Intracellular Folate Levels Research Articles

TMET-04. SENSITIZING DIPGS TO ANTIFOLATES: A PRECLINICAL STUDY ON PURINE BIOSYNTHESIS TARGETS

Abstract Diffuse Intrinsic Pontine Gliomas (DIPGs), among the most devastating pediatric brain tumors, present a desperate need for innovative treatment strategies, given the lack of effective therapies. Our study unveils a significant metabolic vulnerability in DIPGs, rooted in purine biosynthesis, a key facilitator of tumor progression. Contrary to prior reports, we demonstrate DIPG cell lines' sensitivity to methotrexate, an antifolate that targets purine metabolism, underscoring a potential reappraisal of its therapeutic role. Further, our research reveals a promising therapeutic synergy between antifolates, namely methotrexate and LSN-3213128, and SHMT inhibition, offering a potential novel treatment combination. This synergy, demonstrated in a proof-of-concept study, curtailed tumor progression in our DIPG mouse model, underscoring a promising new treatment strategy. Importantly, we identify a paradoxical antifolate resistance mechanism: while typical resistance is linked to dysfunctional folate transport, our findings intriguingly show an upregulation of folate transporters and intracellular folate in DIPGs. This paradox suggests that elevated intracellular folate levels may be effectively outcompeting antifolate metabolites for their targets. Significantly, the failure of physiological concentrations of purines in the cerebrospinal fluid (CSF) to restore DIPG cell viability under antifolate treatment further reinforces our rationale for targeting purine metabolism. The resistance mechanism is further validated by the observed synergy with SHMT inhibition, which disrupts the folate cycle allowing for the antifolates to out-compete the intracellular folates for their intended target. Our findings challenge conventional wisdom, open new avenues in DIPG therapeutic strategy, and highlight a paradoxical antifolate resistance mechanism that necessitates further exploration in the field.

Impaired Function of Solute Carrier Family 19 Leads to Low Folate Levels and Lipid Droplet Accumulation in Hepatocytes.

Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene (SLC19A1) was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of SLC19A1 was evaluated with the aid of an RT2 profiler-array. After shRNA-silencing of SLC19A1 in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in SLC19A1-knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/-1.67-fold decreased risk of developing MAFLD. When SLC19A1 was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional SLC19A1 provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in SLC19A1-KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of SLC19A1 gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.

Folic acid depletion as well as oversupplementation helps in the progression of hepatocarcinogenesis in HepG2 cells

Folate ingestion below and above the physiologic dose has been shown to play a tumorigenic role in certain cancers. Also, excessive folate supplementation after establishment of pre-established lesions led to an advancement in the growth of a few tumors. However, such information has not yet been achieved in the case of HCC. In our study, HepG2 cells were administered with three different concentrations of folic acid i.e. folic acid normal (FN) (2.27 µM), folic acid deficient (FD) (no folic acid), folic acid oversupplementation (FO) (100 µM) for 10 days. Intracellular folate levels were assayed by Elecsys Folate III kit based method. The migratory and invasive abilities were estimated by transwell migration and matrigel invasion methods respectively. FACS was done to evaluate cell viability and apoptosis. Agarose-coated plates were used to access cancer stem cells (CSCs) number. Quantitative RT-PCR and western blotting approaches were used for gene and protein expression of certain tumor suppressor genes (TSGs), respectively. FD cells depicted increased migration, invasion, apoptosis, necrosis and decreased cell viability, CSCs. On the other hand, FO cells showed increased migration, invasion, cell viability and number of CSCs and decreased apoptosis and necrosis. TSGs revealed diminished expression with both FA modulations with respect to FN cells. Thus, FA deficiency as well as abundance enhanced the HCC progression by adapting different mechanisms.

Maternal genotypes of folate/one-carbon metabolism gene variants and nonsyndromic cleft lip with or without cleft palate risk in Chile.

