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PubMed2000

Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA.

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain.

Aminohydrolases↗
PubMed1964

FOLIC ACID METABOLISM IN ANTIFOLIC-RESISTANT MUTANTS OF STREPTOCOCCUS FAECALIS.

Johnson, Alva H. (Sloan-Kettering Institute for Cancer Research, Rye, N.Y.), and Dorris J. Hutchison. Folic acid metabolism in antifolic-resistant mutants of Streptococcus faecalis. J. Bacteriol. 87:786-791. 1964.-Streptococcus faecalis ATCC 8043 and three of its mutants resistant to amethopterin were compared for their quantitative requirements for serine, purines, and thymine; for their quantitative requirements of folic acid (FA) for the synthesis de novo of serine, purines, and thymine; for the susceptibility to amethopterin of each pathway; and for the relative capacity of resting cells of each culture to synthesize N(5)-formyltetrahydrofolate (5-CHOFAH(4)) from FA and serine or FA and formate. The mutants were found to be both qualitatively and quantitatively different from one another and from the wild strain. The growth conditions, specifically the composition of the medium in which each mutant strain was selected, had a marked effect on the metabolic capacities of the mutants. The ability to synthesize serine, purines, and thymine, as observed from the FA requirements, directly reflected the level of resistance of each pathway to amethopterin. The resistant mutants were more efficient than the wild strain in the formation of 5-CHOFAH(4) from FA and formate and, furthermore, this formate activation paralleled their capacities in the synthesis de novo of serine. Alterations in purine and thymine biosyntheses were also observed.

Adenine↗
PubMed1968

Methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A and its repressibility by serine.

The methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A(k) was purified 100-fold. Because it is extremely labile, this enzyme required protection by 1 mm nicotinamide adenine dinucleotide phosphate (NADP(+)) during purification; 0.01 mm EADP(+) with 0.1% bovine plasma albumin stabilized the purified enzyme during storage at -20 C. Although the enzyme has properties of sulfhydryl enzymes, thiol compounds were not stabilizers. Oxidation of methylenetetrahydrofolate, catalyzed by the purified enzyme preparation, is NADP(+)-specific and yields methenyltetrahydrofolate and the reduced pyridine nucleotide. K(m) values for NADP(+) and for 5,10-methylenetetrahydrofolate (prepared as the formaldehyde adduct of biologically synthesized l,l-tetrahydrofolate) were calculated to be 0.021 and 0.026 mm, respectively. Neither purine bases and their derivatives nor serine inhibited the reaction. In growing cultures, the differential rate of synthesis of the methylenetetrahydrofolate dehydrogenase was dependent upon the composition of the medium. A medium which contained acid-hydrolyzed casein, and thus an exogenous source of serine, was repressive for this enzyme. In a serine-free, completely defined medium, the amount of folate added (for serine synthesis de novo) affected the duration of the initial exponential growth phase. At the termination of this phase, which primarily reflected the onset of a decreased rate of serine biosynthesis, synthesis of the methylenetetrahydrofolate dehydrogenase was derepressed. Exogenous serine in the completely defined medium prevented the derepression. Furthermore, physiological concentrations of l-serine were repressive not only for the dehydrogenase but also for the methenyltetrahydrofolate cyclohydrolase and the serine hydroxymethyl-transferase. Concomitantly, the differential rate of synthesis of the formyltetrahydrofolate synthetase of S. faecium var. durans A(k) was increased. Apparently, serine regulates the differential rates of syntheses of these enzymes.

Aminohydrolases↗
PubMed1990

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Aminohydrolases↗
PubMed1978

Availability of monoglutamyl and polyglutamyl folates in normal subjects and in patients with coeliac sprue.

Intestinal folate absorption was assessed in six normal subjects and in four patients with coeliac sprue who were studied before and after treatment by dietary gluten exclusion. Comparisons were made of the luminal disappearance from the perfused jejunum of 3H-pteroylmonoglutamate and pteroyl 14C-glutamylhexaglutamate, and of the 48-hour urinary recovery of each isotope after perfusion and a tissue saturating dose of folic acid. The labelled urinary folates consisted of folic acid, 10-formyltetrahydrofolate, and 5-methyltetrahydrofolate. In each group urinary recovery of 3H was significantly greater than that of 14C, confirming the evidence from jejunal perfusion that the availability of monoglutamyl folate is greater than that of polyglutamyl folate. According to the urinary recovery data, both folates were poorly absorbed in untreated coeliac sprue, but were normally absorbed after treatment. Assuming uniform displacement of the absorbed labelled folates by the parenteral flushing dose, the finding of greater urinary isotope recovery than of luminal folate disappearance from the perfused proximal jejunal segment suggests an adaptation of the distal small bowel for folate absorption in coeliac sprue.

