Differences In Repair Capacity Research Articles

Expression of DNA repair genes in arctic char (Salvelinus alpinus) from Bjørnøya in the Norwegian Arctic

High levels of organochlorines (OCs) have been measured in arctic char (Salvelinus alpinus) from Lake Ellasjøen on Bjørnøya, Norway (74.30°N, 19.0°E). In a nearby lake, Laksvatn, the OC-levels in arctic char were low. A previous study has shown that char from Ellasjøen had significantly higher levels of DNA double strand breaks (DSBs) than char from Lake Laksvatn. Even though there is increasing evidence of the genotoxic effects of OCs, little is known about the effects of OCs on the DNA repair system. The aim of the present study was to determine if the two main DNA DSB repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ), are affected by the higher OC and DSB level in char from Ellasjøen. This was analysed by comparing the transcript level of 11 genes involved in DNA DSB repair in char liver samples from Ellasjøen (n = 9) with char from Laksvatn (n = 12). Six of the investigated genes were significantly upregulated in char from Ellasjøen. As the expression of DNA DSB repair genes was increased in the contaminant-exposed char, it is likely that the DNA DSB repair capacity is induced in these individuals. This induction was positively correlated with the DNA DSB and negatively correlated with one or several OCs for four of these genes. However, the strongest predictor variable for DNA repair genes was habitat, indicating genetic differences in repair capacity between populations. As char from Ellasjøen still had significantly higher levels of DSBs compared to char from Laksvatn, it is possible that chronic exposure to OCs and continued production of DSB has caused selective pressure within the population for fixation of adaptive alleles. It is also possible that DSB production was exceeding the repair capacity given the prevailing conditions, or that the OC or DSB level was above the threshold value of inhibition of the DNA repair system resulting in the rate of DNA damage exceeding the rate of repair.

Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81–88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

Polymorphisms in XPC, XPD and XPG DNA repair genes and leukemia risk in a Tunisian population

Human DNA repair mechanisms protect the genome from DNA damage caused by endogenous and environmental agents. Polymorphisms in DNA repair genes and differences in repair capacity between individuals have been widely reported in different cancers. In this study we aimed to evaluate the associations between XPC Lys939Gln (rs2228001), XPD Lys751Gln (rs13181) and XPG Asp1104His (rs17655) polymorphisms and leukemia risk in a Tunisian population. Genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 206 patients with leukemia and 206 healthy controls. We found increased risk of leukemia among subjects carrying the XPC 939Gln/Gln genotype (odds ratio [OR] = 2.48, 95% confidence interval [CI] = 1.353–4.560, p = 0.0042). Moreover, in subgroup analysis according to clinical types, patients with chronic myeloid leukemia (CML) showed a higher risk than patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (OR = 3.87, 95% CI = 1.820–8.237, p = 0.0003). However, the XPD 751Gln allele may be protective against CML and AML development, and no significant differences in genotype frequencies were observed for the XPG gene between patients and controls. Further studies with larger samples and risk factor information are needed.

Lifespan Differences in Hematopoietic Stem Cells are Due to Imperfect Repair and Unstable Mean-Reversion

The life-long supply of blood cells depends on the long-term function of hematopoietic stem cells (HSCs). HSCs are functionally defined by their multi-potency and self-renewal capacity. Because of their self-renewal capacity, HSCs were thought to have indefinite lifespans. However, there is increasing evidence that genetically identical HSCs differ in lifespan and that the lifespan of a HSC is predetermined and HSC-intrinsic. Lifespan is here defined as the time a HSC gives rise to all mature blood cells. This raises the intriguing question: what controls the lifespan of HSCs within the same animal, exposed to the same environment? We present here a new model based on reliability theory to account for the diversity of lifespans of HSCs. Using clonal repopulation experiments and computational-mathematical modeling, we tested how small-scale, molecular level, failures are dissipated at the HSC population level. We found that the best fit of the experimental data is provided by a model, where the repopulation failure kinetics of each HSC are largely anti-persistent, or mean-reverting, processes. Thus, failure rates repeatedly increase during population-wide division events and are counteracted and decreased by repair processes. In the long-run, a crossover from anti-persistent to persistent behavior occurs. The cross-over is due to a slow increase in the mean failure rate of self-renewal and leads to rapid clonal extinction. This suggests that the repair capacity of HSCs is self-limiting. Furthermore, we show that the lifespan of each HSC depends on the amplitudes and frequencies of fluctuations in the failure rate kinetics. Shorter and longer lived HSCs differ significantly in their pre-programmed ability to dissipate perturbations. A likely interpretation of these findings is that the lifespan of HSCs is determined by preprogrammed differences in repair capacity.

