RESUMEN
BACKGROUND AND HYPOTHESIS: Current treatments for schizophrenia are only partially effective, and there are no medications for negative symptoms or cognitive impairment. Neuromodulation, such as repetitive transcranial magnetic stimulation (rTMS), has potential as a novel intervention for schizophrenia. Prior to clinical use, rTMS should have demonstrated safety in a large schizophrenia population. However, the safety profile of rTMS in schizophrenia is not well characterized, and regulatory agencies have expressed concern about safety in this population. STUDY DESIGN: We conducted a systematic review with meta-analysis of rTMS studies in schizophrenia. We searched PubMed, the Cochrane Library, PsycINFO, and Science Citation Index Expanded for rTMS studies in schizophrenia that reported adverse effects. We extracted the number of participants who experienced an adverse effect and calculated the prevalence of each adverse effect for active or sham rTMS. We tested the difference between the prevalence of events in the active and sham conditions. We assessed risk of bias using the Cochrane Handbook. STUDY RESULTS: The initial search identified 1472 studies. After screening, 261 full-text studies were assessed, and 126 met inclusion criteria (Nâ =â 4122 total subjects). The prevalence of headache or scalp pain, dizziness or syncope, facial twitching, and nausea was higher for active rTMS compared to sham (Pâ <â .05). The prevalence of all other adverse effects, including seizure, was not different between active and sham rTMS. CONCLUSIONS: rTMS is safe and well tolerated for people with schizophrenia. Individuals with schizophrenia are not at increased risk for adverse effects, including seizure, compared to the general population.
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Epidemiological studies have demonstrated the cancer protective effects of dietary agents and other natural compounds isolated from fruits, soybeans, and vegetables on neoplasia. Studies have also revealed the potential for these natural products to be combined with chemotherapy or radiotherapy for the more effective treatment of cancer. In this paper we discuss the potential for targeting the DNA base excision repair enzyme APE1/Ref-1 using dietary agents such as soy isoflavones, resveratrol, curcumin, and the vitamins ascorbate and α-tocopherol. We also discuss the potential role of soy isoflavones in sensitizing cancer cells to the effects of radiotherapy. A comprehensive review of the dual nature of APE1/Ref-1 in DNA repair and redox activation of cellular transcription factors, NF-κB and HIF-1α, is also discussed. Further research efforts dedicated to delineating the role of APE1/Ref-1 DNA repair versus redox activity in sensitizing cancer cells to conventional treatment are warranted.
RESUMEN
Dietary intake of foods rich in antioxidant properties is suggested to be cancer protective. Foods rich in antioxidant properties include grape (Vitis vinifera), one of the world's largest fruit crops and most commonly consumed fruits in the world. The composition and cancer-protective effects of major phenolic antioxidants in grape skin and seed extracts are discussed in this review. Grape skin and seed extracts exert strong free radical scavenging and chelating activities and inhibit lipid oxidation in various food and cell models in vitro. The use of grape antioxidants are promising against a broad range of cancer cells by targeting epidermal growth factor receptor (EGFR) and its downstream pathways, inhibiting over-expression of COX-2 and prostaglandin E2 receptors, or modifying estrogen receptor pathways, resulting in cell cycle arrest and apoptosis. Interestingly, some of these activities were also demonstrated in animal models. However, in vivo studies have demonstrated inconsistent antioxidant efficacy. Nonetheless, a growing body of evidence from human clinical trials has demonstrated that consumption of grape, wine and grape juice exerts many health-promoting and possible anti-cancer effects. Thus, grape skin and seed extracts have great potential in cancer prevention and further investigation into this exciting field is warranted.
