Dimethylformamide-induced changes in the radiation survival of low- and high-passage intestinal epithelial cells (IEC-17) in vitro

The effects of dimethylformamide (DMF) on the radiation response of low- and high-passage intestinal epithelial cells (IEC-17) were examined. The IEC-17 cell line, a rat intestinal epithelial cell line, exhibited a bimodal response to X radiation. The sensitive fraction, which was attributed to a stem cell-like component, had a D0 of 0.90 Gy. The resistant fraction, thought to be the expression of a more mature component, exhibited a D0 of 2.00 Gy. Treatment using a putative cell differentiating agent, N,N-dimethylformamide, increased the resistant fraction of the population from 35 to 80%, suggesting that DMF treatment (100 mM) increased the proportion of mature cells in the IEC-17 cell population. In addition, extended age in culture (greater than 100 passages) resulted in altered morphology, decreased doubling time, increased chromosome number, and loss of anchorage dependence, all features characterizing spontaneously transformed high-passage IEC-17 cells. These high-passage cells also exhibited a bimodal response to X radiation; the sensitive fraction had a D0 of 0.80 Gy while the resistant fraction D0 was 1.50 Gy. DMF increased the resistant fraction from 35 to 55% of the population. Results suggested that the different radiosensitivities of the subpopulations remained throughout the spontaneous transformation of high-passage IEC-17 cells.     

Enhanced recovery from ionizing radiation damage in a lepidopteran insect cell line

TN-368 lepidopteran insect cells display a pronounced resistance to the lethal effects of ionizing radiation and exhibit superior DNA repair capabilities. When a TN-368 cell population entering stationary growth phase is irradiated with 137Cs gamma rays and then incubated for several hours before cell dilution and plating for colony formation, the surviving fraction is increased several-fold over cells diluted and plated immediately after irradiation. Similarly, the survival of cells plated immediately following the second of two equivalent doses separated by several hours is greater than the survival of cells plated immediately following a single dose equal to the sum of the split doses. Both processes exhibit similar biphasic repair kinetics and reach maximal levels by 6 h. The phenomena appear initially to be analogous to confluent-holding and split-dose recovery as described for mammalian cells. However, the survival levels obtained for doses of 61-306 Gy after allowing for these recovery processes to occur are quite high and greatly exceed survival levels for all but relatively low doses less than 50 Gy. For example, while the survival of cells irradiated with 150 Gy is near 0.15, the survival of cells receiving 306 Gy in two equivalent split doses is approximately 0.77. Even if damage induced by the first of the split doses was completely repaired, it might be expected that the survival would be near the level of the second dose alone, or near 0.15. Instead the survival is approximately five times greater, suggesting that the first split dose stimulated a repair system not present in unirradiated cells. The situation for confluent-holding recovery is similar to that for split-dose recovery.     

Loss of repair capacity in density-inhibited cultures of C3H 10T1/2 cells during multifraction irradiation

Multifraction survival curves for slowly cycling, density-inhibited C3H 10T1/2 cells were shown previously to bend toward lower survival levels with increasing total dose, even for doses per fraction as small as about 2.0 Gy. In an attempt to explain this, we tested the capacity of cells to repair potentially lethal damage (PLD) as fractionation progressed. Plateau-phase cultures were exposed to repeated doses of 4.0 Gy of 137Cs gamma rays delivered at 12-hr intervals. After zero, three, five, and seven fractions, some cultures were put aside, incubated for 12 hr at 37 degrees C, irradiated with a single dose of 9.0 Gy, and subsequently returned to a 37 degrees C incubator. At 0, 2, 4, 6, and 12 hr after the 9.0 Gy dose, cultures were trypsinized and plated for a survival assay. Following three fractions of 4.0 Gy, cells were able to repair PLD as well as those receiving a single dose of 9.0 Gy without prior fractionation. Following five fractions, cells were less able to repair PLD, and after seven fractions, only a very small amount of PLD repair was detectable using this method of measurement.     

