Deoxyuridine Suppression Test Research Articles

Abstract 5420: The effect of folate deficiency on chemosensitivity of colon cancer cells to 5-Fluorouracil

Abstract Background: Folate is an essential cofactor in one-carbon transfer reactions including nucleotide biosynthesis, thereby playing an important role in DNA replication and repair. In cancer cells, folate depletion interrupts DNA synthesis, thereby causing cancer cell death. This has been the basis for chemotherapy using antifolates and 5-Fluorouracil (5FU). We determined the effect of folate deficiency on the sensitivity of colon cancer cells to 5FU in a well established in vitro model of folate deficiency. Methods: Three human colon cancer cell lines (HCT116, Caco-2, HT29) were grown in RPMI1640 medium containing 0 (deficient) and 2.3 μM (replete) of folic acid. Proliferation was measured with growth curves every 3 days for 12 days and intracellular folate concentrations were determined at day 0, 6 and 12. Functional folate depletion was determined by the deoxyuridine suppression test. In vitro chemosensitivity of these colon cancer cell lines to 5FU with leucovorin (LV; 5 μM) was determined using a modified SRB protein assay. In HCT116 and Caco-2, thymidylate synthase (TS) activity was measured and TS, p53 and p21 protein expression was determined by immunoblotting. Results: All three colon cancer cell lines grown in the folate-deficient medium exhibited significantly slower growth rates than the corresponding cell lines grown in the folate-replete medium (p<0.05). Intracellular folate concentrations were significantly lower in all three cell lines grown in the folate-deficient medium than those grown in the folate-replete medium (p<0.05). Intracellular folate depletion induced in these cells was functionally significant as indicated by the deoxyuridine suppression test (p<0.05). All cells grown in the folate-deficient medium showed progressively lower intracellular folate concentrations over time (p<0.05). All three cell lines grown in the folate-deficient medium exhibited significantly higher chemosensitivity to 5FU+LV, albeit to a different degree, compared with those grown in the folate-replete medium (p<0.05). Caco-2, but not HCT116, cells grown in the folate-deficient medium showed significantly lower TS activity (p<0.05). Caco-2 cells grown in the folate-deficient medium had significantly lower protein expression of p53 and p21 but not TS. No differences in protein expression of TS, p53, and p21 were observed in HCT116 cells. Conclusion: Our data suggest that folate deficiency significantly enhances the sensitivity of colon cancer cells to 5FU based chemotherapy. Dietary or other strategies to deplete intracellular folate concentrations in colon cancer cells to enhance chemosensitivity to 5FU are worthy of further investigation. Given the dramatically increased dietary intake and blood levels of folate in North America resulting from folic acid fortification and supplementation, whether or not high folate status would affect chemotherapy for colon cancer also warrants future studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5420. doi:1538-7445.AM2012-5420

Neural tube defects induced by folate deficiency in mutant curly tail (Grhl3) embryos are associated with alteration in folate one‐carbon metabolism but are unlikely to result from diminished methylation

Folate one-carbon metabolism has been implicated as a determinant of susceptibility to neural tube defects (NTDs), owing to the preventive effect of maternal folic acid supplementation and the higher risk associated with markers of diminished folate status. Folate one-carbon metabolism was compared in curly tail (ct/ct) and genetically matched congenic (+(ct)/+(ct)) mouse strains using the deoxyuridine suppression test in embryonic fibroblast cells and by quantifying s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) in embryos using liquid chromatography tandem mass spectrometry. A possible genetic interaction between curly tail and a null allele of 5,10-methylenetetrahydrofolate reductase (MTHFR) was investigated by generation of compound mutant embryos. There was no deficit in thymidylate biosynthesis in ct/ct cells, but incorporation of exogenous thymidine was lower than in +(ct)/+(ct) cells. In +(ct)/+(ct) embryos the SAM/SAH ratio was diminished by dietary folate deficiency and normalized by folic acid or myo-inositol treatment, in association with prevention of NTDs. In contrast, folate deficiency caused a significant increase in the SAM/SAH ratio in ct/ct embryos. Loss of MTHFR function in curly tail embryos significantly reduced the SAM/SAH ratio but did not cause cranial NTDs or alter the frequency of caudal NTDs. Curly tail fibroblasts and embryos, in which Grhl3 expression is reduced, display alterations in one-carbon metabolism, particularly in the response to folate deficiency, compared to genetically matched congenic controls in which Grhl3 is unaffected. However, unlike folate deficiency, diminished methylation potential appears to be insufficient to cause cranial NTDs in the curly tail strain, nor does it increase the frequency of caudal NTDs.

