Interaction between radiation and drug damage in mammalian cells. III. The effect of adriamycin and actinomycin-D on the repair of potentially lethal radiation damage.

We have studied the effects of actinomycin-D (AMD) and Adriamycin (ADRM) on the repair of radiation damage in Chinese hamster cells (V79) in plateau phase growth. Suppression of potentially lethal damage repair (PLDR) was observed in the presence of non-toxic levels of AMD and minimally toxic levels of ADRM. The suppression of PLDR by AMD persisted as long as the drug was present. Removal of AMD was followed by prompt repair of potentially lethal injury suggesting that suppression of PLDR by AMD was not accompanied by fixation of injury to a non-repairable state. On the other hand, irradiated cells exposed to ADRM eventually repair potentially lethal injury in the presence of drug after an initial delay. AMD, but not ADRM, inhibited repair of sublethal radiation damage.     

Interaction between radiation and drug damage in mammalian cells. IV. Radiation response of adriamycin-resistant V79 cells

Adriamycin-resistant variants derived from V79 Chinese hamster cells were examined for their radiation response properties. A stable resistant cell line (77A) demonstrated a significant reduction in the extrapolation number of the single-dose radiation survival curve. Second-step mutants from 77A cells exhibited a spectrum of radiation response states including decreased D0 values and large extrapolation numbers. A highly Adriamycin-resistant line (LZ) was found to be radiation sensitive with increased capacity for the accumulation of sublethal radiation injury. LZ cells are known to contain double-minute chromosomes and an amplified gene for the multidrug phenotype and to exhibit multidrug resistant properties. These cells require the presence of Adriamycin in their growth medium to maintain their pleiotropic characteristics. LZ cells became more resistant to radiation following reversion to an intermediate Adriamycin response as the consequence of growth in Adriamycin-free medium. Reverted cells also lost their large capacity for sublethal damage. It is suggested that detailed study of these mutants may provide insight into the identification of radiation-sensitive sites and their relationship to the genetic changes characterizing Adriamycin-resistant cell lines.     

Recombination induced by triple-helix-targeted DNA damage in mammalian cells.

Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also observed. The variety of products suggests that multiple cellular pathways can act on the targeted damage, and data showing that the triple helix can influence these pathways are presented. The ability to specifically induce recombination or gene conversion within mammalian cells by using TFOs may provide a new research tool and may eventually lead to novel applications in gene therapy.     

Choloroquine inhibition of repair of DNA damage induced in mammalian cells by methyl methanesulfonate

Chloroquine (ClQ) inhibited the repair of DNA damage produced in cultured rat liver cells by methyl methanesulfonate (MMS). MMS caused fragmentation of single-strand DNA in alkaline sucrose gradients. Repair of the damage was followed by observing the restoration of the normal sedimentation pattern at intervals after treatment. Repair was significant by 7 h and nearly complete at 24 h. Addition of ClQ during the repair peiod markedly reduced the rate of repair. Also, ClQ increased the lethality of MMS, which could be due to the inhibition of repair. ClQ was found to inhibit protein synthesis, but the effect on repair is probably not due entirely to this action since comparable inhibition of protein synthesis by cycloheximide produced a lesser degree of delay in repair.     

Interaction between radiation and drug damage in mammalian cells. VI. Radiation and doxorubicin age-response function of doxorubicin-sensitive and -resistant Chinese hamster cells.

