Processing ofDNA damage by the nucleotideexcision repair pathway in eukaryotic cells is most likely accomplished by multiprotein complexes. However, the nature of these complexes and the details of the molecular interactions between DNA repair factors are for the most part unknown. Here, we demonstrate both in vivo, using the two-hybrid system, and in vitro, using recombinant proteins, that the human repair factors XPA and ERCC1 specificafly interact. In addition, we report an initial determination of the domains in ERCC1 and XPA that medlate this interaction. These results suggest that XPA may play a role in the iocalation or ld ofan incision complex, composed ofERCC1 and possibly other repair factors, onto a damaged site.Two groups of mutants defective in the early steps of nucleotide-excision repair have been identified in mammalian cells. These are the naturally occurring cell lines obtained from patients with the rare disorders xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy and the laboratory-derived rodent cell lines designated ERCC (excision repair cross-complementing). Eleven genetic complementation groups have been identified in the former series (1, 2) and 11 in the latter (3, 4). Several of the mutants in these two classes fall into the same complementation group; however, the full extent of overlap remains to be determined. Seven of these genes have now been cloned (5-11), setting the stage for investigations into how DNA damage processing is mediated by this system. One aspect of these studies is to determine the nature ofprotein-protein interactions between DNA-repair factors and to define the composition of multifactorial complexes with the eventual goal ofelucidating their function.The yeast two-hybrid system developed by Fields and colleagues (12, 13) is a novel genetic approach that is used for the detection of interacting proteins. In this system, association in yeast between two proteins, one fused to the DNAbinding domain and the second to the activation domain II of GALA, activates promoters containing GALA-binding sites (14). We have used a modification (15, 16) of the original system involving a dual selection scheme for HIS3 and LacZ to screen a human cDNA library for genes whose expression products interact with the human repair factor ERCC1 (5,17). In addition to the isolation of a number of novel genes, this screening also resulted in the identification of the repair protein XPA as a factor that associates with ERCC1.

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombinationindependent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.Alkylating agents were among the first compounds found to be efficacious in cancer therapy and remain important components of many modern chemotherapeutic regimens (38). Many members of this class of drugs have bifunctional groups that can react with both strands of the DNA helix and thus form interstrand and intrastrand cross-links. As profound blocks for both transcription and DNA replication, interstrand cross-links (ICLs) appear to represent the primary cytotoxic lesion induced by most bifunctional alkylating agents.Repair of DNA ICLs has been studied extensively in Escherichia coli (11,12). Both genetic and biochemical evidence has established a combined nucleotide excision repair (NER)-recombination mechanism for the error-free repair of ICLs, in which the gap, created by the Uvr(A)BC excinuclease, is repaired through recA-mediated recombination with a lesionfree homologous chromosome as the donor (10, 37, 41). Although the NER-recombination pathway appears to be the primary mechanism of cross-link repair in E. coli, recent evidence has suggested a recombination-independent pathway for cross-link repair in which the gap created by the uvr(A)BC excinuclease is repaired by translesion bypass in order to circumvent a d...

Maspin, a member of the serpin family of protease inhibitors, is known to have tumor-suppressor functions. However, the association between its expression level and survival has not been demonstrated in human cancer. Using the immunohistochemical technique to examine the expression levels of maspin in 44 cases of oral squamous cell carcinoma (SCC), we found that 66% of the cases expressed low to intermediate levels of maspin and 34% of the cases expressed high levels of maspin. We further examined maspin protein expression in a series of six SCC cell lines from the head and neck, and found that all but one expressed low or no maspin protein. We also compared the clinicopathological features of the oral SCC cases with the maspin expression level, and found that high maspin expression was associated with the absence of lymph node metastasis. More importantly, we showed that higher maspin expression was signi®cantly associated with better rates of overall survival, suggesting that high maspin expression may be a favorable prognostic marker for oral SCC.

