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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Folate Deficiency Induces Cell-Specific Changes in the Steady-State Transcript Levels of Genes Involved in Folate Metabolism and 1-Carbon Transfer Reactions in Human Colonic Epithelial Cells¹

² Department of Nutritional Sciences and ³ Department of Medicine, University of Toronto, Toronto, Ontario, Canada, M5S 1A8; ⁴ Clinical Epidemiology Unit, Sunnybrook Health Sciences Center, Toronto, Ontario, Canada, M4N 3M5; and ⁵ Division of Gastroenterology, St. Michael's Hospital, Toronto, Ontario, Canada, M5B 1W8

^* To whom correspondence should be addressed. E-mail: youngin.kim{at}utoronto.ca.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Intracellular folate homeostasis is essential for the 1-carbon transfer reactions necessary for DNA synthesis and biological methylation reactions in colonic epithelial cells. Perturbed 1-carbon transfer reactions resulting from folate depletion predispose normal colonic epithelial cells to neoplastic transformation while inhibiting the growth of colon cancer cells. Using an in vitro model of folate deficiency, we determined the effects of folate deficiency on the steady-state transcript levels of genes involved in intracellular folate metabolism and 1-carbon transfer reactions in HCT116 and Caco2 human colon adenocarcinoma cells. In HCT116 cells, folate depletion was associated with changes in transcript levels of genes favoring increased folate uptake and intracellular folate retention, the provision of metabolically more effective substrates for folate-dependent enzymes, and reduced folate hydrolysis and efflux. In HCT116 cells, folate depletion was associated with changes in transcript levels of genes favoring the preferential shuttling of the flux of 1-carbon units to the methionine cycle over the nucleotide synthesis pathway. In Caco2 cells, some adaptive responses in response to folate depletion were not as apparent as in HCT116 cells, and in some cases, the direction of change was counterintuitive. In Caco2 cells, the metabolic priority in response to folate depletion was to shuttle the available folate pools to the nucleotide biosynthesis pathway at the expense of biological methylation reactions. In both HCT116 and Caco2 cells, folate depletion was associated with the conservation of the existing pattern and extent of DNA methylation.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

View larger version (27K): [in this window] [in a new window]   Figure 1  Simplified scheme of intracellular folate metabolism and 1-carbon transfer reactions in colonic epithelial cells, highlighting the genes that are involved in intraluminal folate hydrolysis (GCPII), intracellular folate uptake (FR-; RFC), intracellular folate retention (FPGS) and hydrolysis and efflux (GGH), methionine cycle (MTR; MTRR; MTHFR), maintenance of intracellular folate pool (DHFR; SHMT) and nucleotide biosynthesis (TS), DNA methylation (DNMT1, 3a, 3b), and DNA demethylation (MBD2). CH3, methyl group; Hcyt, homocysteine; Met, methionine. Filled circle represents a pteridine ring conjugated to para-aminobenzoic acid. Each filled triangle represents a glutamate, which is linked via a peptide bond to form various chain lengths of polyglutamylated folate.

In the methionine cycle (Fig. 1), 5-methyltetrahydrofolate (5-methylTHF) transfers 1 methyl group to homocysteine to synthesize methionine, thereby ensuring the provision of S-adenosylmethionine (SAM), the primary methyl group donor for most biological methylation reactions, including that of DNA (11,12). The remethylation of homocysteine to methionine is catalyzed by methionine synthase (MTR), a cobalamin-dependent enzyme (13). The reductive methylation of the cobalamin cofactor of MTR to its active state is catalyzed by MTR reductase (MTRR) (14). After donating the methyl group, 5-methylTHF is converted to tetrahydrofolate (THF) and is subsequently converted to 5,10-methyleneTHF by serine hydroxymethyltransferase (SHMT), which catalyzes the reversible interconversion of serine and THF to glycine and 5,10-methyleneTHF (15,16). Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methyleneTHF to 5-methylTHF (17). The substrate 5,10-methyleneTHF is the methyl donor for the nonreversible methylation, catalyzed by thymidylate synthase (TS), of dUMP to dTMP (thymidylate), a precursor for DNA synthesis (1). The synthesis of thymidylate results in the oxidation of 5,10-methyleneTHF to the inactive dihydrofolate, which can be converted back to THF by dihydrofolate reductase (DHFR) (1). Both THF and 5,10-methyleneTHF can enter the purine biosynthesis pathway by the addition of a formyl group (1).

The methylation of cytosine located within the cytosine-guanine dinucleotide (CpG) sequences is an epigenetic modification of mammalian DNA that plays an important role in gene expression, the maintenance of DNA integrity and stability, chromatin modifications, and the development of mutations (11,12,18). DNA methylation is a dynamic process between active methylation, mediated by CpG methyltransferases [DNA methyltransferase (DNMT1) for maintenance methylation and DNMT3a and 3b for de novo methylation] using SAM as the methyl donor (19) and removal of methyl groups from 5-methylcytosine residues by both passive and active mechanisms, including demethylation by a purported demethylase, methyl DNA-binding domain protein 2 (MBD2) (Fig. 1) (20,21).

