Altered thymidylate synthetase in 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells

Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.     

Therapeutic response of leukemic mice treated with fluorinated pyrimidines and inhibitors of deoxyuridylate synthesis

The therapeutic efficacy of combinations of fluorinated pyrimidines and inhibitors of either ribonucleotide reductase or deoxycytidylate deaminase was evaluated for the treatment of the L1210 mouse leukemia in DBA/2 mice. Therapeutic synergisms were observed with optimal combinations of 5-fluor-2'-deoxyuridine and either hydroxyurea or guanazole. In addition, mice treated with guanazole combined with 5-fluorouracil survived longer than was observed with any dose of guanazole or with 5-fluorouracil alone. Tetrahydrodeoxyuridine, a potential prodrug of a transition-state analog of deoxycytidylate deaminase, did not have antitumor activity by itself nor did it improve the therapeutic response of leukemic mice to 5-fluoro-2'-deoxyuridine. These results are consistent with the hypothesis that deoxyuridylate accumulation was limited by inhibition of ribonucleotide reductase but not by administration of tetrahydrodeoxyuridine. It is suggested that combination chemotherapy with fluorinated pyrimidines and inhibitors of deoxyuridylate synthesis may improve the therapeutic response to these drugs.     

Effect of de novo purine synthesis inhibitors on 5-fluorouracil metabolism and cytotoxicity

Methotrexate pretreatment of L1210 cells had been shown previously by us to cause an enhancement of the intracellular accumulation of 5-fluorouracil and of the formation of 5-fluorouracil nucleotides which was correlated with synergistic cytotoxicity. This effect of methotrexate was associated with increases in 5-phosphoribosyl-1-pyrophosphate, the cofactor required for the conversion of 5-fluorouracil to 5-fluorouridine-5'-monophosphate (FUMP). Because these influences on 5-fluorouracil metabolism were most likely mediated by the activity of methotrexate as an inhibitor of purine synthesis, the effects of other agents that inhibit purine synthesis were examined. An inhibitor of amidophosphoribosyltransferase, 6-methylmercaptopurine ribonucleoside, the glutamine antagonists, azaserine and 6-diazo-5-oxo-L-norleucine (DON), and the L-aspartate analogue inhibitor of adenylsuccinate synthetase, L-alanosine, all reduced the incorporation of [1-14C]glycine into adenine and guanine bases isolated from nucleic acids. Each drug also resulted in intracellular elevations of 5-phosphoribosyl-1-pyrophosphate that were 15- to 25-fold greater than control levels. These alterations in de novo purine nucleotide synthesis were associated with enhanced intracellular 5-fluorouracil accumulation and synergistic cytotoxicity.     

Biochemical factors affecting the tightness of 5-fluorodeoxyuridylate binding to human thymidylate synthetase

Ligand binding studies of thymidylate synthetase (5,10-methylenetetrahydrofolate: deoxyuridylate C-methyltransferase, EC 2.1.1.45) isolated from CCRF-CEM human lymphoblastic leukemia cells were conducted to examine the mechanism of 5-fluorodeoxyuridylate (FdUMP) binding to the human enzyme in the presence of L-1-(+)-5,10-methylenetetrahydrofolate (5,10-CH₂H₄PteGlu) and to assess biochemical factors which could account for decreased binding of FdUMP by the enzyme in cells and tissues. Scatchard plots showed one class of binding sites for FdUMP with an apparent dissociation constant (K_D) of 3.1 × 10^?11 M in the absence, and 3.5 × 10^?10 M in the presence, of 80 mM potassium phosphate (P_i). The observed rate constant for FdUMP association (k_on^obs) was dependent on the 5,10-CH₂H₄PteGlu concentration and attained a maximal value of 1.7 × 10⁸M^?1 min^?1 at a concentration of ca. 12μM 5,10-CH₂H₄PteGlu. Increasing the concentration of 5,10-CH₂H₄PteGlu decreased the apparent rate constant of FdUMP dissociation (k_off^obs), although FdUMP had no effect on the rate of 5,10-CH₂H₄PteGlu dissociation. These studies showed that CCRF-CEM thymidylate synthetase has an ordered mechanism of ligand binding and release, with the nucleotide binding first and dissociating last. Incubation of the enzyme-FdUMP-5,10-CH₂H₄PteGlu ternary complex with the substrate dUMP resulted in renewal of enzyme activity at about the same rate as that of FdUMP release. Nucleotides, deoxyuridine (dUrd), and polyoxyanions decreased the rate of FdUMP association but had no significant effect on the rate of FdUMP dissociation. dUMP was the most potent inhibitor of FdUMP binding found, with a binding constant determined from competition experiments of 0.36 μM compared to a Michaelis constant of 2.8 μM. The binding constant for p_i was determined similarly to be 10 mM. The effects of dUMP plus P_i, on decreasing the rate of FdUMP association were additive, whereas the combined effects of dUMP and sub-optimal concentrations of 5,10-CH₂H₄PteGlu were not additive, but multiplicative or greater. The levels of dUMP, P_i, and 5,10-CH₂H₄PteGlu that reportedly are present in some cells and tissues, theoretically could increase the K_Dto more than 10^?6M. These results could account for the coexistence of substantial levels of FdUMP and unbound thymidylate synthetase found in some cells and tissues.     