IntroductionThe orbital subarachnoid space surrounding the optic nerve is continuous with the circulation system for cerebrospinal fluid (CSF) and can be visualized by using magnetic resonance imaging (MRI). We hypothesized that the orbital subarachnoid space width (OSASW) is correlated with and can serve as a surrogate for intracranial pressure (ICP). Our aim was to develop a method for a noninvasive measurement of the intracranial CSF-pressure (CSF-P) based on MRI-assisted OSASW.MethodsThe prospective observational comparative study included neurology patients who underwent lumbar CSF-P measurement and 3.0-Tesla orbital magnetic resonance imaging (MRI) for other clinical reasons. The width of the orbital subarachnoid space (OSASW) around the optic nerve was measured with MRI at 3, 9, and 15 mm behind the globe. The study population was randomly divided into a training group and a test group. After adjusting for body mass index (BMI) and mean arterial blood pressure (MABP), algorithms for the associations between CSF-P and OSASW were calculated in the training group. The algorithms were subsequently verified in the test group. Main outcome measures were the width of the orbital subarachnoid space (OSASW) and the lumbar cerebrospinal fluid pressure (CSF-P).ResultsSeventy-two patients were included in the study. In the training group, the algorithms for the associations between CSF-P and OSASW were as follows: (a) CSF-P = 9.31 × OSASW (at 3 mm) + 0.48 × BMI + 0.14 × MABP-19.94; (b) CSF-P = 16.95 × OSASW (at 9 mm) + 0.39 × BMI + 0.14 × MABP-20.90; and (c) CSF-P = 17.54 × OSASW (at 15 mm) + 0.47 × BMI + 0.13 × MABP-21.52. Applying these algorithms in the independent test group, the measured lumbar CSF-P (13.6 ± 5.1 mm Hg) did not differ significantly from the calculated MRI-derived CSF-P (OSASW at 3 mm: 12.7 ± 4.2 mm Hg (P = 0.07); at 9 mm: 13.4 ± 5.1 mm Hg (P = 0.35); and at 15 mm: 14.0 ± 4.9 mm Hg (P = 0.87)). Intraclass correlation coefficients (ICCs) were higher for the CSF-P assessment based on OSASW at 9 mm and at 15 mm behind the globe (all ICCs, 0.87) than for OSASW measurements at 3 mm (ICC, 0.80).ConclusionsIn patients with normal, moderately decreased or elevated ICP, MRI-assisted measurement of the OSASW appears to be useful for the noninvasive quantitative estimation of ICP, if BMI and MABP as contributing parameters are taken into account.Trial registrationClinical trial registered with the Chinese Clinical Trial Registry: ChiCTR-OCC-11001271

