The relationship between radiosensitivity and repair of potentially lethal damage in human tumor cell lines with implications for radioresponsiveness

The relationship between intrinsic radiosensitivity and repair capacity was studied for 22 human tumor cell lines in vitro. The experimental material was taken from 19 published papers. Parameters from three radiobiological models were used to assess this relationship: the one-hit multitarget model (D0 and n), the linear-quadratic model (alpha and beta), and the mean inactivation dose (D). Data were obtained for cells in three stages: exponentially growing cells (exp), plateau-phase cells plated immediately after irradiation (ip), and plateau-phase cells plated after completion of PLD repair (dp). No significant difference was found between radiosensitivity of exp and ip cells. There was no correlation between repair capacity and intrinsic radiosensitivity assessed with plateau-phase cells plated immediately after irradiation. The correlation studies between intrinsic radiosensitivity or repair capacity and clinical responsiveness were achieved by assigning cell lines to one of three groups of decreasing in vivo radioresponsiveness: highly, medium, and poorly responsive. There was a significant correlation between radiosensitivity and radioresponsiveness, but no correlation between repair capacity and radioresponsiveness. The average repair capacity was about 0.6 Gy, in terms of D. Three parameters, the mean inactivation dose of exponentially growing cells, of plateau-phase cells plated immediately after irradiation, and of plateau-phase cells plated after completion of PLD repair, could be used equally to assess the relationship between in vitro data and radioresponsiveness. The present results are compared to those obtained in a similar study on a group of 48 nontransformed fibroblast cell strains.     

Correlation between PLD repair capacity and the survival curve of human fibroblasts in exponential growth phase: analysis in terms of several parameters

Published data on the in vitro radiosensitivity of 46 nontransformed fibroblasts of different genetic origins studied in plateau phase with immediate or delayed plating were used to investigate to what extent potentially lethal damage repair capacity is related to intrinsic radiosensitivity (i.e., irradiated in exponential growth phase). While most of the survival curve analysis is conducted in terms of D0, Dq, and the mean inactivation dose D, some of the data are also discussed in terms of the linear-quadratic model parameter alpha. Using D it is shown that: (i) the radiosensitivity of human fibroblasts in exponential growth phase does not significantly differ from that of plateau-phase fibroblasts with immediate plating; (ii) the radiosensitivity of plateau-phase cells with delayed plating is correlated to the radiosensitivity of cells with immediate plating: the more radioresistant the cell strain in exponential growth phase, the higher its repair capacity; (iii) the repair capacity of the cell strains is related to their genetic origin. In conclusion, we suggest that the survival curve of growing cells depends on the repair capacity of the cells.     

The radiation response of cells recovering after chronic hypoxia

Experiments were performed to study the influence of hypoxic pretreatment on the radiation response of A431 human squamous carcinoma cells. Reaeration for 10 min after chronic hypoxia (greater than 2 h) was found to enhance the radiosensitivity of A431 cells, and the maximal effect was seen for those cells reaerated after 12 h of hypoxia. The radiosensitivity enhancement for reaerated cells after 12 h of hypoxia was maximized by 5 min after the return to aerobic conditions and reached the control level by 12 h of reaeration. This enhanced radiosensitive state was characterized by a reduced shoulder region and increased slope of the radiation dose-response curve for cells in both the exponential and plateau phases of growth. There was a slight increase in the number of G1 and decrease in the number of S and G2 + M cells for both exponential- and plateau-phase cultures following 12 h hypoxic treatment. Although growth inhibition induced by 12 h of hypoxia was seen for cells in the exponential phase, there was no cell number change in the plateau-phase culture after hypoxia. Plating efficiency (PE) of cells in both growth phases was reduced by 30% after hypoxia. Furthermore, in the exponential-phase culture, the extent of reduction in PE after hypoxia was similar among cells in different phases of the cell cycle. Although S-phase cells in exponentially growing cultures were relatively more resistant to radiation than G1 and G2 + M cells, the cell age-response pattern was the same whether the cells had been aerobic or hypoxic before reaeration and irradiation. Furthermore, the enhancement ratio associated with reaeration after 12 h of hypoxia for these three subpopulations of cells was 1.3. Our results indicate that the increase in radiosensitivity due to reaeration after chronic hypoxia is unlikely to be related to the changes of cell cycle stage and growth phase during hypoxic treatment.     