A comparative study of the radiation and/or doxorubicin (DOX) survival response for synchronous populations of Chinese hamster V79 cells and two DOX-resistant variants (77A and LZ-8) was performed. The greatest cellular radiation sensitivity was observed in mitosis, while the greatest resistance was observed during late S phase for the three cell lines. The variation in radiation response throughout the cell cycle was expressed as a change in the width of the shoulder of the survival curves (Dq) with little change in D0. This suggests that each phase of the cell cycle has a different capacity for accumulation of radiation injury. The radiation age-response function for the three cell lines revealed that 77A and LZ-8 cells were more radiosensitive than V79 cells throughout the cell cycle. Exposure of synchronous populations to DOX (1.84 microM for V79, 9.21 microM for 77A, and 921 microM for LZ-8) for 1 h as a function of cell cycle phase revealed that V79, 77A, and LZ-8 cells exhibited the greatest sensitivity to DOX in mitosis and the most resistance to DOX during S phase, as indicated by the differences in the slope of the initial component of the survival curve. Levels of P-glyco-protein (P-gp) are probably not a factor contributing to DOX age-response function since P-gp levels remain constant throughout the cell cycle in all three cell lines. Synchronous populations of V79, 77A, and LZ-8 cells sequentially treated with DOX and radiation at various cell cycle phases were also analyzed. The results showed that the interaction between radiation and DOX damage resulted in a reduced cellular capacity for the accumulation of radiation damage throughout the cell cycle, as indicated by a decrease in the width of the shoulder of the survival curve. Overall, both DOX-sensitive V79 cells and DOX-resistant 77A and LZ-8 cells exhibited (1) a similar age-response function for radiation or DOX, and (2) no differences in the effects of DOX on radiation-induced damage throughout the cell cycle. These results indicate that acquired resistance to DOX associated with increased levels of P-gp in the cell membrane did not appear to affect the age-response function for radiation or DOX, and the nature of the interaction between damage caused by radiation and DOX was also not affected.     

Mitochondrial DNA repair of oxidative damage in mammalian cells

Nuclear and mitochondrial DNA are constantly being exposed to damaging agents, from endogenous and exogenous sources. In particular, reactive oxygen species (ROS) are formed at high levels as by-products of the normal metabolism. Upon oxidative attack of DNA many DNA lesions are formed and oxidized bases are generated with high frequency. Mitochondrial DNA has been shown to accumulate high levels of 8-hydroxy-2'-deoxyguanosine, the product of hydroxylation of guanine at carbon 8, which is a mutagenic lesion. Most of these small base modifications are repaired by the base excision repair (BER) pathway. Despite the initial concept that mitochondria lack DNA repair, experimental evidences now show that mitochondria are very proficient in BER of oxidative DNA damage, and proteins necessary for this pathway have been isolated from mammalian mitochondria. Here, we examine the BER pathway with an emphasis on mtDNA repair. The molecular mechanisms involved in the formation and removal of oxidative damage from mitochondria are discussed. The pivotal role of the OGG1 glycosylase in removal of oxidized guanines from mtDNA will also be examined. Lastly, changes in mtDNA repair during the aging process and possible biological implications are discussed.     

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.     

DNA damage recognition during nucleotide excision repair in mammalian cells

For the bulk of mammalian DNA, the core protein factors needed for damage recognition and incision during nucleotide excision repair (NER) are the XPA protein, the heterotrimeric RPA protein, the 6 to 9-subunit TFIIH, the XPC-hHR23B complex, the XPG nuclease, and the ERCC1-XPF nuclease. With varying efficiencies, NER can repair a very wide range of DNA adducts, from bulky helical distortions to subtle modifications on sugar residues. Several of the NER factors have an affinity for damaged DNA. The strongest binding factor appears to be XPC-hHR23B but preferential binding to damage is also a property of XPA, RPA, and components of TFIIH. It appears that in order to be repaired by NER, an adduct in DNA must have two features: it must create a helical distortion, and there must be a change in DNA chemistry. Initial recognition of the distortion is the most likely function for XPC-hHR23B and perhaps XPA and RPA, whereas TFIIH is well-suited to locate the damaged DNA strand by locating altered DNA chemistry that blocks translocation of the XPB and XPD components.     

