In vitro DNA synthesis by megaloblastic bone marrow: Effect of folates and cobalamins on thymidine incorporation and de novo thymidylate synthesis

Abstract

The de novo pathway of thymidylate synthesis (i.e., methylation of dUMP to dTMP) is directly folate dependent and indirectly vitamin B12 (cobalamins) dependent. In deficiency of these vitamins, this pathway is impaired, and exogenous deoxyuridine (dU) fails to suppress adequately in vitro incorporation of [3H]thymidine (3H-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 B12 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 3H-TdR incorporation into DNA and thymidine kinase activity (salvage pathway), and on [3H]deoxyuridine (3H-dU) incorporation and dU suppression values (de novo pathway) in cultures of normal and megaloblastic bone marrows. The results showed that 3H-TdR incorporation into DNA and the salvage enzyme, thymidine kinase, activity were greater and 3H-dU incorporation into DNA less in megaloblastic cells as compared with normal cells. The addition of folates significantly reduced 3H-TdR incorporation and thymidine kinase activity and enhanced 3H-dU incorporation in folate and vitamin B12-deficient cells except that 5-CH3-FH4 had no effect on vitamin B12-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 deficiencies 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.

Low holotranscobalamin II is the earliest serum marker for subnormal vitamin B12 (cobalamin) absorption in patients with AIDS

“…The forgetfulness, irritability, and paranoia often seen in folate deficiency reverse within days of starting folate therapy [ 1 1,131. B , , deficiency produces blockade of folate utilization 11 1, 27,39]. The neuropsychiatric stigmata of B deficiency may relate to methyl group deficiency [42].…”

“…100 pg B,,/month by injection was considered marginally adequate therapy for pernicious anemia and most other B,, deficiencies due to poor B,, absorption, with only a few exceptions, such as in certain malignancies [26]. However, we are now seeing AIDS patients whose negative B , , balance, manifested by holotranscobalamin I1 < 20 pg/ml, abnormal ddUST (diagnostic dU suppression test) [39], and elevated granulocyte segmentation were corrected by 1 mg B,, parenterally, but quickly became again abnormal a few weeks later. This suggests they have an AIDS-related (possibly infection-related and/or chronic systemic disease-related) defect in transport and/or delivery of B,, to cells with cell surface receptors for holo-TC 11, and/or a defect in getting B,, into these cells and/or storing it in them and/or delivering it to its apoenzymes and/or a defect in the apoenzymes themselves or in their pathways.…”

Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: Implications for cancer and neuronal damage

“…Low cytosolic levels of N 5 ,N 10 -methylenetetrahydrofolate (the folate cofactor for thymidylate synthase) decreases synthesis of thymidylate (dTMP; ref. 21), increasing the cellular dUMP͞dTMP ratio and DNA polymerase-mediated dUTP misincorporation into DNA (20,(22)(23)(24). Uracil is excised from DNA by uracil-DNA glycosylase and apyrimidinic endonuclease, generating transient singlestrand breaks (nicks) that could result in a less repairable and more hazardous double-strand break if two opposing nicks are formed (25).…”

Megaloblastosis: From Morphos to Molecules

“…A portion of the aspirated bone marrow was collected aseptically in cold heparinized 0.06 M Tris-buffered Hank's balanced salt solution, pH 7.4, and processed into a monolayered suspension for short-term culture; DNA synthesis was monitored by measuring incorporation of 3 H-thymidine ( 3 H-TdR) into DNA, and dU suppression test was done as described by us previously [16,17] . The dU suppression values were calculated as follows: H-TdR incorporation into cellular DNA is a sensitive radionucleoside marker of DNA synthesis.…”