Molecular and cellular effects of folate deficiency contributing to cancer development, progression, and treatment in colonic epithelial cells have been extensively studied and characterized. In contrast, it is largely unknown at present how genes involved in intracellular folate metabolism and 1-carbon transfer reactions in colonic epithelial cells are regulated in response to folate deficiency. Using the previously characterized in vitro model of functional folate deficiency (22,23), we therefore determined the effects of folate deficiency on the steady-state transcript levels of the genes involved in intracellular folate metabolism and 1-carbon transfer reactions in colonic epithelial cells.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Cell lines and culture. Two human colon adenocarcinoma cell lines, HCT116 and Caco-2, were selected for this study. We previously developed and validated an in vitro model of functional folate deficiency using these 2 cell lines (22,23). HCT116 and Caco-2 cells cultured in medium devoid of folic acid were viable but exhibited progressive retarded growth and were associated functional intracellular folate deficiency (22,23). HCT116 and Caco2 cells have different molecular characteristics, representing mismatch repair-deficient and -sufficient colon adenocarcinoma cell lines, respectively (23). Furthermore, Caco2 cells are wild-type, whereas HCT116 cells are heterozygous for the MTHFR C677T polymorphism, which reduces MTHFR activity and increases thermolability of MTHFR, leading to lower levels of 5-methylTHF and an accumulation of 5,10-methyleneTHF (24). HCT116 and Caco2 cells have different phenotypic characteristics; Caco2 cells express phenotypic characteristics of enterocyte differentiation upon reaching confluence and hence make an excellent model of the normal enterocyte that participates in absorptive processes (25). The effect of folate deficiency on methionine cycle intermediates, DNA methylation, and apoptotic and cancer pathway-specific gene expression profile is significantly different between HCT116 and Caco2 cells in this model (22,23).

HCT116 and Caco2 cell lines were obtained from the American Type Culture Collection and were cultured in standard RPMI 1640 medium (Invitrogen) containing 2.3 µmol/L folic acid (control) or in customized RPMI 1640 medium (Invitrogen) free of folic acid (deficient). Growth medium was supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin, and 0.1% fungizone. The folate-deficient (FD) medium therefore contained only the folate present in the dialyzed serum (0.6 nmol/L). This level of folate in medium is physiologically sufficient to sustain growth of both untransformed and transformed mammalian cell lines in culture, including HCT116 and Caco-2 cells (22,23). The cells were maintained at 37°C in 95% humidity and 5% CO₂ and passaged every 4 d. Growth rates were determined by cell counts. Cells were harvested after 20 d of growth and were processed for subsequent analyses.

    Intracellular folate concentrations. Intracellular folate concentrations were determined by a standard microtiter plate assay using Lactobacillus casei (American Type Culture Collection no. 11578) after cellular folate extraction and subsequent treatment with chicken pancreas conjugase, as described previously (26).

    Deoxyuridine suppression test. The deoxyuridine suppression test was used to verify that the intracellular folate depletion was functional, as described previously (22). This test assesses the de novo synthesis of thymidylate on the basis of the competition between 2 pathways: the salvage pathway and the de novo pathway (27). The salvage pathway consists of phosphorylation of thymidine by thymidine kinase. The de novo pathway generates thymidylate by methylating dUMP. Because the enzyme for the latter reaction, TS, requires 5,10-methyleneTHF as a substrate, the deoxyuridine suppression test has been used as a functional assay for determining folate status at the cellular level, including colonic epithelial cells (27). In folate-replete cells, the incorporation of [³H]thymidine into DNA is suppressed by exogenous deoxyuridine, whereas in FD cells, the degree of suppression is less pronounced because of an impaired de novo synthesis of thymidylate and greater use of the salvage pathway (i.e. higher [³H]thymidine incorporation). The percent deoxyuridine suppression was expressed as: ([³H]thymidine incorporation with deoxyuridine/[³H]thymidine incorporation without deoxyuridine) x 100.

