Increase In Radioresistance Research Articles

Effect of insertional mutation in the cspA gene encoding the major cold-shock protein on radiation resistance of Escherichia coli

Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E. coli cells (cspA+). The radioprotective effect of this plasmid is abolished or drastically reduced in mutants recA13 and rpoH15 defective in RecA protein and in induction of the heat-shock protein regulon, respectively. Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to gamma-irradiation of E. coli mutants with an intermediate level of radioresistance (Gamr445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gamr445 mutant. In the chromosome of E. coli with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of gamma-rays and UV light 2.9 and 1.4 times, respectively. These data suggest that the system of cold-shock proteins can modulate resistance of E. coli cells to the lethal effect of gamma-rays and UV light.

Suppression of apoptosis and clonogenic survival in irradiated human lymphoblasts with different TP53 status.

The influence of radiation-induced apoptosis on radiosensitivity was studied in a set of closely related human lymphoblastoid cell lines differing in TP53 status. The clonogenic survival of irradiated TK6 cells (expressing wild-type TP53), WTK1 cells (overexpressing mutant TP53), and TK6E6 cells (negative for TP53 owing to transfection with HPV16 E6) was assessed in relation to the induction of apoptosis and its suppression by caspase inhibition or treatment with PMA as well as after treatment with caffeine. Measurements using the alkaline comet assay and pulsed-field electrophoresis of the induction and repair of DNA strand breaks showed similar kinetics of the processing of early DNA damage in these cell lines. The cytochalasin B micronucleus assay revealed identical levels of residual damage in the first postirradiation mitosis of these cells. Abrogation of TP53-dependent apoptosis in TK6E6 cells resulted in a distinct increase in radioresistance. Further suppression of apoptosis as observed in WTK1 cells overexpressing mutant TP53 apparently was not responsible for the high radioresistance of WTK1 cells, since other means of highly efficient suppression of apoptosis (caspase inhibition or PMA treatment) increased the clonogenic survival of irradiated TK6 cells only to levels similar to those of TK6E6 cells with abrogated TP53-dependent apoptosis. Considering the similar levels of residual chromosomal damage in TK6E6 cells and WTK1 cells, a hitherto unknown mechanism of tolerance needs to be inferred for these TP53 mutant cells. This residual damage tolerance, however, appears to require an intact G2/M-phase checkpoint function since the relative radioresistance of the WTK1 cells was completely lost upon caffeine treatment, which also resulted in a failure of the TK6 and TK6E6 cells to execute apoptosis. In this situation, the cellular response seems to be dominated entirely by TP53-independent mitotic failure.

Correlations between in vivo tumor weight, oxygen pressure, 31P NMR spectroscopy, hypoxic microenvironment marking by β-D-iodinated azomycin galactopyranoside (β-D-IAZGP), and radiation sensitivity

Purpose : The purpose of this study is to evaluate the amount of hypoxic fraction in a rodent tumor by means of polarographic oxygen electrode, phosphorus-31 magnetic resonance spectroscopy ( 31P-MRS), and a newly synthesized hypoxic marker, β-D-iodinated azomycin galactopyranoside (β-D-IAZGP). We also investigated the radiosensitivity for tumors of different weights. Methods and Materials : Murine mammary carcinoma cells, FM3A, were subcutaneously implanted into the back of 5-week-old male C3H/He mice. β-D-IAZGP radiolabeled with 123I or with 125I was injected intravenously into tumor-bearing mice, and marker distribution was measured by nuclear medicine procedures. Radiosensitivity of the tumor was measured by the in vivo/ in vitro clonogenic assay. Tumor oxygenation status was also measured directly by polarographic oxygen electrodes and indirectly estimated from 31P-MR spectra. Results : Higher accumulation of 123I-β-D-IAZGP was observed in the tumors than in normal tissues at 24 h after administration. As to biodistribution of 125I-β-D-IAZGP, the tumor/blood ratio varied widely, but correlated significantly with tumor weight. Mean oxygen pressure (pO 2) values and ratios of nucleoside triphosphate β to inorganic phosphate (β-ATP/Pi) and of phosphocreatine to inorganic phosphate (PCr/Pi) decreased significantly with the increase in tumor volume. As tumor volume increased, the surviving fraction of cells from tumors irradiated in vivo increased significantly. Conclusions : The increase in tumor volume was significantly correlated with a reduction in mean pO 2, a decrease in the ratios of β-ATP/Pi or PCr/Pi, an increase in uptake of β-D-IAZGP, and an increase in radioresistance. Because the uptake of β-D-IAZGP can be measured noninvasively by nuclear medicine techniques, it could be clinically useful for monitoring hypoxia in human tumors.

