Human Prostate Stromal Cell Line Research Articles

CHARACTERISTICS OF A HUMAN PROSTATE STROMAL CELL LINE RELATED TO ITS USE IN A STROMAL–EPITHELIAL COCULTURE MODEL FOR THE STUDY OF CANCER CHEMOPREVENTION

An immortalized human prostate stromal cell line (PS30) was previously established using recombinant retrovirus encoding human papillomavirus 16 gene products. In this study, we further characterize this stromal cell line for its potential use in a stromal-epithelial coculture model for prostate cancer prevention. Using reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunocytochemistry, we examined expression of androgen receptor (AR), vitamin D receptor (VDR), prostate-specific antigen (PSA), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGF) families and their receptors, metalloproteinases (MMP) MMP-2 and MMP-9, as well as the cells' ability to respond to the synthetic androgen R1881. The PS30 stromal cells do not express PSA, confirming their stromal origin. They are positive for both AR messenger ribonucleic acid (mRNA) and protein; however, they do not respond to growth stimulation by the synthetic androgen R1881. The PS30 cells express mRNA for VDR, TGF-betas, IGFs and their receptors, as well as the MMPs. Moreover, they produce significant amounts of TGF-beta1, TGF-beta2, IGFBP-3, and MMP-2 proteins. Our observations confirm the use of PS30 for the study of stromal-epithelial interactions in the modulation of prostate carcinogenesis.

Two for the price of one?

British Journal of Pharmacology (2005) 144, 1–2. doi:10.1038/sj.bjp.0706038

Oxytocin, oxytocin-associated neurophysin and the oxytocin receptor in the human prostate.

Oxytocin has been implicated in the regulation of prostate growth. However, the cellular localisation of oxytocin in the normal and diseased human prostate is not known. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were detected by immunohistochemistry in tissues from patients undergoing routine prostatectomy and in normal human prostate epithelial and stromal cell lines. Western blot analysis detected a single band at 14 kDa with neurophysin antiserum and a 66-kDa band with oxytocin receptor antiserum in epithelial and stromal cell lines. Similar sized bands were also detected in extracts of hyperplastic and adenocarcinomic prostate tissues. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were present in stromal and epithelial cell lines and in tissue from patients with benign prostatic hyperplasia. The peptides were localised predominantly to the epithelial cells, although discrete areas of stromal staining were also observed. There was a significant difference in the intensity of oxytocin-staining between tissue displaying benign prostatic hyperplasia and invasive carcinoma, with less immunoreactivity being present in the malignant epithelial cells. Thus, oxytocin and its neurophysin and receptor are present in epithelial and stromal cells of the human prostate. Oxytocin expression is reduced with tumour progression and may provide a marker for invasive disease.

Neuroepithelial interactions in prostate cancer are enhanced in the presence ofprostatic stroma

Objectives To develop an in vitro model that tests the involvement of prostatic stroma in the active reciprocal interactions between malignant epithelial cells and nerves that occur in perineural invasion. Methods Each of three metastatic prostate cancer cell lines (LnCaP, PC3, and DU-145 at 10 3) was co-cultured in sextuplet experiments with a human prostate stromal cell line (HTS-40C at 10 3) and a mouse dorsal root ganglion in matrigel for 13 days. Carcinoma/ganglia co-cultures (10 6 cells) in the absence of stroma served as controls. Areas of carcinoma cell growth (day 1), neurite growth (days 1 and 3), and perineural invasion (neuroepithelial halo area, day 11) were quantified. Results Mean neurite outgrowth was enhanced in the presence of stroma with LnCaP and PC3, but not with DU-145. Perineural invasion and carcinoma cell growth were enhanced in the presence of stroma in experiments with all three cell lines. The mean cell area (in square millimeters) increased 54.7% with LnCaP in the presence of stroma ( P <0.001). PC3 and DU-145 growth was enhanced 88.5% and 43.4%, respectively, in the presence of stroma. The mean neurite growth (in millimeters) on days 1 and 3 increased 50.8% and 70.8% with LnCaP in the presence of stroma. This enhancement was observed with PC3 by 88.1% and 64.5%. The mean neurite growth decreased in the presence of stroma with DU-145 by 4.9% and 5.4%. Perineural invasion increased 33.8% in the presence of stroma with LnCaP and 24.3% and 26.1% with PC3 and DU-145, respectively. Conclusions These novel findings strongly suggest active stromal participation in perineural invasion. The identification of specific stromal factors may suggest ways of preventing the progression of prostate cancer.

IMMORTALIZATION OF A HUMAN PROSTATE STROMAL CELL LINE USING A RECOMBINANT RETROVIRAL APPROACH

We established an immortalized human prostate stromal cell line with retained markers of cell differentiation and alpha1-adrenergic receptor expression. Primary human prostate stromal explants were infected with an amphotrophic retrovirus encoding the E6/E7 open reading frame of the human papillomavirus type 16. Immunohistochemistry was used to verify the expression of prostate stromal markers. alpha1-Adrenergic receptor expression was investigated using ribonuclease protection assays and radioligand binding. Cell proliferation was measured by the WST-1 assay and cell counting. Clonal isolates of individual prostate stromal cells were isolated and passed in selection media. E6 and E7 expression was verified using reverse transcriptase polymerase chain reaction in the selected cell line. The new prostate stromal cell line PS30 was established which maintains the expression of alpha-smooth muscle actin and expresses 22 fmol./mg. of protein of alpha 1-adrenergic receptors, approximately equal to native human prostate alpha 1-adrenergic receptor expression. However, at a subtype level alpha 1a-adrenergic receptor expression is down-regulated and not detectable by ribonuclease protection assays or radioligand binding, while alpha 1b and alpha 1d-adrenergic receptor expression is enhanced. From a physiological prospective PS30 cells do not form tumors in nude mice and stimulation with phenylephrine does not increase cell proliferation. We successfully established and characterized an in vitro human prostate stromal cell line. This cell line should facilitate studies designed to characterize the role of the adrenergic nervous system in the regulation of prostate growth.