The aim of this study was to evaluate, in a case-control design, the association between maternal genotypes for variants in 23 genes involved in folate/one-carbon metabolism and nonsyndromic cleft lip with or without cleft palate (NSCL/P) in a Chilean population. After applying several filters to an Illumina array, we extracted 175 single nucleotide polymorphisms (SNPs) from 150 mothers of NSCL/P cases and 150 control women. Association was evaluated using computed odds ratio (OR) with a 95% confidence interval (95% CI) in additive, recessive, and dominant models. After multiple comparison correction, only SNP rs4451422 (A>C), located 237bp downstream of the gene encoding the human folylpolyglutamate synthetase (FPGS), maintained a significant association with NSCL/P in the offspring (OR 3.03; 95% CI 1.69-5.26). The variant rs4451422 is associated with a decrease in FPGS expression according to database annotation. Our results lead to a new hypothesis that a lower activity of FPGS enzyme reduces intracellular folate levels and increases the risk of an offspring having NSCL/P.

Ischemia-Reperfusion Injury Reduces Kidney Folate Transporter Expression and Plasma Folate Levels.

Acute or chronic kidney disease can cause micronutrient deficiency. Patients with end-stage renal disease, kidney transplantation or on dialysis have reduced circulating levels of folate, an essential B vitamin. However, the molecular mechanism is not well understood. Reabsorption of folate in renal proximal tubules through folate transporters is an important process to prevent urinary loss of folate. The present study investigated the impact of acute kidney injury (AKI) on folate transporter expression and the underlying mechanism. AKI was induced in Sprague-Dawley rats that were subjected to kidney ischemia (45 min)-reperfusion (24 h). Both male and female rats displayed kidney injury and low plasma folate levels compared with sham-operated rats. The plasma folate levels were inversely correlated to plasma creatinine levels. There was a significant increase in neutrophil gelatinase-associated lipocalin (NGAL) and IL-6 mRNA expression in the kidneys of rats with ischemia-reperfusion, indicating kidney injury and increased inflammatory cytokine expression. Ischemia-reperfusion decreased mRNA and protein expression of folate transporters including folate receptor 1 (FOLR1) and reduced folate carrier (RFC); and inhibited transcription factor Sp1/DNA binding activity in the kidneys. Simulated ischemia-reperfusion through hypoxia-reoxygenation or Sp1 siRNA transfection in human proximal tubular cells inhibited folate transporter expression and reduced intracellular folate levels. These results suggest that ischemia-reperfusion injury downregulates renal folate transporter expression and decreases folate uptake by tubular cells, which may contribute to low folate status in AKI. In conclusion, ischemia-reperfusion injury can downregulate Sp1 mediated-folate transporter expression in tubular cells, which may reduce folate reabsorption and lead to low folate status.

Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells.

Folates are vital cofactors for the regeneration of S-adenosyl methionine, which is the methyl source for DNA methylation, protein methylation, and other aspects of one-carbon (C1) metabolism. Thus, folates are critical for establishing and preserving epigenetic programming. Folypolyglutamate synthetase (FPGS) is known to play a crucial role in the maintenance of intracellular folate levels. Therefore, any modulation in FPGS is expected to alter DNA methylation and numerous other metabolic pathways. To explore the role of polyglutamylation of folate, we eliminated both isoforms of FPGS in human cells (293T), producing FPGS knockout (FPGSko) cells. The elimination of FPGS significantly decreased cell proliferation, with a major effect on oxidative phosphorylation and a lesser effect on glycolysis. We found a substantial reduction in global DNA methylation and noteworthy changes in gene expression related to C1 metabolism, cell division, DNA methylation, pluripotency, Glu metabolism, neurogenesis, and cardiogenesis. The expression levels of NANOG, octamer-binding transcription factor 4, and sex-determining region Y-box 2 levels were increased in the mutant, consistent with the transition to a stem cell-like state. Gene expression and metabolite data also indicate a major change in Glu and GABA metabolism. In the appropriate medium, FPGSko cells can differentiate to produce mainly cells with characteristics of either neural stem cells or cardiomyocytes.-Srivastava, A. C., Thompson, Y. G., Singhal, J., Stellern, J., Srivastava, A., Du, J., O'Connor, T. R., Riggs, A. D. Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells.

