Kenneth Kase

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.

Radiosensitivity of Human Bone Marrow Granulocyte-Macrophage Progenitor Cells and Stromal Colony-Forming Cells: Effect of Dose Rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.

Dosimetric accuracy at low monitor unit settings

Dosimetric accuracies at low monitor units are evaluated for linear accelerators from various manufacturers. A large error is observed in the majority of the accelerators. The error can be positive or negative. Although the error can exceed 20% for the first few monitor units, it is usually less than 5% when more than 10 monitor units are delivered. When low doses are required proper precautions should be taken for dosimetric accuracy including the beam energy, beam flatness and dose per monitor unit.

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.

Blastogenesis of Large Granular Lymphocytes in Nonlymphoid Organs

High numbers of large granular lymphocytes (LGL) accumulate in the livers and peritoneal cavities of mice during the course of viral infection. Accumulation of natural killer (NK) cells at day 3 postinfection (p.i.) was shown to be radiation‐sensitive, implying that proliferation was required for this response. Accumulation occurred in splenectomized mice, indicating that the spleen, known to be an organ for mature NK cell proliferation, was not the major source for liver and peritoneal NK/LGL.