SNF2H interacts with XRCC1 and is involved in repair of H2O2-induced DNA damage.

The protein XRCC1 has no inherent enzymatic activity, and is believed to function in base excision repair as a dedicated scaffold component that coordinates other DNA repair factors. Repair foci clearly represent the recruitment and accumulation of DNA repair factors at sites of damage; however, uncertainties remain regarding their organization in the context of nuclear architecture and their biological significance. Here we identified the chromatin remodeling factor SNF2H/SMARCA5 as a novel binding partner of XRCC1, with their interaction dependent on the casein kinase 2-mediated constitutive phosphorylation of XRCC1. The proficiency of repairing H2O2-induced damage was strongly impaired by SNF2H knock-down, and similar impairment was observed with knock-down of both XRCC1 and SNF2H simultaneously, suggesting their role in a common repair pathway. Most SNF2H exists in the nuclear matrix fraction, forming salt extraction-resistant foci-like structures in unchallenged nuclei. Remarkably, damage-induced formation of both PAR and XRCC1 foci depended on SNF2H, and the PAR and XRCC1 foci co-localized with the SNF2H foci. We propose a model in which a base excision repair complex containing damaged chromatin is recruited to specific locations in the nuclear matrix for repair, with this recruitment mediated by XRCC1-SNF2H interaction.

Differential evaluation of hepatocyte apoptosis and necrosis in acute liver injury.

AIM: The significance of cytokelatin-18 fragment cleaved by caspase-3 (CK-18-fr) and high mobility group box-1 (HMGB-1) were evaluated experimentally and clinically for the differential evaluation of hepatocyte apoptosis and necrosis in patients with acute hepatic injury (AHI). METHODS: In this study, typical apoptosis and necrosis were induced in HepG2 cells by staurosporin (STS) and hydrogen peroxide, respectively. Intracellular generation of CK-18-fr and extracellular leakage of CK-18-fr and HMGB-1 were determined. In the clinical study, serum CK-18-fr and HMGB-1 levels in 84 patients with AHI of varied severity and etiology were measured and compared with conventional liver tests. RESULTS: In the experimental study, CK-18-fr was rapidly increased after STS stimulation, and peaked after 6 h inside the cells but increased in the medium 12 h after stimulation, while hydrogen peroxide stimulation caused no increase either in- or outside the cells. Extracellular HMGB-1 levels markedly increased after hydrogen peroxide stimulation, but did not change after STS stimulation. In the clinical study, serum CK-18-fr increased in correlation with serum aminotransferase, but not other liver tests or markers of disease severity of AHI,. Serum HMGB-1 levels mildly increased without any correlation to liver test or disease severity. Serum HMGB-1 levels in patients with circulation disturbance was significantly higher than that in patients with other etiologies. CONCLUSION: The simultaneous determination of the serum CK-18-fr and HMGB-1 may be useful in the differential diagnosis of hetocellular death in AHI, which is primarily due to apoptosis except in patients with circulation disturbance.

Characterization of human synovial fluid cells of 26 patients with osteoarthritis knee for cartilage repair therapy.

AIM: To investigate the possibility of chondrogenic differentiation and cartilage repair of synovial fluid cells of osteoarthritis (OA) knee. METHODS: Synovial fluids from 26 patients with OA knee were aspirated from each knee joint and cultured in vitro. The morphology of cultured synovial fluid cells, cell proliferation rate, the phenotype, and chondrogenic differentiation were analyzed in in vitro. Also, human autologous synovial fluid cells were transplanted to OA cartilage, and the cells were traced in ex vivo. RESULTS: In 19 of 26 materials, the cells proliferated satisfactorily. The cell proliferation in six materials was very slow and one material contaminated. Culture-expanded synovial fluid cells had a fibroblastic morphology and the phenotype was negative for CD10, CD14, and CD45, and positive for CD13, CD44, and CD105. Pellet culture of synovial fluid cells showed chondrogenic differentiation. In the ex vivo study, autologous transplanted synovial fluid cells were observed in repaired or enhanced regenerative cartilage areas and showed a tendency to infiltrate the original degenerative cartilage of OA. CONCLUSIONS: This study proved the possibility of chondrogenic differentiation of synovial fluid cells of OA knee joints despite the pathologic environment within a diseased joint. Synovial fluid cells were actually heterogeneous cells but they showed chondrogenic differentiation, similar to that of bone marrow-derived mesenchymal progenitor cells (BMMPCs). The Ex vivo study suggested that synovial fluid cells had a tendency to adhere to OA degenerative cartilage in humans.