RESUMEN
Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-kappaB, and the major 5'-endonuclease in base excision repair (BER). We utilized mice containing a heterozygous gene-targeted deletion of APE1/Ref-1 (Apex(+/-)) to determine the impact of APE1/Ref-1 haploinsufficiency on the processing of oxidative DNA damage induced by 2-nitropropane (2-NP) in the liver tissue of mice. APE1/Ref-1 haploinsufficiency results in a significant decline in NF-kappaB DNA-binding activity in response to oxidative stress in liver. In addition, loss of APE1/Ref-1 increases the apoptotic response to oxidative stress, in which significant increases in GADD45g expression, p53 protein stability, and caspase activity are observed. Oxidative stress displays a differential impact on monofunctional (UNG) and bifunctional (OGG1) DNA glycosylase-initiated BER in the liver of Apex(+/-) mice. APE1/Ref-1 haploinsufficiency results in a significant decline in the repair of oxidized bases (e.g., 8-OHdG), whereas removal of uracil is increased in liver nuclear extracts of mice using an in vitro BER assay. Apex(+/-) mice exposed to 2-NP displayed a significant decline in 3'-OH-containing single-strand breaks and an increase in aldehydic lesions in their liver DNA, suggesting an accumulation of repair intermediates of failed bifunctional DNA glycosylase-initiated BER.
Asunto(s)
Proteínas Portadoras/metabolismo , Daño del ADN/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Hígado/metabolismo , Estrés Oxidativo/genética , Animales , Apoptosis , Proteínas Portadoras/genética , Caspasas/metabolismo , ADN Glicosilasas/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Activación Enzimática/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Hígado/efectos de los fármacos , Hígado/patología , Ratones , FN-kappa B/metabolismo , Nitroparafinas/toxicidad , Propano/análogos & derivados , Propano/toxicidad , Unión Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Uracil-ADN Glicosidasa/metabolismoRESUMEN
We previously showed that treatment of prostate cancer cells with soy isoflavones and radiation resulted in greater cell killing in vitro, and caused downregulation of NF-kappaB and APE1/Ref-1. APE1/Ref-1 functions as a redox activator of transcription factors, including NF-kappaB and HIF-1alpha. These molecules are upregulated by radiation and implicated in radioresistance of cancer cells. We extended our studies to investigate the role of HIF-1alpha survival pathway and its upstream Src and STAT3 molecules in isoflavones and radiation interaction. Radiation induced phosphorylation of Src and STAT3 leading to induction of HIF-1alpha. Genistein, daidzein or a mixture of soy isoflavones did not activate this pathway. These data were observed both in PC-3 (AR-) and C4-2B (AR+) androgen-independent cell lines. Pretreatment with isoflavones inhibited Src/STAT3/HIF-1alpha activation by radiation and nuclear translocation of HIF-1alpha. These findings correlated with decreased expression of APE1/Ref-1 and DNA binding activity of HIF-1alpha and NF-kappaB. In APE1/Ref-1 cDNA transfected cells, radiation caused a greater increase in HIF-1alpha and NF-kappaB activities but this effect was inhibited by pretreatment with soy prior to radiation. Transfection experiments indicate that APE1/Ref-1 inhibition by isoflavones impairs the radiation-induced transcription activity of NF-kappaB and HIF-1alpha. This mechanism could result in the inhibition of genes essential for tumor growth and angiogenesis, as demonstrated by inhibition of VEGF production and HUVECs tube formation. Our novel findings suggest that the increased responsiveness to radiation mediated by soy isoflavones could be due to pleiotropic effects of isoflavones blocking cell survival pathways induced by radiation including Src/STAT3/HIF-1alpha, APE1/Ref-1 and NF-kappaB.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de la radiación , Isoflavonas/farmacología , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Western Blotting , Línea Celular Tumoral , ADN-(Sitio Apurínico o Apirimidínico) Liasa/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/efectos de la radiación , Ensayo de Cambio de Movilidad Electroforética , Técnica del Anticuerpo Fluorescente , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , FN-kappa B/efectos de los fármacos , FN-kappa B/efectos de la radiación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/efectos de la radiación , Glycine max/química , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Familia-src Quinasas/efectos de los fármacos , Familia-src Quinasas/efectos de la radiaciónRESUMEN
We previously showed that genistein, the major bioactive component of soy isoflavones, acts as a radiosensitizer and potentiates prostate tumor cell killing by radiation in vitro and in animal tumor models in vivo. However, when given alone in vivo, pure genistein promoted increased lymph node metastasis, which was not observed with a soy isoflavone mixture consisting of genistein, daidzein, and glycitein. In this study, we show that soy inhibit tumor cell growth and potentiates radiation-induced cell killing in vitro like pure genistein. In an orthotopic model, combining soy isoflavones with tumor irradiation inhibited prostate tumor growth. To determine the molecular mechanisms by which soy isoflavones potentiate radiotherapy, we investigated apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) and nuclear factor kappaB (NF-kappaB), two signaling molecules involved in survival pathways. Soy isoflavones