DNA excision repair in permeable human fibroblasts

U.v. irradiation of confluent human fibroblasts activated DNArepair, aspects of which were characterized in the cells afterthey were permeabilized. Incubation of intact cells for 20 minbetween irradiation and harvesting was necessary to obtain amaximum rate of reparative DNA synthesis. Cells harvested immediatelyafter irradiation before repair was initiated displayed onlya small stimulation of DNA synthesis, indicating that permeablecells have a reduced capacity to recognize pyrimidine dimersand activate repair. The distribution of sizes of DNA strandslabeled during 10 min of reparative DNA synthesis resembledthat of parental DNA. However, during a 60-min incubation ofpermeable cells at 37°C, parental DNA and DNA labeled byreparative DNA synthesis were both cleaved to smaller sizes.Cleavage also occurred in unirradiated cells, indicating thatendogenous nuclease was active during incubation. Repair patchessynthesized in permeable cells displayed increased sensitivityto digestion by micrococcal nuclease. However, the change insensitivity during a chase with unlabeled DNA precursors wassmall, suggesting that reassembly of nucleosome structure atsites of repair was impaired. To examine whether this deficiencywas due to a preponderance of incomplete or unligated repairpatches, ³H-labeled (repaired) DNA was purified, then digestedwith exonuclease III and nuclease S₁ to probe for free 3' endsand single-stranded regions. About 85% of the P[³H]DNA synthesizedduring a 10-min pulse resisted digestion, suggesting that amajor fraction of the repair patches that were filled were alsoligated. U.v. light-activated DNA synthesis in permeable cells,therefore, appears to represent the continuation of reparativegap-filling at sites of excision repair activated within intactcells. Gap-filling and ligation were comparatively efficientprocesses in permeable cells, whereas activation of reparativeDNA synthesis and reassembly of native chromatin structure uponcompletion of repair were not.     

Proficient global nucleotide excision repair in human keratinocytes but not in fibroblasts deficient in p53

The p53 tumor suppressor protein is important for many cellular responses to DNA damage in mammalian cells, but its role in regulating DNA repair in human keratinocytes is undefined. We compared the nucleotide excision repair (NER) response of human fibroblasts and keratinocytes deficient in p53. Fibroblasts expressing human papillomavirus 16 E6 oncoprotein had impaired repair of UV radiation-induced cyclobutane pyrimidine dimers in association with reduced levels of p53 and XPC, which is involved in DNA damage recognition. In contrast, keratinocytes expressing E6 alone or concurrently with the E7 oncoprotein, while possessing reduced levels of p53 but normal levels of XPC, continued to repair pyrimidine dimers as efficiently as control cells with normal p53 levels. Despite preservation of DNA repair, E6 and E6/E7 keratinocytes were hypersensitive to UV radiation. E6 fibroblasts exhibited markedly reduced basal and induced levels of mRNA encoding DDB2, another protein implicated in early events in global NER. In contrast, E6 or E6/E7 keratinocytes possessed basal DDB2 mRNA levels that were not significantly altered relative to control cells, although little induction occurred following UV radiation. Intact global NER was also confirmed in SCC25 cells possessing inactivating mutations in p53 as well as in cells treated with pifithrin-alpha, a chemical inhibitor of p53 that decreased sensitivity of cells to UV radiation. Collectively, these results indicate that human keratinocytes, unlike fibroblasts, do not require p53 to maintain basal global NER activity, but p53 may still be important in mediating inducible responses following DNA damage.     

p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts

Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the maximum adduct levels achieved. The fact that this p53-dependent effect is noted at levels of DNA adducts that are commonly found in human tissues (i.e., <100 adducts/10(8) nucleotides) because of environmental factors such as smoking is particularly significant with respect to human carcinogenesis related to environmental exposure.     

p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites

The cyclin-dependent kinase inhibitor p21(waf1/cip1) is known to impair DNA synthesis by binding to PCNA, the co-factor of DNA polymerases delta and epsilon. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA repair efficiency were investigated in p21-null human fibroblasts obtained by targeted homologous recombination. After UV-C irradiation, p21-/- cells showed a threefold reduction in clonogenic survival and an increased susceptibility to apoptosis, as compared with parental p21+/+ cells. Removal of cyclobutane pyrimidine dimers was significantly reduced in p21-/- cells both in the whole genome, and at the level of the rDNA gene cluster, as determined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repair sites in p21-/- fibroblasts, was comparable to that observed in parental p21+/+ cells. However, PCNA remained associated with DNA for a longer period in p21-/- than in p21+/+ cells. These results suggest that in human cells, p21 is required for NER at a step located downstream the recruitment of PCNA to DNA repair sites.     

