Abstract Aims Heart failure (HF) is one of the leading causes of the global burden of disability and mortality. However, the comprehensive epidemic status of HF in China is unclear. Notably, the gender‐specific survey for HF prevalence is lacking. The present study aimed to analyse the gender‐specific prevalence and temporal trend of HF in China and explore the attributable aetiology and risk factors. Methods and results The Global Burden of Diseases, Injuries, and Risk Factors Study 2019 was used to evaluate the age‐standardized prevalence and years lived with disability of HF in China by gender. The temporal trend of HF and attributable risk factors were analysed by Joinpoint regression models from 1990 to 2019. The total age‐standardized prevalence rate of HF steadily decreased over the past two decades from 1079.4 to 1032.8 per 100 000 individuals. Since 2017, the prevalence trend of HF has significantly increased [annual percentage change (APC) of 2.72 for females and 0.61 for males, P < 0.05]. In 2019, the age‐standardized rate of HF prevalence in females surpassed that of males, and hypertensive heart disease was the leading cause of HF for females (42.65% of cases) and males (41.19% of cases). From 2017 to 2019, high systolic pressure contributed to most cases of HF‐related hypertensive heart disease, with an APC of 2.68 for females and 0.48 for males ( P < 0.05). Conclusions Although HF prevalence has steadily decreased over the past two decades, an increasing trend has occurred since 2017, especially for females. The leading cause of HF was hypertensive heart disease. Metabolic risks, particularly high systolic pressure, consistently contribute to the prevalence of heart diseases leading to HF. Promoting HF screening and controlling metabolic risks at the population level are imperative. Gender differences in HF prevalence should be considered.
Dysregulation of the phosphatidylinositol-3-kinase (PI3K) pathway in a wide range of tumors has made PI3K a consensus target to inhibit as illustrated by more than 15 inhibitors now in clinical trials. Our previous work, built on the early pioneering multikinase inhibitor LY294002, resulted in the only PI3K vascular-targeted PI3K inhibitor prodrug, SF1126, which has now completed Phase I clinical trials. This inhibitor has properties that impart more in vivo activity than should be warranted by its enzymatic potency, which in general is much lower than other clinical stage PI3K inhibitors. We embarked on the exploration of scaffolds that retained such properties while simultaneously exhibiting an increased potency toward PI3K. This work resulted in the discovery of the 5-morpholino-7H-thieno[3,2-b]pyran-7-one system as the foundation of a new compound class of potential PI3K inhibitors having improved potency toward PI3K. The synthesis and cancer stem cell-based activity of these compounds are reported herein.
The incidence of thyroid carcinoma is associated with a variety of factors.Radiation is the clear risk factor,the relationship between iodine intake and thyroid carcinoma remains controversial.Researches show that the genetic and epigenetic changes of many signaling pathways are the key of molecular pathogenetic mechanism of thyroid carcinoma.In addition,thyroid stimulating hormone,body mass index and chronic lymphocytic thyroiditis are also associated with thyroid carcinoma.