Mild Methionine Excess Does Not Affect Thymidylate Synthesis or Inflammation Markers Expression in Human Aortic Endothelial Cells

Background: Recent studies in animal models have shown that high methionine intakes induce atherosclerotic changes that may be exacerbated when deficiencies of vitamins B₆, B₁₂ and folate are present. However, the mechanism underlying this possible atherogenic effect remains unknown. The aim of the present study was to evaluate the effects of methionine on the folate-dependent thymidylate-DNA synthesis, as a possible mechanism of atherogenicity, as well as the effect of high methionine/low folate on several key inflammation markers, such as vascular cell adhesion molecule-1 (VCAM-1), receptor for advanced glycation end products (RAGE) and matrix metalloproteinase-9 (MMP-9) in human aortic endothelial cells. Methods: Deoxyuridine suppression test was performed in order to evaluate thymidylate synthesis. To examine the expression of inflammation markers, cells were exposed to high methionine/low folate media for 9 days. Results: The assayed methionine levels (0.1, 0.5 and 5 mM) did not affect the de novo thymidylate-DNA synthesis. Consistent with this result, methionine (1 and 2.5 mM), alone or in combination with folate deficiency, increased homocysteine levels but did not induce the expression of the inflammation markers evaluated. Conclusion: Under the study conditions, methionine was not able to exert the atherogenic mechanism proposed and did not have the hypothesized inflammatory consequences in human aortic endothelial cells.

A fault in the design of the deoxyuridine suppression test.

Clinical & Laboratory HaematologyVolume 1, Issue 1 p. 69-71 A fault in the design of the deoxyuridine suppression test S. N. WICKRAMASINGHE, S. N. WICKRAMASINGHE Department of Haematology and MRC Experimental Haematology Unit, St Mary's Hospital Medical School, University of London, London W12 1 PGSearch for more papers by this authorJ. E. SAUNDERS, J. E. SAUNDERS Department of Haematology and MRC Experimental Haematology Unit, St Mary's Hospital Medical School, University of London, London W12 1 PGSearch for more papers by this author S. N. WICKRAMASINGHE, S. N. WICKRAMASINGHE Department of Haematology and MRC Experimental Haematology Unit, St Mary's Hospital Medical School, University of London, London W12 1 PGSearch for more papers by this authorJ. E. SAUNDERS, J. E. SAUNDERS Department of Haematology and MRC Experimental Haematology Unit, St Mary's Hospital Medical School, University of London, London W12 1 PGSearch for more papers by this author First published: March 1979 https://doi.org/10.1111/j.1365-2257.1979.tb00593.xCitations: 14AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume1, Issue1March 1979Pages 69-71 RelatedInformation

