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Genomic instability consists of a range of genetic alterations within the genome that contributes to tumor heterogeneity and drug resistance. It is a well-established characteristic of most cancer cells. Genome instability induction results from defects in DNA damage surveillance mechanisms, mitotic checkpoints and DNA repair machinery. Accumulation of genetic alterations ultimately sets cells towards malignant transformation. Recent studies suggest that miRNAs are key players in mediating genome instability. miRNAs are a class of small RNAs expressed in most somatic tissues and are part of the epigenome. Importantly, in many cancers, miRNA expression is dysregulated. Consequently, this review examines the role of miRNA dysregulation as a causal step for induction of genome instability and subsequent carcinogenesis. We focus specifically on mechanistic studies assessing miRNA(s) and specific subtypes of genome instability or known modes of genome instability. In addition, we provide insight on the existing knowledge gaps within the field and possible ways to address them., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Recent reports hypothesize that multiple variant DNA repair gene interactions influence cancer susceptibility. However, studies identifying high-risk cancer-related genes use single gene approaches that lack the statistical rigor to model higher order interactions., Methods: To address this issue, we systematically evaluated individual and joint modifying effects of commonly studied polymorphic base and nucleotide excision repair genes relative to prostate cancer (PCA) risk using conventional logistic regression models and multifactor dimensionality reduction (MDR). We hypothesized that inheriting two or more compromised DNA repair loci may increase PCA risk due to altered gene product function. Six genetic alterations were evaluated using germ-line DNA samples from 208 PCA cases and 665 disease-free controls via TaqMan polymerase chain reaction., Results: With the exception of XPD 312, no association existed between individual DNA repair single-nucleotide polymorphisms (SNPs) and PCA. Individuals with the XPD 312 Asn/Asn genotype had an 8.6-fold increase in risk (OR = 8.59; 95% CI = 1.81-40.66). We did not observe any significant single gene or gene-gene interactions based on MDR modeling., Conclusions: Our findings emphasize the importance of utilizing a combination of traditional and advanced statistical tools to identify and validate single gene and multilocus interactions in relation to cancer susceptibility.

2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). In humans, NAT2*4 allele is associated with rapid acetylator phenotype, whereas NAT2*5B allele is associated with slow acetylator phenotype. We hypothesized that rapid acetylator phenotype predisposes humans to DNA damage and mutagenesis from MeIQx. Nucleotide excision repair-deficient Chinese hamster ovary cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected Chinese hamster ovary cell lines. CYP1A1 activity did not differ significantly (P > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had 20-fold significantly higher levels of sulfamethazine N-acetyltransferase (P = 0.0001) and 6-fold higher levels of N-hydroxy-MeIQx O-acetyltransferase (P = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase mutagenesis following MeIQx treatment. Deoxyguanosine-C8-MeIQx was the primary DNA adduct formed and levels were dose dependent in each cell line and in the following order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 and NAT2*5B < transfected with CYP1A1 and NAT2*4. MeIQx DNA adduct levels were significantly higher (P < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism on MeIQx-induced DNA adducts and mutagenesis. The results provide laboratory-based support for epidemiologic studies reporting higher frequency of heterocyclic amine-related cancers in rapid NAT2 acetylators.

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. UvrABC endonuclease, encoded by the UvrA, UvrB, and UvrC genes can incise DNA containing bulky nucleotide adducts and intrastrand cross-links. UvrA, UvrB, and UvrC were cloned from Bacillus caldotenax (Bca)and UvrC from Thermatoga maritima (Tma), and recombinant proteins were overexpressed in and purified from Escherichia coli. Incision activities of UvrABC composed of all Bca-derived subunits (UvrABC(Bca)) and an interspecies combination UvrABC composed of Bca-derived UvrA and UvrB and Tma-derived UvrC (UvrABC(Tma)) were compared on benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE)-adducted substrates. Both UvrABC(Bca) and UvrABC(Tma) specifically incised both BPDE-adducted plasmid DNAs and site-specifically modified 50-bp oligonucleotides containing a single (+)-trans- or (+)-cis-BPDE adduct. Incision activity was maximal at 55-60 degrees C. However, UvrABC(Tma) was more robust than UvrABC(Bca) with 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [(3)H]BPDE-adducted plasmid DNAs. Remarkably, UvrABC(Bca) incised only at the eighth phosphodiester bond 5' to the BPDE-modified guanosine. In contrast, UvrABC(Tma) performed dual incision, cutting at both the fifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDE adduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates were incised more efficiently than trans adducts by both UvrABC(Bca) and UvrABC(Tma). UvrAB-DNA complex formation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrAB binds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABC(Tma) make it a robust tool for DNA adduct studies.