Rearrangement of nucleosome structure during excision repair in xeroderma pigmentosum (group A) human fibroblasts

Rearrangements of chromatin structure during excision repairwere examined in xeroderma pigmentosum (XP; complementationgroup A) human fibroblasts treated with the small-molecule alkylatingagent methyl methanesulfonate (MMS). In agreement with pastreports, we observed normal levels of repair synthesis in thesecells during the first 12 h after exposure to 1.5 mM MMS, incontrast to the near zero incorporation of repair patches followingexposure to 12 J/m² u.v. light. Our results indicate that therelative nuclease sensitivity of newly repaired regions in MMS-treatedXP (group A) cells is quantitatively similar to that of newlyrepaired regions in MMS-treated normal human fibroblasts. Thisenhanced sensitivity is accompanied by a marked under-representationof repair-incorporated nucleotides in isolated nucleosome coreDNA. Pulse-chase experiments demonstrated that these regionsrapidly undergo rearrangements in chromatin structure, and boththe rate and extent of these rearrangements are similar (butnot identical) to those observed in normal cells. This was alsothe case for the rate and extent of ligation of repair patches,as measured by the sensitivity of these regions to exonucleaseIII digestion. If the changes in nuclease sensitivity of newlyrepaired regions in DNA reflect an unfolding of nucleosome structureduring excision repair, then these results indicate that theactivity associated with this unfolding is present in XP (groupA) cells.     

SHORT COMMUNICATION: Negative interference of metal (II) ions with nucleotide excision repair in human cell-free extracts

Inhibition of the nucleotide excision repair (NER) proces isbelieved to cause the potentiation of the genotoxic and mutageniceffects of DNA damaging agents like UV-light or cisplation bymetal ions. However, the precise underlying molecular mechanismof this phenomenon is still unknown. Using in vitro assays,we have determined the potential interference of several metal(II) ions with the lesion recognition and strand incision/displacementsteps of the NER mechanism, independently from the DNA polymerizationstep. When combinations of an optimal Mg²⁺ concentration andconcentrations of various metal ions in a range from 0.1 to1 mM were tested, all combinations, with Mn²⁺ and Ni²⁺ expected,inhibited specifically the incision repair activity by humanprotein extracts. There was a good correlation for Cd²⁺, Co²⁺,Fe²⁺, Fe²⁺, Hg²⁺, Pb²⁺ and Zn²⁺ between an inhibiting effecton the incision activity and a reduced protein binding activityto a damaged DNA probe as assessed by gel mobility shift assay.     

Combined mismatch and nucleotide excision repair defects in a human cell line: mismatch repair processes methylation but not UV- or ionizing radiation-induced DNA damage.

Interaction between long patch mismatch repair (MMR) and persistent DNA O6-methylguanine or 6-thioguanine (6-TG) is implicated in the cytotoxicity of methylating agents and 6-TG, respectively. Human cells with defective MMR tolerate DNA methylation damage and are cross-resistant to 6-TG. To determine whether MMR contributes to the lethal effects of persistent UV-induced DNA lesions, MMR deficiency was introduced into nucleotide excision repair (NER)-defective XP12RO cells. The doubly repair-defective cells, designated XP12ROB4, did not express detectable hMSH2 protein. They had the mutator phenotype, N-methyl-N-nitrosourea and 6-TG resistance typical of MMR-defective cells. Active MMR was not required for the cytotoxicity of UV light, and the hMSH2 defect did not detectably alter the survival of XP12ROB4. The level of spontaneous or UV-induced SCE was also similar in XP12RO and XP12ROB4, indicating that hMSH2 is not required for this recombination process. The combined deficiency in MMR and NER did not confer a significant degree of tolerance to ionizing radiation, and the survival of XP12RO and XP12ROB4 after gamma-radiation was similar. Although it recognizes and processes some persistent damaged or modified DNA base pairs, MMR is unlikely to serve as a general sensor of DNA damage.     

Enhanced nucleotide excision repair in cisplatin resistant human KB carcinoma cells

We previously reported that enhanced active efflux of cisplatin and increased GSH level were observed in KCP-4 cells. In the present study, KCP-4 cells were found to be cross-resistant to ultraviolet (UV) compared with parental KB-3-1 cells. Enhanced nucleotide excision repair (NER) was verified by time-dependent repair of UV-induced DNA damage. In addition, the amount of platinum bound to DNA after exposure to cisplatin decreased in a time-dependent manner in KCP-4 cells and this was reversed by aphidicolin, a DNA polymerase inhibitor. In stationary phase cultures, aphidicolin increased the sensitivity of KCP-4 cells to cisplatin. The expression of xeroderma pigmentosum complementation group F (XPF), an endonuclease involved in NER, was upregulated in KCP-4 cells. In KCP-4 cells the expression of hMSH6, one of the mismatch repair (MMR) factors, was decreased compared to parental KB-3-1 and revertant KCP-4R cells. However, KCP-4 cells were cross-resistant to oxaliplatin, and microsatellite instability was not observed in them. These findings suggest that the enhanced NER activity for DNA damage caused by cisplatin may be involved in cisplatin resistance in KCP-4 cells.     

