Differential growth factor responses of epithelial cell cultures derived from normal human prostate, benign prostatic hyperplasia, and primary prostate carcinoma

Abstract

Because of a lack of information of the optimum nutritional requirements, epithelial cells derived from normal human prostate and prostate tumors have been difficult to propagate in vitro, which hinders research in prostate carcinogenesis. In an effort to establish optimum nutritional conditions and differences in growth characteristics of normal human prostate (NP), benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCA), we have compared the effects of several growth factors on cell proliferation and elucidated growth properties of low passage epithelial cells derived from NP, BPH, and PCA of an African-American patient. Primary and low passage cultures were propagated in serum-free keratinocyte basal medium (KBM) supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 microgram/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (BPE; 50 micrograms/ml), cholera toxin (10 ng/ml), and antibiotics. Almost all NP, BPH, and PCA cells were positive for cytokeratins and prostate-specific antigen (PSA). The NP, BPH, and PCA cells were essentially diploid and lacked mutations in c-K-ras and c-Ha-ras oncogenes, and p53 tumor suppressor gene. However, they exhibited progressively accelerating growth parameters. The population doubling times of NP, BPH and PCA were 51 hr, 37 hr, and 29 hr, respectively; their saturation densities were 2.9 x 10(4)/cm2, 3.3 x 10(4)/cm2, and 7.2 x 10(4)/cm2, respectively. The NP and BPH cells required all of the growth factors in the medium, as deletion of any one of the above factors strongly inhibited their growth. The PCA cells, however, were independent of EGF and hydrocortisone. PC-3, an established human prostate cancer cell line, was independent of the growth factors tested. Fetal bovine serum (FBS) inhibited the growth of NP, BPH and PCA cells. In contrast, FBS stimulated the growth of the PC-3 cells in a concentration-dependent manner. These results indicate that in the absence of any apparent karyotype alterations and mutations in c-K-ras, c-Ha-ras and p53 genes, epithelial cells derived from NP, BPH, and PCA exhibit significant differences in their growth properties and responses to growth factors. These variations may represent early changes involved in prostate cancer, while gene mutations and cytogenetic alterations occur in advanced and/or metastatic tumors.

Role of Notch-1 and E-Cadherin in the Differential Response to Calcium in Culturing Normal versus Malignant Prostate Cells

“…This raises the issue of when using surgical material whether what grows out in lowCa 2+ -SFD medium are truly cancer cells as opposed to highly proliferative transit-amplifying cells derived from basal epithelium of normal glands contaminating the starting cancerous tissue. This latter possibility is supported by the demonstration that (a) such low-Ca 2+ -SFD medium cultures usually are not capable of being propagated beyond 10 passages although they are derived from cancerous tissue (25), (b) they usually lack molecular and karyotypic changes (e.g., 8p…”

“…Based on this success, many groups are using variations of lowCa 2+ -SFD medium containing known growth factors [e.g., PrEBM complete medium or keratinocyte serum-free medium (K-SFM) complete medium from Invitrogen (Carlsbad, CA) in an attempt to grow sufficient numbers of prostate cancer cells from surgical material obtained at radical prostatectomy for molecular analysis and to establish new serially passageable prostate cancer cell lines (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Such a serum-free approach is favored because FCS contains a variety of undefined, and thus hard to standardize, factors in addition to its potent ability to stimulate growth to unwanted prostate stromal cells (i.e., fibroblasts and smooth muscle cells).…”

“…Such a serum-free approach is favored because FCS contains a variety of undefined, and thus hard to standardize, factors in addition to its potent ability to stimulate growth to unwanted prostate stromal cells (i.e., fibroblasts and smooth muscle cells). Using such a low-Ca 2+ -SFD medium approach, it has been claimed that pure populations of prostate cancer cells can be grown and propagated from starting prostate cancer tissue (25)(26)(27)(28). In contrast to these low-Ca 2+ -SFD medium approaches, all of the presently available, serially propagated human prostate cancer cell lines (i.e., DU-145, PC-3, LNCaP, C4-2B, LAPC-4, CWR22-Rv1, CWR-R1, MDA-PCA-2A, MDA-PCA-2B, DuCap, and VCap) were originally established with and are maintained in 10% FCS containing medium whose Ca 2+ is between 650 and 1,860 Amol/L (18).…”

Evidence that Sp1 positively and Sp3 negatively regulate and androgen does not directly regulate functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) gene expression in normal human prostate epithelial cells

“…NHP1-NHP6 cells were cultured in serum-and androgen-free, PrEBM medium (Clonetics) supplemented with insulin, EGF, hydrocortisone, bovine pituitary extract, and cholera toxin, and used at passages 3-7 (Chopra et al, 1996;Tang et al, 1998Tang et al, , 2002Bhatia et al, 2003). PCa cell lines, PPC-1, PC3, LNCaP, and Du145, were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics.…”

Arachidonate 15-Lipoxygenase 2 as an Endogenous Inhibitor of Prostate Cancer Development

“…NHPl, NHP3, NHP4, and NHP6 cells were obtained from Clonetics (Walkersville, MD), and NHP2 and NHP5 cells were generated as previously described (7)(8)(9). These cells were cultured in serum-free, PrEBM medium (Clonetics) …”