Quiescence in 9L cells and correlation with radiosensitivity and PLD repair

The onset of quiescence, changes in X-ray sensitivity, and changes in capacity for potentially lethal damage (PLD) repair of unfed plateau-phase 9L44 cell cultures have been systematically investigated. The quiescent plateau phase in 9L cells was the result of nutrient deprivation and was not a cell contact effect. Eighty-five to 90% of the plateau-phase cells had a G1 DNA content and a growth fraction less than or equal to 0.15. The cell kinetic shifts in the population were temporally correlated with a developing radioresistance, which was characterized by a larger shoulder in the survival curve of the quiescent cells (Dq = 5.71 Gy) versus exponentially growing cells (Dq = 4.48 Gy). When the quiescent plateau-phase cells were refed, an increase in radiosensitivity resulted which approached that of exponentially growing 9L cells. Delayed plating experiments after irradiation of exponentially growing cells, quiescent plateau-phase cells, and synchronized early to mid-G1-phase cells indicated that while significant PLD repair was evident in all three populations, the quiescent 9L cells had a higher PLD repair capacity. Although data for immediate plating indicated that 9L cells may enter quiescence in the relatively radioresistant mid-G1 phase, the enhanced PLD repair capacity of quiescent cells cannot be explained by redistribution into G1 phase. When the unfed quiescent plateau-phase 9L cells were stimulated to reenter the cell cycle by replating into fresh medium, the first G1 was extended by 6 h compared with the G1 of exponentially growing or refed plateau-phase 9L cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resistance of plateau-phase human normal and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light

Clonogenic survival response to 254-nm ultraviolet light was measured in 2 strains of repair-proficient normal human fibroblasts and 4 strains of xeroderma pigmentosum (XP) fibroblasts belonging to complementation groups A, C, D and variant. In all strains except XPA, cells irradiated in plateau phase and subcultured immediately were much more resistant to the lethal effect of UV than cells irradiated in the exponential phase of growth. Typically, 10-20% of plateau-phase cells were extremely resistant. When the cultures were held in plateau phase for 24 h after irradiation and before subculture, there was a further enhance of survival. By use of a UV-specific endonuclease assay, no difference was found in the number of DNA lesions induced in exponentially growing and plateau cultures by the same dose of UV light. Thus plateau-phase cells appear to be more efficient in their DNA-repair capability than cells in exponential growth. XP group A cells were uniquely found to be deficient in the processes which lead to plateau-phase resistance. Since plateau-phase repair was not lacking in XP groups C, D and variant, it may be related to a DNA-repair process different from that which is responsible for the overall UV sensitivity of these cells.     

Dose fractionation effects in plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts

A comparison of gamma-ray dose fractionation effects was made using plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts in an attempt to simulate basically similar populations of cells that differ primarily in their turnover rates. The status of cell populations with respect to their turnover rates may be an important factor influencing dose fractionation effects in early- and late-responding tissues. In this cell culture system, the rate of cell turnover was approximately three times higher for the plateau-phase transformed cultures. While the single acute dose survival curves for log-phase cells were indistinguishable, there were significant differences between the survival curves for plateau-phase cultures of the two cell types. These differences were qualitatively similar to the differences recently postulated for the survival of target cells governing early and late tissue responses. Both cell lines had a similar capacity for repair of sublethal damage, but untransformed cells had a much greater capacity to repair potentially lethal damage in plateau phase. Further, untransformed plateau-phase cultures were much more sensitive to a radiation-induced G1 (or G0 to G1) delay than transformed cultures. Multifraction survival curves were determined for both cell lines for doses per fraction ranging from 9.0 to 0.8 Gy, and from these isoeffect curves of log total dose versus dose per fraction were derived. The isoeffect curve for the slowly cycling, untransformed cells was found to be appreciably steeper than that for the more rapidly cycling transformed cells, a finding consistent with previously reported differences in dose fractionation isoeffect curves for early- and late-responding tissues in vivo.     

The role of DNA polymerase beta in determining sensitivity to ionizing radiation in human tumor cells

Lethal lesions after ionizing radiation are thought to be mainly unrepaired or misrepaired DNA double-strand breaks, ultimately leading to lethal chromosome aberrations. However, studies with radioprotectors and repair inhibitors indicate that single-strand breaks, damaged nucleotides or abasic sites can also influence cell survival. This paper reports on studies to further define the role of base damage and base excision repair on the radiosensitivity of human cells. We retrovirally transduced human tumor cells with a dominant negative form of DNA polymerase β, comprising the 14 kDa DNA-binding domain of DNA polymerase β but lacking polymerase function. Radiosensitization of two human carcinoma cell lines, A549 and SQD9, was observed, achieving dose enhancement factors of 1.5–1.7. Sensitization was dependent on expression level of the dominant negative and was seen in both single cell clones and in unselected virally transduced populations. Sensitization was not due to changes in cell cycle distribution. Little or no sensitization was seen in G₁-enriched populations, indicating cell cycle specificity for the observed sensitization. These results contrast with the lack of effect seen in DNA polymerase β knockout cells, suggesting that polDN also inhibits the long patch, DNA polymerase β-independent repair pathway. These data demonstrate an important role for BER in determining sensitivity to ionizing radiation and might help identify targets for radiosensitizing tumor cells.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on cell killing and interphase chromosome breakage in radiation-sensitive cells

