Eugenia Dogliotti

Microsatellite instability as a marker of prognosis and response to therapy: A meta-analysis of colorectal cancer survival data

BACKGROUND AND METHODS We have reviewed and pooled data from published studies to evaluate the relationship between microsatellite instability (MSI) and colorectal cancer (CRC) prognosis. Thirty-one eligible studies reporting survival in 12782 patients characterised for MSI were pooled using a fixed- or random-effects model. RESULTS The summary odds ratio (OR) estimate for overall survival (OS) associated with MSI was 0.6 (95%CI 0.53-0.69, p<0.0001), with no evidence of heterogeneity. The effect was similar for disease-free survival (DFS) (OR=0.58, 95%CI 0.47-0.72, p<0.0001). In a subset of patients treated with 5-fluorouracil (5-FU)-based chemotherapy a significant improved prognosis was found for microsatellite stable (MSS) tumours (OR=0.52, 95%CI 0.4-0.6, p<0.0001) with no heterogeneity (p=0.53; I(2)=0%). By contrast a large heterogeneity characterised the data relative to 396 patients with MSI tumours (OR=0.69, 95%CI 0.3-1.5, p=0.1; heterogeneity: p=0.03; I(2)=58%). CONCLUSIONS This study confirmed the association between MSI and favourable prognosis as determined by both OS and DFS of CRC patients. A significant beneficial effect of 5-FU therapy was found for MSS tumours whilst no clear conclusion was reached for MSI tumours due to the high inter-study heterogeneity. We propose that this inconclusive result is due to the use of a single marker, such as MSI, that cannot account alone for the complexity of the mechanisms underlying 5-FU cytotoxicity. Future studies to predict response to 5-FU chemotherapy should include additional genome stability markers.

New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA‐distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP‐C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA‐oxidizing agents and this effect is reverted by expression of wild‐type XPC. Upon oxidant exposure, XP‐C primary keratinocytes and fibroblasts accumulate 8,5′‐cyclopurine 2′‐deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8‐hydroxyguanine (8‐OH‐Gua) is also observed. We demonstrate that XPC–HR23B complex acts as cofactor in base excision repair of 8‐OH‐Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC‐HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP‐C patients.

The Mammalian Mismatch Repair Pathway Removes DNA 8-oxodGMP Incorporated from the Oxidized dNTP Pool

Mismatch repair (MMR) corrects replication errors. It requires the MSH2, MSH6, MLH1, and PMS2 proteins which comprise the MutSalpha and MutLalpha heterodimers. Inactivation of MSH2 or MLH1 in human tumors greatly increases spontaneous mutation rates. Oxidation produces many detrimental DNA alterations against which cells deploy multiple protective strategies. The Ogg-1 DNA glycosylase initiates base excision repair (BER) of 8-oxoguanine (8-oxoG) from 8-oxoG:C pairs. The Myh DNA glycosylase removes mismatched adenines incorporated opposite 8-oxoG during replication. Subsequent BER generates 8-oxoG:C pairs, a substrate for excision by Ogg-1. MTH1-an 8-oxodGTPase which eliminates 8-oxodGTP from the dNTP pool-affords additional protection by minimizing 8-oxodGMP incorporation during replication. Here we show that the dNTP pool is, nevertheless, an important source of DNA 8-oxoG and that MMR provides supplementary protection by excising incorporated 8-oxodGMP. Incorporated 8-oxodGMP contributes significantly to the mutator phenotype of MMR-deficient cells. Thus, although BER of 8-oxoG is independent of Msh2, both steady-state and H(2)O(2)-induced DNA 8-oxoG levels are higher in Msh2-defective cells than in their repair-proficient counterparts. Increased expression of MTH1 in MMR-defective cells significantly reduces steady-state and H(2)O(2)-induced DNA 8-oxoG levels. This reduction dramatically diminishes the spontaneous mutation rate of Msh2(-/-) MEFs.

Mammalian base excision repair by DNA polymerases δ and ε

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase  β (Pol  β )-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol β-deleted mouse fibroblasts to separate subfractions containing either Pol δ or Pol ε. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol δ and Pol ε were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol β. Furthermore, our observations indicated, that either Pol δ and/or Pol ε participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol δ and/or Pol ε are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol β in one-gap filling reactions.

Long Patch Base Excision Repair with Purified Human Proteins DNA LIGASE I AS PATCH SIZE MEDIATOR FOR DNA POLYMERASES δ AND ε

Among the different base excision repair pathways known, the long patch base excision repair of apurinic/apyrimidinic sites is an important mechanism that requires proliferating cell nuclear antigen. We have reconstituted this pathway using purified human proteins. Our data indicated that efficient repair is dependent on six components including AP endonuclease, replication factor C, proliferating cell nuclear antigen, DNA polymerases δ or ε, flap endonuclease 1, and DNA ligase I. Fine mapping of the nucleotide replacement events showed that repair patches extended up to a maximum of 10 nucleotides 3′ to the lesion. However, almost 70% of the repair synthesis was confined to 2–4-nucleotide patches and DNA ligase I appeared to be responsible for limiting the repair patch length. Moreover, both proliferating cell nuclear antigen and flap endonuclease 1 are required for the production and ligation of long patch repair intermediates suggesting an important role of this complex in both excision and resynthesis steps.

