Agarose‐selected variants of two human tumor cell lines exhibit altered methionine auxotrophy

Abstract

Our aim was to determine if the selection of human tumor cells with enhanced anchorage-independent growth capacity was associated with alterations in methionine auxotrophy. Cells with an increased ability to form colonies on soft agarose were selected from human melanoma (MeWo) and neuroepithelioma (SK-N-MC) cell lines. In contrast to their respective parental lines, a high proportion of the agarose-selected variants were completely unable to proliferate in methionine-free medium containing its immediate precursor homocysteine. The variants exhibited no significant change in their total DNA 5-methylcytosine content and showed no stimulation of either RNA or DNA synthesis upon the addition of homocysteine when the cells were cultured in methionine-free medium. These variants were unable to synthesize [3H]S-adenosylmethionine from [3H]adenine and homocysteine. The failure to detect the accumulation of [3H]S-adenosylmethionine in these variant lines was not likely due to the enhanced turnover of S-adenosylmethionine but rather to a reduced ability to synthesize methionine from homocysteine and 5-methyltetrahydrofolic acid. These results support our hypothesis that alterations in the metabolism of methionine and/or intracellular transmethylating activities may contribute to, or be associated with, the autonomous growth of malignant human tumor cells.

Changes in cobalamin metabolism are associated with the altered methionine auxotrophy of highly growth autonomous human melanoma cells

“…Methionine synthase activity in melanoma variants Previous results from our laboratory revealed that highly metastatic or anchorage-independent variants of the MeWo human melanoma tumor line were unable to proliferate in methionine-free Hcy-supplemented culture medium, in contrast to the parental MeWo line, which was able to grow under such conditions (Liteplo, 1989;Liteplo and Hipwell, 1989). Since the inability of these HcynRsP variants to proliferate or survive under such conditions appeared to be due to a defect in the in vivo synthesis of methionine from Hcy and 5-methyltetrahydrofolic acid, we examined in vitro methionine synthase activity in cell extracts prepared from MeWo, MeWo-LC1, and D3.12-2 cells.…”

“…The ability of cells to proliferate in either Met+-or Hcy +-medium was routinely determined by measuring the incorporation of [meth~l-~HIthymidine into cellular DNA of cells grown for 5 days in the appropriate medium (Liteplo and Hipwell, 1989;Liteplo, 1989). Similar results were obtained when the cells were counted (Liteplo, 1989).…”

“…We have also found that variants of the MeWo tumor line selected for enhanced anchorage-independent growth also exhibited the HcynRSP phenotype (Liteplo and Hipwell, 1989). Conversely, HcyRsP-revertants obtained from the HcynRsP (MeWo variant) MeWo-LCl cell line were markedly less able to form colonies in soft agarose, compared with MeWo-LC1 cells themselves (Liteplo, 1990).…”

“…Consequently, Liteplo et al (Liteplo and Hipwell, 1989;Liteplo, 1990) adopted the terms "homocysteine-responsive" (HcyR"P) and homocysteine-nonresponsive (HcynR"p) to describe cells which either can or cannot proliferate in culture medium containing Hcy and 5-methyltetrahydrofolic acid in place of methionine.…”

“…1)) the terms "methionine-independent" and "methionine-dependent" (Hoffman, 1985) do not adequately describe the relative ability of cells to salvage methionine from 0 1991 WILEY-LISS, INC. either one of these two pathways. Consequently, Liteplo et al (Liteplo and Hipwell, 1989;Liteplo, 1990) adopted the terms "homocysteine-responsive" (HcyR"P) and homocysteine-nonresponsive (HcynR"p) to describe cells which either can or cannot proliferate in culture medium containing Hcy and 5-methyltetrahydrofolic acid in place of methionine.…”