Background: Cisplatin is a commonly used anti-cancer drug. However, its use is associated with severe side effects including ototoxicity that affects a large fraction of cisplatin-treated patients. Approved therapies that reduce cisplatin-induced ototoxicity are lacking. Among the candidate therapeutics, dexamethasone stands out. There is extensive experience of its use in combination with cisplatin for the prevention of chemotherapy-induced nausea and vomiting indicating that dexamethasone does not affect the anti-cancer effects of cisplatin. Keywords: Cisplatin, ototoxicity, dexamethasone, cancer, chemotherapy, side effects, adverse events.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

Introduction: The p53 tumour suppressor protein plays a pivotal role in the response of mammalian cells to DNA damage. It regulates cell cycle progression, apoptosis and DNA repair mechanisms and is therefore likely to influence response to targeted radionuclide therapy. This study investigated the role of p53 in the cellular response to the Auger-emitting radionuclide indium-111.Methods: Two stable clones of a HT1080 fibrosarcoma cell line, differing only in p53 status due to RNAi-mediated knockdown of p53 expression, were incubated for 1 h with [In-111]-oxinate (0-10MBq/ml). Radiopharmaceutical uptake into HT1080 cells was measured in situ using a non-contact phosphorimager method. Cellular sensitivity and DNA damage were measured by, respectively, clonogenic survival analysis and the single cell gel electrophoresis (Comet) assay.Results: Mean uptake of [In-111]-oxinate in HT1080 cells was unaffected by p53 status, reaching a maximum of 9 Bq/cell. [In-111]-oxinate-induced cytotoxicity was also identical in both clones, as measured by IC50 (0.68 MBq/ml). However the formation of DNA damage, measured immediately after treatment with [In-111]-oxinate, was found to be up to 2.5-fold higher in the p53-deficient HT1080 clone.Conclusions: The increased DNA damage induced in p53-deficient HT1080 cells suggests an early deficiency in the repair of DNA damage during the treatment period. However, the similarity in cellular sensitivity, irrespective of p53 status, suggests that reduced p53 leads to a concomitant reduction in p53-dependent cytotoxicity despite the persistence of DNA damage. The results may provide insight into how tumours that differ in p53 status respond to therapeutic radionuclides. (C) 2013 Elsevier Inc. All rights reserved.

Hypoxia occurs frequently in cancers and can lead to therapeutic resistance due to poor perfusion and loss of the oxygen enhancement effect. 64Cu-ATSM has shown promise as a hypoxia diagnostic agent due to its selective uptake and retention in hypoxic cells and its emission of positrons for PET imaging. 64Cu also emits radiotoxic Auger electrons and β- particles and may therefore exhibit therapeutic potential when concentrated in hypoxic tissue.

Cisplatin is an extremely effective chemotherapeutic agent used for the treatment of testicular and other solid tumours. It induces a variety of structural modifications in DNA, the most abundant being the GpG- and ApG-1,2-intrastrand cross links formed between adjacent purine bases. These cross links account for approximately 90% of cisplatin-induced DNA damage and are thought to be responsible for the cytotoxic activity of the drug. In human cells, the nucleotide excision repair (NER) process removes the intrastrand cross links from the genome, the efficiency of which is likely to be an important determinant of cisplatin cytotoxicity. We have investigated whether the p53 tumour suppressor status affects global NER of cisplatin-induced intrastrand cross links in human cells. We have used a (32)P-postlabelling method to monitor the removal of GpG- and ApG-intrastrand cross links from two human cell models (the 041TR system, in which p53 is regulated by a tetracycline-inducible promoter, together with WI38 fibroblasts and the SV40-transformed derivative VA13) that each differ in p53 status. We demonstrate that the absence of functional p53 leads to persistence of both cisplatin-induced intrastrand cross links in the genome, suggesting that p53 regulates NER of these DNA lesions. This observation extends the role of p53 in NER beyond enhancing the removal of environmentally induced DNA lesions to include those of clinical origin. Given the frequency of p53 mutations in human tumours, these results may have implications for the use of cisplatin in cancer chemotherapy.

