Loss of the Oncogene From Human H-ras-1-Transfected NIH/3T3 Cells Grown in the Presence of Excess Methionine2

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Abstract
Mouse NIH/3T3 cells, transfected with T24 genomic DNA (J10 cells), carry the human H-ras-1 (hu-H-ras-1) oncogene stably integrated in the chromosomal DNA and display the transformed phenotype with a “criss-cross” morphology and capacity for anchorage-independent growth. When these cells were cultured in the presence of 25 mMdl-methionine, they lost their transformed phenotype; the characters of the normal phenotype (before transfection) reappeared, even though cell viability, as measured by the cloning potential on a solid support, was fully conserved. Southern blot analysis showed that cells continuously propagated in methionine-supplemented medium (J10met), unlike cells grown in normal medium, progressively segregated the hu-H-ras-1 gene. The progressively fewer cells still retaining the oncogene could be selected by inoculation of J10met cells into nude (nu/nu) mice of Swiss background. After prolonged methionine treatment, however, no such oncogene-positive cells could be detected, even when the excess methionine was withdrawn from the culture medium after 105 days. Conclusive evidence for the loss of the hu-H-ras-1 oncogene sequence was provided by subcloning the J10met cells. The oncogene sequence could no longer be found, and a fraction of subclones was no longer tumorigenic when assayed in nude mice. Other subclones generated late tumors negative for the hu-H-ras-1 transforming sequence, and a small fraction of subclones gave rise to rapidly growing early tumors that were also negative for the transforming sequence. The question of how methionine can induce the phenotypic and genetic change from malignancy to normality is discussed.