Growth support and toxicity of homocysteine and its effects on methionine metabolism in non-transformed and chemically transformed C3H/10T1/2 cells

Abstract

The effects of homocysteine (Hcy) on one non-transformed (Cl 8) and two malignant clones (Cl 16 and Cl T422) of the C3H/10T1/2 mouse embryo fibroblasts, were examined with regard to toxicity, ability to support growth and effects on methionine (Met) metabolism and glutathione level. Homocysteine in its reduced form (Hcy-SH) was toxic to all cell lines, and the LD90 was estimated to be 1.0 X 10(-4) M for Cl 8 and Cl 16 cells measured by plating efficiency, 0.8 X 10(-4) M for Cl 8 and 0.3 X 10(-4) M for Cl 16 when measured by total cell growth. At toxic concentrations, Hcy-SH showed a drastic effect on cell morphology both in the presence and absence of Met. The same effect was demonstrated with L-cysteine. No toxic effect was seen with homocystine (Hcy-SS-Hcy) or homocysteine thiolactone (Hcy-tl) at similar concentrations. Hcy-tl supported growth of both the non-transformed and malignant cells in Met-deficient medium but with decreasing efficiency in the order Cl 8, Cl 16 and Cl T422. The growth rate constant compared to that of Met-supplemented medium was 0.62 for Cl 8, 0.44 for Cl 16 and 0.38 for Cl T422 cells. The intracellular level of S-adenosylhomocysteine (AdoHcy) increased in all three cell lines in Hcy-tl-supplemented medium. The S-adenosylmethionine (AdoMet) content increased in Cl 8 cells, was constant in Cl 16 cells and decreased in Cl T422 cells under the same conditions. This resulted in a constant ratio of AdoMet/AdoHcy in the non-transformed cells (Cl 8) whereas this ratio decreased by 40% in Cl 16 and by 72% in Cl T422 cells when Hcy-tl replaced Met in the medium. The ability of Hcy-tl to support growth thus seemed to correlate well with alteration in Met metabolism in this cell culture system. The intracellular level of glutathione (GSH) was measured during exponential growth, but showed small variations between non-transformed cells and Cl 16 cells. However, Cl T422 cells showed a distinct lower level of GSH in Met-supplemented medium, and this increased 3- to 4-fold when Met was replaced with Hcy-tl.

Modulation of glutathione content and the effect on methionine auxotrophy and cellular distribution of homocysteine and cysteine in mouse cell lines

“…Stock cultures of non-transformed C3H/10T1/2 Cl 8 (27) and chemkally transformed C3H/10T1/2 Cl T422 (28) mouse embryo fibroblasts were obtained from the laboratory of Dr J.R.Lillehaug, Department of Biochemistry, University of Bergen, and maintained as described previously (9).…”

“…revealed a more complex nature of this phenomenon since some normal cells have been shown to require Met for growth, and different malignant cells have been shown to be Met independent (3)(4)(5)(6)(7)(8)(9). These results indicate that even if Met dependence is a result of an oncogenic transformation process, it may not be an obligatory event for the neoplastic stage.…”

“…We have previously shown that the chemical form in which Hey is administered to the cells is crucial. The reduced form of Hey and of other thiols, including cysteine, is rather toxic to the cells (9,(12)(13)(14), whereas the thiolactone form of homocysteine (Hcy-tl) is non-toxic at the concentration used in the growth experiments (9). Our recent report concerning co-culture of Metindependent fibroblasts with Met-dependent leukemia cells in Met"Hey + medium indicated that inability to utilize Hcy-tl for growth was not due to production and release of toxic factor(s) to the growth medium from the Met-dependent cells (15).…”

“…In a previous report we demonstrated that a transformed cell line with reduced capacity to utilize Hcy-tl for growth showed altered glutathione (GSH) content when transferred to a medium containing Hcy-tl instead of Met (9). Glutathione is the most abundant non-protein thiol in die cells and is involved in cellular detoxication processes (18,19).…”

“…On the other hand, glutathione may be of vital importance in controlling and regulating the level of thiols like Hey and maintaining the balance between the reduced and oxidized forms. Altered GSH metabolism, as in the partly Met-dependent malignant mouse fibroblasts (9), may affect the availability of reduced Hey for Met synthesis.…”