Growth control of androgen-dependent Shionogi carcinoma cells by growth factor(s) produced from androgen-independent Shionogi carcinoma cells in a paracrine mechanism

Androgen-dependent (SC3) and -independent (CADO21) cloned cell lines were established from androgen-dependent mouse mammary tumor (Shionogi carcinoma 115). The effects of conditioned medium (CM) collected from SC3 and CADO21 cells on the anchorage-independent growth of SC3 cells in soft agar were studied. CM prepared from SC3 cells in the absence of testosterone was unable to stimulate the growth of SC3 cells, whereas CM prepared from SC3 cells in the presence of 10(-8) M testosterone stimulated the growth of SC3 cells in a concentration-dependent manner (21 colonies at 10% and 48 colonies at 20%) and this growth-stimulatory effect was not inhibited by 10(-6) M cyproterone acetate. CM prepared from CADO21 cells in the absence of testosterone was also able to stimulate the SC3 cell growth in a concentration-dependent manner (9 colonies at 10% and 19 colonies at 20%). These results suggest that the growth of androgen-dependent SC3 cells is stimulated by androgen-induced growth factor(s) produced from the same cells (autocrine mechanism) and is also regulated by autonomous growth factor(s) produced from androgen-independent cancer cells formed from the dependent cancer cells (paracrine mechanism). A suggested possible mechanism of the progression from androgen-dependent to -independent growth of cancer cells is also discussed.     

Regulation of prostate branching morphogenesis by activin A and follistatin

Ventral prostate development occurs by branching morphogenesis and is an androgen-dependent process modulated by growth factors. Many growth factors have been implicated in branching morphogenesis including activins (dimers of beta(A) and beta(B) subunits); activin A inhibited branching of lung and kidney in vitro. Our aim was to examine the role of activins on prostatic development in vitro and their localization in vivo. Organ culture of day 0 rat ventral prostates for 6 days with activin A (+/- testosterone) inhibited prostatic branching and growth without increasing apoptosis. The activin-binding protein follistatin increased branching in vitro in the absence (but not presence) of testosterone, suggesting endogenous activins may reduce prostatic branching morphogenesis. In vivo, inhibin alpha subunit was not expressed until puberty, therefore inhibins (dimers of alpha and beta subunits) are not involved in prostatic development. Activin beta(A) was immunolocalized to developing prostatic epithelium and mesenchymal aggregates at ductal tips. Activin beta(B) immunoreactivity was weak during development, but was upregulated in prostatic epithelium during puberty. Activin receptors were expressed throughout the prostatic epithelium. Follistatin mRNA and protein were expressed throughout the prostatic epithelium. The in vitro evidence that activin and follistatin have opposing effects on ductal branching suggests a role for activin as a negative regulator of prostatic ductal branching morphogenesis.     

Localization of inhibins and activins in normal endocrine cells and endocrine tumors of the gut and pancreas: an immunohistochemical and in situ hybridization study.

Activins and inhibins, which belong to the TGF beta family, are composed of different combinations of alpha-, betaA-, and betaB-subunits, resulting in inhibin A (alphabetaA), inhibin B (alphabetaB), activin A (betaAbetaA), activin B (betaBbetaB), and activin AB (betaAbetaB). They regulate several cell functions, acting as paracrine/autocrine factors. Their actions, which depend on binding to specific receptors, are also modulated by follistatin. Gastroenteropancreatic (GEP) endocrine cells and endocrine tumors (ETs) produce several growth factors, but it is not well known whether they express follistatin and the various inhibin/activin subunits. We studied their expression in 65 GEP ETs using immunohistochemistry (IHC) and in situ hybridization (ISH). The alpha-subunit and follistatin were not identified in normal GEP endocrine cells and were poorly expressed in ETs. A betaA-subunit immunoreactivity (IR) was detected in A-, G-, EC-, and GIP-cells, while betaB-chain IR was present only in D-cells. The mRNAs encoding for these molecules were poorly expressed in normal tissues. BetaA- and betaB-subunits were identified in several ETs by both IHC and ISH: betaA-subunit mainly in G-cell and A-cell ETs, and betaB-subunit in D-cell, A-cell, and EC-cell ETs. Our results demonstrate a differential expression of activin/inhibin subunits among different types of GEP endocrine cells and related tumors, suggesting a role in modulation of biological functions of these normal and neoplastic endocrine cells.     

Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG)

Activins are multifunctional growth factors belonging to the transforming growth factor-beta superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps. To purify recombinant activin A, we have developed a simple method using the second follistatin domain of an activin-binding protein follistatin-related gene (FLRG). An affinity column was prepared with a partial FLRG fusion protein. The partial FLRG protein contained the second follistatin domain and the C-terminus acidic domain, and was tagged with six histidine residues at its N-terminus. The fusion protein was expressed in Escherichia coli and purified with nickel affinity column. Thereafter, the purified fusion protein was coupled to NHS-activated column. Recombinant activin A was produced in Chinese hamster ovary (CHO) cells, which were stably transfected with rat inhibin/activin betaA-subunit cDNA. After 48-h suspension culture of the cells in a serum free medium, the culture media was recovered and passed through the FLRG-coupled column. After washing with phosphate-buffered saline, bound protein was eluted out with an acidic buffer. Any significant contaminations were not detected when the purified protein was analyzed by SDS-PAGE. Apparent sizes of the protein were 14 and 28 kDa under the reduced and non-reduced conditions, respectively. Western blot analysis confirmed that the purified protein was activin A. The purified recombinant activin stimulated p3TP-lux reporter activity in CHO cells and follicle-stimulating hormone secretion from rat pituitary cells.     

An activin/furin regulatory loop modulates the processing and secretion of inhibin alpha- and betaB-subunit dimers in pituitary gonadotrope cells

Of all ligands of the transforming growth factor beta superfamily, inhibins and activins are a physiologically relevant pair that are functional antagonists of each other. Activin stimulates whereas inhibin blocks follicle-stimulating hormone biosynthesis and secretion from pituitary gonadotrope cells, and together, inhibin and activin control the pituitary gonadal axis essential for normal reproductive function. Sharing a similar beta-subunit, the secretion of inhibin heterodimers (alpha/beta) or activin homodimers (beta/beta) as mature bioactive ligands depends, in part, on the proteolytic processing of precursor proteins. A short loop regulatory pathway controlling precursor processing and dimer secretion was discovered. Activin stimulates endogenous inhibin alpha- and betaB-subunit mRNA, protein, and proteolytic processing. Simultaneously, activin stimulated the proconvertase furin through a Smad2/3-dependent process. The data provide a mechanism where the regulation of furin and inhibin subunits cooperates in an important positive short feedback loop. This regulatory loop augments the secretion of bioactive mature activin B, as well as inhibin B dimers, necessary for local follicle-stimulating hormone beta regulation.     

Integration of proviral sequences, but not at the common integration sites of the FGF8 locus, in an androgen-dependent mouse mammary Shionogi carcinoma.

A retroviral insertional mutation, especially by mouse mammary tumor virus (MMTV), is a major cause of murine mammary tumorigenesis. Prompted by our previous finding that FGF8, an insertionally activated cellular oncogene, is highly expressed in androgen-dependent mouse mammary Shionogi carcinoma cells, we here investigated retroviral integration adjacent to the fgf8 locus in Shionogi carcinoma. In the genomic Southern blots for fgf8 and its 5'-upstream gene npm3, the hybridized fragments were identical to the host DD/Sio mice, the original Shionogi carcinoma 115 tumor, and a pair of cultured Shionogi carcinoma cell lines of SC-3 and SC-4, suggesting that no retroviral integration occurred around either loci. The genomic cloning for the fgf8 locus from SC-3 cells also confirmed no MMTV integration. In addition, npm3, which is usually coactivated with fgf8 by MMTV insertion,was not up-regulated by androgens in SC-3 cells. All these findings led us to conclude that no retroviral insertion was present at the common integration sites adjacent to the fgf8 locus in Shionogi carcinoma although we demonstrated in this study that multiple proviral sequences of MMTV, Moloney murine sarcoma virus and FBJ-murine sarcoma virus are integrated into SC-3 cells in association with their distinct promoter activity in SC-3 cells.     

