Phenotypic selection as the biological mode of epigenetic conversion and reversion in cell transformation

Abstract

Exposure of certain cell lines to methylcholanthrene, X-rays, or physiological growth constraint leads to preneoplastic transformation in all or most of the treated cells. After attaining confluence, a fraction in those cells progress to full transformation, as evidenced by their ability to form discrete foci distinguishable from the surrounding cells by virtue of their higher density. Transformation induced by suspension in agar, an even stronger growth-selective condition than confluence, is reminiscent of all but the final differentiated stage of a normal developmental process, epithelial-mesenchymal transition. Changes associated with transformation are not restricted to focus-forming cells, as the permissiveness for focus formation provided by confluent cells surrounding transformed foci is greater than that of nonselected cells. The neoplastic process can also be reversed in culture. Transformed cells passaged at low density in high serum revert to normal morphology and growth behavior in vitro and lose the capacity for tumor formation in vivo. We propose that transformation and its reversal are driven by a process of phenotypic selection that involves entire heterogeneous populations of cells responding to microenvironmental changes. Because of the involvement of whole cell populations, we view this process as fundamentally adaptive and epigenetic in nature.

Keywords: epigenetics; malignant transformation; phenotypic selection; saturation density.

Fig. 1.

Cellular microenvironment of transformed foci. A culture seeded with 10⁵ cells that had undergone three rounds (3°) of selection at high density (Left) was compared with one that had been diluted 10-fold, leaving 10⁴ cells combined with 10⁵ nontransformed cells (Right). The cultures were assayed in 2% CS and then were fixed and stained at 14 d. Reproduced from ref. .

Fig. 2.

Conversion of NIH 3T3 cells by growth at low cell density in 2% CS and reversion of the cells in 10% CS. Cells produced transformed foci by repeated passage at low density in 2% CS. Transformed cells were isolated from a transformed focus and passaged three times per week at low cell density in 2% CS (○, upper curve). At the indicated passages 100 transformed cells were mixed with 10⁵ nontransformed cells to assay for focus formation in 2% CS. Transformed cells were also passaged in 10% CS and assayed in 2% CS (●, lower curve) (26).

Fig. 3.

Predicted changes in percentage of focus-forming (FF) cells as a function of serum concentration and time for mixtures of FF and nonfocus-forming (NFF) cells. Five hundred FF cells and 5 × 10⁴ NFF cells were mixed together on day 0. One mixture was maintained in 2% CS, another in 10% CS. Based on separate growth rates of FF and NFF cells in 2 and 10% CS the expected number of FF cells, relative to NFF cells, was predicted and is graphed in this figure. The relative number of FF cells (500 cells out of 50,500 total cells) on day 0 is plotted as 1 on the ordinate. The upper line shows the anticipated increase in FF cells, relative to NFF cells, for mixtures maintained in 2% CS. The lower line shows the anticipated decline in FF cells for mixtures maintained in 10% CS. Reproduced from ref. , with permission from Oxford University Press.

Fig. 4.

Reduction in size and density of foci in cultures shifted from 2% CS to 10% CS at 5 wk and retained there for 3 wk. (A) Culture maintained in 10% CS for 8 wk. (B) Culture maintained in 2% CS for 8 wk. (C) Culture maintained in 2% CS for 5 wk and then switched to 10% CS for 3 wk (29).

Fig. 5.

Deadaptation in 10% CS of cells previously adapted to growth in 0.25% CS. Cells adapted to growth in 0.25% CS were deadapted by 0 (+), 2 (◯), 4 (●), 7 (▲), and 10 (∆) serial passages in 10% CS, followed by their growth in 0.25% CS. The latter were also measured in cells that had been maintained exclusively in 10% CS (□) (30).