Systematic analysis of DNA polymerases as therapeutic targets in pan-cancers

Introduction:DNA polymerases are crucial for maintaining genome stability and influencing tumorigenesis.However,the clinical implications of DNA polymerases in tumorigenesis and their potential as anti-cancer therapy targets are not well understood.Methods:We conducted a systematic analysis using TCGA Pan-Cancer Atlas data and Gene Set Cancer Analysis results to examine the expression profiles of 15 DNA polymerases(POLYs)and their clinical correlations.We also evaluated the prognostic value of POLYs by analyzing their expression levels in relation to overall survival time(OS)using Kaplan-Meier survival curves.Additionally,we investigated the correlations between POLY expression and immune cells,DNA damage repair(DDR)pathways,and ubiquitination.Drug sensitivity analysis was performed to assess the relationship between POLY expression and drug response.Results:Our analysis revealed that 14 out of 15 POLYs exhibited significantly distinct expression patterns between tumor and normal samples across most cancer types,except for DNA nucleotidylexotransferase(DNTT).Specifically,POLD1 and POLE showed elevated expression in almost all cancers,while POLQ exhibited high expression levels in all cancer types.Some POLYs showed heightened expression in specific cancer subtypes,while others exhibited low expression.Kaplan-Meier survival curves demonstrated significant prognostic value of POLYs in multiple cancers,including PAAD,KIRC,and ACC.Cox analysis further validated these findings.Alteration patterns of POLYs varied significantly among different cancer types and were associated with poorer survival outcomes.Significant correlations were observed between the expression of POLY members and immune cells,DDR pathways,and ubiquitination.Drug sensitivity analysis indicated an inverse relationship between POLY expression and drug response.Conclusion:Our comprehensive study highlights the significant role of POLYs in cancer development and identifies them as promising prognostic and immunological biomarkers for various cancer types.Additionally,targeting POLYs therapeutically holds promise for tumor immunotherapy.

Biochemical Characterization of Translesion Synthesis by Sulfolobus acidocaldarius DNA Polymerases

To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polBl（Saci_1537） and polB3（Saci_0074） possessed DNA polymerase and 3＇ to 5＇ exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV（Saci_0554） was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn1＋ and Mg2＋. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KC1 with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 ℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.

Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

THE RADIOBIOLOGIC CHARACTERISTICS OF DNA POLYMERASE β IN HEPATOMAS

To investigate the effects of γ rays on DNA polymerase β properties and its DNA repair functions before or after γ rays exposure, DNA polymerase βactivity, gene expression and mRNA levels in SMMC-LTNM hepatomas horn on nude mice or the samples of the liver cancer tissues from 15 patients were measured with 3H-TTP incorporation test, immunocytochemistry and cytoplasmic dot hybridization analysis, respectively.Irradiation was carried out with 60Co-γ rays at ice bath. It was found that DNA polymerase β activity, gene expression and the amount of mRNA were much higher in hepatoma cells than those in normal hepatocytes (P<0.01). In vitro studies, the enzyme activity both in hepatoma and normal liver cells appeared unchanged within 40 Gy γ-ray exposure. Following whole-body exposure of the nude mice bearing SMMC-LTNM with 2 Gy or 4 Gy of γ rays, DNA polymerase β activity in hepatoma increased temorarily at 48 hours postirradiation, and its gene expression seemed more active.The euzyme mRNA increased to 1.76-fold of the control group. 72 hours after exposure, all of these changes returned to normal levels. DNA polymerase βparticipated in DNA repair synthesis and this effect was different between hepatoma and hepatocytes because there were some biologic differences of the enzyme between hepatoma cells and normal liver cells. These data suggested that DNA polymeraseβactivity, its gene expression and mRNA level in hepatomas could increased temporarily after γ rays exposure, which may facilitate the cells to repair DNA damages from radiation.

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.

