Pervin Anklesaria

Expression of transfected recombinant oncogenes increases radiation resistance of clonal hematopoietic and fibroblast cell lines selectively at clinical low dose rate.

To determine the effect of oncogene expression on gamma radiation sensitivity of hematopoietic compared to fibroblastic cells, we selected clonal sublines of an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell line 32D cl 3 and NIH/3T3 embryo fibroblastic cells following transfection with each oncogene linked to the mycophenolic acid resistance gene. Each mycophenolic acid-resistant subclone demonstrated high levels of specific poly(A)+ mRNA for each oncogene. The parent line 32D cl 3 demonstrated similar radiosensitivity at 116 cGy/min (D0 126, n 1.17) compared to 5 cGy/min (D0 123, n 1.65). This pattern was not altered in subclones of 32D cl 3 cells transfected with the epidermal growth factor (EGF) receptor gene and grown in EGF (at 116 cGy/min D0 104, n 0.998, at 5 cGy/min D0 115, n 1.09), or in 32D cl 3 cells expressing the v-sis oncogene (at 116 cGy/min D0 122.4, n 1.79, at 5 cGy/min D0 135, n 1.43). In contrast, expression of the transfected oncogenes v-erb-B, v-abl, or v-src conferred significant radioresistance at 5 cGy/min dose rate (D0 194, n 1.77; D0 165.5, n 1.56; D0 171, n 1.28, respectively). With the exception of v-sis, oncogene expression resulted in nonautocrine factor independence of 32D cl 3 subclones, and production of donor origin tumors in syngeneic new-born or adult mice. Two rare spontaneous factor-independent subclones of 32D cl 3 were also tested. Nonautocrine clone 32D cl 2 demonstrated significantly increased radioresistance at low dose rate (D0 186, n 1.63), while autocrine (IL-3 producing) subclone 32D cl 4 revealed no significant increase in radioresistance at 5 cGy/min. The parent fibroblast cell line NIH/3T3 showed an intrinsic relative radioresistance at low dose rate (at 5 cGy/min D0 157.3, n 1.81, compared to 116 cGy/min D0 134.3, n 1.57). Expression in NIH/3T3 of transfected oncogenes v-abl, v-fms, v-fos, or H-ras increased radioresistance at low dose rate (D0 208.6, n 1.61; D0 206.6, n 1.51; D0 167.5, n 1.85; and D0 206.8, n 1.08, respectively). Thus expression of each of several oncogenes induces resistance to gamma irradiation at 5 cGy/min in hematopoietic and fibroblast cell lines. These data may help explain the clinical recurrence of oncogene-expressing leukemia and lymphoma cells after marrow stem cell ablative doses of low-dose-rate total-body irradiation.

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.

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.

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.

The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.

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.

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.

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.