5-methyldeoxycytidine and Leukemia

A monoclonal antibody against 5-methylcytidine was prepared and characterized. This antibody, termed AMC, was reactive with compounds that had 5-methylcytosine structure (i.e. 5-methyl-2'-deoxycytidine, 5-methylcytidine and 5-methylcytosine). AMC had the highest reactivity to 5-methyl-2'-deoxycytidine among reactive compounds and had no or very slight cross-reactivity to cytidine-related compounds and any other compounds. Analysis of immunoreactive materials in urine revealed that 5-methyl-2'-deoxycytidine rather than 5-methylcytidine was, contrary to our expectation, the major component. Then the inhibition ELISA system using AMC was established and urinary levels of 5-methyl-2'-deoxycytidine in healthy individuals and cancer patients were determined. The mean excretion levels of healthy individuals was 0.90 +/- 0.43 nmol/mumol creatinine and the cut-off value was set at the mean + 2 S.D. of healthy individuals (1.76 nmol/mumol creatinine). Among various types of cancer tested, elevated levels of 5-methyl-2'-deoxycytidine were detected in leukemia patients. From these results, urinary 5-methyl-2'-deoxycytidine might be applicable as a biologic marker for leukemia.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Biomarkers, Tumor; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cross Reactions; Deoxycytidine; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hybridomas; Immunochemistry; Leukemia; Mice; Mice, Inbred BALB C

A simple reversed-phase liquid chromatographic (LC) method for the determination of urinary 5-methyl-2'-deoxycytidine (m5dCyd), recently claimed (on the basis of an imuno-technique) to be a potential marker for leukaemia, has been developed. Sample pre-treatment is based on a microcolumn clean-up step with an average recovery of 79% and a RSD of 3%. Detection limit was 0.2 microg/ml which is about tenfold lower than levels previously measured by an ELISA method in urine of healthy individuals. The creatinine (Cre) excretion, necessary for normalising the m5dCyd excretion, was evaluated by ion-pair liquid chromatography which permitted the simultaneous determination of pseudouridine (psi), a modified nucleoside also potentially useful as a marker for leukaemia. The described LC procedures were applied to the analysis of urine samples from healthy individuals and leukaemia patients. While the urinary psi/Cre ratio was found significantly increased for leukaemia patients, the urinary m5dCyd levels in healthy individuals were below the detection limits and did not increase in presence of the malignant disease.

Topics: Acute Disease; Biomarkers, Tumor; Chromatography, High Pressure Liquid; Deoxycytidine; Female; Humans; Leukemia; Leukemia, Myeloid; Male; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pseudouridine; Sensitivity and Specificity

5-Methylcytosine ( 5MeCyt ) is a possible regulator of eukaryotic gene transcription. We investigated whether this compound could be introduced into DNA from exogenous deoxyribonucleoside 5-methyl-2'-deoxycytidine ( 5MedCyd ). High concentrations of 5MedCyd inhibited the growth of several types of human leukemic cell lines in vitro. However, the effect could be accounted for by dThd, a deamination product of 5MedCyd . We found that radioactivity from [methyl-14C] 5MedCyd and [2-14C] 5MedCyd was incorporated into DNA as thymidylate, and none was present as 5MeCyt . There are two conceivable metabolic pathways from 5MedCyd to thymidylate. The first consists of deoxycytidine or thymidine kinase and deoxycytidylate deaminase, and the second of sequential reactions catalyzed by deoxycytidine deaminase and thymidine kinase. No indication of the first pathway was demonstrable in human leukemic cells. We conclude that the DNA exclusion of 5MeCyt from exogenous 5MedCyd takes place because of powerful deoxycytidine deaminase activity in human malignant hematopoietic cells.

Topics: Cell Division; Cell Line; Cell Survival; Deoxycytidine; Deoxyribonucleosides; Humans; Leukemia; Leukemia, Lymphoid