Key words:
Thyroid neoplasms ; Incidence ; Risk factors
Abstract SF1126 is in development in phase I clinical trials as a single agent and interim results have been presented recently in patients with solid tumors (ASCO, 2009, abstract 2558) and multiple myeloma (ASH, 2009, abstract 3879). SF1126 inhibits all four class I phosphatidylinositol 3-kinase (PI3K) isoforms along with other cancer targets such as mTOR, DNA-PK, PIM1, and PLK1. The current ongoing work reported herein describes translational studies providing support for expanding single agent clinical trials (B-cell malignancies such as CLL and mantle cell lymphoma) and combination trials such as with bortezomib in multiple myeloma. Gene expression profiles from myeloma cell lines exposed to SF1126 have identified that HSP-90AA1 is down regulated in common between 3 different myeloma cell lines, while genes for 14-3-3h, cyclin D1, and EIF4BEP1 are commonly downregulated among 2 of the 3 cell lines tested. Genes that are upregulated in common include Bcl-2, Gab2, and CDK2 further supporting the role of cell cycle regulation as a major effect of PI3K inhibition in myeloma cell lines. Additional data on response to SF1126 exposure will be presented in B-cell, T-cell, and mantle cell lymphoma cell lines and primary patient CLL samples. The in vitro combination of SF1126 with dexamethasone (dex) was examined since dex is so commonly used in myeloma patients. Three day proliferation results on RPMI8266 cells (non-responsive to dexamethasone and KRAS mutated) exposed to 5 uM dex plus 5, 10, or 20 uM SF1126 showed a 12, 46, and 78% decrease, respectively, in cell proliferation as measured by the WST staining method versus a no-SF1126-treatment control. The ability of SF1126 to inhibit hematological cancer stem cells was also evaluated. CD138neg cells were isolated from RPMI8226 cell lines as described in the literature (anti-CD138 antibody-conjugated magnetic beads to serially remove CD138+ cells) to give a highly enriched fraction purported to be cancer stem cell ‘like’. SF1126 at 5, 10, or 20 uM demonstrated an 18, 32, and 50% decrease in CD138neg cell proliferation relative to no-treatment controls. These results compared favorably to lenalidomide in the same experiment which showed no decrease in proliferation. These results show for the first time that SF1126 may have activity towards hematological cells characterized as cancer stem cells. In vitro combinations of SF1126 with bortezomib on several myeloma cell lines have previously been shown to exhibit synergistic effects. SF1126 has also previously been shown to have single agent activity in vivo using MM1R (dexamethasone resistant myeloma) xenograft mouse models. The MM1R xenograft mouse model was thus chosen to study SF1126/bortezomib combinations in vivo. Thirty nude mice with similar MM1R tumor sizes were randomly divided into 5 groups of 6 on day 21 post s.c. tumor cell inoculation and treated as follows (all injections were in 100 uL volume administered iv in the tail vein on Tues/Friday 2X/week schedule): 1) saline control; 2) SF1126 at 20 mg/kg; 3) Bortezomib 0.5 mg/kg (1/2 MTD); 4) Bortezomib followed by SF1126 one hour later; 5) SF1126 followed by bortezomib one hour later. Since bortezomib is given iv in the clinic and SF1126 is given as an iv infusion (90 minutes) in the clinic groups 4 and 5 were designed to determine if there is any difference in order of the agents when given in combination. Tumor volume and body weights were assessed through day 42 (3 weeks of treatment). Groups 2, 3, 4, and 5 showed 49, 47, 74, and 85% inhibition of tumor growth relative to the control (group 1). Both of the combination groups 4 and 5 (regardless of order) showed statistical significance compared to control tumor volume (p<0.005). Excised tumor weights showed the same relationship with groups 2, 3, 4, and 5 showing 23, 22, 56, and 66% less tumor weight versus the group 1 control tumor weight with groups 4 and 5 being significant (p<0.01). No treatment related body weight changes were evident and all groups showed increasing body weight gain over the three week treatment period. A second combination study in MM1R xenograft nude mice was performed but dosing was changed to 2X/week using a two weeks on/one week off schedule followed by two weeks more treatment to mimic the clinical schedule using bortezomib (days 1,4,8,11 of a 21 day cycle) and taking into account the clinical administration of SF1126 (twice weekly with no holidays). Twenty nude mice with similar MM1R tumor size were randomly divided into 4 groups of 5 on day 20 post s.c. tumor cell inoculation and treated as follows (all injections were in 100 uL volume administered iv in the tail vein on a 2X/week schedule): 1) saline control; 2) SF1126 at 20 mg/kg; 3) Bortezomib 0.5 mg/kg (1/2 MTD); 4) SF1126 followed by bortezomib one hour later. Tumor volume and body weights were assessed through day 52. This treatment course represented 2 weeks on, one week off, then finally 2 weeks on. Groups 2 and 3 (single agents) showed 70 and 52% inhibition of tumor growth respectively with the combination (group 3) showing the most inhibition at 82% inhibition of tumor growth relative to the control (group 1). Excised tumor weights showed the same relationship with groups 2, 3, and 4 showing 59, 35 and 71% less tumor weight versus the group 1 control tumor weight. No treatment related body weight changes were evident and all groups showed increasing body weight gain over the three week treatment period although the control group showed the largest weight gain. These results indicate in vivo combination effects and help guide the planning of clinical combination studies using SF1126 and bortezomib in multiple myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-291.
