Transgenic expression of human thymidylate synthase accelerates the development of hyperplasia and tumors in the endocrine pancreas.

Thymidylate synthase (TS) is an essential enzyme for DNA synthesis and repair and elevated levels of TS have been identified as an important prognostic biomarker for colorectal cancer and several other common human malignancies. In addition, TS gene expression has been linked with cell-cycle regulation and cell proliferation through the ability of retinoblastoma protein to repress the transcriptional activation of E2F target genes such as TS. Therefore, overproduction of TS could participate in the progression to a neoplastic phenotype. Consistent with this model, a recent study has suggested that ectopic TS expression can induce a transformed phenotype in mammalian cells. To investigate the role of deregulated TS activity in tumor development, we generated transgenic mice that express high levels of catalytically active human TS (hTS) exclusively in the pancreas and low levels of hTS in multiple other tissues. Analyses of pancreatic tissue in TS transgenic mice revealed abnormalities within the endocrine pancreas, ranging from pancreatic islet hyperplasia to the detection of islet cell tumors. Overexpression of hTS in murine islets provides a new model to study genetic alterations associated with the progression from normal cells to hyperplasia to islet cell tumors, and suggests that this mouse model may be useful for regulating TS activity in vivo for development of cancer prevention and new therapies.

Identification and proposed mechanism of action of thymidine kinase inhibition associated with cellular exposure to camptothecin analogs.

PURPOSE: To investigate the effects of several camptothecin analogs including 9-aminocamptothecin (9-AC), SN38, topotecan, and irinotecan (CPT-11) on the enzymes involved in the pyrimidine salvage pathway including thymidylate synthase (TS). A COMPARE analysis using the NCI 60 cell line drug-screening panel suggested that there were similarities in the mechanisms of action of camptothecin analogs and TS inhibitors. METHODS: TS enzymatic activity was measured by both an in situ tritium release assay using both the H630 colon cancer cell line and the CEM human leukemia cell line, and by a radiolabelled in vitro assay using partially purified human TS as the enzyme source. Thymidine kinase (TK) activity was measure by a radiolabelled in vitro assay using H630 colon cancer cell lysates as the enzyme source. RESULTS: In vitro studies indicated that none of the analogs directly inhibited TS enzymatic activity; however, utilization of a coupled TS/TK in situ assay with radiolabelled deoxyuridine as the precursor revealed marked inhibition by the camptothecin analogs. 9-AC, SN38, and topotecan yielded IC50 values of 1.3, 1.6, and 1.1 microM respectively. In contrast, there was no inhibition detected when deoxycytidine was used as the radiolabelled nucleoside precursor, suggesting that the drug effect was through inhibition of TK, rather than inhibition of TS. In vitro studies using cell lysates from H630 human colon cancer cells to measure TK activity showed no decrease in TK activity after 9-AC treatment. In addition, no changes were detected in the dATP and dTTP nucleotide pools. Permeabilizing the cell membranes with saponin did not abolish the inhibitory effect of the camptothecins indicating that altered cell transport was not responsible for the decreased activity in the in situ assay in intact cells. CONCLUSION: These studies suggest that there is inhibition of TK in intact cells associated with topoisomerase I inhibition by camptothecin analogs, and the inhibition of TK is the result of an indirect effect not related to feedback inhibition by changes in dTTP pools.

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA.

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5'-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434-634. In this paper, a 70 nt RNA sequence, corresponding to nt 480-550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434-634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5'-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434-634 or p644/TS480-550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT-PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480-550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434-634 and p644/TS480-550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.

Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex.

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

The cellular interaction of 5-fluorouracil and cisplatin in a human colon carcinoma cell line.

The combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) has been shown to have synergistic cytotoxicity in human tumours, but the biochemical mechanism for this interaction remains unclear. Therefore, the aim of this study was to investigate the interaction of 5-FU and CDDP in a human colon carcinoma cell line, NCI H548. A 24 h exposure to 5-FU resulted in a 5-FU IC50 value of 24.2 +/- 4.5 microM, Dm 22.6 microM; exposure to CDDP for 2 h resulted in a IC50 value of 20.8 +/- 8.0 microM, Dm 21.9 microM. When cells were exposed simultaneously to 5-FU for 24 h and CDDP for the initial 2 h, or when cells were treated with CDDP for 2 h followed by various concentrations of 5-FU for 24 h, no greater than additive cytotoxicity was observed. In contrast, when cells were treated with 5-FU for 24 h prior to CDDP for 2 h, a greater than additive interaction was noted (5-FU IC50 1.2 +/- 0.6 microM, Dm 1.3 microM, CI 0.45). Thymidine 10 microM partially reversed the growth inhibitory effects of the 5-FU/ CDDP combination. Using both immunological and biochemical assays, no notable differences in the total amount of TS enzyme or the fraction of bound TS enzyme could be detected in the absence or presence of CDDP. No notable differences could be detected in intracellular reduced folate pools, FdUMP or FUTP pools, or 5-FU incorporation into RNA or DNA with the addition of CDDP to 5-FU. DNA fragmentation studies revealed that the combination of 5-FU followed by CDDP demonstrated a greater degree of single-stranded DNA fragments in parental (P = 0.024) and newly synthesised DNA (P = 0.025) compared with the administration of CDDP prior to 5-FU or either drug alone. The increase in smaller DNA fragment size was reversed with the addition of thymidine (10 microM). These findings suggest that the interaction of 5-FU and CDDP is associated with a greater degree of fragmentation of both nascent and parental DNA.

Identification of in vivo target RNA sequences bound by thymidylate synthase.

We developed an immunoprecipitation-RNA-random PCR (rPCR) method to isolate cellular RNA sequences that bind to the folate-dependent enzyme thymidylate synthase (TS). Using this approach, nine different cellular RNAs that formed a ribonucleoprotein (RNP) complex with thymidylate synthase (TS) in human colon cancer cells were identified. RNA binding experiments revealed that seven of these RNAs bound TS with relatively high affinity (IC50 values ranging from 1.5 to 6 nM). One of the RNAs was shown to encode the interferon (IFN)-induced 15 kDa protein. Western immunoblot analyses demonstrated that the level of IFN-induced 15 kDa protein was significantly decreased in human colon cancer H630-R10 cells compared with parent H630 cells. While the level of IFN-induced 15 kDa mRNA expression was the same in parent and TS-overexpressing cell lines, the level of IFN-induced 15 kDa RNA bound to TS in the form of a RNP complex was markedly higher in H630-R10 cells relative to parent H630 cells. These studies begin to define a number of cellular target RNA sequences with which TS interacts and suggest that these TS protein-cellular RNA interactions may have a biological role.

The effect of dose and interval between 5-fluorouracil and leucovorin on the formation of thymidylate synthase ternary complex in human cancer cells.

We examined the importance of dosing interval between leucovorin (LCV) and 5-fluorouracil (5-FU) on intracellular thymidylate synthase (TS) ternary complex, free TS and total TS protein levels in human MCF-7 breast and NCI H630 colon cancer cell lines. A 2- to 3-fold increase in total TS was noted when either cell line was exposed to 5-FU 10 microM plus LCV (0.01-10 microM) compared with a 1.4- to 1.6-fold increase in total TS due to 5-FU 10 microM alone. The amount of TS ternary complex formed was 2- to 3-fold higher in both cell lines treated with the combination of 5-FU and LCV compared with 5-FU alone. TS complex formation and total TS protein increased with LCV dose (0.1-10 microM). In MCF-7 cells, the maximal increase in total TS protein and TS ternary complex formation was observed when 5-FU was delayed for 4 h after the start of LCV exposure. In NCI H630 cells, maximal total TS protein and ternary complex formation occurred when 5-FU was delayed for 18 h after the start of LCV exposure. The amount of free TS did not change in either cell line whether 5-FU was given concurrently with LCV or delayed for up to 24 h. The accumulation rate of intracellular folates in the form of higher glutamates Glu3-Glu5 was rapid in MCF-7 cells (maximal formation after 4 h), whereas in H630 cells accumulation of higher polyglutamates continued to increase up to 18 h. The time of peak folate polyglutamate (Glu3-Glu5) formation coincided with the time of peak TS complex formation and total TS protein in each cell line. In these human carcinoma cell lines, the LCV dose and interval between 5-FU and LCV play a role in increased TS total protein and TS ternary complex; however, the amount of free TS is independent of the interval between 5-FU and LCV. The time-and dose-dependent increases in TS ternary complex and TS total protein are associated with differences in the accumulation of folate polyglutamates in these cell lines.

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.

Thymidylate synthase binds to c-myc RNA in human colon cancer cells and in vitro.

Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.

