In tissue cultures of normal adult and malignant mammalian cells, homocystine has been substituted for methionine in a medium rich in folic acid and cyanocobalamin. Normal adult cells thrive. Three highly malignant cell types from three different species, including man, die.A metabolic defect seen to date only in cancer cells was uncovered during an investigation of transfer RNA methylation using tissue cultures of normal adult and malignant cells (1,2). In several lines of malignant cells we have observed an inability of the cells to survive and grow when methionine is replaced in the medium with homocystine. However, normal adult cell lines derived from mice, rats, and humans survive and grow well with this substitution. The apparent absolute dependence of the malignant cells on preformed methionine is of considerable theoretical interest but may also be of practical importance because it may offer another avenue for treating neoplastic disease.An earlier study (3), confirmed in our laboratory, has clearly indicated that dietary depletion of methionine has an inhibitory effect on the growth of Walker-256 carcinosarcoma. We became interested in this problem through our studies on methylation of transfer RNA in Walker-256 cells, and the present work demonstrates the dependence of this and other malignant cell lines on preformed methionine. In contrast to the malignant cell lines, we show that a number of normal adult cell lines can satisfy their methionine requirements with homocystine in a folic acid-, cyanocobalamin-rich medium. METHODSTissue cultures of Walker-256 (W-256), breast carcinosarcoma of rats; L1210, lymphatic leukemia of mice; J111, monocytic leukemia of humans; as well as liver epithelial and liver fibroblasts of rats, skin fibroblasts of mice, and fibroblasts from human breast and prostate (all concurrently PPLO negative) were grown in a commercially available, specifically modified McCoy's medium. The composition of the medium was routinely checked by amino-acid analysis before use. It was devoid of bactopeptone but reinforced with 15% fetal-calf serum that had been dialyzed for 24 hr at 40 against three changes of a solution containing 8 g of NaCl, 0.4 g of KCl, 1 g of glucose, and 0.35 g of NHCO3 per liter. In addition, the medium contained trace metals (10 ml/liter of medium of a solution containing FeSO4, 1 mg; CuCl2, 100 ,pg; ZnSO4, 100 jAg; MnCl2, 100 pg/liter) vitamin B12, 2-4 mg/liter; folic acid, 5-10 mg/liter; and either gentamycin (50 p&g/mi) or streptomycin sulfate (100 ug/ml) and penicillin 1133 G 100 U/ml. Except for subtractions or additions of methionine and homocystine, there were no other alterations in the medium. Two growth curves of each cell type were run simultaneously. One medium contained L-methionine in the usual concentration (15 mg/liter), while the other medium was devoid of methionine, but instead contained either L-homocystine (15 mg/liter) or DL-homocystine (30 mg/liter). Various amounts of vitamin B12, folic acid, homocystine, homocysteine, or the methyl ...

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In studies of the phosphorylated proteins in rat liver and Walker-256, it was established that the ratio of various fractions of P-N linkages to P-O linkages varies from 0.6 to 3.1. In rat regenerating liver nuclei, the ratio of P-N and P-O varies with time after partial hepatectomy. Using [3H]-lysine and 32Pi, it is shown that phosphoryllysine forms in some new and, presumably, some preexisting H1 molecules. Using [3H]histidine and 32Pi, it is shown that phosphohistidine forms exclusively in preexisting H4. The half-life of H4 phosphohistidine appears to be about 2 h.

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