Mutation of DNA polymerase p in esophageal carcinoma of different regions

AIM： To observe the variation of DNA polymerase β （polβ） in esophageal carcinoma. METHODS： Thirty specimens containing adjacent normal epithelial tissues were collected from patients in Linzhou region （a high risk area for esophageal squamous carcinoma） and 25 specimens were from a non-high risk area. Total RNA was extracted from the samples and reverse transcription polymerase chain reaction （RT-PCR） was performed. PCR products were cloned and sequenced to investigate the po1β gene with DNASIS and OMIGA. Statistical significance was evaluated using the X^2 test. RESULTS： High-inddence area group： Polβ gene variation was detected in 13 of 30 esophageal carcinoma tissue specimens, and only one variation was found in 30 corresponding adjacent normal tissue specimens. Non high-incidence area group： po1β gene variation was detected in 5 of 25 esophageal carcinoma tissue specimens, and no variation was found in 25 corresponding adjacent normal tissue specimens. The incidence of po1β gene variation observed in the high-incidence area group was significantly higher than in the non-high incidence area group. Two mutation hot spots （454-466 and 648-670 nt） and a 58 bp deletion （177-234 nt） were found. CONCLUSION： Variations of polβ perform different functions between the high-incidence areas and the other areas, and may play a more important role in the high-incidence areas.

Possible Role of DNA Polymerase beta in Protecting Human Bronchial Epithelial Cells Against Cytotoxicity of Hydroquinone

Objective To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. Methods DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone （ranged from 10 μmol/L to 120 μmol/L） for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis （SCGE）] were performed respectively to detect the toxicity of hydroquinone. Results assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. Conclusions Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

Biochemical Characterization of Uracil-DNA Glycosylase from Pyrococcus furiosus

We report the characterization of a uracil-DNA glycosylase（UDG） from the hyperthermophilic archaea Pyrococcus furiosus（P, furiosus）. P. furiosus UDG（PfUDG） has high sequence similarity to the families IV and V UDGs（thermostable UDG family and PaUDG-b family）. PfUDG excises uracil from various DNA substrates with the following order： U/T=U/C〉U/G=U/AP=U/-〉U/U=U/I=U/A. The optimal temperature and pH value for uracil exci- sion by PfUDG are 70 ℃ and 9.0, respectively. The removal of U is inhibited by the divalent ions of Fe, Ca, Zn, Cu, Co, Ni and Mn, as well as a high concentration of NaC1. The phosphorothioates near uracil strongly inhibit the exci- sion of uracil by PfUDG. Interestingly, pfuDNA（Pyrococcusfuriosus DNA） polymerase, which tightly binds the ura- cil-carrying oligonucleotide, does not inhibit the excision by Pfl.IDG, suggesting PfUDG in vivo functions as the re- pair enzyme to excise uracil damage in genome.

Effect and mechanism of β-L-D4A on DNA polymerase α

AIM： To investigate the safety of β-L-D4A on DNA polymerase α.METHODS： Ion exchange chromatography was used to separate DNA polymerase cc from crude extract of human Hela cells. Detailed kinetic parameters were determined for β-L-D4A against DNA polymeraseα . RESULTS： DNA polymerase was purified with 4% yield and 31000 units/mg specific activity. The Michaelis constant （Km = 3.22 i^mol/L）, 50% inhibition concentration （IC50 = 178.49 μmol/L） and inhibition constant （Ki = 126 mol/L） of β-L-D4A were determined by kinetic analysis. CONCLUSION： β-L-D4A is a more safe nucleoside for hepatitis B virus （HBV） infection with a lower host toxicity.

Characterization of DNA polymerase δ from deep-sea hydrothermal vent shrimp Rimicaris exoculata

DNA polymerase δ(Polδ)plays a crucial and versatile role in DNA replication and DNA repair processes.Vent shrimp Rimicaris exoculata is the primary megafaunal community living in hydrothermal vents.In this study,the Polδfrom shrimp Rimicaris exoculata was cloned,expressed and characterized.The results showed that the Polδcatalytic subunit(POLD1),852 amino acids in length,shared high homology with crayfish Procambarus clarkii and shrimp Oratosquilla oratoria.The recombinant POLD1 expressed in Escherichia coli showed that the enzyme was active in a range of 20℃ to 40℃ with an optimum temperature at 25℃ and in a wide range of p H with an optimum at pH 6.0.The activities of POLD1 were significantly enhanced in the presence of Triton-X 100,Tween 20 and Mn^(2+).The K_(m)(dNTP)value of POLD1 was 4.7μmol/L.The present study would be helpful to reveal the characterization of Polδof deep-sea vent animals.

