Mariarosaria D’Errico

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.

An altered redox balance mediates the hypersensitivity of Cockayne syndrome primary fibroblasts to oxidative stress.

Cockayne syndrome (CS) is a rare hereditary multisystem disease characterized by neurological and development impairment, and premature aging. Cockayne syndrome cells are hypersensitive to oxidative stress, but the molecular mechanisms involved remain unresolved. Here we provide the first evidence that primary fibroblasts derived from patients with CS‐A and CS‐B present an altered redox balance with increased steady‐state levels of intracellular reactive oxygen species (ROS) and basal and induced DNA oxidative damage, loss of the mitochondrial membrane potential, and a significant decrease in the rate of basal oxidative phosphorylation. The Na/K‐ATPase, a relevant target of oxidative stress, is also affected with reduced transcription in CS fibroblasts and normal protein levels restored upon complementation with wild‐type genes. High‐resolution magnetic resonance spectroscopy revealed a significantly perturbed metabolic profile in CS‐A and CS‐B primary fibroblasts compared with normal cells in agreement with increased oxidative stress and alterations in cell bioenergetics. The affected processes include oxidative metabolism, glycolysis, choline phospholipid metabolism, and osmoregulation. The alterations in intracellular ROS content, oxidative DNA damage, and metabolic profile were partially rescued by the addition of an antioxidant in the culture medium suggesting that the continuous oxidative stress that characterizes CS cells plays a causative role in the underlying pathophysiology. The changes of oxidative and energy metabolism offer a clue for the clinical features of patients with CS and provide novel tools valuable for both diagnosis and therapy.

Role of nucleotide excision repair proteins in oxidative DNA damage repair: an updating

DNA repair is a crucial factor in maintaining a low steady-state level of oxidative DNA damage. Base excision repair (BER) has an important role in preventing the deleterious effects of oxidative DNA damage, but recent evidence points to the involvement of several repair pathways in this process. Oxidative damage may arise from endogenous and exogenous sources and may target nuclear and mitochondrial DNA as well as RNA and proteins. The importance of preventing mutations associated with oxidative damage is shown by a direct association between defects in BER (i.e. MYH DNA glycosylase) and colorectal cancer, but it is becoming increasingly evident that damage by highly reactive oxygen species plays also central roles in aging and neurodegeneration. Mutations in genes of the nucleotide excision repair (NER) pathway are associated with diseases, such as xeroderma pigmentosum and Cockayne syndrome, that involve increased skin cancer risk and/or developmental and neurological symptoms. In this review we will provide an updating of the current evidence on the involvement of NER factors in the control of oxidative DNA damage and will attempt to address the issue of whether this unexpected role may unlock the difficult puzzle of the pathogenesis of these syndromes.

Gene susceptibility to oxidative damage: From single nucleotide polymorphisms to function

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.

The cross talk between pathways in the repair of 8-oxo-7,8-dihydroguanine in mouse and human cells

Although oxidatively damaged DNA is repaired primarily via the base excision repair (BER) pathway, it is now evident that multiple subpathways are needed. Yet, their relative contributions and coordination are still unclear. Here, mouse embryo fibroblasts (MEFs) from selected nucleotide excision repair (NER) and/or BER mouse mutants with severe (Csb(m/m)/Xpa(-/-) and Csb(m/m)/Xpc(-/-)), mild (Csb(m/m)), or no progeria (Xpa(-/-), Xpc(-/-), Ogg1(-/-), Csb(m/m)/Ogg1(-/-)) or wild-type phenotype were exposed to an oxidizing agent, potassium bromate, and genomic 8-oxo-7,8-dihydroguanine (8-oxoGua) levels were measured by HPLC-ED. The same oxidized DNA base was measured in NER/BER-defective human cell lines obtained after transfection with replicative plasmids encoding siRNA targeting DNA repair genes. We show that both BER and NER factors contribute to the repair of 8-oxoGua, although to different extents, and that the repair profiles are similar in human compared to mouse cells. The BER DNA glycosylase OGG1 dominates 8-oxoGua repair, whereas NER (XPC, XPA) and transcription-coupled repair proteins (CSB and CSA) are similar, but minor contributors. The comparison of DNA oxidation levels in double versus single defective MEFs indicates increased oxidatively damaged DNA only when both CSB and XPC/XPA are defective, indicating that these proteins operate in different pathways. Moreover, we provide the first evidence of an involvement of XPA in the control of oxidatively damaged DNA in human primary cells.

The role of CSA and CSB protein in the oxidative stress response

Cockayne syndrome (CS) is a rare hereditary disorder in which infants suffer severe developmental and neurological alterations and early death. Two genes encoding RNA polymerase II cofactors, CSA and CSB, are mutated in this syndrome. CSA and CSB proteins are known to be involved in the transcription-coupled DNA repair pathway but the sensitivity of mutant cells to a number of physical/chemical agents besides UV radiation, such as ionizing radiation, hydrogen peroxide and bioenergetic inhibitors indicate that these proteins play a pivotal role in additional pathways. In this review we will discuss the evidence that implicate CS proteins in the control of oxidative stress response with special emphasis on recent findings that show an altered redox balance and dysfunctional mitochondria in cells derived from patients. Working models of how these new functions might be key to developmental and neurological disease in CS will be discussed.

