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In vitro DNA synthesis by megaloblastic bone marrow: Effect of folates and cobalamins on thymidine incorporation and de novo thymidylate synthesis
Author(s) -
Das Kshitish C.,
Herbert Victor
Publication year - 1989
Publication title -
american journal of hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.456
H-Index - 105
eISSN - 1096-8652
pISSN - 0361-8609
DOI - 10.1002/ajh.2830310103
Subject(s) - thymidylate synthase , thymidine , thymidine kinase , dna synthesis , deoxyuridine , de novo synthesis , biochemistry , nucleotide salvage , microbiology and biotechnology , biology , in vitro , chemistry , dna , enzyme , nucleotide , immunology , fluorouracil , chemotherapy , virus , herpes simplex virus , gene , genetics
The de novo pathway of thymidylate synthesis (i.e., methylation of dUMP to dTMP) is directly folate dependent and indirectly vitamin B 12 (cobalamins) dependent. In deficiency of these vitamins, this pathway is impaired, and exogenous deoxyuridine (dU) fails to suppress adequately in vitro incorporation of [ 3 H]thymidine ( 3 H‐TdR) into DNA via the salvage pathway (i.e., abnormal dU suppression). This abnormality is corrected by the addition of folate compounds (analogues) and/or vitamin B 12 depending on the nature of the underlying deficiency. We studied the effects of addition of PteGlu, 5‐methyl THF (5‐CH3‐FH4), 5‐formyl‐THF (5‐CHO‐FH4), and hydroxy‐cobalamin (OH‐cbl) on 3 H‐TdR incorporation into DNA and thymidine kinase activity (salvage pathway), and on [ 3 H]deoxyuridine ( 3 H‐dU) incorporation and dU suppression values (de novo pathway) in cultures of normal and megaloblastic bone marrows. The results showed that 3 H‐TdR incorporation into DNA and the salvage enzyme, thymidine kinase, activity were greater and 3 H‐dU incorporation into DNA less in megaloblastic cells as compared with normal cells. The addition of folates significantly reduced 3 H‐TdR incorporation and thymidine kinase activity and enhanced 3 H‐dU incorporation in folate and vitamin B 12 ‐deficient cells except that 5‐CH3‐FH4 had no effect on vitamin B 12 ‐deficient cells. None of these additives had any significant effect on normal cells. This study also showed that the addition of the deficient vitamin(s) to the “control tubes” in the dU suppression test is inappropriate, as these vitamins may at least partially correct the defect in cellular DNA synthesis caused by the deficiences of these vitamins and may mask these deficiencies in the results of the in vitro correction of the dU suppression abnormalities in mild cases of megaloblastic anemia.