Cyanocobalamin [c-lactam] Inhibits Vitamin B12 and Causes Cytotoxicity in HL60 Cells: Methionine Protects Cells Completely by J.H. Matthews Blood Volume 89(12):4600-4607 June 15, 1997 ©1997 by American Society of Hematology

The cytotoxic effect of cyanocobalamin [c-lactam] on HL60 cells The cytotoxic effect of cyanocobalamin [c-lactam] on HL60 cells. “Hcy” medium was RPMI 1640 without methionine and without folic acid, containing L-homocysteine thiolactone at 200 μmol/L, 5-methyltetrahydrofolate at 200 nmol/L, vitamin B12 at 3.7 nmol/L, 5%... The cytotoxic effect of cyanocobalamin [c-lactam] on HL60 cells. “Hcy” medium was RPMI 1640 without methionine and without folic acid, containing L-homocysteine thiolactone at 200 μmol/L, 5-methyltetrahydrofolate at 200 nmol/L, vitamin B12 at 3.7 nmol/L, 5% dialyzed human serum, and 5% dialyzed fetal bovine serum. Cyanocobalamin [c-lactam] and additional vitamin B12 were used at 7.4 μmol/L. Cyanocobalamin [c-lactam] caused growth arrest followed by cell death, and this effect was reversed by additional concentrations of vitamin B12 . In nondialyzed serum, cell death did not occur (data not shown). High concentrations of 5-methyltetrahydrofolate (up to 20 μmol/L), did not protect the cells against the inhibitor (data not shown). •, “Hcy” medium alone; ○, “Hcy” plus c-lactam; ▪, “Hcy” plus additional vitamin B12 ; □, “Hcy” plus c-lactam and additional vitamin B12 . J.H. Matthews Blood 1997;89:4600-4607 ©1997 by American Society of Hematology

Methionine protects against the cytotoxic effect of cyanocobalamin [c-lactam] on HL60 cells. “Met” medium was RPMI 1640 without L-homocysteine thiolactone and without folic acid containing methionine at 100 μmol/L, 5-methyltetrahydrofolate at 200 nmol/L, vi... Methionine protects against the cytotoxic effect of cyanocobalamin [c-lactam] on HL60 cells. “Met” medium was RPMI 1640 without L-homocysteine thiolactone and without folic acid containing methionine at 100 μmol/L, 5-methyltetrahydrofolate at 200 nmol/L, vitamin B12 at 3.7 nmol/L, 5% dialyzed human serum, and 5% dialyzed fetal bovine serum. Cyanocobalamin [c-lactam] (7.4 μmol/L) and additional concentrations of vitamin B12 (7.4 μmol/L) had no effect on the doubling time. •, “Met” medium alone; ○, “Met” plus c-lactam; ▪, “Met” plus additional vitamin B12 ; □, “Met” plus c-lactam and additional vitamin B12 . J.H. Matthews Blood 1997;89:4600-4607 ©1997 by American Society of Hematology

Ascorbic acid does not cause cyanocobalamin [c-lactam] to develop a cytotoxic effect on HL60 cells grown in methionine. “Met” medium was RPMI 1640 without L-homocysteine thiolactone and without folic acid, containing methionine at 100 μmol/L, 5-methyltetrah... Ascorbic acid does not cause cyanocobalamin [c-lactam] to develop a cytotoxic effect on HL60 cells grown in methionine. “Met” medium was RPMI 1640 without L-homocysteine thiolactone and without folic acid, containing methionine at 100 μmol/L, 5-methyltetrahydrofolate at 200 nmol/L, vitamin B12 at 3.7 nmol/L, 5% dialyzed human serum, and 5% dialyzed fetal bovine serum. Ascorbic acid was used at 284 μmol/L, and cyanocobalamin [c-lactam] at 7.4 μmol/L. Cyanocobalamin [c-lactam] does not prolong the doubling time in the presence of ascorbic acid, which stabilizes 5-methyltetrahydrofolate. This suggests that spontaneous oxidation of 5-methyltetrahydrofolate to a form that can bypass methionine synthase and enter cellular folate pools directly when the functions of vitamin B12 are inhibited is not the reason why cells continue to proliferate in the presence of cyanocobalamin [c-lactam] and methionine. •, “Met” medium plus ascorbate; ○, “Met” plus ascorbate and c-lactam; ▪, “Met” plus ascorbate and additional vitamin B12 ; □, “Met” plus c-lactam and additional vitamin B12 . J.H. Matthews Blood 1997;89:4600-4607 ©1997 by American Society of Hematology