Possible implications of non-linear radiobiological effects for the estimation of radiation risk at low doses.

Possible implications of the effects of low LET radiation on the induction of cancer at low doses are studied. Low dose hypersensitivity and adaptive response were identified as candidates which may give a non-linear dose effect curve for acute exposures, whereas adaptive response may influence protracted exposures. In this paper acute exposures are studied. Several radiobiological reports on studies with mammalian cell lines have indicated the presence of a hypersensitive region in the radiation survival response at low doses followed by an increase in radioresistance. The two step clonal expansion (TSCE) model for the process of carcinogenesis was adapted in such a way that cell killing after acute radiation induces increased clonal expansion for some time and thus gives a promoting effect of radiation. As a first step, the Radiation Effects Research Foundation (RERF) data on the lung cancer incidence are fitted to study how such a model would influence the assessment of the cancer risk at low doses.

Immune effects of low-dose radiation: short-term induction of thymocyte apoptosis and long-term augmentation of T-cell-dependent immune responses.

We and others have shown that low-dose X or gamma irradiation of mice leads to an increase in their survival after a subsequent lethal high-dose irradiation. The greatest increase in radioresistance appears at a fixed window of dose and time, e.g. 8 weeks after 5-10 cGy or 2 weeks after 50 cGy preirradiation. We show that low-dose irradiation induces thymocyte apoptosis with a maximal level at 6 h postirradiation that returns to background levels after 24 h. At the same time, we observed no morphological alteration of splenocytes and no early modification of the intensity of T-cell-dependent immune responses as measured by plaque-forming cell (PFC) counts. Nevertheless, we found that PFCs were increased 2 weeks after 50 cGy irradiation, which is the same time at which mice expressed the optimal increase in survival after a second lethal irradiation. We also examined thymocyte apoptosis and spleen PFCs in mice subjected to other stress-inducing pretreatments. Our results emphasize the existence of a lag time between the time of low-dose irradiation in vivo and the appearance of radioresistance. A mechanism that interconnects an environmental stimulus with the response of the animal is proposed based on the evidence presented here and reported in the literature.

Enhancement of tumor radiation response by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid

Purpose: 5,6-dimethylxanthenone-4-acetic acid (DMXAA) selectively damages tumor vasculature and is currently in clinical trial as an antitumor agent. Its ability to induce synthesis of tumor necrosis factor (TNF), and its apparent selectivity for poorly-perfused regions in tumors, suggests it possible use in combination with radiotherapy. This investigation examines activity of DMXAA as a radiation modifier using two murine tumors. Methods and Materials: Tumor growth delay was evaluated using i.m. RIF-1 and MDAH-MCa-4 tumors irradiated in unanaesthetised, restrained mice (cobalt-60) using single dose or multiple fractions (8 x 2.5 Gy over 4 days) with DMXAA administered i.p. at various times in relation to irradiation. Results: Administration of DMXAA (80 μmol/kg, i.p.) immediately after radiation resulted in a large increase in tumor growth delay, giving a radiation dose modifying factor of 2.3 for RIF-1 and 3.9 for MDAH-MCa-4. The combination was less active when radiation was given 1–4 h after DMXAA, but was highly active 12–48 h after DMXAA. At the latter times, clamping the tumor blood supply caused a large increase in radioresistance. These studies suggest that cells surviving DMXAA are hypoxic for only a short period. DMXAA increased overall growth delay when administered daily during fractionated irradiation, giving an approximately additive response. Conclusions: The marked synergy between DMXAA and single dose ionising radiation may reflect the complementarity of these agents at the microregional level, with DMXAA preferentially killing hypoxic cells in poorly perfused regions. Despite additional hypoxia shortly after DMXAA treatment, surviving cells appear to reoxygenate quickly which makes it feasible to use DMXAA before and during fractionated radiotherapy. The combination of fractionated radiation and DMXAA appears to be less effective than for single dose radiation (possibly because of the smaller contribution of hypoxia under these conditions), but may be therapeutically useful.

