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W R Mancini

Publications and source records attributed to W R Mancini.

17 recordsLinked to original sources
PubMed1992

Decrease in TTP pools mediated by 5-bromo-2'-deoxyuridine exposure in a human glioblastoma cell line.

The antitumor and radiosensitizing properties of 5-bromo-2'-deoxyuridine (BUdR) appear to be due, in part, to its incorporation into cellular DNA. To optimize conditions for incorporation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP) into DNA, we investigated the metabolism of BUdR to its DNA precursor form, the 5'-triphosphate BrdUTP, in the U251 human glioblastoma cell line. The results demonstrated that BrdUTP accumulated rapidly in this cell line, achieving steady-state values within 2 hr of drug addition. The level of BrdUTP accumulation was proportional to the amount of exogenous BUdR up to a concentration of 100 microM, without apparent saturation. Exposure of glioblastoma cells to BUdR was associated with substantial selective decreases in both the cellular dCTP and TTP pools, the extent of which was dependent on the exogenous BUdR concentration. In the absence of exogenous BUdR, BrdUTP was eliminated rapidly from cells with an initial half-life of approximately 15 min. As the cellular BrdUTP level declined, the dCTP and TTP levels increased to control values. Incorporation of BrdUMP into DNA appeared linear with time as long as the cellular BrdUTP level remained constant. This incorporation was not enhanced by the addition of 5-fluoro-2'-deoxyuridine (FUdR), a potent inhibitor of thymidylate synthetase, which at a concentration of 10 nM had no effect on TTP pools in this cell line. Thus, the decrease in cellular TTP pools mediated by BrdUTP allows the halogenated pyrimidine to enhance its own incorporation into DNA.

Antineoplastic Agents↗
PubMed1992

Localization of the peripheral-type benzodiazepine binding site to mitochondria of human glioma cells.

Subcellular fractionation was performed on human U251 glioblastoma cultures. In all subcellular fractions, the binding of the peripheral benzodiazepine ligand, [3H]PK 11195, correlated with the specific activity of monoamine oxidase (r = 0.95, p less than 0.001) and succinate dehydrogenase (r = 0.93, p less than 0.001), two mitochondrial enzymes. The specific activity of plasma membrane and nuclear markers correlated poorly with the presence of PK 11195 binding sites. These data support the mitochondrion as the primary location of peripheral-type benzodiazepine binding sites (PBBS) in human glioma cells. Mitochondria-rich preparations were then assayed for [3H]Ro5-4964 binding. Six nM [3H]Ro5-4964 failed to specifically bind to human U251 mitochondria, but bound vigorously to mitochondria from rat C6 glioma. These data indicate that the low affinity of Ro5-4864 for PBBS in human glioma cells compared to those in rat is due to interspecies receptor variation rather than impaired drug transport into human cells.

Alkaline Phosphatase↗
PubMed1992

The role of deoxycytidine-metabolizing enzymes in the cytotoxicity induced by 3'-amino-2',3'-dideoxycytidine and cytosine arabinoside.

The cellular metabolism of 3'-amino-2',3'-dideoxycytidine (3'-NH2-dCyd), a cytotoxic agent previously reported to be a poor substrate for purified Cyd/dCyd deaminase (dCydD), was compared with that of cytosine arabinoside (ara-C) in cells that displayed dCydD activity (HeLa) and in cells that did not (L1210). Growth inhibition induced by 3'-NH2-dCyd was dependent on the levels of anabolic enzymes, particularly dCyd kinase (dCydK), whereas cytotoxicity induced by ara-C was dependent on the expression of both anabolic and catabolic enzyme activities. Competition kinetics using purified enzyme revealed that the binding affinity of ara-C to dCydK was 5-fold that of the amino analog. However, this binding advantage is apparently offset in cells that contain high levels of dCydD, since the Ki values for this enzyme were 0.2 and 23 mM for ara-C and 3'-NH2-dCyd, respectively. This was reflected in the decrease in analog sensitivity observed between the two cell lines, whereby the concentrations of ara-C and 3'-NH2-dCyd required to inhibit growth by 50% were 200 and 7 times higher, respectively, in the dCydD-containing HeLa cells as compared with the dCydD-deficient L1210 cells. The metabolic stability and cytotoxicity of 3'-NH2-dCyd was independent of cell number. An unexpected finding was the extent to which the effectiveness of ara-C could be mitigated by the number of dCydD-containing cells. A completely cytotoxic concentration of ara-C was rendered nontoxic by a 10-fold increase in cell number. This observation was supported by an increase in I-beta-D-arabinofuranosyluracil (ara-U) formation, a decrease in ara-C 5'-triphosphate (ara-CTP) accumulation, and a rise in cell viability with increasing cell number. These findings indicate that unlike ara-C, the effectiveness of 3'-NH2-dCyd is independent of the level of deaminase, which suggests its possible utility in situations in which high levels of deaminase are manifest.

