Folate-deficient Medium Research Articles

Uracil misincorporation in human DNA detected using single cell gel electrophoresis.

Poor folate status may be important in the aetiology of several epithelial cell malignancies including cancer of the uterine cervix. Folic acid is essential in the synthesis of purine nucleotides and the pyrimidine nucleoside thymidine and it is probable that imbalances in these DNA precursors negatively effect DNA stability and may ultimately lead to malignant transformation. The development of a modified 'comet assay' using the bacterial DNA repair enzyme uracil DNA glycosylase, to detect misincorporated uracil in human DNA is reported here. The effect of perturbing folic acid and deoxyuridine levels on uracil misincorporation in normal human lymphocytes and cultured human tumour cells was investigated using this assay. HeLa cells and peripheral human lymphocytes incubated as agarose-embedded nucleoids, with 1 unit of uracil DNA glycosylase per microg of DNA, contained low levels of uracil in their DNA. Both HeLa cells and stimulated human lymphocytes cultured in folate-deficient medium were growth arrested. Incubating human lymphocytes in folate-deficient medium significantly increased the level of uracil detected compared with control cells. HeLa cells showed an increase in non-specific DNA damage (strand breaks). Deoxyuridine (100 microM) significantly increased the level of uracil detected in the DNA of both folate-deficient and control HeLa cells. It appears that this modified comet assay specifically detects misincorporated uracil in single human cells. It should, therefore, prove valuable in determining the role of folic acid status in DNA instability and cancer.

Apoptosis of Late-Stage Erythroblasts in Megaloblastic Anemia: Association With DNA Damage and Macrocyte Production

AbstractAn in vitro model of folate-deficient erythropoiesis has been developed using proerythroblasts isolated from the spleens of Friend virus-infected mice fed an amino acid-based, folate-free diet. Control proerythroblasts were obtained from Friend virus-infected mice fed the same diet plus 2 mg folic acid/kg diet. Our previous studies showed that, after 20 to 32 hours of culture in folate-deficient medium with 4 U/mL of erythropoietin, the folate-deficient proerythroblasts underwent apoptosis, whereas control erythroblasts survived and differentiated into reticulocytes over a period of 48 hours. The addition of folic acid or thymidine to the folate-deficient medium prevented the apoptosis of the folate-deficient erythroblasts, thereby implicating decreased thymidylate synthesis as the main cause of apoptosis in the folate-deficient erythroblasts. In the study reported here, we examined intracellular folate levels, uracil misincorporation into DNA, p53 and p21 proteins, and reticulocyte formation in erythroblasts cultured in folate-deficient or control medium. In all experiments, the folate-deficient erythroblasts cultured in folate-deficient medium gave results that varied significantly from folate-deficient erythroblasts cultured in control medium or control erythroblasts cultured in either folate-deficient or control media. Folate-deficient erythroblasts cultured in folate-deficient medium had marked decreases in all coenzyme forms of folate that persisted throughout culture, increased uracil misincorporation into DNA, persistent accumulations of p53 and p21, and decreased reticulocyte production but increased size of individual reticulocytes. A model of folate-deficient erythropoiesis based on apoptosis of late stage erythroblasts is presented. This model provides explanations for the clinical findings in megaloblastic anemia.

New heritable fragile site with spontaneous expression at 1q41.

The report presents a family ascertained through recurrent spontaneous abortions in which a new heritable fragile site located at 1q41 is segregating. The fragile site is present in the mother and her son. It is expressed spontaneously in 100% of the metaphases from lymphocyte culture using standard conditions. The use of folate deficient medium and the addition of FUdR to the medium did not affect the appearance nor the level of expression of the fragile site.

Apoptosis mediates and thymidine prevents erythroblast destruction in folate deficiency anemia.