decreased APE1/Ref-1 expression in vitro, whereas radiation up-regulated it. Pretreatment with soy isoflavones followed by radiation inhibited APE1/Ref-1 expression. APE1/Ref-1 decrease correlated with decreased DNA-binding activity of NF-kappaB mediated by soy isoflavones and radiation, thus promoting cell killing. In vivo treatment of prostate tumors with soy isoflavones and radiation down-regulated APE1/Ref-1 protein expression and NF-kappaB activity, confirming the molecular alterations observed in vitro. The down-regulation of APE1/Ref-1 and NF-kappaB by isoflavones, in vitro and in vivo, supports our hypothesis that these markers represent biological targets of isoflavones. Indeed, a 2-fold increase in APE1/Ref-1 expression, obtained by cDNA transfection, resulted in a 2-fold increase in NF-kappaB DNA-binding activity, and both of which were down-regulated by soy isoflavones, confirming the cross-talk between these molecules and, in turn, causing radiosensitization.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Isoflavonas/farmacología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/radioterapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Genisteína/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Extractos Vegetales/farmacología , Neoplasias de la Próstata/patología , Glycine max/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously reported that genistein, the bioactive isoflavone of soybeans, acts as a radiosensitizer for prostate cancer. Pretreatment of tumor cells with genistein potentiated radiation-induced killing in vitro and in orthotopic models in vivo. However, pure genistein promoted increased lymph node metastasis, when administered alone in vivo. We investigated in vitro and in vivo the effects of soy isoflavones (genistein, daidzein and glycitein) as soy pills of similar composition are used in human interventions but not pure genistein. Soy isoflavones inhibited cell survival and potentiated radiation cell killing in PC-3 tumor cells, in vitro. Increased cell killing correlated with inhibition of antiapoptotic molecules Bcl-xL and survivin, upregulation of proapoptotic Bax molecule and PARP cleavage, suggesting activation of apoptotic pathways. In vivo, using the PC-3 orthotopic metastatic mouse model, soy isoflavones and prostate tumor irradiation led to enhanced control of primary tumor growth and metastasis, as observed with pure genistein and radiation. Interestingly, treatment with soy isoflavones did not increase metastasis to para-aortic lymph nodes in contrast to the consistent increase caused by pure genistein. Histologically prostate tumors, treated with soy isoflavones and radiation, showed tumor destruction and in situ tissue alterations, comparable with genistein and radiation effects. However, genistein, but not soy isoflavones, caused induction of HIF1-alpha in prostate tumors, suggesting that induction of hypoxia by pure genistein could contribute to increased metastasis. Our studies demonstrate the safety and potential role of soy isoflavones for enhancing the therapeutic effect of radiotherapy in prostate cancer.
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Genisteína/farmacología , Glycine max/química , Neoplasias de la Próstata/radioterapia , Radioterapia , Animales , Western Blotting , Línea Celular Tumoral , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND: We have previously reported the potentiation of radiotherapy by the soy isoflavone genistein for prostate cancer using prostate tumor cells in vitro and orthotopic prostate tumor models in vivo. However, when genistein was used as single therapy in animal models, it promoted metastasis to regional para-aortic lymph nodes. To clarify whether these intriguing adverse effects of genistein are intrinsic to the orthotopic prostate tumor model, or these results could also be recapitulated in another model, we used the orthotopic metastatic KCI-18 renal cell carcinoma (RCC) model established in our laboratory. METHODS: The KCI-18 RCC cell line was generated from a patient with papillary renal cell carcinoma. Following orthotopic renal implantation of KCI-18 RCC cells and serial in vivo kidney passages in nude mice, we have established a reliable and predictable metastatic RCC tumor model. Mice bearing established kidney tumors were treated with genistein combined with kidney tumor irradiation. The effect of the therapy was assessed on the primary tumor and metastases to various organs. RESULTS: In this experimental model, the karyotype and histological characteristics of the human primary tumor are preserved. Tumor cells metastasize from the primary renal tumor to the lungs, liver and mesentery mimicking the progression of RCC in humans. Treatment of established kidney tumors with genistein demonstrated a tendency to stimulate the growth of the primary kidney tumor and increase the incidence of metastasis to the mesentery lining the bowel. In contrast, when given in conjunction with kidney tumor irradiation, genistein significantly inhibited the growth and progression of established kidney tumors. These findings confirm the potentiation of radiotherapy by genistein in the orthotopic RCC model as previously shown in orthotopic models of prostate cancer. CONCLUSION: Our studies in both RCC and prostate tumor models demonstrate that the combination of genistein with primary tumor irradiation is a more effective and safer therapeutic approach as the tumor growth and progression are inhibited both in the primary and metastatic sites.