    Real time quantitative RT-PCR. Total cellular RNA was isolated using the RNeasy MidiKit (Qiagen) according to the manufacturer's protocol. cDNA was generated from 5 µg of total RNA using random primers and the SuperScript III RNase H-Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. Primers were designed using Primer Express version 2.0 software (Applied Biosystems) and primer-primer interactions were minimized by analysis using OligoQuant 4.0 software (Molecular Biology Insights). Primers were synthesized by the ACGT. The sequences of primers are as follows: GCPII (forward: 5'-TTGCATGAATTGAAGGCTGAGA-3'; reverse: 5'-TAGGACAGCAAGACATCGTAATGG-3'); RFC (forward: 5'-CTTTGCCACCATCGTCAAGA-3'; reverse: 5'-CAGGATCAGGAAGTACACGGAG-3'); FR- (forward: 5'-GAACGCCAAGCACCACAAG-3'; reverse: 5'-GGTCGACACTGCTCATGCAA-3'); FPGS (forward: 5'-GTGACCCTCAGACACAGTTGGA-3'; reverse: 5'-GGCCATAGCTTCGGAGGAT-3'); GGH (forward: 5'-GCCACAGATACTGTTGACGTGG-3'; reverse: 5'-ATGGAAATTGGCAGTCAGAGG-3'); MTHFR (forward: 5'-ATGGTGAACGAAGCCAGAGG-3'; reverse: 5'-CCGGTCAAACCTTGAGATGAG-3'); MTR (forward: 5'-TGAATGCTGGAAACCTCCCTGT-3'; reverse: 5'-AAGGGCATACTCAAGGCGTTCT-3'); MTRR (forward: 5'-TGAAAAGCGCAGGCTACAGGA-3'; reverse: 5'-AGTGGTGGCTGGCAAGAAGG-3'); DHFR (forward: 5'-ACCTGGTTCTCCATTCCTGAG-3'; reverse: 5'-CCTTGTGGAGGTTCCTTGAGT-3'); TS (forward: 5'-CCAAACGTGTGTTCTGGAAGG-3'; reverse: 5'-GCCTCCACTGGAAGCCATAA-3'); SHMT (forward: 5'-CGAAGCTGATCATCGCAGGA-3'; reverse: 5'-AGCCATGTCCGCCATGAGA-3'); DNMT1 (forward: 5'-TTCCACCAAGCAGGCATCTCT-3'; reverse: 5'-TGACCAGCTTCAGCAGGATGTT-3'); DNMT3a (forward: 5'-CGAGTCCAACCCTGTGATGA-3'; reverse: 5'-GTAATGGTCCTCACTTTGCTGAA-3'); DNMT3b (forward: 5'-ATTGCTGTTGGAACCGTGAAG-3'; reverse: 5'-GCCAATCACCAAGTCAAATGG-3'); MBD2 (forward: 5'-CATCTCAACCCCTCTGCAAAG-3'; reverse: 5'-TTACAGGCAAAAGCCAGTGGA-3'); and 5-aminolevulinate synthase 1 (5-ALAS1; forward: 5'-AAGCAGGTGTCGGTCTGGTG-3'; reverse: 5'-CGAATCCCTTGGATCATGGAG-3').

Real time hot-start PCR was performed at 95°C for 10 min in a 10-µL reaction volume containing 0.5 µmol/L of each primer, 3–4 mmol/L MgCl₂, and 1 µL of cDNA template using the LC FastStart DNA Master SYBR Green 1 kit in the LightCycler rapid thermal cycler system (Roche Diagnostics). Following hot-start PCR, 45 cycles of denaturation at 95°C for 10 s, annealing at 55–56°C for 5 s, and extension at 72°C for 10 s were performed. After amplification, the reaction mixture was cooled to 65°C for 15 s and then temperature was slowly raised to 95°C at 0.1°C/s to generate a dissociation curve to ensure that nonspecific amplification did not occur. All PCR products were analyzed by gel electrophoresis to ensure that the amplicon was of anticipated length.

Standard curves were generated for all target genes and for the endogenous reference gene, human 5-ALAS1, to determine gene product concentrations in the test samples against mRNA expression of 5-ALAS1. Relative quantification was performed using LC Relative Quantification Software version 1.0 (Roche Diagnostics) to compare steady-state mRNA levels between the folate-sufficient (FS; control) and FD groups. The target threshold cycle number (C_t) was defined as the point where the sample fluorescence crossed the threshold, a set level of the fluorescence signal. The target C_t was directly compared with the calibrator C_t and was recorded as containing either more or less mRNA. In this study, the target was the FD group and the calibrator was the control group. Target gene expression of the control and FD cells was normalized to 5-ALAS1 expression to minimize quantification errors confounded by any variation in the amount of cDNA template between the samples. The results of the FD group were expressed as the ratios to those of their corresponding controls.

    Statistical analysis. Analyses for intracellular folate concentrations and deoxyuridine suppression test were performed in triplicate and repeated using 3 independent cell lysates. For real time quantitative RT-PCR, all experiments were performed in triplicate and repeated using RNA extracted from 4–11 independent cultures. Triplicate values were averaged prior to analysis. Comparisons of means between the control and FD groups for intracellular folate concentrations and for the deoxyuridine suppression test were determined by the Student's 2-sample t test using SigmaStat 2.03 for Windows (Access Softek). The real time quantitative RT-PCR results were tested using 1-sample t tests to compare the ratios (FD cell values to the corresponding control cell values) to the null hypothesis value of 1.0. The calculations were performed using Microsoft Excel. For all analyses, the results were considered significant if 2-tailed P-values were <0.05.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Intracellular folate concentrations. HCT116 and Caco2 cells cultured in FD medium demonstrated a significant progressive retarded growth compared with the corresponding cells cultured in FS medium (data not shown). Intracellular folate concentrations of HCT116 and Caco2 cells cultured in FD medium progressively decreased over the study period and by d 18 were 60% lower than corresponding cells grown in FS medium (FS HCT116, 8.1 ± 1.2 vs. FD HCT116, 4.0 ± 0.8 ng folate/10⁶ cells; FS Caco2, 11.2 ± 1.4 vs. FD Caco2, 4.7 ± 0.3 ng folate/10⁶ cells; P < 0.001).