Determinants of trimetrexate lethality in human colon cancer cells.

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.

The effect of reducing reagents on binding of thymidylate synthase protein to thymidylate synthase messenger RNA.

Human thymidylate synthase (TS) protein specifically binds to its own TS mRNA and functions as a translational repressor. In the presence of reducing agents, the RNA binding activity of TS protein is significantly enhanced. In contrast, treatment of TS protein with the oxidizing agent diamide inhibits RNA binding. Scatchard analysis reveals that in the presence of the reducing agent 2-mercaptoethanol, the TS protein/TS mRNA interaction changes from low (Kd = 66 nM) to high (Kd = 2.6 nM) apparent affinity. The catalytic activity of TS is increased by up to 6.5-fold in the presence of 2-mercaptoethanol. These studies demonstrate that the interaction between TS protein and its target TS mRNA is sensitive to the presence of reducing reagents and is dependent upon a reversible sulfhydryl switch mechanism.

Interaction of Pneumocystis carinii dihydropteroate synthase with sulfonamides and diaminodiphenyl sulfone (dapsone).

Dihydropteroate synthase is the target enzyme for the sulfonamide compounds, which are the mainstay of therapy for Pneumocystis carinii pneumonia, a common infection in patients with impaired immunity. The stability of this enzyme, its kinetic constants with respect to substrates, and the 50% inhibitory concentration (IC50) of several sulfonamides and the sulfone dapsone have been characterized using both cell-free and intact organism assay systems. Stability of the enzyme is dependent on storage temperature, reducing reagents, and to a lesser extent, protease inhibitors. The sulfonamides sulfadiazine and sulfamethoxazole were found to be highly potent inhibitors of P. carinii dihydropteroate synthase with IC50s of 0.42 and 0.71 microM, respectively. Dapsone had equivalent potency when compared with the most potent sulfonamides tested in both assay systems. Data suggest that sulfamethoxazole, sulfadiazine and dapsone may represent equivalent choices as P. carinii dihydropteroate synthase inhibitors, assuming an equivalent in vivo drug exposure can be achieved.

Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.

Intracellular metabolism of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate in a human breast-cancer cell line.

This report describes the intracellular metabolism of 5-methyltetrahydrofolate (5-methyl-H4PteGlu) and 5-formyltetrahydrofolate (5-formyl-H4PteGlu) to the various folate forms and their respective polyglutamated states in the MCF-7 human breast-cancer cell line. The intracellular folate distribution observed in MCF-7 cells treated with 5-methyl-H4PteGlu was similar to that seen in cells treated with 5-formyl-H4PteGlu. In cells exposed to 5-formyl-H4PteGlu for 24 h, the folate pool consisted of 103 +/- 10 pmol/mg 10-formyl-H4PteGlu, 120 +/- 18 pmol/mg H4PteGlu, and 71 +/- 18 pmol/mg 5-methyl-H4PteGlu versus 88 +/- 5, 54 +/- 20 and 87 +/- 10 pmol/mg, respectively, for cells exposed to 5-methyl-H4PteGlu. Only the difference seen in H4PteGlu levels between cells exposed to either 5-methyl-H4PteGlu or 5-formyl-H4PteGlu reached statistical significance (P < 0.05). In the absence of vitamin B12, exposure to 5-methyl-H4PteGlu resulted in 154 +/- 17 pmol/mg 5-methyl-H4PteGlu along with only 8 +/- 5 pmol/mg 10-formyl-H4PteGlu and 4 +/- 2 pmol/mg H4PteGlu, thus demonstrating the marked dependence on vitamin B12 for the metabolism of 5-methyl-H4PteGlu to the other intracellular folates. 5-10-Methylene- H4PteGlu (2 +/- 1.3 pmol/mg) was detected only in cells exposed to 5-formyl-H4PteGlu for 24 h, not in cells treated with 5-methyl-H4PteGlu. The profile of polyglutamates detected in cells treated with either 5-formyl-H4PteGlu or 5-methyl-H4PteGlu for 24 h was not significantly different, although cells treated with 5-methyl-H4PteGlu tended to have less conversion to the higher polyglutamates (Glu3-Glu5) as compared with those treated with 5-formyl-H4PteGlu. In 5-methyl-H4PteGlu-treated cells grown in the absence of vitamin B12, the pentaglutamate was the only polyglutamate form detected, accounting for only 11% of the total folate pool. Since there does not appear to be a greater formation of the optimal reduced-folate forms necessary to achieve enhanced thymidylate synthase (TS) inhibition through ternary-complex formation in cells exposed to 5-methyl-H4PteGlu versus 5-formyl-H4PteGlu, these studies suggest that the use of 5-methyl-H4PteGlu would not be advantageous over that of 5-formyl-H4PteGlu in combination regimens with the fluoropyrimidines.