Systemic Overexpression of BCL-2 in the Hematopoietic System Protects Transgenic Mice From the Consequences of Lethal Irradiation

AbstractA new transgenic mouse has been generated in which the proto-oncogene BCL-2 is ubiquitously overexpressed. H2K-BCL-2 transgenic mice overexpress BCL-2 in all cells of the hematolymphoid system and have been used to assess the role of BCL-2 in protecting cells of the hematolymphoid system from the consequences of ionizing radiation. We have expanded on previous studies that have demonstrated protection for specific (lymphoid) cell populations and show that systemic overexpression of BCL-2 can protect the hematopoietic system as a whole, including hematopoietic stem cells (HSC), thus increasing the radioresistance of the animal. The increase in radioresistance in H2K-BCL-2 transgenic mice has two components: an increase in the radioresistance of individual cells and, to a lesser extent, an increase in the size of certain critically important cell populations, such as HSC. Bone marrow transplantation experiments show that the increased radioresistance of the transgenic animals is provided by cells of the hematopoietic system. Protection against the consequences of irradiation is not limited to the increased expression levels of BCL-2 in transgenic mice; levels of endogenous BCL-2 are higher in lymphocyte populations that survive irradiation in wild-type mice. We show that ubiquitous overexpression of BCL-2 in the hematopoietic system can be used to increase the resistance of animals to lethal challenges such as irradiation.

Systemic Overexpression of BCL-2 in the Hematopoietic System Protects Transgenic Mice From the Consequences of Lethal Irradiation

A new transgenic mouse has been generated in which the proto-oncogene BCL-2 is ubiquitously overexpressed. H2K-BCL-2 transgenic mice overexpress BCL-2 in all cells of the hematolymphoid system and have been used to assess the role of BCL-2 in protecting cells of the hematolymphoid system from the consequences of ionizing radiation. We have expanded on previous studies that have demonstrated protection for specific (lymphoid) cell populations and show that systemic overexpression of BCL-2 can protect the hematopoietic system as a whole, including hematopoietic stem cells (HSC), thus increasing the radioresistance of the animal. The increase in radioresistance in H2K-BCL-2 transgenic mice has two components: an increase in the radioresistance of individual cells and, to a lesser extent, an increase in the size of certain critically important cell populations, such as HSC. Bone marrow transplantation experiments show that the increased radioresistance of the transgenic animals is provided by cells of the hematopoietic system. Protection against the consequences of irradiation is not limited to the increased expression levels of BCL-2 in transgenic mice; levels of endogenous BCL-2 are higher in lymphocyte populations that survive irradiation in wild-type mice. We show that ubiquitous overexpression of BCL-2 in the hematopoietic system can be used to increase the resistance of animals to lethal challenges such as irradiation.

Low-Dose Radiation Sensitivity and Induced Radioresistance to Cell Killing in HT-29 Cells Is Distinct from the "Adaptive Response" and Cannot Be Explained by a Subpopulation of Sensitive Cells

Several reports using two different improved assays of clonogenicity have indicated the presence of a hypersensitive region in the radiation survival response at low doses, followed by an increase in radioresistance, in many mammalian cell lines. Mathematical modeling of these responses has suggested that it is unlikely that this effect can be explained by the presence of a small subpopulation of sensitive cells; however, this possibility cannot be excluded solely on the basis of those results. A second explanation has been offered which hypothesizes that a radiation-induced mechanism causes an increase in cellular radioresistance. This proposal has led to speculation that the substructure observed at low doses in these cell lines is related to the adaptive response, which hypothesizes the induction of a repair mechanism after a small priming dose of radiation which can protect cells against a larger second dose given several hours later. We have investigated these proposals with a study of priming doses using human tumor HT-29 cells, which we have previously shown to exhibit low-dose hyper-radiosensitivity. Our results provide significant evidence that this effect cannot be explained by a subpopulation of sensitive cells. However, the results also suggest that the radiation-induced increase in radioresistance observed in this cell line is distinct from the adaptive response.