Deficiency of the vitamin folic acid causes pancytopenia by decreasing the production of new blood cells. Although impaired DNA synthesis and destruction of hematopoietic cells have been implicated, the mechanism by which folate deficiency decreases blood cell production is uncertain. An in vitro model of folate-deficient erythropoiesis was developed by using proerythroblasts isolated from folate-deficient mice that were infected with Friend leukemia virus. Proerythroblasts from folate-deficient mice had one-tenth the total folate as did proerythroblasts from control mice. The folate-deficient proerythroblasts underwent apoptosis, a form of programmed cell death, after 20-32 h in culture in folate-deficient medium. At the time of apoptosis the cells had differentiated into the later erythroblast stages and some had begun hemoglobin synthesis. Addition of either folic acid or thymidine, but not deoxycytidine or inosine, to the folate-deficient medium prevented the apoptosis and permitted proliferation and differentiation of the proerythroblasts into reticulocytes. The prevention of apoptosis by thymidine indicates (i) that decreased thymidylate synthesis plays a role in erythroblast apoptosis and the anemia of folate deficiency and (ii) that DNA cleavage is likely to be a primary event in the apoptosis of folate-deficient erythroblasts. Apoptosis of erythroblasts in the late stages of differentiation leads to decreased erythrocyte production and to anemia. The increased erythropoietin produced in response to the anemia increases the number of erythroid progenitor cells in the differentiation stages preceding those in which the cells undergo apoptosis. This population shift to earlier stage erythroblasts and proerythroblasts is characteristic of bone marrows of individuals with folate deficiency anemia.

Changes in folate and pterin metabolism after disruption of the Leishmania H locus short chain dehydrogenase gene.

The short chain dehydrogenase gene ltdh, which is derived from the H locus of Leishmania, confers high level resistance to the dihydrofolate reductase inhibitor methotrexate via a novel mechanism. Resistance is correlated with LTDH overproduction. High level resistance of ltdh transfectants was observed even in poor media where reduced folates are essential for the synthesis of thymidylate precursors. The ltdh transfectants were also capable of growing for several passages, at slightly reduced rates, in unsupplemented folate-deficient medium (fdDME-L), unlike the wild-type cells that could grow only in fdDME-L if supplemented with various folates or pterins. An homozygous ltdh mutant was obtained by gene targeting. This mutant became hypersensitive to methotrexate, but unlike wild-type cells, methotrexate toxicity could not be circumvented by the addition of thymidine. Although the homozygous mutant was capable of growing in fdDME-L supplemented with folate derivatives, it lost its ability to thrive in fdDME-L supplemented with pterins. Our results support the model in which LTDH is a key enzyme responsible for the conversion of a pterin derivative into an essential cofactor. This essential cofactor can also be converted into reduced folates at sufficient levels for growth when LTDH is overproduced, rendering dihydrofolate reductase dispensable. The novelty and uniqueness of LTDH opens the possibility of developing parasite-specific inhibitors.

Altered folate-binding protein mRNA stability in KB cells grown in folate-deficient medium

Folate-binding protein (FBP), a high-affinity folate receptor, is responsible for cellular accumulation of folate and folate analogs such as methotrexate in human KB (nasopharyngeal carcinoma) cells. Both FBP and FBP mRNA increase 3- to 5-fold when KB cells are grown in folate-deficient (less than 10 nM folate) medium (KB-FD), compared with growth in standard folate-replete medium containing at least 2μM folate (KB-FR). The possible mechanisms of enhanced FBP gene expression in KB-FD were examined in this study. Southern blot analysis revealed no significant change in the FBP gene organization or copy number in the KB-FD DNA. While hypomethylation of the FBP gene was observed in KB-FD DNA, relative to KB-FR DNA, exposure of KB-FR to the DNA methylation inhibitors did not result in elevated FBP mRNA levers. The transcriptional rate of the FBP gene was the same in KB-FR and KB-FD. RNA half-life studies indicated that the half-life of FBP mRNA in KB-FD was increased approximately 2.5-fold, compared with KB-FR. Thus, the increase in the steadystate levels of FBP mRNA in KB-FD can be attributed partly to increased FBP mRNA stability.

Folate-sensitive and aphidicolin-inducible fragile sites are expressed in the genome of the domestic cat

Peripheral blood lymphocytes from three clinically normal domestic cats were cultured for folate-sensitive and aphidicolin-inducible fragile site expression. Induction of folate-sensitive fragile sites was accomplished by culturing cells with trimethoprim plus caffeine. Chromosomes from all cats expressed both folate-sensitive and aphidicolin-inducible breaks and gaps. There were no significant differences between the two methods of fragile site induction in the percentage of cells expressing chromosome breaks and gaps or the mean number of breaks and gaps per cell. All three cats expressed specific chromosome breaks resembling fragile sites at A1q21–22, A1p22, and B1q32. All three sites were induced by aphidicolin. The sites at A1q21–22 and B1q32 were also induced in folate-deficient medium. This is the first report of the induction of chromosomal fragile sites in a feline species.

Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.

KB cells express a folate-binding protein that is anchored to the plasma membrane by a glycosylated phosphatidylinositol (GPI) tail and these cells can grow in medium containing a very low folate concentration (1 nM). In contrast, mouse 3T3 cells do not express a membrane-associated folate-binding protein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-binding protein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-binding protein similar to KB cells, 1 and 1.4 ng/micrograms protein, respectively. The capacity for binding [3H] folate to the surface of transfected 3T3 cells cultured in folate-deficient medium is 7.7 pmol/10(6) cells, and this is approximately 50% of the surface binding capacity of KG cells under similar culture conditions. Moreover, after treatment of the transfected 3T3 cells with phospholipase C specific for phosphatidylinositol, the binding of [3H] folate to the surface of these cells is reduced by 90%, indicating that, like the KB cells, the folate-binding protein is anchored to the plasma membrane by a GPI tail. Although the doubling time of wild-type 3T3 cells markedly increases after 13 d of culture in folate-deficient medium, the doubling time of both the transfected 3T3 cells and KB cells do not change. The results of these experiments indicate that the GPI-anchored folate-binding protein provides a mechanism to maintain a level of folate that permits the folate-dependent metabolic functions necessary for cell survival under low folate conditions.

Biopterin conversion to reduced folates byLeishmania donovani promastigotes

The ability of Leishmania donovani promastigotes to proliferate in folate-deficient medium supplemented with pterins suggests that pterins can serve as a source of folate in these parasites [16]. Using reversed-phase high-performance liquid chromatography, the ability of intact L. donovani to transform [3H]biopterin into tetrahydrofolates was demonstrated. Radioactivity was primarily associated with 5-methyltetrahydrofolate and 10-formyltetrahydrofolate. A mutant strain of L. donovani, MTXA5, that was genetically deficient in folate transport capacity and incapable of growing in pterin-supplemented folate-deficient growth medium, exhibited a greatly reduced capacity to metabolize [3H]biopterin to reduced folates. These data indicated that wild-type L. donovani promastigotes, unlike mammalian cells, were able to convert biopterin to tetrahydrofolates and supported the hypothesis that folate transport deficiency in mutant organisms is associated with an inability to transform pterins to reduced folates.

Folate‐deficient human lymphoblasts: changes in de novo purine and pyrimidine synthesis and phosphoribosylpyrophosphate

Peripheral blood lymphocytes from healthy volunteers cultured with phytohaemagglutinin in a folate-deficient medium exhibit megaloblastic maturation and reduced cellular folate content. For these cells, changes in de novo purine and pyrimidine synthesis and cellular phosphoribosylpyrophosphate (PRPP) have been determined. Mitogen-stimulated cells cultured with undialyzed pooled human serum (PHS) exhibit undetectable de novo purine synthesis, a three-fold increase in PRPP content and augmented de novo pyrimidine synthesis; these changes are independent of cellular folate status. Folate-replete cells cultured with PHS which was dialyzed to reduce purine compounds concentrations show markedly increased de novo purine synthesis. The PRPP content and pyrimidine synthesis rates of these cells are similar to those of folate-replete cells cultured with undialyzed PHS. Folate-deficient cells cultured in dialyzed PHS show a 10-fold reduction in purine synthesis and a corresponding increase in PRPP levels. Pyrimidine synthesis was moderately reduced. The purine bases hypoxanthine and adenine markedly reduced the augmented purine synthesis of folate-replete cells or the increased PRPP content of folate-deficient cells cultured with dialyzed PHS. These findings suggest that cellular folate status is critical for de novo purine synthesis only when coupled with purine bases restriction and that the latter are efficient regulators of de novo purine synthesis.

