PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

Radiation protection of stimulated human lymphocytes by nicotinamide

Nicotinamide (NA) when added to human lymphocytes in vitro together with a mitogen, protected against the inhibition by gamma and UV radiation of stimulated cell growth. When stimulated by phytohemagglutinin (PHA), concanavalin A (Con A) or pokeweed mitogen (PWM) maximum protection has been observed with approximately 1 mM NA (dose reduction factor of 2-3). To obtain protection the cells had to be stimulated immediately after irradiation in the presence of NA. It is suggested that the intracellular level of NAD+ may be rate limiting for excision repair in human lymphocytes irradiated in the G0 phase. This level is presumably increased by exogenously supplied NA, leading to enhanced repair of DNA damage and increased survival.     

Chromatin decondensed by acetylation shows an elevated radiation response

V-79 Chinese hamster lung fibroblasts exposed to 5 mM n-sodium butyrate were irradiated with 60Co gamma rays and cell survival was determined by the cell colony assay. In a separate set of experiments the acetylated chromatin obtained from these cells was irradiated and the change of molecular weight of the DNA was evaluated by alkaline sucrose density centrifugation. At a survival level of 10(-2) to 10(-4) cells exposed to butyrate were found to be 1.3-1.4 times more radiosensitive than control cells. Exposure of isolated chromatin to 100 Gy of 60Co gamma irradiation generated 0.9 +/- 0.03 single-strand breaks (ssb) per 10 Gy per 10(8) Da and 2.0 +/- 0.3 ssb/10 Gy/10(8) Da for control and acetylated chromatin, respectively. The elevated radiation sensitivity of chromatin relaxed by acetylation is in good agreement with previous results on chromatin expanded by histone H1 depletion [Heussen et al., Radiat. Res. 110, 84-94 (1987)]. Packing and accessibility of DNA in chromatin appear to be major factors which influence the radiation sensitivity. The intrinsic radiation sensitivity of chromatin in various packing states is discussed in light of the variation of radiation sensitivity of whole cells in the cell cycle which incorporates repair.     

Response of X-ray-sensitive CHO mutant cells to gamma radiation. I. Effects of low dose rates and the process of repair of potentially lethal damage in G1 phase

X-ray-sensitive CHO mutants (xrs-5 and xrs-6) were exposed to isoleucine-deficient (IL-) medium for 24-36 h to accumulate G1-phase cells. Cells exposed to IL- medium for up to 5 days did not show significant changes in plating efficiency when returned to normal medium. Nearly confluent cultures of IL- -treated cells were irradiated with either 60Co gamma rays (75 cGy/min) or 137Cs gamma rays (2.7, 6.0, or 15.3 cGy/h). A significant reduction (approximately 2.5-fold) in the radiation sensitivity of the parental CHO K-1 cells was observed for chronic low-dose-rate radiation exposure compared to the results obtained for acute high-dose-rate exposure. However, no noticeable differences were observed in the survival curves of either xrs-5 or xrs-6 cells when low-dose-rate and acute exposures were compared. CHO K-1 cells exhibited potentially lethal damage repair while held in IL- medium after gamma irradiation, whereas no repair was observed in either of the radiation-sensitive mutant lines examined at similar survival levels.     

Cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with accelerated 56Fe ions

We examined cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with high-energy 56Fe ions. Cells were irradiated with graded doses of 56Fe ions (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The survival curves for cells plated 1 h after irradiation (immediate plating) showed little or no shoulder. However, the survival curves for cells plated 24 h after irradiation (delayed plating) had a small initial shoulder. The RBE for 56Fe ions compared to 137Cs gamma rays was 1.99 for immediate plating and 2.73 for delayed plating at the D10. The repair ratio (delayed plating/immediate plating) was 1.67 for 137Cs gamma rays and 1.22 for 56Fe ions. The dose-response curves for initially measured and residual chromatid fragments detected by the Calyculin A-mediated premature chromosome condensation technique showed a linear response. The results indicated that the induction frequency for initially measured fragments was the same for 137Cs gamma rays and 56Fe ions. On the other hand, approximately 85% of the fragments induced by 137Cs gamma rays had rejoined after 24 h of postirradiation incubation; the corresponding amount for 56Fe ions was 37%. Furthermore, the frequency of chromatid exchanges induced by gamma rays measured 24 h after irradiation was higher than that induced by 56Fe ions. No difference in the amount of chromatid damage induced by the two types of radiations was detected when assayed 1 h after irradiation. The results suggest that high-energy 56Fe ions induce a higher frequency of complex, unrepairable damage at both the cellular and chromosomal levels than 137Cs gamma rays in the target cells for radiation-induced lung cancers.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