Effects of irradiation on intestinal cells in vivo and in vitro

The effects of irradiation on intestinal epithelial cells were analyzed in vivo and in vitro. The in vivo study was carried out on the rat small intestine and for the in vitro study the intestinal crypt cell-line IEC-6 was used. Rat intestine and IEC-6 cells were irradiated with X-ray doses ranging between 1-16 Gy. Energy-dispersive X-ray microanalysis was used for detection of the elemental changes in the cells. Cell morphology was investigated in the scanning electron microscope, DNA-synthesis by autoradiography of 3H-thymidine incorporating nuclei and proliferation by cell counting. Our results indicate that in vivo, in the crypt cells, the increasing doses of irradiation led to increased sodium and lowered potassium and phosphorus concentrations. Corresponding ion shifts were found in the irradiated IEC-6 cells. Cells continued to proliferate up to the dose of 8 Gy, although the proliferation rate became lower with increasing dose of irradiation. The increasing dose of irradiation significantly reduced DNA-synthesis (16 Gy decreased DNA-synthesis by 50%) which resulted in a complete inhibition of cell proliferation. Analysis of goblet cells also showed characteristic radiation-dependent elemental changes. Scanning electron microscopical investigation of cells in culture revealed that most of the control cells were flat and had rather smooth cell membranes. Irradiation led to the appearance of numerous different membrane manifestations (microvilli of varying length and distribution, and blebs). Frequency of differences in the topology of the cells was related to the dose of irradiation. Our study clearly demonstrates that even low doses of irradiation cause changes in the ionic composition of the cells and inhibit DNA-synthesis and cell proliferation. The effects observed in the crypt cells in vivo were the same as in the intestinal cell line in vitro, which indicates that IEC-6 cells can be used for investigation of side effects of radiation to the abdomen.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

Radiosensitivity and proliferative activity of vertebral CFU-S surviving in mice injected with oncogenic activities of 239Pu or 241Am

Survival, radiosensitivity and capability to produce differentiated progeny were followed in CFU-S from lumbar vertebrae of mice injected with 198.6 kBq 239Pu/kg or 208.6 kBq 241Am/kg. The CFU-S assay and 59Fe uptake into spleen colonies were used. The number of CFU-S from treated mice was significantly lower than in controls. Higher radiosensitivity of CFU-S from 239Pu- or 241Am-treated mice was demonstrated using additional exposure to 0.5 Gy X-rays 1, 24, 48, 72 hrs after cell transplantation and expressed more precisely by survival curves obtained 1 hr after the marrow cell injection. The effect of 239Pu on CFU-S was characterized by Do 0.58 Gy (n = 0.91) and that of 241Am by Do 0.64 Gy (n = 0.91); corresponding control values were Do 0.89 Gy, n = 1.11. Lower iron utilization due not only to the decreased CFU-S numbers, but also to the defective production of erythroid cells per one CFU-S was found. Complexity of radiation effect on hemopoietic stem cells was demonstrated by the present study.     

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.     

Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.     

Cell-cycle-dependent repair of potentially lethal damage in the XR-1 gamma-ray-sensitive Chinese hamster ovary cell

Repair of potentially lethal damage (PLD) was investigated in a gamma-ray-sensitive Chinese hamster cell mutant, XR-1, and its parent by comparing survival of plateau-phase cells plated immediately after irradiation with cells plated after a delay. Previous work indicated that XR-1 cells are deficient in repair of double-strand DNA breaks and are gamma-ray sensitive in G1 but have near normal sensitivity and repair capacity in late S phase. At irradiation doses from 0 to 1.0 Gy (100 to 10% survival), the delayed- and immediate-plating survival curves of XR-1 cells were identical; however, at doses greater than 1.0 Gy a significant increase in survival was observed when plating was delayed (PLD repair), approaching a 20-fold increase at 8 Gy. Elimination of S-phase cells by [3H]thymidine suicide dramatically increased gamma-ray sensitivity of plateau-phase XR-1 mutant cells and reduced by 600-fold the number of cells capable of PLD repair after a 6-Gy dose. In contrast, elimination of S-phase cells in plateau-phase parental cells did not alter PLD repair. These results suggest that the majority of PLD repair observed in plateau-phase XR-1 cells occurs in S-phase cells while G1 cells perform little PLD repair. In contrast, G1 cells account for the majority of PLD repair in plateau-phase parental cells. Thus, in the XR-1 mutant, a cell's ability to repair PLD seems to depend upon the stage of the cell cycle at which the irradiation is delivered. A possible explanation for these findings is discussed.     