DNA damage and repair in mammalian cells exposed to p-hydroxyphenylpyruvic acid

Tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine metabolism in which the fumarylacetoacetate hydrolase enzyme is defective. This disease is clinically heterogeneous and a chronic and acute form is discerned. Characteristic of the chronic form is the development of cellular hepatocarcinoma. Although p-hydroxyphenylpyruvic acid (pHPPA) is used as one of the diagnostic markers of this disease, it was suggested that it is unlikely to be involved in the pathophysiology of HT1 as it is present in other disorders that does not have hepatorenal symptoms. It was the aim of this study to investigate the possible effect of pHPPA on DNA damage and repair in mammalian cells. The comet assay was used to establish the genotoxicity of pHPPA in human peripheral blood lymphocytes and isolated rat hepatocytes after their exposure to pHPPA. At first glance the damage to DNA caused by pHPPA seemed reparable in both cell types, however, after challenging the DNA repair capacity of metabolite-treated cells with treatment with H(2)O(2), a marked impairment in the DNA repair capability of these cells was observed. We suggest that the main effect of pHPPA is the long-term impairment of the DNA repair machinery rather than the direct damage to DNA and that this effect of pHPPA, together with the other characteristic metabolites, e.g., FAA and MAA, causes cellular hepatocarcinoma to develop in the chronic form of HT1.     

The regulation of DNA repair processes in mammalian cells. II. The repair of DNA radiation damage in NIH 3T3 murine cells transformed by the v-myc oncogene]

The kinetics of repair of the ionizing radiation-induced DNA single- and double-strand breaks in the normal NIH 3T3 mouse cells and in those transformed with virus oncogenes v-myc has been investigated. The incubation of non-transformed cells for 18 hours in serum-free medium results in significant decrease in the rate of the single-strand DNA breaks repair during the first minutes of post-irradiation incubation. This effect is absent in transformed cells. The DNA double-strand breaks repair is more efficient in transformed NIH 3T3 cells as compared to that in the non-transformed ones both after their incubation in the medium with 10% fetal bovine serum or without serum. However, more significant differences in the rate of elimination of these DNA lesions was found in the serum-free medium. Hence, the presence of v-myc sequences in the transformed cells prevented from a decrease in the efficiency of DNA repair due to incubation of cell culture in serum-free medium. These results agree with the assumption that c-myc gene product may be a mediator in regulation of DNA repair by the epidermal growth factor. These data also show that the c-myc gene expression in an important condition providing a high efficiency of the constitutive DNA repair process.     

Pesticide induced DNA damage and its repair in cultured human cells.

The effects of pesticides on the induction of unscheduled DNA synthesis in SV-40 transformed human cells (VA-4) in culture with and without metabolic activation by liver microsomes was studied. Results showed that ten of the thirteen compounds examined either directly or upon metabolic activation induced unscheduled DNA synthesis in the human cell system used. The DNA repair kinetics and size of the repaired regions resulting from treatment with four of the chemicals (Carbaryl, Chlordane, Dieldrin and 2.4-D Fluid) were studied by 313 nm photolysis of repaired regions containing bromodeoxyuridine (BUdR). The size of the repaired regions differed between compounds but could generally be classified as either of the X-ray (short) or UV-type (long).     

Induction and subsequent repair of DNA damage by fast neutrons in cultured mammalian cells

The induction and repair of DNA damage were studied by a DNA unwinding method in mouse L5178Y cells exposed to fast neutrons. DNA lesions induced by fast neutrons were classified into three types from their repair profiles: fast-reparable breaks (T1/2 = 3-5 min), slow-reparable breaks (T1/2 = 70 min), and nonreparable breaks. The repair rates of both fast-reparable and slow-reparable breaks were almost the same as those of corresponding damage induced by low-LET radiation. Neutrons induced a smaller amount of fast-reparable damage, an almost equal amount of slow-reparable damage, and a larger amount of nonreparable damage than those induced by equal doses of gamma rays or X rays. RBEs for fast- and slow-reparable damage were 0.3 and 0.9, respectively. The RBE for nonreparable damage was dose dependent and was 1.4 at the level of 100 breaks/10(12) Da DNA. Among the three types of lesions, only the nonreparable damage levels correlated with the linear-quadratic shape of the survival curves and with the enhanced killing effectiveness of neutrons (RBE = 1.7 at D0).     