Effect of gamma radiation on the survival of fungal and actinomycetal florae contaminating medicinal plants

This study evaluates the effect of gamma radiation on the viability of fungi and actinomycetes that contaminate medicinal plants. The relationship between the total lipids of some fungi and actinomycetes and their sensitivity to gamma radiation is also investigated. The data reveal that the viable counts of these florae decrease approximately exponentially with the radiation dose, the effective dose for the elimination of these microorganisms being about 5 kGy for all the medicinal plants under study. Response of pure cultures of fungi and actinomycetes isolated from medicinal plants to increasing absorbed doses of gamma radiation indicate that an increase in radioresistance is in the following order: Streptomyces rimosus, Fusarium solani, Nocardia kuroishii. F. oxysporum, A. fumigatus, A. flavus, A. parasiticus and A. ochraceus. The total lipid contents of molds and actinomycetes have been reported to be increased by increasing the radio-resistance of microorganisms, and hence there is a relationship between the radio-sensitivity of microorganisms and the total lipid mass of flora mycelia.

Low-Dose Hypersensitivity and Increased Radioresistance in a Panel of Human Tumor Cell Lines with Different Radiosensitivity

It is well known that cells of human tumor cell lines display a wide range of sensitivity to radiation, at least a part of which can be attributed to different capacities to process and repair radiation damage correctly. We have examined the response to very low-dose radiation of cells of five human tumor cell lines that display varying sensitivity to radiation, using an improved assay for measurement of radiation survival. This assay improves on the precision of conventional techniques by accurately determining the numbers of cells at risk, and has allowed us to measure radiation survival to doses as low as 0.05 Gy. Because of the statistical limitations in measuring radiation survival at very low doses, extensive averaging of data was used to determine the survival response accurately. Our results show that the four most resistant cell lines exhibit a region of initial low-dose hypersensitivity. This hypersensitivity is followed by an increase in radioresistance over the dose range 0.3 to 0.7 Gy, beyond which the response is typical of that seen in most survival curves. Mathematical modeling of the responses suggests that this phenomenon is not due to a small subpopulation of sensitive cells (e.g. mitotic), but rather is a reflection of the induction of resistance in the whole cell population, or at least a significant proportion of the whole cell population. These results suggest that a dose-dependent alteration in the processing of DNA damage over the initial low-dose region of cell survival may contribute to radioresistance in some cell lines.

Small Doses of High-Linear Energy Transfer Radiation Increase the Radioresistance of Chinese Hamster V79 Cells to Subsequent X Irradiation

Chinese hamster V79 cells show a complex X-ray survival response which is characterized by hypersensitivity followed by increased resistance as the dose increases to 1 Gy. This hypersensitivity can be eliminated by pretreating cells with X rays or hydrogen peroxide. Accordingly, the protective effect that results from the priming treatments could be considered analogous to the "adaptive response" induced by low-linear energy transfer (LET) radiation and some chemical agents in human lymphocytes. Indeed, no hyper-radiosensitive response after single treatments in V79 cells or adaptive response in human lymphocytes has been reported after exposure to high-LET radiation. To investigate this further, we measured the survival after X irradiation of V79-379A cells previously irradiated with small priming doses of high-LET radiation. After a 0.2-Gy priming dose of neutrons followed by a 1-Gy 250 kVp X-ray dose given 4 h later, survival was 1.08 +/- 0.04 compared to 0.73 +/- 0.03 when the doses were given concurrently. Increases in survival were also observed from 0.80 +/- 0.03 to 0.96 +/- 0.05 after a 0.2-Gy priming treatment with 250 kVp X rays and from 0.78 +/- 0.03 to 0.84 +/- 0.03 with a priming dose of Bragg-peak negative pi mesons. The results indicate that a protective effect, as measured by an increase in radioresistance, is induced by high-LET neutrons, as well as by Bragg-peak pi mesons and X rays, and that a threshold level of damage is required for adaptation to occur.