THE INHIBITORY EFFECT OF EXTRACT OF CAMELLIA SINENSIS AND EXTRACT OF CAMELLIA PTILOPHYLLA CHANG ON DNA POLYMERASE OF EHRLICH ASCITES CARCINOMA CELLS

Objective: To detect the effect of extract of Camellia Sinensis (ECS) and extract of Camellia Ptilophylla Chang (ECPC) on DNA polymerase (Pol) of Ehrlich ascites tumor cells Methods: Referring to the method of K Ono, Pol was extracted from Ehrlich ascites tumor cells in mice Pol α, β, and γ were separated by phosphocellulose column chromatography and were identified The effect of ECPC and ECS on Pol was studied Results: ECPC and ECS were shown to inhibit the activity of Pol α, β, and γ IC 50 values of ECS on Pol α , β, and γ were 10 2μg/ml, 9 9μg/ml and 28 9μg/ml respectively IC 50 values of ECPC on Pol α, Pol β and Pol γ were 5 6μg/ml, 15μg/ml and 14 7μg/ml respectively The modes of inhibition of ECPC on Pol α, Pol β and Pol γ were noncompetitive with respect to template DNA The Ki values of ECPC on Pol α , β, and γ were 2 68±0 12μg/ml, 2 24±0 12μg/ml , 2 56±0 18μg/ml Conclusion: ECPC and ECS were shown to have inhibitory effect on DNA polymerase of tumor cells The mode of inhibition of ECPC on Pol α, Pol β and Pol γ were noncompetitive with respect to template DNA

SPECIFIC UP-REGULATION OF DNA POLYMERASE BY HUMAN PAPILLOMAVIRUS 16

Obje, etive To analyze how the infection of human papillomavirus 16 （ HPV16 ） affects expression of DNA poly- merase β（DNA polB） with the aim of probing the mechanism of over-expression of DNA polB in human cancers. Methods Four fragments of human DNA polB promoter were amplified and constructed into luciferase reporter vec- tor pGL-Basic, generating pGL-BP, pGL-BMH, pGL-BMS, and pGL-BAT constructs respectively, and co-transfected with HPV16 or HPV6 into Hep2 cells. Luciferase activity was assayed 48 hours after transfectiorL Semi-quantitative RT- PCR was used to measure mRNA expression of endogenous DNA polB. Immunohistochemistry and in situ hybridization were used to analyze DNA polB expression and HPV16 or HPV6 infection in 38 cases of cervical lesions respectively. Results With co-transfection of HPV16 and DNA polB promoter-driving reporters into Hep2 cells, pGL-BP re- porter in full-length DNA polB promoter presented markedly elevated luciferase activities （ P 〈 0.05 ）. However, the other three mutant reporters： pGL-BMH, pGL-BMS, and pGL-BAT, generated no reporting activities in the presence of HPV16 （P 〉0.05 ）. On the contrary, all of polB promoter reporters were little stimulated in co-transfection of HPV6 （P 〉0.05 ）. The transfection of HPV16 could enhance the endogenous polB mRNA expression compared with that of HPV6 （3.42 vs. 0.80, P 〈0.05）. The DNA polB expression was found in 8 of 10 HPV16-positive cervical intraepi- thelial neoplasia grade Ⅲ（ CIN Ⅲ） cases, while was only found in 3 of 11 HPV6-positive condyloma accuminatum cases, but was negative in all chronic cervicitis cases. The correlation of DNA polB expression with HPV16 infection in cervical lesions was significant （ P 〈0.05 ）. Conclusions HPV16 is able to specifically stimulate the expression of DNA polB in human epithelial cells through interaction with the core upstream regulatory sequences of DNA polB promoter. Over-expression of DNA polB might be an explanation for the molecular mechanism underlying HPV-related human cancers.