Adult↗
PubMed1974

Folates in human serum.

Constituent serum folates have been shown to be altered and in some cases raised in diseases in which total folate levels are known to be frequently decreased. Absorption experiments showed that orally administered folate analogues affect the folate pool in different ways. The stability of the 10-formyltetrahydrofolate level in normal subjects is demonstrated.

Administration, Oral↗
PubMed1976

Biopterin derivatives in human body fluids and tissues.

Levels of biopterin derivatives in urine, serum, milk, cerebrospinal fluid, brain, and liver have been measured with the Crithidia fasciculata assay. Normal levels in serum and urine have been given and compared with those in a number of benign and malignant proliferative disorders, phenylketonuria, kidney disease, Parkinson's disease, schizophrenia, controlled epilepsy, rheumatoid arthritis, and pernicious anaemia. The active component of Crithidia factor in serum was 7,8-dihydrobiopterin. Tissue, urine, and some serum samples contained two active materials, the principal one being 7,8-dihydrobiopterin; a minor constituent was probably tetrahydrobiopterin. Serum biopterin levels following methotrexate administration were raised and subsequent administration of folic acid and 5-formyltetrahydrofolic acid further increased serum levels of biopterin derivatives; this was in contrast to the total absence of response to oral folates without prior methotrexate and to 5-methyltetrahydrofolic acid either with or without methotrexate being given.

Adolescent↗
PubMed1986

Pteridines and mono-amines: relevance to neurological damage.

Patients with phenylalanine hydroxylase deficiency show increased concentrations of biopterins and neopterins, and reduced concentrations of serotonin and catecholamines, when phenylalanine concentrations are raised. The pterin rise reflects increased synthesis of dihydroneopterin and tetrahydrobiopterin, and the amine fall a reduction in amine synthesis due to inhibition by phenylalanine of tyrosine and tryptophan transport into neurones. The pterin and amine changes appear to be independent of each other and are present in the central nervous system as well as the periphery; they disappear when phenylalanine concentrations are reduced to normal. Patients with arginase deficiency show a similar amine disturbance but have normal pterin levels. The amine changes probably contribute neurological symptoms but pterin disturbance is not known to affect brain function. Patients with defective biopterin metabolism exhibit severely impaired amine synthesis due to tetrahydrobiopterin deficiency. Pterin concentrations vary with the site of the defect. Symptoms include profound hypokinesis and other features of basal ganglia disease. Neither symptoms nor amine changes are relieved by controlling phenylalanine concentrations. Patients with dihydropteridine reductase (DHPR) deficiency accumulate dihydrobiopterins and develop secondary folate deficiency which resembles that occurring in patients with defective 5,10-methylene tetrahydrofolate reductase activity. The latter disorder is also associated with Parkinsonism and defective amine and pterin turnover in the central nervous system, and a demyelinating illness occurs in both disorders. In DHPR deficiency cerebral calcification may develop in a similar distribution to that seen in congenital folate malabsorption and methotrexate toxicity. Symptoms are ameliorated by therapy with 5-formyltetrahydrofolate but exacerbated by folic acid.

Amines↗
PubMed1988

The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immunologically distinct from the mouse NAD-dependent [corrected] bifunctional enzyme.

Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2',5'-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase-cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.

Aminohydrolases↗
PubMed1980

Utilization of administered folacin derivatives by rats fed a diet low in methionine and folacin.

Rats fed a basal diet low in folacin and methionine or the basal supplemented with 1.5% methionine were injected with either tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, or folic acid. The level of formiminoglutamic acid (FIGLU) excretion was used as an indicator of the animal's metabolically available folacin. Rats fed the basal diet had no decline in FIGLU excretion after dosing. The methionine-supplemented group had significant decreases in FIGLU excretion after dosing with the folacin derivatives. When rats eating the basal diet were dosed with methionine or homocysteine, FIGLU excretions were again decreased.

Animals↗
PubMed1987

Folate transport by human intestinal brush-border membrane vesicles.