Abstract Aims Fibroblast growth factor 23 (FGF23) has been implicated in the occurrence of atrial fibrillation (AF), but its prognostic value in AF patients remains unclear. Methods and results A total of 35 197 AF patients with available follow-up data (3.56, 0.47–8.92 years) from the UK Biobank were included. Clinical association between serum FGF23 and AF-related outcomes including mortality, heart failure (HF), ischaemic stroke, and dementia were analysed using multivariable Cox regression. In those passed quality control for array sequencing, polygenic score for FGF23 (PGSFGF23) was calculated as genetic instrument, and the association between PGSFGF23 and the occurrence of endpoints after first AF diagnosis were further explored. In 886 patients who diagnosed AF at or prior to the enrolment, elevated serum FGF23 levels were significantly associated with an increased risk of all-cause (37% increase per standard deviation) and cardiovascular (40% increase per standard deviation) mortality and HF (43% increase per standard deviation). A total of 35 197 patients were available for genetic array sequencing data. Using polygenic score including seven independent SNPs reaching genome-wide significance threshold, genetic association analysis indicated that increased PGSFGF23 is associated with reduced risk of HF but increased risk of all-cause mortality and ischaemic stroke. Conclusion Our findings suggest that FGF23 is a potential biomarker for accessing AF-related outcomes. The paradoxical association between genetic FGF23 and serum FGF23 level highlights the need for further investigation to elucidate the underlying mechanisms.
To study the inhibitory effect of flavonoids from Glycyrrhiza uralensis on thioacetamide-induced chonic hepatic fibrosis in rats and the effect on the protein expressions of transforming growth factor-β1 (TGF-β1) and Caspase-3 in livers.Male Sprague-Dawley rats were randomly divided into totally seven groups: the normal control group, the model group, LF groups s (400, 200, 100, 50 mg · kg(-1) · d(-1)) and the silymarin positive control group (30 mg · kg(-1) · d(-1)). The hepatic fibrosis model was induced in the rats through intraperitoneal injection with 3% thioacetamide (TAA) at a dose of 150 mg · kg(-1) body weight twice a week for 12 weeks. During the course, the control group and the model group were orally administered with saline (1 mL · kg(-1) · d(-1)). After the modeling and drug intervention, the pathologic changes and fibrosis in liver tissues were observed by HE staining and Masson's Trichrome staining. The serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and liver hydroxyproline (HYP) contents were assayed by biochemical process. The serum hyaluronic acid (HA) was assessed by radioimmunoassay. In addition, the protein expressions of liver TGF-β1 and Caspase-3 were examined by immunohistochemical method. The mRNA expression of TGF-β1 in hepatic tissues was examined by quantitative Real-time PCR analysis.Compared with the model group, flavonoids can protect the integrity of the structure of liver tissues, significantly reduce the hepatic cell degeneration and necrosis and the proliferation of fibrous tissues, notably reduce the serum AST, ALT, ALP and HA and HYP in hepatic tissues and down-regulate the protein expressions of liver TGF-β1 and Caspase-3 and the mRNA expression of TGF-β1 in hepatic tissues.The licorice flavonoids can resist the thioacetamide-induced hepatic fibrosis in rats. Its mechanism may be related to the down-regulation of the protein expressions of TGF-β1 and Caspase-3.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe yet rare inherited arrhythmia disorder. The cornerstone of CPVT medical therapy is the use of β-blockers; 30% of patients with CPVT do not respond well to optimal β-blocker treatment. Studies have shown that flecainide effectively prevents life-threatening arrhythmias in CPVT. Flecainide is a class IC antiarrhythmic drug blocking cardiac sodium channels. RyR2 inhibition is proposed as the principal mechanism of antiarrhythmic action of flecainide in CPVT, while it is highly debated. In this article, we review the current progress of this issue.
