DNA synthesis in isolated lymphocyte nuclei. Effects of megaloblastic anaemia due to folate or vitamin B-12 deficiency or antimetabolite drugs.
DNA synthesis has been studied in nuclei isolated from phytohaemagglutinin-stimulated lymphocytes from normal subjects and patients with megaloblastic anaemia. Lymphocytes were incubated for 72 h, nuclei isolated and incorporation of tritiated deoxythymidine triphosphate ([3H]TTP) into DNA measured, usually over a 10 min incubation period. Preincubation of normal phytohaemagglutinin-stimulated lymphocytes with methotrexate (1 - 10(-5) M, 48--72 h), 5-fluorouracil (1 - 10(-6) M, 70--72 h), and 1-beta-D-arabinofuranosyl cytosine (cytosine arabinoside) (4 - 10(-5) M, 71--72 h) caused a mean rise in [3H]TTP incorporation of 1.7 (P less than 0.01), 1.7 (P less than 0.05) and 2.4 (P less than 0.0025) fold, respectively. Hydroxyurea (3 - 10(-4) M, 48--72 h) in two experiments caused a mean increase of 1.6 fold. Untreated vitamin B-12- and folate-deficient cells showed a 2.0-fold (P less than 0.05) increase above the incorporation when the deficiencies were corrected by addition of vitamin B-12 and folic acid between 0 and 72 h in vitro. The mean percentages of the incorporation due to ATP-independent synthesis in nuclei from normal untreated cells, 5-fluorouracil-treated, cytosine arabinoside treated and vitamin B-12- or folate-deficient cells were 56 +/- 7% S.E., 41 +/- 7%, 84 +/- 3% and 28 +/- 6%, respectively. 5-Fluorouracil caused a two-fold increase in the cytoplasmic fraction of DNA polymerase when added to phytohaemagglutinin-stimulated lymphocytes between 48 and 72 h of culture but had no significant effect when added between 70 and 72 h.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000328:Adult; D000368:Aged; D000748:Anemia, Macrocytic / Q000378:metabolism*; D000749:Anemia, Megaloblastic / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003561:Cytarabine / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology; D005494:Folic Acid Deficiency / Q000378:metabolism*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008727:Methotrexate / Q000494:pharmacology; D008875:Middle Aged; D013942:Thymine Nucleotides / Q000378:metabolism; D014806:Vitamin B 12 Deficiency / Q000378:metabolism*
D037102:Lectins; D013942:Thymine Nucleotides; D003561:Cytarabine; D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005033:Ethylmaleimide; D005472:Fluorouracil; D006918:Hydroxyurea; D008727:Methotrexate