Abstract 4791: Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted antifolates

Abstract Folates participate in one-carbon (C1) transfer reactions in normal and cancer cells. Antifolate therapeutics disrupt cytosolic C1 metabolism, and have long been a mainstay for the therapy of a number of cancers. There are three major folate transporter systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). RFC is broadly expressed in tissues and tumors and is characterized by a neutral pH optimum. PCFT has more limited tissue distribution but is widely expressed in human solid tumors and exhibits an acidic pH optimum. FRα is expressed in a subset of solid tumors including epithelial ovarian cancer (EOC) but shows limited expression in most normal tissues. We previously discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl compounds typified by AGF94 which show selective cellular uptake by FRα and/or PCFT over RFC, in contrast to the classical antifolates methotrexate (MTX) and pemetrexed (PMX) which are transported by both PCFT and RFC, and to a lesser extent by FRα. We showed that AGF94 had enhanced antitumor activities by targeting cytosolic C1 metabolism to PCFT-expressing tumors but lacking RFC, reflecting contraction of cellular tetrahydrofolate pools. In EOC cells, AGF94 showed substantial in vitro inhibition of cell proliferation independent of FRα expression, as long as PCFT was present. We systematically explored the impact of folate transporter redundancies among PCFT, FRα and RFC in determining anti-tumor efficacy of novel tumor-targeted and classical antifolates. Towards this goal, we engineered cell line models from PCFT-, FR-, and RFC-null HeLa cells to express doxycycline (DOX)-inducible FRα or RFC. We used these models to constitutively express PCFT together with DOX-inducible FRα or RFC. These were characterized for transporter expression and function, as well as intracellular folate levels, with/without DOX induction. The relative contributions of RFC, PCFT and FRα to transport function were evaluated from pH 5.5 to 7.4, with radiolabeled MTX and AGF347 which targets both cytosolic and mitochondrial C1 metabolism. We assessed antiproliferative activities of classical antifolates (PMX, MTX, PT523) versus tumor-targeted compounds (e.g., AGF94, AGF102, AGF347) in these models with/without DOX. Our results establish that co-expression of the major folate transporters can have variable and surprisingly disparate and substantial impacts on anti-tumor efficacies of both classical and tumor-targeted antifolates. Our study identified critical determinants of anti-tumor activity with classical and tumor-targeted antifolates, including relative levels of folate transporter expression and transporter specificity, and the impact of intracellular folate levels and extracellular pH. Citation Format: Zhanjun Hou, Carrie O’Connor, Changwen Ning, Adrianne Wallace-Povirk, Josephine Frühauf, Md. Junayed Nayeen, Aleem Gangjee, Larry H. Matherly. Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4791.

High-fat diet consumption reduces hepatic folate transporter expression via nuclear respiratory factor-1.

Folate is an essential micronutrient for biological function. The liver, a primary organ for folate metabolism and storage, plays an important role in folate homeostasis. Proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC) are the major folate transporters responsible for folate uptake at basolateral membrane of hepatocytes. Low serum folate levels are frequently associated with obesity. We investigated the mechanism that regulated folate status in a mouse model with diet-induced obesity. Mice (C57BL/6J) were fed a high-fat diet (60% kcal fat) for 8weeks. Mice displayed increased hepatic lipid accumulation and decreased folate levels in the liver and serum compared to mice fed a normal chow diet (10% kcal fat). High-fat diet-fed mice had low expression of PCFT and RFC and decreased nuclear respiratory factor-1 (NRF-1)/DNA-binding activity. Treatment with NRF-1 siRNA or palmitic acid reduced folate transporter expression in hepatocytes. Inhibition of NRF-1 mediated folate transporter expression significantly reduced intracellular folate levels. These results suggest that chronic consumption of high-fat diets impairs folate transporter expression via NRF-1-dependent mechanism, leading to reduced hepatic folate storage. Understanding the regulation of folate homeostasis in obesity may have an important implication in current guideline of folate intake. KEY MESSAGES: Serum and liver folate levels are decreased in diet-induced obese mice. Chronic high-fat diet consumption impairs expression of hepatic PCFT and RFC. NRF-1 regulates hepatic folate transporters expression and folate levels.