Megaloblastosis: From Morphos to Molecules

Objective: Megaloblastosis (i.e., megaloblastic transformation of erythroid precursor cells in the bone marrow) is the cytomorphological hallmark of megaloblastic anemia resulting from vitamin B₁₂ and folate deficiency. It is characterized by a finely stippled lacy pattern of nuclear chromatin, which is believed to be an expression of deranged cellular DNA synthesis. However, the molecular basis of these cytomorphological aberrations still remains obscure. The current presentation describes the results of our studies on some molecular events associated with the development of megaloblastosis. Methods: Transmission electron microscopy was used to study megaloblasts as well as DNA fibers extracted from megaloblastic and normoblastic bone marrows with and without treatment with proteinase K during the extraction procedure; cellular DNA synthesis in bone marrow cultures was studied by incorporation of ³H-thymidine and deoxyuridine suppression test, while histone biosynthesis in bone marrow cells was studied by in vitro incorporation of ³H-tryptophan, ³H-lysine and ³H-arginine into histones. Results: Derangement of DNA synthesis occurred due to an impaired de novo pathway of thymidylate synthesis in both vitamin-B₁₂- and folate-deficient human megaloblastic bone marrows as well as in the bone marrows of rhesus monkeys and rats with experimentally induced folate deficiency. Interestingly, folate-deficient monkeys developed frank megaloblastic bone marrows, but folate-deficient rats did not. On the other hand, megaloblastic changes in the bone marrow of human patients with myelodysplastic syndrome and erythroleukemia were not associated with this DNA synthetic abnormality. Biosynthesis of predominantly arginine-rich histones in megaloblastic bone marrows was markedly reduced as compared to normoblastic bone marrows, which was consistently associated with elongation and despiralization of chromosomes and finely stippled nuclear chromatin in megaloblasts. Conclusion: The impaired biosynthesis of predominantly arginine-rich nuclear histones appeared to be a common molecular event (a denominator) underlying the development of megaloblastosis with or without abnormal DNA synthesis.

Gene expression profiles of folate deficient colon cancer cells

Folate is a B vitamin that inversely modulates colorectal carcinogenesis. Folate status is also an important determinant in chemotherapeutic response. Mediating the transfer of onecarbon moieties is the sole biochemical function known for folate, and in this role folate plays a critical role in DNA synthesis, repair and methylation This study determined the gene expression profiles induced by tolate deficiency in 2 human colon adenocarcinoma cell lines, HCT116 and Caco2. Cells were grown in either standard (2.3 ~tM Mate) or folate deficient (0 ~M) RPMI medium for 20 days. Folate-deficient HCTll6 and Caco2 cells showed sigmficantly reduced growth rates and had sigmficantly decreased (by 80-90%) intracellular folate concentrations compared with corresponding folate-sufficient ceils (P<O.001 ). The deoxyuridine suppression test confirmed functionally significant intracellular folate depletion in folate-deficlent cells (P<0.01). Total RNA from folate-deficient and sufficient ceils of each cell line was hybridized onto 2 commercially available human cancer pathway (a panel of key genes involved in 6 biological pathways frequently altered during transformation and tumongenesis) and apoptosis (a panel of key genes involved in apoptosis) cDNA arrays and differentially expressed genes were identified RT-PCR analysis was used to confirm the results of cDNA array. A total of 13 and 22 cancer pathway genes were differential expressed in folate-defictent HCT116 and Caco2 cells, respectively, compared with corresponding folate-sufficient cells; 4 genes were differentially expressed in both folatedeficient ceils Most notably affected changes were upregulated ATM and downregulated MDM2 in folate-deficient Caco2 cells, upregulated p21 in folate-deficient HCT 116 cells and upregulated b-catenin and VEGF in both folate-deficient ceils. A total of 5 and 12 apoptosis genes were difterentially expressed in folate-deficient HCT 116 and Caco2 cells, respectively, compared with corresponding folate-sufficient cells; 1 gene was differentially expressed in both fofate-deficient cells. Most significantly altered changes were downregulated MDM2 and BAX in folate-deficient Caco2 cells and upregulated B1RC3 in both folate-deficient HCT116 and Caco2 cells. These data suggest that folate deficiency affects the expression of key genes that are related to cell cycle control, DNA damage and repair, apoptosis, signal transduction and angiogenesis in colon cancer cells in a cell-specific manner.

