ISOLATION AND ANALYSIS OF MELANOMA CELL MUTANTS RESISTANT TO THE ANTI-PROLIFERATIVE ACTION OF RETINOIC ACID

  • 1 January 1983
    • journal article
    • research article
    • Vol. 43  (6) , 2868-2875
Abstract
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 were derived from the S91-C-2 cells by exposing them to the mutagen ethyl methanesulfonate and plating in soft agarose in the presence of 1 .mu.M .beta.-all-trans-retinoic acid. Under such conditions, the nonmutagenized S91-C-2 cells failed to grow; however, 2 .times. 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 mo., in the absence of retinoic acid, were characterized further. They were 3- to > 1000-fold and 100- to > 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. 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.