The effect of BrdU incorporation on cell radiosensitivity as well as on the induction of chromosome damage by radiation was studied in plateau-phase xrs-5 cells using the premature chromosome condensation (PCC) method. It is well known that xrs-5 cells are sensitive to ionizing radiation and defective in the repair of radiation-induced DNA double-strand breaks, chromosome damage, and potentially lethal damage (PLD). Compared to repair-proficient CHO 10B cells, a reduction was observed in the overall BrdU-mediated radiosensitization in plateau-phase xrs-5 cells for the same degree of thymidine replacement. This finding is interpreted with a model for BrdU-induced radiosensitization advanced previously, in which two distinct components act to produce the overall radiosensitization observed. One component involves processes associated with the increase in initial damage (DNA and chromosome) production per unit absorbed dose and causes an increase in the slope of the survival curve, while the second component involves enhanced fixation of radiation-induced damage (PLD) and causes a reduction in the width of the shoulder of the survival curve. It is suggested that in plateau-phase xrs-5 cells, the deficiency in the repair of radiation-induced damage compromises BrdU-mediated radiosensitization by leaving active only the radiosensitization component that is associated with an increase in damage induction. Enhancement of cell killing by BrdU in plateau-phase xrs-5 cells resulted in a decrease in D0, the relative value of which was similar to the relative increase in the production of chromosome damage as measured by the PCC method. The relative values for the change in D0 and the production of chromosome aberrations were similar in plateau-phase CHO 10B and xrs-5 cells, suggesting that the physicochemical and/or biochemical processes associated with this phenomenon are the same in the two cell lines. Radiosensitization of a magnitude similar to that observed in exponentially growing CHO 10B cells was induced by BrdU in exponentially growing xrs-5 cells. This effect is attributed to a partial expression of the repair gene (transiently during S phase in all cells, or throughout the cycle in a fraction of cells) that permits some repair of radiation-induced damage and which is compromised by BrdU.     

Ataxia-telangiectasia homo- and heterozygous cells show a normal repair and fixation response to anisotonic NaCl treatment after irradiation

The effect of anisotonic NaCl treatment on fixation and repair of radiation-induced potentially lethal damage (PLD) was tested in normal human cells and in three homozygous ataxia-telangiectasia (A-T) and two heterozygous A-T cell strains. Fixation of radiation-induced PLD occurred in all cell strains exposed to 0.05, 0.5, or 1.5 mole/liter NaCl solutions immediately after irradiation. This effect was observed in both plateau-phase and exponentially growing normal and A-T cells. When an incubation period at 37 degrees C was introduced between irradiation and the subsequent anisotonic treatment, recovery was observed in both normal and A-T cells strains. These data show that A-T cells are as proficient as normal cells in repairing PLD that is sensitive to anisotonic NaCl treatment. It is proposed that two PLD repair systems may exist, one that is expressed after irradiation in proliferatively arrested cells and another that occurs in plateau-phase as well as exponentially growing cells, and is expressed by the postirradiation treatments described here and by Raaphorst and Azzam (Radiat. Res. 86, 52-66 (1981].     

Comparison of endogenous TP53 genomic status with clonogenicity and different modes of cell death after X irradiation

Although extensive data indicate that the tumor suppressor TP53 modifies the radiation responses of human and rodent cells, the exact relationship between TP53 and radiation responsiveness remains controversial. To elucidate the relevance of endogenous TP53 genomic status to radiosensitivity in a cell-type-independent manner, different cells of 10 human tumor cell lines with different tissues of origin were examined for TP53 status. The TP53 status was compared with radiation-related cell survival parameters (D(q), D(0), SF2) and with the mode of cell death. Different modes of cell death were examined by measuring radiation-induced micronucleation, apoptosis and abnormal cells. Alterations of the TP53 gene were detected in eight cell lines. No splicing mutation was found. Five cell lines showed codon 68 polymorphism. Codon 72 alterations were found in four cell lines. "Hot spot" alterations were detected in only two of 10 cell lines. Although the cells differed widely in survival parameters (D(q), D(0), SF2) and modes of cell death (micronucleation/apoptosis/abnormal cells) after irradiation, significant cell-type-independent correlations were obtained between the multiple cell death parameter micronucleation/apoptosis/abnormal cells and SF2 (P < 0.001) and D(q) (P = 0.003). Moreover, cells with a wild-type TP53 gene were more resistant to X rays than cells with a mutated TP53 gene or cells that were TP53-deficient. The alterations within exons 5-10 of the TP53 correlated with a enhanced radiosensitivity. For the first time, we demonstrated a correlation between endogenous genetic alterations within exons 5-10 of TP53 and radiation-related cell survival and cell death. This indicates a new molecular relevance of TP53 status to intrinsic cellular radiosensitivity.     