A mathematical model of the kinetics of 5-fluorouracil and its catabolites in freshly isolated rat hepatocytes

A mathematical model for the kinetics of 5-fluorouracil (FUra) catabolism in liver cells is proposed. It is based on published data for the metabolism of FUra by isolated rat hepatocytes. The model relies on biochemical knowledge of the catabolic pathway. The key-steps are: the cellular uptake and the conversion of the unchanged drug to dihydrofluorouracil (FUH2) and subsequently to alpha-fluoro-beta-alanine (FBAL); the cellular fluxes of the 2 catabolites, FUH2 and FBAL. Water is partitioned between the extracellular and intracellular spaces. The first step is described by Michaelis-Menten kinetics and the other processes by first-order kinetics. Satisfactory fitting of the model validates these simplifications and provides values for the parameters describing the process. The model indicates that the kinetics of FUra disappearance are non linear, the Vmax of the first step being between 3.1 and 5.0 microM/min and the Km between 12 and 37 microM; the rate limiting step is the degradation of FUH2 (the major intracellular catabolite) with a rate constant of 0.1 to 0.02 min-1; the FUH2 transmembrane exchange is active; the exchange of the final catabolite FBAL is by diffusion.     

Modulation of 5-fluorouracil cytotoxicity through thymidylate synthase and NF-kappaB down-regulation and its application on the radiolabelled iododeoxyuridine therapy on human hepatoma cell

The inhibition of thymidylate synthase (TS) by 5-fluorouracil (5-FU) was known to increase the incorporation of radiolabelled iododeoxyuridine (IdUrd) into DNA. The relatively non-toxic compounds such as thiol-containing antioxidant pyrrolidinodithiocarbamte (PDTC) or aromatic fatty acid phenylbutyrate (PB) had been reported to enhance the cytotoxic efficacy of 5-FU. We designed a novel strategy through triplet combination of PB, PDTC and 5-FU to increase the radiolabelled IdUrd uptake and investigated the underlying mechanisms. The growth inhibition and [(125)I]IdUrd-DNA incorporation by PB, PDTC, 5-FU in different combinations were tested on parent or p21(Waf1) transfected Hep3B cells. The combination of PB and PDTC was more effective in enhancing 5-FU cytotoxicity than either drug alone. The combination of PB/PDTC and 5-FU blocked cells in S-phase and resulted in 8.5-fold increase of radiolabelled IdUrd-DNA incorporation. The transfection of p21(Waf1) did not change the general pattern of enhancement. Intriguingly, the combination of PB and PDTC effectively down-regulated NF-kappaB and TS and prevented their up-regulation from 5-FU treatment than either drug alone through a p21(Waf1)-independent mechanism. Based on this strategy, the 3-drug combination offered potential for improved radiolabelled IdUrd molecular radiotherapy for hepatoma treatment.     