DNA interstrand cross-links (ICLs) are the most cytotoxic lesions to eukaryotic genome and are repaired by both homologous recombinationdependent and -independent mechanisms. To better understand the role of lesion bypass polymerases in ICL repair, we investigated recombination-independent repair of ICLs in REV3 and REV1 deletion mutants constructed in avian DT40 cells and mouse embryonic fibroblast cells. Our results showed that Rev3 plays a major role in recombination-independent ICL repair, which may account for the extreme sensitivity of REV3 mutants to cross-linking agents. This result raised the possibility that the NER gap synthesis, when encountering an adducted base present in the ICL repair intermediate, can lead to recruitment of Rev3, analogous to the recruitment of polymerase during replicative synthesis. Indeed, the monoubiquitination-defective Proliferating Cell Nuclear Antigen (PCNA) mutant exhibits impaired recombination-independent ICL repair as well as drastically reduced mutation rate, indicating that the PCNA switch is utilized to enable lesion bypass during DNA repair synthesis. Analyses of a REV1 deletion mutant also revealed a significant reduction in recombination-independent ICL repair, suggesting that Rev1 cooperates with Rev3 in recombination-independent ICL repair. Moreover, deletion of REV3 or REV1 significantly altered the spectrum of mutations resulting from ICL repair, further confirming their involvement in mutagenic repair of ICLs.Bifunctional alkylating agents generate DNA interstrand cross-links (ICLs), 2 which prevent strand separation required for essential DNA functions such as replication, transcription, and recombination. Because an ICL compromises both strands of the double helix, recombination with an undamaged homologous sequence is required for error-free repair. This has been shown in both prokaryotic and eukaryotic systems (1, 2). However, ICL repair also occurs in a recombination-independent fashion. Our investigations in mammalian cells have suggested an NER-and translesion synthesis-based errorprone mechanism of ICL repair in which the gap created by the NER dual incisions is resynthesized through participation of lesion bypass DNA polymerases (3, 4). This error-prone mechanism may account for the mutagenic impact of ICLs. A similar mechanism has been demonstrated in budding yeast, suggesting that recombination-independent ICL repair may be a highly conserved mechanism in eukaryotes (5, 6).The budding yeast mutants REV3 and REV1 are characterized by their marked reduction in UV-induced mutability (7) and profound sensitivity to ICLs during G 1 or stationary phases. REV3 encodes the catalytic subunit of DNA polymerase (Pol), a member of the B-type lesion bypass polymerase (8). Mouse embryonic fibroblasts (MEFs) derived from REV3 Ϫ/Ϫ embryos and an avian REV3 Ϫ/Ϫ mutant exhibited severe sensitivity to DNA cross-linking agents (2, 9 -11), suggesting an important role for Pol in the repair of DNA ICLs. REV1 encodes a deoxycytidyltransferase (12, 13), which has been ...

Stromal cell-derived factor-1 (SDF-1) is a potent chemokine for bone marrow-derived stromal stem cells (BMSCs) that express CXCR4, the receptor for SDF-1. SDF-1 is considered to play an important role in the trafficking of BMSCs. We investigated the contribution of SDF-1 to the recruitment of BMSCs to the wound area and its promotion of wound repair and neovascularization. BMSCs were pretreated with or without anti-CXCR4 blocking antibody and combined with CM-DiI label, and injected via the tail vein into mice with full-thickness skin wounds on the dorsum. Simultaneously, anti-SDF-1 antibody was injected into local wounds in another group of mice. The results show that blockade of CXCR4 on either infused BMSCs or SDF-1 in the host wounds (1) dramatically impaired the number of infused BMSCs being recruited to the injured tissue, (2) reduced the expression of growth factors involved in the repair of injured tissue such as vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor beta 1, (3) decreased the resultant neovascularization, and (4) retarded wound healing. Taken together, the findings indicate that the SDF-1/CXCR4 signal pathway facilitates wound healing through augmenting BMSC recruitment to wound tissues, responsive secretion of growth factors by BMSCs and neovascularization in the wound area.

No abstract

DNA interstrand cross-linking agents have been widely used in chemotherapeutic treatment of cancer. The majority of interstrand cross-links (ICLs) in mammalian cells are removed via a complex process that involves the formation of double strand breaks at replication forks, incision of the ICL, and subsequent error-free repair by homologous recombination. How double strand breaks effect the removal of ICLs and the downstream homologous recombination process is not clear. Here, we describe a plasmid-based recombination assay in which one copy of the CFP gene is inactivated by a site-specific psoralen ICL and can be repaired by gene conversion with a mutated homologous donor sequence. We found that the homology dependent recombination (HDR) is inhibited by the ICL. However, when we introduced a double strand break adjacent to the site of the ICL, the removal of the ICL was enhanced and the substrate was funneled into a HDR repair pathway. This process was not dependent on the nucleotide excision repair pathway, but did require the ERCC1-XPF endonuclease and REV3. In addition, both the Fanconi anemia pathway and the mismatch repair protein MSH2 were required for the recombinational repair processing of the ICL. These results suggest that the juxtaposition of an ICL and a DSB stimulates repair of ICLs through a process requiring components of mismatch repair, ERCC1-XPF, REV3, Fanconi anemia proteins, and homologous recombination repair factors.