Thymidine, folates

At least nine fragile sites are present in human chromosomes. The fragility refers to chromosomal sites that break easily in the presence of certain compounds such as aphidocolin, methotrexate, and high doses of caffeine (Sutherland, 1979a, b). These sites are generally (GCC) stretches and in most cases are protected if folate, thymidine, or folinic acid is in the medium (Sutherland, 1979a, b,... 

Methotrexate (MTX, chemical structure shown in Fig. 1.) competitively inhibits the dehyrofolate reductase, an enzyme that plays an essential role in purine synthesis. The dehydrofolate reductase regenerates reduced folates when thymidine monophosphate is formed from deoxyuridine monophosphate. Without reduced folates cells are unable to synthesize thymine. Administration of N-5 tetrahydrofolate or N-5 formyl-tetrahydrofolate (folinic acid) can bypass this block and rescue cells from methotrexate activity by serving as antidote. 

Ralitrexed is a folate analog with greater selectivity. It easily crosses the cell membrane and undergoes polyglutamation. Within tissues, ralitrexed may be stored up to 29 days. It directly inhibits thymidylate synthase, the key enzyme for synthesizing thymidine triphosphate (TTP). The drug has been described to induce apoptosis in tumor cells. Ralitrexed is used for the treatment of colon carcinomas. 

In 1995, Horie et al. described a polymorphic tandem repeat found in the 5 -un-translated region of the thymidylate synthase gene [70]. Thymidylate synthase (TS TYMS) catalyzes the intracellular transfer of a methyl group to deoxyuridine-5-monophosphate (dUMP) to form deoxythymidine-5-monophosphate (dTMP), which is anabolized in cells to the triphosphate (dTTP). This pathway is the only de- novo source of thymidine, an essential precursor for DNA synthesis and repair. The methyl donor for this reaction is the folate cofactor 5,10-methylenetetrahydro-folate (CH2-THF) (Figure 24.4). 

These one-carbon groups, which are required for the synthesis of purines, thymidine nucleotides and for the interconversion some amino acids, are attached to THF at nitrogen-5 (N5), nitrogen-10 (N10) or both N5and N10. Active forms of folate are derived metabolically from THF so a deficiency of the parent compound will affect a number of pathways which use any form of THF. 

TFIF is formed from the vitamin folate through two reductions catalyzed by dihydrofolate reductase shown in Figure 1-17-4. It picks up a one-carbon unit from a variety of donors and enters the active one-carbon pool. Important pathways lequirii forms of THF from this pool include the synthesis of all purines and thymidine, wfakh in turn are used for DNA and RNA synthesis during cell growth and division. 

Inhibition of nucleobase synthesis (2). Tetrahydrofolic acid (THF) is required for the synthesis of both purine bases and thymidine. Formation of THF from folic acid involves dihydrofolate reductase (p. 272). The folate analogues aminopterin and methotrexate (ame-thopterin) inhibit enzyme activity as false substrates. As cellular stores of THF are depleted, synthesis of DNA and RNA building blocks ceases. The effect of these antimetabolites can be reversed Ltillmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of iicense. 

A depressed uptake of deoxyuridine brought about by a deficiency of vitamin B12 can be reversed as reflected by a reduction in labeled thymidine uptake by the addition of as little as 1 xg of the vitamin. Folic acid added to a concentration of 50 p.g/ml of the culture will correct abnormal results due to folate deficiency and may partially correct abnormal results due to vitamin B12 deficiency. Abnormal results may occur in bone marrow from patients with iron deficiency and from patients being treated with 5-fluorouracil (B7). 

By growing cells in the presence of increasing concentrations of aminopterin a number of resistant cells lines have been isolated (Hakala and Ishihara, 1962 Littlefield, 1969). These have been characterised as having either an altered permeability to the drug or an altered folate reductase or an increased rate of synthesis and hence increased amounts of the enzyme (Alt et al., 1976), resulting, at least in part, from a selective amplification of the dihydrofolate reductase gene (Alt et al., 1978 Schimke et al., 1988). The problem is considered in more detail in 11.8.1. The importance of the antifolates lies in their role in the HAT selection technique ( 13.5) devised by Szybalski (1962) (see also Szybalski et al., 1962 and Littlefield, 1964) for the isolation of hybrids between mutant cells defective on the one hand in thymidine kinase and on the other hand... 