The p53 tumour suppressor protein plays a pivotal role in the response of mammalian cells to DNA damage. In addition to its regulatory role in cell cycle progression, p53 regulates apoptosis and can therefore influence cellular survival in response to DNA damage. More recent work has revealed that p53 is also involved in the nucleotide excision repair (NER) of structurally diverse types of DNA damage. The relative influence of p53 on NER and cellular sensitivity to DNA damage was investigated in this study using cells that differ in p53 status. Two cell models were selected: 041 TR fibroblasts in which the expression of p53 is regulated by a tetracycline-inducible promoter, and WI38 primary lung fibroblasts together with their isogenic derivative VA13, in which p53 is abrogated post-translationally by SV40 transformation. Cells were exposed to the clinically and environmentally relevant DNA-damaging agents cisplatin (0-5 microM, 2 h), (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (0-0.5 microM, 30 min) and UV-C (0-5 J/m2), each of which induce structurally distinct types of DNA damage known to be subject to p53-dependent NER. Sensitivity of the p53-proficient and p53-deficient cells to this DNA damage was then compared at each dose of DNA-damaging agent using the clonogenic survival assay and the colorimetric MTT assay. p53-proficient cells were more sensitive than p53-deficient cells to cisplatin, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and UV-C; these differences in cellular sensitivity were more apparent in the 041 TR cells (up to 3.6-, 5.8- and 1.9-fold, respectively) than the WI38/VA13 cells (up to 2.3-, 1.4- and 1.4-fold, respectively). Thus, despite the well-documented persistence of DNA damage in p53-deficient fibroblasts due to impaired NER, loss of p53 results in reduced DNA damage-mediated cytotoxicity.

The most versatile cellular pathway for dealing with a large variety of structurally-unrelated DNA alterations is nucleotide excision repair (NER). Most genomic damage, if not repaired, may contribute to mutagenesis and carcinogenesis, as well as to cellular lethality. There are two subpathways of NER, termed global genomic repair (GGR) and transcription-coupled repair (TCR); While GGR deals with all repairable lesions throughout the genome, TCR is selective for the transcribed DNA strand in expressed genes. Proteins involved in the initial recognition of lesions for GGR as well as for TCR (i.e. RNA polymerase) may sometimes initiate gratuitous repair events in undamaged DNA. However, the damage recognition enzymes for GGR are normally maintained at very low levels unless the cells are genomically stressed. Following UV irradiation in human fibroblasts the efficiency of GGR is upregulated through activation of the p53 tumor suppressor gene. The transactivation role of p53 includes control of expression of the genes, XPC and XPE, which are implicated in GGR but not TCR. These inducible responses are essential for the efficient repair of the most prominent lesion produced by UV, the cyclobutane pyrimidine dimer (CPD). They are also clinically relevant, as we have shown them to operate upon chemical carcinogen DNA damage at levels to which humans are environmentally exposed (e.g. through smoking). Thus, for benzo(a)pyrene (at 10–50 adducts per 108 nucleotides) repair was essentially complete within 1 day in p53+/+ human fibroblasts while no repair was detected within 3 days in p53−/− cells. The levels of all four DNA adducts formed by benzo(g)chrysene, also exhibited p53–dependent control in human fibroblasts. However, unlike humans most rodent tissues are deficient in the p53-dependent GGR pathway. Since rodents are used as surrogates for humans in environmental cancer risk assessment it is very important that we determine how they differ from humans with respect to DNA repair and oncogenic responses to environmental genotoxins.

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.

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 M ( )-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to 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 M resulted in a level of 1100 total adducts/10 nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 M, the maximum level of total adducts at 24 h was 150/10 nucleotides in p53-proficient cells and 210 adducts/10 nucleotides in p53-deficient cells. A concentration of 0.01 M resulted in a maximum of 20 adducts/10 nucleotides in p53-proficient cells at 4 h, but 40 adducts/10 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 M or 0.01 M, 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 nucleotides) because of environmental factors such as smoking is particularly significant with respect to human carcinogenesis related to environmental exposure.

Thymine glycols are formed in DNA by exposure to ionizing radiation or oxidative stress. Although these lesions are repaired by the base excision repair pathway, they have been shown also to be subject to transcription-coupled repair. A current model for transcription-coupled repair proposes that RNA polymerase II arrested at a DNA lesion provides a signal for recruitment of the repair enzymes to the lesion site. Here we report the effect of thymine glycol on transcription elongation by T7 RNA polymerase and RNA polymerase II from rat liver. DNA substrates containing a single thymine glycol located either in the transcribed or nontranscribed strand were used to carry out in vitro transcription. We found that thymine glycol in the transcribed strand blocked transcription elongation by T7 RNA polymerase ∼50% of the time but did not block RNA polymerase II. Thymine glycol in the nontranscribed strand did not affect transcription by either polymerase. These results suggest that arrest of RNA polymerase elongation by thymine glycol is not necessary for transcription-coupled repair of this lesion. Additional factors that recognize and bind thymine glycol in DNA may be required to ensure RNA polymerase arrest and the initiation of transcription-coupled repairin vivo.