Animals↗
PubMed1991

Variability of 5-bromo-2'-deoxyuridine incorporation into DNA of human glioma cell lines and modulation with fluoropyrimidines.

Human glioma-derived cell lines were found to vary in their ability to incorporate the radiosensitizer 5-bromo-2'-deoxyuridine (BrdUrd) into DNA after one cell doubling. The U-251 cell line was the best incorporator of BrdUrd, whereas U-118 and D-54 demonstrated poor incorporation with respective C50 (BrdUrd concentration required for 50% of the maximum amount of BrdUrd incorporation into DNA) values of 2.8- and 6-fold greater than that of U-251 (P less than 0.001). Modulation of radiosensitizer uptake into DNA could be achieved using the thymidylate synthase inhibitors 5-fluorouracil or 5-fluoro-2'-deoxyuridine (FdUrd). Incorporation into U-251 cells increased only slightly in the presence of the fluoropyrimidines. The BrdUrd concentration required for 50% of the maximum amount of BrdUrd incorporation into DNA changed (P less than 0.001) from 1.8 +/- 0.11 microM (SD) in the absence of a modulator to 1.1 +/- 0.09 or 1.1 +/- 0.16 microM in the presence of 10 microM 5-fluorouracil or 5 nM FdUrd, respectively. The D-54 cell line, which was the worst incorporator of BrdUrd, was found to have an extensive amount of BrdUrd into DNA following biomodulation. The C50 in the absence of modulation was 7.3 +/- 1.3 microM, which was reduced (P less than 0.001) to 0.62 +/- 0.04 and 0.32 +/- 0.13 microM, respectively, in the presence of 10 microM 5-fluorouracil and 5 nM FdUrd. This represents a 12- to 22-fold reduction in the concentration of radiosensitizer required to achieve the same level of BrdUrd incorporation into DNA. Furthermore, this enhancement of BrdUrd DNA incorporation seen in the presence of the fluoropyrimidines is observed at clinically achievable concentrations. The degree of radiosensitization was solely dependent upon the amount of BrdUrd incorporated into DNA. D-54 cells grown in the presence of 0.18 microM BrdUrd plus 5 nM FdUrd or 2.8 microM BrdUrd alone yielded a similar level of BrdUrd incorporation into DNA and radiosensitization, though a 15-fold lower BrdUrd concentration was used in the presence of FdUrd. The combined use of a radiosensitizer with a fluoropyrimidine may overcome poor incorporation of BrdUrd into DNA that may exist among resistant subpopulations of cells within malignant glioma.

Bromodeoxyuridine↗
PubMed1991

Synthesis and anticancer and antiviral activities of various 2'- and 3'-methylidene-substituted nucleoside analogues and crystal structure of 2'-deoxy-2'-methylidenecytidine hydrochloride.