5-氮胞苷对贵州小型猪淋巴细胞DNA损伤及修复的影响

目的　研究贵州小型猪淋巴细胞对化学物或药物引起的DNA损伤及修复影响的反应。方法　用单细胞凝胶电泳技术检测比较 5 氮胞苷对PHA刺激和未刺激淋巴细胞的DNA损伤及其修复过程。结果　 5 氮胞苷引起未刺激淋巴细胞明显的DNA泳动 (彗星尾 ) ,经修复孵育 2h后 ,DNA泳动与孵育前比较无显著差异 ,而 5 氮胞苷引起的刺激细胞DNA泳动经 2h修复孵育后与孵育前比较显著减少。结论　 5 氮胞苷引起贵州小型猪未刺激淋巴细胞DNA损伤经 2h孵育未能修复 ,而刺激细胞的DNA损伤明显修复。     

Critical periods of the mitotic cycle: influence of aminopterin and thymidine on production of chromosomal aberrations by radiation in Crepis capillaris.

The influence of aminopterin (AP), tritiated thymidine ([3H] TdR) and "cold" thymidine (TdR) on production of chromosomal aberrations in meristematic cells of Crepis capillaris irradiated in different stages of the mitotic cycle with 300 rad of 63Co gamma-rays was studied. All the chemical treatments increased most of all the frequency of aberrations induced during two "critical periods" localized before the stage of DNA synthesis (fixation 9 h after irradiation) and before that of mitosis (4 h). Treatments with TdR and [3H]TdR increased most of all the frequency of chromatid aberrations when irradiation was performed in G1, and the frequency of gaps when irradiated in G2. Treatment with AP increased the yield of different types of aberration more uniformly. The modifying effect of the chemicals tested appeared to be independent of replicative synthesis. The "critical periods" are suggested to be the stages when regular "proof reading" and correction of spontaneous errors takes place [9,13]. In addition to this regular mechanism, radiation induces an "emergency" mechanism of repair. AP inhibits the mechanism of regular repair; in addition TdR and [3H] TdR suppress the lateral spread of primary injuries across the chromosome.     

Measurements of alkali-labile DNA damage and protein-DNA crosslinks after 2450 MHz microwave and low-dose gamma irradiation in vitro

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA-protein or DNA-DNA crosslinks in C3H 10T(1/2) cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T(1/2) cells were irradiated with 4 Gy of gamma rays ((137)Cs). Immediately after gamma irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA-protein crosslinks. To measure DNA-protein crosslinks independent of DNA-DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and gamma irradiation, positive controls for DNA-protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T(1/2) cells irradiated with 4 Gy gamma rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by gamma rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with gamma rays. When DNA-protein crosslinks were specifically measured, we found no evidence for the induction of DNA-protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA-DNA or DNA-protein crosslinks.     

Pyridine nucleotide coenzyme levels in phytohemagglutinin-stimulated human lymphocytes

A radioisotopic, enzymatic cycling procedure was used to measure NAD, NADH, NADP and NADPH in cultured human lymphocytes at 0, 24 and 48 h after exposure to phytohemagglutinin (PHA). During the 0–24 h period after PHA addition NAD and NADH were increased in both control and test cultures leading to a decrease in the NAD: NADH ratio. During the 24–48 h period increases in NAD and NADH occurred in test cultures in parallel with increased incorporation of [³H]TdR. No change in the NAD: NADH ratio was seen. The results indicate that the levels of NAD and NADH may be affected by the culture conditions and that increases in these compounds occur in stimulated cells during a time period in which DNA turnover is elevated and cell volume is increased but before extensive cell division.     

Radiosensitization by hypoxic pretreatment with misonidazole: an interaction of damage at the DNA level

Prolonged exposures to misonidazole (MISO) in vitro under hypoxic conditions result in radiosensitization which is characterized by a decrease in the size of the radiation survival curve shoulder for cells irradiated under hypoxic or aerobic conditions after drug removal. Although intracellular glutathione (GSH) was depleted during hypoxic exposures to MISO, this could not account for the dose-additive radiosensitization (decrease in shoulder size) since GSH depletion by diethylmaleate had no effect on the sensitivity of cells irradiated in air. The alkaline elution assay was used to measure DNA strand breaks and their repair after exposure to MISO, graded doses of X rays, and the combination of MISO pretreatment with X rays. The elution rate of DNA from irradiated cells increased linearly with X-ray dose, with and without MISO pretreatment. However, the DNA elution rates measured after MISO pretreatment were greater by a constant amount at all X-ray doses greater than 1 Gy. In terms of both cell survival and DNA elution rate, MISO-pretreated cells behaved as though they had received an extra 1.5 Gy. Although the initial damage after X rays was greater in MISO-pretreated cells, there was no effect of MISO pretreatment on the rate of repair of radiation-induced DNA strand breaks. The agreement between the differences in survival levels and DNA elution rates for irradiated control and MISO-pretreated cells and absence of an effect on DNA repair rates suggest that the pretreatment sensitization is due to an additive interaction of damage at the DNA level.     

