CATALYTIC CLEAVAGE OF THE ANDROGEN RECEPTOR MESSENGER-RNA AND FUNCTIONAL INHIBITION OF ANDROGEN RECEPTOR ACTIVITY BY A HAMMERHEAD RIBOZYME

Androgen receptor (AR) plays a key role in cell growth both in the nor mal prostate and in prostate cancer. Androgen ablation and prolonged a ntiandrogen therapy can give rise to AR-dependent prostate tumors, whi ch nonetheless can grow in the androgen-deprived milieu. Here we descr ibe the ribozyme approach to selectively degrading the AR mRNA and the reby inhibiting AR function. A trans-acting hammerhead ribozyme was de signed to cleave the rat AR mRNA at the position +1827/1828, a region predicted to be minimally involved in generating stable secondary stru ctures. Using AR mRNA fragments as substrates, it was established that this ribozyme can specifically cleave the RNA target in a sequence-sp ecific manner. Kinetic experiments determined a K-m for the substrate of 77 nM and a k(cat)/K-m value of 1.8 x 10(7) M-1.min(-1), suggesting a catalytic efficiency similar to that of protein enzymes such as the relatively nonspecific ribonuclease A and a sequence-specific endonuc lease EcoRI. Transient cotransfections of prostate-derived PC3 cells w ith three plasmids, an AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR expression vector, and a ribozyme expression vec tor, showed that the ribozyme was capable of reducing the functional a ctivity of AR. At an equimolar ratio of the AR expression plasmid to r ibozyme expression plasmid, androgen-inducible CAT activity was inhibi ted 70%. Similar extents of inhibition were also observed at the cellu lar mRNA level using ribonuclease protection assays, indicating that t he ribozyme functioned as an AR mRNA cleaving enzyme in cellule. Immun ocytochemical examination revealed a decline of AR immunoreactivity in ribozyme-transfected cells. In addition, no morphologically detectabl e cellular abnormalities were associated with ribozyme expression, ind icating the absence of deleterious side effects. These results offer a new avenue for the control of AR function and cell growth, especially in the case of androgen-resistant, but AR-dependent, prostate cancer cells.