An acidic extracellular environment reduces the fixation of radiation damage

Chinese hamster ovary cells in exponential growth were irradiated with gamma or X rays in an acidic or alkaline extracellular environment. Incubation in acid medium during or after irradiation reduced the degree of fixation of potentially lethal lesions. Fixation and repair of acid-modified damage occurred with a halftime of 9 to 10 min. The rate of repair of sublethal damage was unaffected by hydrogen ion concentration and progressed with a half-time of 30 min. An acid environment modified only survival of exponentially growing cells; unfed plateau-phase cultures were unaffected.     

The effect of pH on potentially lethal damage recovery in A549 cells

The radiation sensitivity and potentially lethal damage recovery (PLDR) capacity of A549 human lung carcinoma cells have been studied. For unfed monolayer cultures, radiation sensitivity was greater in plateau phase than in log phase of growth. PLDR was observed when plateau-phase cells were held in their own spent medium postirradiation, such that the dose-response curve with 24 h holding was similar to that for log-phase cells plated immediately after irradiation. The high PLDR capacity of A549 plateau-phase cells (recovery factor between 40 and 70 for 24 h holding after 10 Gy) was reduced 10-fold or more by alkalinizing the pH of the spent medium immediately after irradiation from a value of 6.5 +/- 0.1 to a value of 7.6. Medium alkalinization resulted in an increase in the rate of glycolysis, with subsequent reacidification to a pH of 7.3 within 2 h of the pH adjustment. No change in cell cycle distribution was observed in the plateau-phase cultures up to 32 h after change of medium pH, and no increase in cell density was found after 48 h. A slight increase in the rate of incorporation of radiolabeled thymidine into acid-precipitable material was observed at 4 and 24 h after alkalinization of the medium. While it is not possible at present to define a mechanism for this pH effect, our results demonstrate that, at least for this cell line, variables such as medium pH and glucose concentration can profoundly influence the observation of PLDR.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

Salivary gland-sparing prophylactic pilocarpine treatment has no effect on tumor regrowth after irradiation

Radiotherapy of head and neck cancer frequently damages the salivary glands. Prophylactic administration of the muscarinic receptor agonist pilocarpine reduces subsequent radiation damage to the salivary glands in rats, but its effects on tumor cell radiosensitivity and tumor regrowth after irradiation had not been assessed. In the current study, we first tested the effect of pilocarpine on clonogenic cell survival in vitro. No effect of pilocarpine on radiosensitivity was observed in a panel of cell lines either with or without expression of muscarinic receptors. Second, a single dose of pilocarpine known to protect salivary gland tissue from radiation damage was given to rats transplanted with subcutaneously growing rhabdomyosarcomas 1 h prior to irradiation with a single dose of 35 Gy. No alterations in growth delay were detected (26 +/- 2 days for controls compared to 26 +/- 2 days for pilocarpine treatment). Our data indicate that pilocarpine pretreatment, which has been shown previously to protect salivary glands from radiation, does not protect tumor cells or tumors. Use of this drug therefore may lead to therapeutic gain in the treatment of head and neck cancer.     

Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

Possible importance of PLD repair in the modulation of BrdUrd and IdUrd-mediated radiosensitization in plateau-phase C3H10T1/2 mouse embryo cells

