Human cytosolic and mitochondrial folylpolyglutamate synthetase are electrophoretically distinct. Expression in antifolate-sensitive and -resistant human cell lines.

Folylpolyglutamate synthetase (FPGS) activity in CCRF-CEM human leukemia cells was found in the cytosolic ( approximately 67% of total) and mitochondrial ( approximately 22%) fractions. A polyclonal antipeptide antibody (430Ab) to human FPGS specifically recognized distinct immunoreactive bands ( approximately 60 kDa) present in each subcellular fraction. Human cytosolic FPGS (hcFPGS) migrated more rapidly than mitochondrial FPGS (hmFPGS); their estimated difference in molecular mass was 1 kDa. The human K562 acute nonlymphocytic leukemia and the A253 and FaDu head and neck cancer cell lines also expressed the two FPGS isoforms, and the ratio of hcFPGS to hmFPGS protein in each cell line was similar. Since K562 and A253 cells are intrinsically resistant to pulse methotrexate (MTX) exposure relative to CCRF-CEM and FaDu cells, respectively, because of decreased MTX polyglutamate synthesis (despite having similar levels of total FPGS activity expression), these data suggest that the natural difference in drug sensitivity cannot be explained by compartmentalization of FPGS activity. Higher expression of hmFPGS relative to hcFPGS was observed in some sublines of CCRF-CEM with acquired MTX resistance suggesting that differential expression of the hmFPGS isoform may contribute to MTX resistance caused by decreased FPGS activity.

Cellular aspects of folate and antifolate membrane transport.

Folates--one carbon carriers--take part in the metabolism of purine, thymidylate and some amino acids. Internalization of these compounds employs several mechanisms of transport systems. Reduced folate carriers and folate receptors play the most important role in this process. The physiological role of these molecules in normal and neoplastic cells is described regarding changes in transport activity and connection of transport systems with resistance to antifolates and cancer development.

Synergistic effect of 5-fluorodeoxyuridine and quinazoline antifolates on murine leukemia self-cultured in vitro.

The effect of thymidylate synthase inhibitors, fluorodeoxyuridine (FdUrd) and its two sulphonamide derivatives was examined in the culture of murine leukemia cells -- 5178Y (parental subline) and its fluorodeoxyuridine resistant subline 5178Y/F. A synergistic effect of the antimetabolites on cell survival was observed on exposure of the culture of either line to a slightly inhibitory concentration of FdUrd (1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroylsulphogluta mate or 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroylsulphoglyci ne. This effect was accompanied by a marked reduction, in both cell lines of intracellular concentration of 5,10-methylenetetrahydro-pteroyl-polyglutamate, although its concentration in the resistant subline was 3 times as high as in the parental line. The inhibitory effect of combined drugs on the cellular pool of folates in 5178Y line depended also on the sequence of drug addition, whereas in the FdUrd resistant line this sequence was without any effect. The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by antifolates is intensified by a prior use of FdUrd.

The effects of combined antifolates on inhibition of growth of murine leukemia cells cultured in vitro.

The synergistic effect of trimetrexate (TMTX) and sulphonamide derivatives of quinazoline on the cultured 5178Y murine leukemia cells was examined. On exposure to the slightly inhibitory concentrations of TMTX (0.1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroyl-sulphoglyc ine (DMPDDSF) (0.02 microM) a synergistic inhibitory effect of the antifolates on cell growth was observed. These two drugs in the same combination caused also synergistic inhibition of de novo synthesis of thymidylate in intact cells as measured by tritium release from [5-(3)H]deoxyuridylate. This was accompanied by a marked reduction in intracellular concentration of 5,10-methylenetetrahydro-pteroyl-polyglutamate (5,10CH2H4PteGlu(n)) (0.2 microM) and dihydropteroyl-polyglutamate (0.12 microM). In these conditions de novo biosynthesis of purine was decreased by 50%. These observations show that growth inhibition by combined antifolates is mediated by intracellular depletion of the substrate of thymidylate synthase -- 5,10CH2H4PteGlu(n). The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by DMPDDSF is intensified by prior application of TMTX -- an inhibitor of dihydrofolate reductase.