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.     

D-ribose inhibits DNA repair synthesis in human lymphocytes

D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.     

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.     

Alterations in histone acetylation following exposure to <Superscript>60</Superscript>Co γ-rays and their relationship with chromosome damage in human lymphoblastoid cells

Chromosome damage is related to DNA damage and erroneous repair. It can cause cell dysfunction and ultimately induce carcinogenesis. Histone acetylation is crucial for regulating chromatin structure and DNA damage repair. Ionizing radiation (IR) can alter histone acetylation. However, variations in histone acetylation in response to IR exposure and the relationship between histone acetylation and IR-induced chromosome damage remains unclear. Hence, this study investigated the variation in the total acetylation levels of H3 and H4 in human lymphocytes exposed to 0–2 Gy ⁶⁰Co γ-rays. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to modify the histone acetylation state of irradiated cells. Then, the total acetylation level, enzyme activity, dicentric plus centric rings (dic?+?r) frequencies, and micronucleus (MN) frequencies of the treated cells were analyzed. Results indicated that the acetylation levels of H3 and H4 significantly decreased at 1 and 24 h, respectively, after radiation exposure. The acetylation levels of H3 and H4 in irradiated groups treated with SAHA were significantly higher than those in irradiated groups that were not treated with SAHA. SAHA treatment inhibited HDAC activity in cells exposed to 0–1 Gy ⁶⁰Co γ-rays. SAHA treatment significantly decreased dic?+?r/cell and MN/cell in cells exposed to 0.5 or 1.0 Gy ⁶⁰Co γ-rays relative to that in cells that did not receive SAHA treatment. In conclusion, histone acetylation is significantly affected by IR and is involved in chromosome damage induced by ⁶⁰Co γ-radiation.     

DNA-repair capacity and lipid peroxidation in chronic alcoholics

The possible impact of long-term overexposure to ethanol was studied in a group of chronic alcoholics in the psychiatric hospital. The level of DNA methylation and unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU) in lymphocytes and lipid peroxidation (LPO) in plasma were used as markers of injury caused by alcohol abuse. The data were correlated with plasma levels of some natural antioxidants (vitamins A, C and E) and vitamin B12. The following results were obtained. The degree of DNA methylation by MNU in lymphocytes was the same in the exposed and control groups under our experimental conditions. The DNA excision-repair capacity of lymphocytes measured as UDS was decreased in alcoholics (p less than 0.01) and LPO in plasma was significantly higher (p less than 0.01) as a consequence of alcohol overconsumption. By the simple regression method, a correlation was found between LPO and vitamin C levels (LPO = -0.078 x vit. C + 1.9; p less than 0.05) and between UDS and LPO values (UDS = -0.384 x LPO + 4.1; p less than 0.05). These results support the hypothesis of a connection of cell membrane status and DNA damage and repair and the possible role of active oxygen species in cell damage caused by ethanol.     

Analysis of DNA-damage response to ionizing radiation in serum-shock synchronized human fibroblasts

Many aspects of cellular physiology, including cellular response to genotoxic stress, are related to the circadian rhythmicity induced by the molecular clock. The current study investigated if the cellular response to DNA damage is in relation to endogenous expression levels of the PER2 protein, a key component of the molecular regulatory system that confers rhythmicity in mammalian cells. Human normal fibroblasts (CCD-34Lu) were subjected to serum shock to induce circadian oscillations of the PER2 protein and then irradiated with γ- rays at times corresponding to the trough and peak expression of the PER2 protein. To better examine cellular response to DNA damage, the experiments performed in this study were carried out in non-proliferating CCD-34Lu fibroblasts in order to maintain the cell and circadian cycles separated while they were being exposed to genotoxic stress. Study results demonstrated that clonogenic cell survival, double-strand break repair kinetics, and TP53 protein levels were affected in the cells irradiated at the trough than in those irradiated at peak expression of the PER2 protein.     

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.     

Effect of dose rate on the survival of irradiated human skin fibroblasts.

The survival of cells in density-inhibited, confluent cultures maintained at 37 degrees C was examined following exposure to 137Cs gamma rays at low dose rates (0.023 or 0.153 Gy/h) or to 60Co gamma rays at a single high dose rate (0.70-0.75 Gy/min). Cells from an ataxia telangiectasia (AT) homozygote showed no dose-rate effect, whereas a three- to fivefold increase in D0 was observed for all other cell strains exposed at low dose rates. The magnitude of the dose-rate effect did not differ significantly among cells from persons with hereditary retinoblastoma, basal cell nevus syndrome, or AT-heterozygote compared with normal cell strains, and was not related to the size of the shoulder (extrapolation number) of the survival curve. Furthermore, no differences in the capacity for the repair of potentially lethal damage during confluent holding were observed among these latter cell strains.     

Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of [3H]thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h.