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Antineoplásicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/radioterapia , Genisteína/farmacología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/radioterapia , Animales , Terapia Combinada , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Ratones DesnudosRESUMEN
A trend in investigating the use of several nutritional compounds for cancer chemoprevention has revealed that phytochemicals demonstrated anti-cancer properties by inhibiting signal transduction pathways essential for cancer cell proliferation, tumor growth, invasion and metastasis. Emerging evidence suggests that the anti-proliferative and anti-oxidant effects of some of these dietary agents could be utilized to both potentiate the response of cancer cells to radiotherapy and reduce radiation-induced toxicity to normal surrounding tissues. Using pre-clinical orthotopic models of prostate cancer, studies on the combination of soy isoflavones with tumor irradiation demonstrate a synergistic anti-cancer effect between these two modalities and emphasize the potential and safety of dietary factors to improve conventional radiotherapy for a better control of tumor growth and metastasis. The goal of this review is to focus on the role of soy isoflavones as potent radiosensitizers for prostate cancer and other malignancies. We will discuss molecular pathways regulated by soy isoflavones that inhibit survival pathways activated by radiation and ultimately drive the cells to cell death both in vitro and in vivo in pre-clinical models.
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Glycine max/química , Neoplasias de la Próstata/radioterapia , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Radioterapia , Animales , Humanos , Masculino , Neoplasias de la Próstata/patologíaRESUMEN
Pretreatment with genistein, a bioactive component of soy isoflavones, potentiated cell killing induced by radiation in human PC-3 prostate cancer cells in vitro. Using an orthotopic xenograft in nude mice, we demonstrated that genistein combined with prostate tumor irradiation caused greater inhibition of primary tumor growth and increased control of spontaneous metastasis to para-aortic lymph nodes, increasing mouse survival. Paradoxically, treatment with genistein alone increased metastasis to lymph nodes. This observation is of concern in relation to soy-based clinical trials for cancer patients. To address whether this observation is because nude mice have an impaired immune system, these studies were repeated in orthotopic RM-9 prostate tumors in syngeneic C57BL/6 mice. The combination of genistein with radiation in this model also caused a greater inhibition of primary tumor growth and spontaneous metastasis to regional para-aortic lymph nodes, whereas treatment with genistein alone showed a trend to increased lymph node metastasis. Data from the syngeneic and xenograft models are comparable and indicate that the combination of genistein with radiotherapy is more effective and safer for prostate cancer treatment than genistein alone, which promotes metastatic spread to regional lymph nodes.
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Supervivencia Celular/efectos de la radiación , Modelos Animales de Enfermedad , Genisteína/administración & dosificación , Neoplasias de la Próstata/radioterapia , Neoplasias de la Próstata/secundario , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Quimioterapia Adyuvante , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/efectos de la radiación , Metástasis Linfática , Masculino , Ratones , Neoplasias de la Próstata/patología , Dosis de Radiación , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Resultado del TratamientoRESUMEN
Young (4- to 6-month-old) and aged (24- to 28-month-old) mice were exposed to 2-nitropropane (2-NP), a DNA oxidizing agent, and the ability to induce DNA polymerase beta (beta-pol) and AP endonuclease (APE) was determined. In contrast to the inducibility of these gene products in response to oxidative damage in young mice, aged mice showed a lack of inducibility of beta-pol and APE. APE protein level and endonuclease activity were both reduced 40% (p<.01) in response to 2-NP. Accordingly, the accumulation of DNA repair intermediates in response to 2-NP differed with age. Young animals accumulated 3'OH-containing DNA strand breaks, whereas the aged animals did not. A role for p53 in the difference in DNA damage response with age is suggested by the observation that the accumulation of p53 protein in response to DNA damage in young animals was absent in the aged animals. Our results are consistent with a reduced ability to process DNA damage with age.