Site of action 5-FU per se is devoid of antineoplastic activity and must be converted to the corresponding deoxynucleotide (5-FdUMP, Figure 38.9), which competes with deoxyuridine monophosphate (dUMP) for thymidylate synthetase. 5-FdUMP acts as a pseudosubstrate and is entrapped with the enzyme and its N5,N10-methylene tetrahydrofolic acid coenzyme in a ternary complex that cannot proceed to products. DNA synthesis decreases due to lack of thymidine, leading to imbalanced cell growth and cell death. [Note Leucovorin is given with 5-FU because the reduced folate coenzyme is required in the thymidylate synthetase reaction. Lack of sufficient coenzyme reduces the effectiveness of the antipyrimidine.] 5-FU is also incorporated into RNA and low levels have been detected in DNA. 

Tetrahydrofolic acid (THF) is a coenzyme in the synthesis of purine bases and thymidine. These are constituents of DNA and RNA and are required for cell growth and replication. Lack of THF leads to inhibition of cell proliferation. Formation of THF from dihydrofolate (DHF) is catalyzed by the enzyme dihydrofolate reductase. DHF is made from folic acid, a vitamin that cannot be synthesized in the body but must be taken up from exogenous sources. Most bacteria do not have a requirement for folate, because they are capable of synthesizing it-more precisely DHF-ffom precursors. Selective interference with bacterial biosynthesis of THF can be achieved with sulfonamides and trimethoprim. 

In addition to the methods described here, measurement of urinary ac-etamido p-aminohenzoyl glutamate will reflectfolate turnover (Section 10.2.3) and incorporation of uracil, instead of thymidine into the DNA in leukocytes or lymphocytes may provide a sensitive index of folate status. 

In normal cells, the incorporation of [ H] thymidine into DNA after preincubation with dUMP is 1.4% to 1.8% of that without preincubation. By contrast, cells that are deficient in folate form little or no thymidine from dUMP and incorporate nearly as much of the [ H] thymidine after incubation with dUMP as they do without preincubation. 

The inability to absorb Vitamin B12 occms in pernicious anemia. In pernicious anemia intrinsic factor is missing. The anemia results from impaired DNA synthesis due to a block in purine and thymidine biosynthesis. The block in nucleotide biosynthesis is a consequence of the effect of vitamin B12 on folate metabolism. When vitamin B-12 is deficient essentially all of the folate becomes trapped as the N -methyltetrahydrofolate derivative as a result of the loss of functional methionine synthase. This trapping prevents the synthesis of other tetrahydrofolate derivatives. required for the purine and thymidine nucleotide biosynthesis pathways. 

Inhibition of the Reductase affects folate metabolism leading to decreased glycine formation from serine and decreased purine synthesis which requires CH3-THF. To facilitate normal cells folinic acid (Leucovorin) is given along with methotrexate. This acid aids normal cells by its conversion to the coenzyme of Thymidyiate S)mthetase, thus bypassing the block. Since the thymidine nucleotide requirements of rapidly proliferating cells are much greater than for quiescent cells folinic acid cannot meet the demands of the cancer cells. 

Folate deficiency can result in mutations at posiliorts in DNA normally occupied by thymidine. Low levels of folate in the cell may result in accumulation of the substrate for thymidylate synthase (dUMP), and a decline in its product (dTMP) (see Folate section). This dUMP, after conversion to dUTP, is mistakenly recognized by DNA polymerase, and incorporated into DNA during replication. When DNA polymerase makes this mistake, it catalyzes the incorporation of U opposite A, as shown here ... 

Once inside the cell, folates participate in a number of interconnected metabolic pathways involving (1) thymidine and purine biosynthesis necessary for DNA synthesis, (2) methionine synthesis via homocysteine remethylation, (3) methylation reactions involving S-adenosylmethionine (AdoMet), (4) serine and glycine interconversion, and (5) metabolism of histidine and formate (see Figure 8). Via these pathways. 

Hattori Y, Maitani Y (2005) Folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase. Cancer Gene Ther 12 796-809... 

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