Method for probing cells in radiation-induced G2 arrest: demonstration of potentially lethal damage repair

Chinese hamster ovary cells were arrested in the G2 phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G2, for the investigation of G2 arrest. Comparisons were made of the number of cells retrieved from G2 arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. The retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. The repair curves (surviving fraction vs time) were each described by a single exponential. G2 cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

Induction and rejoining of DNA double-strand breaks in bladder tumor cells

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC-3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24). The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this.     

Repair of potentially lethal X-ray damage in fibroblasts derived from patients with hereditary and D-deletion retinoblastoma

We examined X-ray induced potentially lethal damage repair (PLDR) in density inhibited plateau phase cultures of six fibroblast strains derived from patients with hereditary retinoblastoma and two patients with D-deletion retinoblastoma and compared them to three normal controls. PLD was measured in hereditary retinoblastoma (7 Gy exposure) and normal cells (7 and 9 Gy exposure) after 24 h repair time. PLD survival curves were performed at 2-9 Gy on six retinoblastoma and three normal control cell strains. Thus, PLDR was compared at equitoxic survival levels as well as after exposure to equal doses of radiation. Some retinoblastoma strains showed normal PLDR whereas others were possibly deficient. Implications of PLDR for susceptibility to radiation-induced and spontaneous tumours in hereditary retinoblastoma patients are discussed.     

Nonlinear survival curves for cells of solid tumors after large doses of fast neutrons and gamma rays.

We have previously proposed that survival curves for cells of murine NFSa fibrosarcomas after exposure to fast neutrons might demonstrate curvature when the neutron doses reach a level high enough to cure the fibrosarcomas. We report here that this is the case. Murine NFSa fibrosarcomas growing in the hind legs of syngeneic mice were exposed to either gamma rays or fast neutrons. The tumors were removed and retransplanted into fresh recipients to obtain 50% tumor cell doses, from which the dose-cell survival relationship was constructed. Survival curves showed continuous bending down to 10(-7), and were well fitted using the linear-quadratic model. The alpha and beta values for neutrons were larger than those for gamma rays. When the surviving fractions at experimental TCD50 doses were calculated using these values, comparable figures were obtained for neutrons and gamma rays. The RBEs for neutrons were comparable for the TCD50 and TD50 assays. Neutron RBE was independent of dose within a range of 5-28 Gy. The capacity of the tumors to repair the damage caused by large doses of neutrons was identical to that for small doses of neutrons, indicating that cells retained the capacity to repair neutron damage irrespective of the size of the dose.     

ADPRT抑制剂对不同辐射敏感性人肿瘤细胞株的作用比较

从细胞的克隆形成能力和细胞DNA双链断裂及修复几方面分析了两个人卵巢癌细胞株HOC8和A2780对电离辐射的敏感性并探讨了ADP-核糖基转移酶（ADPR）的特异性抑制剂3-氨基苯甲酰胺（3－AB）对二者的辐射增敏效应,结果表明A2780细胞的辐射敏感性大大高于HOC8细胞,其D0值分别为0．9和2．5Gy;γ射线所致两株细胞的初始DNA双链断裂水平没有显著差异,但A2780细胞对DNA双链断裂的修复能力比HOC8细胞低．3AB能降低受照细胞的克隆形成能力及细胞对双链断裂的修复能力,其中对HOC8细胞的作用更为明显．     

