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Differential regulation of cyclin-dependent kinase inhibitors in monolayer and spheroid cultures of tumorigenic and nontumorigenic fibroblasts.

The Ras proto-oncogene has been implicated in the in vivo development of tumors and in the in vitro transformation of cultured cell lines. In both of these conditions, Ras-mediated disruption of cell cycle-regulatory mechanisms leads to unregulated cellular proliferation, although the exact mechanisms by which Ras accomplishes this are not clear. Using as a model the M1 and MR1 rat fibroblast cell lines, which differ in the expression of a regulated Ras (M1 cells) versus a constitutively active Ras (MR1 cells), we examined the role of Ras in the control of cellular proliferation in two-dimensional (monolayer) and three-dimensional (spheroid) cell cultures. These cell lines are very similar in their monolayer growth characteristics, but M1 cells will arrest their cell cycle progression in aggregate culture, whereas MR1 cells proliferate normally as small spheroids. We report here that G1-phase arrest in plateau-phase monolayer cultures of both M1 and MR1 cells correlates with up-regulated expression of the cyclin-dependent kinase (CDK) inhibitor p18INK4c. Enhanced p18INK4c expression was also observed in G1-arrested M1 cells cultured as multicellular spheroids but was not induced in small proliferating MR1 multicellular spheroids. The kinetics of G1 arrest in M1 cells after inoculation into aggregate culture correlated well with the induction of p18INK4c expression. Conversely, resumption of proliferation in monolayer culture of arrested M1 cells isolated from spheroids coincided with the loss of expression of p18INK4c. After extended culture, cells in the inner region of MR1 spheroids arrested in the G1 phase without any up-regulation of p18INK4c expression. In this case, the CDK inhibitor p21(Cip1/Waf1) was selectively induced in the inner regions of large MR1 spheroids, concomitant with a decrease in cyclin and CDK expression. Thus, Ras-dependent regulation of p18INK4c expression seems to control the ability of rat embryo fibroblasts to proliferate as small multicellular aggregates, whereas p21(Cip1/Waf1) expression seems to regulate the G1-phase arrest induced by the stressful microenvironment found within the inner region of large spheroids.