Granulopoiesis longevity in continuous bone marrow cultures and factor-dependent cell line generation: significant variation among 28 inbred mouse strains and outbred stocks.

Longevity of granulopoiesis in corticosteroid-supplemented continuous bone marrow cultures and generation of permanent WEHI-3 dialyzed conditioned medium (DCM)-dependent hematopoietic progenitor cell lines were evaluated for 28 inbred mouse strains and outbred stocks. Cultures from AKR/J, NZW, C58/J, DBA/2J, and C57BL/KsJ mice generated granulocytes for 45-58 weeks and released more than 10(5) murine sarcoma virus (MuSV) rescue units of endogenous retrovirus; nonadherent (NA) cells from 50% or more cultures produced permanent WEHI-3 DCM-dependent cell lines. Cultures from 129/J, BALB/cJ, SJL/J, RF/J, CD-1 Swiss, C57BR/cdJ, C57BL/Ka, CBA/J, RFM/Sn, NZB, C3H/HeJ, NIH Swiss [N:NIH(S)], C57BL/10J, C57L/J, SWR/J, HRS/J, and C57BL/6J mice generated granulocytes for 12-38 weeks; some released 10(1)-10(2) MuSV rescue units of retrovirus, and NA cells from less than 50% of cultures from all but SWR/J and C57L/J mice produced WEHI-3 DCM-dependent cell lines. Thus longevity of granulopoiesis in mouse continuous bone marrow cultures varies significantly among strains.

Murine leukemia viruses: induction of macrophage production of granulocyte-macrophage colony-stimulating factor in vitro.

Infection in vitro of freshly explanted N:NIH(S) mouse bone marrow with ectopic murine leukemia viruses produced an increase over control uninfected cultures in the 50 or more cell granulocyte-macrophage (GM) colonies and 10-49 cell clusters detected after 7 days of incubation in 0.3% agar at 37 degrees C and 7% CO2. This effect was observed only at plating densities above 5.0 X 10(4) cells/ml and was not observed with macrophage-depleted populations of colony-forming units of GM progenitor cells (GM-CFUc) purified by isopyknic density gradient centrifugation of nonadherent cells harvested from long-term bone marrow cultures. Fewer virus-infected, compared to uninfected, peritoneal exudate macrophages were required to stimulate the same number of GM colonies and clusters in a given number of purified GM-CFUc. In contrast, murine leukemia virus infection of T-lymphocytes or NIH/3T3 embryo fibroblasts did not stimulate release of GM-CFUc coloney-stimulating factor (CSF). Single Cell suspensions of virus-infected freshly explanted whole bone marrow grown in CSF concentrated from L929 or WEHI-3 cell-conditioned medium produced more GM-CFUc colonies and GM clusters/1 X 10(5) cells compared to single cell suspensions of uninfected marrow. This phenomenon suggests that the colonoy-forming cells responding to CSF from virus-infected marrow may have been different from those responding to L929 or WEHI-3 cell CSF. The data indicate that increased granulopoiesis observed following retrovirus infection in vivo or in long-term marrow cultures was attributable in part to virus stimulation of production of CSF by adherent marrow stromal cells including macrophages.

Pool size of pluripotential hematopoietic stem cells increased in continuous bone marrow culture by Friend spleen focus-forming virus.

Continuous mouse bone marrow cultures were infected with Friend murine leukemia virus. Production of nonadherent (NA) and adherent cells, granulocyte-macrophage colony-forming unit(s) of progenitor cells (GM-CFUc), pluripotential hematopoietic stem cells (CFUs), the self-renewal potential (Rs) of CFUs, and generation of factor-dependent (FD) multipotential and committed permanent stem cell cloned lines were measured. Uninfected marrow cultures from C57BL/6J, C57BL/6JUt, B6.S, C3H/HeJ, (C57BL/6J x DBA/2J)F1, CD- 1 Swiss, or N:NIH(S) mice generated NA cells, GM-CFUc, and CFUs for 20-41 weeks; cultures infected with Rauscher or other helper viruses generated them for 35-45 weeks. GM-CFUc and CFUs production in SFFV-positive cultures persisted for over 65 weeks and exceeded control levels by twentyfold to fiftyfold. The Rs of CFUs in SFFV-positive cultures was not detectably increased above control cultures. Multipotential (erythroid-neutrophil-mast cell-basophil-eosinophil) permanent FD cell clones were derived from control and SFFV-positive cultures. Thus SFFV amplifies the stem cell pool in vitro without detectably increasing the Rs capacity of CFUs.