Replication study of polymorphisms associated with response to methotrexate in patients with rheumatoid arthritis

About 70 genetic studies have already addressed the need of biomarkers to predict the response of patients with rheumatoid arthritis (RA) to methotrexate (MTX) treatment. However, no genetic biomarker has yet been sufficiently validated. Here, we aimed to replicate a selection of 25 SNPs in the largest collection of patients up to date, which consisted of 915 patients treated with MTX. The change in disease activity (measured as ΔDAS28) from baseline was considered the primary outcome. In addition, response according to widely used criteria (EULAR) was taken as secondary outcome. We considered consistency between outcomes, P values accounting for the number of SNPs, and independence from potential confounders for interpretation of the results. Only the rs1801394 SNP in MTRR fulfilled the high association standards. Its minor allele was associated with less improvement than the major allele according to ΔDAS28 (p = 0.0016), and EULAR response (p = 0.004), with independence of sex, age, baseline DAS28, smoking, seropositivity, concomitant corticosteroid use or previous treatments. In addition, previous evidence suggests the association of this SNP with response to MTX in another autoimmune disease, juvenile idiopathic arthritis, and with high intracellular folate levels, which could contribute to poor response.

Contrasting roles of the ABCG2 Q141K variant in prostate cancer

ABCG2 is a membrane transport protein that effluxes growth-promoting molecules, such as folates and dihydrotestosterone, as well as chemotherapeutic agents. Therefore it is important to determine how variants of ABCG2 affect the transporter function in order to determine whether modified treatment regimens may be necessary for patients harboring ABCG2 variants. Previous studies have demonstrated an association between the ABCG2 Q141K variant and overall survival after a prostate cancer diagnosis. We report here that in patients with recurrent prostate cancer, those who carry the ABCG2 Q141K variant had a significantly shorter time to PSA recurrence post-prostatectomy than patients homozygous for wild-type ABCG2 (P=0.01). Transport studies showed that wild-type ABCG2 was able to efflux more folic acid than the Q141K variant (P<0.002), suggesting that retained tumoral folate contributes to the decreased time to PSA recurrence in the Q141K variant patients. In a seemingly conflicting study, it was previously reported that docetaxel-treated Q141K variant prostate cancer patients have a longer survival time. We found this may be due to less efficient docetaxel efflux in cells with the Q141K variant versus wild-type ABCG2. In human prostate cancer tissues, confocal microscopy revealed that all genotypes had a mixture of cytoplasmic and plasma membrane staining, with noticeably less staining in the two homozygous KK patients. In conclusion, the Q141K variant plays contrasting roles in prostate cancer: 1) by decreasing folate efflux, increased intracellular folate levels result in enhanced tumor cell proliferation and therefore time to recurrence decreases; and 2) in patients treated with docetaxel, by decreasing its efflux, intratumoral docetaxel levels and tumor cell drug sensitivity increase and therefore patient survival time increases. Taken together, these data suggest that a patient's ABCG2 genotype may be important when determining a personalized treatment plan.

Folate deficiency enhances the inflammatory response of macrophages

B-vitamin deficiency is a risk factor for vascular disease. The mechanism by which the deficiency impacts on disease risk is unclear. We have analysed whether the inflammatory response of mononuclear cells can be modified by cellular folate status in vitro.We show that the mouse monocyte cell line RAW264.7 grown under folate restriction displays a decrease in intracellular folate levels and a reduced growth rate. The cells also show a 2- to 3-fold increase in expression of the inflammatory mediators, IL1β, IL6, TNFα and MCP1 at the RNA and protein level (p<0.01) under conditions of folate deficiency. In contrast the production of the vaso-protective mediator nitric oxide is significantly reduced under these conditions. These metabolic changes are independent of the concentration of homocysteine in the medium and occur in the absence of significant changes in global DNA methylation. Folate deficiency may therefore exacerbate cardiovascular disease by augmenting pro-inflammatory signals in the monocyte-macrophage lineage.