Measuring and Interpreting Holo-Transcobalamin (Holo-Transcobalamin II)

At one time, diagnosing cobalamin deficiency was fairly simple, usually involving a patient with clinical problems, and was usually settled by determining whether the serum cobalamin concentration was low or not. As reviewed elsewhere (1), things began to change after sensitive metabolic tests were introduced and as attention extended to the cobalamin status of asymptomatic persons. Metabolic studies confirmed that most low cobalamin concentrations in asymptomatic patients and seemingly healthy persons represented subclinical cobalamin insufficiency, but ∼30–40% of these cobalamin concentrations did not represent insufficiency and thus could be considered “falsely low”. The diagnostic reliability of serum cobalamin was further challenged by metabolic demonstrations of “falsely normal” cobalamin concentrations; these too occurred most often, but not exclusively, in asymptomatic persons. The search continues for the optimal test to diagnose deficiency because the metabolic tests also have disadvantages. Increased plasma total homocysteine is too nonspecific; methylmalonic acid determination, although posing fewer problems of specificity, is complex and expensive; and the deoxyuridine suppression test is too unwieldy for practical use. Interest has been drawn to the possible benefit of measuring only the cobalamin attached to transcobalamin (TC; also called TC II; the TC-cobalamin complex is called holo-TC or holo-TC II) rather than the total cobalamin content of plasma. The concept, suggested by Lindemans et al. in 1983 (2), is simple and attractive: holo-TC contains the biologically available cobalamin because only TC promotes specific uptake of its cobalamin by all cells. The much larger fraction of serum cobalamin carried by haptocorrin (HC; also called TC I, R binder, or sometimes cobalophilin) is considered metabolically inert because no cellular receptors exist for holo-HC (also called holo-TC I). However, the physiologic cycle of holo-TC is quite complex: ( a ) ileal holo-TC enters the portal circulation, carrying cobalamin absorbed from the gut (3); ( b ) some of the …

The history of folic acid.

The history of folic acid.

Current concepts in cobalamin deficiency.

The application of sensitive metabolic tests, such as the deoxyuridine suppression test and measurement of homocysteine and methylmalonic acid, to cobalamin status has identified the entity of mild, preclinical cobalamin deficiency. This state, common in the elderly, responds to cobalamin therapy. Preclinical deficiency may exist within the nervous system as well, although this requires further study. Nevertheless, it is well to remember that not all low cobalamin levels and not all abnormal metabolite results reflect cobalamin deficiency. Interpretation of metabolic results still requires caution, as do proposals to raise the cut-off point for low cobalamin levels to capture some normal levels that are associated with metabolic abnormality. The recognition of mild, preclinical deficiency has opened up many important issues. These include identifying its causes, what should be done about it, and what the clinical impact of the hyperhomocysteinemia itself is. Although malabsorptive disorders, especially food-cobalamin malabsorption, underlie about half of all cases of preclinical deficiency, no cause can be found in the remainder of these cases; poor dietary intake appears to be uncommon. In addition, unusual states of neurologically symptomatic cobalamin deficiency are being recognized, such as nitrous oxide exposure in patients with unrecognized deficiency and severe deficiency in children of mildly deficient mothers. All of these have broadened and complicated the picture of cobalamin deficiency while providing greater opportunities for prevention.

Cobalamins and folates as seen through inborn errors of metabolism: A review and perspective

This chapter discusses the inborn errors of metabolism that helps to understand the normal metabolism of folates and cobalamins, especially how they relate to the normoblastic development of the bone marrow, and megaloblastic anemia. The chapter provides information about the metabolism of these two vitamins and reviews some of the research techniques and their limitations. Selective patients with inborn errors of folate and cobalamin metabolism, and the concepts evolving from such errors, are presented in the chapter. Most biochemical studies done on megaloblastic anemia in the past were on bone marrow aspirates. These studies focused on the deoxyuridine suppression test. Dihydrofolate (DHF), an end product of de novo thymidine synthesis, is not reduced to tetrahydrofolate in the bone marrow as it has been shown that most of the dividing bone marrow cells have little or no dihydrofolate reductase activity. It is suggested that the constant supply of reduced folates to the bone marrow is derived from plasma formyltetrahydrofolates (formylTHFs). The inborn errors involving 5,10-methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) reactions support this conclusion. An alternative route could be the formylation of THF.

Embryonic folate metabolism and mouse neural tube defects.