Transport of folic acid (Pte-Glu) across the brush-border membrane of human intestine was studied using brush-border membrane vesicle (BBMV) technique. The transport of Pte-Glu was higher in BBMV prepared from the jejunum than those prepared from the ileum (0.70 +/- 0.05 and 0.14 +/- 0.02 pmol X mg protein-1 X 10 s-1, respectively). The transport of Pte-Glu appeared to be carrier mediated and was pH dependent and increased with decreasing incubation buffer pH; saturable (Kt = 1.69 microM, Vmax = 4.72 pmol X mg protein-1 X 10 s-1); inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate, methotrexate, and 5-formyltetrahydrofolate (Ki = 2.2, 1.4 and 1.4 microM, respectively); not affected by inducing a relatively positive or negative intravesicular compartment; independent of Na+ gradient; and inhibited by 4,4'-diisothiocyanatostlibene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor. The increase in Pte-Glu transport on decreasing incubation buffer pH appeared to be in part mediated through a direct effect of acidic pH on the transport carrier and in part through the pH gradient imposed by activating Pte-Glu-:OH- exchange and/or Pte-Glu-:H+ co-transport mechanisms. The important role of an acidic extravesicular environment in Pte-Glu transport is consistent with a role for the intestinal surface acid microclimate in folate transport. These results demonstrate that Pte-Glu transport in human BBMV occurs by a carrier-mediated system that is similar to that described for rat and rabbit intestinal BBMV.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Reduced folate derivatives are endogenous substrates for cMOAT in rats.

We examined the role of the canalicular multispecific organic anion transporter (cMOAT) in the biliary excretion of reduced folate derivatives in vivo and in vitro using normal [Sprague-Dawley rats (SDR)] and mutant [Eisai hyperbilirubinemic rats (EHBR)] rats whose cMOAT is hereditarily deficient. In vivo, the biliary excretion of endogenous tetrahydrofolate (H4PteGlu), 5-methyltetrahydrofolate (5-CH3-H4PteGlu), and 5,10-methylenetetrahydrofolate (5, 10-CH2-H4PteGlu) in EHBR was reduced to 8.2%, 1.9%, and 5.5% of those in SDR, respectively, whereas that of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) was detected only in SDR and not in EHBR. Bile drainage caused reduction of endogenous plasma folate concentrations in SDR but not in EHBR. In vitro, significant ATP-dependent uptake of 3H-labeled 5-CH3-H4PteGlu into canalicular membrane vesicles was observed only in SDR. This ATP-dependent uptake was saturable with a Michaelis constant (Km) value of 126 microM, which was comparable with its inhibitor constant (Ki) value of 121 microM for the ATP-dependent uptake of a typical cMOAT substrate, 2,4-dinitrophenyl-S-glutathione (DNP-SG). Vice versa, DNP-SG inhibited the uptake of 5-CH3-H4PteGlu with a Ki of 35 microM, which was similar to its Km value. In addition, H4PteGlu and 5, 10-CH2-H4PteGlu also inhibited the ATP-dependent uptake of DNP-SG. These results indicate that 5-CH3-H4PteGlu and other derivatives are transported via cMOAT. Therefore, reduced folate derivatives are the first endogenous substrates for cMOAT that do not contain glutathione, glucuronide, or sulfate moieties.

Animals↗
PubMed2001

Ontogeny of hepatic enzymes involved in serine- and folate-dependent one-carbon metabolism in rabbits.

Serine occupies a central position in folate-dependent, one-carbon metabolism through 5,10-methylenetetrahydrofolate (MTHF) and 5-formyltetrahydrofolate (FTHF). We characterized the ontogeny of the specific activity of key enzymes involved in serine, 5,10-MTHF, and 5-FTHF metabolism: methenyltetrahydrofolate synthetase (MTHFS), MTHF reductase (MTHFR), the glycine cleavage system (GCS), methionine synthase (MS), and serine hydroxymethyltransferase (SHMT) in rabbit liver, placenta, brain, and kidney. In liver, MTHFS activity is low in the fetus (0.36 +/- 0.07 nmol. min(-1). mg protein(-1)), peaks at 3 wk (1.48 +/- 0.50 nmol. min(-1). mg protein(-1)), and then decreases to adult levels (1.13 +/- 0.32 nmol. min(-1). mg protein(-1)). MTHFR activity is highest early in gestation (24.9 +/- 2.4 nmol. h(-1). mg protein(-1)) and declines rapidly by birth (4.7 +/- 1.3 nmol. h(-1). mg protein(-1)). MS is highest during fetal life and declines after birth. Cytosolic SHMT activity does not vary during development, but mitochondrial SHMT peaks at 23 days. GCS activity is high in the fetus and the neonate, declining after weaning. In placenta and brain, all activities are low throughout gestation. Cytosolic and mitochondrial SHMT activities are low in kidney and rise after weaning, whereas MTHFS is low throughout development. These data suggest that the liver is the primary site of activity for these enzymes. Throughout development, there are multiple potential sources for production of 5,10-MTHF, but early in gestation high MTHFR activity and low MTHFS activity could reduce 5,10-MTHF availability.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