Various 2'- and 3'-methylidene-substituted nucleoside analogues have been synthesized and evaluated as potential anticancer and/or antiviral agents. Among these compounds, 2'-deoxy-2'-methylidene-5-fluorocytidine (22) and 2'-deoxy-2'-methylidenecytidine (23) not only demonstrated potent anticancer activity in culture against murine L1210 and P388 leukemias, Sarcoma 180, and human CCRF-CEM lymphoblastic leukemia, producing ED50 values of 1.2 and 0.3 microM, 0.6 and 0.4 microM, 1.5 and 1.5 microM, and 0.05 and 0.03 microM, respectively, but also were active in mice against murine L1210 leukemia. Of all the tested drug dosage levels (25, 50, and 75 mg/kg, respectively) compound 23 had no toxic deaths and compound 22 yielded only one toxic death at the highest dosage level. On the contrary, in the same study, 1-beta-D-arabinofuranosylcytosine (ara-C) resulted in 2/5, 5/5, and 5/5 toxic deaths, respectively. Both compounds 22 and 23 have shown better anticancer activity than ara-C, yielding higher T/C x 100 values and some long-term survivors (greater than 60 days). In addition, compounds 22 and 23 were found to have, respectively, approximately 130 and 40 times lower binding affinity for cytidine/deoxycytidine deaminase derived from human KB cells compared to ara-C, suggesting that the two 2'-methylidene-substituted analogues may be more resistant to deamination. Cytoplasmic deoxycytidine kinase (dCK) was required for compounds 22 and 23 action. Furthermore, compounds 14, 22, 23, and 24 also have antiherpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) activity in cell culture. In addition, the crystal structure of 2'-deoxy-2'-methylidenecytidine hydrochloride (23-HCl) was determined by X-ray crystallography.

Animals↗
PubMed1991

Effect of verapamil on the uptake and efflux of etoposide (VP16) in both sensitive and resistant cancer cells.

The effect of calcium antagonist verapamil on the uptake and efflux of Etoposide (VP16), a semi-synthetic derivative of podophylotoxin and a broad spectrum antineoplastic agent, has been investigated and compared in sensitive (UM-UC-2) and resistant (UM-UC-9) human bladder cancer cells, and L1210 leukemia cells, by using both radioisotope (3[H]-VP16) liquid scintillation and high performance liquid chromatography assay with electrochemical detection. The uptake of VP16 was rapid in all three cell lines, showing an initial rapid linear phase followed by a second slower phase, but at steady state the ratios of intracellular to extracellular VP16 concentrations were only 0.004-0.006. No significant difference in drug uptake was observed in sensitive UM-UC-2 and resistant UM-UC-9 cells at all concentrations studied. Verapamil at a concentration of 10 microM enhanced the intracellular VP-16 levels in all sensitive and resistant cell lines. The increments were 21.5% for UM-UC-2, 11.8% for UM-UC-9, and 31.0% for L1210 cells after 30 minutes incubation with 1 microM VP16. A slower efflux of VP16 was observed in verapamil treated cells in all three cell lines. There was a small increase in the nonexchangeable components in verapamil treated cells, although only 5-10% of VP16 was retained. No peak other than that of VP16 was detected in the HPLC chromatogram of extracts from both cell pellet and influx or efflux medium.

Animals↗
PubMed1988

Specific inhibition of DNA biosynthesis induced by 3'-amino-2',3'-dideoxycytidine.

3'-Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) produced an S-phase-specific block in exponentially growing L1210 leukemia cells. The monophosphate and triphosphate forms of the drug were detected within a few hours of 3'-NH2-dCyd treatment of intact cells. No significant change in the deoxynucleoside triphosphate levels was observed during the early stages of treatment. However, by 24 h a 2-fold increase in the amount of the deoxynucleoside triphosphates was seen. The triphosphate form of the drug competitively inhibited dCTP incorporation into calf thymus DNA using highly purified DNA polymerase alpha. The Ki was determined to be 9.6 microM with respect to dCTP. Incorporation of the analogue into DNA was not detected. On the other hand, sucrose gradient analysis suggested that incorporation of the analogue into actively synthesized DNA may account for the biological activity of this compound. Treatment with 3'-NH2-dCyd induced single-strand breaks in actively synthesized DNA, but no double-strand breaks were observed in the presence of the analogue. The data indicate that 3'-amino-2',3'-dideoxycytidine specifically interferes with DNA replication at the level of DNA polymerase by inhibiting chain elongation.

Animals↗
PubMed1988

Isoquinoline and peripheral-type benzodiazepine binding in gliomas: implications for diagnostic imaging.