The deoxyuridine suppression test showed that the FD cells were less suppressed by exogenous deoxyuridine (by 20% in HCT116 cells and by 10% in Caco2 cells; P < 0.01) compared with the corresponding FS cells, thereby confirming that the observed intracellular folate depletion in the FD cells was functionally important. Preincubation of these cells with 100 µmol/L folinic acid (5-formylTHF, a precursor of 5,10-methyleneTHF) resulted in a correction of the deoxyuridine suppression test in the FD cells, whereas the values remained unchanged in the FS cells (data not shown). This demonstrates that repletion of cellular folate pool in the FD cells resulted in a normal synthesis of thymidylate through the de novo pathway and therefore indicates that the abnormal deoxyuridine suppression observed in the FD cells was due to a cellular deficiency of folate.

    Steady-state levels of GCPII, RFC, and FR- transcripts. The GCPII gene, which is responsible for hydrolysis of the polyglutamate chain of folates for absorption in the small intestine (28,29), was not expressed in either the FD or FS HCT116 or the Caco2 cell lines. The steady-state level of FR- transcript was 100% higher in the FD HCT116 cells than the FS HCT116 cells (P < 0.001; Table 1). In contrast, the steady-state level of FR- transcript was 27% lower in the FD Caco2 cells than the FS Caco2 cells (P = 0.057; Table 1). The steady-state level of RFC transcript was 43% lower in the FD HCT116 cells than the FS HCT116 cells (P < 0.001; Table 1). However, the steady-state level of RFC transcript did not differ between the FD and FS Caco2 cells.

    Steady-state levels of DHFR, cytoplasmic SHMT, and TS transcripts. The steady-state level of DHFR transcript was 61% lower in the FD HCT116 cells and 29% lower in the FD Caco2 cells than in the corresponding FS cells (P < 0.001 and P = 0.011, respectively; Table 1). The steady-state level of TS transcript was 57% lower in the FD HCT116 cells than in the FS HCT116 cells (P < 0.001; Table 1). In contrast, the steady-state level of TS transcript did not differ between the FD and FS Caco2 cells. The steady-state level of cSHMT transcript was 59% lower in the FD HCT116 cells than in the FS HCT116 cells (P < 0.001; Table 1). In contrast, the steady-state level of cSHMT transcript was 20% higher in the FD Caco2 cells than in the FS Caco2 cells (P = 0.004; Table 1).

    Steady-state levels of MTR, MTRR, and MTHFR transcripts. The steady-state level of MTR transcript was 36% lower in the FD HCT116 cells and 24% lower in the FD Caco2 cells than in the corresponding FS cells (P < 0.001 and P = 0.002, respectively; Table 1). The steady-state level of MTRR transcript was 46% lower in the FD HCT116 cells and 35% lower in the FD Caco2 cells than in the corresponding FS cells (P < 0.001; Table 1). The steady-state level of MTHFR transcript was 49% higher in the FD HCT116 cells than in the FS HCT116 cells (P = 0.001; Table 1). In contrast, the steady-state level of MTHFR transcript was 16% lower in the FD Caco2 cells than in the FS Caco2 cells (P = 0.019; Table 1).

    Steady-state levels of DNMT and MBD2 transcripts. The steady-state level of DNMT1 transcript was 3.3-fold higher in the FD HCT116 cells and 3-fold higher in the FD Caco2 cells than in the corresponding FS cells (P = 0.009 and P = 0.029, respectively; Table 1). The steady-state level of DNMT3a transcript was 25% lower in the FD HCT116 cells and 29% lower in the FD Caco2 cells than in the corresponding FS cells (P = 0.005 and P < 0.001, respectively; Table 1). The steady-state level of DNMT3b transcript was also 47% lower in the FD HCT116 cells and 37% lower in the FD Caco2 cells than in the corresponding FS cells (P = 0.004 and P = 0.007, respectively; Table 1).

The steady-state level of MBD2 transcript was 46% lower in the FD HCT116 cells than in the FS HCT116 cells (P < 0.001; Table 1). However, the steady-state level of MBD2 transcript did not differ between the FD and FS Caco2 cells.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
We hypothesized that folate depletion would upregulate genes involved in intracellular folate uptake to ensure that the critical intracellular folate concentrations required for 1-carbon transfer reactions were maintained. As expected, FR- was significantly upregulated in the FD HCT116 cells, consistent with the major function of FR-, which is to ensure adequate intracellular folate concentrations for cell survival in conditions of folate deficiency (30–32) and with prior observations made in several human and murine noncolonic normal and tumor cell lines (30,33–38). However, the lack of upregulation of FR- and a paradoxical nonsignificant trend toward downregulation in the FD Caco2 cells were an unexpected finding. This suggested a possible cell-specific FR- regulation in response to folate depletion. Another possible explanation relates to the observation that FR- is already highly expressed in Caco2 cells and further upregulation may not be necessary to increase intracellular folate uptake. In contrast to the observed upregulation of RFC in several human cancer cell lines and in rat small intestine and colon (7,39,40) in response to folate depletion, RFC was significantly downregulated in the FD HCT116 cells, whereas RFC transcript level was not significantly changed in the FD Caco2 cells. One possible explanation for this seemingly paradoxical downregulation of RFC in HCT116 is that RFC located in the basolateral membrane may be downregulated to reduce folate efflux. This is an intriguing possibility that requires further studies.