Effects of 5-fluorouracil substitution on the RNA conformation and in vitro translation of thymidylate synthase messenger RNA.

In vitro transcribed thymidylate synthase (TS) mRNA which is 100% substituted with 5-fluorouracil (FUra) was analyzed for changes in mRNA secondary structure, for alterations in translational efficiency, and for evidence of translational miscoding in vitro. FUra substitution in TS mRNA results in an altered migration pattern in non-denaturing RNA gels and in decreased hyperchromicity in RNA melting temperature studies, consistent with a change in mRNA secondary structure. However, no change in the translational efficiency of FUra-substituted TS mRNA is seen compared to control TS mRNA in either rabbit reticulocyte lysate or wheat germ extract in vitro translation systems. Analysis of the in vitro translation product of FUra-substituted TS mRNA by Western immunoblotting, isoelectric focusing, 5-fluoro-2'-deoxyuridine 5'-monophosphate binding, and TS catalytic activity experiments shows no difference compared to control TS mRNA. We conclude that the in vitro translation products of FUra-substituted and control TS mRNA are identical. Our findings do not support the hypothesis that changes in the mRNA template are responsible for the RNA-directed cytotoxicity of FUra.

Inhibition of Pneumocystis carinii dihydropteroate synthetase by para-acetamidobenzoic acid: possible mechanism of action of isoprinosine in human immunodeficiency virus infection.

Isoprinosine has been reported to decrease progression to AIDS, primarily by preventing Pneumocystis carinii pneumonia (PCP), in human immunodeficiency virus-infected patients, but the mechanism of action is unknown. para-Acetamidobenzoic acid (PAcBA), one component of isoprinosine, is structurally related to para-aminobenzoic acid (PABA), a precursor of de novo folate synthesis. This pathway is known to be important for P. carinii because sulfonamides, which are effective anti-P. carinii agents, inhibit incorporation of PABA into folate precursors by the enzyme dihydropteroate synthetase (DHPS). Inhibition of P. carinii DHPS by PAcBA was investigated by using two assays. In short-term cultures of P. carinii from rats, [3H]PABA incorporation into reduced folates was inhibited by both isoprinosine (mean +/- standard error 50% inhibitory concentration [IC50], 20 +/- 8.4 microM) and PAcBA free acid (IC50, 240 +/- 100 microM); a soluble PAcBA salt was more potent than PAcBA free acid alone (IC50, 29 +/- 48 microM). The activity of PAcBA free acid was confirmed in a cell-free DHPS inhibition assay (IC50, 120 +/- 120 microM). Inosine and dimethylaminopropanol, two other components of isoprinosine, were poor inhibitors of PABA incorporation (IC50, > 1,000 microM). PAcBA free acid also showed activity in inhibiting the DHPS of Toxoplasma gondii, but was a poor inhibitor of the DHPSs of Escherichia coli and Saccharomyces cerevisiae. In a rat model of PCP, the PAcBA salt administered intraperitoneally demonstrated no activity against established PCP either alone or when used in combination with trimethoprim; the lack of efficacy in this model may be due to the rapid metabolism of the drug. Prevention of PCP by PaCBA through inhibition of P. carinii DHPS may explain the activity of isoprinosine in decreasing the progression to AIDS in human immunodeficiency virus-infected patients.

Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro.

Dihydrofolate reductase (DHFR) is a critical enzyme in de novo purine and thymidylate biosynthesis. An RNA gel mobility shift assay was used to demonstrate a specific interaction between human recombinant DHFR protein and its corresponding DHFR mRNA. Incubation of DHFR protein with either its substrates, dihydrofolate or NADPH, or with an inhibitor, methotrexate, repressed its ability to interact with DHFR mRNA. An in vitro rabbit reticulocyte lysate translation system was used to show that the addition of exogenous human recombinant DHFR protein to in vitro translation reactions specifically inhibited DHFR mRNA translation. These studies suggest that the direct interaction between DHFR protein and its mRNA may be a mechanism for regulation of DHFR synthesis.