Comparative effects of X irradiation on the testes of adult Sprague-Dawley and Wistar rats

The response of the testes of two strains of adult rats (Sprague-Dawley and Wistar) to graded single doses and split doses of 230 kVp X rays has been investigated. A marked difference was noted between the strains in the response of the clonogenic spermatogonia to irradiation, as measured histologically by the repopulation index. Single-dose response curves derived for these cells in the Sprague-Dawley strain had a much larger shoulder (up to about 4-5 Gy) than for the Wistar (less than 2 Gy). Split-dose studies revealed that this difference may partly be explained by a greater repair capacity in the cells of the Sprague-Dawley strain. Changes in serum FSH concentrations mirrored the changes in clonogenic spermatogonial survival following split doses of radiation.     

Method For Probing Cells In Radiation-Induced G2Arrest: Demonstration of Potentially Lethal Damage Repair

Abstract. Chinese hamster ovary cells were arrested in the G₂ phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G₂, for the investigation of G₂ arrest. Comparisons were made of the number of cells retrieved from G₂ arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. the retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. the repair curves (surviving fraction us time) were each described by a single exponential. G₂ cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

Split-dose exposure to N-methyl-N'-nitro-N-nitrosoguanidine in BALB/3T3 C1 a31-1-1 cells: evidence of DNA repair by alkaline elution without changes in cell survival, mutation and transformation rates

Dose fractionation of a direct-acting chemical carcinogen, the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was studied for its concurrent effects on survival, DNA damage and repair, ouabain resistance (Ouar) mutations and neoplastic transformation, in the mouse embryo cell line BALB/3T3 C1A31-1-1. MNNG doses of 0.5, 1 and 2 micrograms/ml were added to the cells either as a single exposure or in two equal fractions separated by 1, 3 or 5 h intervals. No significant difference in cytotoxicity was found when single and split-dose treatments were compared. No recovery from sublethal damage was therefore found in this cell line by split-dose administration of MNNG, although such an effect was found when the same cell line was treated with single and split doses of X-rays. Repair of DNA damage as measured by alkaline elution was studied up to 24 h after a single MNNG exposure (0.5 micrograms/ml). DNA repair was rapid during the first 5 h after treatment and slow thereafter. DNA damage detected after split doses of MNNG at 1 and 5 h intervals was significantly lower than after a corresponding single dose. With both single and split doses, rejoining of single-strand breaks (ssb) was nearly complete after 24 h of repair time. Ouar mutation and neoplastic transformation frequencies were determined for single and split doses of MNNG with the second treatment being given during (1 h) or after (5 h) the period of rapid DNA repair. No significant differences in either effect were detected for dose splitting at any tested dose.     

Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0-30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the gammaH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.     

Tissue repair and repopulation in the tumor bed effect

These experiments were designed to study the kinetics and magnitude of cell repair and repopulation in tissues whose damage results in the tumor bed effect. The right hind thighs of mice were irradiated with single doses or two equal gamma-ray fractions. Interfraction intervals ranging from 30 min to 24 h (to measure the kinetics of repair from sublethal damage) and 6 and 12 weeks (to determine the extent of repopulation) were used. One day after the second radiation dose 5 X 10(5) FSA tumor cells were inoculated into the center of the irradiated field. Radiation dose-response curves were obtained by calculating the time required for tumors to reach 12 mm diameter. No recovery occurred within 6 h of the radiation delivery as measured by this assay. Some recovery, 3.2-4.6 Gy above a single radiation dose, occurred when the interval between two fractions was 24 h. With increasing interfraction intervals of 6 and 12 weeks further dose sparing occurred in the amount of 5.0-6.9 and 7.5-8.3 Gy, respectively. The data suggest that repopulation is the major contributor to the radiation dose-sparing recovery of stromal tissue and that some proliferative response may occur as early as 1 day after the first irradiation.