Differences in nucleotide excision repair capacity between newly diagnosed colorectal cancer patients and healthy controls

Alteration of DNA integrity is a potential cause of cancer and it is assumed that reduced DNA repair capacity and accumulation of DNA damage may represent intermediate markers in carcinogenesis. In this case-control study, DNA damage and nucleotide excision repair capacity (NER-DRC) were assessed in association with sporadic colorectal cancer (CRC). Both parameters were quantified by comet assay in blood cells of 70 untreated incident patients and 70 age-matched healthy controls. mRNA expression and polymorphisms in relevant NER genes were concurrently analyzed. The aim of this study was to characterize incident CRC patients for NER-DRC and to clarify possible relations between investigated variables. Comet assay and mRNA expression analysis showed that CRC patients differ in repair capacity as compared to controls. Patients had a lower NER-DRC and simultaneously they exhibited higher endogenous DNA damage (for both P < 0.001). Accumulation of DNA damage and decreasing NER-DRC behaved as independent modulating parameters strongly associated with CRC. Expression levels of 6 out of 9 studied genes differed between groups (P ≤ 0.001), but none of them was related to DRC or to any of the studied NER polymorphisms. However, in patients only, XPC Ala499Val modulated expression levels of XPC, XPB and XPD gene, whereas XPC Lys939Gln was associated with XPA expression level in controls (for all P < 0.05). This study provides evidence on altered DRC and DNA damage levels in sporadic CRC and proposes the relevance of the NER pathway in this malignancy. Further, alterations in a complex multigene process like DNA repair may be better characterized by functional quantification of repair capacity than by quantification of individual genes transcripts or gene variants alone.

Simultaneous measurement of benzo[a]pyrene‐induced Pig‐a and lacZ mutations, micronuclei and dna adducts in mutaTMmouse

In this study we compared the response of the Pig-a gene mutation assay to that of the lacZ transgenic rodent mutation assay, and demonstrated that multiple endpoints can be measured in a 28-day repeat dose study. Muta™Mouse were dosed daily for 28 days with benzo[a]pyrene (BaP; 0, 25, 50 and 75 mg/kg body weight/day) by oral gavage. Micronucleus (MN) frequency was determined in reticulocytes (RETs) 48 hr following the last dose. 72 h following the last dose, mice were euthanized, and tissues (glandular stomach, small intestine, bone marrow and liver) were collected for lacZ mutation and DNA adduct analysis, and blood was evaluated for Pig-a mutants. BaP-derived DNA adducts were detected in all tissues examined and significant dose-dependent increases in mutant Pig-a phenotypes (i.e., RETCD24- and RBC CD24-) and lacZ mutants were observed. We estimate that mutagenic efficiency (i.e., rate of conversion of adducts into mutations) was much lower for Pig-a compared to lacZ, and speculate that this difference is likely explained by differences in repair capacity between the gene targets, and differences in the cell populations sampled for Pig-a versus lacZ. The BaP doubling doses for both gene targets, however, were comparable, suggesting that similar mechanisms are involved in the accumulation of gene mutations. Significant dose-related increases in % MN were also observed; however, the doubling dose was considerably higher for this endpoint. The similarity in dose response kinetics of Pig-a and lacZ provides further evidence for the mutational origin of glycosylphosphatidylinositol (GPI)-anchor deficiencies detected in the Pig-a assay. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc.