Folate‐deficient human lymphoblasts: Changes in deoxynucleotide metabolism and thymidylate cycle activities

Peripheral blood lymphocytes of healthy volunteers cultured with phytohaemagglutinin in folate-deficient medium exhibit megaloblastic maturation with reduced intracellular folate content. We have employed this in vitro model for megaloblastic maturation to determine accompanying changes in cellular thymidylate cycle activities and deoxynucleotide levels. Folate-deficient cells exhibit a two-fold increase in thymidine kinase and thymidylate synthase activities. These increased activities were reduced to those of folate-replete cells by co-culture of folate-deficient cells with thymidine. Folate deficiency was accompanied by reduced cellular levels of thymidine triphosphate (TTP) and deoxyguanosine triphosphate (dGTP). Exogenous deoxyuridine produced no increase in the reduced levels of TTP of folate-deficient cells but effected a two-fold increase in cellular deoxycytidine triphosphate. Exogenous thymidine increased the reduced TTP levels of folate-deficient cells and corrected the reduced dGTP level; the increase in cellular TTP accompanying exogenous thymidine was more pronounced in folate-deficient cells. These in vitro findings are compatible with a block in de novo thymidylate synthesis and explain in part the reported in vivo changes for the deoxynucleotide pool in megaloblastic marrow cells due to folate or vitamin B12 deficiency.

Nutritional requirements of wild-type and folate transport-deficient Leishmania donovani for pterins and folates

The nutritional requirements for folates and pterins were assessed for two strains of Leishmania donovani promastigotes, a wild-type (D1700) parental strain and a mutant derivative (MTXA5) which possesses a markedly diminished capacity to transport both [ 3H]folate and [ 3H]methotrexate (MTX) [14]. Both L. donovani strains have an absolute growth requirement for an exogenous pterin, since their proliferation could not be sustained in completely defined medium lacking either a pterin or a folate. Supplementation of the growth medium by many of a spectrum of folates and pterins could support growth of the wild-type cell line. Surprisingly, however, the MTXA5 strain could not thrive in folate-deficient medium fortified with any of the pterins that promoted wild-type cell division, including biopterin and neopterin. The relationship between the incapacity of MTXA5 cells to transport folate and methotrexate and their inability to multiply in folate-deficient medium supplemented with pterins was evaluated using genetic approaches. First, an independently generated MTX-resistant mutant, MTXB4, was isolated in 1 mM MTX. The phenotype of the MTXB4 cells was like that of MTXA5 cells since they were unable to transport [ 3H]MTX and [ 3H]folate or grow in folate-deficient medium supplemented with pterins. Second, three revertants of the MTXA5 cells were selected for their ability to grow in 1 μM biopterin. All three revertants had regained [ 3H]folate and [ 3H]MTX transport capability concomitant with growth sensitivity to methotrexate toxicity. These observations provide genetic evidence that the competence of L. donovani to transport [ 3H]folate and [ 3H]MTX and the capability of the organisms to utilize pterins as a nutritional factor are influenced by a common genetic locus.

Nutritional folate-deficiency in Chinese hamster ovary cells: Chromosomal abnormalities associated with perturbations in nucleic acid precursors

Folate deficiency is known to induce chromosomal abnormalities. We used a nutritionally folate-deficient Chinese hamster ovary (CHO) cell culture system to examine modulation of chromosome damage by purine or pyrimidine supplementation. The cells were cultured in folate-deficient (Fol −) medium or Fol − medium supplemented with thymidine (dT) or hypoxanthine (Hx) until population growth arrest. The cultures were then switched to complete medium, permitting the cells to begin cell division. Cell-cycle progression was followed by flow cytometry to identify the first mitosis, when samples for analysis were collected. The mitotic index, frequency of chromosomal aberrations in mitotic cells, and relative distribution of different types of aberrations were determined. Cells grown in Fol − medium supplemented with Hx entered the G 2/M phase of the cell cycle at 14 hours after media change as compared with 16 hours for Fol − cultures or 24 hours for Fol − cultures supplemented with dT. Cells cultured in Fol − medium alone or supplemented with dT showed similar frequencies of damage, averaging 20–22%, as compared with 2% for control cultures. In contrast, cells grown in Hx-supplemented medium exhibited a lower frequency of damaged mitoses (15%), as well as a reduction in certain types of abnormalities. This latter finding is surprising in light of our previous work showing that the presence of Hx during folate deficiency produced a more severe perturbation of phenotype (cellular enlargement) and growth control (S-phase delay and cell killing) than did dT supplementation or the absence of both nucleotide precursors.