Effect of nitrogen starvation on DNA repair in Saccharomyces cerevisiae.

The gamma radiation response of diploid yeast Saccharomyces cerevisiae was studied under the condition of nitrogen starvation. An increase in radioresistance was observed when the cells were grown either on solid or in liquid media containing low nitrogen supply. Most of the induced resistance was obtained in the first 1 h of incubation in the stress medium. Cells incubated in synthetic liquid medium with limited nitrogen source showed an increase in radioresistance with time as the nitrogen supply in the medium became depleted. Diploid strains carrying a mutation at the RAD52 locus did not show increased radioresistance in synthetic low nitrogen medium. The frequencies of gene conversion at a tryptophan locus and reverse mutation at an isoleucine locus were studied for the strain D7 under nitrogen stress. Our results suggest that nitrogen starvation induces DNA repair which is essentially error free and is mediated through activated recombination pathways.

Intrinsic Radiosensitivity and Chromosome Aberration Analysis Using Fluorescence In Situ Hybridization in Cells of Two Human Tumor Cell Lines

The survival curves for cells of two human tumor cell lines, HT29 and MeWo, have been defined using a Dynamic Microscopic Imaging Processing Scanner (DMIPS). There are two major differences between these two cell lines: (a) HT29 is more radioresistant than MeWo (surviving fraction at 2 Gy of 74 and 27%, respectively) and (b) HT29 presents a marked multiphasic survival curve with hypersensitivity at low doses (< 0.5 Gy) followed by an increase in radioresistance at higher doses which we have interpreted as "induced radioresistance"; this phenomenon is much less pronounced for the more radiosensitive cell line MeWo. We have now measured in these two cell lines the stable chromosomal aberrations and fragments, with the method of fluorescence in situ hybridization (FISH). We have analyzed chromosome 4, which does not have spontaneous translocations in either of these two cell lines. A dose-effect relationship was studied for radiation doses up to 5 Gy. At all doses, both translocations and breaks are more frequent in the radiosensitive cell line MeWo compared to the radioresistant cell line HT29. The correlation between survival and translocations is different for HT29 and MeWo, thus indicating that another factor(s) may be involved in cell killing in these lines.

Influence of an autocrine multitherapy resistance factor on radiation responses of melanoma cells

An autocrine multitherapy resistance factor (MTRF) produced by a radioresistant subclone of S91 mouse melanoma (S91/I3) causes an increase in radioresistance of a radiosensitive subclone (S91/amel). MTRF has no effect on the survival of S91/I3, which is already relatively resistant to gamma-irradiation. In this study, we examined the effect of MTRF in the form of S91/I3 conditioned medium or as S91/I3 heavily-irradiated cells (I3-HRCells) on cellular responses of S91/amel cells after exposure to gamma-rays. Target S91/amel cells retained more than half of their ability to respond to rescue by MTRF on day 4 after exposure to 3 Gy. Continuous presence of MTRF during colony formation was necessary for maximum plating efficiency. Although the extent of double strand DNA breakage and repair was the same in S91/amel and S91/I3, split-dose recovery experiments with MTRF revealed previously undetected repair of sublethal damage in S91/amel cells. MTRF did not alter the extent of potentially lethal damage repair (PLDR) in S91/I3 or S91/amel. S91/amel cells were more responsive to MTRF if they had been harvested from confluent dishes, while S91/I3 cells produced a more effective factor if they had been harvested in exponential phase. These findings demonstrate that MTRF has unique properties. It does not appear to be involved in genome repair since it does not alter the extent of PLDR and it is effective when added to cells after complete split-dose recovery has occurred.

Erythropoietin increases the radioresistance of a clonal hematopoietic progenitor cell line expressing a transgene for the erythropoietin receptor.