Folic acid prevents 70 percent of human neural tube defects (NTDs) but its mode of action is unclear. The deoxyuridine suppression test detects disturbance of folate metabolism in homozygous splotch (Pax3) mouse embryos that are developing NTDs in vitro. Excessive incorporation of [3H]thymidine in splotch embryos indicates a metabolic deficiency in the supply of folate for the biosynthesis of pyrimidine. Exogenous folic acid and thymidine both correct the biosynthetic defect and prevent some NTDs in splotch homozygotes, whereas methionine has an exacerbating effect. These data support a direct normalization of neurulation by folic acid in humans and suggest a metabolic basis for folate action.

Effect of methotrexate and 5-fluorouracil on de novo thymidylate synthesis in human colon carcinoma cell line, Caco-2

Although colon cancers respond poorly to chemotherapeutic agents, 5-fluorouracil (5-FU) is considered the most effective single agent in the treatment of advanced colon cancer, whereas methotrexate has been reported as an ineffective agent. But both 5-FU and methotrexate inhibit de novo thymidylate synthesis. In the present study we assessed the sensitivity of folate-dependent thymidylate synthesis to methotrexate and 5-FU in Caco-2, a human colon carcinoma cell line. Sensitivity was assessed indirectly, by the deoxyuridine suppression test and directly, by the degree of inhibition of [ 3H]deoxyuridine incorporation into deoxyribonucleic acid (DNA). Methotrexate or 5-fluorodeoxyuridine resulted in a significant decrease in suppression of [ 3H]thymidine incorporation by exogenous deoxyuridine. [ 3H]deoxyuridine incorporation was also inhibited by the two agents. Inhibition was dose dependent and 50% inhibition occurred at about 2.5 μmol/L methotrexate and 25 μmol/L 5-FU. In a second study, the effect of methotrexate and 5-FU on [ 3H]deoxyuridine incorporation into DNA was assessed under conditions in which a Caco-2 cell monolayer was exposed to the agents either at the apical or at the basolateral membrane side. Under these conditions, inhibition was also dose dependent and cells were more sensitive to basolateral exposure to both methotrexate and 5-FU ( P < 0.05). The data suggest that both methotrexate and 5-FU are effective inhibitors of thymidylate synthesis in Caco-2 cells. Determining the degree of inhibition of deoxyuridine incorporation into DNA is an effective method for evaluating these agents' effect on de novo thymidylate synthesis. Further studies are required to determine if these inhibitory effects also hold for colon cancer tissue biopsies and whether the reported differences in therapeutic efficacy between the two agents can be manifested in vitro.

Limited value of serum holo-transcobalamin II measurements in the differential diagnosis of macrocytosis.

To study the value of serum holo-transcobalamin II (holo-TCII) measurements in the differential diagnosis of macrocytosis. Holo-TCII concentrations were measured in serum samples from 50 healthy non-vegetarian subjects and 30 patients with macrocytosis, using a technique based on the adsorption of holo-TCII with amorphous, precipitated silica. Deoxyuridine (dU) suppression tests were performed on the bone marrow cells of all the patients. Haematological diagnoses were made using standard criteria. The causes of macrocytosis were cobalamin (Cbl) deficiency due to pernicious anaemia or following partial gastrectomy (10 patients), dietary folate deficiency with/without Cb1 deficiency (four patients), chronic alcoholism (four patients), myelodysplastic syndrome (five patients), treatment with methotrexate or azathioprine (three patients), and congenital dyserythropoietic anaemia (CDA) (four patients). Undetectable or low holo-TCII concentrations were found in all patients with Cb1 deficiency and in some or all patients from each of the other diagnostic categories. There was also no correlation between the dU suppressed value and the holo-TCII concentration: all 15 patients with high dU suppressed values and nine of 15 with normal dU suppressed values, including four patients with CDA, had low holo-TCII concentrations. Measurements of serum holo-TCII concentrations by the silica adsorption method are not of value in the differential diagnosis of macrocytosis. The finding of low serum holo-TCII concentrations in patients with macrocytosis due to causes other than Cb1 deficiency may result not only from a state of negative Cb1 balance but also from other factors, such as increased utilisation of holo-TCII as a consequence of erythroid hyperplasia.