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Envejecimiento/fisiología , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Estrés Oxidativo , Factores de Edad , Análisis de Varianza , Animales , Western Blotting , Daño del ADN , Reparación del ADN/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/efectos de los fármacos , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos , Nitroparafinas/farmacología , Probabilidad , Propano/análogos & derivados , Propano/farmacología , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
BACKGROUND: New cancer therapeutic strategies must be investigated that enhance prostate cancer treatment while minimizing associated toxicities. We have previously shown that genistein, the major isoflavone found in soy, enhanced prostate cancer radiotherapy in vitro and in vivo. In this study, we investigated the cellular and molecular interaction between genistein and radiation using PC-3 human prostate cancer cells. METHODS: Tumor cell survival and progression was determined by clonogenic analysis, flow cytometry, EMSA analysis of NF-kappaB, and western blot analysis of cyclin B1, p21WAF1/Cip1, and cleaved PARP protein. RESULTS: Genistein combined with radiation caused greater inhibition in PC-3 colony formation compared to genistein or radiation alone. Treatment sequence of genistein followed by radiation and continuous exposure to genistein showed optimal effect. Cell cycle analysis demonstrated a significant dose- and time-dependent G2/M arrest induced by genistein and radiation that correlated with increased p21WAF1/Cip1 and decreased cyclin B1 expression. NF-kappaB activity was significantly decreased by genistein, yet increased by radiation. Radiation-induced activation of NF-kappaB activity was strongly inhibited by genistein pre-treatment. A significant and striking increase in cleaved PARP protein was measured following combined genistein and radiation treatment, indicating increased apoptosis. CONCLUSION: A mechanism of increased cell death by genistein and radiation is proposed to occur via inhibition of NF-kappaB, leading to altered expression of regulatory cell cycle proteins such as cyclin B and/or p21WAF1/Cip1, thus promoting G2/M arrest and increased radiosensitivity. These findings support the important and novel strategy of combining genistein with radiation for the treatment of prostate cancer.
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Anticarcinógenos/farmacología , Apoptosis , Fase G2 , Genisteína/farmacología , FN-kappa B/metabolismo , Neoplasias de la Próstata , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular , Terapia Combinada/métodos , Ciclina B/metabolismo , Ciclina B1 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales/métodos , Fase G2/efectos de los fármacos , Fase G2/efectos de la radiación , Humanos , Masculino , FN-kappa B/efectos de los fármacos , FN-kappa B/efectos de la radiación , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/radioterapiaRESUMEN
The mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase beta (beta-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce beta-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, beta-pol-haploinsufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young beta-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and beta-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for beta-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases.
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ADN Polimerasa beta/genética , Deficiencia de Ácido Fólico/genética , Alimentación Animal , Animales , Western Blotting , Núcleo Celular/metabolismo , ADN/metabolismo , Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , Suplementos Dietéticos , Ácido Fólico/metabolismo , Heterocigoto , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Químicos , Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Uracilo/química , Uracilo/metabolismo , Uracil-ADN GlicosidasaRESUMEN
Apurinic/apyrimidinic (AP) endonuclease (APE) is a multifunctional protein possessing both DNA repair and redox regulatory activities. In base excision repair (BER), APE is responsible for processing spontaneous, chemical, or monofunctional DNA glycosylase-initiated AP sites via its 5'-endonuclease activity and 3'-"end-trimming" activity when processing residues produced as a consequence of bifunctional DNA glycosylases. In this study, we have fully characterized a mammalian model of APE haploinsufficiency by using a mouse containing a heterozygous gene-targeted deletion of the APE gene (Apex(+/-)). Our data indicate that Apex(+/-) mice are indeed APE-haploinsufficient, as exhibited by a 40-50% reduction (p < 0.05) in APE mRNA, protein, and 5'-endonuclease activity in all tissues studied. Based on gene dosage, we expected to see a concomitant reduction in BER activity; however, by using an in vitro G:U mismatch BER assay, we observed tissue-specific alterations in monofunctional glycosylase-initiated BER activity, e.g. liver (35% decrease, p < 0.05), testes (55% increase, p < 0.05), and brain (no significant difference). The observed changes in BER activity correlated tightly with changes in DNA polymerase beta and AP site DNA binding levels. We propose a mechanism of BER that may be influenced by the redox regulatory activity of APE, and we suggest that reduced APE may render a cell/tissue more susceptible to dysregulation of the polymerase beta-dependent BER response to cellular stress.