Cell killing and DNA damage induced in cultured mammalian cells by some tetrahydrobenzopsoralenquinones

The mechanism of action of two tetrahydrobenzopsoralenquinones: 4-methyl-tetrahydrobenzopsoralenquinone (compound 3) and 4-hydroxymethyltetrahydrobenzopsoralenquinone (compound 4) was studied in mammalian cells. These agents differ structurally from earlier benzo and tetrahydrobenzopsoralen derivatives 4-hydroxymethylbenzopsoralen (compound 1) and 4-hydroxymethyltetrahydrobenzopsoralen (compound 2) by the replacement of the benzopyranone with a quinonepyranone. In this study, we evaluated the antiproliferative activity of such derivatives in normal human lymphocytes and CHO cells cultivated in vitro. Compound 4 showed a noticeable antiproliferative activity. Studying the induction of chromosomal aberrations and of SCEs, we demonstrated that compound 4 has a clastogenic effect on mammalian cells. By means of DNA filter elution and protein precipitation techniques we evaluated the DNA damage produced by the tested compounds. Some experiments performed in presence of a DNA synthesis inhibitor showed that ongoing DNA synthesis is involved in cell killing by derivative 4. All data obtained suggest that compound 4 can interfere with the activity of topoisomerase II. Catalytic studies carried out with purified topoisomerase II and bacteriophage DNA confirmed this hypothesis.     

Distinct roles of Ape1 protein in the repair of DNA damage induced by ionizing radiation or bleomycin

Ionizing radiation (IR) and bleomycin (BLM) are used to treat various types of cancers. Both agents generate cytotoxic double strand breaks (DSB) and abasic (apurinic/apyrimidinic (AP)) sites in DNA. The human AP endonuclease Ape1 acts on abasic or 3'-blocking DNA lesions such as those generated by IR or BLM. We examined the effect of siRNA-mediated Ape1 suppression on DNA repair and cellular resistance to IR or BLM in human B-lymphoblastoid TK6 cells and HCT116 colon tumor cells. Partial Ape1 deficiency (～30% of normal levels) sensitized cells more dramatically to BLM than to IR cytotoxicity. In both cases, expression of the unrelated yeast AP endonuclease, Apn1, largely restored resistance. Ape1 deficiency increased DNA AP site accumulation due to IR treatment but reduced the number of DSB. In contrast, for BLM, there were more DSB under Ape1 deficiency, with little change in the accumulation of AP sites. Although the role of Ape1 in generating DSB was greater for IR, the enzyme facilitated removal of AP sites, which may mitigate the cytotoxic effects of IR. In contrast, BLM generates scattered AP sites, and the DSB have 3'-phosphoglycolate termini that require Ape1 processing. These DSB persist under Ape1 deficiency. Apoptosis induced by BLM (but not by IR) under Ape1 deficiency was partially p53-dependent, more dramatically in TK6 than HCT116 cells. Thus, Ape1 suppression or inhibition may be a more efficacious adjuvant for BLM than for IR cancer therapy, particularly for tumors with a functional p53 pathway.     

Interaction between X-ray and alpha-particle damage in V79 cells

V79 cells have been exposed to X-rays or 238Pu alpha-particles or to X-rays following priming alpha-particle doses of 0.5, 2 or 2.5 Gy. The survival curve for exposure to alpha-particles was exponential with a D0 of 0.89 Gy. Following exposure to priming alpha-particle doses the resulting X-ray survival curves had the same slope as the single dose X-ray curve, but a reduced shoulder. For alpha-particle priming doses of 0.5 and 2 Gy this reduction was the same as for the same X-ray doses. 2.5 Gy alpha-particles reduced the subsequent X-ray curve Dq to almost zero. alpha-particles do cause damage capable of interacting with X-ray damage.