Pteroylglutamic (folic) acid fails to protect HL60 cells against cyanocobalamin [c-lactam]. “PGA-Hcy” medium was RPMI 1640 without methionine and without folic acid, containing L-homocysteine thiolactone at 200 μmol/L, pteroylglutamic acid at 200 nmol/L, vi... Pteroylglutamic (folic) acid fails to protect HL60 cells against cyanocobalamin [c-lactam]. “PGA-Hcy” medium was RPMI 1640 without methionine and without folic acid, containing L-homocysteine thiolactone at 200 μmol/L, pteroylglutamic acid at 200 nmol/L, vitamin B12 at 3.7 nmol/L, and dialyzed serum. Cyanocobalamin [c-lactam] at 7.4 μmol/L caused growth arrest followed by cell death, and this effect was reversed by additional concentrations of vitamin B12 (7.4 μmol/L). High concentrations of pteroylglutamic acid (up to 20 μmol/L) did not reverse the cytotoxicity (data not shown). Pteroylglutamic acid, a nonmethylated folate, fails to prevent the cell death caused by cyanocobalamin [c-lactam]. Methyl group trapping is, therefore, not the cause of the cytotoxic effect of the inhibitor. •, “PGA-Hcy” medium alone; ○, “PGA-Hcy” plus c-lactam; ▪, “PGA-Hcy” plus additional vitamin B12 ; □, “PGA-Hcy” plus c-lactam and additional vitamin B12 . J.H. Matthews Blood 1997;89:4600-4607 ©1997 by American Society of Hematology

The effects of various concentrations of methionine on the growth inhibitory effects of cyanocobalamin [c-lactam] on HL60 cells. “Hcy” culture medium was RPMI 1640 without methionine and without folic acid, containing L-homocysteine thiolactone at 200 μmol/... The effects of various concentrations of methionine on the growth inhibitory effects of cyanocobalamin [c-lactam] on HL60 cells. “Hcy” culture medium was RPMI 1640 without methionine and without folic acid, containing L-homocysteine thiolactone at 200 μmol/L, 5-methyltetrahydrofolate at 200 nmol/L, vitamin B12 at 3.7 nmol/L, 5% dialyzed human serum and 5% dialyzed fetal bovine serum, to which was added methionine to give the concentrations shown. Cyanocobalamin [c-lactam] was used at 7.4 μmol/L. HL60 cells were grown for 4 weeks under these conditions, and cell counts were performed every 48 hours. The doubling time was calculated from the slope of the regression line between the log of the cell count and the duration of culture. The proliferation rate was calculated as the number of doubling times per 24 hours. Because small concentrations of methionine, equivalent to those present in nondialyzed serum, protect the cells against the inhibitor, its cytotoxicity is caused by methionine deficiency. J.H. Matthews Blood 1997;89:4600-4607 ©1997 by American Society of Hematology