Synergistic effect of 5-fluorouracil and N-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in a human mammary carcinoma

The biological effects of $\text{N-(}\text{phosphonacetyl}\text{)-}\text{l}\text{-}\text{aspartate}$ (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10^?7 to 1.5 × 10^?5M) and PALA (6.0 × 10^?5 to 3.6 × 10^?3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [³H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [¹⁴C]deoxyguanosine and [³H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10^?5M PALA and 1 × 10^?6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10^?6M [³H]-5-FU into poly(A)RNA in comparison to [³H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10^?5 M PALA and 1 × 10^?6 M [³H]-5-FU resulted in a 285 per cent increase in the incorporation of [³H]-5-FU into non-poly(A)RNA. The incorporation of either [¹⁴C]deoxyguanosine or [³H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [³H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.     

5-Benzylacyclouridine and 5-benzyloxybenzylacyclouridine,potent inhibitors of uridine phosphorylase

Various pyrimidine acyclonucleosides (l-(2'-hydroxyethoxymethyl)uracils) are specific inhibitors of uridine phosphorylase [Niedzwicki et al., Biochem. Pharmac.30, 2097 (1981)]. 5-Benzyluracils have also been shown to inhibit this enzyme [Baker and Kelley. J. med. Chem.13, 461 (1970); Woodman et al., Biochem. Pharmac. 29,1059 (1980)]. We have synthesized the acyclonucleoside analogs of 5-benzyluracil (BU) and 5-benzyloxybenzyluracil (BBU). These compounds, 5-benzyl-1-(2'-hydroxy-ethoxymethyl) uracil (BAU) and 5-(m-benzyloxybenzyl)-1-(2'-hydroxyethoxymethyl)uracil (BBAU), are potent inhibitors of uridine phosphorylase. K_i values of 98 and 32 nM were estimated for BAU and BBAU respectively. These compounds are better inhibitors of uridine phosphorylase than BU (K_i = 1575 nM), BBU (K_i = 270 nM), and all other compounds previously tested, and they have no effect on thymidine phosphorylase, uridine-cytidine kinase, or thymidine kinase. Potential chemotherapeutic applications of BAU and BBAU are discussed.     

5'-deoxy-5'-methylthioadenosine phosphorylase--II. Role of the enzyme in the metabolism and antineoplastic action of adenine-substituted analogs of 5'-deoxy-5'-methylthioadenosine

The biological activities of several previously synthesized [J. A. Montgomery et al., J. med. Chem. 17, 1197 (1974)] adenine-substituted analogs of 5'-deoxy-5'-methylthio- or 5'-deoxy-5'-ethyl-thioadenosine, including the 2-fluoroadenine, 2-chloroadenine, 2,6-diaminopurine, 8-azaadenine, and 4-aminopyrazolo [3,4-d]pyrimidine-containing derivatives, have been reexamined. It is demonstrated that many of these analogs are cleaved to their respective free base analogs by 5'-deoxy-5'-methyl-thioadenosine phosphorylase (MTAPase), an enzyme associated with polyamine biosynthesis, and that this reaction is necessary for the cytotoxic action of these MTA analogs to be fully expressed. Evidence to support this includes: (1) the growth of two MTAPase-containing human colon carcinoma cell lines (the HCT-15 and DLD-1 lines) was inhibited by these analogs, whereas an MTAPase-deficient cell line, the CCRF-CEM human T-cell leukemia, was relatively insensitive to their cytotoxic action; (2) extracts of the MTAPase-containing colon carcinoma cell lines were able to cleave these analogs to their respective free base analogs; in contrast, extracts of MTAPase-deficient CCRF-CEM cells were unable to cleave these analogs; (3) intact colon carcinoma cells converted these MTA analogs to their corresponding 5'-phosphorylated analog nucleotides, whereas CCRF-CEM cells did not, at least to detectable levels; and (4) the MTA analog, 5'-deoxy-5'-ethylthio-4-aminopyrazolo [3,4-d]pyrimidine ribonucleoside, which is not a substrate of MTAPase, did not form analog nucleotides and was essentially noncytotoxic to all cell lines tested, whereas the corresponding adenine analog, 4-aminopyrazolo [3,4-d]pyrimidine, readily formed analog nucleotides and was highly cytotoxic to all the lines. It is postulated that the corresponding adenine analog 5'-phosphorylated nucleotides are the primary active metabolites of these MTA analogs, having been formed by the cleavage of these nucleosides to free adenine analogs by MTAPase, followed by the conversion of these base analogs to analog nucleotides by adenine phosphoribosyltransferase and the enzymes of adenine nucleotide phosphorylation. This pathway represents a novel drug-activation system for the synthesis of analog nucleotides and has the potential to be exploited chemotherapeutically.     

