ISOLATION AND CHARACTERIZATION OF PROPAGABLE CELL-LINES (HUNC) FROM THE ANDROGEN-SENSITIVE DUNNING R3327H RAT PROSTATIC ADENOCARCINOMAS

The Dunning H rat prostate tumor (R3327H) is a widely used experimenta l model of human prostatic adenocarcinoma (CaP), The Dunning H tumor h as been characterized as androgen-sensitive, androgen-receptor (AR) po sitive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the ch aracteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma th at maintained androgen sensitivity and expression of AR, PSA and PAP, Tissue harvested from an in vivo R3327H tumor was dissociated with col lagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC-E) and a vimentin-po sitive stromal cell line (HUNC-S) were generated from the primary cult ure, subcultured continuously for >300 days, and passaged >50 times. S urvival of the HUNC-E cell line in vitro depended on several media sup plements, including nicotinamide, insulin, transferrin, selenium and e pidermal growth factor (EGF), HUNC-E cells expressed AR and produced P SA and PAP throughout the culture period, as confirmed by immunocytoch emistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which dem onstrated the androgen-sensitive nature of the cells in vitro, When HU NC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subc utaneously into syngeneic male hosts, tumors formed in 2/3 animals wit h an average latency of 7 months. RT-PCR and immunocytochemical charac terization of the HUNG cell lines revealed that the cells expressed se veral growth factors and their cognate receptors, including HGF, TGF-a lpha and the TGF-beta s, indicating the establishment of potential aut ocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell l ines, which retain the characteristics of the epithelial and stromal c omponents of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcin oma.