Effect of combined inhibitors of thymidylate synthase-5-fluorodeoxyuridine and quinazoline antifolates on murine leukemia cells cultured in vitro.

The synergistic effect of two different inhibitors of thymidylate synthase-FdUrd and sulphonamide derivatives on murine leukemia cells-5178Y (parental subline) and 5178Y/F (its fluorodeoxyuridine resistant subline) in culture was examined. Upon the exposure of cultures from both lines to a slightly inhibitory concentration of FdUrd (1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroylsulphogluta mine or -glycine a synergistic effect of antimetabolites on cell growth was observed. This was accompanied by a marked reduction in intracellular concentration in both cell lines of 5,10CH2H4PteGlu; the intracellular concentration of 5,10CH2H4PteGlu(n) in the resistant subline was 3 times higher than in parental line. The inhibitory effect of combined drugs on the cellular pool of 5178Y of the two antimetabolites also depends on the sequence of their addition; however in the FdUrd resistant cell-line the dependence on the sequence of the addition was not observed. The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by antifolates is intensified by proprior use of FdUrd.

2-Thio derivatives of dUrd and 5-fluoro-dUrd and their 5'-monophosphates: synthesis, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

A convenient synthesis of 5-fluoro-2-thiouracil (11) is based on hydrolytic deamination of 5-fluoro-2-thiocytosine (9). Lewis acid-catalyzed condensation of di-TMS-5-fluoro-2-thiouracil (13) or di-TMS-2-thiouracil (14) with 2-deoxy-3,5-di-O-p-toluyl-D-ribofuranosyl chloride (15) led to mixtures of the beta- and alpha-anomers of 3',5'-toluylated 2'-deoxy-5-fluoro-2-thiouridine (16 and 18) or 2'-deoxy-2-thiouridine (17 and 19), each of which was deblocked with MeOH-NH3 to give the desired free anomeric nucleoside pairs 1, 5 and 3, 7, respectively. These were selectively converted to the corresponding 5'-monophosphates 2, 6 and 4, 8, with the aid of the wheat shoot phosphotransferase system. Conformations of the nucleosides 1, 3, 5, 7 are deduced from 1H NMR spectra, and circular dichroism spectra for nucleotide anomeric pairs 2, 6 and 4, 8 are reported. Whereas beta-2-thio-dUMP (4) was a good substrate (Km approximately 10(-5) M), beta-5-fluoro-2-thio-dUMP (2) proved to be a potent competitive, slow-binding inhibitor (Ki approximately 10(-8) M) of the purified enzymes from Ehrlich ascites carcinoma and L1210 cells. The alpha-anomer 6 was a weak inhibitor, with Ki in the mM range, and its congener 8 hardly interacted with the enzyme. The beta-anomer 1 exhibited antitumor activity in a mouse leukemic cell line L5178Y (IC50 approximately 10(-6) M), hence 40-100-fold weaker than 5-fluoro-dUrd. Its alpha-anomer 5 was 10-fold less active, but exhibited at least 10-fold higher selectivity with respect to the tumor cells than the beta-anomer 1.

At least four regulatory genes control sulphur metabolite repression in Aspergillus nidulans.

Mutations in four genes: sconA (formerly suA25meth, mapA25), sconB (formerly mapB1), sconC and sconD, the last two identified in this work, relieve a group of sulphur amino acid biosynthetic enzymes from methionine-mediated sulphur metabolite repression. Exogenous methionine has no effect on sulphate assimilation in the mutant strains, whereas in the wild type it causes almost complete elimination of sulphate incorporation. In both mutant and wild-type strains methionine is efficiently taken up and metabolized to S-adenosylmethionine, homocysteine and other compounds, scon mutants also show elevated levels of folate-metabolizing enzymes which results from the large pool of homocysteine found in these strains. The folate enzymes appear to be inducible by homocysteine and repressible by methionine (or S-adenosylmethionine).

