FIG. 8.

MEKK3 reverses cell-transforming activities of oncogenic Ras. NIH 3T3 [MEKK3-ER] cells were infected with a recombinant retrovirus expressing constitutively active RasQ61L and used as a pool of infected cells for all experiments. (A) Uninfected (left panels) or RasQ61L-infected (right panels) NIH 3T3 [MEKK3-ER] cells were seeded at low density in the absence of E2 (top panels) or when confluent in the presence of E2 (bottom panels). After 4 days when cells minus E2 had reached confluency as well, photographs were taken with Hoffmann optics, which gives a three-dimensional image of the cell surface. Ras-transformed cells show the characteristic rounded and spindle-shaped morphology (top right panel) which is largely reverted to a flat morphology in the presence of E2 (bottom panel right). (B) NIH 3T3 [MEKK3-ER] cells transformed with RasQ61L were tested for anchorage-independent growth in the presence (bottom panel) or absence (top panel) of E2, corresponding to active or inactive MEKK3-ER, respectively. Two weeks after seeding photographs were taken and the percentage of colonies consisting of at least five cells was determined in a total of 20 different fields from four different dishes and in two independent experiments, each with or without E2. (C) Untransformed (MEKK3) or RasQ61L-transformed (MEKK3 + ras) NIH 3T3 [MEKK3-ER] cells were starved in the absence of serum for 3.5 days with or without E2 and then either stimulated or not stimulated with 10% FCS for 15 h, with or without the continued presence E2, as indicated. Detergent lysates (100 μg) were separated by SDS-PAGE and immunoblotted with antibodies against cyclin D1 (cycD1), cyclin A, endogenous MEKK3, or the MEKK3-ER fusion protein, as indicated on the left. Ras-transformed cells express the G₁-phase cyclin D1 even in the absence of serum, which is suppressed in the presence of E2 and active MEKK3-ER. Serum stimulation further increases cyclin D1 expression, but only in the absence of E2.