Localization of X-ray cross complementing gene 1 protein in the nuclear matrix is controlled by casein kinase II-dependent phosphorylation in response to oxidative damage.

Base excision repair/single strand break repair (BER/SSBR) of damaged DNA is a highly efficient process. X-ray cross complementing protein 1 (XRCC1) functions as a key scaffold protein for BER/SSBR factors. Recent work has shown that XRCC1 forms dense foci at sites of DNA damage in a manner dependent on casein kinase II (CK2) phosphorylation. To investigate the mechanism underlying foci formation, we analyzed the subnuclear localization and phosphorylation status of XRCC1 during the repair process by biochemical fractionation of HeLa cellular proteins. The localization was also verified by in situ extraction of the fixed cells. In unchallenged cells, XRCC1 was primarily found in the chromatin fraction in a highly phosphorylated form; in addition, a minor population (10-15%) existed in the nuclear matrix (NM) with no or marginal phosphorylation. After hydrogen peroxide treatment, hyperphosphorylated XRCC1 appeared in the NM and accordingly, those in the chromatin fraction decreased. Foci formation and changes in XRCC1 distribution could be abolished by the knockdown of CK2, the expression of a non-phosphorylatable version of XRCC1, or the inhibition of poly-ADP ribosylation at the damage sites. Other BER factors, like DNA polymerase beta, were also found to accumulate in the NM after hydrogen peroxide-induced DNA damage, although its association with the NM seemed relatively weak. Our results suggest that the constitutive phosphorylation of XRCC1 in the chromatin and its DNA damage-induced recruitment to the NM are critical for foci formation, and that the core reactions of BER/SSBR may occur in the NM.

Inhibition of ocular angiogenesis by diced small interfering RNAs (siRNAs) specific to vascular endothelial growth factor (VEGF).

The effects of diced small interfering RNAs (siRNAs) designed for vascular endothelial growth factor (VEGF) on the expression of VEGF in human retinal pigment epithelial cell line ARPE-19 cells in vitro and on corneal angiogenesis in vivo were examined. The exposure to diced siRNAs significantly reduced the VEGF mRNA expression in ARPE-19 cells with minimal toxicity. In suture-induced corneal angiogenesis models, diced siRNAs minimized the severity of angiogenesis. Histological analysis displayed no particular tissue damage in the conjunctiva where siRNA was injected. The approach using diced siRNAs can be a new tool for various neovascular ocular diseases.

Possible association of the X-ray cross complementing gene 1 (XRCC1) Arg280His polymorphism as a risk for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the invasion of synovial cells into cartilage and bone, exhibiting certain features of transformed cells. To examine whether retardation of DNA repair pathway of oxidative damage is a possible mechanism in altered phenotypes of these cells, we analyzed SNPs of the base excision repair (BER) protein, X-ray repair cross complementing gene 1 (XRCC1), among RA patients. Genomic DNA was extracted from blood cells of 40 RA patients and SNPs of the three allele of the XRCC1 coding region (codons 194, 280 and 399) were determined by PCR, followed by sequencing. Of the three polymorphisms, only the XRCC1 Arg280His allele was associated with increased RA risk (odds ratio 13; 95% confidence interval 1.1-147) after adjustment for smoking. These data provide evidence for the first time that BER, which is involved in the recovery from oxidative damage, may correlate with RA.

Inhibitory effect of triamcinolone acetonide on corneal neovascularization.

