Pamela S. Adams

Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate

SummaryThe proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin andeither EGFor prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF, purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their homologues).

Heparin-binding growth factor/prostatropin attenuates inhibition of rat prostate tumor epithelial cell growth by transforming growth factor type beta

SummaryNormal rat prostate epithelial cell growth requires both epidermal growth factor and heparin-binding growth factor/prostatropin. In contrast, epithelial cells derived from the transplantable Dunning R3327H rat tumor require either epidermal growth factor or heparin-binding growth factor/prostatropin. Transforming growth factor type beta inhibited normal epithelial cell growth. Transforming growth factor beta inhibited epidermal growth factor-dependent growth of tumor epithelial cells, independent of epidermal growth factor concentrations. Transforming growth factor beta increased the effective dose of heparin-binding growth factor type 1 required to support tumor epithelial cell growth by 10-fold but saturating levels of heparin-binding growth factor type 1 (290 pM) completely attenuated the inhibitory effect of transforming growth factor beta. These results suggest that prostate tumor epithelial cells may escape the inhibitory effect of transforming growth factor beta as a consequence of alteration of the concurrent requirement for both epidermal growth factor (or homologues) and heparin-binding growth factors.

Production and significance of TGF-β in AT-3 metastatic cell line established from the Dunning rat prostatic adenocarcinoma

A colony formation assay using NRK-49F cells revealed that a metastatic cell line, AT-3, established from the Dunning prostatic carcinoma could produce TGF-beta in a latent form. TGF-beta at a concentration as low as 0.05 ng/ml either stimulated the attachment or detachment of AT-3 cells depending on the kind of culture media. Acid extracts from conditioned medium (5 micrograms/ml) showed the activity comparable to that of TGF-beta (5 ng/ml). The detached cells were able to grow in suspension. TGF-beta (0.1 ng/ml) could also stimulate the growth of MC3T3-El osteoblasts established from mouse calvaria. These results suggest that TGF-beta is a key growth factor for osteoblastic bony metastasis of prostate cancer.

Modified nutrient medium MCDB 151, defined growth factors, cholera toxin, pituitary factors, and horse serum support epithelial cell and suppress fibroblast proliferation in primary cultures of rat ventral prostate cells

nuclei to the culture, which may obscure metaphase spreads and possibly may interfere with the mitotic stimulation. In addition this separates leukocytes from any lipid component in the blood that may be significant, particularly in situations where control over feeding may not be possible (e.g., wild raptor speciesk The amount of blood required is similar to that used by Zartman (4L being much less than earlier methods (2,3}. Finally, the culture conditions more closely approximate the average physiological state of birds than any previously reported method. In our limited experience of material from birds of 10 different orders, the use of phytohemagglutinin is far superior to other mitotic stimulants. Pokeweed mitogen, as suggested by Zartman (4), seems to be less suitable for most avian species, with the possible exception of the Falconiformes ( I lL Inasmuch as leukocyte cultures are perhaps the easiest and most convenient method of obtaining chromosome preparations, the method reported here should facilitate cytogenetic investigations of avian species, particularly at the population level. In addition, it may find application in the genetic sexing of monotypic birds for captive propagation programs.

Potential Role of HBGF (FGF) and TGF-Beta on Prostate Growth

We review in this paper the role of heparin-binding growth factor (HBGF*) or fibroblast growth factor (FGF*), rat prostate cancer cells produce TGF-beta, IGF-II* and OGF*. Of these growth factors, TGF-beta and unknown labile factor with 19 kDa are the most probable candidates responsible for osteoblastic bony metastasis of prostate cancer. In vitro experiments suggest that TGF-beta modulates cell detachment of prostate cancer cells together with nutritional factors. HBGF-dependent growth of the prostate tumor epithelial cells is free from inhibition by TGF-beta, whereas normal prostate epithelial cells are sensitive to TGF-beta inhibition. Transfection experiments suggest that HBGF-2 (basic FGF) might be closely related to the malignant growth of prostate cancer, in addition to tumor angiogenesis.

The androgen-dependent rat prostate protein, probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-1

SummaryRat prostate extracts contain an abundant 20–22 kilodalton heparin-binding protein with near identical chromatographic properties, but only 0.2–1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor). Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val-tyr-leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro) and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein “probasin”.

Heparin-binding fibroblast growth factors and prostate cancer.

Studies of model rat prostate tissue and derived cells indicate the insulin-like (IGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-beta) and heparin-binding fibroblast growth factor (HBGF) families and their receptors may play important roles in regulation of normal prostate cell growth. Tumor cells at different levels in the progression from slow-growing, hormone-dependence to fast-growing, hormone-independence exhibit distinct alterations in expression of specific growth factors and their receptor phenotype. Distinct IGF-I and HBGF mRNAs are constitutively expressed in the mesenchymal cells of slow-tumors, but alteration in HBGF receptor phenotype occurs in the epithelial cells. Fast-tumors exhibit even higher constitutive expression of multiple HBGFs. Splice variants in cDNA for the HBGF receptor in fast-tumors suggest constitutive expression of an intracellular receptor, that together with intracellular HBGFs, may constitute an intracellular autocrine system that is independent of exogenous hormones and growth factors.

Heparin-Binding (Fibroblast) Growth Factor/Receptor Gene Expression in the Prostate

Heparin-binding (fibroblast) growth factors (HBGF) play both autocrine and paracrine roles in growth of normal and tumor prostate epithelial and mesenchymal cells. In the rat, expression of HBGF-1 dominates in normal tissue and slow-growing Dunning prostate tumor tissues. Both HBGF-1 and HBGF-2 are expressed in fast-growing, malignant variants of the Dunning tumor. Expression of HBGF-1 occurs in young normal prostate epithelial cells and disappears with age. Slow-growing Dunning tumor mesenchymal cells, not epithelial cells, constitutively express HBGF-1. HBGF requirement and receptor phenotype is altered in tumor epithelial cells. In contrast to IGF-1 and the Elg/Bek/Cek gene product, a candidate HBGF receptor, neither HBGF-1 nor HBGF-2 expression in normal and tumor tissues increases during androgenstimulated growth. Flg/Bek/Cek is expressed in young prostates, during androgen-stimulated growth of adult prostate, constitutively in tumors, and appears limited to the stromal cell fraction of the slow-growing tumors. Expression is elevated in the fast-growing, highly malignant tumors. Normal and slow-growing tumor epithelial cells have receptor sites for HBGF that may differ from Flg/Bek/Cek. Similar to rat prostate tissue, HBGF-1 expression dominates in young human prostate tissue. However, both HBGF-1 and HBGF-2 mRNA as well as the Flg/Bek/Cek gene are detectable in adult human prostate tissue. These differences in rat and human tissue may reflect differences in the epithelial:stromal cell ratio in prostates of the two species.

Assay of growth factors for prostate epithelial cells

The maintenance and proliferation of isolated prostate epithelial cells is androgen independent, but requires multiple other hormones and hormonelike growth factors. Methods are described for isolation and characterization of epithelial cells from normal rat prostate and the androgen-responsive transplantable Dunning R3327 rat tumor. Pure tumor-derived cell lines can be established by serial culture techniques. The normal primary and serially cultured cell lines are then used to assay growth factors. Cell proliferation is quantitated by computerized videometry.