The effect of cyanocobalamin [c-lactam] on the incorporation of 5-[14CH3]-tetrahydrofolate into TCA-precipitable material by intact HL60 cells. “Hcy” medium was RPMI 1640 without methionine and without folic acid containing L-homocysteine thiolactone at 200... The effect of cyanocobalamin [c-lactam] on the incorporation of 5-[14CH3]-tetrahydrofolate into TCA-precipitable material by intact HL60 cells. “Hcy” medium was RPMI 1640 without methionine and without folic acid containing L-homocysteine thiolactone at 200 μmol/L, 5-methyltetrahydrofolate at 200 nmol/L, vitamin B12 at 3.7 nmol/L, 5% nondialyzed human serum, and 5% nondialyzed fetal bovine serum. Cyanocobalamin [c-lactam] and additional vitamin B12 were used at 7.4 μmol/L. “Met” medium was the same, except that methionine at 100 μmol/L was used instead of L-homocysteine thiolactone. Nondialyzed serum was used to maintain the viability of cells grown with the inhibitor. The cells were cultured in the specified medium for several weeks until the time of assay, when they were washed and processed in the assay medium as described in the text. Results represent the mean from five experiments. (a) “Hcy” medium alone, (b) “Hcy”+c-lactam, (c) “Hcy”+additional vitamin B12, (d) “Hcy”+c-lactam+additional vitamin B12 , (e) “Met” medium alone, (f ) “Met”+c-lactam, (g) “Met”+additional vitamin B12 , (h) “Met”+c-lactam+additional vitamin B12 . P < .05 (t-test) for (a) versus (b), (b) versus (d), (e) versus (f ), (f ) versus (h), and (a) versus (e). ▪, nonmethionine-suppressible uptake of label; T, 1 SEM: n = 5. J.H. Matthews Blood 1997;89:4600-4607 ©1997 by American Society of Hematology

The effect of cyanocobalamin [c-lactam] on the incorporation of [1-14C] propionic acid into TCA-precipitable material by intact HL60 cells. “Hcy” medium was RPMI 1640 without methionine and without folic acid, containing L-homocysteine thiolactone at 200 μm... The effect of cyanocobalamin [c-lactam] on the incorporation of [1-14C] propionic acid into TCA-precipitable material by intact HL60 cells. “Hcy” medium was RPMI 1640 without methionine and without folic acid, containing L-homocysteine thiolactone at 200 μmol/L, 5-methyltetrahydrofolate at 200 nmol/L, vitamin B12 at 3.7 nmol/L, 5% non-dialyzed human serum, and 5% nondialyzed fetal bovine serum. Cyanocobalamin [c-lactam] and additional vitamin B12 were used at 7.4 μmol/L. “Met” medium was the same, except that methionine at 100 μmol/L was used instead of L-homocysteine thiolactone. The medium was supplemented with nondialyzed serum to maintain the viability of cells grown with the inhibitor. The cells were grown for several weeks and then assayed in their culture medium. The results are the mean from three experiments. (a) “Hcy” medium alone, (b) “Hcy” + c-lactam, (c) “Hcy” + additional vitamin B12, (d) “Hcy” + c-lactam + additional vitamin B12 , (e) “Met” medium alone, (f ) “Met” + c-lactam, (g) “Met” + additional vitamin B12, (h) “Met” + c-lactam + additional vitamin B12 . P < .05 (Wilcoxon) for (a) versus (b) and (b) versus (d) only. J.H. Matthews Blood 1997;89:4600-4607 ©1997 by American Society of Hematology

The methylfolate trap hypothesis. The methylfolate trap hypothesis. Reaction (a) is held to be irreversible under physiological conditions so that reaction (b) is the only metabolic escape for methylfolate. When reaction (b) is inhibited by either a deficiency of vitamin B12 or by cyanocobalamin [c-lactam], folate should be trapped as methylfolate. S-adenosyl methionine has a negative effect on reaction (a) so that inhibition of reaction (b) should effectively increase the flow of 5,10-methylene tetrahydrofolate towards methylfolate. CH3-THF, 5-methyl tetrahydrofolate; 5,10-CH2-THF, 5,10-methylene tetrahydrofolate; THF, tetrahdrofolate. Reaction (a), methylene tetrahydrofolate reductase. Reaction (b), methionine synthase. J.H. Matthews Blood 1997;89:4600-4607 ©1997 by American Society of Hematology