UMLS:C0005684 - FACTA Search

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Query: UMLS:C0005684 (bladder cancer)
16,431 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

5' Amino-5'-deoxythymidine (5'-AdThd) has been demonstrated previously to antagonize dTTP-mediated feedback inhibition of purified thymidine kinase from 647V, a human bladder cancer cell line. Low concentrations of 5'-AdThd (3-30 microM) have also been shown to stimulate cellular uptake of iododeoxyuridine (IdUrd) in 647V cells at clinically relevant IdUrd concentrations (2 microM). We report that the combination of 30 microM 5'-AdThd plus 2 microM IdUrd results in a significant increase of IdUrd replacement of thymidine (dThd) (18%) in the DNA of 647V cells over that obtained by exposure to 2 microM IdUrd alone (7.9%). However, increasing the 5'-AdThd concentration to 300 microM inhibited the incorporation of IdUrd into DNA (3%). IdUrd-induced radiosensitization of 647V cells, as measured by clonogenic survival, was enhanced by coincubation with 30 microM 5'-AdThd, while 300 microM 5'-AdThd reduced the IdUrd radiosensitization. Additionally, radiation-induced single strand break generation when IdUrd was incorporated into 647V DNA, as measured by rapid alkaline elution, was also enhanced by coincubation with 30 microM 5'-AdThd, while 300 microM 5'-AdThd resulted in a decrease in the number of single strand breaks produced. In T24, another bladder cancer cell line, and SV-HUC-TT1, a tumorigenic cell line derived from SV-HUC, 3-10 microM 5'-AdThd was also able to enhance IdUrd replacement of dThd in DNA. However, no stimulation of dThd replacement by 5'-AdThd occurred in SV-HUC, a prototypic "normal" bladder urothelial cell line. Since 5'-AdThd is not a substrate for mammalian thymidine kinase and has little or no cytotoxicity in vitro and in vivo, it may be a selective modulator of IdUrd radiosensitization of human bladder carcinoma and should be tested in vivo.
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PMID:Modulation of IdUrd-DNA incorporation and radiosensitization in human bladder carcinoma cells. 219 32

Fluorodeoxyuridine (FdUrd) is a cytotoxic analogue of thymidine which requires activation by thymidine kinase to FdUMP. FdUMP inhibits thymidylate synthetase and, thus, the synthesis of dTTP. 5'-Aminothymidine (5'-AdThd) can antagonize the feedback inhibition exerted by dTTP on thymidine kinase activity and thereby stimulate FdUrd phosphorylation. This provided a novel approach to assess the degree to which end product inhibition regulates the phosphorylation of FdUrd. We used 5'-AdThd to investigate the effects of dThd and IdUrd on the regulation of FdUrd uptake in intact 647V cells, a human bladder cancer cell line. Contributions from catabolic processes were found not to be important in our system. We detected no nucleoside phosphorylase activity in the 647V cells or any effect of 5'-AdThd on the breakdown of 5-fluorodeoxyuridine monophosphate to FdUrd by crude preparations from these cells. Thus, phosphorylation by thymidine kinase determined FdUrd uptake (phosphorylation). In the absence of added nucleosides the rate of FdUrd uptake increased in a time dependent fashion. Diminished feedback inhibition of thymidine kinase appeared to be an important factor, as evidenced by a decrease in intracellular dTTP pools and a time dependent loss in the ability of 5'-AdThd to stimulate FdUrd uptake. Thymidine and iododeoxyuridine inhibited FdUrd phosphorylation (uptake) by two mechanisms: competition for the active site of thymidine kinase and increased feedback inhibition. Increased feedback inhibition was indicated by stimulation of FdUrd uptake by 5'-AdThd. The effects of IdUrd on FdUrd uptake were also time dependent, presumably reflecting accumulation of iododeoxyuridine triphosphate and dTTP pools. FdUrd cytotoxicity was modulated by dThd, IdUrd, and 5'-AdThd in parallel to their perturbation of FdUrd uptake. Individually they reduced the growth inhibitory properties of FdUrd. These results show that the regulation of FdUrd uptake is critically dependent on the presence of dThd and IdUrd and emphasize the potential importance of circulating levels of these nucleosides in mediating FdUrd activation and cytotoxicity.
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PMID:Regulation of the activation of fluorodeoxyuridine by substrate competition and feedback inhibition in 647V cells. 252 Dec 99

