Defective DNA synthesis in megaloblastic anaemia. Studies employing velocity sedimentation in alkaline sucrose gradients.
1. Autoradiographic experiments revealed that the average size of the replicating unit (replicon) in human phytohaemagglutunin-stimulated lymphocytes is 45 (+/- 1.3) micron. 2. A 5 min pulse of [3H]thymidine labelled DNA chains of approximately 40 S (15 micron) in control lymphocytes as revealed by velocity sedimentation in alkaline sucrose density gradients. Upon chasing in the absence of [3H]-thymidine the labelled DNA increased in size. By 6 h the bulk of the label co-sedimented with full-sized chromosomal DNA. 3. In untreated lymphocytes from patients with megaloblastic anaemia due to vitamin B-12 or folate deficiency or lymphocytes treated with methotrexate (10(-5) M) or hydroxyurea (5 . 10(-4) M) the increase in size of pulse-labelled DNA was slower than in control cells. 4. The block in maturation of pulse-labelled DNA to bulk DNA was not permanent. At 24 h of chase 75-80% of the pulse-label in both control and megaloblastic lymphocytes co-sedimented with bulk DNA. 5. We conclude that the lesions seen in DNA synthesis in megaloblastic anaemia due to folate or vitamin B-12 deficiencies occur through impaired biosynthesis of nucleotide precursors of DNA. Possible explanations of why the defects in DNA synthesis cause altered morphology of proliferating cells in megaloblastic anaemia are suggested.
D000748:Anemia, Macrocytic / Q000378:metabolism*; D000749:Anemia, Megaloblastic / Q000378:metabolism*; D001345:Autoradiography; D002499:Centrifugation, Density Gradient; D004261:DNA Replication; D005494:Folic Acid Deficiency / Q000150:complications; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D014806:Vitamin B 12 Deficiency / Q000150:complications
D006918:Hydroxyurea; D008727:Methotrexate