DNA polymerase delta interacting protein 3 facilitates the activation and maintenance of DNA damage checkpoint in response to replication stress

Background:Replication stress response is crucial for the maintenance of a stable ge-nome.POLDIP3(DNA polymerase delta interacting protein 3)was initially identified as one of the DNA polymeraseδ(Polδ)interacting proteins almost 20 years ago.Using a variety of in vitro biochemical assays,we previously established that POLDIP3 is a key regulator of the enzymatic activity of Polδ.However,the in vivo function of POLDIP3 in DNA replication and DNA damage response has been elusive.Methods:We first generated POLDIP3 knockout(KO)cells using the CRISPR/Cas9 technology.We then investigated its biological functions in vivo using a variety of biochemical and cell biology assays.Results:We showed that although the POLDIP3-KO cells manifest no pronounced defect in global DNA synthesis under nonstress conditions,they are sensitive to a va-riety of replication fork blockers.Intriguingly,we found that POLDIP3 plays a crucial role in the activation and maintenance of the DNA damage checkpoint in response to exogenous as well as endogenous replication stress.Conclusion:Our results indicate that when the DNA replication fork is blocked,POLDIP3 can be recruited to the stalled replication fork and functions to bridge the early DNA damage checkpoint response and the later replication fork repair/restart.

Expression of Human Cytomegalovirus DNA Polymerase in Insect Cells Using Baculovirus Expression System：Purification and Biochemical Characterizations

Human Cytomegalovirus (HCMV) DNA polymerase gene was overexpressed in insect cells using the baculovirus transfer system. A6. 2 kb HCMV Rsr Ⅱ-EcoRI DNA fragment with intact HCMV pci gene coding sequence was engineered into NheI site of vector pBlueBac under the control of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPV carried HCMV pci gene was generated by cotransfection of Spodoptera frugiperta cell (SF21) with AcNPV DNA and baculovirus transfer vector with HCMV pci gene. In fection of SF21 cell with recombinant virus lead to the expression of 140 kD peptide of HCMV specific DNA polymerase at the level approximately 2 mg per 108 cells. The polypeptide was purified from the infected SF21 cells by a series of column chromatography to homogeneity. The purified enzyme had a molecular weight of 140 kD and reacted with antiserum specific for HCMV DNA polymerase. It exhibited both 3'-5'and 5'-3' exonuclease activities.This enzyme is also sensitive to phosphono acetate.

POLE2 Regulates Apoptosis of Oral Squamous Cell Carcinoma Cells through the PI3K/AKT Signaling Pathway

Objective Oral squamous cell carcinoma(OSCC)is the most common malignant tumor of the head and neck,but its occurrence and progression mechanisms remain unclear.In addition-there is a lack of effective targeting drugs.The second major subunit of DNA polymerase(POLE2)catalyzes the prolongation of new strand replication and modifies exonuclease domain activity.Our previous study found that POLE2 was associated with OSCC progression,but the mechanism remains unclear.Methods The expression of POLE2 in OSCC tissues was detected using immunological assays.Mann-Whitney U analysis was used to investigate the relationship between POLE2 gene expression and tumor classification and prognosis of OSCC.POLE2 expression was inhibited in OSCC cells,and the effects of gene and protein expression were detected using RT-PCR and Western blotting.The POLE2 knockout model was constructed by transfecting a lentiviral vector.Cell proliferation,apoptosis,and migration were detected using various assays including colony formation,MTT,flow cytometry,wound healing assay,Transwell assay,and the Human Apoptosis Antibody Array.The animal model of OSCC was established by subcutaneous injection of transfected HN6 into 4-week-old female nude mice.After 30 days,tumors were removed under anesthesia and tumor weight and dimension were recorded.Tumor cell proliferation was analyzed using Ki67 staining.Results POLE2 gene levels were significantly higher in the OSCC tissues than in the normal tissues.In addition,POLE2 gene levels were statistically correlated with tumor classification and prognosis.Silencing POLE2 inhibited the proliferation of oral cancer cells and promoted apoptosis in vitro.Animal experiments also supported a positive correlation between POLE2 and OSCC tumor formation.We further demonstrated that POLE2 could upregulate the expression of apoptosis-related proteins such as caspase-3,CD40,CD40L,DR6,Fas,IGFBP-6,p21,and SMAC.In addition,POLE2 regulated OSCC development by inhibiting the PI3K/AKT signaling pathway.Conclusion POLE2 is closely related to the progression of OSCC.Thus,POLE2 may be a potential target for OSCC treatment.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Sequence Analysis of Attachment Gene of Lumpy Skin Disease and Sheep Poxviruses