We have previously reported that 5'-aminothymidine (5'-AdThd), an antagonist of the feedback inhibition exerted by dTTP that regulates thymidine kinase, enhances the uptake and cytotoxicity of 5-iododeoxyuridine in various human bladder cancer cell lines but not in normal human urothelial cells (HU) propagated in vitro. In this work we have analyzed the factors that could potentially account for the differential effect of 5'-AdThd among various cell types: 647V (a human bladder cancer cell line); HU; SV-HU (a SV40-transformed human urothelial cell line), and C3H/10T1/2 mouse embryo fibroblasts (10T1/2) cells. 5'-AdThd enhanced the uptake of IdUrd in SV-HU cells (greater than 400%), similar to what we have observed before for 647V cells. However, in 10T1/2 and HU cells, 5'-AdThd only minimally increased the uptake of 5-iododeoxyuridine (about 160%). Thymidine kinases purified from the different sources were similarly sensitive to inhibition by dTTP or 5'-AdThd and to deinhibition of the dTTP-induced regulation of enzyme activity by 5'-AdThd. Furthermore, [3H]-5'-AdThd permeated and accumulated intracellularly in all cell types. In none of these cultures was nucleoside phosphorylase activity detected, as indicated by the inability of the cells to produce thymine or iodouracil after exposure to the appropriate nucleosides. Also, 5'-AdThd did not affect the breakdown of dTMP by crude preparations of cytosolic 5'-nucleotidase from the different cells. We found that intracellular dTTP pools in the various cell types were substantially high (15-26 microM) compared to the sensitivity of thymidine kinase to inhibition by dTTP (IC50 2-4 microM). This suggests that thymidine kinase is in a strongly inhibited state in situ. To test the sensitivity of thymidine kinase (in situ) to regulation by dTTP we investigated: (a) the effect of depleting intracellular dTTP pools with methotrexate on the uptake of thymidine (dThd); and (b) the effect of pH on the uptake of dThd and its perturbation by 5'-AdThd, since the inhibition of thymidine kinase activity by dTTP is known to be pH dependent. We found that a 47% reduction of dTTP pools by methotrexate in 10T1/2 and HU cells did not result in an increase in thymidine kinase activity, as indicated by the lack of an effect on the uptake of dThd. However, we have previously shown that, under similar conditions, 647V cells show a substantial increase in dThd uptake.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Basis for the differential modulation of the uptake of 5-iododeoxyuridine by 5'-aminothymidine among various cell types. 270 29

We have studied the effect of pH on the interactions between thymidine kinase, thymidine triphosphate, and 5'-amino-2',5'-dideoxythymidine (5'-AdThd) in purified preparations of the enzyme and in intact 647V cells, a human bladder cancer cell line. Thymidine kinase is competitively inhibited by 5'-AdThd. dTTP feedback inhibits in a noncompetitive fashion. However, 5'-AdThd partially reverses the inhibition produced by dTTP resulting in enhanced enzyme activity. We have found that dTTP (pKa = 7.5) is a much more potent inhibitor of purified preparations of thymidine kinase activity at low pH conditions. For example, 2.5 microM dTTP inhibited thymidine kinase activity by 50, 85, and 95% at pH values of 8.0, 7.5, and 6.5, respectively. The interaction of 5'-AdThd (pKa = 8.5) at either the active (competitive) or the regulatory (deinhibition) site is not altered significantly over a pH range of 6.5 to 9.5. To extend these findings to intact cells, we studied the perturbation of the uptake of thymidine by 5'-AdThd in 647V cells incubated in media buffered at various pH values. In cells exposed to media buffered at pH 8.5 or 7.5, 5'-AdThd maximally stimulated thymidine uptake about 250 and 300% at 10 and 30 microM, respectively. However, at pH 6.5, 300 microM 5'AdThd was required to produce maximal stimulation of about 500%. These observations are consistent with the greater sensitivity of thymidine kinase (in situ) to feedback inhibition by dTTP at the lower pH conditions. Intracellular dTTP pool sizes were not affected by variations in pH during the short time course of our experiments. However, after 1 h, the intracellular concentration of 5'-AdThd was twice that of the extracellular medium in conditions at pH 7.5 and 8.5 but was equimolar across the membrane at pH 6.5. This does not account for the differences in the perturbation of thymidine uptake by 5'-AdThd at various pH values. In general, our results indicate that regulation of thymidine kinase by dTTP is pH dependent, while its modulation by 5'-AdThd is not, and that regulation of thymidine kinase in situ is sensitive to alterations in pH.
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PMID:Modulation of the feedback regulation of thymidine kinase activity by pH in 647V cells. 279 Jul 82

