Dihydrofolate Reductase mRNA Expression Research Articles

Daily supplementation with 5 mg of folic acid in Brazilian patients with hereditary spherocytosis

Patients with hereditary spherocytosis (HS) have increased rates of erythropoiesis and higher folate requirements. In a case-control study of patients with HS, we evaluated the associations between the use of...

A Daily Dose of 5 mg Folic Acid for 90 Days Is Associated with Increased Serum Unmetabolized Folic Acid and Reduced Natural Killer Cell Cytotoxicity in Healthy Brazilian Adults

The effects of high-dose folic acid (FA) supplementation in healthy individuals on blood folate concentrations and immune response are unknown. Objective: The aim of the study was to evaluate the effects of daily consumption of a tablet containing 5 mg FA on serum folate; number and cytotoxicity of natural killer (NK) cells; mRNA expression of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), interferon γ (IFNG), tumor necrosis factor α (TNFA), and interleukin 8 (IL8) genes; and concentrations of serum inflammatory markers. Methods: This prospective clinical trial was conducted in 30 healthy Brazilian adults (15 women), aged 27.7 y (95% CI: 26.4, 29.1 y), with a body mass index (in kg/m2) of 23.1 (95% CI: 22.0, 24.3). Blood was collected at baseline and after 45 and 90 d of the intervention. Serum folate concentrations were measured by microbiological assay and HPLC-tandem mass spectrometry [folate forms, including unmetabolized folic acid (UMFA)]. We used real-time polymerase chain reaction to assess mononuclear leukocyte mRNA expression and flow cytometry to measure the number and cytotoxicity of NK cells. Results: Serum folate concentrations increased by ∼5-fold after the intervention (P < 0.001), and UMFA concentrations increased by 11.9- and 5.9-fold at 45 and 90 d, respectively, when compared with baseline (P < 0.001). UMFA concentrations increased (>1.12 nmol/L) in 29 (96.6%) participants at day 45 and in 26 (86.7%) participants at day 90. We observed significant reductions in the number (P < 0.001) and cytotoxicity (P = 0.003) of NK cells after 45 and 90 d. Compared with baseline, DHFR mRNA expression was higher at 90 d (P = 0.006) and IL8 and TNFA mRNA expressions were higher at 45 and 90 d (P = 0.001 for both). Conclusion: This noncontrolled intervention showed that healthy adults responded to a high-dose FA supplement with increased UMFA concentrations, changes in cytokine mRNA expression, and reduced number and cytotoxicity of NK cells. This trial was registered at www.ensaiosclinicos.gov.br as RBR-2pr7zp.

Elevated Serum Folic Acid Concentrations Were Associated with Higher mRNA Expression of DHFR Gene in Patients with Hereditary Spherocytosis

