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1.
J Clin Pediatr Dent ; 41(3): 219-224, 2017.
Article in English | MEDLINE | ID: mdl-28422591

ABSTRACT

OBJECTIVE: To compare the effect of Papacarie and Atraumatic Restorative Treatment (ART) on pain and discomfort during caries removal among children. STUDY DESIGN: Fifty healthy, 4-8 year-old children were equally and randomly allocated to Papacarie and ART to remove caries from decayed primary teeth. A randomized, controlled, blinded, two parallel-arms clinical trial was conducted in the clinic of the Pediatric Dentistry and Dental Public Health Department, Alexandria University, Egypt in March 2014. Pain and discomfort were assessed blindly by two independent investigators watching videotaped treatment sessions using the Sound, Eye and Motor scale (SEM). Their reliability was assessed using Kappa statistics. The effect of caries removal methods, time spent to remove caries and other confounders on SEM score was assessed using regression analysis. RESULTS: Mean time to remove caries using Papacarie and ART was 5.8 and 4.8 minutes, P= 0.005. Median Paparie and ART scores for the S, E and M components were 1, 1, 1 and 3, 2, 3. Adjusted mean SEM score= 3.6 and 7.8, P <0.0001. Method of caries removal was the only factor significantly affecting pain and discomfort. CONCLUSION: Papacarie is associated with minimal pain during caries removal from primary teeth compared to ART, although it has longer working time.


Subject(s)
Dental Atraumatic Restorative Treatment/methods , Dental Caries/therapy , Dental Cavity Preparation/methods , Pain Measurement , Papain , Child , Child, Preschool , Female , Humans , Male
2.
Cancer Chemother Pharmacol ; 45(4): 312-9, 2000.
Article in English | MEDLINE | ID: mdl-10755320

ABSTRACT

PURPOSE: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468. METHODS: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed lusing neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases alpha and delta). RESULTS: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 microM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase alpha enzyme while the DNA polymerase delta seems to be resistant to the inhibitory effect of that drug. CONCLUSIONS: Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase alpha and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cytarabine/pharmacology , DNA Replication/drug effects , DNA, Neoplasm/biosynthesis , Antigens, Polyomavirus Transforming/metabolism , Arabinofuranosylcytosine Triphosphate/pharmacology , Breast Neoplasms/metabolism , Cell Division/drug effects , DNA Polymerase I/biosynthesis , DNA Polymerase III/biosynthesis , Humans , Replicon/drug effects , Tumor Cells, Cultured
3.
Cancer Chemother Pharmacol ; 45(4): 320-8, 2000.
Article in English | MEDLINE | ID: mdl-10755321

ABSTRACT

PURPOSE: Gemcitabine (dFdC) and cytarabine (araC) are both analogs of deoxycytidine. Gemcitabine is a relatively new drug that has been shown in both clinical trials and in vitro systems to have more potent antitumor activity than araC. We have previously isolated a fully functional multiprotein DNA replication complex from human cells and termed it the DNA synthesome. Using the DNA synthesome, we have successfully examined the mechanism of action of several anticancer drugs that directly affect DNA synthesis. In this study, we compared the effects of dFdC and araC on in vitro DNA synthesis mediated by the DNA synthesome with the effects of these drugs on intact MCF7 cell DNA synthesis. METHODS: We examined the effects of dFdC and araC on intact MCF7 cell DNA synthesis and clonogenicity. We also performed in vitro SV40 replication assays mediated by the MCF7 cell-derived DNA synthesome in presence of dFdCTP and araCTP. The types of daughter molecules produced in the assay were analyzed by neutral and alkaline agarose gel electrophoresis. Finally, we examined the effects ofdFdCTP and araCTP on the synthesome-associated DNA polymerase alpha and delta activities. RESULTS: Our results showed that dFdC was more potent than araC at inhibiting intact MCF7 cell DNA synthesis and clonogenicity. [3H]Thymidine incorporation was inhibited by 50% at a dFdC concentration of 10 microM, which was about tenfold lower than the concentration of araC required to inhibit intact cell DNA synthesis by the same amount. As examined by clonogenicity assay, dFdC was also significantly more cytotoxic than araC after a 24-h incubation. In vitro SV40 replication assays using the DNA synthesome derived from MCF7 cells demonstrated that the formation of full-length DNA along with replication intermediates were inhibited by dFdCTP in a concentration-dependent manner. Full-length DNA was produced in the in vitro DNA replication assay even when the dFdCTP was incubated in the assay at concentrations of up to 1 mM. We observed that in the presence of 10 microM dCTP, 3 microM dFdCTP and 60 microM araCTP were required to inhibit in vitro SV40 DNA synthesis by 50%. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there was no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome-associated DNA polymerases alpha and delta. It was found that the drug concentrations required to inhibit 50% of the synthesome-associated DNA polymerase delta activity were much higher than those required to inhibit 50% of DNA polymerase alpha activity for both dFdCTP and araCTP. CONCLUSION: Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase alpha activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Breast Neoplasms/metabolism , Cytarabine/pharmacology , DNA, Neoplasm/biosynthesis , Deoxycytidine/analogs & derivatives , Antigens, Polyomavirus Transforming/biosynthesis , Antigens, Polyomavirus Transforming/genetics , Arabinofuranosylcytosine Triphosphate/pharmacology , Breast Neoplasms/genetics , Cloning, Molecular , DNA Polymerase I/metabolism , DNA Polymerase III/metabolism , Deoxycytidine/pharmacology , Humans , Replicon/drug effects , Replicon/genetics , Tumor Cells, Cultured , Gemcitabine
4.
Nucleic Acids Res ; 27(19): 3799-804, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10481018

ABSTRACT

The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase alpha, and that this role for DNA polymerase epsilon cannot be modeled by SV40 DNA replication.


Subject(s)
DNA Polymerase II/metabolism , DNA Replication , DNA, Viral/biosynthesis , Simian virus 40/genetics , Animals , Antibodies/immunology , Bromodeoxyuridine/metabolism , Catalytic Domain , Cattle , Cell Line , DNA Polymerase II/antagonists & inhibitors , DNA Polymerase II/immunology , Fibroblasts/cytology , HeLa Cells , Humans , Neutralization Tests , Rabbits , Simian virus 40/physiology , Virus Replication
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