Alcohol-associated folate disturbances result in altered methylation of folate-regulating genes

Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The steady-state accumulation of folate seems to depend on the activity of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamate residues, and gamma-glutamyl hydrolase (GGH), which removes them, enabling it to be transported across the biological membranes. Overexpression of GGH and downregulation of FPGS would be expected to decrease intracellular folate in its polyglutamylated form, thereby increasing efflux of folate and its related molecules, which might lead to resistance to drugs or folate deficiency. The study was sought to delineate the activity of GGH and expression FPGS in tissues involved in folate homeostasis during alcoholism and the epigenetic regulation of these enzymes and transporters regulating intracellular folate levels. We determined the activity of GGH and expression of FPGS in tissues after 3 months of ethanol feeding to rats at 1 g/kg body weight/day. The results showed that there was not any significant change in the activity of folate hydrolyzing enzyme GGH in ethanol-fed rats while there was significant down regulation in the expression of FPGS. Ethanol feeding decreased the total as well as polyglutamated folate levels. There was tissue-specific hyper/hypo methylation of folate transporter genes viz. PCFT and RFC by chronic ethanol feeding. Moreover, hypermethylation of FPGS gene was observed in intestine and kidney without any change in methylation levels of GGH in the ethanol-fed rats. In conclusion, the initial deconjugation of polyglutamylated folate by GGH was not impaired in ethanol-fed rats while the conversion of monoglutamylated folate to polyglutamylated form might be impaired. There was tissue-specific altered methylation of folate transporter genes by chronic ethanol feeding.

The prevalence of folate-remedial MTHFR enzyme variants in humans

Studies of rare, inborn metabolic diseases establish that the phenotypes of some mutations in vitamin-dependent enzymes can be suppressed by supplementation of the cognate vitamin, which restores function of the defective enzyme. To determine whether polymorphisms exist that more subtly affect enzymes yet are augmentable in the same way, we sequenced the coding region of a prototypical vitamin-dependent enzyme, methylenetetrahydrofolate reductase (MTHFR), from 564 individuals of diverse ethnicities. All nonsynonymous changes were evaluated in functional in vivo assays in Saccharomyces cerevisiae to identify enzymatic defects and folate remediability of impaired alleles. We identified 14 nonsynonymous changes: 11 alleles with minor allele frequencies <1% and 3 common alleles (A222V, E429A, and R594Q). Four of 11 low-frequency alleles affected enzyme function, as did A222V. Of the five impaired alleles, four could be restored to normal functionality by elevating intracellular folate levels. All five impaired alleles mapped to the N-terminal catalytic domain of the enzyme, whereas changes in the C-terminal regulatory domain had little effect on activity. Impaired activity correlated with the phosphorylation state of MTHFR, with more severe mutations resulting in lower abundance of the phosphorylated protein. Significantly, diploid yeast heterozygous for mutant alleles were impaired for growth, particularly with lower folate supplementation. These results suggested that multiple less-frequent alleles, in aggregate, might significantly contribute to metabolic dysfunction. Furthermore, vitamin remediation of mutant enzymes may be a common phenomenon in certain domains of proteins.

