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Skin Aging manifests primarily with wrinkles, dyspigmentations, texture changes, and loss of elasticity. During the skin aging process, there is a loss of moisture and elasticity in skin resulting in loss of firmness finally leading to skin sagging. The key molecule involved in skin moisture is hyaluronic acid (HA), which has a significant water-binding capacity. HA levels in skin decline with age resulting in decrease in skin moisture, which may contribute to loss of firmness. Clinical trials have shown that topically applied ROL effectively reduces wrinkles and helps retain youthful appearance. In the current study, ROL was shown to induce HA production and stimulates the gene expression of all three forms of hyaluronic acid synthases (HAS) in normal human epidermal keratinocytes monolayer cultures. Moreover, in human skin equivalent tissues and in human skin explants, topical treatment of tissues with a stabilized-ROL formulation significantly induced the gene expression of HAS mRNA concomitant with an increased HA production. Finally, in a vehicle-controlled human clinical study, histochemical analysis confirmed increased HA accumulation in the epidermis in ROL-treated human skin as compared to vehicle. These results show that ROL increases skin expression of HA, a significant contributing factor responsible for wrinkle formation and skin moisture, which decrease during aging. Taken together with the activity to increase collagen, elastin, and cell proliferation, these studies establish that retinol provides multi-functional activity for photodamaged skin.

To mount highly specific and adapted immune responses, B lymphocytes assemble and diversify their antibody repertoire through mechanisms involving the formation of programmed DNA damage. Immunoglobulin class switch recombination (CSR) is triggered by DNA lesions induced by activation-induced cytidine deaminase, which are processed to double-stranded DNA break (DSB) intermediates. These DSBs activate the cellular DNA damage response and enroll numerous DNA repair factors, involving poly(ADP-ribose) polymerases Parp1, Parp2, and Parp3 to promote appropriate DNA repair and efficient long-range recombination. The macroParp Parp9, which is overexpressed in certain lymphomas, has been recently implicated in DSB repair, acting together with Parp1. Here, we examine the contribution of Parp9 to the resolution of physiological DSBs incurred during V(D)J recombination and CSR by generating Parp9-/- mice. We find that Parp9-deficient mice are viable, fertile, and do not show any overt phenotype. Moreover, we find that Parp9 is dispensable for B-cell development. Finally, we show that CSR and DNA end-joining are robust in the absence of Parp9, indicating that Parp9 is not essential in vivo to achieve physiological DSB repair, or that strong compensatory mechanisms exist.

Background Environmental factors such as solar ultraviolet (UV) radiation and other external aggressors provide an oxidative challenge that is detrimental to skin health. The levels of endogenous antioxidants decrease with age, thus resulting in less protection and a greater potential for skin damage. The NF-E2-related factor-2 (Nrf2) – antioxidant response element (ARE) pathway is a primary defense mechanism against oxidative stress, and induces the expression of antioxidant, detoxification and repair genes. Activation of ARE-Nrf2 can help restore oxidative homeostasis of the skin and play a role in inflammatory response and DNA repair mechanisms. Objective To evaluate the role of a purified parthenolide-depleted Feverfew (PD-Feverfew) extract on the ARE-Nrf2 pathway and DNA repair in skin cells. Methods These studies were undertaken in primary human keratinocytes or KB cells using Luciferase Promoter assay, siRNA transfection studies, Western blot analyses, Immunofluorescence microscopy, comet assay and quantitative real-time PCR. Results PD-Feverfew was found to induce Nrf2 nuclear translocation and to increase ARE activity in a dose dependent manner. Furthermore, knockdown of Nrf2 resulted in suppression of PD-Feverfew-induced ARE activity. PD-Feverfew was also found to induce phosphorylation of Akt, a kinase downstream of PI3K. Inhibition of PI3K via pre-treatment with the selective pharmacological inhibitor, LY294002, abolished PD-Feverfew-induced Nrf2/ARE activation. PD-Feverfew also reduced UV-induced DNA damage in a PI3K and Nrf2-dependent manner. Conclusions Therefore, by increasing endogenous defense mechanisms and aid in DNA repair of damaged skin cells via activation of a PI3K-dependent Nrf2/ARE pathway, PD-Feverfew may help protect the skin from numerous environmental aggressors.