In Egypt,protection of cattle against lumpy skin disease (LSD) was carried out using a sheep poxvirus (Kenyan strain) vaccination strategy.In the present study 15 skin nodules from LSD suspected cows and 5 scab samples from sheep pox (SP) suspected sheep were collected.Hyperimmune rabbit sera to Lumpy skin disease virus (LSDV)/Ismailyia88 strain and sheep pox virus (SPV)/ Kenyan vaccinal strain were prepared.The causative agent in the collected samples was identified using immunoflourescence (IF) and immunoperoxidase techniques.Of the 15 skin nodules suspected of LSD,10 showed a positive reaction and 3 out of 5 skin scabs suspected of sheeppox were found to be positive.An antigenic correlation between field skin isolate of LSDV,tissue culture adapted LSDV/Ismailyia88 strain,field skin isolate of SPV and SPV/Kenyan vaccinal strain was studied using prepared hyperimmune sera.Also,nucleotide sequence of the PCR amplified attachment gene fragments of field skin isolate of LSDV,tissue culture adapted LSDV/Ismailyia88 strain,field skin isolate of SPV and SPV /Kenyan vaccinal strain were compared.The results revealed that the four used viruses were antigenically identical.Sequence analysis indicated that field skin LSDV isolate is more related to tissue culture adapted LSDV/Ismailyia88 strain than to vaccinal SPV/ Kenyan strain and the skin isolate of SPV is more closely related to field skin isolate of LSDV than to SPV/Kenyan vaccinal strain.Thus,further study should be applied on the advantage of a LSD vaccine prepared from LSDV in protection of cattle against LSD compared to the commonly used sheep pox vaccine.

IGF-1 Accelerates Cell Aging by Inhibiting POLD1 Expression

Objective The individual cascades of the insulin-like growth factor-1(IGF-1)signaling pathway and the molecular mechanism of aging have not been fully clarified.In the current study,we explored the effect of DNA polymerase delta 1(POLD1)on the IGF-1 signaling pathway in cell aging.Methods First,we analyzed the relationship between IGF-1 and POLD1 expression in aging.To investigate the effect of IGF-1 on POLD1 expression and aging,the 2BS cells were incubated with youngage or old-age human serum,IGF-1 protein,or linsitinib.Next,the effect of IGF-1 on aging was examined in the 2BS cells with increased or decreased POLD1 expression to clarify the molecular mechanism.Results In this study,we found that IGF-1 expression increased and POLD1 expression decreased with aging in human serum and hippocampal tissues of SAMP8 mice,and a negative relationship between IGF-1 and POLD1 expression was observed.Furthermore,the cells cultured with old-age human serum or IGF-1 showed lower POLD1 expression and more pronounced senescence characteristics,and the effect could be reversed by treatment with linsitinib or overexpression of POLD1,while the effect of linsitinib on cell aging could be reversed with the knockdown of POLD1.Conclusion Taken collectively,our findings demonstrate that IGF-1 promotes aging by binding to IGF-1R and inhibiting the expression of POLD1.These findings offer a new target for anti-aging strategies.

Expression of Polt in tissues and cell lines of transitional cell carcino-ma

Objective：To explore the expression of DNA polymerase iota in transitional cell carcinoma ceils and tissues; Methods： RT-PCR was applied to detect the expression of polymerase iota in BIU87 and T24 ceils, then the expression of polymerase iota was also detected in the same way in transitional cell car- cinoma which was derived from clinical bladder carcinoma and renal pelvic carcinoma. Results ： The expres- sion of Poh was low in bladder normal membrana mucosa but significantly elevated in transitional cell car- cinoma ceils. Compared with the expression of polymerase iota in bladder normal mucous membranes, the expression of polymerase iota was significantly increased in transitional cell carcinoma tissue （P〈0. 01 ） and associated with the grade of transitional cell carcinoma. Conclusion： The significantly increased ex- pression of polymerase iota may be associated with the generation and development of transitional cell carcinoma, even with its high heterogenicity.