Joel S. Greenberger

STROMAL CELL-MEDIATED STIMULATION OF OSTEOCLASTOGENESIS

Abstract In the bone marrow microenvironment, stromal cells or their products are known to regulate proliferation and differentiation of hematopoietic stem cells. The purpose of this investigation was to characterize stroma-mediated effects of differentiation-inducing factors on osteoclastogenesis in defined murine cultures. Hematopoietic progenitors (derived from long-term bone marrow cultures, LTBMCs) were cocultured with cloned stromal cell lines to demonstrate the indirect effects of various differentiation-inducing factors. Osteoclastogenesis was compared in three murine marrow systems (whole bone marrow, progenitors cultured alone, and cocultures of progenitors with stromal cell lines) by analysis of multinuclearity and tartrate-resistant acid phosphatase (TRAP) activity. The cultures were treated for two weeks with murine recombinant GM-CSF (5 U/ml), 1,25-dihydroxyvitamin D3 (10-8 M), or parathyroid hormone (PTH, 10-8 M). In whole bone marrow cultures, osteoclast differentiation was stimulated by GM-CSF, PTH and 1,25-dihydroxyvitamin D3. With progenitors alone, only GM-CSF promoted osteoclastogenesis. Each agent stimulated osteoclastogenesis in cocultures of progenitors with a stromal cell line (GBLneor). Thus, the coculture system is a partially defined model for whole bone marrow cultures. In contrast, progenitors that were cocultured with a stromal cell line derived from an osteopetrotic op/op mouse failed to differentiate in the presence of PTH or 1,25-dihydroxyvitamin D3. These results indicate that stimulation of osteoclastogenesis by PTH or 1,25-dihydroxyvitamin D3 is mediated indirectly through factors present in normal marrow stromal cells and that an osteopetrotic stromal cell line failed to support differentiation.

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.

Expression of p210 BCR ABL increases hematopoietic progenitor cell radiosensitivity

PURPOSEnThe 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.nnnMETHODS AND MATERIALSnWe, 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.nnnRESULTSnThe 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.nnnCONCLUSIONnThese 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.

Recombinant murine GM-CSF increases resistance of some factor dependent hematopoietic progenitor cells to low-dose-rate gamma irradiation

The effects of murine recombinant IL-3 (multi-CSF) and murine recombinant GM-CSF (granulocyte-macrophage colony stimulating factor) on the radiation biology of clonal hematopoietic progenitor cell lines were evaluated. Four clonal cell lines with growth response to either IL-3 or GM-CSF (FDCP-1JL26, and bg/bg d64) or exclusively dependent on IL-3 (32D cl 3 and B6SUtA), were pre-incubated in IL-3, or GM-CSF, for 7 days prior to gamma irradiation, then washed and irradiated at 5 cGy/min, or 116 cGy/min, and transferred to semisolid medium supplemented with either IL-3, or GM-CSF, for assay of 7 day greater than or equal to 50 cell colonies. The cell lines demonstrated similar radiosensitivity and lack of a detectable dose-rate effect when grown in IL-3 (FDCP-1JL26: D0 154, n 1.05 at 5 cGy/min, and D0 138, n 1.16 at 116 cGy/min; bg/bg d64: D0 95.7, n 1.16 at 5 cGy/min, and D0 97.7 n .993 at 116 cGy/min; B6SUtA: D0 101, n 1.29 at 5 cGy/min, D0 100, n 1.27 at 116 cGy/min; and cell line 32D cl 3: D0 123, n 1.65 at 5 cGy/min, and D0 126, n 1.17 at 116 cGy/min). In contrast, FDCP-1JL26 cells demonstrated a significant relative radioresistance at low-dose-rate when grown in recombinant GM-CSF, (D0 217, n 1.27 at 5 cGy/min, D0 138, n 1.34 at 116 cGy/min, p less than .005). The increase in radioresistance of FDCP-1 cells at low-dose-rate was induced either by preincubation in GM-CSF with transfer to IL-3, or by preincubation in IL-3 and transfer to recombinant GM-CSF. Growth factor independent malignant subclones of lines B6SUtA and FDCP-1JL26 demonstrated a significant increase in radioresistance at low-dose-rate (B6SUtA EL4JL: D0 187, n 1.39 at 5 cGy/min, and D0 133, n 1.73 at 116 cGy/min (p. less than .05); and FDCP-1JL26 F7 cl 2: D0 191, n 1.17 at 5 cGy/min, and D0 150, n 1.31 at 116 cGy/min [p less than .05]). Thus, some hematopoietic progenitor cell lines are induced by GM-CSF to grow after irradiation at low-dose-rate similar to the growth of clonal malignant cell lines. The data may have implications for the radiation biology of normal hematopoietic progenitor cells in two circumstances: (a) selective survival of GM-CSF responsive cells after total body irradiation, and (b) selective survival of some hematopoietic progenitors in vivo during clinical recombinant GM-CSF infusion.