Binding of the isoquinoline PK 11195 and of the benzodiazepines Ro5-4864 and flunitrazepam was compared in glioma cells and tissues. In human and rat glioma cell cultures [3H]PK 11195 bound with higher affinity (Kd = 14.01 and 15.76 nM, respectively) than either Ro5-4864 (Ki = 1200 and 84.9 nM, respectively) or flunitrazepam (Ki greater than 10,000 and = 848 nM, respectively). Autoradiograms of postmortem human brain sections containing glioma revealed that [3H]PK 11195 bound specifically to intact tumor cells and not to cells of normal cerebral cortex or necrotic areas of the tumor. Total [3H]Ro5-4864 or [3H]flunitrazepam binding to these sections was indistinguishable from nonspecific binding, and regions of tumor and normal brain could not be delineated. These results support the use of radiolabeled PK 11195 for clinical trials of imaging human gliomas by positron emission tomography.

Benzodiazepines↗
PubMed1988

Presence of peripheral-type benzodiazepine binding sites on human erythrocyte membranes.

A nanomolar affinity peripheral-type benzodiazepine binding site is described in human erythrocyte membranes. [3H]PK 11195 is displaced from this binding site by unlabeled drugs with the rank order PK 11195 greater than Ro 5-4864 greater than flunitrazepam much greater than clonazepam. Neither GABA nor a non-hydrolyzable analog of GTP have an effect on binding parameters. These data provide evidence that a peripheral-type benzodiazepine binding site, pharmacologically similar to the intracellular binding site described in other tissues, is present in the plasma membrane of human erythrocytes.

Benzodiazepinones↗
PubMed1986

Production of herpes simplex virus type 1 thymidine kinase in the presence of thymidine analogues.

Treatment of herpes simplex virus type 1 (HSV-1) infected Vero, BHK, BHKtk- and LMtk- cells with 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd) caused increased synthesis of ICP36 and an increase in HSV-1 thymidine kinase (tk) activity at late times of infection. The overproduced ICP36 was identified as the HSV-1 encoded tk protein by immunoprecipitation. Whereas the thymidine analogue 5'-amino-5'-deoxythymidine (AdThd) caused an increase in HSV-1 tk synthesis and activity in wild type Vero and BHK cells, 5-iodo-2'-deoxyuridine (IdUrd) caused a similar increase only in tk- cells (LMtk-, BHKtk-). In vivo and in vitro stabilization studies using a [35S]methionine pulse-chase experiment or heat inactivation studies with purified HSV-1 tk revealed that stabilization of tk by the analogues could not account for the extent of the observed increase. Since overproduction of tk is observed only at late times of infection, it is suggested that the presence of these thymidine analogues in either the viral DNA or the cellular nucleotide pools is responsible for the observed differential effects.

Animals↗
PubMed1984

Physical and biological consequences of incorporation of antiviral agents into virus DNA.

The molecular basis for the antiviral activity is discussed for a variety of nucleoside compounds approved for clinical use in the U.S.A. (5-iodo-2'-deoxyuridine, 5-trifluoromethyl-2'-deoxyuridine, 9-beta-D-arabinofuranosyladenine, 9-(2-hydroxyethoxymethyl)guanine), or in clinical trial (E-5-(2-bromovinyl)-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinosyl)-5-iodocytosine, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), or of specific interest to our laboratory (5-iodo-5'-amino-2',5'-dideoxyuridine, 5'-amino-5'-deoxythymidine). The consequence of incorporation of idoxuridine, the 5'-amino analog of thymidine or the 5'-amino analog of idoxuridine into the DNA of herpes simplex virus type 1 on transcription and translation is emphasized.

Acyclovir↗
PubMed1983

Ribo- and deoxyribonucleoside effect on 3'-amino-2',3'-dideoxycytidine-induced cytotoxicity in cultured L1210 cells.

