Survival and repair of potentially lethal radiation damage in confluent vascular smooth muscle cell cultures

Confluent cultures of rat aortic smooth muscle cells demonstrated dose-dependent potentially lethal damage repair (PLDR), with an average repair factor of 4 for a 10 Gy dose. PLDR occurred with a half-time of 2 hours and was nearly maximal by 8 h. Among other factors, PLDR may contribute to the radioresistance of large blood vessels.     

Enhancement of anti-cancer effect by potential lethal damage repair(PLDR)-inhibitors

Nucleoside analogues (3'-dG, Ara-A) have recently been found to be an effective chemical substance for inhibiting the recovery of potential lethal damage caused by radiation and anti-cancer drugs. By combination of radiation and inhibitors, both Ara-A and 3'-dG produced a higher sensitivity effect closer to the center of "Spheroid". In the experimental tumor S-180, combination of adriamycin , cyclophosphamide and Ara-A produced an eminent sensitivity effect amounting to the enhancement ratio of 1.24 and 1.36. By combination of 5-FU and 3'-dG, an enhancement ratio of 1.39 was obtained. These findings indicate the possibility that these inhibitors may be used as a chemosensitizer and also suggest that they would be useful for chemotherapy in the future.     

Importance of cell proliferative state and potentially lethal damage repair on radiation effectiveness: implications for combined tumor treatments (review).

The dependence of parameters of the linear-quadratic (LQ) model on cell proliferation kinetics of tumors in relation to potentially lethal damage (PLD) and its repair is evaluated. The influence of sensitizing agents on these parameters during fractionated radiotherapy is assessed. Suggestions for scheduling of radiation combined of with sensitizing agents are derived. The parameters alpha and beta of the linear-quadratic model for dose dependence of cell reproductive inactivation, derived from experimental and clinical data, are evaluated to assess their dependence on cell proliferative state, on PLD repair and on the action of various sensitizing agents. PLD contributes to the linear as well as to the quadratic component of the LQ model. PLD is less effectively repaired in proliferating (P) cells than in clonogenic (G0) cells of the quiescent (Q) cell compartment. PLD is influenced by various agents applied during, as well as after irradiation. The parameters alpha and beta are affected differently by the proliferative state of cells, by some of the sensitizing agents, and by radiation quality. The relative fractions of P cells and Q cells can change during fractionated treatments. If recruitment is effective, the fraction of G0 cells decreases in the latter part of a treatment schedule. PLD from subsequent radiation doses is then repaired less and the effectiveness of radiation combined with sensitizing agents may be enhanced. The analyses using the LQ model show differences in PLD and its repair between P cells and G0 cells in tumors. If due to recruitment the compartment of clonogenic G0 cells diminishes during treatment, the combination of radiation with sensitizing agents and the application of high-LET radiation should be scheduled to take this factor into account. For poorly differentiated tumors with high labeling indices (LI), benefit from combined treatments is expected from early in the course of fractionated radiotherapy. Well differentiated tumors with low LI are suggested to benefit most from irradiation combined with sensitizing agents in the latter part of a treatment schedule. New methods are required to assess the clonogenic G0 cells in the Q cell compartment and to monitor recruitment of these cells into the P cell compartment.     

Inhibition of potentially lethal radiation damage repair in normal and neoplastic human cells by 3-aminobenzamide: an inhibitor of poly(ADP-ribosylation)

The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, on potentially lethal damage repair (PLDR) was investigated in normal human fibroblasts and four human tumor cell lines from tumors with varying degrees of radiocurability. The tumor lines selected were: Ewing's sarcoma, a bone tumor considered radiocurable and, human lung adenocarcinoma, osteosarcoma, and melanoma, three tumors considered nonradiocurable. PLDR was measured by comparing cell survival when cells were irradiated in a density-inhibited state and replated at appropriate cell numbers at specified times following irradiation to cell survival when cells were replated immediately following irradiation. 3AB was added to cultures 2 hr prior to irradiation and removed at the time of replating. Different test radiation doses were used for the various cell lines to obtain equivalent levels of cell survival. In the absence of inhibitor, PLDR was similar in all cell lines tested. In the presence of 8 mM 3AB, differential inhibition of PLDR was observed. PLDR was almost completely inhibited in Ewing's sarcoma cells and partially inhibited in normal fibroblast cells and osteosarcoma cells. No inhibition of PLDR was observed in the lung adenocarcinoma or melanoma cells. Except for the osteosarcoma cells, inhibition of PLDR by 3AB correlated well with radiocurability.     

