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

Abstract

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, 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 characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC-E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry 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 demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.

Isolation and partial characterization of an epithelial cell line (RPE‐F344) from the regenerating prostate of a normal adult male rat

“…19 The goal of the present study was to isolate normal rat prostatic epithelial cells that could be propagated in vitro without the introduction of immortalizing genes, to serve as a normal counterpart to transformed rat prostate cell lines, such as the HUNC-E derived from the Dunning rat prostate cancer model. 20 The RPE-F344 cells were isolated from involuted prostatic remnants four days after administration of exogenous testosterone; a time when the purported stem-like cells are proliferative and a signi®-cant number of transitional cells are present. 27 While ®ve epithelial-like colonies were selected from the original culture, only one colony gave rise to an easily propagable cell line that retained epithelial morphology and characteristics.…”

“…Four days after administration of testosterone rats were euthanized and the prostate remnants excised, pooled, digested enzy-matically with 0.5 mg/ml collagenase (Worthington Biochemical Corporation, Lakewood, NJ), and placed in culture in media supplemented as described previously. 20 Prostate growth media (PGM) consists of Richter's Improved MEM (Irvine Scienti®c, Santa Ana, CA) supplemented with 10 mM nicotinamide, 20 ng/ml epi-dermal growth factor (EGF), 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ ml selenium, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml fungizone and 2% fetal bovin serum or serum replacement (CPSR-2, Sigma, St Louis, MO). In some experiments a basal media was used (BPM), which is PGM without EGF, serum, or phenol red (0.1% bovine serum albumin (BSA) was in-cluded in BPM to maintain cell attachment).…”

“…20 Brie¯y, cells were cultured on chamber slides, ®xed in 95% ethanol, and subjected to immunocytochemical staining based on antibody manufacturer's protocols. Primary antibodies included anti-AR, anti-Bcl-2, anti-EGFR, anti-c-met, and anti-c-kit (all from Santa Cruz, Santa Cruz, CA), anti-pancytokeratins (Sigma, St Louis, MO), anti-keratin 903 (Enzo Diagnostic, Farmingdale, NY), anti-prostatic acid phosphatase (DAKO, Carpinteria, CA), and anti-p27kip1 (Transduction Laboratories, Lexington, KY).…”

“…22 Reverse transcription and PCR were carried out as described previously. 20 PCR reactions were conducted in EasyStart 1 PCR reaction tubes (Molecular Bioproducts, San Diego, CA) using primers speci®c for AR, PAP, TGFbs and TGFb receptors, IGFRs, EGFR, and c-met. Actin was ampli®ed from each sample to ensure quality of the reverse transcription and even loading between samples.…”

“…This is in sharp contrast to a transformed rat prostate epithelial cell line (HUNC-E) isolated in our laboratory, which is characterized by autocrine production of several growth factors concomitant with expression of cognate receptors. 20 Androgen response of RPE-F344 cells RPE-F344 cells were evaluated for androgen sensitivity by evaluation of growth kinetics in BPM or PGM media with or without 15 nM DHT supplementation. The stimulatory Isolation and partial characterization of RPE-F344 SC Presnell et al effects of BPM androgen were evident at day 5, when the RPE-F344 cultures were expanding rapidly and had not reached con¯uence ( Figure 3A).…”