Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimentalmodel of human prostatic adenocarcinoma (CaP). The Dunning H tumor has beencharacterized as androgen-sensitive, androgen-receptor (AR) positive,prostate-specific antigen and prostatic acid phosphatase (PAP) positive. Todate, the tumor has been maintained by serial passage in vivo because ofthe lack of an in vitro cell line that retains the characteristics of thein vivo tumor. The objective of the present study was to establish apropagable cell line from R3327H adenocarcinoma that maintained androgensensitivity and expression of AR, PSA and PAP. Tissue harvested from an invivo R3327H tumor was dissociated with collagenase and placed intoRichter's improved media (with supplements). A cytokeratin-positiveepithelial cell line (HUNC- E) and a vimentin-positive stromal cell line(HUNC-S) were generated from the primary culture, subcultured continuouslyfor >300 days, and passaged >50 times. Survival of the HUNC-E cellline in vitro depended on several media supplements, includingnicotinamide, insulin, transferrin, selenium and epidermal growth factor(EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout theculture period, as confirmed by immunocytochemistry and Western blotanalyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT)to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independentgrowth and PSA production, which demonstrated the androgen-sensitive natureof the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1ratio and introduced subcutaneously into syngeneic male hosts, tumorsformed in 2/3 animals with an average latency of 7 months. RT-PCR andimmunocytochemical characterization of the HUNC cell lines revealed thatthe cells expressed several growth factors and their cognate receptors,including HGF, TGF-alpha and the TGF-betas, indicating the establishment ofpotential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-SCaP cell lines, which retain the characteristics of the epithelial andstromal components of the in vivo R3327H tumor, will allow a more thoroughand informative molecular and biological analysis of prostaticadenocarcinoma.