased erythroid precursors in the spleens.