High folic acid increases cell turnover and lowers differentiation and iron content in human HT29 colon cancer cells

Folate, a water-soluble B vitamin, is a cofactor in one-carbon metabolism and is essential for DNA synthesis, amino acid interconversion, methylation and, consequently, normal cell growth. In animals with existing pre-neoplastic and neoplastic lesions, folic acid supplementation increases the tumour burden. To identify processes that are affected by increased folic acid levels, we compared HT29 human colon cancer cells exposed to a chronic supplemental (100 ng/ml) level of folic acid to cells exposed to a normal (10 ng/ml) level of folic acid, in the presence of vitamin B12 and other micronutrients involved in the folate-methionine cycle. In addition to higher intracellular folate levels, HT29 cells at 100 ng folic acid/ml displayed faster growth and higher metabolic activity. cDNA microarray analysis indicated an effect on cell turnover and Fe metabolism. We fully confirmed these effects at the physiological level. At 100 ng/ml, cell assays showed higher proliferation and apoptosis, while gene expression analysis and a lower E-cadherin protein expression indicated decreased differentiation. These results are in agreement with the promoting effect of folic acid supplementation on established colorectal neoplasms. The lower expression of genes related to Fe metabolism at 100 ng folic acid/ml was confirmed by lower intracellular Fe levels in the cells exposed to folic acid at 100 ng/ml. This suggests an effect of folate on Fe metabolism.

Association of Methylenetetrahydrofolate Reductase Gene Polymorphisms and Sex-Specific Survival in Patients With Metastatic Colon Cancer

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating intracellular folate levels, which affects DNA synthesis and methylation. Two MTHFR gene polymorphisms, C677T and A1298C, are linked to altered enzyme activity. Several studies have shown these two polymorphisms to be associated with response to fluorouracil (FU) -based treatment in advanced colon cancer patients, but data are inconsistent and contradictory. Meanwhile, epidemiologic studies demonstrated that these MTHFR polymorphisms were associated with cancer risk in a sex-specific manner. We tested the hypothesis of whether these two polymorphisms are associated with sex-specific clinical outcome in metastatic colon cancer patients treated with FU-based chemotherapy. This study included 318 patients (177 men and 141 women) with metastatic colon cancer treated between 1992 and 2003 at the University of Southern California/Norris Comprehensive Cancer Center or Los Angeles County/University of Southern California Medical Center. Peripheral blood samples were collected from each patient, and genomic DNA was extracted from WBCs. Two MTHFR gene polymorphisms (C677T and A1298C) were tested by fluorogenic 5'-nuclease assay. The A1298C polymorphism showed statistically significant differences in overall survival (OS) in female, but not male, patients with metastatic colon cancer (log-rank test, P = .038). Among females, OS was greater for patients with the A/A genotype (n = 67; median OS, 18.4 months) compared with patients with the A/C genotype (n = 50; median OS, 13.9 months) or C/C genotype (n = 10; median OS, 15.6 months). Although preliminary, these data support the role of the A1298C polymorphism in MTHFR as prognostic marker in female patients with metastatic colon cancer. Further studies are needed to confirm these findings.

Molecular genetic analysis of the human dihydrofolate reductase gene: relation with plasma total homocysteine, serum and red blood cell folate levels

Disturbances in folate metabolism may increase the risk of certain malignancies, congenital defects and cardiovascular diseases. The gene dihydrofolate reductase (DHFR) is primarily involved in the reduction of dihydrofolate, generated during thymidylate synthesis, to tetrahydrofolate in order to maintain adequate amounts of folate for DNA synthesis and homocysteine remethylation. In order to reveal possible variation that may affect plasma total homocysteine (tHcy), serum folate and red blood cell (RBC) folate levels, we sequenced the DHFR coding region as well as the intron-exon boundaries and DHFR flanking regions from 20 Caucasian individuals. We identified a 9-bp repeat in the 5'-upstream region that partially overlapped with the 5'-untranslated region, and several single-nucleotide polymorphisms, all in non-coding regions. We screened subjects for the 9-bp repeat (n=417), as well as the recently reported 19-bp deletion in intron 1 (n=330), and assessed their associations with plasma tHcy, serum and RBC folate levels. The 19-bp del/del genotype was associated with a lower plasma tHcy (-14.4% [95% confidence interval: -23.5 to -4.5], P=0.006) compared with the wild-type genotype. This may suggest that intracellular folate levels are affected.

