FIG. 2.

Effects of several cell cycle inhibitors on NIH 3T3 cellular transformation by Ras. (A) Photograph of foci obtained after cotransfection of NIH 3T3 cells with pMZNT-17 (expressing oncogenic N-ras N61) and the empty vector pCR3.1 (left) or pMZNT-17 and pHM414 (expressing p15^INK4b) (right). (B) Suppression activity of p15^INK4b and other cell cycle inhibitors (p16^INK4a and p27^Kip1) on focus formation by Ras (left panel) or Rgr (right panel) in NIH 3T3 cells. Cells were cotransfected with pMZNT-17 (Ras) or pNM11 (Rgr) and the expression plasmid for p15^INK4b, p16^INK4a, or p27^Kip1. The empty vector or the vector containing p15^INK4b in the opposite orientation (pHM411) was used as a control. Results are represented as percentages of the number of foci obtained with Ras or Rgr and the empty vector (taken as 100%). The mean and standard error were calculated from three independent experiments. (C) Effect of p15^INK4b expression on anchorage-independent growth of Ras-transformed NIH 3T3 cells. Transformed cells expressing or not expressing p15^INK4b were seeded in agar-containing medium and grown for 2 weeks. The presence of this cell cycle inhibitor was accompanied by a decrease in the number and size of colonies in two separate experiments. A sample of the colonies obtained in cells expressing Ras or Ras plus p15^INK4b is shown. (D) Focus formation by oncogenic Ras in NIH 3T3 cells containing a p15^INK4b genomic fragment. The same number of NIH 3T3, NIH 3T3/CRpmp15, or NIH 3T3/MM134 cells was transfected with pMZNT-17, and the number of foci was scored after 15 days. Results are presented as percentages of the number of foci obtained with Ras in NIH 3T3 cells (taken as 100%). The mean and standard error were calculated from three independent experiments.