Virus and cell requirements for Friend virus granulocytic leukemogenesis in long-term bone marrow cultures of NIH swiss [N:NIH(S)] mice.

The effect of hematopoietic stem cell age on leukemogenesis in vitro was tested in nonrecharged, corticosterold-supplemented NIH Swiss [N:NIH(S)] mouse long-term bone marrow cultures infected with Friend murine leukemia virus of anemia-inducing strain (F-MuLV-A) or spleen focus-forming virus (SFFV) [Rauscher murine leukemia virus (R-MuLV)], a pseudotype virus derived by rescue of the SFFV genome from SFFV-Balb/3T3 clone A31 nonproducer cells with clonal helper R-MuLV. Cultures at 33 degrees C derived from 10-day-old or adult mouse marrow generated colony-forming unit culture granulocytic macrophage (CFUc) progenitor cells for over 20 weeks and colony-forming unit spleen cells for 14 weeks and generated permanent granulocytic leukemia cell lines after infection with F-MuLV-A at week 1, 2, or 4 but not at week 8. Leukemia lines were of granulocyte phenotype whether induced by F-MuLV-A or SFFV (R-MuLV) and synthesized myeloperoxidase and lysozyme but were restricted in ability to generate superoxide in response to phorbol myristate acetate stimulation. Cultures (31 degrees C) infected with temperature-sensitive (ts) helper virus mutant pseudotypes of SFFV as well as SFFV (R-MuLV) generated granulocytic leukemia lines, whereas only SFFV (R-MuLV) pseudotype virus-infected cultures became leukemic at 37 degrees C. R-MuLV wild type or ts mutant helper virus infection alone increased cell proliferation and numbers of CFUc but did not generate leukemia. These data indicated that gene(s) specific to F-MuLV-A or a virus rescued from SFFV-Balb/3T3 clone A31 nonproducer cells are required for transformation in vitro of a hematopoietic stem cell present in early but absent in late bone marrow cultures.

Biologic effects of prolonged melphalan treatment of murine long-term bone marrow cultures and interleukin 3-dependent hematopoietic progenitor cell lines.

The mechanism of alkylating agent-induced leukemia is unknown. For the determination of whether chronic alkylating agent treatment of hematopoietic stem cells in vitro was detectably leukemogenic, murine long-term bone marrow cultures (LTBMC) and clonal interleukin 3 (IL-3)-dependent multipotential hematopoietic progenitor cell lines [B6SUtA clone (cl) 27 and Ro cl 3-1] derived from LTBMC were chronically pulse treated in vitro with the alkylating agent melphalan [L-phenylalanine mustard (L-PAM)]. Weekly treatment of C3H/HeJ or CD-1 Swiss mouse LTBMC with 3 X 10(-6)M L-PAM significantly decreased cumulative production of nonadherent granulocytes and granulocyte-macrophage progenitor cells responsive to L-cell or WEH1-3 cell colony-stimulating factor compared to the production seen in untreated control cultures; it also significantly reduced the hematopoietic longevity (13 wk compared to greater than 20 wk for untreated control cultures). Weekly, twice weekly, or daily (3 X 10(-6)M) L-PAM treatment of IL-3-dependent cell lines induced gradual L-PAM adaptation in the absence of a detectable change in the maximum binding capacity of 125I-labeled IL-3. No leukemogenic variants of line B6SUtA cl 27 were detectably induced. However, 3 stably expressed marker chromosomes were induced after 12 months of L-PAM treatment of line B6SUtA cl 27. Thus IL-3-dependent hematopoietic progenitor cells slowly adapt to L-PAM when in suspension culture in vitro. Physiologic expression of drug toxicity in LTBMC may prevent this hematopoietic cell gradual adaptation.

Effects of murine leukemia virus infection on long-term hematopoiesis in vitro emphasized by increased survival of bone marrow cultures derived from BALB/Mo mice.

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 nonadherent 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 hematopoiesis to 41 weeks. Granulocyte-macrophage colony-forming unit cultures were produced for 61 weeks in BALB/Mo cultures, 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 differentiated to mature granulocytes following cocultivation with purified marrow or thymic stromal cells. There was no detectable 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 granulocyte progenitor cells in both primary marrow culture and in suspension culture in WEHI-3 dialyzed conditioned medium.