Neutrophil nuclear segmentation in mild cobalamin deficiency: relation to metabolic tests of cobalamin status and observations on ethnic differences in neutrophil segmentation.

Neutrophil hypersegmentation is considered the most sensitive peripheral blood cell marker of cobalamin deficiency. However, its diagnostic value in the mild deficiency states that accompany most low cobalamin levels and its relation to metabolic test of cobalamin status are unknown. The authors compared neutrophil lobe averages and percent neutrophils with 5 or more lobes (%5+ lobes) in 169 subjects with their mean corpuscular volume (MCV) and serum cobalamin, methylmalonic acid (MMA), homocysteine, and folate levels and, in 65 cases, with the deoxyuridine suppression test (dUST). Only 9 subjects had hypersegmentation by lobe average and 20 subjects by %5+ lobes. They were not more often cobalamin-deficient than subjects without hypersegmentation. Moreover, only one of 34 subjects with dUST results diagnostic for cobalamin deficiency had neutrophil hypersegmentation. Both indices of neutrophil segmentation in the 169 subjects correlated significantly with homocysteine levels. They also showed weak inverse correlation with cobalamin levels, but did not correlate with MMA, folate, or MCV values. Cobalamin therapy for 6 months did not significantly change neutrophil lobe averages in 35 subjects with mild deficiency, compared with 8 nondeficient controls, and only marginally improved the %5+ lobes. A surprising, incidental observation was that blacks had significantly greater neutrophil segmentation by both criteria than did whites and others. This difference was unrelated to cobalamin or folate status. Our results indicate that dUST abnormalities precede all morphologic changes of deficiency, including hypersegmentation. Although a tendency exists for neutrophil segmentation to increase very slightly as some serum values, especially homocysteine, start to worsen in mild cobalamin deficiency, the metabolic changes precede overt hypersegmentation. Neutrophil nuclear segmentation is insufficiently sensitive in relation to metabolic evidence of deficiency to be used as a clinical tool in the diagnosis of mild cobalamin deficiency.

Subtle Cobalamin Deficiency

Subtle Cobalamin Deficiency

Cobalamin‐dependent metabolism in chronic myelogenous leukemia determined by deoxyuridine suppression test and the formiminoglutamic acid and methylmalonate excretion in urine

The cobalamin metabolism in chronic myelogenous leukemia (CML) was evaluated in 18 newly diagnosed and untreated patients by formiminoglutamic acid (FiGlu) and methyl malonic acid excretion (MMA) tests. A deoxyuridine (dU) suppression test of bone marrow cells was compared in patients with acute myelogenous leukemia (N = 5), myelodysplastic disease (N = 3), untreated pernicious anemia (N = 16), folate deficiency (N = 7), and a hospital reference group without signs of cobalamin or folate deficiency (N = 22). All had normal MMA excretion but 3 of 15 patients had increased FiGlu excretion. In vitro thymidine uptake in bone marrow cells of CML patients were lower (mean 40 fmol/106 cells) than pernicious anemia patients (115 fmol/106 cells). Methotrexate (MTX) increased the uptake in all cases. Addition of formyl-THF, methyltetrahydrofolate (methyl-THF), and pteroylglutamic acid (PGA) tended to normalize the effect of MTX. In pernicious anemia methyl-THF only decreased the uptake in combination with CN-Cbl. dU suppression values were significantly higher (6.3%) in CML than in the reference group (4.4%), but significantly lower than in pernicious anemia (41.6%) and folate deficiency (28.5%). The dU suppression values in bone marrow cells of CML patients correlated significantly with the transferrin saturation. In buffy coat cells dU suppression values were even higher (9.3%) than in bone marrow cells of the same CML patients. Addition of folate forms and CN-Cbl did not change the dU suppression values in CML, as it did in pernicious anemia. MTX increased dU suppression values significantly in all patients, but more in CML (64.5%) than in pernicious anemia (48.6%) and controls (49.8%). The MTX effect was to some extent neutralized by folate analogues with formyl-THF as the most effective followed by methyl-THF and lastly PGA. Methyl-THF also neutralized MTX in pernicious anemia, but its effect was certainly enhanced by addition of CN-Cbl. Thymidine uptake and dU suppression patterns were not significantly changed in CML after treatment with busulfan for 1 week or in accelerated phase. We concluded that signs of cobalamin or folate deficiency (apart from one patient) cannot be demonstrated in untreated CML. However, dU suppression was significantly increased and more so in circulating myeloid cells than in bone marrow. This indicates a deranged metabolism of deoxynucleotides which is independent of cobalamin and folates, and a difference between bone marrow cells and circulating cells. dU suppression is a valuable indicator of cobalamin deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Deoxyuridine suppression test on isolated rat bone marrow cells and the in vitro effect of bidisomide