Poly(ADP‐ribosyl)ation is a post‐translational modification of proteins that is mediated by poly(ADP‐ribose) polymerases (PARPs). Although the existence and nature of the nucleic acid‐like molecule poly(ADP‐ribose) (PAR) has been known for 40 years, understanding its biological functions—originally thought to be only the regulation of chromatin superstructure when DNA is broken—is still the subject of intense research. Here, we review the mechanisms controlling the biosynthesis of this complex macromolecule and some of its main biological functions, with an emphasis on the most recent advances and hypotheses that have developed in this rapidly growing field.

Previous studies using rodent cells indicate that a deficiency in XRCC1 results in reduced single-strand break repair, increased sensitivity to DNA-damaging agents, and elevated levels of sister chromatid exchange (SCE). Epidemiological studies have suggested an association of certain human XRCC1 polymorphisms with genetic instability and cancer susceptibility. However, investigations on the molecular functions of XRCC1 in human cells are limited. To determine the contributions of this nonenzymatic scaffold protein, we suppressed XRCC1 levels in several human cell lines using small interfering RNA (siRNA) technology. We report that XRCC1 down-regulation in HeLa cells leads to a concomitant decrease in the DNA ligase 3 protein level and an impaired nick ligation capacity. In addition, depletion of XRCC1 resulted in a significantly increased sensitivity to the alkylating agent methyl methanesulfonate and the thymidine base analog 5-hydroxymethyl-2'-deoxyuridine, a slightly increased sensitivity to ethyl methanesulfonate and 1,3-bis(2-chloroethyl)-1-nitrosourea, and no change in the response to camptothecin. We also discovered that a 70-80% reduction in XRCC1 protein leads to an elevated level of SCE in both HeLa cells and normal human fibroblasts, but does not affect chromosome aberrations in the diploid fibroblasts. Last, XRCC1 siRNA transfection led to an approximately 40% decrease in the survival of BRCA2-deficient cells, supporting a model whereby the accumulation of unrepaired SSBs leads to the accumulation of cytotoxic DNA double strand breaks following replication fork collapse in cells defective in homologous recombination.

Lead is a widespread environmental toxin, found in contaminated water sources, household paints, and certain occupational settings. Classified as a probable carcinogen by the International Agency for Research on Cancer (IARC), lead promotes mutagenesis when combined with alkylating and oxidizing DNA-damaging agents. We previously reported that lead inhibits the in vitro repair activity of Ape1, the major endonuclease for repairing mutagenic and cytotoxic abasic sites in DNA. We investigated here whether lead targets Ape1 in cultured mammalian cells. We report a concentration-dependent inhibition of apurinic/apyrimidinic (AP) site incision activity of Chinese hamster ovary (CHO) AA8 whole cell extracts by lead. In addition, lead exposure results in a concentration-dependent accumulation of AP sites in the genomic DNA of AA8 cells. An increase in the oxidative base lesion 8-oxoguanine was observed only at high lead levels (500 microM), suggesting that non-specific oxidation plays little role in the production of lead-related AP lesions at physiological metal concentrations--a conclusion corroborated by "thiobarbituric acid reactive substances" assays. Notably, Ape1 overexpression in AA8 (hApe1-3 cell line) abrogated the lead-dependent increase in AP site steady-state levels. Moreover, lead functioned cooperatively to promote a further increase in abasic sites with agents known to generate AP sites in DNA (i.e., methyl methansulfonate (MMS) and hydrogen peroxide (H2O2), but not the DNA crosslinking agent mitomycin C. Hypoxanthine guanine phosphoribosyltransferase (hprt) mutation analysis revealed that, whereas lead alone had no effect on mutation frequencies, mutagenesis increased in MMS treated, and to a greater extent lead/MMS treated, AA8 cells. With the hApe1-3 cell line, the number of mutant colonies in all treatment groups was found to be equal to that of the background level, indicating that Ape1 overexpression reverses MMS- and lead-associated hprt mutagenesis. Our studies in total indicate that Ape1 is a member of an emerging group of DNA surveillance proteins that are inhibited by environmental heavy metals, and suggest an underlying mechanism by which lead promotes co-carcinogenesis.