Induction of malignant transformation of cocultivated hematopoietic stem cells by X-irradiation of murine bone marrow stromal cells in vitro.

X-irradiation of purified primary cultures of mouse bone marrow stroma or permanent cloned marrow stromal cell lines in plateau phase decreases production of macrophage progenitor cell-specific colony-stimulating factor to a plateau minimum of 40% of control levels after doses of 50 to 500 Gy delivered at 2 Gy/min. After 50 Gy there is increased bioavailability of another growth factor(s) that is distinct from macrophage progenitor cell-specific colony-stimulating factor, granulocyte-macrophage progenitor cell colony-stimulating factor, or colony-stimulating factor for multipotential hematopoietic stem cells (interleukin 3). Liquid-phase cocultivation of irradiated stromal cells with either nonadherent cells from continuous marrow cultures or cloned dual granulocyte-macrophage progenitor cell colony-stimulating factor/interleukin 3-dependent hematopoietic progenitor cell lines induces evolution over 5 weeks of factor-independent colony-forming cells. Subcultured factor-independent colonies generated clonal malignant cell lines with multiple distinct karyotypic alterations. Inoculation of 10(6) cells s.c. from factor-independent clones into syngeneic mice produces local granulocytic monomyeloid tumors with spread to spleen, lymph nodes, and bone marrow. These data provide the first demonstration in vitro of indirect X-irradiation leukemogenesis through cells of the marrow stroma.

Effects of monoclonal antibody and complement treatment of human marrow on hematopoiesis in continuous bone marrow culture.

Long-term bone marrow cultures were established from single-cell suspensions of human bone marrow that had been treated with monoclonal antibodies and complement. Each treated cell suspension was evaluated for production of hematopoietic stem cells over 20 weeks. Treatment with antibody to HLA-DR (Ia), B1, J2, or J5 did not remove adherent cells including those differentiating to adipocytes in 17-hydroxycorticosteroid. In contrast, treatment with monoclonal antibody directed against human beta 2-microglobulin reduced adipocyte numbers by 100-fold and reduced the total adherent cell density over 70%. Cumulative total nonadherent cell and granulocyte-macrophage colony-forming units (GM-CFUc) production over 20 weeks was not significantly altered by one cycle of anti-Ia plus complement or up to three cycles of treatment with complement and anti-J2, -J5, or -B1. However, one cycle of treatment with anti-beta 2-micro-globulin depressed production of both GM-CFUc and nonadherent cells by over 100-fold compared to other treatment groups. While one cycle of treatment of anti-Ia and complement killed all detectable cells forming CFU-erythroid-granulocyte-megakaryocyte-macrophage, blast-forming units (erythroid), and GM-CFUc, GM cluster-forming cells survived. Treatment of marrow with three cycles of anti-Ia and complement removed all detectable GM colony- and GM cluster-forming cells; however, this marrow produced fewer cumulative Ia-positive GM-CFUc. Long-term bone marrow cultures may prove to be an interesting system for in vitro analysis of the effects of new immunotherapeutic agents including other monoclonal antibodies prior to clinical use.

Expression of M-CSF and its receptor (C-FMS) during factor-independent cell line evolution from hematopoietic progenitor cells cocultivated with gamma irradiated marrow stromal cell lines.