Structural analogs of 5'-methylthioadenosine as substrates and inhibitors of 5'-methylthioadenosine phosphorylase and as inhibitors of human lymphocyte transformation

5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase was purified 13.4-fold from human peripheral lymphocytes. The enzyme demonstrated normal Michaelis-Menten kinetics with Km values of 26 microM and 7.5 mM for the two substrates, MTA and phosphate, respectively. The rate of MTA degradation was temperature dependent, 47 degrees being the optimum temperature. Five structural analogs served as alternative substrates with Km values ranging from 31 to 53 microM while two compounds, 5'-deoxy-5'-methylthiotubercidin (MTT) (Ki = 31 microM) and adenine (Ki = 172 microM), were inhibitory. These same analogs were examined as inhibitors of mitogen-induced human lymphocyte blastogenesis. MTT was found to be the most effective inhibitor of lymphocyte transformation with an I50 of 80 microM.     

Human tissues degrade uridine much less than thymidine. Possible consequence for 5-fluorouracil therapy

In view of clinical trials to improve FUra chemotherapy of cancer by combined application with Urd and dThd, we investigated the capacity of human tissues to split these nucleosides.All human normal and neoplastic tissues gave a uridine-splitting activity which can be inhibited by β-L-pTdR and behaves in this respect as uridine-deoxyuridine phosphorylase (EC 2.4.2.3). dThd splitting, however, which is 2–9-fold higher than that of Urd, is insensitive towards β-L-pTdR, confirming earlier results that it is due to thymidine phosphorylase (EC 2.4.2.4).On the other hand, tissues, e.g. spleen of rats and mice, in which dThd and Urd are split by uridine-deoxyuridine phosphorylase, degrade 2–5-fold more Urd than dThd. Thus, free pyrimidine base competing with FUra for degradation and thus prolonging the life time of the drug in the body, will be formed mainly from dThd in the human body but more so from Urd in the rat or mouse.     

Cellular transformation of the investigational new anticancer drug NB1011, a phosphoramidate of 5-(2-bromovinyl)-2'-deoxyuridine,results in modification of cellular proteins not DNA

NB1011 [E-5-(2-bromovinyl)-2'-deoxyuridine-5'-(L-methylalaninyl)-phenylphosphoramidate], a phosphoramidate prodrug of E-5-(2-bromovinyl)-2'-deoxyuridine-5'-monophosphate (BVdUMP), is an investigational new anticancer drug. NB1011 targets thymidylate synthase (TS), which catalyzes the transformation of BVdUMP into cytotoxic reaction products. Due to the elevated levels of TS expression in tumor cells compared to normal cells, these cytotoxic products are preferentially generated inside tumor cells, and, as expected, NB1011 is more toxic to cells with higher levels of TS expression. Therefore, NB1011 therapy should kill tumor cells without severely damaging normal cells. Radiolabeled NB1011 was used to determine the intracellular fate of NB1011 reaction products and, possibly, the mechanism of action of this investigational new drug. We found significant incorporation of the radiolabel into cellular macromolecules. In contrast to our expectations that NB1011 product(s) would be incorporated into DNA, we discovered that cellular proteins were the labeled macromolecular fraction. Herein, we report that the intracellular transformation of NB1011 involves formation of the corresponding monophosphate, TS-dependent transformation into highly reactive intermediates, and subsequent incorporation into cellular proteins. TS itself appears to escape irreversible inactivation. Our data suggest that protein modification not DNA incorporation accounts for the therapeutic effect of NB1011. The proposed mechanism is rather unexpected for a nucleotide analogue and could lead to the discovery of new cellular protein targets for future drug design.     

