Biomedical subjects
Publications and source records attributed to A R Bapat.
A new DNA polymerase and DNase activity were identified from cells infected with human B-lymphotropic herpesvirus (HBLV). DNA polymerase associated with HBLV infection was similar in its sensitivity to inhibition by ppi analogs as other herpesvirus-specific DNA polymerases but was dissimilar in its inhibition by certain nucleoside triphosphates.
5'-Deoxy-4',5-difluorouridine (4'-F-5'-dFUrd) (10) has been synthesized on the basis of the rationale that the labilization of the glycosidic linkage caused by the 4'-fluoro substituent might allow this compound to be a better prodrug form of the anticancer drug 5-fluorouracil (FUra) than is the widely studied fluoropyrimidine 5'-deoxy-5-fluorouridine (5'-dFUrd). The rate of solvolytic hydrolysis of the glycosidic linkage of 4'-F-5'-dFUrd at pH 1 was about 500-fold faster than that of 5'-dFUrd. Since uridine phosphorylase is thought to be the enzyme that causes degradation of 5'-dFUrd in vivo to generate FUra, we compared the substrate interactions of 5'-dFUrd and 4'-F-5'-dUrd with this enzyme. The Vmax for hydrolysis of 4'-F-5'-dFUrd to FUra by uridine phosphorylase was about 5-fold greater than that of 5'-dFUrd, whereas the Km value of 4'-F-5'-dFUrd was 10-fold lower. The combination of these two factors results in 4'-F-5'-dFUrd having a 50-fold higher value of V/K than does 5'-dFUrd. Against L1210 cells in culture, the IC50 value for growth inhibition by 4'-F-5'-dFUrd was 3 X 10(-7) compared to 3 X 10(-6) for 5'-dFUrd.
Recently, 2-halogenated deoxyadenosine analogs (F, Cl, and Br) have been shown to have antitumor activity. These analogs are phosphorylated by cells and are believed to exert their cytotoxic action at the nucleoside triphosphate level. In this work the interaction of these nucleoside triphosphate analogs with potential targets, such as DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase was examined in detail. All of these compounds competitively inhibited the incorporation of dAMP into DNA by DNA polymerase alpha, beta, or gamma. F-dATP was able to completely substitute for dATP using DNA polymerase alpha and gamma, but not with DNA polymerase beta. Cl-dATP and Br-dATP substituted poorly for dATP using DNA polymerase alpha and beta. Extension of a 32P-labeled primer by DNA polymerase alpha, beta, or gamma on a single-stranded M13 template showed that these compounds were incorporated into the 3' end of the growing DNA chain and that elongation beyond the incorporated analogs was significantly retarded for Cl-dATP and Br-dATP using either DNA polymerase alpha or beta. DNA primase using poly(dC) as template was inhibited by these compounds at a concentration 4 to 5 times greater than that required for 2-F-araATP. The 2-halogenated dATP analogs were potent inhibitors of ADP reduction by ribonucleotide reductase. In conclusion, the cytotoxic action of 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine may partially be mediated through the mechanism of "self-potentiation," by depression of the deoxynucleoside triphosphate pools due to inhibition of ribonucleotide reductase, which would facilitate their incorporation into DNA and result in the inhibition of DNA synthesis.
Herpes simplex virus type 2 (HSV-2) induces a novel ribonucleotide reductase (RR) composed of two subunits (140 and 38 kDa) in infected cells. Other investigators have developed a monoclonal antibody, A6, against the 140-kDa subunit of RR and have found, in about 1% of the cases, an inability to detect this protein in cells infected with clinical isolates of HSV-2. We therefore investigated whether in such cases the clinical isolates were capable of inducing viral RR activity and whether the lack of detection of the 140-kDa protein by the monoclonal antibody was due to an alteration in the antigenic site of this protein. Six such isolates were examined and were found to induce RR activity, similar to HSV-2 (strain 333) RR, which did not require ATP for CDP reduction. Western blot analyses using A6 failed to detect the protein. However, R1, a polyclonal antibody raised against viral RR was capable of detecting this subunit. In addition, R1 was also capable of neutralizing RR activity induced by all the isolates and HSV-2 (strain 333). In conclusion, the lack of detection of the large subunit of RR was not due to the lack of induction but was due to an alteration in the antigenic site recognized by A6; this alteration did not appear to affect the properties of the induced RR activity.
