SPECIFICITY OF GLUCURONOSYLTRANSFERASE ACTIVITY IN THE HUMAN CANCER CELL-LINE LNCAP, EVIDENCE FOR THE PRESENCE OF AT LEAST 2 GLUCURONOSYLTRANSFERASE ENZYMES

Recent findings obtained by our group showed that incubation of LNCaP cells with labeled steroids leads to the formation of 3- and 17-hydrox ysteroid glucuronides. In this study, the specificity and the kinetic properties of 3-hydroxy-C(19)steroid uridine diphospho-glucuronosyltra nsferase (3-OH-UGT) and 17-hydroxy-C-19 steroid UGT (17-OH-UGT) activi ties in LNCaP cells were investigated. Results indicate that the UGT h as a high affinity for testosterone, dihydrotestosterone (DHT), andros terone (ADT) and androstane-3 alpha,17 beta-diol (3 alpha-DIOL), with K-m values ranging from 0.25 to 0.68 mu M. The K-m values are approx. 10-fold higher for androst-5-ene-3 beta,17 beta-diol (5-ene-DIOL) and androstane-3 beta,17 beta-diol (3 beta-DIOL). The relative specificiti es (V-max/K-m) also showed higher turnover rates for testosterone, DHT , ADT and 3 alpha-DIOL with values ranging from 2.93 to 5.71, than for 3 beta-DIOL and 5-ene-DIOL with ratios of 0.41 and 1.10, respectively . Dixon plot and Cornish-Bowden analysis demonstrate that testosterone , DHT, ADT, and 3 alpha-DIOL inhibit the glucuronidation of DHT and AD T in a competitive fashion. In contrast, when the studies are performe d with 3 beta-diol and 5-ene-DIOL the inhibition of ADT glucuronidatio n is uncompetitive while the glucuronidation of DHT is inhibited compe titively, suggesting the presence of two UGT enzymes, one for glucuron idation of the 17 beta-OH group and a second for the 3 alpha-OH group. Further evidence for the presence of two UGTs in LNCaP cells was obta ined by incubation with a variety of 3 beta-OH-C-19 steroids which cau sed a marked inhibition of DHT-G formation but had no effect on the gl ucuronidation of ADT. In summary, our data demonstrate the presence of at least two UGTs in the human prostate cancer cell line LNCaP. The r elative specificity of the 17-OH-UGT in LNCaP cells is 3 alpha-DIOL > DHT > testosterone, while ADT is glucuronidated by the 3-OH-UGT.