We hypothesized that folate depletion would upregulate FPGS and downregulate GGH, with consequent intracellular retention of polyglutamylated folate for the provision of efficient substrates for the enzymes necessary for critical 1-carbon transfer reactions (1). Consistent with this hypothesis and prior observations made in human and murine tumor cells in response to folate depletion in both in vitro and in vivo xenograft models (41–43), folate depletion significantly upregulated FPGS in both HCT116 and Caco2 cells. Consistent with our hypothesis, folate depletion was associated with a nonsignificant trend toward downregulation of GGH in HCT116 cells but had no significant effect on GGH in Caco2 cells.

Folate depletion significantly downregulated MTR and MTRR in both HCT116 and Caco2 cells. The net effect of this downregulation would be decreased remethylation of homocysteine to methionine and consequent reduced SAM and impaired biological methylation reactions (Fig. 1). Furthermore, this would lead to the "methyl trap" in which the methyl group is trapped in the form of 5-methylTHF, because the conversion of 5-methyTHF to THF is compromised (Fig. 1). This methyl trap would also lead to impaired biosynthesis of purines and thymidylate because of reduced THF (Fig. 1).

The effect of folate depletion on MTHFR was markedly different between HCT116 and Caco2 cells. MTHFR catalyzes the irreversible conversion of 5,10-methyleneTHF to 5-methylTHF, thereby committing 1-carbon units to the methionine cycle (Fig. 1) (17). 5,10-methyleneTHF is a key substrate in folate metabolism, which can be directed toward nucleotide biosynthesis or toward methionine regeneration (44). Several lines of evidence indicate that limited methyl group availability shifts the flux of 1-carbon units among folate-dependent pathways such that folate cofactors are preferentially shuttled to the methionine cycle to protect SAM-dependent methylation reactions at the expense of DNA synthesis (44,45). Consistent with this, folate depletion significantly upregulated MTHFR in HCT116 cells, which was associated with significantly increased SAM concentrations and SAM to S-adenosylhomocysteine (SAH) ratios, as previously reported by our group (22). In contrast, folate depletion downregulated MTHFR in Caco2 cells and we previously showed a significant reduction in SAM concentrations and SAM to SAH ratios in the FD Caco2 cells compared with the FS Caco2 cells (22).

The effect of folate depletion on cSHMT was also markedly different between HCT116 and Caco2 cells. The change in cSHMT regulation in response to folate depletion in both HCT116 and Caco2 cell lines was in the opposite direction to the change in MTHFR regulation. Folate depletion significantly downregulated cSHMT in HCT116 cells, whereas it significantly upregulated cSHMT in Caco2 cells. SHMT catalyzes the reversible transfer of the hydroxymethyl group of serine to THF to form 5,10-methyleneTHF and glycine (Fig. 1) (15,16). This reaction is a major source of THF-activated 1-carbon units in mammalian cells and serves as a major entry point for 1-carbon units into the nucleotide synthesis pathway (15,16). In contrast to MTHFR that directs available folate toward the methionine cycle over nucleotide synthesis (17), cSHMT acts as a metabolic switch that directs folate toward nucleotide synthesis at the expense of homocysteine remethylation by providing 5,10-methyleneTHF to TS for thymidylate synthesis and by increasing the cytoplasmic availability of THF for conversion to 10-formylTHF and use in purine synthesis (Fig. 1) (46,47). Simultaneously, cSHMT inhibits homocysteine remethylation by decreasing the availability of 5,10-methyleneTHF to MTHFR and by sequestering 5-methylTHF and depleting cellular levels of SAM (47). This appears to be the case in the FD Caco2 cells; folate depletion significantly upregulated cSHMT and downregulated MTHFR. The steady-state level of TS transcript in the FD Caco-2 cells was not significantly different from that in controls, suggesting that in response to folate depletion, TS utilizes the available 5,10-methyleneTHF pools for thymidylate synthesis in Caco2 cells. A previous study indeed demonstrated that increased cSHMT expression results in increased rates of de novo thymidylate synthesis, indicating that cSHMT shuttles 5,10-methyleneTHF to TS through production of 5,10-methyleneTHF from serine (46). In contrast, in the FD HCT116 cells with upregulated MTHFR, downregulated cSHMT, and increased SAM concentrations and SAM to SAH ratios (22), TS was significantly downregulated. This suggests that in HCT116 cells, the available 5,10-methyleneTHF pools were shuttled to the methionine cycle pathway at the expense of thymidylate synthesis. Interestingly, in both FD HCT116 and Caco2 cells, DHFR was significantly downregulated. This observation is consistent with the metabolic priority of the methionine cycle pathway in the FD HCT116 cells but is inconsistent with the metabolic priority of the nucleotide synthesis pathway in the FD Caco2 cells.