Exclusion of exogenous 5-methyl-2'-deoxycytidine from DNA in human leukemic cells. A study with [2(-14)C]- and [methyl-14C]5-methyl-2'-deoxycytidine

Modification of DNA-cytosine by a 5-methyl group is thought to be an important mechanism which regulates the expression of eukaryotic genes. This modification takes place after semiconservative replication. There is very little evidence, if any, that 5MeCyt could be naturally incorporated into mammalian DNA in semiconservative replication. We have clarified the possibility of incorporating 5MedCyd pharmacologically into human leukemic cells in vitro. To this end, we developed a novel small-scale synthesis method for 14C-labeled 5MedCyd starting from commercially available [14C]dThd derivatives. Particular attention was focused upon possible incorporation of radioactive 5MedCyd derivatives into the acid-soluble cellular fraction as well as into nucleic acids and protein in human cells. The results showed that [2(-14)C]- and [methyl-14C]5MedCyd were incorporated into human leukemic cells to a similar extent. The radioactivity originating from these compounds was incorporated mainly into the acid-soluble pool and nucleic acids. The exact nature of the intracellular radioactive molecules in RNA is not known, but the radioactive label in DNA hydrolyzate co-chromatographed exclusively with thymine. Hence, 5MedCyd is deaminated to thymidine before incorporating into DNA. This deamination had taken place already (partially) in the culture medium. Human leukemic cells do effectively protect their DNA from incorporation of exogenous 5MedCyd.     

Substrate properties of 5-fluorouridine diphospho sugars detected in hepatoma cells

Nucleotide sugars derived from 5-fluorouridine were studied in cultured AS-30D hepatoma cells as well as in kinetic enzyme assays in vitro in comparison with the physiologic uridine diphospho sugars. Hepatoma cells converted 5-fluoro [¹⁴C]uridine to 5-fluorouridine diphospho (FUDP) glucose, FUDP-galactose, FUDP-N-acetylglucosamine, FUDP-N-acetylgalactosamine, and trace amounts of FUDP-glucuronate, as analyzed by different systems of high-performance liquid chromatography. 5-Fluoro[¹⁴C]uridine and [¹⁴C]uridine, at concentrations of 5 μM in the culture medium, were phosphorylated by the cells during 60 min to similar amounts of FUTP and UTP, respectively, while the synthesis of [¹⁴]FUDP-sugars was reduced to 14% as compared to that of [¹⁴C]UDP-sugars.FUDP-sugars, synthesized by chemical and enzymatic procedures, were assayed in vitro as substrates for enzymes of UDP-sugar metabolism. K_m and V values in a range comparable to that of the respective UDP-sugars were determined for FUDP-sugars in the reactions catalyzed by UDP-glucose pyrophosphorylase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, UDP-N-acetylglucosamine 2-epimerase, glycogen synthase, and UDP-glucose dehydrogenase.Our experiments in hepatoma cells and with enzymes in vitro have revealed additional reactions of FUDP-sugar metabolism demonstrating a metabolite pattern analogous to that of UDP-sugars. The amounts of FUDP-sugars formed relative to UDP-sugars in intact cells were smaller than suggested on the basis of their kinetic comparison in vitro.     

In vivo inactivation by acivicin of carbamoylphosphate synthetase II in rat hepatoma

The antitumor drug acivicin, l-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, in vivo irreversibly inactivated carbamoyl-phosphate synthetase II (glutamine-dependent) (EC 6.3.5.5), the first and rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, in transplantable rat hepatoma and host liver. With two injections of 0.5 mg acivicin per 100 g body weight to one group and two injections of 5 mg to another group, enzyme activity decreased to 20 and 1% in hepatoma and to 99 and 31% in liver respectively. Aspartate carbamoyltransferase (EC 2.1,3.2) activity was not affected, Acivicin in vitro selectively inactivated glutamine-dependent activity of the synthetase II from the hepatoma and liver, with an inactivation constant (K_inact) of 90 μM and a minimum inactivation halftime (T) of 0.7 min. The inactivation velocity with 10 μM acivicin was 5.0-fold stimulated by 2mM MgATP and 18.4-fold by 2 mM MgATP plus 16.7 mM bicarbonate. MgATP at 0.5 mM caused halfmaximum stimulation of the inactivation velocity. Under in vitro conditions, l-glutamine (1 mM) protected the enzyme from inactivation by 10 μM acivicin. The synthetase activity was protected in vitro by 6mM concentrations of glycine (84%). l-glutamate (59%) and l-aspartate (51%) and by 0.5 mM UTP (35%) from inactivation by 20 μM acivicin. The results are compatible with the suggestion that acivicin is an active site-directed affinity analog of l-glutamine.     