The global genomic repair of DNA adducts formed by the human carcinogen (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) has been studied by 32P-postlabeling in human fibroblasts in which p53 expression can be regulated. At low BPDE adduct levels (10-50 adducts/10(8) nucleotides), repair was rapid and essentially complete within 24 h in p53+ cells, whereas no repair was detected within 72 h in similarly treated p53- cells. At 10-fold higher BPDE adduct levels, repair under both conditions was rapid up to 8 h, after which a low level of adducts persisted only in p53- cells. These results demonstrate a dependence on p53 for the efficient repair of BPDE adducts at levels that are relevant to human environmental exposure and, thus, have significant implications for human carcinogenesis.

The global genomic repair of DNA adducts formed by the human carcinogen (6)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) has been studied byP-postlabeling in human fibroblasts in which p53 expression can be regulated. At low BPDE adduct levels (10–50 adducts/10 8 nucleotides), repair was rapid and essentially complete within 24 h in p53 cells, whereas no repair was detected within 72 h in similarly treated p53 cells. At 10-fold higher BPDE adduct levels, repair under both conditions was rapid up to 8 h, after which a low level of adducts persisted only in p53 cells. These results demonstrate a dependence on p53 for the efficient repair of BPDE adducts at levels that are relevant to human environmental exposure and, thus, have significant implications for human carcinogenesis.

In the thirty-third Cold Spring Harbor Symposium, mycolleagues and I reported on our demonstration of "Repair replication of DNA in vivo" (Hanawalt et al. 1969).We began with the cautious statement, "It has been postulated that damaged DNA in bacteria and in some othercellular systems is subject to repair by a mechanism thatinvolves the replacement of the defective single-strandregions with a short stretch of oligonucleotide utilizingthe complementary base pairing information in the intactstrand." That symposium was held just a few years following the discovery of excision repair in Escherichiacoli (Boyce and Howard-Flanders 1964; Pettijohn andHanawalt 1964; Setlow and Carrier 1964). At that meeting, we also learned of an endonuclease activity in extracts of T4-bacteriophage-infected E. coli that incisesUV-irradiated DNA (Takagi et al. 1969). The responsibleenzyme, later designated T4 endoV, was to figure prominently in our research in subsequent years as a highly specific and sensitive probe for UV-induced cyclobutanepyrimidine dimers (CPDs) (Ganesan 1973; van Zeelandet al. 1981; Bohr et al. 1985). The fledgeling DNA repairfield received an important stimulus that same year withthe discovery that repair replication is defective in cellsfrom victims of the rare autosomal recessive hereditarydisease, xeroderma pigmentosum (XP), characterized bysevere sensitivity to sunlight and a several thousandfoldincrease in cutaneous cancers (Cleaver 1968). For manyyears thereafter, it has been considered that nucleotideexcision repair (NER) is of importance primarily for dealing with UV-induced DNA photoproducts—a view reinforced by the fact that the incidence of internal cancers inXP patients is not remarkably enhanced...

The role of metal ion–DNA interactions in the Fenton reaction-mediated formation of putative intrastrand cross-links, 8-hydroxydeoxyguanosine (8-OHdG) and single- and double-strand breaks was investigated. Salmon sperm DNA and pBluescript K+ plasmid were incubated with hydrogen peroxide and either copper(II), iron(II), or nickel(II), which differ in both their affinity for DNA and in the spectrum of oxidative DNA damage they induce in Fenton reactions. EDTA was included in these incubations according to two different strategies; the first (strategy 1) in which DNA and metal ions were mixed prior to the addition of EDTA, the second (strategy 2) in which EDTA and metal ions were mixed prior to the addition of DNA. The formation of the putative intrastrand cross-links, monitored by 32P-postlabelling, was not affected by the addition of between 10 μM and 5 mM EDTA to the copper(II) Fenton reaction according to strategy 1. In contrast, the level of cross-links declined significantly upon inclusion of 20 μM EDTA and above when added according to strategy 2. Similarly, formation of these lesions declined in the iron(II) Fenton reaction more dramatically upon addition of 5 mM EDTA when added according to strategy 2 compared to strategy 1, while the yield of cross-links formed in the nickel(II) Fenton reaction declined equally with both strategies with up to 25 mM EDTA. The formation of single- and double-strand breaks was investigated in plasmid DNA by agarose gel electrophoresis and subsequent densitometry. The formation of linear DNA in the iron(II) Fenton reaction decreased dramatically upon inclusion of EDTA according to strategy 2, while no such decline was observed using strategy 1. In contrast, the formation of linear DNA in the copper(II) Fenton reaction decreased upon inclusion of EDTA according to both strategies. A decrease in the formation of open-circular DNA was also observed upon inclusion of EDTA according to both strategies; however this decrease occurred at a lower EDTA concentration in strategy 2 (100 μM) compared to strategy 1 (200 μM), and the level of open-circular DNA reached a lower level (8.5% compared to 24.2%). The nickel(II) Fenton reaction generated only open-circular DNA, and this was completely inhibited upon addition of 25 μM EDTA according to both strategies. There was less formation of 8-OHdG in the copper(II) and iron(II) Fenton reactions when EDTA was added according to strategy 2 than according to strategy 1. These results suggest that a site-specific mechanism is involved in the formation of double-strand breaks and, to a lesser extent, 8-OHdG and the putative intrastrand cross-links, while the formation of single-strand breaks is more likely to involve generation of hydroxyl radicals in solution.