The ends of linear chromosomes are capped and protected by protein-DNA complexes termed telomeres. Consequences of telomere dysfunction include genomic instability that can contribute to neoplastic transformation and progression. Telomere binding proteins interact with numerous proteins involved in DNA repair, underscoring the importance of regulating DNA repair pathways at telomeres. Telomeric DNA is particularly susceptible to oxidative damage, and such damage is repaired primarily via the base excision repair (BER) pathway. Using a screen for potential interactions between telomere repeat binding factor 2 (TRF2) and proteins involved in BER of oxidized bases in vitro, we found that TRF2 physically bound DNA polymerase β (Pol β) and flap endonuclease 1 (FEN-1). The interactions with endogenous proteins in human cell extracts were confirmed by coimmunoprecipitation experiments. The primary binding sites for both Pol β and FEN-1 mapped to the TRF2 NH2-terminal and COOH-terminal domains. We further tested the ability of TRF2 to modulate BER protein partners individually on a variety of substrates in vitro. TRF2 stimulated Pol β primer extension DNA synthesis on telomeric and nontelomeric primer/template substrates, resulting in up to a 75% increase in the proportion of longer products. TRF2 also stimulated Pol β strand displacement DNA synthesis in reconstituted BER reactions and increased the percent of long-patch BER intermediates on both telomeric and nontelomeric substrates. Potential roles of TRF2 in cooperation with BER proteins for DNA repair pathways at telomeres, as well as other genomic regions, are discussed. (Cancer Res 2006; 66(1): 113-24)

Base excision repair (BER) is the major corrective pathway for most spontaneous, oxidative, and alkylation DNA base and sugar damage. X-ray cross-complementing 1 (XRCC1) has been suggested to function at nearly every step of this repair process, primarily through direct protein-protein interactions. Using whole cell extract (WCE) repair assays and DNA damage measurement techniques, we examined systematically the quantitative contribution of XRCC1 to specific biochemical steps of BER and single-strand break repair (SSBR). Our studies reveal that XRCC1-deficient Chinese hamster ovary WCEs exhibit normal base excision activity for 8-oxoguanine (8-OH-dG), 5-hydroxycytosine, ethenoadenine, and uracil lesions. Moreover, XRCC1 mutant EM9 cells possess steady-state levels of endogenous 8-OH-dG base damage similar to those of their wild-type counterparts. Abasic site incision activity was found to be normal in XRCC1-deficient cell extracts, as were the levels of abasic sites in isolated chromosomal DNA from mutant cells. While one- and five-nucleotide gap filling was not affected by XRCC1 status, a significant approximately 2-4-fold reduction in nick ligation activity was observed in EM9 WCEs. Our results herein suggest that the primary biochemical defect associated with XRCC1 deficiency is in the ligation step of BER/SSBR, and that XRCC1 plays no significant role in endogenous base damage and abasic site repair, or in promoting the polymerase gap-filling step.

X-ray cross complementing 1 (XRCC1) protein has been suggested to bind to DNA single-strand breaks (SSBs) and organize protein interactions that facilitate efficient DNA repair. Using four site-specifically modified human XRCC1 mutant expression systems and functional complementation assays in Chinese hamster ovary (CHO) XRCC1-deficient EM9 cells, we evaluated the cellular contributions of XRCC1s proposed N-terminal domain (NTD) DNA binding and DNA polymerase beta (POLbeta) interaction activities. Results within demonstrate that the interaction with POLbeta is biologically important for alkylating agent resistance and SSB repair, whereas the proposed DNA binding function is not critical to these phenotypes. Our data favor a model where the interaction of XRCC1 with POLbeta contributes to efficient DNA repair in vivo, whereas its interactions with target DNA is biologically less relevant.