Iododeoxyuridine (IdUrd) potentiated the lethal but not the growth inhibitory properties of fluorouracil (FUra) and fluorodeoxyuridine (FdUrd) in human bladder cancer cells (T24). The rate of incorporation of IdUrd into DNA was enhanced by both fluoropyrimidines, but to a significantly greater extent by FdUrd. Both inhibition of iododeoxyridylate dehalogenation and the depletion of thymidine triphosphate pools contributed to the increased incorporation rate. Inhibition of dehalogenation accounted for 67% of the observed stimulation in the case of FUra, but only 37% of the increase produced by FdUrd. The depletion of dTTP pools, both in the presence and absence of IdUrd, was greater after FdUrd than FUra exposure. The observed increase in the rate of incorporation of IdUrd appears to account for the enhanced toxicity seen with FdUrd, but other factors may be involved in the case of FUra. Since FUra and IdUrd appear to be mutually potentiating and do not share a dependence on thymidine kinase activity, this drug combination warrants further investigation.
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PMID:Modulation of 5-iodo-2'-deoxyuridine metabolism and cytotoxicity in human bladder cancer cells by fluoropyrimidines. 293 42

5'-Aminothymidine represents a novel class of compounds capable of antagonizing the feedback inhibition which normally regulates thymidine kinase. As a consequence, the uptake of iododeoxyuridine, a substrate of thymidine kinase, can be substantially increased by 5'-aminothymidine. in this study the phosphorylation, uptake, and cytotoxicity of iododeoxyuridine was markedly enhanced by 5'-aminothymidine in three human bladder cancer cell lines, T24, HT1197 and 647V. In contrast, neither the uptake nor the toxicity of iododeoxyuridine was increased by 5'-aminothymidine in normal human urothelial cells propagated in vitro. Although 30 microM 5'-aminothymidine increased the uptake of 3 microM iododeoxyuridine 4- to 5-fold in the HT1197 and 647V cells and 2.5-fold in the T24 cells, no stimulation was produced in the normal urothelial cells. The modulation of iododeoxyuridine uptake was associated with parallel changes in the inhibition of cellular replication. The cytotoxicity of iododeoxyuridine was strongly augmented by 5'-aminothymidine in the HT1197 and 647V cells, modestly increased in the T24 cells, and unchanged in the normal urothelial cells. The degree to which iododeoxyuridine phosphorylation was stimulated did not correlate with cellular replication rates or with intracellular thymidine triphosphate concentrations. Iododeoxyuridine uptake was markedly increased in the HT1197 (doubling time = 52 h) and 647V (doubling time = 24 h) cells, moderately in the rapidly growing cells T24 (doubling time = 20 h), and minimally in the normal urothelial cells which doubled every 32 h. In exponentially growing cells the thymidine triphosphate pools were approximately 18 microM in the normal cells and about 21, 24, and 35 microM in the HT1197, T24, and 647V cells, respectively. The use of 5'-aminothymidine and other compounds capable of antagonizing feedback inhibition may provide a new means of increasing the efficacy of cytotoxic nucleosides.
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PMID:Preferential stimulation of iododeoxyuridine phosphorylation by 5'-aminothymidine in human bladder cancer cells in vitro. 373 Nov 5

The design of compounds which disrupt the regulation of key enzymes is a new strategy for drug development. 5'-AdThd, which can antagonize the feedback inhibition that normally regulates thymidine kinase, is representative of this novel class of agents. The ability of 5'-AdThd to deinhibit thymidine kinase markedly increases the capacity of some cells to phosphorylate thymidine and IdUrd and, thereby, enhance their cytotoxicity. This effect is evident in three human bladder cancer cell lines, but not in normal human urothelial cells. Thus, 5'-AdThd may be able to increase the therapeutic selectivity of IdUrd. Intracellular dTTP pools are critically involved in mediating the effects of 5'-AdThd, but differences in these pools from cell type of cell type may not account for the selective stimulation of IdUrd uptake produced in the cancer cells.
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PMID:Perturbation of thymidine kinase regulation: a novel chemotherapeutic approach. 381 83

We have reported that noncytotoxic concentrations of 3'-azido-3'-deoxythymidine (AZT) increase the cytotoxicity of ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporation into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5'-deoxythymidine triphosphate (dTTP) pools, which compete with AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furthermore, the inhibition of TS would increase the activity of both thymidine kinase (TK) and thymidylate kinase (TdK). Each of these consequences of TS inhibition would favor more incorporation of AZT into DNA. Thus, we reasoned that other TS inhibitors should also result in increased AZT incorporation into DNA and, perhaps, in increased cytotoxicity. N6-[4-(Morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. This potent TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder cancer cells. A 24-h exposure to 5 microM AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 in MGH-U1 cells resulted in an enhanced antitumor effect relative to that of each agent alone; 50 microM AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies of this combination revealed that simultaneous treatment with 5 microM AG-331 plus 1.8 microM [3H]-AZT produced as much as a 68% +/- 7% increase in AZT incorporation into DNA. This observation was associated with an increase in DNA single-strand breaks, measured as comet tail moment, of up to 6.6-fold. These studies support our original premise that TS inhibition favors increased incorporation of AZT into DNA and that the combination causes more cell kill than either drug alone in MGH-U1 cells.
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PMID:Cytotoxic and biochemical implications of combining AZT and AG-331. 785 Sep 19