Radio sensitivity of human prostate cancer and malignant melanoma cell lines

Abstract The relative radioresponsiveness of human prostate cancer compared to malignant melanoma is well known. The effects of β-estradiol or testosterone on the X-irradiation survival of several human cell lines were studied, including: human prostate carcinoma cell lines PC3 and DU145 and human malignant melanoma cell lines A375 and A875. Lines PC3 and DU 145 demonstrated 55–61 fmol per 10 6 cells of androgen receptor with no detectable estrogen or progesterone receptor. Cells were irradiated at 120 cGy/min dose rate. There was no detectable toxicity of up to 10 −4 M testosterone or β-estradiol on PC3 or DU 145 cells in the absence of X-irradiation. At plating efficiencies from 11–13%, and plating densities of 1 × 10 4 cells per 60 cm 2 flask, cell lines PC3 and DU145 demonstrated a D o of 108.5 ± 6.5, n 2.1 ± 0.7 cGy, and D o of 143.5 ± 1.5 cGy, n 2.4 ± 0.5, respectively. The addition of testosterone or β-estradiol at 10 −4 to 10 −10 M prior to or after, X-irradiation did not alter radiosensitivity. At the same dose rate of 120 cGy/min, malignant melanoma cell lines A375 and A875 had a D o of 125 ± 2.5 cGy, n 1.56 ± 0.8 SF 2 0.65 ± 0.03 and line A875 demonstrated a D o of 129 ± 4.5 cGy, n 1.58 ± 0.4 SF 2 0.55 ± 0.04, respectively. The radiosensitivity of melanoma cell lines did not decrease at low dose rate 5 cGy/min. Thus, the in vitro radiosensitivity of androgen receptor positive prostate cancer cell lines is not necessarily altered by the presence of androgen before or after irradiation. The data support the concept that all malignant melanoma cell lines do not show a broad-shouldered cell survival curve in vitro and intrinsic cellular radioresistance.

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.

FUTURE DIRECTIONS IN CLINICAL BONE MARROW TRANSPLANTATION: INTERESTS CONVERGE ON THE BONE MARROW MICROENVIRONMENT

In comparison to the tremendous volume of literature on the role of the niultipoteritial haeinatopoietic stem cell in clinical marrow transplantation, little attention has been directed to cells of the bone marrow microenvironment. Interest has been recently aroused concerning effects of preparatory or conditioning regimens on the transplant recipients stroinal cells that may cxplain failure of cngraftment or persistent throm bocytopenia. Furthermore, interest in the pathophysiology of several categories of haematological disease thought to be attributable tu direct or indirect marrow strornal cell defects (myelofibrosis, aplastic anaemia and osteopetrosis) is apparent. As a third avenue of interest, moleculer cloning and expression ofthe genes for haematopoietic cell growth k t o r s has revealed that cells of the marrow stroma produce such factors and map regulate haematopoiesis through hurmurial as well as cell contact mechanisms. Xon-haematopoietic cells of the bone marrow are clasitied in multiple lineages, based on their in situ morphology and ultrastructure. Fibroblasts, reticular adventitial cells. niiicrophages, sinus lining cells, endothelial cells and fat-containing cells have been described. Continuous marrow culture techniques for murine and human marrow reveal several of these cell types in vitro (Dexter & Testa, 197h: Gartner & Kaplan, 19XOj. Murine and human long-term niarrow cultuire experiments conclusively show that adherent stromal cells sustain self-renewal of rnultiputeritial haematopoietic stem cells in vitro. The regulativn by stromal cells of haeniatopoictic cell growth and differentiation is altered by X-irradiation and chemotherapeutic alkylating agents delivered under conditions similar to that sustained by marrow in the transplant recipient (Greeriberger r t a / . 1984. 1985 ). Enthusiastic research directed toward understanding the subtle interact.ion between cells of the haematopoietic microenvironment appears vital to development of new protocols in clinical marrow transplmtation. Kelapse of lcukaemia in donor marrow cells, selective failure of engraftment of specific haeniatopoietic cellular lineages such as the nicgakaryocyte repopulating ccll and recent observations that T-lymphocyte depleted histoincompatible marrow may be rejected by the allograft recipients stromal cells that persist despite irradiation arid chemotherapy. serve a s stimuli of this research effort. Current bone marrow transplantation protocols involve ablation of recipient haematopoietic stem cells using al:ents that have not 9et been effectively studied for their deleterious effects on marrow stroma. Recent compelling evidence from tissue culture systems suggests that plateau-phase (non-dividing) cells of the stromal microenvironment express physiological damage following irradiation and chemotherapy (Greenberger et nl , 1984; Gualtieri ct d.

Biochemical Purification of a CSF-1 like Molecule Released during Malignant Transformation of IL-3 Dependent Hematopoietic Progenitor cell lines Cocultivated with Gamma Irradiated Clonal Marrow Stromal cell lines

Cocultivation of IL-3 dependent hematopoietic progenitor cell line FDC-P1JL26 with 5000 cGy irradiated clonal bone marrow stromal cell line D2XRII has been demonstrated to stimulate selection of factor independent hematopoietic cell lines that produce tumors in vivo (1,2). Hematopoietic stem cell specific and stromal cell specific variables in this experiment have been described (3). The precise molecular mechanism of the malignant transformation of hematopoietic cells and the growth factor or cell membrane contact which is responsible for the transformation have not yet been elucidated. Biochemical purification of several liters of conditioned medium from D2XRII cells revealed a 75,000 molecular weight protein that was neutralized by a polyclonal antiserum to M-CSF. This growth factor stimulated formation of macrophage colonies in fresh mouse bone marrow cells in vitro. A biochemical purification scheme utilizing a Pellicon cassette system concentration, followed by lentil lectin chromatography, ion exchange high pressure liquid chromatography, gel filtration high pressure liquid chromatography, and reverse phase HPLC yield biological activity using tritiated thymidine incorporation into microwell cultures of FDC-P1JL26 cells (4). Active fractions were run out on NaDodSO4/PAGE gel electrophoresis and revealed a band consistent in size with 75,000 molecular weight.