The salvage metabolism of
5-methyldeoxycytidine 5'-monophosphate (
5MedCMP) was studied in human promyelocytic
leukemia (HL-60) cells and in PHA-stimulated human lymphocytes. To this end [5'-32P]
5MedCMP was synthesized by a novel postlabeling procedure. At low substrate concentrations (less than 100 microM), the
enzyme(s) present in crude HL-60 whole-
cell extract deaminated
5MedCMP faster than they did
dCMP. Although the phosphorylation of
dCMP to
dCDP was easily demonstrable with both kinds of
cell extracts, no phosphorylation of
5MedCMP to 5MedCDP (5-methyldeoxycytidine 5'-diphosphate) was observed. This phenomenon was confirmed using HL-60 cells made permeable to
nucleotides with
Tween 80. In view of the substantial 5MeCyt (5-methylcytosine) content of
DNA and the degradation of
DNA that occurs in cells, it is conceivable that 5MedCyd (5-methyl-2'-deoxycytidine) and
5MedCMP are available for reutilization in
DNA synthesis. This would have devastating effects on cellular control and gene expression. The results of the present investigation indicate that rapid deamination at the monophosphate level and, in particular, stringent discrimination of
5MedCMP by cellular monophosphokinase(s) are the key mechanisms by which reutilization of
DNA 5MeCyt is prevented in human hematopoietic cells.