The fragile X‐chromosome: an evaluation of the results in a routine cytogenetic laboratory in the period 1981–1986

We report on the cytogenetic studies, performed in a routine cytogenetic laboratory between 1981 and 1986, on 428 subjects: 291 probands with non-specific mental retardation, 101 first-degree relatives of fra(X) positive patients and 36 non-retarded patients, referred for other reasons. As a rule 50 cells, cultured in folate-deficient medium were analysed. The results are compared with data collected from the literature and with the data expected from prior genetic risks and fra(X) penetrance. If no more than 50 cells were analysed, the fragile site was found in a lower than expected proportion (69.2%) of the retarded first-degree male relatives. The conclude that in a substantial number of unselected patients with mental retardation the diagnosis fra(X) syndrome will only be confirmed cytogenetically if at least 100 cells are analysed. Five percent of the male and 10% of the female index patients showed a fragile X-like abnormality, probably not associated with the Martin-Bell syndrome.

Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter.

From a mutagenized population of wild type Leishmania donovani promastigotes, a clone was isolated in a single step by virtue of its resistance to 1 mM methotrexate, a potent inhibitor of dihydrofolate reductase. This methotrexate-selected cell line, MTXA5, was cross-resistant to aminopterin but just as sensitive to growth inhibition caused by pyrimethamine, trimethoprim, and cytotoxic purine and pyrimidine analogs. Unlike previously characterized methotrexate-resistant Leishmania (Coderre, J. A., Beverley, S. M., Schimke, R., and Santi, D. V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2132-2136), resistance to the antimetabolite was not due to gene amplification or increased dihydrofolate reductase activity. The genetic defect in MTXA5 cells appeared to be in the methotrexate-folate transport system. The rate of uptake and transport of [3H]methotrexate and [3H]folate into MTXA5 cells was less than 1% of that of wild type parental cells. Neither wild type nor MTXA5 cells could multiply in folate-deficient medium, and thymine and thymidine at concentrations which circumvented methotrexate toxicity, did not restore the ability of Leishmania to grow. The concentration of exogenous folate that restored growth of wild type and mutant cells, however, was virtually identical, although MTXA5 cells, unlike parental cells, could not proliferate in folate-deficient medium supplemented with 10 microM biopterin. Interestingly, methotrexate and aminopterin could stimulate the growth of both leishmanial strains in folate-deficient medium, suggesting that these antifolate analogs were serving as a pteridine source for the parasite. These somatic cell genetic studies of folate transport in Leishmania provide genetic evidence for a specific folate permease in L. donovani promastigotes and have important implications concerning the mechanisms by which these parasites utilize exogenous pteridines and folates and by which they might become resistant to parasite-directed chemotherapeutic regimens.

Fragile sites at 4q23 and 7q11.23 unique to bone marrow cells

Fragile sites in chromosome bands 4q23 and 7q11.23 were discovered in bone marrow cells. Expression of these fragile sites was induced by treatment of the cells sequentially with 10 −7 M methotrexate and then 10 −5 M thymidine. No expression was observed in bone marrow cells without treatment. The fragile sites at 4q23 and 7q11.23 were seen individually, together, and in the homozygous state in a total of 20 bone marrow samples. The bone marrow karyotypes were normal in all cases. Expression of these common fragile sites at 4q23 and 7q11.23 could not be induced in blood lymphocytes from two subjects using methotrexate and thymidine, or by other means including the addition of bromodeoxyuridine and fluorodeoxyuridine or growth in folate-deficient medium. Because the fragile sites at 4q23 and 7q11.23 have never been observed in lymphocytes treated with methotrexate and thymidine for high-resolution chromosome analysis, it appears that these fragile sites are not expressed under these conditions in T lymphocytes. We propose that the differential expression of these fragile sites in bone marrow cells reflects genes active in bone marrow cells but not in blood lymphocytes.

The effect of hydroxyurea on the expression of the common fragile site at 3p14.