The folate pathway is a target for resistance to the drug para-aminosalicylic acid (PAS) in mycobacteria.

The increasing rate of multidrug-resistant tuberculosis has led to more use of second-line antibiotics such as para-aminosalicylic acid (PAS). The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined. We have isolated PAS-resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels. BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway. We also find that mutations in thyA are associated with clinical PAS resistance. We have identified PAS-resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme. Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.

Marked suppression of the activity of some, but not all, antifolate compounds by augmentation of folate cofactor pools within tumor cells

1 Folates have been co-administered with some antifolates to diminish host toxicity; however, the extent to which this will reduce antitumor activity is not known. To further clarify this issue, studies were undertaken to characterize and quantitate the impact of alterations in intracellular folate levels on the activities of a variety of antifolates in L1210 murine leukemia cells. Intracellular folate cofactor levels increased almost in proportion to the increase in extracellular 5-formyltetrahydrofolate (5-CHO-THF) over a concentration range that encompassed physiological levels of 5-methyltetrahydrofolate. This resulted in a spectrum of increases in the ic 50 values of antifolates upon continuous exposure to drugs [Lometrexol (DDATHF) (70x) > trimetrexate (TMQ) (30x), multitargeted antifolate, LY231514 (ALIMTA) (30x) > Raltitrexed, Tomudex (ZD1694) (10x), 6 R-2′,5′-thienyl-5,10-dideazatetrahydrofolic acid (LY309887) (10x) > methotrexate (MTX) (6x) > (2 S)-2-{ o-fluoro- p-[ N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl)- N-(prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl) butyric acid (ZD9331) (3x), N α-(4-amino-4-deoxypteroyl)- N δ-hemiphthaloyl-l-ornithine (PT523) (3x)]. Upon a 4-hr pulse exposure to drug, the ic 50 values for DDATHF and ALIMTA were increased > 180- and 5-fold, respectively, with only a 2.5-fold increase in the extracellular 5-CHO-THF level within the physiological range. The reductions in drug sensitivities could be attributed to decreases in accumulation of polyglutamate derivatives of ALIMTA and DDATHF. Hence, in these studies, natural folates diminished the activity of agents that undergo polyglutamation by suppression of the formation of these active congeners at the level of folylpolyglutamate synthetase. For inhibitors of dihydrofolate reductase, the suppressive effect of endogenous folates appears to be due to competition between the antifolate and dihydrofolate at the level of the target enzyme. These data should be carefully considered in the design of regimens with antifolates, which incorporate co-administration of folates.

Folate Deficiency Induces a Cell Cycle-Specific Apoptosis in HepG2 Cells

The human hepatoma HepG2 cell line was chosen as a representative of solid tissue-derived cell systems in which folate metabolism and apoptosis induction have not been thoroughly investigated. HepG2 cells were cultivated in the control or folate-deficient media (control media lacking of folate, glycine, thymidine and hypoxanthine) for 4 wk. This resulted in a decrease in intracellular folate levels to 32% of the control within 1 wk, which was followed by growth arrest and greater cell death rates. These disturbances of folate deficiency coincided with apoptotic induction, as characteristically shown by nucleosomal DNA fragmentation of 180–200 base pair multimers, nuclear chromatin condensation and positive terminal transferase-mediated dUTP nick end labeling assay. Apoptosis coincided with an accumulation of cells in S-phase, a subsequent G2/M phase block and a significant increase in mean protein content as evaluated by flow cytometric analyses employing a double-staining method. The growth and cell cycle arrest under folate-deficient conditions was independent of a change of p53 expression as measured by an enzyme-linked immunosorbent assay. Supplementation of 2 μmol/L folate normalized cell cycles and diminished DNA fragmentation. Taken together, these data indicate that HepG2 cells cultivated in folate-deficient medium have a low folate concentration, decreased growth and viability, and increased apoptotic propensity. This occurrence of apoptosis was associated with a cell cycle-specific mechanism and independent of p53-mediated pathway.

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.