Functional effects of Arg228Gln and Val2343Leu XPA polymorphisms on benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide-(+/-)-anti (BPDE) survival and repair were investigated in SV40 immortalized XP12RO cells complemented with wild type and polymorphic XPA cDNAs in an inducible cDNA expression system. In contrast to previous studies showing little impact of XPA polymorphisms on UV survival and repair, cells complemented with polymorphic XPAs displayed improved BPDE survival and repair as compared to wild type XPA-complemented cells. Survival after BPDE treatment was measured using AlamarBlue reduction and colony forming ability. Cells expressing low levels of either polymorphic XPA had equivalent or improved survival compared to wild type XPA-complemented cells (XPAwt cells). XPA induction improved BPDE survival in Arg228Gln (R228Q cells) and Val234Leu (V234L cells) complemented cells, but not XPAwt cells. BPDE-induced DNA damage repair was measured both by reactivation after transfection of a luciferase reporter plasmid reacted with BPDE in vitro, and by removal of adducts from genomic DNA of BPDE-treated cells. BPDE-induced DNA damage repair in R228Q and V234L cells expressing XPA at very low levels was similar to repair in XPAwt cells expressing XPA at normal levels. XPA induction improved repair in R228Q and V234L cells but not in XPAwt cells. Our findings suggest that both Arg228Gln and Val234Leu XPAs function better than wild type XPA for BPDE adduct removal. These observations differ from UV repair results suggesting that the differences are lesion specific. The location of the polymorphisms within the putative poly(ADP-ribose) binding domain suggests that poly(ADP-ribose) interaction is important in repair.

Polymorphisms in DNA repair genes may contribute to defects in DNA repair and increased susceptibility to cancer. The xeroderma pigmentosum group A (XPA) gene is required for nucleotide excision repair (NER) and mutations in XPA highly predispose humans to skin cancer. We examined DNA samples from 189 individuals for polymorphisms in the XPA gene. First, SSCP analysis was used to examine each of the six exons and their intron boundaries. One frequent single nucleotide polymorphism (SNP) in the untranslated region of exon 1 and two rare SNPs which produce the changes Arg228Gln and Val234Leu in the coding region of exon 6 were identified. Quite surprisingly, no sequence variants were found within the coding regions or the adjacent intron boundaries of exons 1-5. Ecdysone-inducible expression vectors containing wild type XPA cDNA or cDNAs representing the two polymorphisms that we identified in exon 6 were created and independently introduced into the XPA deficient cell line XP12RO-SV. Transcription-coupled repair (TCR), global genome repair (GGR) and cell survival following UV irradiation were studied in each cell line in the absence or presence of the ecdysone hormone analog, ponasterone A. No substantial difference in repair or cell survival was found in cells complemented with wild type or polymorphic alleles of XPA. A 10-fold increase in the expression of XPA by addition of ponasterone A resulted in faster removal of 6-4 photoproducts from the total genomes of cells complemented with wild type or polymorphic alleles of XPA but had no significant impact on TCR or global genome repair of cyclobutane pyrimidine dimers (CPDs). Since our SSCP analysis failed to detect significant numbers of polymorphisms we directly sequenced exons 4-6 in a subset of our samples. One additional rare SNP, which produces the change Leu252Val was found in exon 6 and four rare SNPs and one rare single nucleotide deletion were found in intron 4. Hence, the XPA gene appears to be a cold spot for genetic variation and rare polymorphisms in the coding region of the gene do not reduce NER or cell survival after UV irradiation., (Copyright 2002 Elsevier Science B.V.)

Cells from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair (TCR), which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS (of which there are two complementation groups, CSA and CSB) do not demonstrate an elevated incidence of cancer. Recently, we demonstrated that disruption of the CSB gene reduces the spontaneous tumor rate in cancer predisposed Ink4a/ARF-/- mice as well as causing their embryo fibroblasts to proliferate more slowly and be more sensitive to UV-induced apoptosis. In the present study we characterized phosphorothioate backbone antisense oligodeoxynucleotides (AOs) that reduced the levels of CSB mRNA in A2780/CP70 ovarian carcinoma cells. The AOs caused the cells to proliferate more slowly and made them more sensitive to either cisplatin or oxaliplatin. The AOs also enhanced the cytotoxicity of hydrogen peroxide and gamma-radiation, both of which can induce oxidative DNA lesions, which are subject to TCR. The AOs did not potentiate the cytotoxicity of topotecan, which induces DNA strand breaks. Chemically modified () AOs (MBOs) targeting CSB were able to potentiate the anti-tumor effect of cisplatin against A2780/CP70 tumor xenografts formed in nude mice. The MBOs enabled a non-toxic (3 mg/kg) dose of cisplatin to have the same degree of anti-tumor efficacy as a more toxic (5 mg/kg) cisplatin dose. Collectively, these results suggest that the CSB gene product may be viewed as an anti-cancer target.

The capacity of human and other eukaryotic cells to recognize a disparate variety of damaged sites in DNA, and selectively excise and repair them, resides in a deceptively small simple protein, a 38-42 kDa zinc-finger binding protein, XPA (xeroderma pigmentosum group A), that has no inherent catalytic properties. One key to its damage-recognition ability resides in a DNA-binding domain which combines a zinc finger and a single-strand binding region which may infiltrate small single-stranded regions caused by helix-destabilizing lesions. Another is the augmentation of its binding capacity by interactions with other single-stranded binding proteins and helicases which co-operate in the binding and are unloaded at the binding site to facilitate further unwinding of the DNA and subsequent catalysis. The properties of these reactions suggest there must be considerable conformational changes in XPA and associated proteins to provide a flexible fit to a wide variety of damaged structures in the DNA.

The ability of an XPA minigene construct to complement the DNA repair defect in xeroderma pigmentosum group A (XP-A) cells was demonstrated. XP-A cells (XP12BE-SV) were stably transformed with an XPA minigene linked to a neomycin resistance (neor) expression cassette. The G418-resistant clone XAN1 was isolated and its DNA repair phenotype compared with XP12BE-SV cells transformed with a cosmid containing a human chromosome 8 gene and a neo(r) cassette and selected for G418 resistance (2-0-A2), DNA repair-normal human fibroblasts and untransfected XP12BE-SV cells. Colony forming ability after UV-irradiated reactivation of a UV-irradiated chloramphenicol acetyltransferase (CAT) expression vector and UV-induced mutagenesis in a supF tRNA shuttle vector (pSP189) were all restored to normal levels in XAN1 cells. In addition, mutation spectra in the supF gene of pSP189 after replication in all four cell lines were compiled at low (100 J/m2) and high (1000 J/m2) UV doses. The majority of mutations were point mutations and these were predominately G:C-->A:T transitions regardless of dose for all cell lines. Dose-dependent differences were observed in the positions of mutation hot spots in pSP189 mutation spectra after replication in all four cell lines. Mutation spectra for XAN1 and GM0637 cells had only minor differences. An increase in the proportion of transversions was observed only in plasmids irradiated with a low UV dose and replicated in XAN1 cells. 2-0-A2 cells were reported to have partial restoration of DNA repair that was later suggested to be caused by a reversion. 2-0-A2 cells were nearly identical to XP12BE-SV cells in all aspects investigated, indicating that transformation to neor had no effect on DNA repair in these cells.

Cytochromes P450 catalyze the bioactivation of many carcinogens. In particular, cytochrome P450 1A1 (CYP1A1) catalyzes the conversion of polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, into potent mutagenic agents. Human skin fibroblasts, both DNA repair deficient (xeroderma pigmentosum group A: XPA) and DNA repair normal have been co-transformed with a chimeric gene construct containing human CYP1A1 coding sequences controlled by the cadmium (Cd) ion inducible mouse metallothionein-I promoter and pRSV-NEO, a dominant selectable marker for G418 resistance. Individual G418 resistant colonies were cloned and analyzed for Cd inducible CYP1A1 activity. Six clones of DNA repair deficient cells and five clones of DNA repair proficient cells have been isolated which express Cd inducible CYP1A1. Benzo[a]pyrene-trans-7,8-diol (BPD) is cytotoxic in Cd induced CYP1A1 expressing cells. The cytotoxicity can be inhibited by 10 microM alpha-napthoflavone. Differential cytotoxicity between the DNA repair deficient and proficient CYP1A1 expressing transformants is observed. BPD is cytotoxic to Cd induced CYP1A1 expressing XPA cells at > 10-fold lower doses than it is to Cd induced CYP1A1 expressing DNA repair normal cells. These data indicate that BPD is metabolized to a DNA damaging agent by induced CYP1A1. In contrast, benzo[a]pyrene-trans-7,8-diol-9,10-epoxide added to the media is only slightly more cytotoxic to DNA repair deficient than to proficient cells regardless of CYP1A1 expression. These studies demonstrate the usefulness of the CYP1A1 transformed fibroblasts in examining the cytotoxic effects of benzo[a]pyrene metabolites and suggest the future usefulness in examining the toxic effects of polycyclic aromatic hydrocarbons and other xenobiotics bioactivated by CYP1A1.