Effects of 5'-iododeoxyuridine on the repair of radiation induced potentially lethal damage interphase chromatin breaks and DNA double strand breaks in Chinese hamster ovary cells.

The effect of 5'-iododeoxyuridine incorporation into DNA on radiation sensitivity, cellular repair capability, induction and repair of interphase chromatin breaks, as well as induction and repair of DNA double strand breaks was investigated in plateau-phase Chinese hamster ovary cells exposed to X rays. Repair of potentially lethal damage, as measured by delayed plating plateau-phase cells, was used to assay cellular repair capacity. Induction and repair of interphase chromatin breaks were assayed by means of premature chromosome condensation, whereas induction and repair of DNA double strand breaks were assayed by pulsed field gel electrophoresis. Incorporation of 5'-iododeoxyuridine into DNA sensitized cells to radiation. Radiosensitization increased with increasing percent thymidine replacement and was accompanied by an increase in the number of chromatin breaks scored per Gy and a small increase in the number of DNA double strand breaks produced. Cells grown in the presence of 5'-iododeoxyuridine were able to repair potentially lethal damage. When the comparison was made at equal doses, the extent of this repair was higher but its rate slower in 5'-iododeoxyuridine containing cells. At equal survival levels, cells that had incorporated IdU also repaired PLD to a slightly higher extent than control cells grown in IdU free medium. The magnitude of potentially lethal damage repair increased as cells "aged" in the plateau-phase, particularly for high 5'-iododeoxyuridine concentrations (8 microM). Incorporation of 5'-iododeoxyuridine reduced the rate of repair of interphase chromatin breaks and the rate of repair of DNA double strand breaks (both the fast and the slow component). The results suggest that reduction in the efficiency of repair of DNA double strand breaks and chromatin breaks, produced by radiation in 5'-iododeoxyuridine containing cells, is one determinant of the radiosensitization observed.     

Induction and repair inhibition of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells

Compounds of nickel(II) and cadmium(II) are carcinogenic to humans and to experimental animals. One frequently discussed mechanism involved in tumor formation is an increase in reactive oxygen species by both metals with the subsequent generation of oxidative DNA damage. In the present study we used human HeLa cells to investigate the potential of nickel(II) and cadmium(II) to induce DNA lesions typical for oxygen free radicals in intact cells and the effect on their repair. As indicators of oxidative DNA damage, we determined the frequencies of DNA strand breaks and of lesions recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg protein), including 7,8- dihydro-8-oxoguanine (8-hydroxyguanine), a pre-mutagenic DNA base modification. Nickel(II) caused a slight increase in DNA strand breaks at 250 microM and higher, while the frequency of Fpg-sensitive sites was enhanced only at the cytotoxic concentration of 750 microM. The repair of oxidative DNA lesions induced by visible light was reduced at 50 microM and at 100 microM nickel(II) for Fpg-sensitive sites and DNA strand breaks, respectively; the removal of both types of lesions was blocked nearly completely at 250 microM nickel(II). In the case of cadmium(II), DNA strand breaks occurred at 10 microM and no Fpg- sensitive sites were detected. However, the repair of Fpg-sensitive DNA lesions induced by visible light was reduced at 0.5 microM cadmium(II) and higher, while the closure of DNA strand breaks was not affected. Since oxidative DNA damage is continuously induced during aerobic metabolism, an impaired repair of these lesions might well explain the carcinogenic action of nickel(II) and cadmium(II).      

A linear-quadratic model of cell survival considering both sublethal and potentially lethal radiation damage

We assessed the dose-dependence of repair of potentially lethal damage in Chinese hamster ovary cells x-irradiated in vitro. The recovery ratio (RR) by which survival (SF) of the irradiated cells was enhanced increased exponentially with a linear and a quadratic component, namely xi and psi: RR = e xi D + psi D2. Survival of irradiated cells can thus be expressed by a combined linear-quadratic model considering four variable, namely alpha and beta for the capacity of the cells to accumulate sublethal damage, and xi and psi for their capacity to repair potentially lethal damage: SF = e(xi - alpha)D + (psi - beta)D2.     

Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein

Genistein, an in vitro inhibitor of topoisomerase II and tyrosine kinases, suppressed growth and induced differentiation in HL-205 cells, a clonal population of the human promyelocytic HL-60 leukemia cells, and in K-562-J cells, a clonal population of the human erythroid K-562 leukemia cells. Maturing HL-205 cells acquired either granulocytic or monocytic markers, namely, reactivity with the murine OKM1 monoclonal antibody, expression of nitroblue tetrazolium dye reduction, and staining for nonspecific esterase. The maturing K-562-J cells stained with benzidine, which indicates the presence of hemoglobin, an erythroid maturation marker. Although the acquisition of the maturation markers in both HL-205 and K-562-J cells was time dependent up to 6 days, the kinetics of this induction differed between the two cell types. Despite the in vitro inhibitory effect of genistein, treatment of either HL-205 or K-562-J cells with 150 micrograms/ml genistein for up to 16 h did not alter topoisomerase II activity (as determined by the unknotting assay) in their nuclear extracts. Analysis with the anti-phosphotyrosine PY-20 murine monoclonal antibody indicated that treatment of K-562-J cells with genistein decreased the reactivity of the antibody with two of the cellular proteins. However, no reactivity with the PY-20 antibody was detected in untreated or genistein-treated HL-205 cells. An early event in the HL-205 and K-562-J cells, occurring after only 1 h of treatment with 30-200 micrograms/ml genistein, was the induction of DNA damage as measured by an alkaline elution assay. This damage may be a contributing factor in the genistein-induced cell differentiation in the HL-205 and K-562-J cells.     

UVC-induced expression of a tumor-associated antigen in human cell hybrids (HeLa x skin fibroblasts): repair of potentially lethal and potentially transforming damage

The UVC-induced expression of a cell surface protein which correlateswith the induction of tumorigenicity has been observed in humancell hybrids (HeLa x skin fibroblasts). A dose-response curvefor the induction of this marker of neoplastic transformationhas been obtained. Studies of the repair of potentially lethaland potentially transforming damage indicate a slow repair processwhich results in enhanced survival and decreased neoplastictransformation over the period 6–24 h post-treatment.The kinetics of this repair process are consistent with previouslypublished work on the kinetics of removal of thymine dimersfrom UVC-irradiated human skin fibroblasts. During the firstfew hours post-irradiation (0–6 h) there are fluctuationsin transformation frequency while survival remains essentiallyconstant. It is during this period that 6–4 photoproductsare removed from UV-irradiated DNA. It is possible that repairof these lesions may lead to an increase in transformation frequencyand have no impact in terms of survival.     

叠氮胸苷对人胶质瘤细胞辐射后DNA损伤单链修复的影响

目的:观察逆转录酶抑制剂叠氮胸苷(AZT,3'-azido-3'-deoxythmidine)对人脑胶质瘤细胞U251放射性DNA单链损伤(singlestrand break,SSB)修复的影响,探讨其放射增敏的机制。方法:实验分为四组:1)空白组:未行AZT与γ射线处理;2)放射组:细胞接受2Gyγ射线单次照射;3)加药组:细胞培养液中加入终浓度为0·8mmol/L的AZT作用24h;4)药放组:细胞经过0·8mmol/L的AZT作用24h后再行2Gyγ射线单次照射。用碱性单细胞凝胶电泳方法检测辐射后DNA SSB的尾矩。结果:空白组与加药组细胞无慧尾,两组差异无统计学意义,F=0·238,P=0·628,表明AZT本身不会导致DNASSB;而放射组和药放组均出现明显的彗星图象,药放组与放射组的初始SSB相比差异无统计学意义,P=0·628,但前者的修复速度较慢(15min、30min、1h和2h时间点两组的SSB差异有统计学意义,F值分别为4·652、4·160、7·134和4·715,P值分别为0·038、0·049、0·011和0·037),照射后2h放射组的SSB基本修复完全(放射组与空白组比较,P=0·099),而药放组仍有显著残留(药放组与空白组比较,P<0·0001)。结论:端粒酶抑制剂AZT放射增敏可能与其抑制DNASSB的修复有关。肿瘤防治杂志,2005,12(20):1525-1529     