The deoxyuridine suppression test was performed on isolated rat bone marrow cells in order to study the effect of bidisomide, a new Class I antiarrhythmic agent, on folate-dependent DNA synthesis. Methotrexate and 5-fluorouracil, two known inhibitors of DNA synthesis, were included in the study to validate the test system. Methotrexate and 5-fluorouracil, at a concentration of 5.5 μ m, decreased thymidine incorporation into DNA by way of the de novo pathway (thymidylate synthase activity). The salvage pathway of DNA synthesis (thymidine kinase activity), however, was not affected by these anticancer drugs. Bidisomide up to 1 m m did not affect the folate-dependent thymidylate synthase activity, nor the thymidine kinase activity of isolated rat bone marrow cells.

Bone marrow cells from vitamin B12- and folate-deficient patients misincorporate uracil into DNA

Bone marrow cells from 15 patients with normal deoxyuridine (dU) suppression test results, 3 healthy subjects, and 11 patients with megaloblastic anemia caused by vitamin B12 or folate deficiency were examined for misincorporation of uracil into DNA. Cells were incubated with [5–3H] uridine for 2 hours and their DNA extracted. The DNA was hydrolyzed to deoxyribonucleosides with DNase 1, phosphodiesterase and alkaline phosphatase, and any dU present was separated from other deoxyribonucleosides by Aminex A6 chromatography. The quantity of dU/mg DNA and the radioactivity in the dU peak/mg DNA were then calculated. The results clearly showed that there was markedly increased uracil misincorporation into the DNA of vitamin B12- or folate-deficient marrow cells. Misincorporation of uracil into DNA may be an important biochemical lesion underlying both the megaloblastic change and the ineffectiveness of hematopoiesis in vitamin B12 and folate deficiency.

Bone Marrow Cells From Vitamin B12- and Folate-Deficient Patients Misincorporate Uracil Into DNA

AbstractBone marrow cells from 15 patients with normal deoxyuridine (dU) suppression test results, 3 healthy subjects, and 11 patients with megaloblastic anemia caused by vitamin B12 or folate deficiency were examined for misincorporation of uracil into DNA. Cells were incubated with [5-3H] uridine for 2 hours and their DNA extracted. The DNA was hydrolyzed to deoxyribonucleosides with DNase 1, phosphodiesterase and alkaline phosphatase, and any dU present was separated from other deoxyribonucleosides by Aminex A6 chromatography. The quantity of dU/mg DNA and the radioactivity in the dU peak/mg DNA were then calculated. The results clearly showed that there was markedly increased uracil misincorporation into the DNA of vitamin B12- or folate-deficient marrow cells. Misincorporation of uracil into DNA may be an important biochemical lesion underlying both the megaloblastic change and the ineffectiveness of hematopoiesis in vitamin B12 and folate deficiency.

Dr Chanarin's comments on our study of the deoxyuridine suppression test in vitamin B12 and folate deficiency were not unexpected. In answer to the specific questions:

Dr Chanarin's comments on our study of the deoxyuridine suppression test in vitamin B₁₂ and folate deficiency were not unexpected. In answer to the specific questions: