Angelo Calcagnile

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.

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.

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.

UV mutation signature in tumor suppressor genes involved in skin carcinogenesis in xeroderma pigmentosum patients

Molecular analysis of p53 and patched (PTCH), two candidate tumor suppressor genes for non-melanocytic skin cancer, was performed in skin tumors from six patients affected by the cancer-prone disease xeroderma pigmentosum (XP). UV-specific p53 mutations were detected at a frequency of 38–50% in all the tumor types analysed, including melanomas. Additional analysis of PTCH mutations in the subset of eight basal call carcinomas (BCC) revealed a very high mutation frequency of this gene (90%) which exceeded that detected in the p53 gene in the same tumors (38%). PTCH mutations were predominantly UV-specific C>T transitions. This mutation pattern is different from that reported in BCC from normal donors where PTCH mutation frequency is 27% and mutations are frequently deletions and insertions. These findings suggest that PTCH mutations represent an earlier event in BCC development than p53 alterations and that the inability of XP patients to repair UV-induced PTCH mutations might significantly contribute to the early and frequent appearance of BCC observed in these patients.

Apoptosis and efficient repair of DNA damage protect human keratinocytes against UVB.

Since the eighties it is well known that keratinocytes are more resistant to the lethal effects of UV light than fibroblasts, but the mechanism behind this phenomenon is still unknown. In the present study we investigated cell survival, apoptosis, cell cycle progression, UV photoproduct induction and repair, p53 gene response following UVB exposure in primary cultures of keratinocytes, and we compared the response with that of primary fibroblasts from the same skin biopsy. Keratinocytes are the primary target for UVB-induced human cutaneous malignancies, thus epidermal cells might have specific strategies to mantain genomic integrity. Nucleotide excision repair (NER) is a major defense mechanism against the deleterious effects of pyrimidine dimers (CPD) and 6-4 photoproducts (6-4 PP), the most biologically relevant damage induced by UV into DNA. The NER system has two distinct subpathways: global genome repair (GGR) that repairs lesions throughout the genome, and transcriptioncoupled repair (TCR) that operates on lesions in the transcribed strand of active genes (reviewed in Balajee and Bohr). To efficiently repair damage, cells transiently arrest their growth at different points of the cell cycle (reviewed in Bartek and Lukas). To limit the survival in the presence of irreparable DNA damage, cells die by apoptosis (reviewed in Kulms and Schwarz). This phenomenon is evident in skin with the appearance of sunburn cells. In keratinocytes, UV-induced apoptosis is p53-dependent. The colony-forming ability of primary keratinocytes and fibroblasts from two independent skin biopsies was measured after UVB exposure (Figure 1a). Keratinocytes were more resistant to the lethal effects of UVB than the fibroblasts (D37 of 1000 J/m and 500 J/m for keratinocytes and fibroblasts, respectively). Cell death can occur via different mechanisms including apoptosis. Apoptosis was measured by TUNEL assay at different times after cell exposure to 1000 J/m of UVB (Figure 1b). The number of apoptotic keratinocytes increased significantly at 24 and 72 h after UVB exposure whereas at the same dose fibroblasts were completely refractory to apoptosis. The activation of an apoptotic response by UVB in keratinocytes was confirmed by fluorimetric detection of caspase-3 (data not shown). Therefore, keratinocytes although more resistant to the lethal effects of UVB are more susceptible to UVB-induced apoptosis than fibroblasts. The differential sensitivity to UVB of keratinocytes and fibroblasts might be because of differences in the level or repair of DNA damage in the two cell types. Fibroblasts and keratinocytes were exposed to 1000 J/m of UVB and the amount of CPD and 6-4 PP was determined on the extracted DNA by ELISA using the specific antibodies. The yield of both DNA lesions was approximately 1.5-fold higher in fibroblasts than in keratinocytes but the ratio of CPD to 6-4 PP was similar in the two cell types (data not shown). In general, the loss of 6-4 PP was more rapid than that of CPD, as expected on the basis of their half-life (Figure 1c) (reviewed in Balajee and Bohr). The repair rate of 6-4 PP was similar in both cell types. In contrast, CPD were repaired at a significant faster rate in keratinocytes than in fibroblasts. After 24 h irradiation, only 20% of the initial CPD were left in keratinocyte DNA whereas over 50% of the initial lesions remained in fibroblast DNA. The higher efficiency in repair of CPD by keratinocytes cannot be ascribed to the lower level of initial DNA damage since the repair kinetics in fibroblasts following a dose of 500 J/m was similar to that reported after a dose of 1000 J/m UVB (data not shown). To address the question of whether cell cycle progression is differentially affected in the two cell types, cells were exposed to UVB and cell cycle position was determined 24 and 48 h after irradiation. A representative cell cycle distribution at 24 h post-irradiation is displayed in Figure 1d. In fibroblasts, at both UVB doses, a G1–S phase arrest was observed whereas in keratinocytes the cell cycle distribution was substantially unaltered. The level of the stress response protein p53 was determined after irradiation. Both fibroblasts and keratinocytes responded to UVB damage (1000 J/m) with stabilization of the p53 protein (Figure 1e). However, while in fibroblasts p53 displayed a significant increase at 12 h after irradiation and continued to accumulate up to 24 h, in keratinocytes p53 level reached a peak at 6 h and then drastically decreased to background at 12 h. Moreover, higher levels of UVB-induced p53 protein were observed in fibroblasts than in keratinocytes. In keratinocytes, a rapid but transitory p53 response to UVB was observed also after 2000 J/m (data not shown). From this study, apoptosis and an efficient DNA repair machinery for UV photoproducts emerged as the major defense mechanisms of keratinocytes against the deleterious effects of UVB. Keratinocytes undergo apoptosis at UVB doses that are ineffective in fibroblasts. It has been proposed that the stalling of the transcription machinery at CPD leads to activation of p53, thus initiating apoptosis. This model is strongly supported by the finding that in TCR-defective fibroblasts, derived from patients with Cockayne syndrome, p53 and apoptosis are induced at UVC doses that are Cell Death and Differentiation (2003) 10, 754–756 & 2003 Nature Publishing Group All rights reserved 1350-9047/03

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

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N-methyl-N-nitrosourea-induced mutations in human cells : effects of the transcriptional activity of the target gene

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.

Characterization of the ultraviolet B and X-ray response of primary cultured epidermal cells from patients with disseminated superficial actinic porokeratosis

In this study we addressed the question as to whether the mutagenesis by methylating agents is affected by the transcriptional activity of the damaged gene. An Epstein-Barr virus (EBV)-derived shuttle vector system was developed where the genetic target for mutation analysis, the bacterial gpt gene, is under the control of an eukaryotic inducible promoter in plasmid pF1-EBV and lacks the eukaryotic promoter in plasmid pF2-EBV. Two human cell lines that episomically maintain these shuttle vectors were established. In clone 6NT cells, which contain pF1-EBV plasmid, the gpt gene is actively transcribed and the transcription rate is regulated by zinc ions. In clone 3 cells, which harbor pF2-EBV plasmid, the gpt gene is not transcribed. Following treatment of both cell lines with the potent alkylating carcinogen N-methyl-N-nitrosourea (MNU), G.C to A.T transitions were the major mutagenic event, consistent with the miscoding potential of O6-methylguanine. The mutations were predominantly generated in the non-transcribed DNA strand of the active gpt gene. The same strand-bias was observed when the gpt gene was transcriptionally inactive, indicating that MNU-induced strand-specific formation of mutations is not due to transcription. Our data identify as major determinants of this phenomenon the sequence-specificity of MNU mutagenesis and the conformational properties of the target protein. Differences in mutation distribution were observed between the transcriptionally active and inactive gpt gene. This finding suggests that the organization of active genes in chromatin might modulate DNA alkylation and/or DNA repair.