Inhibition of mammalian thymidylate synthase by 10-formyltetrahydropteroylpolyglutamate.

Reduced derivatives of 10-formylfolate have been evaluated as inhibitors of mammalian thymidylate synthase (EC 2.1.1.45) from H35 hepatoma cells. With 5,10-methylenetetrahydrofolylheptaglutamate as the substrate, 10-formyltetrahydrofolylmonoglutamate is a competitive inhibitor with a Ki of 2.4 microM, which is reduced to 0.1 microM for the heptaglutamate derivative. 10-Formyldihydrofolylmono- and -heptaglutamate are approximately threefold less inhibitory than the tetrahydro analog. The concentrations of 10-formyltetrahydrofolate and 10-formyldihydrofolate were measured in dividing hepatoma cells by a novel enzymatic assay and were found to be 5 microM and undetectable, respectively. These results suggest that the concentration of 10-formyltetrahydrofolate within the dividing cells has the potential to severely inhibit or modulate thymidylate biosynthesis.

Role of substrate depletion in the inhibition of thymidylate biosynthesis by the dihydrofolate reductase inhibitor trimetrexate in cultured hepatoma cells.

The effects of the lipid-soluble dihydrofolate reductase inhibitor, trimetrexate, on the inhibition of thymidylate biosynthesis as a result of perturbation in cellular folate pools in H35 hepatoma cells in vitro has been investigated. Exposure of the cultures to increasing concentrations of trimetrexate between 2 and 20 nM causes a marked reduction in de novo thymidylate biosynthesis and a concomitant decrease in (6R)5,10-methylenetetrahydropteroylpolyglutamate (5,10-CH2H4PteGlun) from 2.0-0.2 microM, respectively. This is accompanied by an increase in H2PteGlun from 1.2 microM in control cultures to 4.7 microM in cultures exposed to 20 nM trimetrexate. The dependency of de novo thymidylate biosynthesis on intracellular 5,10-CH2H4PteGlun in trimetrexate-treated cells is compared with (a) the relationship of thymidylate biosynthesis on intracellular levels of 5,10-CH2H4PteGlun in folate-depleted cells supplemented with increments of folic acid and (b) the substrate (5,10-CH2H4PteGlun) dependence of purified thymidylate synthase from the same source. All three results are nearly identical demonstrating that trimetrexate-dependent inhibition of de novo thymidylate biosynthesis is primarily a result of substrate depletion. These results coupled with the weak inhibitory properties of H2PteGlun for thymidylate synthase Ki = 5.0 microM) suggest that H2PteGlun accumulation is not the major determinant in inhibiting thymidylate synthase following trimetrexate inhibition but under certain conditions has the potential to enhance the inhibition caused by substrate depletion.

Interrelated regulation of sulphur-containing amino-acid biosynthetic enzymes and folate-metabolizing enzymes in Aspergillus nidulans.

In Aspergillus nidulans homocysteine can be metabolized both to cysteine and methionine. Mutants impaired in the main pathway of cysteine synthesis or in the sulphate assimilation pathway show a low pool of glutathione and elevated levels of homocysteine synthase and of the homocysteine-to-cysteine pathway enzymes. On the other hand, the level of methionine synthase and other enzymes of folate metabolism is depressed in these mutants. This anticoordinated regulation provides a mechanism controlling the partition of homocysteine between the two diverging pathways. Homocysteine synthase was found derepressed, along with folate enzymes, in a strain carrying a mutation which suppresses mutations in metA, metB and metG genes. These results indicate that homocysteine synthase can be regarded as the enzyme of an alternative pathway of methionine synthesis and strongly suggest that the regulatory mechanisms governing sulphur-containing amino acid and folate metabolisms are interrelated.