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ADN Polimerasa beta/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/deficiencia , Animales , Disparidad de Par Base , Sitios de Unión , Encéfalo/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Haplotipos , Heterocigoto , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Especificidad de Órganos , Oxidación-Reducción , Testículo/metabolismoRESUMEN
The base excision repair pathway (BER) is believed to maintain genomic integrity by repairing DNA damage arising spontaneously or induced by oxidizing and alkylating agents. To establish the role of DNA polymerase beta (beta-pol) in BER and beta-pol-dependent BER in maintaining genomic stability, we have measured the impact of a gene-targeted disruption in the beta-pol gene on DNA repair capacity and on in vivo sensitivity to carcinogens. We have extensively phenotyped the DNA beta-pol heterozygous (beta-pol(+/-)) mouse as expressing approximately 50% less beta-pol mRNA and protein and as exhibiting an equivalent reduction in the specific activity of beta-pol. We measured BER activity by in vitro G:U mismatch and (8-OH)G:C repair and find that there is a significant reduction in the ability of extracts from beta-pol(+/-) mice to repair these types of DNA damage. In vivo, the beta-pol(+/-) mice sustain higher levels of DNA single-strand breaks as well as increased chromosomal aberrations as compared with beta-pol(+/+) littermates. Additionally, we show that reduction in beta-pol expression and BER activity results in increased mutagenicity of dimethyl sulfate as evidenced by a 2-fold increase in LacI mutation frequency. Importantly, the beta-pol(+/-) mice do not exhibit increased sensitivity to DNA damage induced by N-nitroso-N-ethylurea, ionizing radiation, or UV radiation, which induce damage processed by alternative repair pathways, demonstrating that this model is specifically a BER-deficient model.
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Carcinógenos/toxicidad , Daño del ADN , ADN Polimerasa beta/deficiencia , Reparación del ADN/fisiología , Animales , ADN Polimerasa beta/biosíntesis , ADN Polimerasa beta/genética , Masculino , Ratones , Organismos Libres de Patógenos EspecíficosRESUMEN
Caloric restriction is a potent experimental manipulation that extends mean and maximum life span and delays the onset and progression of tumors in laboratory rodents. While caloric restriction (CR) clearly protects the genome from deleterious damage, the mechanism by which genomic stability is achieved remains unclear. We provide evidence that CR promotes genomic stability by increasing DNA repair capacity, specifically base excision repair (BER). CR completely reverses the age-related decline in BER capacity (P<0.01) in all tissues tested (brain, liver, spleen and testes) providing aged, CR animals with the BER phenotype of young, ad libitum-fed animals. This CR-induced reversal of the aged BER phenotype is accompanied by a reversal in the age-related decline in DNA polymerase beta (beta-pol), a rate-limiting enzyme in the BER pathway. CR significantly reversed the age-related loss of beta-pol protein levels (P<0.01), mRNA levels (P<0.01) and enzyme activity (P<0.01) in all tissues tested. Additionally, in young (4-6-month-old) CR animals a significant up-regulation in BER capacity, beta-pol protein and beta-pol mRNA is observed (P<0.01), demonstrating an early effect of CR that may provide insight in distinguishing the anti-tumor from the anti-aging effects of CR. This up-regulation in BER by caloric restriction in young animals corresponds to increased protection from carcinogen exposure, as mutation frequency is significantly reduced in CR animals exposed to either DMS or 2-nitropropane (2-NP) (P<0.01). Overall the data suggest an important biological consequence of moderate BER up-regulation and provides support for the hormesis theory of caloric restriction.