Determination of apoptosis, uracil incorporation, DNA strand breaks, and sister chromatid exchanges under conditions of thymidylate deprivation in a model of BER deficiency

Thymidylate synthase (TS) is an important target of several chemotherapeutic agents. During TS inhibition, dTTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). BER has been hypothesized to play a role in the response to thymidylate deprivation, despite a lack of direct evidence. We previously found that beta-pol null murine fibroblasts were approximately six-fold more resistant than wild-type cells to raltitrexed, a folate-based inhibitor specific for TS. In this study, a number of endpoints were determined to understand the influence of BER and beta-pol during raltitrexed treatment. Raltitrexed induced apoptosis in wild-type cells to a greater extent than in beta-pol null cells. A PARP inhibitor decreased the sensitivity to raltitrexed, although the extent was not different between wild-type and beta-pol null cells. No evidence was seen for extensive strand break formation that preceded apoptosis, although raltitrexed induced more sister chromatid exchanges in wild-type cells. Increased levels of uracil in DNA were detected following treatment in wild-type and beta-pol null cells. However, uracil levels were only approximately two-fold higher in DNA from treated cells compared to untreated. Uracil DNA glycosylase activity was slightly higher in beta-pol null cells, although not sufficiently different to explain the difference in sensitivity to raltitrexed. Taken together, the data suggest that the sensitivity of the wild-type cells to raltitrexed is not associated with activation of PARP-1 dependent BER, extensive uracil incorporation into DNA and persistent strand breaks, but rather with changes suggestive of DNA recombination.     

Purine and pyrimidine salvage pathways in Leishmania donovani

Leishmania donovani, grown in culture, salvaged radiolabeled purine bases which were distributed into adenine and guanine ribonucleotides and into the RNA of these cells. De novo synthesis of purines in L. donovani does not occur [J. J. Marr, R. L. Berens and D. J. Nelson, Biochim. biophys. Acta544, 360 (1978)]. [8-¹⁴C]Adenine was rapidly deaminated to hypoxanthine via the action of an adenine aminohydrolase (EC 3.5.4.2). [8-¹⁴C]Guanine was also rapidly deaminated by guanase (EC 3.5.4.3) to form xanthine in these cells. Therefore, the formation of nucleotides of hypoxanthine and xanthine are the first committed steps of purine salvage in L. donovani. While purines are efficiently conserved by this parasite, the salvage of pyrimidines is not so dramatic. [2-¹⁴C]Orotic acid was converted to OMP and then incorporated into the pyrimidine nucleotides and into RNA, indicating the existence of the later steps of de novo pyrimidine synthesis. [6-¹⁴C]Thymidine was salvaged by L. donovani, being incorporated into the thymine deoxyribonucleotides and into DNA. The major pathway of thymidine metabolism in this parasite, however, was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (EC 2.4.2.4).     

Effects of antifolates on the binding of 5-fluoro-2'-deoxyuridine monophosphate to thymidylate synthase