BACKGROUND: Corneal neovascularization (NV) plays an important role in the pathogenesis of corneal disorders. Recently, triamcinolone acetonide (TA) has been reported as a potential treatment for ocular angiogenesis. However, there are no reports on the inhibitory effect of TA on the corneal NV. METHODS: Triamcinolone acetonide (2 mg) was administered to four rabbits' eyes by a subconjunctival injection immediately after a basic fibroblast growth factor (bFGF)-pellet was placed into the cornea. As a control, four eyes received an injection of distilled water. Four weeks later, the inhibition of corneal NV was evaluated as the percentage ratio of the vessel invasion area to the area that was sandwiched between the pellet and the limbus cornea. To identify the characteristic appearance of new corneal vessels, the control cornea was examined by using the antibody of vascular endothelial growth factor (VEGF). To confirm TA concentration in TA-treated corneas, the TA level was measured using high-performance liquid chromatography. RESULTS: Neovascularization from the limbus to the pellet was detected in control eyes 4 weeks after the bFGF pellet implantation. TA-treated eyes demonstrated the inhibition of the neovascular response to the pellet. The severity of NV as compared between control and TA-treated eyes was statistically significant (P<0.05). Morphologically, new vessel growth was shown in the control cornea, and endothelial cells of new vessels were positively stained with the antibody of VEGF. TA concentration in TA-treated corneas at 2 weeks showed 63.5+/-42.8 microg/g (n=4, mean +/- SD), while TA was not detected in control and TA-treated corneas at 4 weeks. The level of TA was effectively maintained for at least 2 weeks after the subconjunctival injection. CONCLUSION: We have demonstrated that subconjunctival TA administration inhibited rabbit corneal NV. This agent may prove useful in the treatment of corneal angiogenic disorders.

Hyaluronan synthases, hyaluronan and its CD44 receptors in the posterior segment of rabbit eye.

To understand the possible roles of the hyaluronan synthetase (HAS)/hyaluronan (HA)/CD44 signaling system in the posterior eye segment, we investigated the expression of rabbit HAS isoforms and CD44 mRNA by RT-PCR and the level of HA by using HA assay and immunohistochemistry. HA was detectable in vitreous, retina and choroid. The expression of three HAS isoforms was clearly detected in both retina and choroids. Rabbit choroid showed a significant increase of the HAS2 and HAS3 expression compared with rabbit retina (HAS2 p = 0.0014 < 0.05; HAS3 p = 0.0006 < 0.05). Similarly, mRNA expression of CD44 was detected in both retina and choroids. This evidence may suggest that the HAS/HA/CD44 signaling system is important in maintaining the functional structure of retina and choroid.

The Arg280His polymorphism in X-ray repair cross-complementing gene 1 impairs DNA repair ability.

The contribution of three single nucleotide polymorphisms (SNPs) that substitute amino acids in the X-ray repair cross-complementing gene 1 (XRCC1) protein, Arg194Trp (R194W), Arg280His (R280H), and Arg399Gln (R399Q), to the risk of various types of cancers has been extensively investigated by epidemiological researches. To investigate whether two of these polymorphisms directly influence their repair ability, we established Chinese hamster ovary (CHO) EM9 cell lines transfected with XRCC1(WT), XRCC1(R194W), or XRCC1(R280H) genes and analyzed the DNA repair ability of these cells. The EM9 cells that lack functional XRCC1 proteins exhibit severe sensitivity to methyl methanesulfonate (MMS). Introduction of the human XRCC1(WT) and XRCC1(R194W) gene to EM9 cells restored the MMS sensitivity to the same level as the AA8 cells, a parental cell line. However, introduction of the XRCC1(R280H) gene partially restored the MMS sensitivity, resulting in a 1.7- to 1.9-fold higher sensitivity to MMS compared with XRCC1(WT) and XRCC1(R194W) cells at the LD(50) value. The alkaline comet assay determined diminished base excision repair/single strand break repair (BER/SSBR) efficiency in XRCC1(R280H) cells as observed in EM9 cells. In addition, the amount of intracellular NAD(P)H decreased in XRCC1(R280H) cells after MMS treatment. Indirect immunofluorescence staining of the XRCC1 protein showed an intense increase in the signals and clear foci of XRCC1 in the nuclei of the XRCC1(WT) cells, but a faint increase in the XRCC1(R280H) cells, after MMS exposure. These results suggest that the XRCC1(R280H) variant protein is defective in its efficient localization to a damaged site in the chromosome, thereby reducing the cellular BER/SSBR efficiency.

