[CANCER RESEARCH 41, 3556-3565, September 1981] 0008-5472/81/0041-OOOOS02.00 Effects of Murine Leukemia Virus Infection on Long-Term Hematopoiesis in Vitro Emphasized by Increased Survival of Bone Marrow Cultures Derived from BALB/Mo Mice1 Joel S. Greenberger,2 Richard K. Shadduck, Rudolf Jaenisch, Abdul Waheed, and Mary Ann Sakakeeny Joint Center for Radiation Therapy. Department of Radiation Therapy. Harvard Medical School and Sidney Farber Cancer Institute, Boston. Massachusetts 02115 [J. S. G., M. A. S.I; Heinrich Fette Institute. Hamburg. Germany [R. J.]; and Montefiore Hospital, Pittsburgh. Pennsylvania 15213[R. K. S., A. WJ ABSTRACT The effect of infection with Moloney murine leukemia virus (Mo-MuLV) on long-term bone marrow cultures was studied. Cultures were derived from the bone marrow of BALB/Mo mice, which carry Mo-MuLV as an endogenous virus, or from BALB/c, 129/J, or BALB/c x 129/J mice that were infected with Mo-MuLV in vitro. The following parameters were tested: longevity of generation of granulocytes; biological properties of nonadherent cells in colony-forming assays for pluripotential stem cells and committed granulocyte-macrophage colony- forming unit culture, erythroid, or metachromasia-positive mast cell-basophil colony-forming cells; differentiation of nonadher ent cells following cocultivation with thymic or bone marrow stromal cells; and generation of WEHI-3 dialyzed conditioned medium-dependent permanent cell lines. Granulocytes were generated for 65 weeks in BALB/Mo marrow cultures, 31 weeks for BALB/c, 22 weeks in 129/J, and 28 weeks in BALB/c x 129/J cultures. Exogenous infection of BALB/c cultures with Mo-MuLV increased the longevity of hemato- poiesis to 41 weeks. Granulocyte-macrophage colony-forming unit cultures were produced for 61 weeks in BALB/Mo cul tures, 25 to 40 weeks in Mo-MuLV-infected cultures, and 19 to 33 weeks in uninfected control cultures. Nonadherent cells harvested from BALB/Mo marrow cultures generated cloned permanent WEHI-3 dialyzed conditioned medium-dependent, nonleukemogenic granulocyte-macrophage colony-forming unit culture cell lines at greater efficiency than did Mo-MuLV- infected or uninfected BALB/c cultures. The cell lines differ entiated to mature granulocytes following cocultivation with purified marrow or thymic stromal cells. There was no detect able differentiation of nonadherent cells to lymphocytes or mast cells. Thus, Mo-MuLV does not detectably transform granulocyte progenitor cells in vitro to granulocytic leukemia. However, Mo-MuLV replication stimulates self-renewal of gran ulocyte progenitor cells in both primary marrow culture and in suspension culture in WEHI-3 dialyzed conditioned medium. INTRODUCTION Moloney leukemia virus induces thymic T-cell lymphocytic leukemia in susceptible mouse strains following a latent period of several months (34, 38). The molecular parameters of leu- kemogenesis have been studied in animals infected as new- borns with virus or in mice carrying the virus in their germ line, the BALB/Mo strain (25). The preleukemic phase in both situations is characterized by an increase of viral-specific DMA copies in hematopoietic cells, such as spleen, thymus, and bone marrow (27, 28). A second increase of viral copies occurs in tumor cells during the process of malignant transformation. The available evidence suggests that the increase in viral- specific DNA sequences is a consequence of superinfection of susceptible target cells (37), representing a necessary but not sufficient precondition of malignant transformation. The development of a long-term culture system for murine bone marrow has facilitated studies on the effects of Friend, Rauscher, and Abelson murine leukemia virus infection on hematopoiesis (7, 12, 13, 15-18, 21-23). Bone marrow cul tures maintained in horse serum or corticosteroid-supple- mented PCS3 (11 ) generate predominantly granulocyte colony- forming cells and mature neutrophilic granulocytes with rela tively little erythroid, megakaryocytic, or lymphocytic differen tiation (12, 21 ). However, CFU-T have been identified in murine marrow cultures, suggesting the presence of available target cells for thymic lymphocyte leukemogenesis in vitro (30). In vivo experiments have indicated that factors specific to the thymic microenvironment are required for T-cell transformation, implying that induction of T-cell leukemias in bone marrow cultures might be impossible (3, 31). The present experiments were carried out to delineate the effects of Mo-MuLV on hemopoiesis in long-term bone marrow cultures. We specifically sought to determine whether germ line integration of the Mo-MuLV genome in the marrow of BALB/Mo mice produced effects distinct from exogenous in fection with Mo-MuLV and whether virus-infected hematopoi etic progenitor cells could be induced to form leukemogenic cell lines in vitro. MATERIALS AND METHODS Mice. AKR/J, C3H/HeJ, BALB/cJ, and 129/J mice were obtained from The Jackson Laboratory, Bar Harbor, Maine; NIH Swiss mice were from the animal colonies of the NIH, Bethesda, Md.; Swiss CD-1 mice were from the Charles River Breeding Laboratories, Inc., Wil mington, Mass. BALB/Mo and control BALB/c x 129/J (BALB/129) mice, the strain in which BALB/Mo mice were derived (25), were from the Heinrich Rette Institute, Hamburg, Germany. All mice were 30 to 33 g. ' Supported by National Cancer Institute Grants CA25412-02 and CA26785- 01. by CA-15237-05, a grant from the Deutsche Forschungsgemeinschaft, and by a research contract from the National Cancer Institute, Bethesda, Md. 1 To whom requests for reprints should be addressed, at Joint Center for Radiation Therapy, 50 Binney Street, Boston, Mass. 02115. Received February 23. 1981; accepted June 16, 1981. 3 The abbreviations used are: FCS, fetal calf serum; CFU-T, colony-forming unit T-lymphocyte; Mo-MuLV, Moloney murine leukemia virus; DMEM, Dul- becco's modified Eagle's medium; CM, conditioned medium; DCM, dialyzed conditioned medium; R-MuLV, Rauscher murine leukemia virus; SFFV, spleen focus-forming virus; GM, granulocyte-macrophage; CFUc, colony-forming unit culture; CFUe, colony-forming unit erythroid; CFU-L, colony-forming unit B-lym- phocyte; CFU-meta. colony-forming unit metachromasia; CPUs, colony-forming unit spleen; CSF, colony-stimulating factor. 3556 CANCER RESEARCH VOL. 41 Research. on November 25, 2021. © 1981 American Association for Cancer cancerres.aacrjournals.org Downloaded from