ABSTRACT
BACKGROUND: In this study, defined culture conditions were used to examine the effects of recombinant TGFbeta1 on prostatic stromal cells and to determine the role of endogenous TGFbeta produced by these cells. METHODS: Cells were grown +/- recombinant TGFbeta1 and cell population sizes in replicate cultures determined. In other experiments, TGFbeta1 production by prostatic stromal cells was examined and the effects of neutralization of this activity on cell population sizes and apoptosis evaluated. RESULTS: At > 1 ng/ml, TGFbeta1 reduced cell population sizes while at 0.01 ng/ml cell numbers were increased cf. controls. Stromal cells produced up to 10 ng/ml/48 hr of latent TGFbeta1 of which < 0.2% was biologically active. When cells were treated with anti-TGFbeta1 antibodies, cell numbers decreased cf. controls and the proportion of apoptotic cells increased. CONCLUSIONS: These observations suggest that TGFbeta1 is an autocrine factor made by prostatic stromal cells in which it inhibits apoptosis at the activity levels produced.
Subject(s)
Prostate/physiology , Transforming Growth Factor beta/physiology , Antibodies/pharmacology , Apoptosis/drug effects , Culture Media, Serum-Free , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Male , Prostate/drug effects , Prostate/metabolism , Prostatic Hyperplasia/pathology , Recombinant Proteins/pharmacology , Stromal Cells/cytology , Stromal Cells/drug effects , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1ABSTRACT
BACKGROUND: Epithelial cells are critically dependent upon cell-matrix and cell-cell adhesion for growth and survival. Anoikis is programmed cell death caused by disruption of cell-substrate adhesion in normal epithelial cells. METHODS: We studied the induction of anoikis in vitro in two cell lines; HaCaT and SW742. PI3K, JAK2 and PKC are key elements in signalling pathways regulating cell survival, and using specific inhibitors we also examined their potential role in the induction of anoikis. RESULTS: When prevented from adhesion by culture on polyHEMA, HaCaT cells underwent apoptosis selectively from the proliferating population; surviving cells underwent cell cycle arrest. In SW742 cells anoikis also occurred, but was balanced by increased cycling. The effects of specific kinase inhibitors indicated that both Janus kinase 2 and protein kinase C partially protect HaCaT cells from anoikis through inducing cell cycle arrest of surviving nonadherent cells; inhibition of Phosphatidylinositol 3-kinase did not induce cycling in HaCaTs prevented from adhesion but did stimulate anoikis. SW742 cells showed markedly different responses: Janus kinase 2 inhibition activated apoptosis directly, Phosphatidylinositol 3-kinase inhibition stimulated both cell cycling and apoptosis, while protein kinase C inhibition stimulated cycling but inhibited apoptosis. CONCLUSIONS: Susceptibility to cell death in adhesion-prevented epithelial cells may thus be regulated by signalling pathways involving Phosphatidylinositol 3-kinase, Janus kinase 2 and protein kinase C. The ability of epithelial tumour cells to invade and metastasize may therefore result from disruption of these pathways.
Subject(s)
Anoikis/physiology , Carcinoma/metabolism , Colonic Neoplasms/metabolism , Epithelial Cells/metabolism , Keratinocytes/metabolism , Proto-Oncogene Proteins , Apoptosis , Bromodeoxyuridine/metabolism , Bromodeoxyuridine/pharmacokinetics , Carcinoma/drug therapy , Carcinoma/pathology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Cycle/drug effects , Cell Line , Cell Survival/drug effects , Cell Survival/physiology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Epithelial Cells/cytology , Epithelial Cells/drug effects , Humans , Janus Kinase 2 , Keratinocytes/cytology , Keratinocytes/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Polyhydroxyethyl Methacrylate/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
BACKGROUND: The majority of the overgrowth in benign prostatic hyperplasia (BPH) specimens is comprised of connective tissue. Factors that control stromal growth in the prostate are poorly understood; however, members of the transforming growth factor beta (TGFbeta) family may be of particular importance in the etiology of BPH. METHODS: Thirty-two low-passage stromal cultures were generated from human prostatectomy specimens. Their stromal origin was confirmed and expression of TGFbetas analyzed by duplex reverse transcription-polymerase chain reaction (RT-PCR). Challenge experiments were designed to study the effects of exogenous TGFbeta1 on stromal cell growth and synthesis of extracellular matrix components. RESULTS: The expression of TGFbetas 1, 2, and 3 was demonstrated in all 32 cell strains. The stromal origin of the cell lines was confirmed. Exogenous TGFbeta1 added to stromal cultures resulted in inhibition of cell growth and increased production of type I collagen. CONCLUSIONS: The prostatic stromal cell strains we have developed are a reliable mod- el for investigating prostatic connective tissue biology. The challenge experiments with TGFbeta1 provide further evidence for the involvement of TGFbetas in prostatic enlargement, as modulators of the extracellular matrix in the absence of growth stimulation.