Cancer has become significant comorbidity in patients with atrial fibrillation (AF). However, little is known about the efficacy and safety of AF ablation, the first-line rhythm control strategy, in patients with cancer. This study aims to evaluate the incidence and risk of AF recurrence and safety endpoints in patients with cancer compared to the non-cancer group after ablation.From August 2011 to December 2020, we consecutively enrolled cancer patients in the China-AF cohort. We used propensity score matching (1:3) to select the control group and assessed the risk of AF recurrence and adverse events after ablation in cancer patients using a multivariable Fine and Gray competing risk model.A total of 203 patients with cancer were enrolled and 21 of them were active cancer, with a median follow-up of 12.3 months. The cumulative incidence of AF recurrence was comparable between patients with and without cancer (43.8% vs. 51.1%; p = .88). No difference in the risk of AF recurrence, thromboembolism, major bleeding, and mortality was observed after adjusting confounders. Active cancer was not associated with an increased risk of AF recurrence compared to the stable disease (SHR = 1.32; 95% CI 0.72-2.43; p = .46). Cancer was associated with a low risk of cardiovascular hospitalization (SHR, 0.54; 95% CI, 0.36-0.81; p = .01). Subgroup analysis found that hematological malignancy was associated with a high risk of AF recurrence (SHR, 5.68; 95% CI, 3.00-10.8; p < .001).This study suggests that catheter ablation could be feasible for rhythm control of AF patients with concomitant cancer.
Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G1/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs+)), as measured by [3H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs−) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs+ cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs− cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs+cells but not in DNA-PKcs− cells. Reversal of UV-induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA-containing fraction from UV-irradiated DNA-PKcs+ cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs− cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation. Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G1/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs+)), as measured by [3H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs−) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs+ cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs− cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs+cells but not in DNA-PKcs− cells. Reversal of UV-induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA-containing fraction from UV-irradiated DNA-PKcs+ cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs− cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation. replication protein A proliferating cell nuclear antigen ataxia-telangiectasia mutant phosphatidylinositol phosphate-buffered saline ammonium sulfate DNA-dependent protein kinase DNA-PK catalytic subunit severe combined immune deficiency Cells exposed to UV irradiation are predominantly arrested in S-phase rather than at the G1/S boundary while repair occurs (1Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (775) Google Scholar). The molecular mechanism of damage-induced S-phase arrest is not known; however, the effects of UV irradiation during S-phase on subsequent cell cycles are magnified in repair-deficient cells (2Orren D.K. Petersen L.N. Bohr V.A. Cell. 1997; 8: 1129-1142Google Scholar), indicating that these effects may be initiated by DNA damage itself. 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Res. 1997; 9: 295-302PubMed Google Scholar,61Muller C. Salles B. Oncogene. 