Deficient nucleotide excision repair capacity enhances human prostate cancer risk

Prostate cancer (CaP) is the most commonly diagnosed non-skin cancer and the second leading cause of cancer death in American men. The etiology of CaP is not fully understood. Because most of the DNA adducts generated by some CaP-related carcinogens, including polycyclic aromatic hydrocarbons, heterocyclic amines, and pesticides, are removed by the nucleotide excision repair (NER) pathway, we pilot tested the hypothesis that CaP is associated with deficient NER capacity (NERC), measured by a plasmid-based host reactivation assay. Using cryopreserved lymphocytes collected in an ongoing, clinic-based case-control study, our results showed that the mean NERC was significantly lower (P = 0.03) in 140 cases (mean +/- SD, 8.06 +/- 5.17) than in 96 controls (9.64 +/- 5.49). There was a significant association between below-median NERC and CaP risk: odds ratio (OR), 2.14; 95% confidence interval (CI), 1.19-3.86, after adjustment for age, race/ethnicity, smoking history, benign prostatic hyperplasia, and family history. This association was stronger in younger (<60 years of age) subjects (OR, 3.98; 95% CI, 1.13-14.02) compared with older (> or = 60) subjects (OR, 1.74; 95% CI, 0.90-3.37). When we stratified NERC values by quartiles of controls, there was a significant dose-dependent association between lower NERC and elevated CaP risk (p (test for linear trend), 0.01). Compared with the highest quartile of NERC as the referent group, the adjusted ORs for the 75th, 50th, and 25th quartiles were: 1.09 (95% CI, 0.46-2.59); 1.81 (95% CI, 0.77-4.27); and 2.63 (95% CI, 1.17-5.95), respectively. This pilot study is the first direct evidence associating deficient NERC with human CaP risk.     

Impaired nucleotide excision repair in UV-irradiated human oral keratinocytes immortalized with type 16 human papillomavirus genome

We previously reported that 'high risk' human papillomaviruses (HPV) induce genetic instability in human oral keratinocytes. To understand the mechanisms of HPV-induced genetic instability, we determined the nucleotide excision repair (NER) capacity of normal (NHOK) and human papillomavirus type-16 immortalized oral keratinocytes (HOK-16B) by strand-specific removal of UV-induced cyclobutane pyrimidine dimers (CPDs) from a 16 Kb fragment of the p53 gene. In NHOK the NER activity was initiated in both DNA strands immediately, although the process in the non-transcribed strand was notably slower than that of the transcribed strand. In HOK-16B cells the initiation of CPDs removal was delayed for at least 8 h in both DNA strands, and the process was significantly slower than that in NHOK. UV-irradiation enhanced the p53 protein level more than 30-fold in NHOK, but it did not significantly alter the protein level in the HOK-16B cells. UV-irradiation also increased the p21WAF1/CIP1 protein level only in NHOK. These data indicate that 'high risk' HPV induces genetic instability by impairing NER capacity of cells. Impaired NER activity of HOK-16B cells may be implicated with their inability to enhance active p53 when challenged by genotoxic stress.     

Excision of tamoxifen-DNA adducts by the human nucleotide excision repair system

The antiestrogen tamoxifen is used in the treatment of breast cancer and has recently been recommended as a chemopreventive drug for women at high risk for breast cancer. However, women treated with the drug have an increased incidence of endometrial cancer. It has been suggested that this endometrial cancer might result from mutagenic DNA adducts, which are formed by electrophilic tamoxifen species generated by metabolic activation of the drug. Because the frequency of damage-induced mutations is strongly dependent on the repairability of the lesion, we investigated the repair of the major tamoxifen-DNA adducts by the human nucleotide excision repair system. Using the reconstituted human excision repair system and synthetic DNA substrates, we found that the four types of tamoxifen-DNA adducts detected in the endometrium were repaired with moderate to poor efficiency by nucleotide excision repair. It is concluded that individual variations in repair capacity may play a role in the development of tamoxifen-induced endometrial cancer.     

