Isolation and analysis of melanoma cell mutants resistant to the antiproliferative action of retinoic acid.

Retinoic acid inhibits both the anchorage-dependent and the anchorage-independent growth of the murine melanoma S91-C-2 cells. To explore the mechanism of these effects, several mutant cell clones resistant to retinoic acid-induced growth inhibition have been derived from the S91-C-2 cells by exposing them to the mutagen ethyl methane sulfonate and plating in soft agarose in the presence of 1 microM beta-all-trans-retinoic acid. Under such conditions, the nonmutagenized S91-C-2 cells failed to grow; however, 2 X 10(-6) of the mutagenized cells did form colonies. These colonies were isolated, expanded in culture, and recloned in agarose containing retinoic acid. Five cell clones that retained their drug-resistant phenotype after repeated subculture for 3 months, in the absence of retinoic acid, were characterized further. They were found to be 3- to greater than 1000-fold and 100- to greater than 100-fold resistant to retinoic acid-induced inhibition of anchorage-independent and anchorage-dependent growth relative to the wild-type C-2 cells, respectively. The rate of uptake of [3H]-retinoic acid by the resistant cell clones was similar to that of the sensitive C-2 cells, indicating that resistance is not the result of reduced uptake. Analysis of cytoplasmic retinoic acid-binding protein revealed that it is present in the most resistant clones in amounts that are similar to or even greater than those found in the sensitive S91-C-2 cells. These results indicate that resistance is not the result of the absence of the binding protein. The retinoic acid-resistant mutants exhibited cross-resistance to related retinoids such as 13-cis-retinoic acid and all-trans-retinol as well as to the arotinoid p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl) propenyl]benzoic acid suggesting that they all share a similar mechanism of action. These resistant mutants may provide a useful system for further studies of the molecular processes through which retinoic acid exerts its antiproliferative effects.