3'Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) is a potent inhibitor of the replication of cultured L1210 cells, with an IC50 of 1 microM. When ribo- and deoxyribonucleosides were examined for their effects on 3'-NH2-dCyd-induced cytotoxicity, only dCyd could both prevent and reverse these effects. Furthermore, even when the maximum increase in modal cell volume was allowed to develop (24 hr) in the presence of 2.5 microM 3'-NH2-dCyd, the addition of 25 microM dCyd to the medium containing 3'-NH2-dCyd reduced the modal cell volume nearly to control levels within 24 hr. Examination of the viability of these cells by colony formation in soft agar, following as much as a 9.5-hr exposure of 1, 2.5 and 10 microM 3'-NH2-dCyd, revealed that the lethal effects of the 3'-NH2-dCyd treatment were not observed only when 25 microM dCyd was added to the medium during this time. However, the lethality of a 24-hr exposure of 2.5 and 10 microM 3'-NH2-dCyd could not be prevented either by removal of the drug from the medium or by a 24-hr exposure of the medium containing 3'-NH2-dCyd to 25 microM dCyd. When the effect of 3'-NH2-dCyd on DNA biosynthesis in L1210 cells was examined, it was found that radiolabeled dAdo incorporation decreased by approximately 60, 80 or 90% following a 2.5-hr exposure to 2.5, 10 or 20 microM 3'-NH2-dCyd respectively. The addition of 25 microM dCyd under the same conditions resulted in a greater amount of dAdo incorporation compared to the unrescued cultures. Deamination of 3'-NH2-dCyd by partially purified human cytidine-deoxycytidine deaminase was about 2.5% that of either Cyd or dCyd deamination. The deaminated derivative, 3'-amino-2',3'-dideoxyuridine, was significantly less cytotoxic even at 50 microM.

Animals↗
PubMed1983

The relationship between incorporation of E-5-(2-Bromovinyl)-2'-deoxyuridine into herpes simplex virus type 1 DNA with virus infectivity and DNA integrity.

E-5-(2-Bromovinyl)-2'-deoxyuridine (BrvdUrd) produced a dose-dependent shift in the density of herpes simplex virus type 1 (HSV-1) DNA at concentrations which yielded potent inhibition of virus replication in cultured Vero cells. Although the density of cellular DNA was not altered by these concentrations of BrvdUrd, incorporation of this analogue into cellular DNA of HSV-1-infected cells has been previously observed in this laboratory. The degree of inhibition correlated with the amount of BrvdUrd substituted for thymidine in HSV-1 DNA. BrvdUrd-substituted DNA was more labile as determined by a dose-dependent increase in single strand breaks when examined by centrifugation in alkaline sucrose gradients. Thus, the potent antiviral action of BrvdUrd observed in cell culture correlates not only with its incorporation into HSV-1 DNA but also with an altered stability of this DNA.

Bromodeoxyuridine↗
PubMed1983

Synthesis and antineoplastic activity of 3'-azido and 3'-amino analogues of pyrimidine deoxyribonucleoside.

Several new 3'-azido and 3'-amino nucleosides (8, 9, 12, and 13) have been synthesized and their biological activities evaluated. Among them, 3'-amino-2',3'-dideoxycytidine (13) was found to exhibit potent cytotoxic activity against both L1210 and S-180 cells in vitro with an ID50 of 0.7 and 4.0 microM, respectively. Furthermore, 13 has also shown antitumor activity against L1210 tumor bearing mice with a T/C X 100 value of 283.

Animals↗
PubMed1983

Synthesis and biological activity of various 3'-azido and 3'-amino analogues of 5-substituted pyrimidine deoxyribonucleosides.

Various new 5-substituted 3'-azido- and 3'-amino derivatives of 2'-deoxyuridine and 2'-deoxycytidine have been synthesized and biologically evaluated. Among these compounds, 3'-amino-2',3'-dideoxy-5-fluorouridine (3), 3'-amino-2',3'-dideoxycytidine (7a), and 3'-amino-2',3'-dideoxy-5-fluorocytidine (7c) were found to be the most active against murine L1210 and sarcoma 180 neoplastic cells in vitro, with an ED50 of 15 and 1 microM, 0.7 and 4 microM, and 10 and 1 microM, respectively. The 3'-azido derivatives, 2 and 6c, were less active in comparison with their 3'-amino counterparts. In addition, the 5-fluoro-3'-amino nucleosides, 3 and 7c, were tested against L1210 leukemia bearing CDF1 mice. Our preliminary findings indicate that compound 7c (6 X 200 mg/kg) was as active as the positive control, 5-fluorouracil (6 X 20 mg/kg), yielding a T/C X 100 of 146 and 129, respectively. However, 3 was found to be inactive in this experiment.

Animals↗