Rescue effect of exogenous reduced folates on methotrexate polyglutamylation and dihydrofolate reductase activity in L1210 cells.

L1210 can be rescued from exposure to an inhibitory effect of methotrexate (MTX) by subsequent addition of 5-formyltetrahydrofolate, 5-methyltetrahydrofolate or dihydrofolate. All folates listed caused marked reduction of long-chain MTX polyglutamates content and increased the activity of dihydrofolate reductase. This indicates that the rescue is a result of interaction of the reduced folates with two processes-polyglutamylation of MTX and generation of dihydrofolate.

Dihydrofolate-mediated reversal of methotrexate toxicity to hepatoma cells in vitro.

H35 hepatoma cells can be rescued from exposure to an inhibitory pulse of methotrexate (MTX) by subsequent addition of folinic acid, dihydrofolate or thymidine. Both folinic acid and dihydrofolate cause the dissociation of methotrexate--dihydrofolate reductase complex although dihydrofolate rescues less effectively than folinic acid. Thymidine does not cause a measurable dissociation of the enzyme--inhibitor complex. The results suggest that the rescue of MTX treated cells by reduced folate coenzymes can be mediated at least in part by the generation of dihydrofolate which by itself can partially reverse MTX inhibition of cell growth.

Synthesis and retention of methotrexate polyglutamates in Ehrlich ascites carcinoma cells.

The formation of methotrexate (MTX) polyglutamates in Ehrlich ascites carcinoma parental and 5-fluorodeoxy-uridine (FdUrd)-resistant cells was examined. The prolonged retention of methotrexate in noneffluxable-polyglutamate form and the pharmacological application of this process is discussed.

Regulation of methotrexate polyglutaminate formation in Ehrlich ascites carcinoma cells by endogenous folate pool.

The conversion of methotrexate to poly-gamma-glutamyl derivatives in Ehrlich ascites carcinoma cells which are characterized by different pools of endogenous folates is described. The cells in which folate pool was high (the 5-fluorodeoxy-uridine-resistant cell line) the ability to convert methotrexate to its polyglutamate derivatives was much lower than in the cells in which folate pool was smaller (the parental cell line). When the cellular folate pool was reduced by treatment of the cells with lysolecithin, a similar methotrexate polyglutamate concentration in both cell lines was observed. These data suggest that cellular folate pool has a regulatory effect on methotrexate polyglutamate synthesis.

Methionine synthase assay based on coupling with thymidylate synthase reaction.

Methionine synthase reaction may be coupled with thymidylate synthase-catalysed tritium release from [5-3H]dUMP via non-enzymatic reaction of formaldehyde with tetrahydrofolate. A convenient and sensitive assay of methionine synthase activity, based on this principle, is described.

Studies of formation and efflux of methotrexate polyglutamates with cultured hepatic cells.

Methotrexate polyglutamates are extensively synthesized when cultured hepatocytes and H35 hepatoma cells are exposed to micromolar concentrations of methotrexate. The predominant species found within the cell have from two to four additional gamma-linked glutamate residues. When either cell type containing a mixture of methotrexate and its polyglutamate derivatives is exposed to medium lacking methotrexate, there is a rapid release of methotrexate. This release has a T1/2 of 2 to 4 min and is apparently complete within 30 to 60 min. Methotrexate polyglutamates leave the cells much more slowly and appear to do so by two mechanisms. Although cleavage to methotrexate and subsequent efflux appears to be quantitatively the more important pathway, there is also a slow, finite loss of intact methotrexate polyglutamates from cells which exclude trypan blue. The T1/2 for the loss of methotrexate polyglutamates by both cell types, when placed in medium lacking methotrexate, is approximately 6 to 8 hr. These results, together with those of an earlier study (Galivan, J. (1980) Mol. Pharmacol. 17:105-110), suggest that the polyglutamate derivatives are forms of methotrexate which are as cytotoxic as methotrexate but which offer a potentially greater capacity for cellular destruction because they are retained longer in the tissue.