A polymorphism in the hMLH1 gene (-93G↷A) associated with lung cancer susceptibility and prognosis

Polymorphisms in DNA repair genes may be associated with differences in repair capacity of DNA damage and may thereby influence an individual's susceptibility to lung cancer. We investigated the association between the -93G-->A polymorphism in the mismatch repair hMLH1 gene for its role in the susceptibility and survival of non-small cell lung cancer (NSCLC) patients. Using a case-control study design, 165 NSCLC patients and 193 controls with similar range for age, gender and smoking habit distributions were subjected to genotype analysis. The risk of lung cancer was estimated by logistic regression analysis. The Kaplan-Meier method was used to estimate the probability of survival and the log-rank test was used to assess the significance of the difference between survival probabilities. The homozygous variant A/A genotype was associated with a significantly increased risk for lung cancer compared with the other genotypes (Crude analysis P=0.003, Adjusted analysis P=0.011, using the logistic regression model). The patients with a homozygous variant A/A genotype had a trend toward poorer prognoses compared with other patients, especially smoking (P=0.05, by log-rank test), male (P=0.06), or squamous carcinoma (P=0.08) patients. This is the first case-control study to show a significant association between the hMLH1-93G-->A polymorphism and the susceptibility to and prognosis of lung cancer. The results herein may be useful for risk assessment and disease monitoring of NSCLC.

Dietary and genetic modulation of DNA repair in healthy human adults

The DNA in all cells of the human body is subject to damage continuously from exogenous agents, internal cellular processes and spontaneous decomposition. Failure to repair such damage is fundamental to the development of many diseases and to ageing. Fortunately, the vast majority of DNA damage is detected and repaired by one of five complementary DNA repair systems. However, recent studies have shown that even in healthy individuals there is a wide inter-individual variation in DNA repair capacity. Part of this variation can be accounted for by polymorphisms in the genes encoding DNA repair proteins. However, it is probable that environmental factors, including dietary exposure as well as diet-gene interactions, are also responsible for much of the difference in repair capacity between individuals. Whilst there is some evidence from human studies that generalised malnutrition or low intakes of specific nutrients may affect DNA repair, as yet there is limited understanding of the molecular mechanisms through which nutrients can modulate this key cellular process.

Repair of oxidative DNA damage: assessing its contribution to cancer prevention

DNA repair is a crucial factor in maintaining a low steady-state level of oxidative DNA damage and protecting us from cancer. Cancer case-control studies, using indirect assays in which chromosome breakage in lymphocytes is taken as a measure of failure to repair DNA, indicate an association between poor repair and cancer risk, but case-control studies can be misleading. Surprisingly little is known of the variations in repair capacity within healthy human populations. It is likely that differences in repair enzyme activity result from genetic polymorphisms in repair genes, which have been shown, in some cases, to be linked to cancer. There is a need for prospective studies, in which genotype is analysed (for a range of repair and related genes) and repair activity measured before cancer has developed. Using a new method to measure repair in an extract of lymphocytes, based on a modification of the comet assay, we are seeking answers to the following questions: what is the normal range of repair activities in healthy humans; do differences in repair capacity correlate with genetic variations; is low repair capacity associated with a high risk of cancer; how important is DNA repair rate in determining the steady-state level of damage; what is the extent of intra-individual variation; is repair modulated by environmental factors or induced by damage; are there differences in repair capacity between men and women; what is the association of DNA repair with ageing?

ROLE OF DNA REPAIR IN PARTICLE- AND FIBER-INDUCED LUNG INJURY

Lung injury after particle exposure is associated with the generation of reactive oxygen species (ROS), leading to increased levels of oxidative DNA damage. Furthermore, both stable and unstable DNA lesions may arise after metabolization of polycyclic aromatic hydrocarbons (PAH) frequently bound to particles. Oxidative DNA damage is predominantly removed by base excision repair (BER), which is usually comparatively fast and efficient. Nevertheless, even without exogenous exposure a measurable steady-state level of oxidative DNA lesions exists due to oxygen metabolism, and repair capacities may be exceeded upon particle exposure. Stable DNA adducts induced by PAHs are eliminated by nucleotide excision repair (NER), which is unequally distributed in the genome and incomplete in transcriptionally inactive DNA regions. Thus, even low adduct levels due to environmental exposure lead to persistent lesions in the human lung. Other factors that need to be considered are potential repair inhibitions by associated metal compounds and interindividual differences in repair capacities. Taken together, the extent of DNA repair is an important determinant in particle-induced genotoxicity and carcinogenicity.