1997; 15: 2343-2348Crossref PubMed Scopus (41) Google Scholar). These results suggest that DNA-PK not only senses DNA damage but also functions as a transmitter of signals that allow repair of damaged DNA and protects cells from apoptosis. Previous studies with UV-irradiated HeLa cells suggested a role for RPA in UV-induced inhibition of replication because this event was reversed by the addition of RPA (3Carty M.P. Zernik-kobak M. McGrath S. Dixon K. EMBO J. 1994; 13: 2114-2123Crossref PubMed Scopus (169) Google Scholar). In this report, we investigated a role for DNA-PK in replication arrest following UV irradiation. We present evidence that DNA-PK plays an essential role in UV-induced replication arrest, such that DNA-PK, upon UV irradiation, acts to induce replication arrest without affecting DNA repair activity. SV40 replication origin-containing plasmid, pSV01ΔEP, and SV40 T-antigen were prepared as described previously (62Stigger E. Dean F.B. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (59) Google Scholar). Antipolymerase α monoclonal antibody (SJK237) and anti-RPA (p34 and p70) polyclonal antibodies (from rabbits) were described previously (62Stigger E. Dean F.B. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (59) Google Scholar). An anti-DNA-PKcs antibody was a kind gift from Dr. C. Anderson (Brookhaven National Laboratory), and an anti-PCNA antibody was purchased from Calbiochem. DNA-PK holoenzyme was purified from HeLa cells according to the procedure described previously (44Lees-Miller S.P. Chen Y.-R. Anderson C.W. Mol. Cell. Biol. 1990; 10: 6472-6481Crossref PubMed Scopus (357) Google Scholar). Two malignant glioblastoma cells, M059K (DNA-PK+) and M059J (DNA-PK-) were obtained from Dr. M. J. Allalunis-Turner (Cross Cancer Institute, Edmonton, Canada), mouse SCID-st cells were from Drs. J. M. Brown and C. Kirchgessner (Stanford University School of Medicine, Palo Alto, CA), and NIH3T3 was from Dr. M. Marshall (Indiana University School of Medicine, Indianapolis, IN). Monolayer culture of HeLa cells, M059K, and M059J were grown in tissue culture dishes (150 × 25 mm) in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum, and mouse SCID-st and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C in a CO2 incubator. Culture dishes with 80% confluence were washed twice with 10 ml of phosphate-buffered saline (PBS) and were exposed to UV-C light (GE, G30T8) in the presence of 5 ml of PBS. After adding fresh medium, UV-irradiated cells were further incubated for the indicated amount of time at 37 °C in a CO2 incubator. Nonirradiated cells were also prepared the same way without UV irradiation. To study the effect of wortmannin, cells were pretreated with wortmannin (1.0 μm) for 1 h prior to UV irradiation and continued to grow in the presence of wortmannin until time of harvest. Cells (5 × 105/60-mm dish) were incubated with 0.5 μCi/ml [3H]thymidine (75 Ci/mmol) for 1 h prior to UV irradiation at 10 J/m2. At the indicated time points, cell metabolism was stopped by the addition of 0.1 volume of 2.3m citric acid. After washing the cells with PBS, DNA was precipitated with 10% trichloroacetic acid at 4 °C for 2 h, followed by acid-insoluble radioactivity measurement. Cytosolic cell extracts were prepared according to the procedure originally described by Li and Kelly (63Li J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6973-6977Crossref PubMed Scopus (352) Google Scholar). Briefly, monolayer cells were washed twice with PBS and hypotonic buffer. After removing the excess amount of buffer, the swollen cells were scraped into a Dounce homogenizer (approximate volume of 0.2 ml/dish) and dounced 5–8 times on ice. Cell lysates were then centrifuged at 14,000 rpm for 30 min at 4 °C to remove nuclear pellets and the insoluble materials. Ammonium sulfate (AS) fractionation of cytosolic cell extracts was done as described previously (9Wobbe C.R. Weissbach L. Borowiec J.A. Dean F.B. Murakami Y. Bullock P.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1834-1838Crossref PubMed Scopus (257) Google Scholar). Immunoblotting was performed as described previously (62Stigger E. Dean F.B. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (59) Google Scholar). Protein fractions were separated on a 12% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose (Millipore Corp.), and immunoblotted with either monoclonal or polyclonal antibodies. After incubation with either125I-protein A or 125I-protein G, proteins were visualized by autoradiography. Replication reactions were carried out as described previously (26Lee S.