Gamma irradiation of plateau-phase clonal bone marrow stromal cell lines produces factor-independent growth of cocultivated clonal interleukin-3/granulocyte-macrophage colony-stimulating factor-dependent hematopoietic progenitor cell lines. The process is associated with three biologic changes including: (i) adherence of hematopoietic cells to stromal cells forming 'cobblestone islands'; (ii) an intermediate stage [during which the cells show proliferation in suspension in the presence in leukemogenic stromal factor (LSF), a factor similar to macrophage colony-stimulating factor (M-CSF) released by irradiated stromal cells, and transient hematopoietic cell surface expression of MAC-1, and c-fms (M-CSF receptor)]; and (iii) a third stage of factor-independence. A monoclonal antibody to M-CSF receptor inhibited proliferation of intermediate stage but not all factor-independent cell subclones. In the present studies, a subclonal factor-independent malignant subline of FDC-P1JL26 derived by cocultivation with gamma-irradiated stromal cells as well as the parent clone and intermediate stage cells were shown to express significant levels of M-CSF polyA+ mRNA and M-CSF of at least two sizes (23 and 15 kDa) as detected by 35S-methionine labelling and immunoprecipitation with polyclonal anti-M-CSF antiserum. There was no significant difference in intracellular M-CSF protein size between cells at each of the three stages of biologic change. This M-CSF was not detected on the cell surface by fluorescence-activated cell sorting (FACS). In contrast, c-fms expression at the cell surface was detected by FACS analysis and c-fms polyA+ mRNA was only detected during the intermediate stage of induction of factor-independence. FDC-P1JL26 parent cells, the subclone stimulated by LSF, and the factor-independent subclone, showed little or no detectable autophosphorylation of the c-fms receptor at tyrosine. There was no detectable rearrangement of the M-CSF or c-fms genes by Southern analysis between clonal lines during the three stages. While we cannot rule out an autocrine mechanism or mutated c-fms receptor mechanism, the data also suggest that evolution of hemopoietic cell factor-independence during cocultivation with irradiated stromal cells may involve a mechanism distal to the c-fms receptor/M-CSF interaction.

Radiosensitivity of cloned permanent murine bone marrow stromal cell lines: nonuniform effect of low dose rate.

The x-irradiation biology of supportive stromal cells of the bone marrow microenvironment was investigated by using cloned permanent cell lines that were established from hematopoietically active murine long-term bone marrow cultures. X-irradiation survival curves were derived for each cell line at either 120 cGy/min or clinical low dose rate (LDR) (5 cGy/min) that is used in total body irradiation protocols prior to bone marrow transplantation. Four cell lines, MBA-1, MBA-13, 14F2.1, and D2XRII (Group I) demonstrated a significant increase in D0 at 5 cGy/min (280 cGy, 270 cGy, 210 cGy, and 240 cGy, respectively) compared to 120 cGy/min (215 cGy, 210 cGy, 157 cGy, and 210 cGy, respectively) (p less than 0.05). In contrast, three other clonal cell lines, +/+ #2 cl 4, S1d #3, and GPI alpha-1 (Group II) showed no significant dose rate dependent change in D0 at 5 cGy/min (164 cGy, 174 cGy, and 159 cGy, respectively) compared to 120 cGy/min (159 cGy, 167 cGy, and 143 cGy, respectively). Group I and II cell lines could not be distinguished by differences in synthesis of extracellular matrix proteins including laminin, collagen types I and IV, or histochemically detectable enzymes. Three Group I lines (MBA-1, MBA-13, and D2XRII) and one Group II line, Sld #3, showed decreased support capacity for cocultivated hematopoietic stem cells in vitro. All seven lines had detectable polyA+ mRNA for monocyte colony-stimulating factor (M-CSF) as detected by molecular hybridization; one had detectable levels of polyA+ mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) (D2XRII); one, detectable levels of polyA+ mRNA for interleukin 1 (IL-1); and none of the seven had detectable polyA+ mRNA for granulocyte colony-stimulating factor (G-CSF) or IL-3 (multi-CSF). The data indicate that some cells of the hematopoietic microenvironment may not be selectively protected by clinical low dose rate irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecularly cloned and expressed murine T-cell gene product is biologically similar to interleukin-3.

Clonal lines of mouse inducer ly1+ly2- inducer T-lymphocytes that depend for growth upon interleukin-2 have been demonstrated to produce a factor that stimulates colony formation by bone marrow granulocyte-macrophage (GM-CFUc) progenitor cells and replication of factor-dependent mast cell/basophil and multipotential hematopoietic cell lines in vitro. The molecularly cloned and expressed gene product for this growth factor demonstrates the following activities in vitro: using fresh bone marrow or purified subpopulations of nonadherent cells from murine continuous bone marrow cultures as target cells: stimulation of colony formation by GM-CFUc, mast cell progenitor cells, multipotential granulocyte/erythroid/megakaryocyte/macrophage progenitor cells (CFU-GEMM) colonies, erythroid progenitor cells forming macroscopic bursts (BFUe), and megakaryocyte progenitor cells (CFU-mega). The gene product also supports growth of previously reported mast cell growth-factor-dependent cell lines and several classes of interleukin-3 (IL-3)-dependent hematopoietic progenitor cell lines that are multipotential (neutrophil/basophil/eosinophil or neutrophil/basophil/erythroid); or committed to granulocyte-macrophage, or mast cell/basophil differentiation. The gene product does not detectably support replication of IL-2-dependent murine T-cell lines. The biologic activity of the gene product was inhibited greater than or equal to 90% by rabbit antisera prepared against purified interleukin-3. The data indicate that this T-cell derived lymphokine gene product is biologically very similar to interleukin-3.