Deficient nucleotide excision repair activity in protein extracts from normal human lymphocytes

DNA repair activity in human peripheral blood lymphocytes (PBL)has been investigated by various techniques. Here, we reportthe use of an in vitro assay in order to assess nucleotide excisionrepair activity (NER). The mechanism of this major repair processrelies on two broad steps: first, recognition, incision andexcision of the damaged DNA; second, repair synthesis on thegapped DNA. Briefly, damaged plasmids were incubated with wholecell extracts which allows one to quantify DNA repair synthesis.When NER was determined on plasmid DNA damaged with UV-lightor cisplatin, PBL extracts showed no repair synthesis for unstimulatedlymphocytes. Using a new in vitro assay measuring only the damage-specificDNA incision activity in cell extracts, we found that the incisionstep in the repair reaction was blocked in unstimulated PBL.By mixing PBL with XP (group A, B, C, D) extracts, no restorationof NER activity was observed. In addition, these lymphocytesalso lacked DNA replication activity as determined with pre-incisedplasmid substrate. However, a phytohemagglutinin treatment ofPBL led to an extent of repair synthesis similar to that observedwith extracts from lymphoblastoid cells. When lymphocytes wereincubated in 20% serum medium with and without phytohemagglutinin,the repair activity increased dramatically after 24 h. Duringthe activation of lymphocytes, the extent of repair synthesiswas proportional to the percentage of cells in S phase of thecell cycle. Our results suggest that the blockage of the cellcycle in G0/G1 in PBL may be responsible for their lack of NERactivity.     

Mutations of the ING1 tumor suppressor gene detected in human melanoma abrogate nucleotide excision repair

Epidemiological evidence indicates that ultraviolet radiation (UVR) is the primary environmental cause of the rapid increase in the incidence of human cutaneous melanoma observed in the past decades. However, the genetic changes caused by UVR that lead to melanoma formation remain unclear. The ING1 (inhibitor of growth 1) tumor suppressor plays an important role in cellular stress response to UVR. To further investigate whether ING1 is involved in melanoma development, we examined the mutational status of the ING1 gene in 46 human cutaneous melanoma biopsies and characterized the biological importance of ING1 mutations in nucleotide excision repair. Single-strand conformation polymorphism and DNA sequencing were used to detect the mutational status of the ING1 gene. The host-cell-reactivation assay and radioimmunoassay were used to determine the role of ING1 mutations in nucleotide excision repair. We show that 20% of the melanoma primaries contained missense mutations in the SAP30-interacting domain and PHD finger motif of the ING1 gene with the R102L and N260S alterations observed more than once. Furthermore, our data indicate that patients that harbor ING1 mutations in the tumors have a higher risk to die from the disease within 5 years (50%) compared to patients with no ING1 mutation (18%). Moreover, we demonstrated that mutations at codon 102 or 260 as well as deletion of the PHD finger motif are detrimental to p33ING1-mediated enhancement of DNA repair. Taken together, our data indicate that ING1 mutations abrogate its enhancement in nucleotide excision repair.     

The function of DNA topoisomerases in UV-induced DNA excision repair: studies with specific inhibitors in permeabilized human fibroblasts.

Fifteen specific inhibitors of DNA topoisomerases I and II were used to elucidate whether these enzymes participate in the excision repair of UV-induced DNA damage, monitoring DNA repair synthesis in confluent saponin-permeabilized human fibroblasts. To achieve a sufficient degree of accuracy dose--response experiments were performed, analysed by linear regression, and the concentrations at which repair activity was reduced to 50% were calculated and designated K50. Camptothecin, a specific inhibitor of topoisomerase I did not markedly diminish DNA repair synthesis. Similarly, when combined with topoisomerase II inhibitors [nalidixic acid, oxolinic acid, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside) (etoposide), 4'-demethylepipodophyllotoxin-thenylidene-beta-D-glucoside (teniposide), 1,4-dihydroxy-5,8-bis ((2-[(2-hydroxyethyl)amino]ethyl)amino)-9,10-anthracenedione (mitoxantrone), 5-(N-phenyl-carboxamido)-2-thiobarbituric acid (merbarone) or 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)], it did not lower K50 values determined for topoisomerase II-specific drugs in separate experiments. The effects observed can be classified according to the mechanism of action the inhibitors exhibit. (i) Novobiocin and coumermycin, inhibitors of the ATPase subunit of topoisomerase II, completely reduced DNA repair synthesis. (ii) Inhibition of repair was also found for ethidium bromide, quinacrine and distamycin, drugs known to modify the DNA substrate by intercalation or binding to the DNA minor groove. (iii) Inhibitors acting through intercalation and, simultaneously, binding to the cleavable DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) also suppressed reparative DNA synthesis. (iv) Only small effects were observed for etoposide, nalidixic acid and oxolinic acid, whereas teniposide caused marked inhibition of DNA repair synthesis. (v) Merbarone, a novel type of topoisomerase II inhibitor, blocked UV-induced DNA repair drastically. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kDa form of topoisomerase II is the main target enzyme for inhibitors which suppressed DNA excision repair and that this isozyme is involved in steps preceding repair-specific DNA incision.