The effects of de novo dTMP inhibition by N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2- thenoyl)-L-glutamic acid (D1694) or N6-[4-(morpholinosulfonyl)benz]-N6-diaminobenz[cd]indole glucuronate (AG-331) on clonogenic survival, thymidylate synthase (TS) and thymidine kinase (TK) activity, and expression of S-(p-nitrobenzyl)-6-thioinosine-sensitive nucleoside transporter (NT) sites were addressed in the human bladder cancer cell line, MGH-U1. These two TS inhibitors are structurally diverse. D1694 is a folate-based TS inhibitor, whereas AG-331 is a novel agent that inhibits the cofactor binding site of the enzyme. They also differ with respect to their cytotoxic effects in this cell line; D1694 cytotoxic 50% inhibitory concentration (IC50) and IC90 were 6.0 and 9.0 nM, respectively and IC50 and IC90 for TS inhibition were 2.5 and 4.8 nM, respectively. In contrast, AG-331 cytotoxic IC50 could not be achieved even at concentrations of up to 20 microM for 24-h exposures, and IC50 and IC90 for TS inhibition were 0.7 and 3.0 microM, respectively. Similar effects for D1694 and AG-331 were observed in their modulation of TK activity and NT expression. 5-(SAENTA-x8)-Fluorescein, a highly modified form of adenosine incorporating a fluorescein molecule which binds with a 1:1 stoichiometry to S-(p-nitrobenzyl)-6-thioinosine-sensitive NT sites, was used to investigate the expression of NT following exposure of cells to D1694 and AG-331. TK activity was addressed by the metabolism of [3H]thymidine to [3H]TMP by cellular extracted protein and by an alternative flow cytometric method using a modified form of thymidine incorporating a fluorescent molecule, dansyl-5-amino-2-deoxyuridine. Results obtained by both methods were comparable. At concentrations of 5 and 10 nM, D1694 increased TK activity 2.3-4.5-fold and NT expression 34-39-fold. AG-331, at concentrations of 5 and 10 microM, increased TK activity 1.8-2.5-fold and NT expression 22-31-fold, respectively. These data suggest that TK activity and NT expression have a common regulatory mechanism which is sensitive to endogenous dTTP pools and that the salvage pathway is a complex system of kinases coordinated with transport of nucleosides.
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PMID:Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression. 788 59

These studies were undertaken to determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase (ADV/RSV-tk) and the prodrug ganciclovir (GCV) in an orthotopic murine bladder cancer model. We utilized a replication-defective adenoviral construct containing the beta-galactosidase gene as a control and the herpes simplex virus thymidine kinase gene as the therapeutic vector under the transcription control of the Rous sarcoma virus long terminal repeat promoter. Intravesically created, orthotopic bladder tumors were established in syngeneic C3H/He female mice. India ink injection and beta-galactosidase studies were performed to determine if transurethral administration, direct tumor injection, or the combination was the most efficient route of virus administration. Optimal dosing of ADV/RSV-tk was determined by direct tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate but similar controlled experiments. Growth curve studies demonstrated reliable tumor formation by 14 days. Direct transvesical tumor injection resulted in the best distribution and intratumor gene expression as measured by X-gal staining. Dose-ranging experiments demonstrated an optimal viral dose of 5 x 10(8) plaque-forming units and a greater than twofold reduction in tumor growth for the animals treated with ADV/RSV-tk compared to controls. Efficacy studies demonstrated a greater than threefold reduction in tumor growth. No clinical or gross pathologic toxicity was detected. Long-term survival results suggested a survival benefit for the treatment animals compared to controls. We conclude that ADV/RSV-tk in combination with GCV provides effective therapy for orthotopic murine bladder cancer by significantly inhibiting tumor growth with limited toxicity to the host. These data provide further support for testing this suicide gene therapy strategy in human Phase I trials.
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PMID:In vivo adenovirus-mediated suicide gene therapy of orthotopic bladder cancer. 1098 51

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