X-ray Repair Cross Complementing 1 (XRCC1) is thought to function as a scaffolding protein in both base excision repair and single-strand break repair (SSBR), since it interacts with several proteins participating in these related pathways and has no known enzymatic activity. Moreover, studies indicate that XRCC1 possesses discrete G1 and S phase-specific functions. To further define the contribution of XRCC1 to DNA metabolism, we determined the in vivo localization pattern of this protein and searched for novel protein interactors. We report here that XRCC1 co-localizes with proliferating cell nuclear antigen (PCNA) at DNA replication foci, observed exclusively in the S phase of undamaged HeLa cells. Furthermore, fluorescence resonance energy transfer (FRET) analysis and co-immunoprecipitation indicate that XRCC1 and PCNA are in a complex and likely physically interact in vivo. In vitro biochemical analysis demonstrated that these two proteins associate directly, with the interaction being mediated by residues between amino acids 166 and 310 of XRCC1. The current evidence suggests a model where XRCC1 is sequestered via its interaction with PCNA to sites of DNA replication factories to facilitate efficient SSBR in S phase.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins that is mediated by poly(ADP-ribose) polymerases (PARPs). Although the existence and nature of the nucleic acid-like molecule poly(ADP-ribose) (PAR) has been known for 40 years, understanding its biological functions--originally thought to be only the regulation of chromatin superstructure when DNA is broken--is still the subject of intense research. Here, we review the mechanisms controlling the biosynthesis of this complex macromolecule and some of its main biological functions, with an emphasis on the most recent advances and hypotheses that have developed in this rapidly growing field.

The Cockayne syndrome B (CSB) protein--defective in a majority of patients suffering from the rare autosomal disorder CS--is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP-DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2'-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.

There is currently a great interest in the efficiency of micronutrients against age-associated disorders. The present study aimed to evaluate the efficacy of beta-carotene on the incidence of lymphoid neoplasia, a fatal pathology associated with OFI mouse ageing. Beta-carotene, given as a water-dispersible preparation to 8-month-old mice, on a four month follow-up study, significantly reduced the incidence of neoplasm (12.5% versus 50% for controls). Evaluation of the parameters of oxidative stress showed a highly-significant reduction of the antioxidant defenses in the liver of cancer mice when compared to healthy controls (78% decrease in GSH-Px activity and 47% decrease of the ratio GSH/GSSG). Liver GSH-Px activity was 35% higher in old than in young mice, which correlated with higher (41%) plasma Se level. In conclusion beta-carotene improved the antioxidant status of the mice, causing a 4.5-fold increase in the liver GSH/GSSG ratio, an effect which was probably responsible for the lowered incidence of neoplasia observed.

Over the years, several lines of evidence have emerged supporting the role of oxidative stress in the development of diabetic complications. This could involve the increase in the production of reactive oxygen species and the decrease in antioxidative defense systems. Modulation of the level of intracellular reactive oxygen species is likely to affect the intracellular redox homeostasis, which is crucial for numerous biological events such as the transcriptional activation of genes. In this work we studied the binding of the redox transcription factors Sp1 and NF-kappaB extracted from kidney and liver of streptozotocin diabetic (STZ) and fructose-fed rats using electrophoretic mobility shift (EMSA) assay. In addition, the level in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) was assessed within DNA by high performance liquid chromatography with electrochemical detection (HPLC-EC). A decrease in the affinity of Sp1 to DNA was observed in the kidney of STZ rats and fructose-fed rats (15% +/- 8.3 and 54% +/- 6.9, respectively, versus control group set to 100%). This was also found to occur to a lower extent, in the liver. Interestingly, higher levels of 8-oxodGuo, a biomarker of DNA oxidation, were measured in the kidney of diabetic rats. Therefore, the modification in the binding efficiency of Sp1 or NF-kappaB could be related to reactive oxygen species-mediated DNA damage.

Mononuclear cells generate a variety of hormone-like proteins termed growth factors that are instrumental in the evolution and resolution of inflammatory reactions. Many of these growth regulatory molecules have multifunctional properties. For example, the mononuclear cell-derived growth factors, platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-beta), are potent leukocyte chemoattractants. In addition, TGF-beta, a product of platelets, T lymphocytes, and monocytes, appears to induce the transcription of other monocyte-derived growth hormone genes. In this regard, picomolar concentrations of TGF-beta stimulate peripheral blood monocytes to transcribe the genes for PDGF (c-sis), basic fibroblast growth factor (FGF), interleukin 1 (IL-1), and tumor necrosis factor (TNF). Furthermore, levels of mRNA for TGF-beta, which is constitutively expressed in resting monocytes, are also increased by exogenous TGF-beta. Each of these monocyte products exhibits a plethora of biological activities on other cell types. T lymphocytes, in response to antigen, contribute to this network by secreting growth factors and lymphokines that regulate monocyte growth factor production.