C3H10T1/2 mouse embryo cells exhibiting strong contact inhibition of growth at confluency were grown in the presence of 5-bromodeoxyuridine (BrdUrd) or 5-iododeoxyuridine (IdUrd) (0-1.2 microM) with daily refeeding and exposed to gamma-rays (6 Gy) either in the logarithmic or the plateau phase of growth. Sensitization to radiation was observed in both growth states with increasing concentration of BrdUrd or IdUrd but the degree of sensitization achieved was lower for plateau-phase cells. Because the degree of [H3]BrdUrd incorporation was found to be similar in exponentially growing and plateau-phase cells, it is hypothesized that the radiosensitization caused by pyrimidine analogues may be affected by the physiological state of the cells at the time of irradiation. Delayed plating of plateau-phase cells (6 h) caused an increase in survival, indicating repair of potentially lethal damage (PLD). A greater increase in cell survival was observed in cells that had been grown in the presence of BrdUrd and IdUrd and it was found to increase with increasing concentrations. This analogue-concentration dependent PLD repair activity resulted in an almost complete loss of the radiosensitizing effect in delayed plated plateau-phase cells up to a concentration of about 0.6 microM of BrdUrd and IdUrd. Both compounds, but especially BrdUrd, caused a relaxation in the mechanism of contact inhibition and led to higher cell densities in the plateau phase. The results suggest that repair and/or expression of PLD might be involved in the mechanism underlying BrdUrd and IdUrd-mediated radiosensitization and point out the potential importance of PLD repair in the modulation of the radiosensitizing effect of these compounds in their clinical application.     

Persistent production of colony-stimulating factor (CSF-1) by cloned bone marrow stromal cell line D2XRII after X-irradiation

The adherent stromal layer in long-term bone marrow cultures (LTBMC) provides the cellular environment necessary for the in vitro proliferation and differentiation of pluripotential hematopoietic stem cells. The role of humoral hematopoietic growth factors, colony-stimulating factors (CSF) in the regulation of hematopoietic cell production in this system is poorly understood. We have recently isolated and cloned an adherent cell line, D2XRII, derived from murine LTBMC. Plateau phase 25 cm2 cultures of 2 X 10(6) D2XRII cells in 8.0 ml produced CSF-1 (M-CSF) at around 100-150 units/0.1 ml medium. Following X-irradiation there was a dose-dependent decrease in the production of CSF-1 to a plateau of 50% of control levels at 10,000 rad. Higher doses did not produce a further decrease. The X-ray dose reducing CSF-1 production to 50% was 100-fold above the lethal dose as measured by clonagenic survival following trypsinization and replating. Trypsinized replated viable adherent but nondividing X-irradiated D2XRII cells were maintained for up to 8 weeks after irradiation and demonstrated continuous production of CSF-1. The data indicate significant divergence of two biologic effects of X-irradiation on plateau-phase marrow stromal cells: physiologic function of adherence and CSF-1 production, versus proliferative integrity. This divergence of effects may be very relevant to understanding the mechanism of X-irradiation-associated marrow suppression and leukemogenesis.     

Radiobiology of ultrasoft X rays. IV. Flat and round-shaped hamster cells (CHO-10B, HS-23)

The results reported earlier in this series indicated that the relative biological effectiveness (RBE) of ultrasoft X rays decreases with decreasing cell thickness, approaching unity for the thinnest cells used, plateau-phase human skin fibroblasts (HSF). The possible dependence of RBE on the configuration of the cell nucleus is investigated further in this paper using two CHO cell lines that attach well and have similar intrinsic radiosensitivities to 60Co gamma rays. One of the lines forms monolayers similar to V79 cells, while the other remains more spherical during growth. We find an increasing RBE with decreasing X-ray energy for both of these cell lines, consistent with our results using V79 cells. Also consistent with our results obtained with 10T1/2 and HSF cells, we find an increasing RBE with increasing cell thickness. The possible dependence of RBE on radiosensitivity and the use of the concept of mean dose for ultrasoft X rays is discussed.     

The level of induced DNA double-strand breaks does not correlate with cell killing in X-irradiated mitotic and G1-phase CHO cells

The neutral (pH 9.6) filter elution technique was used to evaluate DNA damage induced in CHO cells irradiated at mitosis or in G1-phase under various incubation and postirradiation treatment conditions. Mitotic and G1/S border cells were more sensitive to radiation than G1 cells with respect to cell killing, but showed similar (G1/S) or lower (M) DNA elution dose--response curves. Similar cell survival and DNA/elution dose--response curves were obtained with plateau-phase cultures containing mainly G1-cells, as well as with G1 cells obtained after division of mitotic cells in either fresh or conditioned medium. However, survival of plateau-phase cells could be modified substantially by delayed-plating or postirradiation treatment with araA. These results, together with previously published observations, indicate that induction of DNA dsb cannot be invoked as an explanation for the variations in radiosensitivity observed through the cycle, or as an explanation for the formation of the survival curve shoulder. It is proposed that repair and fixation of radiation-induced DNA damage, expressed at the cell survival level as repair and fixation of alpha-PLD, are responsible for these effects.