The response of enzymes critical for the homeostasis of DNA methylation to folate depletion was similar in HCT116 and Caco2 cells. DNMT1, which is responsible for maintenance of CpG methylation, was significantly upregulated, whereas DNMT3a and 3b, which are responsible for de novo CpG methylation, were significantly downregulated in both the FD HCT116 and Caco2 cells. This suggests that the limited available pool of SAM for CpG DNA methylation may be preferentially utilized for maintaining CpG DNA methylation over de novo DNA methylation. Folate depletion significantly downregulated MBD2, the purported DNA demethylase (20,21), in HCT116 but not in Caco2 cells. This observation in HCT116 cells suggests a compensatory downregulation of MBD2 to maintain DNA methylation.

In summary, in HCT116 cells, folate depletion induced an adaptive regulation favoring increased folate uptake (FR- upregulation) and intracellular folate retention (FPGS upregulation), the provision of metabolically more effective substrates for folate-dependent enzymes (FPGS upregulation), and reduced folate hydrolysis (GGH downregulation) and efflux (possible basolateral RFC downregulation). Furthermore, in HCT116 cells, folate depletion appeared to preferentially shuttle the flux of 1-carbon units to the methionine cycle (MTHFR upregulation) to protect methylation reactions and thereby suppress DNA synthesis (cSHMT, TS, and DHFR downregulation). Folate depletion upregulated maintenance DNA methylation and downregulated de novo DNA methylation and demethylation in HCT116 cells, which would result in maintaining the existing pattern and extent of DNA methylation. In Caco2 cells, some adaptive responses in response to folate depletion were not as apparent as in HCT116 cells and in some cases, the direction of change was counterintuitive (e.g. FR- downregulation). In Caco2 cells, the metabolic priority in response to folate depletion was to shuttle the available folate pools to the nucleotide biosynthesis pathway (cSHMT upregulation and maintenance of TS) at the expense of the methionine cycle (MTHFR downregulation).

Our data suggest that folate depletion poses different metabolic stresses in HCT116 and Caco2 cells, resulting in different adaptive regulation of MTHFR and cSHMT to prioritize the shuttling of a limited pool of 5,10-methyleneTHF to either the nucleotide synthesis or biological methylation pathway. Our study suggests that in contrast to prior observations (44,45), SAM synthesis is not the absolute metabolic priority over thymidylate biosynthesis in response to folate deficiency. Consistent with a recent observation (47), our study indicates that in response to folate depletion, DNA synthesis may become the metabolic priority at the expense of homocysteine remethylation in certain cells. The cell-specific effect of folate depletion on the regulation of MTHFR and cSHMT between HCT116 and Caco2 cells may be in part related to differences in intracellular folate concentrations and distributions, molecular and phenotypic characteristics (23,24), growth rates, concentrations of various amino acid substrates, and the presence and relative activity of several enzymes involved in methionine cycle and purine biosynthesis (48–50). Future studies are warranted to determine whether the flux of 1-carbon units parallels the differential changes in MTHFR and cSHMT transcript levels observed in these cell lines. The observations made in the HCT116 and Caco2 colon cancer cell lines cannot be extrapolated to normal colonic epithelial cells, nor can they be generalized to other cancer cell lines. As such, the effect of folate deficiency and supplementation on genes involved in folate metabolism and 1-carbon transfer reactions at the transcriptional, translational, and posttranslational levels as well as their functional ramifications need to be determined in normal colonic epithelial cells in in vivo studies.

	FOOTNOTES

 
¹ Supported by a grant from the Canadian Institutes of Health Research (grant no. MOP-14126 to Y-I. K.) and by an open fellowship from the University of Toronto (to I. H.).

⁶ Abbreviations used: 5-ALAS1, 5-aminolevulinate synthase 1; CpG, cytosine-guanine dinucleotide; C_t, cycle number; DHFR, dihydrofolate reductase; DNMT, DNA methyltransferase; FD, folate-deficient; FPGS, folylpolyglutamate synthase; FR, folate receptors; FS, folate-sufficient; GCPII, glutamate carboxypeptidase II; GGH, -glutamyl hydrolase; 5-methylTHF, 5-methyltetrahydrofolate; MBD2, methyl DNA-binding domain protein 2; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; RFC, reduced folate carrier; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; TS, thymidylate synthase.

Manuscript received 31 August 2006. Initial review completed 15 November 2006. Revision accepted 19 December 2006.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

1. Shane B. Folate chemistry and metabolism. In: Bailey LB, editor. Folate in health and disease. New York: Marcel Dekker; 1995. p. 1–22.