Asunto(s)
Envejecimiento/genética , Restricción Calórica , Reparación del ADN/fisiología , Propano/análogos & derivados , Envejecimiento/metabolismo , Animales , ADN/efectos de los fármacos , ADN Polimerasa beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutágenos/farmacología , Mutación , Nitroparafinas/farmacología , Propano/farmacología , Ratas , Ratas Endogámicas F344 , Ésteres del Ácido Sulfúrico/farmacología , Regulación hacia ArribaRESUMEN
Base excision repair (BER) is the DNA repair pathway primarily responsible for repairing small base modifications and abasic sites caused by normal cellular metabolism or environmental insult. Strong evidence supports the requirement of DNA polymerase beta (beta-pol) in the BER pathway involving single nucleotide gap filling DNA synthesis in mammalian systems. In this study, we examine the relationship between oxidative stress, cellular levels of beta-pol and BER to determine whether oxidizing agents can upregulate BER capacity in vivo. Intraperitoneal injection of 2-nitropropane (2-NP, 100 mg/kg), an oxidative stress-inducing agent, in C57BL/6 mice was found to generate 8-hydroxydeoxyguanosine (8-OHdG) in liver tissue (4-fold increase, P < 0.001). We also observed a 4-5-fold increase in levels of DNA single strand breaks in 2-NP treated animals. The protein level of the tumor suppressor gene, p53 was also induced in liver by 2-NP (2.1-fold, P < 0.01), indicating an induction of DNA damage. In addition, we observed a 2-3-fold increase in mutant frequency in the lacI gene after exposure to 2-NP. Interestingly, an increase in DNA damage upregulated the level of beta-pol as well as BER capacity (42%, P < 0.05). These results suggest that beta-pol and BER can be upregulated in response to oxidative stress in vivo. Furthermore, data show that heterozygous beta-pol knockout (beta-pol(+/-)) mice express higher levels of p53 in response to 2-NP as compared with wild-type littermates. While the knockout and wild-type mice display similar levels of 8-OHdG after 2-NP exposure, the beta-pol(+/-) mice exhibit a significant increase in DNA single strand breaks. These findings suggest that in mice, a reduction in beta-pol expression results in a higher accumulation of DNA damage by 2-NP, thus establishing the importance of the beta-pol-dependent BER pathway in repairing oxidative damage.
Asunto(s)
ADN Polimerasa beta/metabolismo , Reparación del ADN/fisiología , Estrés Oxidativo/fisiología , Propano/análogos & derivados , Animales , ADN/efectos de los fármacos , ADN/metabolismo , Daño del ADN , ADN Polimerasa beta/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Mutación , Nitroparafinas/farmacología , Propano/farmacologíaRESUMEN
The biological mechanisms responsible for aging remain poorly understood. We propose that increases in DNA damage and mutations that occur with age result from a reduced ability to repair DNA damage. To test this hypothesis, we have measured the ability to repair DNA damage in vitro by the base excision repair (BER) pathway in tissues of young (4-month-old) and old (24-month-old) C57BL/6 mice. We find in all tissues tested (brain, liver, spleen and testes), the ability to repair damage is significantly reduced (50-75%; P<0.01) with age, and that the reduction in repair capacity seen with age correlates with decreased levels of DNA polymerase beta (beta-pol) enzymatic activity, protein and mRNA. To determine the biological relevance of this age-related decline in BER, we measured spontaneous and chemically induced lacI mutation frequency in young and old animals. In line with previous findings, we observed a three-fold increase in spontaneous mutation frequency in aged animals. Interestingly, lacI mutation frequency in response to dimethyl sulfate (DMS) does not significantly increase in young animals whereas identical exposure in aged animals results in a five-fold increase in mutation frequency. Because DMS induces DNA damage processed by the BER pathway, it is suggested that the increased mutagenicity of DMS with age is related to the decline in BER capacity that occurs with age. The inability of the BER pathway to repair damages that accumulate with age may provide a mechanistic explanation for the well-established phenotype of DNA damage accumulation with age.