BackgroundFolic acid (FA) is the synthetic form of folate, a B complex vitamin which plays an important role in several reactions in the body. Folate deficiency produces several clinical complications including anemia and is associated with an increased risk of cancer, cardiovascular disease and neural tube defects. With the advent of FA and iron fortification of wheat and corn flour (150 µg of folic acid and 4.2 mg of iron/100 grams) in Brazil since 2004, the population was exposed to amounts of folate beyond that normally present in foods. The form present in fortified food is taken up by cells and reduced by the enzyme dihydrofolate reductase (DHFR) first to dihydrofolate (DHF) and then tetrahydrofolate (THF). DHF is the preferred natural substrate. Amounts of FA higher than the defined tolerable upper intake of 1 mg/day could impair the ability of DHFR to convert DHF to THF. Some patients with hemolytic anemia, such as hereditary spherocytosis (HS), need larger amounts of FA to compensate for their increase in erythropoiesis and have been receiving 5mg/day of supplemental FA, the only formulation available in Brazil, in addition to being exposed to compulsory food fortification with FA.ObjectiveThe aim of this study was to compare the effects of 5mg/day FA on serum folate levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes; and cytotoxicity of lymphocytes and NK cells in patients with HS and healthy individuals not receiving supplemental FA.Material and MethodsTwenty-five patients with HS exposed to mandatory fortification and in use or not of 5 mg/day of FA were included in this study. Forty-five healthy people were recruited as a control group, and matched with HS patients according to age, gender, body mass index and self-reported skin color. Blood count, including reticulocytes, C-reative protein and lactic dehydrogenase (LDH) were performed. Serum folate (SF) and vitamin B12 were determined by a microbiological method. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed in duplicate, using Real Time PCR. Cytotoxicity of lymphocytes and NK cells were also carried out using a flow cytometric assay.ResultsEight HS patients did not use FA 5 mg everyday (4 declared no use anytime and 4 reported intermittent use) and none of control group used FA supplementation. Reticulocytes and LDH were higher in HS group (P<0.05), however no difference was found between levels of C-reative protein (P=0.173) and vitamin B12 (P= 0.699) when compared with control group. The HS group had higher SF levels and elevated mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 when compared with controls (P<0.05, Figure 1). It is not clear whether FA use or underlying disease was be responsible for increasing of mRNA expression of these genes. To verify the effect of SF levels on HS patients, this group was classified into two subgroups according to median SF (< 46.6 and ≥ 46.6 nmol/L). Interestingly, the subgroup with higher SF levels showed significantly elevated DHFR mRNA expression but no difference was found in the mRNA expression of the other genes studied. The two subgroups were similar according to WBC, RBC, hemoglobin, MCV, reticulocytes, LDH, C-reative protein, vitamin B12 and cytotoxic capacity of lymphocytes and NK cells.ConclusionsElevated SF concentrations were associated with higher mRNA expression of DHFR gene in HS patients, suggesting that the use of higher amounts of FA might influence the expression and activity of DHFR and thus affect folate metabolism in these patients. It is not known whether normal subjects receiving similar high doses of FA show the same effects.Financing: FAPESP 2012/12912-1 and CNPq 4826412012-6 [Display omitted] The line in each graphic is the median.The median and percentiles 25 and 75 (P25 – P75) for control and hereditary spherocytosis groups were, respectively: A- serum folate: 22.1 (14.5 – 36.6 nmolL) and 51.5 (19.5 – 95.2 nmolL), B- DHFR mRNA expression: 1.59 (1.00 – 2.48) and 35.8 (19.0 – 52.7). In Figure C, the median and percentiles 25 and 75 (P25 – P75) for DHFR mRNA expression, according to serum folate levels (SF <46.6 and SF ≥ 46.6 nmol/L), were, respectively: 19.6 (1.4 – 38.6) and 50.3 (23 – 66.8). DisclosuresNo relevant conflicts of interest to declare.

Abstract 4531: Molecular determinants of sensitivity to pralatrexate in a panel of human cancer cell lines

Abstract Background: Pralatrexate (FOLOTYN®), an antifolate inhibiting dihydrofolate reductase (DHFR), is a high affinity substrate for cellular uptake via reduced folate carrier-1 (RFC-1) and polyglutamation by folyl-polyglutamyl synthetase (FPGS). This study evaluated the sensitivity of human cancer cell lines to pralatrexate and methotrexate, and investigated molecular determinants of primary sensitivity and acquired resistance. Materials and Methods: Anti-proliferative activity was evaluated in a panel (N=15) of human cancer cell lines using the MTT assay. mRNA expression was analyzed by qRT-PCR and protein expression by Western blot. Results: Two groups of cell lines with at least 100-fold difference in sensitivity to pralatrexate were identified. Pralatrexate was 3- to 19-fold (mean: 9-fold) more potent than methotrexate in the sensitive lines (N=9). Sensitivity to pralatrexate correlated with higher FPGS mRNA expression. Two pralatrexate-resistant cell lines (DU-PDX &amp; HEP-PDX) and two methotrexate-resistant cell lines (DU-MTX &amp; HEP-MTX) were established from DU-145 (prostate) and HEP2 (head &amp; neck) cell lines. DU-PDX and HEP-PDX cells were cross-resistant to methotrexate. DU-MTX and HEP-MTX cells showed partial cross-resistance to pralatrexate but remained more sensitive to pralatrexate compared to methotrexate (7.5- and 11-fold, resp.). Acquired resistance to pralatrexate was associated with decreased RFC-1 expression in both DU-PDX and HEP-PDX cell lines, suggesting that reduced pralatrexate uptake may be an important mechanism of acquired resistance. No change in DHFR mRNA expression was found in the pralatrexate-resistant cells. In contrast, significant increases in DHFR mRNA and protein expression were seen in the methotrexate-resistant HEP-MTX cells, suggesting different mechanisms of resistance for these two antifolates. Conclusions: Consistent with prior findings, pralatrexate was more potent than methotrexate against a panel of human cancer cell lines. Primary sensitivity to pralatrexate was associated with higher FPGS expression whereas acquired resistance was associated with decreased RFC-1 expression. Increased DHFR expression correlated with resistance to methotrexate, but not pralatrexate. The marked differences in potency and molecular determinants of tumor response observed in these preclinical models provide rationale for development of pralatrexate in methotrexate-sensitive cancers as well as in settings of acquired methotrexate resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4531. doi:10.1158/1538-7445.AM2011-4531