叠氮胸苷对人胶质瘤细胞辐射后DNA损伤单链修复的影响

目的：观察逆转录酶抑制剂叠氮胸苷（AZT，3＇-azido-3＇-deoxythmidine）对人脑胶质瘤细胞U251放射性IONA单链损伤（single strand break，SSB）修复的影响，探讨其放射增敏的机制。方法：实验分为四组：1）空白组：未行AZT与γ射线处理；2）放射组：细胞接受2Gyγ射线单次照射；3）加药组：细胞培养液中加入终浓度为0．8mmol／1，的AZT作用24h；4）药放组：细胞经过0．8mmol／L的AZT作用24h后再行2Gyγ射线单次照射。用碱性单细胞凝胶电泳方法检测辐射后DNA SSB的尾矩。结果：空白组与如药组细胞无慧尾，两组差异无统计学意义，F=0．238，P=0．628，表明AZT本身不会导致DNA SSB；而放射组和药放组均出现明显的彗星图象，药放组与放射组的初始SSB相比差异无统计学意义。P=0．628．但前者的修复速度较慢（15min、30min、1h和2h时间点两组的SSB差异有统计学意义，F值分别为4．652、4．160、7．134和4．715，P值分别为0．038、0．049、0．011和0．037），照射后2h放射组的SSB基本修复完全（放射组与空白组比较，P=0．099），而药放组仍有显著残留（药放组与空白组比较．P〈0．0001）。结论：端粒酶抑制剂AZT放射增敏可能与其抑制DNA SSB的修复有关。     

The effect of 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation induced DNA double strand damage and repair in V79 cells

Radiation induced DNA double strand breaks are believed to be important lesions involved in processes related to cell killing, induction of chromosome aberrations and carcinogenesis. This paper reports the effects of the radioprotector 2-[(aminopropyl)amino]ethanethiol (WR-1065) on radiation-induced DNA damage and repair in V79 cells using the neutral elution method performed at pH 7.2 or pH 9.6. WR-1065 (4 mM) was added to the culture medium either 30 minutes prior to and during irradiation with Cobalt-60 gamma rays (for dose response experiments) or during the repair times tested (for DNA rejoining experiments). The results indicate that WR-1065 is an effective protector against the formation of radiation-induced double-strand breaks in DNA as measured using a neutral elution technique at either pH. The protector reduced the strand scission factors by 1.44 and 1.77 in experiments run at pH 9.6 and pH 7.2, respectively. The kinetics of DNA double-strand rejoining were dependent upon the pH at which the neutral elution procedure was performed. Unlike the results obtained with alkaline elution, rejoining of DNA breaks was unaffected by the presence of WR-1065 at either pH.     

Comparative DNA strand breakage induced by FUra and FdUrd in human ileocecal adenocarcinoma (HCT-8) cells: relevance to cell growth inhibition.

Although several mechanisms of 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) cytotoxicity have been postulated, their effects on cellular DNA integrity have not been fully investigated. Cytotoxicity and the induction of DNA single and double strand breaks (SSB and DSB) were evaluated in the human ileocecal adenocarcinoma cell line, HCT-8, following 2 hr of exposure to these agents. Alkaline and neutral elution techniques were utilized to quantitate the amounts of DNA SSB and DSB induced by FdUrd and FUra. FdUrd, but not FUra, induced a high level of DNA SSB and DSB. These effects were concentration and time dependent, reaching a maximum at 10 microM FdUrd and at 12 hr post-treatment. Minimal amounts of DNA damage were observed in HCT-8 cells exposed to FUra, even at a concentration of 300 microM that produced greater than 99% inhibition of cell growth. In order to delineate the mechanisms associated with the effects observed following FUra and FdUrd treatment of HCT-8 cells, the effects of thymidine and leucovorin were evaluated. DNA SSB and DSB induced by FdUrd were reversed by thymidine. The effects of thymidine were time dependent. Complete reversal of DNA damage and cytotoxicity was achieved when 10 microM thymidine was added at up to 12 hr after treatment, the time of appearance of maximum DNA damage. These results suggest that the extensive DNA damage induced by FdUrd (but not by FUra) was an important determinant of FdUrd cytotoxicity in HCT-8 cells.     