Changes in microstructure and gene expression of articular chondrocytes cultured in a tube under mechanical stress.

OBJECTIVE: The objective of this study was to clarify the effects of mechanical stress on chondrocytes cultured in a tube. Centrifugal pressure was applied to chondrocytes cultured in a tube for 28 days, and the effect of this stress was evaluated using a molecular biological method. DESIGN: Articular cartilage was harvested from a rabbit. A cell suspension was then prepared, and transferred in 1 ml aliquots to polypropylene tubes. After 48 h of incubation, centrifugal pressure (6.9 MPa) was applied every 24 h. Changes in morphology, expression of messenger RNA (mRNA) for insulin-like growth factor-I (IGF-I) and type II collagen, cell number, wet weight and protein concentration were evaluated. RESULTS: Microscopically, formation of chondrocyte clusters was seen in the cultures subjected to stress. Ultrastructurally, collagen fibers were seen to run parallel to the cytoplasmic surface of the stressed chondrocytes. The peak of IGF-I mRNA expression was seen on day 5, whereas type II collagen mRNA expression peaked on day 14. Cell number, wet weight and protein concentration were significantly increased in the stressed cultures. CONCLUSIONS: These results suggest that mechanical stress might affect the arrangement of collagen fibers and the IGF-I activity of chondrocytes cultured in a tube, thus influencing chondrocyte proliferation and increasing the volume of the extracellular matrix. Furthermore, mechanical stress may also affect the metabolism of articular cartilage in vitro.

Changes in serum levels of heat shock protein 70 in preterm delivery and pre-eclampsia.

AIM: The aim of this study was to investigate heat-shock protein (Hsp)70 as a novel marker to evaluate the curative effects of treatment for preterm delivery high-risk patients and pre-eclampsia. METHODS: After obtaining informed consent, serum samples were collected from 31 preterm delivery high-risk patients with a tocolysis index of three points or above (A), seven pre-eclampsia patients (P), 46 normal pregnant women (B), and seven non-pregnant women (C). Of the 31 preterm delivery high-risk patients, 15 had preterm delivery (Ap) and 16 had full-term delivery (Af). The levels of Hsp70 were measured using enzyme-linked immunosorbent assay. RESULTS: The Hsp70 levels in normal pregnant women were 8.6 +/- 1.9 ng/mL (first trimester), 5.5 +/- 1.0 ng/mL (second trimester) and 5.5 +/- 0.7 ng/mL (third trimester). There was no statistical difference in the Hsp70 levels between the three trimesters. The mean Hsp70 levels were 21.9 +/- 5.3 ng/mL (A), 35.3 +/- 9.6 ng/mL (Ap), 9.4 +/- 2.2 ng/mL (Af), 24.4 +/- 3.6 ng/mL (P), 6.1 +/- 0.6 ng/mL (B), and 2.4 +/- 0.6 ng/mL (C). Group Ap had significantly higher Hsp70 levels than group Af (P = 0.0112) and group B (P <0.0001). The duration of pregnancy after hospitalization for group Ap was significantly shorter than that for group Af (P=0.0088) and group B (P <0.0001). Group P also had significantly higher Hsp70 levels than group B (P <0.0001). CONCLUSION: Because Hsp70 levels were particularly high in treatment-resistant preterm delivery cases, Hsp70 may prove to be a useful marker for evaluating the curative effects of treatment for preterm delivery.

The effect of indocyanine green on cultured retinal glial cells.