Influence of Reduced Folate Carrier and Dihydrofolate Reductase Genes on Methotrexate-Induced Cytotoxicity

The aim of this study is to investigate the effect of genetic variations and the expression of the reduced folate carrier (RFC) and dihydrofolate reductase (DHFR) on the drug sensitivity to methotrexate (MTX) in different cancer cell lines. We examined the six human cancer cell lines (MCF-7, AGS, A549, NCI-H23, HCT-116 and Saos-2). The cytotoxicity of MTX was measured by sulforhodamine B (SRB) assay. The expressions of the DHFR and RFC were evaluated by real-time PCR and western blotting. Four single nucleotide polymorphisms (SNPs) of the DHFR and two SNPs of the RFC were genotyped. The IC₅₀s of MTX was in an extensively broad range from 6.05±0.81 nM to>1,000 nM in the cell lines. The Saos-2 (>1,000 nM) and MCF-7 (114.31±5.34 nM) cells were most resistant to MTX; in contrast, the AGS and HCT-116 cells were highly sensitive to MTX with an IC(50) of 6.05±0.81 nM and 13.56±3.76 nM, respectively. A reciprocal change of the RFC and DHFR mRNA expression was found between the MTX-sensitive AGS and MTX-resistant Saos-2 cells. There was no significant difference in the expression levels of RFC protein in both the AGS and Saos-2 cells, whereas DHFR protein was more increased in the MTX-resistant Saos-2 cells treated with MTX. The genotype of the MTX-sensitive AGS cells were mutant variants of the DHFR; in contrast, the Saos-2 cells had the wild-type of the DHFR. In conclusion, this study showed that inverse change of the RFC and DHFR mRNA and protein expression was associated with RFC and DHFR polymorphisms and it is postulated that this phenomenon might play an important role in sensitivity of certain cancers to MTX.

Reduced Folate Carrier and Dihydrofolate Reductase Expression in Acute Lymphocytic Leukemia May Predict Outcome: A Children's Cancer Group Study

Methotrexate is a major component of current treatment regimens for children with acute lymphocytic leukemia (ALL). Potential mechanisms of methotrexate resistance include impaired drug uptake, decreased drug retention, and dihydrofolate reductase (DHFR) amplification. The purpose of this study was to assess whether reduced folate carrier (RFC) and DHFR expression in untreated leukemic blasts correlated with outcome. Quantitative real-time RT-PCR was used to measure RFC and DHFR mRNA expression in leukemic blasts from 40 newly diagnosed patients with ALL obtained in a blinded fashion from Children's Cancer Group studies. Low RFC expression at diagnosis correlated significantly with an unfavorable event free survival. Surprisingly, low, not high, DHFR expression correlated significantly with an unfavorable event-free survival. Proliferative cell nuclear antigen (PCNA) expression demonstrated a weak inverse relationship between sample PCNA and DHFR or RFC expression, suggesting that DHFR and RFC expression may be markers for factors other than drug resistance. These results suggest that impaired transport may be an important mechanism of intrinsic methotrexate resistance in ALL, and DHFR expression also may be an important prognostic factor in ALL. Additional studies are necessary to clarify the mechanism for the correlation of low DHFR expression with poor outcome.

Similarities between human ataxia fibroblasts and murine SCID cells: high sensitivity to gamma rays and high frequency of methotrexate-induced DHFR gene amplification, but normal radiosensitivity to densely ionizing alpha particles.

Two gamma-ray hypersensitive cell lines, human ataxia telangiectasia (AT) and murine severe combined immune deficiency (SCID) cells, proved to be very competent in amplifying their dihydrofolate reductase (DHFR) gene under methotrexate selection stress. Over a period of months, methotrexate-resistant clones were obtained which were able to grow in progressively increasing methotrexate concentrations up to 1 mM. By then methotrexate-resistant AT and SCID cells had amplified their DHFR gene 6- and 30-fold, respectively, and showed very high DHFR mRNA expression. In contrast, related cells with normal radiosensitivity (human GM637 and mouse BALB/c fibroblasts) did not show DHFR gene amplification under comparable conditions. This correlation of the capacity of DHFR gene amplification and gamma-ray hypersensitivity in AT and SCID cells suggests that gene amplification may have a mechanism(s) in common with those involved in repair of gamma-radiation-induced damage. No difference in cell killing could be observed following exposure to densely ionizing alpha particles: AT and SCID cells exhibited comparable survival rates to GM637 and BALB/c cells, respectively.

A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.