2. Choi SW, Mason JB. Folate status: effects on pathways of colorectal carcinogenesis. J Nutr. 2002;132:S2413–8.[Abstract/Free Full Text]

3. Kim YI. Folate and carcinogenesis: evidence, mechanisms, and implications. J Nutr Biochem. 1999;10:66–88.[Medline]

4. Lamprecht SA, Lipkin M. Chemoprevention of colon cancer by calcium, vitamin D and folate: molecular mechanisms. Nat Rev Cancer. 2003;3:601–14.[Medline]

5. Kim YI. Role of folate in colon cancer development and progression. J Nutr. 2003;133:S3731–9.[Abstract/Free Full Text]

6. Kamen B. Folate and antifolate pharmacology. Semin Oncol. 1997;24 Suppl 18:S30–9.

7. Backus HH, Pinedo HM, Wouters D, Padron JM, Molders N, van Der Wilt CL, van Groeningen CJ, Jasen G, Peters GJ. Folate depletion increases sensitivity of solid tumor cell lines to 5-fluorouracil and antifolates. Int J Cancer. 2000;87:771–8.[Medline]

8. Chandler CJ, Wang TT, Halsted CH. Pteroylpolyglutamate hydrolase from human jejunal brush borders. Purification and characterization. J Biol Chem. 1986;261:928–33.[Abstract/Free Full Text]

9. Antony AC. Folate receptors. Annu Rev Nutr. 1996;16:501–21.[Medline]

10. Sirotnak FM, Tolner B. Carrier-mediated membrane transport of folates in mammalian cells. Annu Rev Nutr. 1999;19:91–122.[Medline]

11. Kim YI. Folate and DNA methylation: a mechanistic link between folate deficiency and colorectal cancer? Cancer Epidemiol Biomarkers Prev. 2004;13:511–9.[Abstract/Free Full Text]

12. Kim YI. Nutritional epigenetics: impact of folate deficiency on DNA methylation and colon cancer susceptibility. J Nutr. 2005;135:2703–9.[Abstract/Free Full Text]

13. Chen LH, Liu ML, Hwang HY, Chen LS, Korenberg J, Shane B. Human methionine synthase. cDNA cloning, gene localization, and expression. J Biol Chem. 1997;272:3628–34.[Abstract/Free Full Text]

14. Leclerc D, Wilson A, Dumas R, Gafuik C, Song D, Watkins D, Heng HH, Rommens JM, Scherer SW, et al. Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc Natl Acad Sci USA. 1998;95:3059–64.[Abstract/Free Full Text]

15. Girgis S, Nasrallah IM, Suh JR, Oppenheim E, Zanetti KA, Mastri MG, Stover PJ. Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene. Gene. 1998;210:315–24.[Medline]

16. Stover PJ, Chen LH, Suh JR, Stover DM, Keyomarsi K, Shane B. Molecular cloning, characterization, and regulation of the human mitochondrial serine hydroxymethyltransferase gene. J Biol Chem. 1997;272:1842–8.[Abstract/Free Full Text]

17. Ueland PM, Hustad S, Schneede J, Refsum H, Vollset SE. Biological and clinical implications of the MTHFR C677T polymorphism. Trends Pharmacol Sci. 2001;22:195–201.[Medline]

18. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002;3:415–28.[Medline]

19. Li E, Jaenisch R. DNA methylation and methyltransferases. In: Ehrlich M, editor. DNA alterations in cancer: genetic and epigenetic changes. Natick (MA): Eaton Publishing; 2000. p. 351–65.

20. Bhattacharya SK, Ramchandani S, Cervoni N, Szyf M. A mammalian protein with specific demethylase activity for mCpG DNA. Nature. 1999;397:579–83.[Medline]

21. Ng HH, Zhang Y, Hendrich B, Johnson CA, Turner BM, Erdjument-Bromage H, Tempst P, Reinberg D, Bird A. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet. 1999;23:58–61.[Medline]

22. Stempak JM, Sohn KJ, Chiang EP, Shane B, Kim YI. Cell and stage of transformation-specific effects of folate deficiency on methionine cycle intermediates and DNA methylation in an in vitro model. Carcinogenesis. 2005;26:981–90.[Abstract/Free Full Text]

23. Novakovic P, Stempak JM, Sohn KJ, Kim YI. Effects of folate deficiency on gene expression in the apoptosis and cancer pathways in colon cancer cells. Carcinogenesis. 2006;27:916–24.[Abstract/Free Full Text]

24. Sohn KJ, Croxford R, Yates Z, Lucock M, Kim YI. Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate. J Natl Cancer Inst. 2004;96:134–44.[Abstract/Free Full Text]

25. Engle MJ, Goetz GS, Alpers DH. Caco-2 cells express a combination of colonocyte and enterocyte phenotypes. J Cell Physiol. 1998;174:362–9.[Medline]

26. Tamura T. Microbiological assay of folates. In: Piccairo MF, Stokstad R, Gregory JF, editors. Folic acid metabolism in health and diseases. New York: Wiley-Liss; 1990. p. 121–37.