Production of colony-stimulating factor(s) for granulocyte-macrophage and multipotential (granulocyte/erythroid/megakaryocyte/macrophage) hematopoietic progenitor cells (CFU-GEMM) by clonal lines of human IL-2-dependent T-lymphocytes.

Human T-lymphocyte lines that were selected for recognition of HLA-DR6 antigen and were dependent for growth in vitro on an added source of interleukin-2 (IL-2) were derived from the peripheral blood of normal individuals. Each was tested for production of a lymphokine(s) with properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) using as target cells nonadherent cells from human long-term bone marrow cultures (LTBMC) or fresh marrow. Each of eight T-lymphocyte lines that were OKT3, OKT4, and HLA-DR positive produced GM-CSF that stimulated colony formation by both LTBMC cells and fresh marrow. Individually examined single-cell-derived bone marrow colonies growing in T-cell GM-CSF contained peroxidase-positive neutrophils, and macrophage-monocytes (GM-CFUc). Supernatant from a single-cell-derived T-cell clonal line designated F1 stimulated formation of granulocyte-macrophage colonies, megakaryocyte colonies, macroscopic erythroid bursts, and multipotential colonies containing erythroid cells, megakaryocytes, neutrophilic and eosinophilic granulocytes, and monocyte-macrophages (CFU-GEMM) in the presence of added erythropoietin. These data indicate that human IL-2-responsive T-lymphocytes produce lymphokine(s) that stimulate proliferation of primitive as well as committed hematopoietic stem cells, and implicate human T-lymphocytes in regulation of human multipotential hematopoietic stem cells in vivo.

Biological characterization of cloned permanent stromal cell lines from anemic Sl/Sld mice and +/+ littermates.

Permanent cloned bone marrow stromal cell lines, designated Sld1, Sld2, Sld3, were derived from long-term bone marrow cultures (LTBMC) of Sl/Sld mice and littermate WCB6F1 mice (designated +/+1, +/+2, +/+1.10, and +/+2.4). Production of extracellular matrix proteins by these cell lines was detected using specific antibodies. Fibronectin, laminin, and collagen type IV were detected in all clonal cell lines tested. Each cloned stromal cell line or parent stromal cell culture was also tested in vitro for support of engrafted hemopoietic stem cells. Engrafted hemopoietic stem cells from C57BL/6J LTBMCs forming CFU-GEMM and BFUe colonies were supported at 76% and 44% efficiency by lines +/+2.4 and +/+1.10, respectively, compared to the number recovered from parent uncloned C57BL/6J stromal cell cultures. In contrast, cell lines Sld1 and Sld3 were significantly less supportive of CFU-GEMM progenitor cells (14% and 18% efficiency, respectively) and erythroid progenitors (6% and less than 0.1% efficiency, respectively). Some Sld lines also showed reduced support compared to +/+ cell lines of a clonal multipotential erythroid cell line B6SUtA. These permanent stromal cell lines should be useful in elucidation of the specific microenvironmental factors required for support of multilineage and erythroid progenitor cells.

Expression of a selectable gene transferred by a retroviral vector to hematopoietic stem cells and stromal cells in murine continuous bone marrow cultures.