Association between Integrin-Dependent Migration Capacity of Neural Stem Cells in Vitro and Anatomical Repair Following Transplantation

In previous transplantation studies using neural stem cell lines immortalized by the temperature-sensitive SV40 large T-antigen, we have shown that animals with experimental hippocampal lesions resulting from four vessel occlusion recover spatial memory functions more effectively when grafted with the MHP36 cell line than with the MHP15 cell line [Gray et al. (1999). Philos. Trans. R. Soc. London Biol. Sci. 354: 1407–1421]. In the present study, we have investigated the cellular and molecular basis of these differences in repair capacity both in vivo and in vitro. Using the same model of hippocampal damage we have shown that following transplantation MHP36 cells migrate and align within the damaged CA1 of the ipsilateral hippocampus. MHP15 cells, in contrast, migrate in a more indiscriminate pattern that does not reflect the anatomy of the region. To analyze the migratory properties of these two cell lines in more detail, we performed migration assays at a nonpermissive temperature on the extracellular matrix substrates laminin, fibronectin, and vitronectin. These showed that MHP36 cells have a greater migration potential than the MHP15 cells. While the pattern of cell surface extracellular matrix receptors of the integrin family was identical in both cell lines, the different degrees of migration on vitronectin were both blocked by inhibitors of αV integrins. Differences in integrin signaling therefore contribute to the greater migration potential of the repairing MHP36 cell line.

Clinical results of unconventional fractionation radiotherapy in central nervous system tumors.

Malignant brain tumors (primary and metastatic) are apparently resistant to most therapeutic efforts. Several randomized trials have provided evidence supporting the efficacy of radiation therapy. Attempts at improving the results of external beam radiotherapy include altered fractionation, radiation sensitizers and concomitant chemotherapy. In low-grade gliomas, all clinical studies with radiotherapy have employed conventional dose fractionation regimens. In high-grade gliomas, hypofractionation schedules represent effective palliative regimens in poor prognosis subsets of patients; short-term survival in these patients has not allowed to evaluate late toxicity. In tumors arising within the central nervous system, hyperfractionated irradiation exploits the differences in repair capacity between tumour and late responding normal tissues. It may allow for higher total dose and may result in increased tumor cell kill. Accelerated radiotherapy may reduce the repopulation of tumor cells between fractions. It may potentially improve tumor control for a given dose level, provided that there is no increase in late normal tissue injury. In supratentorial malignant gliomas, superiority of accelerated hyperfractionated over conventionally fractionated schedules was observed in a randomized trial; however, the gain in survival was less than 6 months. At present no other randomized trial supports the preferential choice for altered fractionation irradiation. Also in pediatric brainstem tumors there are no data to confirm the routine use of hyperfractionated irradiation, and significant late sequelae have been reported in the few long-term survivors. Shorter treatment courses with accelerated hyperfractionated radiotherapy may represent a useful alternative to conventional irradiation for the palliation of brain metastases. Different considerations have been proposed to explain this gap between theory and clinical data. Patients included in dose/effect studies are not stratified by prognostic factors and other treatment-related parameters. This observation precludes any definite conclusion about the relative role of conventional and of altered fractionation. New approaches are currently in progress. More prolonged radiation treatments, up to higher total doses, could delay time to tumor progression and improve survival in good prognosis subsets of patients; altered fractionation may be an effective therapeutic tool to achieve this goal.