BACKGROUND: Recently, indocyanine green (ICG) has been utilized to visualize inner limiting membrane in vitreous surgery. However, the safety of ICG injected into the vitreous has not been well established. The possible toxicity of ICG on Muller cells was investigated using cultured rat retinal glial cells (RGCs). METHODS: Rat RGCs were cultured in Dulbecco modified Eagle medium supplemented with 20% fetal calf serum. The cytotoxicity of ICG was assayed with viable cell number and resazurin metabolic assay. The expression of the apoptosis-related gene bcl-2 was examined with real-time polymerase chain reaction analysis. RESULTS: The effects of ICG on the viability of rat RGCs were tested at two different concentrations (0.05% and 0.5%). ICG significantly decreased the viable cell number of RGCs at 0.5%, while there was no significant effect at 0.05%. Similarly, the metabolic activity to resazurin was significantly decreased by exposure to 0.5% ICG. However, ICG showed little effects on resazurin metabolism at 0.05%. The expression levels of bcl-2 mRNA were higher in cells treated with 0.5% ICG than in those treated with 0.05% ICG and untreated control cells. CONCLUSION: The data suggest that ICG initiates the death of RGCs at high concentrations, in part, through apoptosis-related signal pathways.

Efficient repair of DNA damage induced by heavy ion particles in meiotic prophase I nuclei of Caenorhabditis elegans.

The effects of heavy ion particle irradiation on meiosis and reproductive development in the nematode Caenorhabditis elegans were studied. Meiotic pachytene nuclei are significantly resistant to particle irradiation by the heavy ions carbon and argon, as well as to X-rays, but not UV, whereas diplotene to diakinesis stage oocytes and early embryonic cells are not. Chromosomal abnormalities appear in mitotic cells and in maturing oocytes irradiated with heavy ion particles during the diplotene to the early diakinesis stages, but not in oocytes irradiated during the pachytene stage. The pachytene nuclei of ced-3 mutants, which are defective in apoptosis, are similarly resistant to ionizing radiation, but pachytene nuclei depleted for Ce-atl-1 (ataxia-telangiectasia like 1) or Ce-rdh-1/rad-51 are more sensitive. Pachytene nuclei thus appear to effectively repair heavy ion-induced DNA damage by the meiotic homologous recombination system.

Reduced expression and loss of heterozygosity of the SDHD gene in colorectal and gastric cancer.

Mitochondrial DNA (mtDNA) mutations occur in a variety of human cancers, suggesting a possible role for mitochondrial respiratory functions in tumorigenesis. Recent studies have demonstrated that SDHD, a nuclear gene encoding one of the mitochondrial complex II subunits, acts as a tumor-suppressor for hereditary paragangliomas and pheochromocytomas. In order to determine whether the SDHD function plays a wider role in human malignancies, we examined SDHD gene alterations in 52 colorectal and 59 gastric cancers and 7 cancer cell lines. Loss of heterozygosity (LOH) at the SDHD gene locus was found in 5 of 35 (14%) colorectal and 5 of 40 (13%) gastric cancers. Reduced SDHD gene expression, which was partly associated with SDHD gene LOH, was observed in 15 of 19 (79%) colorectal cancers examined. Unlike classical tumor-suppressor genes, however, partial loss rather than complete loss of the SDHD gene expression was preferentially observed and the reduced expression could not result from CpG-island methylation or coding mutation. Interestingly, the mtDNA mutations (12 cases) and the SDHD gene LOH (6 of 7 cases) did not occur in the same cancers, suggesting that these alterations might have similar functional effects in tumorigenesis. We suggest that SDHD alterations can affect mitochondrial respiratory chain functions and play a role in colorectal and gastric cancers as a distinct type of tumor suppressor.

Caenorhabditis elegans Ce-rdh-1/rad-51 functions after double-strand break formation of meiotic recombination.