Retroviral-mediated gene transfer to multipotent and committed hematopoietic stem cells and marrow stromal cells was evaluated in long-term bone marrow cultures (LTBMCs). The retroviral vector pZIP-Neo(SV)(X) carrying the bacterial neomycin resistance (neor) gene that confers resistance to the neomycin analog G418 in mammalian cells was packaged in a Moloney envelope either as a replication-competent or replication-defective virus. Virus was introduced by infection of long-term marrow cultures at day 7. During a period of 12 weeks in culture, 10%-50% of harvested hematopoietic progenitor cells that formed differentiated CFU-GEMM colonies in response to pokeweed mitogen-containing spleen cell-conditioned medium (SCCM) and erythropoietin expressed the neor gene. In contrast, 1%-10% of hematopoietic progenitor cells that formed colonies in agar in response to WEHI-3B- or L-cell-conditioned medium expressed resistance to G418. The percentage of resistant progenitors was not detectably enhanced when replication-competent Moloney murine leukemia virus (M-MuLV) was present as helper virus, even though M-MuLV infected greater than 90% of cells in the long-term marrow cultures. In a separate CFU-F assay, 12%-17% of the adherent stromal cells in LTBMCs were found to express the neor gene. Thus gene transfer is limited by the fraction of progenitor cells that can integrate and express the transferred genetic sequences, rather than by the fraction of cells that are initially infected by the vector.

Hematopoietic stem cell- and marrow stromal cell-specific requirements for gamma irradiation leukemogenesis in vitro.

The hematopoietic and stromal cell-specific properties of the cells involved in gamma irradiation leukemogenesis in vitro were defined. Cocultivation of clonal factor-dependent (FD), interleukin 3 (IL-3)-dependent cell lines 32D cl 3 or B6SUtA, or dual IL-3-/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell lines FDC-P1 or bg/bg d64 was carried out with clonal stromal cell lines D2XRII, GB1/6, +/+ 2.4, or Sld3. FD cell lines were added to control or 5000-cGy-irradiated plateau phase monolayer cultures of each stromal cell line, and parameters of stem cell engraftment and malignant transformation in vitro were quantitated. Cobblestone island formation by FD cells, cumulative production of nonadherent hematopoietic cells, and evolution of tumorigenic factor-independent (FI) subclonal lines were quantitated over 5-8 weeks. There was no detectable evolution of FI sublines with 32D cl 3, B6SUtA, or bg/bg d64 cells cocultivated with control or irradiated Sld3 stromal cells. IL-3-dependent cell lines 32D cl 3 or B6SUtA formed small 10- to 49-cell cobblestone "clusters" at low frequency on control or irradiated D2XRII, showed limited proliferation for less than 1 week, and showed no detectable evolution of FI cell lines. Subclones of 32D cl 3 derived by transfection and expression of recombinant oncogenes v-sis, or c-myc, or the epidermal growth factor receptor remained factor dependent and did not transform to factor independence after cocultivation with irradiated stromal cell lines. In contrast, cell line bg/bg d64, and each of seven subclonal lines of FDC-P1, including subclones selected for growth in GM-CSF, formed abundant cobblestone island colonies of greater than or equal to 50 cells on irradiated D2XRII stromal cells, produced non-adherent cells over 5-8 weeks, and showed evolution of tumorigenic FI subclonal lines. The data provide evidence for stable biological differences in both the hematopoietic and stromal cell components of the in vitro model of gamma irradiation leukemogenesis.

Effects of low dose rate irradiation on plateau phase bone marrow stromal cells in vitro: demonstration of a new form of non-lethal, physiologic damage to support of hematopoietic stem cells.

The clinical use of low dose rate (LDR) (5-25 rad/min) total body irradiation in bone marrow transplantation patients is well established. We have developed an in vitro system for study of the effects of LDR irradiation on bone marrow stromal cells. Purified mouse bone marrow stromal cell cultures in plateau phase with no detectable hematopoiesis were prepared and were then "engrafted" in vitro by addition of purified nonadherent hematopoietic cells from continuous bone marrow cultures. Hematopoietic cells were added in liquid medium or suspended in an overlay of semisolid 0.4% agar-containing medium. Other agar overlays contained Interleukin-3-dependent cloned multipotential hematopoietic stem cell line B6SUtA. In parallel experiments, a cloned permanent bone marrow stromal cell line D2XRII was used in place of purified stromal cell cultures. Stromal cultures were irradiated at 5 rad/min, 20 rad/min, or 200 rad/min, 24 hours or 3 weeks prior to "engraftment." Two classes of irradiation damage were demonstrated following 1000 rad irradiation at 200 rad/min: 1) Decreased clonagenic survival of trypsinized replated marrow stromal cells (lethal effect); and 2) decreased production by marrow stromal cells or D2XRII cells of colony stimulating factors (CSF)s for granulocyte-macrophage progenitor cells and B6SUtA cells (physiologic effect). Holding the cultures in plateau phase for 3 weeks after irradiation was associated with significantly more repair of the lethal effect compared to the physiologic effect. Cultures irradiated at 5 rad/min or 20 rad/min to doses producing significantly less lethal effect showed a complex alteration of production of growth factors. Cumulative cell production by hemopoietic stem cells added in liquid culture was comparably decreased for all three dose rates. These data demonstrate a distinct physiologic expression of irradiation damage to bone marrow stromal cells that affects cell to cell interaction, responds differently to changes in dose rate, and is repaired with kinetics different from those of the lethal effect of irradiation. The present system should prove valuable for investigation of cellular interactions in hematopoietic stem cell engraftment that are altered by total body irradiation.