Mutation spectra analysis suggests that N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli

To determine the influence of some bacterial DNA repair pathways on the mutagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr-ada+ogt+, and uvr-ada-ogt-). Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains. One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced. All mutants were characterized by single base-pair substitutions, most of which (more than 96%) were GC-->AT transitions (the mutated G being almost exclusively preceded 5' by a purine). Mutation distribution was not random. Position 160 (5'-GGT-3', nontranscribed (NT) strand) was a uvr+ada+ogt(+)-specific hot-spot. Position 123 (5'-GGG-3', NT strand) was a common hot-spot but significantly more mutable in repair-proficient strains than in repair-deficient strains. Conversely, position 168 (5'-GGA-3', transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains. By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr- strains, whereas in the uvr- background, no difference was observed between mutation spectra in ada+ogt+ versus ada-ogt- strains. Our results are consistent with the hypothesis that O6-alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of GC-->AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr- strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E. coli.

Resistance to chemotherapeutic antimetabolites: a function of salvage pathway involvement and cellular response to DNA damage.

The inherent or acquired (induced) resistance of certain tumours to cytotoxic drug therapy is a major clinical problem. There are many categories of cytotoxic agent: the antimetabolites, e.g. methotrexate (MTX), N-phosphonacetyl-L-aspartate (PALA), 5-fluorouracil (5-FU), 6-mercaptopurine (6-TG), hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (AraC); the alkylating agents, e.g. the nitrogen mustards and nitrosoureas; the antibiotics, e.g. doxorubicin and mitomycin C; the plant alkaloids, e.g. vincristine and vinblastine; and miscellaneous compounds, such as cisplatin. There are also many mechanisms of drug resistance elucidated principally from in vitro studies. These include mutation of target genes, amplification of target and mutated genes, differences in repair capacity, altered drug transport and differences in nucleoside and nucleobase salvage pathways (Fox et al, 1991). The aim of the present review is to evaluate in detail the mechanisms of response of both normal and tumour cells to three chemotherapeutic antimetabolites, MTX, PALA and 5-FU, which are routinely used in the clinic either alone or in combination to treat some of the commonest solid tumours, e.g. breast, colon, gastric and head and neck. The normal and tumour cell response to these agents will be discussed in relation to the operation of the known alternative 'salvage pathways' of DNA synthesis and current theories of DNA damage response.

Radiosensitivity and double-strand break rejoining in tumorigenic and non-tumorigenic human epithelial cell lines

Radiosensitivity and repair of DNA damage induced by ionizing radiation and restriction enzymes were investigated in three human epithelial cell lines: two tumorigenic squamous carcinoma cell lines (SCC-4 and SCC-25), and a nontumorigenic epidermal keratinocyte cell line (RHEK-1). Sensitivity to ionizing radiation was determined using a clonogenic cell survival assay, which showed SCC-4 to be more radiosensitive than SCC-25 and RHEK-1, which in turn displayed about equal sensitivity. Using DNA precipitation under alkaline conditions for the analysis of induction and repair of DNA single-strand breaks (ssb), an increased level of ssb induction was found for SCC-4 while the efficiency of ssb repair was about equal in all three cell lines. Using pulsed-field gel electrophoresis (PFGE) for the measurement of induction and repair of DNA double-strand breaks (dsb), no consistent differences were detected between the three cell lines. A plasmid reconstitution assay was used to determine the capacity to rejoin restriction enzyme-induced dsb in whole-cell extracts prepared from the three cell lines. In these experiments, dsb rejoining was shown to be significantly reduced in the most radiosensitive SCC-4 cell line while it was about equal in RHEK-1 and SCC-25. The results indicate that plasmid reconstitution in cell-free extracts is a sufficiently sensitive assay to detect differences in repair capacity among tumour cell lines of different radiosensitivity which remain undetectable by DNA precipitation and PFGE.

Measurement of DNA repair deficiency in workers exposed to benzene.

We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m2 UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repaired normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m2 and 350 J/m2 were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (< 0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay.

Suppressed DNA repair capacity of peripheral lymphocytes in pregnant women

The ability to repair potentially carcinogenic lesions was measured for peripheral lymphocytes isolated from women in the first, second and third trimesters of pregnancy and 6 weeks post-partum. Freshly isolated lymphocytes were damaged with 254 nm ultraviolet radiation and allowed to perform repair in their autologous plasma. DNA repair capacity during pregnancy was over 50% lower than that measured 6 weeks post-partum. No significant difference in repair capacity was observed among the three trimesters. Mitogen-stimulated DNA synthesis was also measured for cells cultured in their autologous plasma. There was no significant difference in thymidine incorporation between cells collected during pregnancy (1.02 ± 0.17 cpm/10 6 cells) and cells collected 6 weeks postpartum (1.45 ± 0.94 cpm/10 6 cells). The results are, however, consistent with alterations in the immune response during pregnancy.