During meiotic prophase 1, homologous recombination is accompanied by dynamic chromosomal changes. The Ce-rdh-1/rad-51 gene is the only bacterial recA-like gene in the nematode C. elegans genome. Upon depletion of Ce-rdh-1/rad-51 using the RNA interference method, abnormal 'kinked' chromosomes can be observed in mature oocytes at diakinesis, whereas synapsis between homologous chromosomes during the pachytene stage is normal. Following fertilization, Ce-rdh-1/rad-51-depleted embryos die early in embryogenesis, and their nuclei exhibit abnormal chromosome fragments and bridges. From epistasis analyses with Ce-spo-11 defective mutant and ionizing radiation, it is indicated that Ce-rdh-1/rad-51 functions after double-strand break (DSB) formation of meiotic recombination. Under the Ce-chk-2 defective condition, whose meiotic synapsis and meiotic recombination between homologous chromosomes are completely inhibited, the Ce-rdh-1/rad51 is normally expressed in the gonadal cells. Moreover, it seems that exogenous DSBs in the Ce-chk-2 defective nuclei at the pachytene stage can be repaired between sister chromatids in a Ce-rdh-1/rad-51-dependent manner. These results indicate that Ce-rdh-1/rad51 functions after both endogenous and exogenous DSB formation during meiosis, but not as 'pairing centers' for meiotic synapsis.

Independent roles of XRCC1's two BRCT motifs in recovery from methylation damage.

XRCC1 is known to be involved in base excision repair (BER)/single-strand break repair (SSBR) through interaction with other BER enzymes. Hypersensitivity of XRCC1-deficient cells against alkylating agents has been explained by loss of interaction with BER proteins. XRCC1 is a unique DNA repair protein containing two BRCT motifs, recently identified in several DNA repair and cell cycle regulating proteins. To study the function(s) of the two BRCT motifs of the XRCC1 protein, we established CHO EM9 (XRCC1-null) cells expressing XRCC1 protein altered in either one of the two BRCT motifs. Colony-forming ability after methyl methanesulfonate (MMS) treatment was dependent on the BRCT-a motif, but not on the BRCT-b motif. Surprisingly, reduced BER/SSBR rate in vivo, measured by an alkaline comet assay, was observed in the BRCT-b motif-deficient cells, while the BRCT-a motif-deficient cells showed the repair rate comparable with the wild-type (WT) cells. The BRCT-a motif-mutated cells, instead, showed deficiency in initiation of DNA replications after MMS treatment. Furthermore, we found that XRCC1 is multiply phosphorylated in vivo and hyperphosphorylation of XRCC1 after MMS treatment is dependent on the BRCT-a motif. These data suggest a new function dependent on the integrity of the BRCT-a motif of XRCC1 in recovery from MMS-induced damage.

Induction of osteogenic protein-1 expression by interleukin-1beta in cultured rabbit articular chondrocytes.

To elucidate the effects of interleukin-1beta (IL-1beta) on osteogenic protein-1 (OP-1) gene expression in a polylayer culture of rabbit articular chondrocytes, we measured rabbit OP-1 mRNA using quantitative TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Rabbit articular chondrocytes were isolated and cultured in minimum essential medium eagle alpha modification containing 10% fetal bovine serum for 7 days. IL-1beta was then added and cultures were continued for 48 or 96 hours. OP-1 gene expression was detected in cell cultures both with and without addition of IL-1beta. However, the level of expression was very low in the control group. OP-1 gene expression was significantly increased about 450- to 800-fold in IL-1beta-treated groups (0.1, 1, and 10 ng/ml) versus the control group. Evaluation of serial changes in OP-1 expression after addition of IL-1beta (10 ng/ml) revealed that OP-1 gene expression increased rapidly after addition of IL-1beta, reaching a peak at 48 hours, and then decreasing. Simultaneous assay of CD44 expression demonstrated a rapid increase, similar to that of OP-1 expression, following addition of IL-1beta: this was followed by a more gradual increase. Assay of hyaluronan synthase-2 (HAS-2) expression following addition of IL-1beta showed an increase after OP-1 expression had already reached a peak. Our results demonstrate that OP-1 expression is induced by IL-1beta and suggest that this expression, like that of HAS-2, may play a role as a protective mechanism against inflammatory cytokines.