The influence of methylenetetrahydrofolate reductase polymorphism, nutritional status and anticancer drugs on one-carbon metabolic fluxes pathway and DNA methylation

Abstract

基因的遺傳多型性對於腫瘤的發生和預防扮演一個重要的角色。亞甲基四氫葉酸還原酶的多型性和某些種類癌症發生的風險率有關，但是其機制仍然需要進一步的研究。 亞甲基四氫葉酸還原酶催化 5,10-亞甲基四氫葉酸的還原反應，而這個反應的產物為 5-甲基四氫葉酸。 5-甲基四氫葉酸可以提供甲基給同半胱胺酸，使同半胱胺酸轉變成甲硫胺酸。已知亞甲基四氫葉酸還原酶多型性中TT同型合子的會導致酵素活性的下降並且改變細胞內葉酸形式的組成。在本研究論文裡，我們研究此葉酸形式的重新分配是否會導致同半胱胺酸的再甲基化下降或/且提高嘌呤或胸腺嘧啶的生合成作用；而DNA甲基化及核酸生合成的調控和腫瘤的發生機制有密切關係。我們利用表現不同亞甲基四氫葉酸還原酶多型性的淋巴母細胞來研究同半胱胺酸的再甲基化和嘌呤及胸腺嘧啶的生合成作用及代謝改變情形。在不同的營養狀態下，以特定之穩定同位素分別追蹤代謝路徑，並利用氣相層析質譜儀來測定胞內胺基酸和核苷酸代謝路徑的變化。我們數據指出在細胞內葉酸充足情形之下，亞甲基四氫葉酸還原酶的基因多型性並不會影響依賴葉酸的同半胱胺酸再甲基化相對流量。 但在細胞內葉酸不充足的情形之下，亞甲基四氫葉酸還原酶基因突變型其同半胱胺酸(葉酸依賴的)再甲基化的代謝會顯著較正常型來的低；推測可能是因為甲基化形式的葉酸來源受到限制。 當葉酸狀態充足的情形之下，亞甲基四氫葉酸還原酶基因突變型細胞的嘌呤的生合成作用其相對流量顯著較正常型為高；推測可能是因為可被利用的甲醯化型式的葉酸增加的緣故。 然而當葉酸不充足時，兩個基因型之間的嘌呤的生合成作用似乎並無不同；推測突變型細胞其嘌呤生合成的優勢只有在葉酸充足的情形之下才存在。我們的數據亦顯示出亞甲基四氫葉酸還原酶基因的多型性的影響和葉酸營養狀態有密切的關係，此種關係因而可能會影響和疾病發生及蔓延過程中相關代謝途徑的調控。亞甲基四氫葉酸還原酶基因突變型細胞的嘌呤生合成作用的優勢或許可以解釋何以在某些流行病學研究顯示此種基因型可降低某些癌症發生的風險，即此種基因型對於某些癌症發生具保護作用，但在其他的情形下如葉酸營養狀態不理想時，反而觀察到其可能有增加癌症發生風險。本論文提出此細胞模型，以研究人類亞甲基四氫葉酸還原酶基因變異和癌症發生及與營養狀況交互作用的相關代謝途徑。

Genetic predisposition plays a significant role in tumorigenesis and cancer prevention. A common polymorphism 677C->T in methylenetetrahydrofolate reductase (MTHFR) is associated with altered risk of certain types of cancer, but the mechanism remains to be determined. MTHFR catalyzes the reduction of 5, 10-methylene- tetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for the synthesis of methionine from homocysteine. The homozygous C677T mutation in the MTHFR gene results in reduction in enzyme activity and changes in cellular distribution of folates. In the present study we investigate if the folate form redistribution results in compromised homocysteine remethylation and elevated de novo purine synthesis, both of which may be potential regulatory pathways involved in tumorigenesis. Metabolic fluxes of homocysteine remethylation and de novo purine synthesis were studied in Epstein-Barr virus transformed lymphoblasts expressing common polymorphic allele 677 C and 677T of the MTHFR gene. Using different stable isotopic tracers, specific enrichments in intracellular amino acid pool and nucleotides were measured by Gas Chromatography /Mass Spectrometry under various nutritional conditions. Our preliminary data indicated that when cellular folate was adequate, the MTHFR genotype did not affect the folate dependent homocysteine remethylation pathways (relative enrichments of methionine m+1 specie labeled from 13C-serine tracer in CC vs. TT=0.45 + 0.28 vs. 0.40 + 0.11, p=0.83). Only when cells became folate deficient, the folate dependent remethylation pathway in TT genotype was significantly reduced compared to the CC genotypes (CC vs. TT = 0.048 + 0.011 vs. 0.008 + 0.006, p=0.043), presumably due to limited methylated folates. Using 13C-serine as the tracer, de novo purine synthesis in TT genotype was found to be elevated when folate status is adequate (relative enrichments in the purine +1 specie in CC vs. TT = 0.48 + 0.11 vs. 0.71 + 0.06, p<0.0001), presumably due to increased availability of formylated folates. When folate was restricted, no difference in purine synthesis was found between the 2 genotypes, suggesting that the advantage in purine synthesis only exists when folate supply is adequate. Our data suggest that the impacts of MTHFR 677T polymorphism are closely related to nutritional conditions, and such alterations may modulate metabolic pathways involved in disease onset/progression. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in certain type of cancers. These transformed cells are potential models for studying the consequences of human genetic variation in MTHFR and their relationship to pathways involved in cancer pathogenesis or progression.