Accumulation of the Oxidative Base Lesion 8-Hydroxyguanine in DNA of Tumor-Prone Mice Defective in Both the Myh and Ogg1 DNA Glycosylases

The OGG1 and MYH DNA glycosylases prevent the accumulation of DNA 8-hydroxyguanine. In Myh−/− mice, there was no time-dependent accumulation of DNA 8-hydroxyguanine in brain, small intestine, lung, spleen, or kidney. Liver was an exception to this general pattern. Inactivation of both MYH and OGG1 caused an age-associated accumulation of DNA 8-hydroxyguanine in lung and small intestine. The effects of abrogated OGG1 and MYH on hepatic DNA 8-hydroxyguanine levels were additive. Because there is an increased incidence of lung and small intestine cancer in Myh−/−/Ogg1−/− mice, these findings support a causal role for unrepaired oxidized DNA bases in cancer development.

Genome-wide expression profile of sporadic gastric cancers with microsatellite instability

Gastric cancers with mismatch repair (MMR) inactivation are characterised by microsatellite instability (MSI). In this study, the transcriptional profile of 38 gastric cancers with and without MSI was analysed. Unsupervised analysis showed that the immune and apoptotic gene networks efficiently discriminated these two cancer types. Hierarchical clustering analysis revealed numerous gene expression changes associated with the MSI phenotype. Amongst these, the p53-responsive genes maspin and 14-3-3 sigma were significantly more expressed in tumours with than without MSI. A tight immunosurveillance coupled with a functional p53 gene response is consistent with the better prognosis of MSI cancers. Frequent silencing of MLH1 and downregulation of MMR target genes, such as MRE11 and MBD4, characterised MSI tumours. The downregulation of SMUG1 was also a typical feature of these tumours. The DNA repair gene expression profile of gastric cancer with MSI is of relevance for therapy response.

The role of CSA in the response to oxidative DNA damage in human cells

Cockayne syndrome (CS) is a rare genetic disease characterized by severe growth, mental retardation and pronounced cachexia. CS is most frequently due to mutations in either of two genes, CSB and CSA. Evidence for a role of CSB protein in the repair of oxidative DNA damage has been provided recently. Here, we show that CSA is also involved in the response to oxidative stress. CS-A human primary fibroblasts and keratinocytes showed hypersensitivity to potassium bromate, a specific inducer of oxidative damage. This was associated with inefficient repair of oxidatively induced DNA lesions, namely 8-hydroxyguanine (8-OH-Gua) and (5′S)-8,5′-cyclo 2′-deoxyadenosine. Expression of the wild-type CSA in the CS-A cell line CS3BE significantly decreased the steady-state level of 8-OH-Gua and increased its repair rate following oxidant treatment. CS-A cell extracts showed normal 8-OH-Gua cleavage activity in an in vitro assay, whereas CS-B cell extracts were confirmed to be defective. Our data provide the first in vivo evidence that CSA protein contributes to prevent accumulation of various oxidized DNA bases and underline specific functions of CSB not shared with CSA. These findings support the hypothesis that defective repair of oxidative DNA damage is involved in the clinical features of CS patients.

Polymorphic DNA repair and metabolic genes: a multigenic study on gastric cancer

Risk factors for gastric cancer (GC) include inter-individual variability in the inflammatory response to Helicobacter pylori infection, in the ability of detoxifying DNA reactive species and repairing DNA damage generated by oxidative stress and dietary carcinogens. To evaluate the association between polymorphic DNA repair genes and GC risk, a case-control study including 314 histologically confirmed GC patients and 548 healthy controls was conducted in a GC high-risk area in Tuscany, Italy. Polymorphic variants of base excision repair (APE1-D148E, XRCC1-R194W, XRCC1-R399Q and OGG1-S326C), nucleotide excision repair (XPC-PAT, XPA-23G>A, ERCC1-19007T>C and XPD-L751Q), recombination (XRCC3-T241M) and alkylation damage reversal (MGMT-L84F) were tested for their potential role in the development of GC by using logistic regression models. The same population was also characterised for GSTT1 and GSTM1 variant alleles to search for possible functional interactions between metabolic and DNA repair genotypes by two-way interactions using multivariate logistic models. No significant association between any single DNA repair genotype and GC risk was detected with a borderline association with the XPC-PAT homozygous genotype [odds ratio (OR) =1.42; 95% confidence interval (CI) 0.94-2.17]. Gene-gene interaction analysis revealed combinations of unfavourable genotypes involving either multiple DNA repair polymorphisms or DNA repair and GST-specific genotypes. The combination of the XPC-PAT and the XPA variant alleles significantly increased GC risk (OR=2.15; 95% CI 1.17-3.93, P=0.0092). A significant interaction was also found between the APE1 wild-type genotype and either the single GSTT1 (OR=4.90; 95% CI 2.38-10.11, P=0.0079) or double GSTM1-GSTT1 null (OR=7.84; 95% CI 3.19-19.22, P=0.0169) genotypes or the XPA-mutant allele (OR=3.56; 95% CI 1.53-8.25, P=0.0012). These findings indicate that a complex interaction between host factors such as oxidative stress, antioxidant capacity and efficiency of multiple DNA repair pathways underlies the inter-individual variability in GC risk.

Human base excision repair complex is physically associated to DNA replication and cell cycle regulatory proteins

It has been hypothesized that a replication associated repair pathway operates on base damage and single strand breaks (SSB) at replication forks. In this study, we present the isolation from the nuclei of human cycling cells of a multiprotein complex containing most of the essential components of base excision repair (BER)/SSBR, including APE1, UNG2, XRCC1 and POLβ, DNA PK, replicative POLα, δ and ɛ, DNA ligase 1 and cell cycle regulatory protein cyclin A. Co-immunoprecipitation revealed that in this complex DNA repair proteins are physically associated to cyclin A and to DNA replication proteins including MCM7. This complex is endowed with DNA polymerase and protein kinase activity and is able to perform BER of uracil and AP sites. This finding suggests that a preassembled DNA repair machinery is constitutively active in cycling cells and is ready to be recruited at base damage and breaks occurring at replication forks.