-H. Kim D.K. J. Biol. Chem. 1995; 270: 12801-12807Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, reaction mixtures (40 μl) contained 40 mm creatine phosphate-di-Tris salt (pH 7.7); 1 μg of creatine kinase; 7 mmMgCl2; 0.5 mm dithiothreitol; 4 mmATP; 200 μm UTP, GTP, and CTP; 100 μm dTTP, dGTP, and dCTP; 20 μm [α-32P]dATP (specific activity of 30,000 cpm/pmol); 0.8 μg of SV40 T-antigen; 0.3 μg of SV40 origin-containing DNA (pSV01ΔEP); and the indicated amounts of RPA. The reaction mixtures were incubated for 60 min at 37 °C and then stopped with 40 μl of stop solution containing 20 mm EDTA, 1% SDS, and Escherichia coli tRNA (0.5 mg/ml). One-tenth of the reaction mixture was used to measure the acid-insoluble radioactivity. Replication products in the remaining reaction mixture were analyzed electrophoretically, separating the isolated DNA in a 1% agarose gel with TBE buffer. The gel was subsequently dried and exposed to x-ray film. Repair of UV-damaged DNA was carried out according to the published procedure (64Stigger E. Drissi R. Lee S.-H. J. Biol. Chem. 1998; 273: 9337-9343Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Reaction mixtures (50 μl) contained 0.2 μg of UV-irradiated (450 J/m2) pBS (3 kilobase pairs) and nonirradiated p5A (4.5 kilobase pairs); 40 mm creatine phosphate-di-Tris salt (pH 7.7); 1 μg of creatine kinase; 50 mm HEPES-KOH (pH 7.8); 70 mm KCl; 7.5 mm MgCl2; 0.5 mm dithiothreitol; 0.4 mm EDTA; 2 mm ATP; 20 μm dGTP, dCTP, and TTP; 8 μm [α-32P]dATP (25,000 cpm/pmol), 5 μg of bovine serum albumin; and the indicated amounts of the whole cell extracts. After incubation for 3 h at 30 °C, DNA was isolated from the reaction mixtures, linearized with BamHI, and separated on a 1% agarose gel electrophoresis in the presence of 0.5 μg/ml ethidium bromide. The DNA and repair products were analyzed by both fluorography and autoradiography. Reaction mixtures (20 μl) contained kinase buffer (50 mm HEPES (pH 7.5), 2 mm EGTA, 0.1 mm EDTA, 100 mm KCl, 10 mmMgCl2, and 125 μm [γ-32P]ATP (specific activity, 30,000 cpm/pmol)), 150 μm substrate peptide (44Lees-Miller S.P. Chen Y.-R. Anderson C.W. Mol. Cell. Biol. 1990; 10: 6472-6481Crossref PubMed Scopus (357) Google Scholar), 0.4 μg of calf-thymus activated DNA, and increasing amounts of cell lysates (1 and 2 μg). After a 30-min incubation at 30 °C, the reaction mixtures were stopped by the addition of 30% acetic acid. Reaction mixtures (5 μl) were spotted onto a P-81 strip, and after extensive washing radioactivity was measured. DNA-PK activity was shown as pmol of 32P transferred to the substrate peptide. To understand the molecular mechanism of UV-induced replication arrest, we examined whether DNA-PK plays a role in this regulatory event. For this, two malignant glioblastoma human cells (M059K (DNA-PKcs+) and M059J (DNA-PKcs−)) (65Lees-Miller S.P. Godbout R. Chan D.W. Weinfeld M. Day III, R.S. Barron G.M. Allalunis-Turner M.J. Science. 1995; 267: 1183-1185Crossref PubMed Scopus (502) Google Scholar) were labeled with [3H]thymidine (0.5 μCi/ml) and examined for in vivo DNA synthesis at various time points following a low dose of UV irradiation (10 J/m2). The amounts of DNA synthesis in asynchronously grown M059K (DNA-PKcs+) and M059J (DNA-PKcs−) cells were similar in the absence of UV irradiation, but DNA synthesis was significantly inhibited following UV irradiation (Fig. 1). Most importantly, much tighter replication arrest was observed with DNA-PKcs+cells compared with that with DNA-PKcs− cells up to 8 h following UV irradiation (Fig. 1), suggesting a possible role for DNA-PKcs (or its holoenzyme) in UV-induced replication arrest. It should be pointed out, however, that the inhibition of replication following UV damage in DNA-PKcs+ cells could be due to the G1 checkpoint arrest that results in fewer cells traversing the G1/S boundary. To further investigate a role for DNA-PKcs in UV-induced replication arrest, we prepared cell extracts from M059K (DNA-PKcs+) and M059J (DNA-PKcs−) cells at various