Melanin Reduces Ultraviolet-Induced DNA Damage Formation and Killing Rate in Cultured Human Melanoma Cells

Epidermal melanin pigment is believed to prevent development of ultraviolet (UV)-induced skin cancer by shielding cell nuclei and reducing DNA damage formation. It has not been experimentally proved, however, whether melanin reduces UV-induced DNA damage, because published experiments have been inconclusive. The present study was carried out to determine whether intracellular melanin protected cultured cells against UV-induced DNA damage and killing. Three human melanoma cell lines containing different amounts of melanin were used. Absorption spectrum, subcellular localization of melanin, and melanin concentration were examined in the three cell lines. Two types of DNA damage, cyclobutane pyrimidine dimers and (6-4)photoproducts, were detected by an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies specific for these photolesions. We found that melanin reduced the induction rates of both types of DNA damage in pigmented cells irradiated with low doses of UV in a melanin concentration-dependent manner. Almost no differences in repair capacity for the two types of photolesions were observed among the three melanoma cell lines. We also found that the more highly melanotic melanoma cell lines were more UV resistant than the less melanotic melanoma cell lines. These results suggest that intracellular melanin plays an important role in preventing UV-induced cell killing by reducing the formation of two types of DNA damage.

Cell inactivation by heavy charged particles.

The inactivation of cells resulting in lethal or aberrant effects by charged particles is of growing interest. Charged particles at extremely high LET are capable of completely eliminating cell-type and cell-line differences in repair capacity. It is still not clear however whether the repair systems are inactivated, or merely that heavy-ion lesions are less repairable. Studies correlating the particle inactivation dose of radioresistant cells with intact DNA analyzed with pulse field gel electrophoresis and other techniques may be useful, but more experiments are also needed to assess the fidelity of repair. For particle irradiations between 40-100 keV/microns there is however evidence for particle-induced activation of specific genes in mammalian cells, and certain repair processes in bacteria. New data are available on the inactivation of developmental processes in several systems including seeds, and cells of the nematode C. elegans. Future experimental and theoretical modeling research emphasis should focus on exploring particle-induced inactivation of endpoints assessing functionality and not just lethality, and on analyzing molecular damage and genetic effects arising in damaged but non-inactivated survivors. The discrete nature of selective types of particle damage as a function of radiation quality indicates the value of accelerated ions as probes of normal and aberrant biological processes. Information obtained from molecular analyses of damage and repair must however be integrated into the context of cellular and tissue functions of the organism.

Sublethal damage repair in squamous cell carcinoma cell lines.

Squamous cell carcinoma (SCC) cell lines recently established from head and neck tumors were studied for their capability of repairing sublethal radiation induced damage (SLDR). Total doses of 1.25, 2.50, 5.00, and 7.50 Gy were used either as a single dose or split in two equal fractions with a 24 h interval. Cell survival was determined using a 96-well plate clonogenic assay based on limiting dilution. Survival data was fitted by linear quadratic model, and the area under the survival curve (AUC) was obtained with numerical integration. The amount of SLDR was expressed as AUC-ratio comparing survivals from split dose vs single dose experiments. SLDR capacity was observed to vary markedly between individual cell lines (AUC-ratios 1.0-1.5). The relatively radiation-sensitive cell lines had a tendency toward higher SLDR proficiency (correlation coefficient 0.85). The differences in repair capacity could not be explained by the differences in doubling times of the cell lines. The inverse relationship between SLDR capacity and inherent radiosensitivity could explain the poor radiocurability of the sensitive donor tumors. Two of the most resistant cell lines were found SLDR deficient. This is a novel finding, since SLDR has been previously reported in all studied cells.