Hydroxyurea (HU) is an inhibitor of DNA synthesis, which can inhibit the enzyme ribonucleotide reductase, reduce the syntheses of all four deoxyribonucleoside diphosphates (dNDP), and disturb the balance of the dNTP pool. We have studied the effect of HU on the common fragile site at 3p14 (FRA3B) and have found that G2 treatment with HU increased not only the frequency of chromosomal aberration but also the expression of FRA3B in both complete and folate deficient media. There is a synergistic effect between HU and growth in folate deficient medium on the induction of FRA3B. Our results suggest that the inhibition of DNA repair, including the inhibition in G2 phase, plays an important role in the expression of FRA3B, supporting other authors' data on the effect of other DNA repair inhibitors, such as aphidicolin, caffeine, 1-beta-D-arabinofuranosylcytosine, and 5-fluorodeoxyuridine, on the expression of FRA3B.

Phosphate-Induced Phosphoribosylpyrophosphate Elevations to Assess Deranged Folate and Purine Nucleotide Metabolism

Phosphoribosylpyrophosphate (PRPP) levels increase several-fold in HL-60 cells adapted to folate deficiency either by continuous passage in folate-deficient medium or by short-term incubation with 10(-8) M methotrexate (MTX). The addition of folic acid (PteGlu) or 5-formyltetrahydrofolic acid (5-CHO-H4PteGlu) in the form of Leucovorin normalizes this effect. The reactions for measuring PRPP levels are time and temperature dependent and are influenced by PRPP-reacting substances in undialyzed serum. Inorganic phosphate (PO4), when added to the assay, markedly stimulates PRPP levels in HL-60 cells and can be used to stress folate-dependent PRPP utilization for purine synthesis. The integrity of the folate-dependent pathways of purine-synthesizing cells can be sensitively assessed by measurement of PRPP levels during a 2-hr assay in the presence of PO4 in medium free of folate but containing dialyzed serum. In HL-60 cells that are folate deficient or in the presence of MTX (as low as 2 X 10(-9) M), PO4-stimulated PRPP levels remain elevated due to ineffective utilization unless folate is added to the incubation mixture. The sensitivity of this PRPP assay to metabolically assess the integrity of folate-dependent reactions in purine synthesis is comparable to that of the deoxyuridine suppression assay. Inorganic phosphate can also be used to stimulate the incorporation of purine analogs, such as 6-mercaptopurine, into intact red blood cells which may have therapeutic implications for targeting drug delivery.

The effect of 1-beta-D-arabinofuranosyl-cytosine on the expression of the common fragile site at 3p14.

The effect of the G2-treatment of 1-beta-D-arabino-furanosyl-cytosine (araC) on the expression of the common fragile site at 3p14 (FRA3B) was studied. A significantly increased frequency of FRA3B induced by G2 treatment of araC was found in the lymphocytes grown in folate-deficient medium (positive rate 100%). A relatively low frequency of FRA3B was also induced in the cultures with folate in four of the seven subjects. There is a synergistic effect between araC and growth in folate-deficient medium on the induction of FRA3B. The results suggest that the DNA lesions related to the expression of FRA3B induce the long-patch repair and that the low DNA polymerase alpha activity and inefficient repair process during G2 phase is involved in the expression of FRA3B.

Analysis of fragile X-mental retardation families using flanking polymorphic DNA probes.

Fragile-X mental retardation (FRAX-MR) is one of the more common X-linked disorders affecting 1 in 1,500 newborn males. This disease is characterized by the expression of fragile site in the region q27.3 of the X-chromosome of affected boys when their lymphocytes are cultured in folate deficient medium. In most patients there is macroorchidism postpubertally. The clinical diagnosis of carrier females based on the expression of fragile site in Xq27.3 is usually difficult and sometimes impossible. About half of the carrier females escape diagnosis by this method. Furthermore, prenatal diagnosis is not always feasible. Using Restriction Fragment Length Polymorphism (RFLP) and cloned DNA segments from the region Xq27-Xqter as probes, we have investigated Swedish families with FRAX-MR in three generations. Interesting observations, previously unreported to our knowledge, have been made in some patients and carrier mothers, using one of the probes which is localized to the distal end of Xq. The significance of these findings and the linkage of the disease locus to the different probes used in this study is presented.