FIG. 5.

Spontaneous acquisition of anchorage-independent proliferation is associated with frequent loss of p16^INK4A expression. (A) Colonies of cells expressing dnp53 and Ras that proliferated in soft agar were picked, dissociated, and expanded in culture. A total of 4 × 10³ cells were reseeded in agarose. Two weeks later the colonies were photographed. The parental polyclonal dnp53/Ras-expressing cells are shown adjacent to three clones. Assays were carried out in duplicate, and a representative of three independent assays is shown. (B) Immunoblots of lysates prepared from 10 dnp53/Ras-expressing clones that had acquired the ability to proliferate efficiently in soft agar. p16^INK4A mRNA levels were determined by semiquantitative RT-PCR.