Folate based inhibitors of thymidylate synthase (TS) might facilitate binding of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) to TS similar to the natural reduced folate 5,10-methylenetetrahydrofolate (CH(2)-H(4)-folate). We studied the lipophilic, non-polyglutamatable antifolates Nolatrexed (NTX) and AG331 and antifolates, that can have a polyglutamate side chain like the natural folate CH(2)-H(4)-folate; GW1843U89, Raltitrexed (RTX) and Multi-targetted antifolate (MTA) and pentaglutamates (RTX-Glu(5) and MTA-Glu(5)). The capacity of these compounds to facilitate the binding of [(3)H]FdUMP to Lactobacillus casei TS and an ammoniumsulphate precipitate of human TS was investigated. Only NTX, RTX-Glu(5) and MTA-Glu(5) facilitated FdUMP binding to L. casei TS and their dissociation constant K(d) (0.2-0.7 microM) was low compared to CH(2)-H(4)-folate (2.0 microM). The small lipophilic molecule NTX was favorable to the larger AG331. Polyglutamylation, as indicated by the difference in effect of RTX vs. RTX-Glu(5) and MTA vs. MTA-Glu(5), seems to be important for a classical antifolate to facilitate binding of FdUMP to bacterial TS. Effects of antifolates on FdUMP binding to human TS were different. At a low concentration (0.05 microM) NTX, RTX-Glu(5) and MTA-Glu(5) facilitated 3-5 times higher binding of [(3)H]FdUMP to TS than CH(2)-H(4)-folate. At higher concentrations (0.3-5 microM) of NTX, RTX-Glu(5) and MTA-Glu(5) the FdUMP binding decreased. The complex remained stable in the absence of (anti)folate for at least 24hr. The K(d) values of the antifolates for human TS varied from 19 to 387 nM, while the K(d) of CH(2)-H(4)-folate for human TS was 351 nM. The Hill coefficients, which indicated the type of cooperativity of the antifolates in the binding of FdUMP to TS were positive (0.58-0.99) at low concentrations (<0.3 microM) and negative (-0.35 to -0.81) at concentrations >0.3 microM except for GW1843U89, which only showed negative cooperativity (-1.70). It was shown with [(14)C]NTX that when the binding of FdUMP decreased at high NTX concentrations, the binding of NTX to TS still increased. This also held for the natural substrate dUMP. The negative cooperativity of the antifolates was clearly concentration dependent. The difference between human and L. casei TS in the FdUMP binding assays with antifolates can possibly be explained by interaction of the two subunits of human TS, which was absent in L. casei TS. The binding of antifolates to one of the two subunits induced a conformational change of the other subunit. This change no longer allowed the binding of FdUMP or dUMP at the active site. In conclusion this study showed that antifolates enhanced the binding of FdUMP to TS, especially at low antifolate concentrations, that are also clinically achievable, e.g. in human plasma.     

Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression

Pemetrexed (ALIMTA, MTA) is a novel thymidylate synthase (TS) inhibitor and has shown activity against colon cancer, mesothelioma and nonsmall-cell lung cancer. We induced resistance to Pemetrexed in the human colon cancer cell line WiDr by using a continuous exposure to stepwise increasing Pemetrexed concentrations (up to 20 microM) as well as a more clinically relevant schedule with intermittent exposure (up to 50 microM) for 4 hr every 7 days, resulting in WiDr variants WiDr-cPEM and WiDr-4PEM, respectively. However, using the same conditions, it was not possible to induce resistance in the WiDr/F cell line, a variant adapted to growth under low folate conditions. Mechanisms of resistance to Pemetrexed were determined at the level of TS, folylpolyglutamate synthetase (FPGS) and reduced folate carrier (RFC). WiDr-4PEM and WiDr-cPEM showed cross-resistance to the polyglutamatable TS inhibitor Raltitrexed (6- and 19-fold, respectively) and the nonpolyglutamatable TS-inhibitor Thymitaq (6- and 42-fold, respectively) but not to 5-fluorouracil. The ratios of TS mRNA:beta actin mRNA in WiDr-4PEM and WiDr-cPEM were 5-fold (P=0.01) and 18-fold (P=0.04) higher, respectively, compared to WiDr (ratio: 0.012). In addition, TS protein expression in the resistant WiDr variants was elevated 3-fold compared to WiDr, while the catalytic activity of TS with 1 microM dUMP increased from 30 pmol/hr/10(6) cells in WiDr cells to 2201 and 7663 pmol/hr/10(6) cells in WiDr-4PEM and WiDr-cPEM, respectively. The activity of FPGS was moderately decreased, but not significantly different in all WiDr variants. Finally, no evidence was found that decreased catalytic activity of RFC was responsible for the obtained Pemetrexed resistance. Altogether, these results indicate that resistance to Pemetrexed in the colon cancer cell line WiDr was solely due to upregulation of TS of which all related parameters (mRNA and protein expression and TS activity) were increased, rather than alterations in FPGS or RFC activity.     

Toxicity of combinations of arabinosylcytosine and 3-deazauridine toward neoplastic cells in culture

The antiproliferative effects of 3-deazauridine and arabinosylcytosine toward cells in culture (HeLa and RPMI 6410 lymphoblastoid cells) were potentiated when the agents were present together. The conversion of arabinosylcytosine to nucleotide metabolites in RPMI 6410 cells was greatly enhanced in the presence of deazauridine: this enhancement is thought to be the basis of the potentiation in antiproliferative activity. These drug combination effects did not occur in two RPMI 6410 sublines which were resistant to deazauridine and deficient in uridine kinase activity.