The formation of 8-hydroxydeoxyguanosine (8-OHdG) and both single- and double-strand breaks in DNA by Fenton-type reactions has been investigated. Salmon sperm DNA was exposed to hydrogen peroxide (50 mM) and one of nine different transition-metal ions (25 microM-1 mM). Modified DNA was isolated and subjected to analysis by liquid chromatography coupled to an electrochemical detection system (LC-ECD), to evaluate the formation of 8-OHdG. The highest yield of 8-OHdG was obtained following treatment of DNA with the chromium(III) Fenton reaction (a maximum of 19 400/10(6) nucleotides), followed by iron(II) (13 600), vanadium(III) (5800), and copper(II) (5200). The chromium(VI) Fenton reaction generated a moderate yield of 8-OHdG (3600/10(6) nucleotides), while the yield obtained in DNA treated with cobalt(II), nickel(II), cadmium(II), and zinc Fenton reactions was not significantly higher than in control incubations of DNA with hydrogen peroxide alone. Similar treatment of the double-stranded plasmid pBluescript K+ with hydrogen peroxide (1 mM) and each transition-metal ion (1-100 microM) followed by quantitative agarose gel electrophoresis demonstrated that open-circle DNA, resulting from single-strand breaks, was generated in Fenton reactions involving all nine metal ions. In contrast, linear DNA was only formed in Fenton reactions involving chromium(III), copper(II), iron(II), and vanadium(III) ions. Formation of linear DNA, under conditions that generated relatively few single-strand breaks, suggests that these four transition-metal ions partake in Fenton reactions to generate true double-strand breaks. Furthermore, the generation of 8-OHdG exhibits a good correlation with the formation of double-strand breaks, suggesting that they arise by a similar mechanism.

Generation of putative intrastrand cross-links and strand breaks was investigated in salmon sperm DNA exposed to Fenton-type oxygen radical-generating systems. 32P-Postlabeling analysis of DNA treated with hydrogen peroxide and either copper(II), chromium(VI), cobalt(II), iron(II), nickel(II), or vanadium(III) resulted in the detection of between four and eight radioactive TLC spots that are probably hydroxyl radical-mediated oxidative DNA lesions. The copper Fenton system generated the highest total yield of these DNA lesions (75.6 per 10(8) nucleotides), followed by cobalt (47.5), nickel (26.2), chromium (25.1), iron (21.7), and vanadium (17.1). Two spots, common to all these Fenton systems, were the major oxidation products in each case. Similar Fenton-type treatment of the purine dinucleotides dApdG and dApdA resulted in products that were chromatographically identical on anion-exchange TLC and on reverse-phase HPLC to the two major products generated in DNA. These results extend our earlier studies suggesting that these products were the result of a free radical-mediated intrastrand cross-linking reaction. Incubations involving cadmium(II), chromium(III), or zinc(II) ions with hydrogen peroxide did not generate DNA oxidation products at levels greater than in incubations with hydrogen peroxide alone. Generation of the putative intrastrand cross-links increased in a concentration-dependent manner up to 1 mM cobalt, nickel, or chromium(VI) ions. However, in experiments with copper, iron, or vanadium ions, maximum levels were obtained at 250, 150, and 150 microM, respectively, and the yield declined with higher concentrations of these three metal ions. Agarose gel electrophoresis demonstrated extensive DNA strand breakage with copper, iron, chromium(III), or vanadium, but not with nickel, chromate(VI), cobalt, cadmium, or zinc Fenton systems. The results demonstrate that generation of the putative intrastrand cross-links and strand breaks in DNA, mediated by Fenton reactions, occurs by independent mechanisms.