Mary Ann Sakakeeny

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

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.

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.

Role of stromal and hematopoietic stem cells in Friend spleen focus forming virus effects in continuous bone marrow culture

Replication of the Friend spleen focus forming virus (SFFV) in C3H/HeJ or C57BL/6J mouse continuous bone marrow cultures is associated with an increased cumulative production of pluripotential/hematopoietic stem cells (CFUs), granulocyte-macrophage progenitor cells (GM-CFUc), and total granulocytes, compared to uninfected or helper virus infected control cultures. The site(s) of action of the virus are not known. To determine whether viral effect(s) occurred in adherent stromal and/or non-adherent hematopoietic stem cells, purified cell populations, comprised exclusively of cells from each compartment, were separated from C57BL/6J (Fv-1bb) or C3H/HeJ (Fv-1nn) marrow cultures and were left uninfected or were infected with host range replication restricted B-tropic or N-tropic helper pseudotype viruses of SFFV respectively. Cell populations were then mixed to establish continuous hematopoiesis in allogeneic or syngeneic combinations. Virus host range restriction between compartments was maintained in allogeneic reconstituted cultures which showed active hematopoiesis for 16-17 weeks and no significant differences between: (1) virus infected and uninfected groups or (2) groups comprised of stromal cells from one or the other strain. Thus, these data indicate that the prolongation of hematopoiesis in undisturbed long-term marrow cultures by SFFV occurs through an interaction of adherent hematopoietic stem cells with the marrow stroma.

Infection of hematopoietic and stromal cells in human continuous bone marrow cultures by a retroviral vector containing the neomycin resistance gene.

Stability and expression of the bacterial neomycin resistance gene (neor) transferred to human continuous marrow cultures by a retroviral vector [pZIP-NeoSV(X)] was evaluated over 4 weeks. Following infection of long-term human marrow cultures with pZIP-NeoSV(X), 10-15% of the stromal cells demonstrated high replating efficiency in a dose of the neomycin analogue G418 that was toxic to stromal cells from uninfected cultures. In contrast, G418 resistance was detected in less than or equal to 1% of GM-CFUc and CFU-GEMM derived from the same virus-infected compared to control cultures. Infection of human CFU-GEMM enriched 100 X by monoclonal antibody selection with pZIP-NeoSV(X) did not increase the percentage of neor progenitors. Marrow cells from cultures infected with pZIP-NeoSV(X) and a replication competent amphotropic virus transferred the vector and G418 resistance to HeLa cells at a frequency of 1/10(5) for nonadherent and 1/10(4) for adherent cells. Two established human hematopoietic (HL60 and K562) and one stromal cell line (KM101) stably expressed the neor gene. Thus, a higher efficiency of infection and expression of a gene transferred by pZIP-NeoSV(X) to permanent human hematopoietic tumor cell lines and fresh marrow stromal cells contrasts with a lower level of expression in fresh CSF-dependent human hematopoietic stem cells.

Friend virus-infected long-term bone marrow cultures produce colony stimulating factor dependent and independent granulocyte-macrophage progenitor cells for over four years in vitro.

C3H/HeJ mouse long-term bone marrow cultures infected at initiation with a cloned polycythemic strain of Friend spleen focus forming virus in a cloned N-tropic murine leukemia virus helper virus coat, persistently produced: colony-forming unit spleen (CFUs) for 55 weeks that formed macroscopic spleen colonies in syngeneic or allogeneic C57B10.Br/J mice; and L-cell or WEHI-3 cell conditioned medium-dependent granulocyte-macrophage colony forming unit culture (GM-CFUc); and morphologically normal granulocytes for over 245 weeks. Colony stimulating factor (CSF)-independent colony forming progenitor cells were first detectably produced in vitro at 75 weeks, and when subcultured generated karyotypically distinct permanent factor-independent tumorigenic cell lines. Nonadherent cells removed from long-term marrow cultures at 19 but not at 77 weeks reconstituted donor origin hematopoiesis in C57B10.Br/J mice as measured by B-cell lineage surface immunoglobin allotype. Nonadherent cells removed at 77 weeks produced lethal splenomegaly and marrow infiltration with culture origin cells in C57B10.Br/J mice. Despite generation of clonal malignant cell lines, L-cell DSF (CSF-1, M-CSF) responsive GM-CFUc that were simultaneously produced over 4 years in the same long-term marrow cultures, grew to 7 day colonies in semisolid medium and terminally differentiated. Thus, adherent stromal cells in Friend virus-infected long-term bone marrow cultures simultaneously support CSF-responsive and malignant CSF-independent hematopoietic progenitor cells.

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.

Establishment of Bone Marrow Stromal Cell Cultures and Permanent Clonal Stromal Cell Lines from Osteopetrotic (mi/mi) and Steel Mutant (Sl/Sld) Mice: Studies of Bone Resorption by Engrafted Hemopoietic Stem Cells In Vitro

The cellular defect in osteopetrosis is unknown. The disorder has been cured by total body irradiation and marrow transplantation in the mi/mi mouse mutant. The etiology has been presumed due to a defect in the ability the marrow stem cells to differentiate into normally functioning osteoclasts. Data are not available to prove that replacing only the osteoclast precursor (an unidentified cell) will cure the disorder. To test whether marrow stromal cells may be responsible for the cure and transmission of this disorder, we found that C57BL/6J marrow cultures contain elements capable of degrading or resorbing 45Ca radiolabelled bone particles (60±5.2% of bone added to the culture). Uncloned and cloned marrow stromal cell lines can be engrafted with stem cells from continuous C57BL/6J mouse spleen cultures and bone is degraded or resorbed by these cultures at a level equal to whole long-term marrow cultures (59 ± 6.2%). Uncloned stromal cells from 6–8 week old osteopetrotic mi/mi mutants were engrafted with nonadherent cells (containing stem cells) from continuous spleen cell cultures of C57BL/6J, mi/+ heterozygote and mi/mi mice. The release of 45Ca from bone particles added was 2.2 ± .08% with each stem cell source. In contrast, cultures of C57BL/6J stromal cells engrafted with mi/mi non-adherent spleen cells showed 45Ca release 96% of normal. In cultures of C57BL/6 stromal cells alone, 45Ca release from bone was not higher than background 45Ca release in the absence of cells. Permanent clonal marrow stromal cell lines were substituted for primary uncloned stromal cell cultures. Clones derived from mi/mi mice blocked stem cell mediated 45Ca resorption compared to clonal lines from mi/+, steel mutation Sl/Sld, or control WCB6F1 (+/+) mice. These data suggest that marrow cells from mi/mi mice may be involved in the reduction of osteoclast function seen in this mutant and provide a focus for studying the role of marrow stroma in the genesis of osteopetrosis.