It has been suggested that herpes simplex virus (HSV) type 1 may induce a virus-specific DNA topoisomerase activity which copurifies with virus-induced DNA polymerase. We have examined DNA topoisomerase (TOPO) I and II activities in HSV-2-infected HeLa S3 cells. Both activities were partially purified using DEAE-cellulose, phosphocellulose and double-stranded DNA cellulose column chromatography. It was found that both activities could be separated from HSV-2-specific DNA polymerase. Throughout the purification TOPO I could be immunologically detected with a monoclonal antibody developed against human TOPO I. Regardless of the source, mock- or HSV-2-infected human cells, both types of topoisomerase were equally tolerant of 200 mM-KCl. There appeared to be no apparent heterogeneity of TOPO I in HeLa S3 cells through the course of the HSV-2 infection. We conclude that host cell topoisomerases are quite stable in HSV-2-infected HeLa S3 cells and that there is no evidence that HSV-2 is capable of inducing HSV-2-specific TOPO I and TOPO II activities.
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5-Fluoro-2',3'-dideoxy-3'-fluorouridine (3'-FFdUrd) and 5-fluoro-2',3'-dideoxy-3'-fluorouridine 5'-phosphate (3'-FFdUMP) have been synthesized, and their interactions with thymidine (dThd) phosphorylase and thymidylate (dTMP) synthetase, respectively, have been examined. 3'-FFdUrd is not a substrate for dThd phosphorylase, but is a weak, noncompetitive inhibitor (Ki = 1.7 mM). 3'-FFdUMP inhibits dTMP synthetase competitively with deoxyuridylate (Ki = 0.13 mM) when both the substrate and inhibitor are present simultaneously. However, in the presence of 5,10-methylenetetrahydrofolate, the inhibition increases with time in a first-order manner (konobsd = 0.029 s-1). A complex is formed between [6-3H]3'-FFdUMP and dTMP synthetase, which is isolable on nitrocellulose filters, and has a dissociation rate (koffobsd = 1.4 X 10(-2) min-1) similar to that of the potent inhibitor 5-fluoro-2'-deoxyuridylate (koffobsd = 1.3 X 10(-2) min-1) from its ternary complex with dTMP synthetase. These results are explained in terms of a two-stage model involving the initial formation of a reversible adsorption complex, followed by a slow conversion to a tight-binding catalytic complex.
A line of human lymphocytic leukemia cells (CCRF-CEM) has been obtained which is 140-fold resistant to the potent cell growth inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). The cells were also 11-fold cross-resistant to 5-fluorouracil. In contrast to several previous studies involving FdUrd-resistant mouse cells, thymidylate synthetase levels were not substantially elevated in these FdUrd-resistant human leukemic cells. Thymidine kinase activity was also unchanged in the resistant cells, although the levels of 5-fluoro-2'-deoxyuridylate (FdUMP), the potent inhibitor of thymidylate synthetase, generated at equimolar doses of FdUrd were about 40% lower than in the sensitive cells. Studies of the kinetics of FdUMP binding to thymidylate synthetase isolated from the FdUrd-resistant cells disclosed a considerably higher dissociation constant (Kd = 1.0 X 10(-9) M) for the ternary covalent enzyme . FdUMP . 5,10-methylene tetrahydrofolate complex compared to the value obtained with enzyme from sensitive cells (Kd = 4.4 X 10(-11) M). The thymidylate synthetase from the FdUrd-resistant cells also showed 17-fold weaker binding of 2'-deoxyuridylate, even though the Km value for 2'-deoxyuridylate was 3-fold lower compared to the enzyme from FdUrd-sensitive cells. The turnover number of the altered enzyme was 1.8-fold higher than that for the normal enzyme but the rate constants for the release of FdUMP from the ternary complex, which is also an enzyme-catalyzed reaction, were identical for both enzymes. Electrophoresis of the radiolabeled ternary complexes on nondenaturing gels showed small but reproducible differences in migration rates. These results demonstrate that the mechanism of resistance to FdUrd in this cell line involves an alteration in the target enzyme, thymidylate synthetase, which causes it have a lower affinity for nucleotides.
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The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed. Glyceraldehyde-3-phosphate dehydrogenase was the only enzyme inhibited by all three Pt compounds tested, with K2PtCl4 being the most effective and cis-Pt(NH3)2Cl2 the least effective inhibitor. Semilogarithmic plots of residual activity versus inhibition time indicated that the inhibition reactions were not simple first-order processes, except for the inhibition of glucose-6-phosphate dehydrogenase by K2PtCl4 which appeared to be first-order with respect to enzyme concentration.
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