Caspase-dependent and -independent cell death pathways after DNA damage (Review)

Apoptosis is known to be an important phenomenon in exerting antitumor response to cancer therapy, which is regulated by Bcl-2 family proteins through mitochondrial permeability transition (MPT). Insertion by the activated Bax/Bak in response to DNA damage induces mitochondrial membrane permeabilization (MMP) via an anion channel, VDAC in mitochondrial outer membrane that plays a crucial role in releasing small molecules such as cytochrome c, Smac/DIABLO, Omi/HtrA2, AIF, and endonuclease G leading to cell death. The released small molecules are involved in caspase-dependent and -independent cell death pathway that is inhibited by Bcl-2/xL. Despite the fact that the pancaspase inhibitor, zVAD-fmk inhibited the caspase cascade, cell death mediated by caspase-independent pathway was not blocked. Similarly, although etoposide induced-apoptosis was inhibited in Bax(-/-)/Bak(-/-)mouse embryonic fibroblasts, autophagy was not inhibited, which was regulated by Bcl-xL. It appears that the cross-talk between caspase-dependent and -independent apoptotic cell death including autophagic cell death that was mediated by MPT affects overall tumor response to anticancer treatment. In this review, to assist a comprehensive understanding of MPT-mediated cell death pathway for exploring appropriate targets in cancer therapy, role of the caspase-dependent and -independent cell death pathway in the interaction of these pathways is discussed.     

In vitro oxygen-dependent survival of 2 human cell lines after radiation combined with tirapazamine (SR-4233) and cisplatin]

Recent data have shown that the in vitro and in vivo cytotoxicity of bioreductive drugs could be significantly increased by combination with ionising radiation or chemotherapy. Various parameters such as oxygen tension and timing of administration of the drugs could play a crucial role in the efficacy of combined treatment modalities. The aim of this study was to define the oxygen dependency of cell survival after in vitro irradiation and incubation with tirapazamine, a bioreductive drug, and cisplatin given alone or simultaneously. Two human cell lines were studied: one cell line sensitive to tirapazamine, Na11+, a pigmented melanoma with a high percentage of hypoxic cells, and a less sensitive cell line to tirapazamine, HRT18, a rectal adenocarcinoma. Gas changes were made to study cell survival at four different oxygen concentrations (pO2): air (20.9% O2), 10.2 and 0.2% O2. Cells were incubated with tirapazamine and cisplatin alone or combined for one hour at 37 degrees C, then irradiated and cultured. For Na11+, cell survival after irradiation was comparable in air and at 10% oxygen with the two drugs given alone or combined. At 2 and 0.2% oxygen, cell killing was largely increased by tirapazamine and was not modified by the addition of cisplatin. For HRT18, cell survival was not modified when cisplatin was added to radiation, whatever the oxygen partial pressure. At low pO2 (2 and 0.2%) the cytotoxic effect of tirapazamine was not significantly decreased by the addition of cisplatin. When cytotoxic and bioreductive drugs are combined to radiation, the magnitude of the observed effect is highly dependent on the partial oxygen pressure and on the sensitivity of the cell line to the individual drugs. This has very important implications for clinical strategies based on combined chemo-radiotherapy.     

Interaction of ICRF 159 with radiation, and its effect on sub-lethal and potentially lethal radiation damage in vitro

ICRF 159 has been shown to increase the X-radiation sensitivity of exponentially growing EMT6 mouse tumour cells in vitro. This was found only with ICRF 159 exposure times longer than 10 h and only when the drug was given before irradiation. The increase in radiation sensitivity was expressed as a reduction of the shoulder of the radiation survival curve. As ICRF 159 was shown to have no effect on the repair of sub-lethal radiation damage, it was concluded that the drug reduced the capacity to accumulate such damage. ICRF 159 was also shown to have no effect on the repair of potentially lethal radiation damage in late plateau cells.