The Effect of Replacement of Methionine by Homocystine on Survival of Malignant and Normal Adult Mammalian Cells in Culture

Abstract

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 ...

Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference

“…It has been suggested that growth arrest by methionine restriction may be related to the role of methionine as a methyl donor for methylation of DNA, RNA and proteins (Lu and Epner, 2000). Interestingly, normal cells are able to grow in culture when methionine is substituted with homocysteine because mammalian cells can convert homocysteine to methionine (Stern and Hoffman, 1986), but most cancer cells fail to grow in the absence of methionine, even when supplemented with homocysteine (Halpern et al, 1974;Mecham et al, 1983;Stern and Hoffman, 1986), which may result from the elevated levels of transmethylation in cancer cells as compared with normal cells (Tisdale, 1980;Stern and Hoffman, 1984;Judde et al, 1989). It was recently reported that telomeric recombination was elevated by a decrease in methylation of sub-telomeric regions in mouse embryonic stem (ES) cells deficient for DNA Figure 5 Correlation among TRF1, TRF2, TIN2 and RAP1 foci in IIICF/c cells.…”

Downregulation of Cdc6 and pre-replication complexes in response to methionine stress in breast cancer cells

“…By contrast, availability of homocysteine does not rescue cancer cells from their methionine dependence. [5][6][7][8] Importantly, remethylation of homocysteine to generate methionine appears to be unaffected in cancer cells. 9 Cancer cell methionine dependency is well-documented.…”

“…When the same cells are cultured in methionine stress conditions, methionine-free media supplemented with homocysteine (Met-Hcy+), after 1 wk only normal cells exist in the culture dish. 6 In vitro studies of prostate cancer cells indicate that they suffer a specific cell cycle arrest and eventually undergo apoptosis when cultured in Met-Hcy+ media. 10 Consistent with a specific effect of methionine restriction non-transformed cells but leads to cell proliferation block in many cancer cells.…”

“…10 Consistent with a specific effect of methionine restriction non-transformed cells but leads to cell proliferation block in many cancer cells. 6,7,10,11 Relatively little was known about methionine dependency of breast cancer cells. We therefore analyzed growth of various breast cancer cell lines in growth medium with (Met+) or without (Met-Hcy+) methionine (Fig.…”

Investigations of tissue folates in normal and malignant tissues

“…The Walker 256 carcinosarcoma in cell culture shows methionine auxotrophy (Halpern et al, 1974) which cannot be explained by the lack of methionine synthetase (Hoffman & Erbe, 1976). In these experiments no 5-CH3THF derivatives were found in Walker 256 carcinosarcoma extracts except shortly after administration of FA.…”