Erythropoietin (Epo) is a serum glycoprotein growth factor required for the survival, proliferation and differentiation of committed erythroid progenitor cells. In the present study, we sought to determine whether the action of Epo via its receptor is also implicated in the repair of radiation-induced cell damage. Overexpression of the Epo receptor (Epo-R) was achieved as a result of transfection of the 32D cl 3 clonal hematopoietic cell line. These clonal lines allowed us to investigate the effects of Epo on the radiation sensitivity in vitro of a clonal murine hematopoietic progenitor cell line. Low level expression of Epo-R on many hematopoietic cell types was thus circumvented. Ligand binding of Epo resulted in increased radioresistance of 32D cl 3 subclonal lines expressing the Epo-R transgene. The D0 of 32D Epo-R cells at 1.49 Gy/min was 1.33 Gy and n was 1.39. The D0 of parental clonal cell line 32D cl 3 cells at 1.49 Gy/min was 1.36 Gy and n was 1.39. In contrast, at the low dose rate of 0.0595 Gy/min, the D0 of 32D Epo-R cells was 2.0 Gy and n was 1.24, while parental clonal line 32D cl 3 showed a D0 of 1.35 Gy and n was 1.39. The increased radioresistance was statistically significant at low dose rate (p < 0.05). Combined exposure to Epo and interleukin 3 (IL-3) increased proliferation of 32D Epo-R cells but did not induce a detectable further increase in radioresistance. Temporal dissociation between growth factor-activated tyrosine phosphorylation of intracellular substrates, and the radioprotective effect was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased Radiation Resistance in Transformed and Nontransformed Cells with Elevated ras Proto-Oncogene Expression

The cellular Ha-ras oncogene, activated by missense mutations, has been implicated in intrinsic resistance to ionizing radiation. This study shows that the overexpression of the unmutated gene (proto-oncogene) may also be involved in how the cells respond to radiation. The experimental system consisted of mouse NIH 3T3-derived cell lines which carry multiple copies of a transcriptionally activated human c-Ha-ras proto-oncogene. Both tumorigenic (RS485) and revertant nontumorigenic subclones (PR4 and 4C3) which have high levels of ras expression exhibited a marked increase in radioresistance as measured by D0 compared to control NIH 3T3 cells. Other nontransformed cells with elevated levels of ras (phenotypically revertant line 4C8-A10) also had a significantly increased resistance to radiation, further indicating an association between ras and radioresistance. The increased radioresistance of the RS485 and phenotypic revertants could not be explained by a differential expression of the myc or metallothionein I genes or by variations in cell cycle. The correlation between increased ras proto-oncogene expression and radioresistance suggests that the ras encoded p21, a plasma membrane protein, may participate in the cellular responses to ionizing radiation.

The influence of a seventy-two hour gap on the acute response of mouse skin and oesophagus

The sparing effect of a 72 h gap, inserted after the third, sixth or ninth fraction of a 12 fraction X-ray schedule, was investigated in two early responding normal tissues: mouse skin and oesophagus. Acute skin reactions and body weight loss were used as assays to quantify changes in the radiation tolerance of these tissues. In skin, no evidence of compensatory proliferation was seen if a gap was inserted, whatever its position in the schedule. In oesophagus, a small but significant increase in radioresistance was observed if the gap was positioned 1/4 of the way through treatment compared with the schedule delivered without an interruption (12F/12 days). Although an increase in normal tissue tolerance is not observed by increasing the overall time from 12 to 15 days, provided tumour clonogen proliferation does not occur during the weekend split, the insertion of a gap makes continuous accelerated clinical regimes, currently given without an interruption at weekends, easier to introduce into wider clinical practice.

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.

The Increase in Radioresistance of Chinese Hamster Cells Cultured as Spheroids Is Correlated to Changes in Nuclear Morphology

Chinese hamster V79 cells grown as spheroids in roller culture are more radioresistant than those grown as monolayers. The supercoiled structure of chromatin, as salt-extracted nucleoids, has been examined using flow cytometry. Irradiated viable cells from spheroid culture contain restraints to supercoil relaxation that are absent in monolayer cells. Further analysis of the chromatin organization from each growth form shows that the radioresistant spheroid cells contain a DNA-protein matrix that is more resistant to detergent-induced degradation. The increase in structural integrity may be due to the retention of a 55-60 kDa protein that is apparent in the nucleoids of spheroid, but not monolayer cells. The increase in structural integrity of the spheroid cells may explain their greater radioresistance by providing a more stable platform for high-fidelity DNA damage repair.