ATYPICAL ANDROGEN RECEPTOR IN THE HUMAN-MELANOMA CELL-LINE IIB-MEL-J

To evaluate the presence of androgen receptors in the human melanoma c ell line IIB-MEL-J, a Scatchard plot analysis was performed. Cells in culture revealed a single binding component with an apparent dissociat ion constant (K-D) at 37 degrees C of 11 nM and a binding capacity of 326 fmol/mg protein when measured with [H-3]-R1881. Competition analys is revealed an atypical relaxation of specificity, since not only andr ogen (testosterone, dihydrotestosterone [DHT], R1881) and antiandrogen (hydroxy-flutamide [OH-FLU]) competed for [H-3]-R1881 binding, but al so estradiol, progesterone, and cortisol at 500-fold excess concentrat ion. Binding of [H-3]-estradiol and [H-3]-R5020 in the absence of unla beled DHT were completely suppressed in its presence. Immunohistochemi stry of androgen receptor with a monoclonal antibody showed that nucle i were vigorously stained. Different doses of flutamide (FLU) and OH-F LU tested on cultured IIB-MEL-J cells in the presence of serum inhibit ed significantly cell proliferation in a dose-dependent manner. When c ells were incubated with 10 nM DHT and 1% charcoal-adsorbed serum, a s ignificant stimulation of growth that was observed was inhibited by 4 mu M OH-FLU. DHT stimulation was completely reversed by the antiestrog en tamoxifen. In addition, male nude mice transplanted with IIB-MEL-J tumor were treated with FLU when tumors were palpable. FLU was effecti ve in diminishing tumor growth and increasing survival rate of the ani mals. As a conclusion, the presence of functional androgen receptors i n these cells has been demonstrated by growth inhibition in vitro and in vivo with antiandrogens, and their atypical nature is suggested by binding cross-reactivity and competition studies.