times following UV irradiation (10 J/m2) and examined in vitro DNA replication activity using SV40 origin-containing DNA. Replication activity of DNA-PKcs+ cells sharply declined within 2 h following UV irradiation, whereas DNA-PKcs− cells were only slightly affected by UV irradiation (Fig.2 A, lanes 1–3 versus lanes 6–8). A striking difference between DNA-PKcs+ and DNA-PKcs− was observed 12–24 h after UV irradiation, such that the reversal of inhibition of replication was observed in DNA-PKcs+ cells, but not in DNA-PKcs− cells (Fig. 2 A, lane 5 versus lane 10). Treatment of cells with wortmannin (1.0 μm), an inhibitor of DNA-PK, abolished both rapid replication arrest and the reversal of the arrest in UV-irradiated DNA-PKcs+ cells but showed a gradual decrease in replication activity similar to that observed in DNA-PKcs−cells (Fig. 2 A, lanes 11–15). Reversal of replication arrest in DNA-PKcs+ cells occurred in a UV dose-dependent manner, which requires low UV dosage (10 J/m2) (Fig. 2 B). With high dose UV irradiation, DNA-PK+ cells may induce apoptotic signal without DNA replication. In contrast to replication arrest, UV irradiation had no effect on nucleotide excision repair activity regardless of DNA-PKcs presence or absence (Fig. 2 D). Taken together, these results strongly suggest that DNA-PKcs plays a crucial role in UV-induced replication arrest, and that may also be necessary for the reversal of the arrest.Figure 2Replication and repair activities of DNA-PKcs + and DNA-PKcs − cells following UV irradiation. A, extracts were prepared from UV-irradiated (10 J/m2) DNA-PKcs+ (M059K) and DNA-PKcs− (M059J) cells at various time points (0, 2, 4, 8, and 24 h) and were examined for their replication activity. Where indicated, cells (DNA-PKcs+) were treated with 1 μm of wortmannin for 1 h prior to UV irradiation. Reaction mixtures contained SV40 origin-containing DNA (pSV01ΔEP), 200 μg of cell extracts, and 0.8 μg of SV40 T-antigen. After a 60-min incubation at 37 °C, DNA was isolated and analyzed on 1.0% neutral agarose gel electrophoresis. B and C, replication arrest in DNA-PKcs+ (B) and DNA-PKcs− (C) cells in response to various UV doses. The extent of DNA synthesis (dAMP incorporated, in pmol) was quantitated by acid-insoluble radioactivity. D, nucleotide excision repair activity of DNA-PKcs+ and DNA-PKcs− cells. Where indicated, 150 μg (lanes 1, 3, 5,7, 9, and 11) and 300 μg (lanes 2, 4, 6,8, 10, and 12) of whole cell extracts were added. The top panel indicates a fluorograph of the gel, and the bottom panel is an autoradiogram.View Large Image Figure ViewerDownload Hi-res image Download (PPT) If DNA-PKcs (or DNA-PK holoenzyme) is directly involved in UV-induced replication arrest, we may also see stimulation of replication with cell extracts from UV-irradiated DNA-PKcs+ cells by blocking DNA-PK kinase activity. To examine this, cell extracts from either nonirradiated or UV-irradiated DNA-PKcs+ cells were preincubated with wortmannin at 37 °C for 30 min and examined for replication activity (Fig.3 A). Replication arrest caused by UV irradiation of DNA-PKcs+ cells was reversed up to 80% by incubating cell extracts with wortmannin (Fig. 3 A, lanes 5–7) under conditions where replication activity of nonirradiated DNA-PKcs+ cells was only slightly stimulated (Fig. 3 A, lanes 2–4). In contrast, replication activity of cell extracts from DNA-PKcs− cells was unaffected by wortmannin in the presence or absence of UV irradiation (Fig. 3 B). The amount of wortmannin (1.0 μm) used in this experiment was sufficient to inhibit more than 95% of DNA-PK kinase activity present in cell extracts (Fig. 3 C). Reversal of UV-induced replication arrest required preincubation of extracts with wortmannin at 37 °C prior to replication reaction (Fig. 3 D), suggesting that DNA-PK is involved in replication arrest through a modulation of target protein. Together, this in vitro result is consistent with the in vivo observations (Figs. 1 and 2) that DNA-PKcs (or its holoenzyme) plays a crucial role in UV-induced replication arrest. It is sti
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