Overexpression of parkin rescues the defective mitochondrial phenotype and the increased apoptosis of Cockayne Syndrome A cells

The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated, CSA causes the Cockayne Syndrome group A (CS-A), a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells features include ROS hyperproduction, accumulation of oxidative genome damage, mitochondrial dysfunction and increased apoptosis that may contribute to the neurodegenerative process. In this study, we show that CSA localizes to mitochondria and specifically interacts with the mitochondrial fission protein dynamin-related protein (DRP1) that is hyperactivated when CSA is defective. Increased fission is not counterbalanced by increased mitophagy in CS-A cells thus leading to accumulation of fragmented mitochondria. However, when mitochondria are challenged with the mitochondrial toxin carbonyl cyanide m-chloro phenyl hydrazine, CS-A fibroblasts undergo mitophagy as efficiently as normal fibroblasts, suggesting that this process remains targetable to get rid of damaged mitochondria. Indeed, when basal mitophagy was potentiated by overexpressing Parkin in CSA deficient cells, a significant rescue of the dysfunctional mitochondrial phenotype was observed. Importantly, Parkin overexpression not only reactivates basal mitophagy, but plays also an anti-apoptotic role by significantly reducing the translocation of Bax at mitochondria in CS-A cells. These findings provide new mechanistic insights into the role of CSA in mitochondrial maintenance and might open new perspectives for therapeutic approaches.

The Molecular Basis of Skin Carcinogenesis

The skin is constituted by two different tissues: the epidermis, which is composed mostly of squamous epithelial cells (called keratinocytes), and the underlying dermis, which is composed predominantly of dermal fibroblasts. The epidermis is organized in four distinct cell layers. The innermost basal layer consists of actively proliferating keratinocytes, which are characterized by a relatively dispersed network of keratin filaments, primarily keratin 5 (K5) and K14 (1,2). As basal keratinocytes migrate toward the skin surface, proliferation ceases and terminal differentiation begins. Cells of the suprabasal spinous layer are metabolically active and express high amounts of two differentiation-specific keratins, K1 and K10 (1,3). They also produce glutamine and lysine-rich envelope proteins, such as involucrin, which are crosslinked by epidermal transglutaminase and deposited at the inner surface of the cellular membrane, to form the cornified envelope (4,5). In the overlying granular layer, cells contain numerous lipid-containing granules, that are released in the intercellular space. Keratinocytes at this step synthesize filaggrin and loricrin, which also contribute to the cornified envelope formation (6,7). The outermost layer of the epidermis is the cornified layer. Here cells are metabolically inactive, lack a nucleus and cytoplasmic organelles, and are full of keratin filaments.

Characterization of DNA polymerase β splicing variants in gastric cancer: the most frequent exon 2-deleted isoform is a non coding RNA

DNA repair polymerase beta (Pol beta) gene variants are frequently associated with tumor tissues. In this study a search for Pol beta mutants and splice variants was conducted in matched normal and tumor gastric tissues and blood samples from healthy donors. No tumor associated mutations were found while a variety of alternative Pol beta splicing variants were detected with high frequency in all the specimens analysed. Quantitative PCR of the Pol beta variant lacking exon 2 (Ex2Delta) and the isoforms with exon 11 skipping allowed to clarify that these variants are not tumor- neither tissue-specific and their levels vary greatly among different individuals. The most frequent Ex2Delta variant was further characterized. We clearly demonstrated that this variant does not encode protein, as detected by both western blotting and immunofluorescence analysis of human AGS cells expressing HA-tagged Ex2Delta. The lack of translation was confirmed by comparing the DNA gap-filling capacity and alkylation sensitivity of wild type and Pol beta null murine fibroblasts expressing the human Ex2Delta variant. We showed that the Ex2Delta transcript is polyadenylated and its half-life is significantly longer than that of the wild type mRNA as inferred by treating AGS cells with actinomycin D. Moreover, we found that it localizes to polyribosomes suggesting a role as post-transcriptional regulator. This study identifies a new type of DNA repair variants that do not give rise to functional proteins but to non-coding RNAs that could either modulate target mRNAs or represent unproductive splicing events.

MIR-200a Modulates the Expression of the DNA Repair Protein OGG1 Playing a Role in Aging of Primary Human Keratinocytes

Oxidative DNA damage accumulation may induce cellular senescence. Notably, senescent cells accumulate in aged tissues and are present at the sites of age-related pathologies. Although the signaling of DNA strand breaks has been extensively studied, the role of oxidative base lesions has not fully investigated in primary human keratinocyte aging. In this study, we show that primary human keratinocytes from elderly donors are characterized by a significant accumulation of the oxidative base lesion 8-OH-dG, impairment of oxidative DNA repair, and increase of miR-200a levels. Notably, OGG1-2a, a critical enzyme for 8-OH-dG repair, is a direct target of miR-200a and its expression levels significantly decrease in aged keratinocytes. The 8-OH-dG accumulation displays a significant linear relationship with the aging biomarker p16 expression during keratinocyte senescence. Interestingly, we found that miR-200a overexpression down-modulates its putative target Bmi-1, a well-known p16 repressor, and up-regulates p16 itself. miR-200a overexpression also up-regulates the NLRP3 inflammasome and IL-1β expression. Of note, primary keratinocytes from elderly donors are characterized by NRPL3 activation and IL-1β secretion. These findings point to miR-200a as key player in primary human keratinocyte aging since it is able to reduce oxidative DNA repair activity and may induce several senescence features through p16 and IL-1β up-regulation.