27. Cravo ML, Mason JB, Selhub J, Rosenberg IH. Use of the deoxyuridine suppression test to evaluate localized folate deficiency in rat colonic epithelium. Am J Clin Nutr. 1991;53:1450–4.[Abstract/Free Full Text]

28. Chang SS, Heston WD. The clinical role of prostate-specific membrane antigen (PSMA). Urol Oncol. 2002;7:7–12.[Medline]

29. Renneberg H, Friedetzky A, Konrad L, Kurek R, Weingartner K, Wennemuth G, Tunn UW, Aumuller G. Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread. Urol Res. 1999;27:23–7.[Medline]

30. Kamen BA, Capdevila A. Receptor-mediated folate accumulation is regulated by the cellular folate content. Proc Natl Acad Sci USA. 1986;83:5983–7.[Abstract/Free Full Text]

31. Luhrs CA, Raskin CA, Durbin R, Wu B, Sadasivan E, McAllister W, Rothenberg SP. Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium. J Clin Invest. 1992;90:840–7.[Medline]

32. Bottero F, Tomassetti A, Canevari S, Miotti S, Menard S, Colnaghi MI. Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo. Cancer Res. 1993;53:5791–6.[Abstract/Free Full Text]

33. Matsue H, Rothberg KG, Takashima A, Kamen BA, Anderson RG, Lacey SW. Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate. Proc Natl Acad Sci USA. 1992;89:6006–9.[Abstract/Free Full Text]

34. Kane MA, Elwood PC, Portillo RM, Antony AC, Najfeld V, Finley A, Waxman S, Kolhouse JF. Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells. J Clin Invest. 1988;81:1398–406.[Medline]

35. Brigle KE, Spinella MJ, Westin EH, Goldman ID. Increased expression and characterization of two distinct folate binding proteins in murine erythroleukemia cells. Biochem Pharmacol. 1994;47:337–45.[Medline]

36. Jansen G, Kathmann I, Rademaker BC, Braakhuis BJ, Westerhof GR, Rijksen G, Schornagel JH. Expression of a folate binding protein in L1210 cells grown in low folate medium. Cancer Res. 1989;49:1959–63.[Abstract/Free Full Text]

37. Jhaveri MS, Wagner C, Trepel JB. Impact of extracellular folate levels on global gene expression. Mol Pharmacol. 2001;60:1288–95.[Abstract/Free Full Text]

38. Hsueh CT, Dolnick BJ. Altered folate-binding protein mRNA stability in KB cells grown in folate-deficient medium. Biochem Pharmacol. 1993;45:2537–45.[Medline]

39. Said HM, Chatterjee N, Haq RU, Subramanian VS, Ortiz A, Matherly LH, Sirotnak FM, Halsted C, Rubin SA. Adaptive regulation of intestinal folate uptake: effect of dietary folate deficiency. Am J Physiol Cell Physiol. 2000;279:C1889–95.[Abstract/Free Full Text]

40. Subramanian VS, Chatterjee N, Said HM. Folate uptake in the human intestine: promoter activity and effect of folate deficiency. J Cell Physiol. 2003;196:403–8.[Medline]

41. Mendelsohn LG, Gates SB, Habeck LL, Shackelford KA, Worzalla J, Shih C, Grindey GB. The role of dietary folate in modulation of folate receptor expression, folylpolyglutamate synthetase activity and the efficacy and toxicity of lometrexol. Adv Enzyme Regul. 1996;36:365–81.[Medline]

42. Gates SB, Worzalla JF, Shih C, Grindey GB, Mendelsohn LG. Dietary folate and folylpolyglutamate synthetase activity in normal and neoplastic murine tissues and human tumor xenografts. Biochem Pharmacol. 1996;52:1477–9.[Medline]

43. van der Wilt CL, Cloos J, de Jong M, Pinedo HM, Peters GJ. Screening of colon tumor cells and tissues for folylpolyglutamate synthetase activity. Oncol Res. 1995;7:317–21.[Medline]

44. Green JM, MacKenzie RE, Matthews RG. Substrate flux through methylenetetrahydrofolate dehydrogenase: predicted effects of the concentration of methylenetetrahydrofolate on its partitioning into pathways leading to nucleotide biosynthesis or methionine regeneration. Biochemistry. 1988;27:8014–22.[Medline]

45. Scott JM, Dinn JJ, Wilson P, Weir DG. Pathogenesis of subacute combined degeneration: a result of methyl group deficiency. Lancet. 1981;2:334–7.[Medline]

46. Oppenheim EW, Adelman C, Liu X, Stover PJ. Heavy chain ferritin enhances serine hydroxymethyltransferase expression and de novo thymidine biosynthesis. J Biol Chem. 2001;276:19855–61.[Abstract/Free Full Text]

47. Herbig K, Chiang EP, Lee LR, Hills J, Shane B, Stover PJ. Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses. J Biol Chem. 2002;277:38381–9.[Abstract/Free Full Text]

48. Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem. 1990;1:228–37.[Medline]

49. Selhub J. Homocysteine metabolism. Annu Rev Nutr. 1999;19:217–46.[Medline]

50. Field MS, Szebenyi DM, Stover PJ. Regulation of de novo purine biosynthesis by methenyltetrahydrofolate synthetase in neuroblastoma. J Biol Chem. 2006;281:4215–21.[Abstract/Free Full Text]

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