IN-VITRO REVERSION OF TRANSFORMED PHENOTYPE IN MOUSE C3H 10T1/2 CELLS- MODIFICATION IN GENOMIC 5-METHYLCYTOSINE CONTENT/

Spontaneous tumor regression is still one of the most puzzling events in human cancer. A cell culture model of malignant transformation desi gned to permit the study of this phenomenon in vitro was applied to ex amine reversion and re-expression of the transformed phenotype in two X-ray transformed mouse 10T1/2 cell clones. By alternating cell passag es at low and high seeding density, the expression of cell contact inh ibition and tumorigenic capacity were both reverted and restored. Grow th of non-transformed wild-type cells was not affected by seeding dens ity. This reversion of the transformed phenotype was associated with a modification in genomic 5-methylcytosine content. Initially, the tran sformed clones were hypomethylated, as occurs in most human tumors. Af ter only four passages at low seeding density, the phenotype was rever ted to that of non-transformed 10T1/2 cells and genomic 5-methylcytosi ne content was significantly increased to levels measured in non-trans formed C3H/10T1/2 mouse cells. Thus, hypomethylation induced by ionizi ng radiation was not a permanent feature of malignantly transformed 10 T1/2 cells. Although genomic 5-methylcytosine content returned to norm al levels during low density passaging, the methylation pattern of the c-myc gene specifically was not associated with cell passages either at low or high seeding density. In an attempt to identify genes involv ed in this process, expression of the tumor suppressor gene p53 was me asured. Western blot analysis failed to detect any correlation between expression of p53 protein and reversion of the transformed phenotype. The results of this study indicate that the transformed phenotype is not permanently associated with the malignant transformation of C3H/1O T1/2 cells, and can be modulated by growth conditions in vitro. We pro pose that modulation of genomic 5-methylcytosine levels may be involve d in this process.