Expression of p210 bcr/abl increases hematopoietic progenitor cell radiosensitivity.

PURPOSE: The cytogenetic finding of the Ph1+ chromosome and its molecular biologic marker bcr/abl gene rearrangement in cells from patients with chronic myeloid leukemia are associated with a proliferative advantage of the Ph1+ clone in vivo. Although the transition to the acute terminal phase or blastic crisis is often associated with additional cytogenetic abnormalities, the molecular events which correlate the initial cytogenetic lesion with the terminal phase are poorly understood. Defective cellular DNA repair capacity is often associated with chromosomal instability, increased mutation frequency, and biologic alterations. METHODS AND MATERIALS: We, therefore, tested whether the protein product of the bcr/abl translocation (p210) could alter DNA repair after gamma-irradiation of murine cell lines expressing the bcr/abl cDNA. RESULTS: The 32D cl 3 parent, 32D cl 3 pYN (containing the control vector plasmid) and each of two sources of 32D cl 3 cells expressing p210 bcr/abl cDNA (32D-PC1 cell line and 32D-LG7 subclone) showed a D0 of 1.62, 1.57, 1.16, and 1.27 Gy, respectively. Thus, expression of the p210 bcr/abl product induced a significant (p < 0.05) increase in radiosensitivity at the clinically relevant radiation therapy dose-rate (1.16 Gy/min). The increased radiosensitivity of p210 bcr/abl expressing cells persisted if cells were held before plating in a density-inhibited state for 8 hr after gamma-irradiation, indicating little effect on the repair of potentially lethal gamma-irradiation damage. The IL-3 dependent parent 32D cl 3 cells demonstrated programmed cell death in the absence of growth factor or following gamma-irradiation to 200 cGy. Expression of bcr/abl cDNA in the 32D-PC1 and 32D-LG7 sub clones abrogated IL-3 requirement of these cell lines and inhibited gamma-irradiation induced programmed cell death. CONCLUSION: These data suggest a role for bcr/abl p210 in amplifying gamma-irradiation DNA damage or broadly inhibiting DNA repair, conditions that may stimulate further cytogenetic alterations in hematopoietic cells.

Alteration in hematopoietic stem cell seeding and proliferation by both high and low dose rate irradiation of bone marrow stromal cells in vitro.

The mechanism of physiologic alteration by high (HDR) or low dose rate (LDR) (5 or 120 cGy/min) irradiation of plateau-phase bone marrow stromal cell cultures was investigated using a technique of in vitro bone marrow transplantation. Purified stromal cell cultures from C57BL/6J, C3H/HeJ, or (C57BL/6J X DBA2/J)F1 (B6D2F1) mouse marrow were irradiated to doses of 2.5 to 10 Gy at LDR or 10-100 Gy at HDR and were then engrafted in vitro with nonadherent hematopoietic cells from murine continuous bone marrow cultures. Parameters of engraftment quantitated included: (1) numbers of adherent proliferating hematopoietic cell colonies, "cobblestone islands" (2) cumulative production of nonadherent hematopoietic cells over 8 weeks after engraftment, (3) M-CSF, GM-CSF and multi-CSF (IL-3) dependent hematopoietic progenitor cells forming greater than or equal to 50 cell colonies in semisolid medium, (4) cumulative production of CFUs, and (5) number of adherent stromal cells positive for detectable extracellular laminin or collagen type IV (markers of endothelial cells, reticular adventitial cells, or sinus lining cells). There was a decrease in cobblestone island formation between 5 and 10 Gy and this parameter possibly increased at doses of 50 and 100 Gy. There was no difference between HDR and LDR irradiation to 10 Gy. Irradiation to doses above 10 Gy decreased support of engrafted cells forming CFU-GM and CFU-GEMM. Measures of CFUs after 10 Gy were variable but indicated a possible increase with HDR and no effect of LDR at 1 week and a decrease in both HDR and LDR groups at 3 weeks after engraftment. Thus, LDR and HDR irradiation in vitro alter several specific parameters of marrow stromal cell support for engrafted hematopoietic stem cells.