Enterohepatic circulation kinetics of bile-active folate derivatives and folate homeostasis in rats.

We examined the effect of enteric infusion of 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlu), tetrahydrofolate (H4PteGlu), 10-formyltetrahydrofolate (10-HCO-H4PteGlu), and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) on concentrations of plasma 5-CH3-H4PteGlu in rats. Concentrations of plasma 5-CH3-H4PteGlu rapidly decreased during continuous bile diversion, whereas initial levels of plasma 5-CH3-H4PteGlu were maintained by means of enteric infusion of 5-CH3-H4PteGlu at a dose of 3 nmol/h, which was approximately equal to total secretion of bile folates. Plasma 5-CH3-H4PteGlu levels were also maintained by the infusion of 5,10-CH2-H4PteGlu, H4PteGlu, or 10-HCO-H4PteGlu at the same dose as 5-CH3-H4PteGlu. The results indicate that folates secreted into the bile are reabsorbed through the intestine to regulate plasma concentrations of 5-CH3-H4PteGlu. We examined the secretion kinetics of bile folates after the intravenous injection of 5-CH3-H4PteGlu or folic acid (PteGlu) at 1 mg/kg body wt. When 5-CH3-H4PteGlu was injected, the bile secretion of folates other than 5-CH3-H4PteGlu increased by < 5 times the base level, whereas that of bile 5-CH3-H4PteGlu markedly increased by approximately 30 times. When PteGlu was given, the bile secretion of nonmethylated tetrahydrofolates markedly increased by 15-27 times, whereas that of 5-CH3-H4PteGlu increased by approximately 7 times. The results suggest that oxidized folates may be one of the sources for bile nonmethylated tetrahydrofolates. It is concluded that nonmethylated tetrahydrofolates in bile play an important role in folate homeostasis through enterohepatic circulation, together with 5-CH3-H4PteGlu.

Animals↗
PubMed1992

Folate transport and binding by cultured human proximal tubule cells.

Because mechanisms for the renal regulation of folate excretion are poorly understood, a cell culture system representative of the human proximal tubule (HPT) was used for studies of renal folate transport. After confluent cultures of HPT cells were incubated with 3H-labeled folic acid (PteGlu), binding to the apical membrane was determined by an acid removal procedure, and transport was subsequently measured in solubilized cells. Although PteGlu binding was almost all specific (suppressed by excess unlabeled PteGlu), HPT cells transported PteGlu by specific and nonspecific processes. Specific PteGlu binding and transport were both saturable processes, reaching maxima of 0.5 and 0.1 pmol/mg protein, respectively, with half-maximal constants of 12 and 50 nM, respectively. The PteGlu analogues methotrexate and 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) inhibited both the binding and transport of PteGlu, with 5-CH3-H4PteGlu being more potent (lower half-maximal inhibitory concentration). In contrast, 5-formyltetrahydrofolic acid significantly reduced PteGlu transport at concentrations (100-250 nM) that had no effect on binding. These data suggest that the HPT cells will serve as a good model for studies of renal folate reabsorption. Initial characterization of the transport of folate by HPT cells suggests two distinct processes, binding to a high-affinity membrane folate-binding protein followed by a structurally specific transfer into the cell.

Biological Transport↗
PubMed2004

Selective preservation of pemetrexed pharmacological activity in HeLa cells lacking the reduced folate carrier: association with the presence of a secondary transport pathway.

A methotrexate (MTX)-resistant HeLa subline (R5), developed in this laboratory, with impaired transport due to a genomic deletion of the reduced folate carrier (RFC) was only 2-fold resistant to pemetrexed (PMX), but 200- and 400-fold resistant to raltitrexed (ZD1694) and N(alpha)-(-4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-1-ornithine (PT523), respectively, compared with parental HeLa cells when grown with 2 microM folic acid. When folic acid was replaced with the more physiological 25 nM 5-formyltetrahydrofolate, R5 cells were 2-fold collaterally sensitive to PMX but still 40- and 200-fold resistant to ZD1694 and PT523, respectively. Sensitivity to PT523 and PMX could be completely restored, and sensitivity to ZD1694 nearly restored, by transfection of RFC cDNA into R5 cells, indicating that the defect in drug transport was the only, or major, factor in resistance. The preserved PMX activity in R5 cells could not be related to the very low expression of folate receptors. Rather, retained PMX activity in R5 cells was associated with residual transport by another process that exhibits good affinity for PMX (Kt = 12 microM) with much lower affinities for ZD1694, MTX, and PT523 (Kis of approximately 90, 100, and 250 microM, respectively). PMX transported by this route was rapidly converted to higher polyglutamates and, when grown with 25 nM 5-formyl-tetrahydrofolate, the rate of formation of these derivatives and their net accumulation in R5 cells was comparable to that of wild-type cells. These data suggest that selective preservation of PMX pharmacological activity in RFC-null R5 cells is due, in part, to partial preservation of transport by secondary process with a higher affinity for PMX than the other antifolates evaluated.

Antimetabolites, Antineoplastic↗
PubMed2004

A prominent low-pH methotrexate transport activity in human solid tumors: contribution to the preservation of methotrexate pharmacologic activity in HeLa cells lacking the reduced folate carrier.

Whereas the major folate transporter, the reduced folate carrier (RFC), has a physiological pH optimum, transport activities for folates and antifolates have been detected with low pH optima. Because the interstitial pH in solid tumors is generally acidic, the mechanisms by which antifolates are transported at low pH could be an important determinant of drug activity under these conditions. The current study quantitated the low pH methotrexate (MTX) transport activity in human solid tumor cell lines from the National Cancer Institute tumor panel and other sources. MTX influx at pH 5.5 was equal to, or greater than, influx at pH 7.4 in 29 of 32 cell lines. To assess the role of RFC in transport at low pH in one of these cell lines, a HeLa clonal line (R5) was selected for MTX resistance due to a genomic deletion of the carrier gene. MTX influx was depressed by 70% in R5 versus wild-type HeLa cells at pH 7.4. At pH 6.5, influx in these two lines was similar; as the pH was decreased to 5.5 influx increased in both cell lines. Similarly, whereas net MTX uptake over 1 h was markedly decreased in R5 cells at pH 7.4, net uptake in HeLa and R5 cells was comparable at pH 6.5. Also, as compared with MCF7 breast cancer cells, MTX uptake was markedly decreased at pH 7.4, but only minimally at pH 6.5, in the MDA-MB-231 human breast cancer cell line that lacks RFC expression. When grown with folic acid (2 micro M) at pH 7.4, the IC(50) for MTX was 14-fold higher in R5 as compared with wild-type HeLa cells; the difference was only 4-fold when cells when grown at pH 6.9; the IC(50)s were identical at this pH when the medium folate was 25 nM 5-formyltetrahydrofolate. These data demonstrate that transport activity at low pH is prevalent in human solid tumors, is RFC-independent in R5 cells and MDA-MB-231 breast cancer cells, and can preserve MTX activity in the absence of RFC at an acidic pH relevant to solid tumors in vivo.

Antimetabolites, Antineoplastic↗
PubMed1980

[Radiational basis and practice of the citrovorum factor (Leucovorin) protection after high-dose methotrexate therapy. High-dose methotrexate/leucovorin].

The effects of methotrexate on the DNA metabolism of human lymphoblast cultures and bone marrow cells were estimated. The ratio of the 3H-deoxyuridine and 3H-thymidine incorporation rates ia a good parameter for the methotrexate effect. Citrovorum factor (5-formyltetrahydrofolic acid) reverses methotrexate toxicity only when its concentration exceeds that of methotrexate at least ten times. Guidelines for the practice of high-dose methotrexate therapy and the following citrovorum factor rescue, especially for patients with retarded methotrexate elimination are presented. A new simple formula enables the exact calculation of the citrovorum factor dose for individual patients according to their methotrexate serum levels. This formula, derived from the experimental data of the bone marrow and lymphoblast culture cells, gives a rational basis for the high-dose rescue with citrovorum factor in cases with expected or manifest toxicity, and means, moreover, a safety factor for the practice of high-dose methotrexate therapy.

Bone Marrow↗