Gamma-irradiation response of cocultivated bone marrow stromal cell lines of differing intrinsic radiosensitivity.

There is evidence for differences in the gamma-irradiation response of different cellular lineages within the bone marrow microenvironment. We previously reported that heterogeneity is demonstrable in the gamma-irradiation response of five clonal stromal cell lines, derived from one human bone marrow specimen, despite morphological, histochemical, cytogenetic, and functional similarity. In the present study we tested whether one stromal cell line could affect the intrinsic radiosensitivity of another. Two clonal stromal cell lines, which display distinct gamma-irradiation responses relative to dose rate were used: KM 101, which shows the same radiosensitivity at a low dose rate of 5 cGy/min (LDR) and a high dose rate of 120 cGy/min (HDR) and KM 104 which shows significant gamma-irradiation resistance at LDR. To facilitate the study of the gamma-irradiation response of each cell line during cocultivation, we derived stable subclones of each, expressing the transfected neomycin resistance (neo-r) gene, which confers resistance to the neomycin analog: G 418. Introduction of the neo-r gene did not alter cell lines radiosensitivity. The results show that cocultivation of stromal cell lines before, during, and after gamma-irradiation induces changes in repair of radiation-induced damage, with a dominant effect of a resistant cell line at LDR. In fact, the radiation survival curves of cocultivated stromal cell lines were always characteristic of KM 104, and a dose rate effect was observed, even when KM 101 was present in large excess. Moreover, our results are consistent with preferential killing of the more radiosensitive stromal cell line: both LDR and HDR Do values of the neo-r KM 101, cocultivated with the parent KM 104 for 24 hr before, and during gamma irradiation were significantly lower compared to the neo-r subclone irradiated alone. The LDR Do value of the neo-r KM 104 cocultivated for 24 hr before, and during gamma irradiation with excess of parent KM 101, was significantly higher, compared to the neo-r cells irradiated alone.

Radiosensitivity of permanent human bone marrow stromal cell lines: effect of dose rate.

In contrast to the dose-rate independent X ray killing observed with human bone marrow hematopoietic stem cells, bone marrow adherent stromal cells from the same fresh marrow harvests demonstrate increased radiation resistance at low dose rate (LDR) (5 cGy/min), compared to high dose rate (HDR) irradiation (120-200 cGy/min). Physiologic changes observed in plateau phase bone marrow cells after LDR irradiation in vivo and in vitro suggested that marrow stromal cells might be heterogeneous in LDR irradiation repair. Five permanent clonal bone marrow stromal lines were derived from a single human marrow donor. Each cell line was positive for markers of fibroblasts including: immunohistochemically detectable fibronectin, collagen, acid phosphatase, and nonspecific esterase, and was negative for Factor VIII, alkaline phosphatase, lysozyme and several markers of marrow macrophages. The x-irradiation survival curve of each cell line was determined at LDR and HDR in vitro. Cell lines KM102, KM103, KM104, and KM105 each demonstrated a significant (p less than .05) increase in radioresistance at LDR (D0 = 142, n = 2.9; D0 = 131, n = 2.5; D0 = 145, n = 2.1 and D0 = 127, n = 2.1 respectively) compared to HDR: (D0 = 111, n = 2.1; D0 = 94, n = 3.5; D0 = 99, n = 3.5 and D0 = 95, n = 2.1 respectively). In contrast, cell line KM101 demonstrated no significant change in radiosensitivity relative to dose rate at LDR (D0 = 113, n = 3.3) compared to HDR, D0 = 114, n = 3.3. Cell line KM101 was more supportive than the other lines of cocultivated hemopoietic cells in vitro. Subclones of KM101 and KM104 selected by retroviral vector transfer of the neor gene for growth in the antibiotic neomycin-analogue G418, maintained the stably associated radiobiologic properties of each parent clonal line. These data indicate significant heterogeneity in the LDR irradiation response of clonal stromal cell lines derived from human bone marrow.