Characterization of androgen uptake by purified nuclei from an androgen-dependent and two androgen-independent cell lines of the shionogi mouse mammary carcinoma

Properties of androgen uptake by nuclei prepared from an androgen-dependent and two androgenindependent tumour cell lines from the Shionogi mouse mammary carcinoma were characterized. It was found that:1. Maximal uptake of dihydrotestosterone (DHT) by the androgen-dependent tumour cell line occurred after 2 h incubation at 20°C; 2. Nuclei from three tumour cell lines displayed similar affinity for DHT but the two androgen-independent cell lines had less than one-quarter the number of uptake sites; 3. Loss of label from nuclei which had been pre-incubated with [³H]-DHT for 18 h at 20°C was greater from the AD cell line nuclei than from the androgen-independent cell lines; 4. Whole cell contamination of the nuclear preparations did not contribute to specific DHT uptake.     

Three cell lines showing androgen-dependent, -independent,and-suppressed phenotypes,established from a single tumor of androgen-dependent shionogi carcinoma 115

Summary We investigated the heterogeneity of cells in terms of androgen responsiveness within a single tumor mass of Shionogi carcinoma SC-115 showing androgen-dependent growth. After cloning of the tumor by the limiting dilution method in the presence of androgen, we isolated 40 clones at random. Twenty-two clones required androgen for growth (androgen-dependent phenotype), 16 did not (androgen-independent phenotype), and the remaining two clones showed growth inhibition when androgen was added (androgen-suppressed phenotype). In addition, 22 androgen-dependent clones showed heterogeneity in growth factor sensitivity in the absence of androgen. All clones were sensitive to both acidic and basic fibroblast growth factor (FGF), 7 of 22 clones were sensitive to epidermal growth factor (EGS) and transforming growth factor (TGF)-α, and 2 of 22 clones were sensitive to TGF-β. This preexisting heterogeneity may be partly responsible for the growth of androgen-dependent tumor under hormone-deprived circumstances. Three typical clones, SC2G, SC1G, and SC4A, were selected from androgen-dependent, -independent, and-suppressed phenotypic groups, respectively. These clones, as well as original solid tumors, were found to produce heparin-binding growth factors of heterogeneous elution positions. The molecular nature of these growth factors is not yet known. Neither anti-basic FGF antibody nor anti-EGF antibody inhibited the cell growth when added in cell culture, suggesting the factors were distinct from basic-FGF and EGF.     

Effects of intermittent androgen suppression on the stem cell composition and the expression of the TRPM-2 (clusterin) gene in the Shionogi carcinoma

The proportion of turnorigenic stem cells and the expression of the apoptosis-related gene, TRPM-2 (clusterin), were studied in populations of Shionogi carcinoma cells subjected to multiple cycles of androgen withdrawal and replacement (intermittent androgen suppression). The parent androgen-dependent cell line was initially transplanted into a male mouse which was castrated when the estimated weight of the resultant tumour became approximately 3 g. After the tumour had regressed to 40% or less of the original weight, it was transplanted into the next non-castrated male. This was repeated for four cycles of transplantation and castration-induced apoptosis before the tumour progressed to an androgen-independent state. The proportion of total stem cells in the tumour, as determined by in vivo limiting dilution assays in male mice, was constant during the first three cycles but increased 15-fold between the third and fourth cycles. In the parent androgen-dependent tumour before androgen ablation, the androgen-independent stem cell population formed 0.8% of the total stem cell compartment. After the fourth cycle this population increased to 47%; a population of similar size (33%, P = 0.8) was found in the androgen-independent recurrent form of the tumour induced by one-time castration. Whether androgen withdrawal therapy was intermittent or continuous, conversion to androgen independence thus occurred when one-third to one-half of the total stem cell compartment was populated by androgen-independent stem cells. The androgen-repressed TRPM-2 (clusterin) gene was actively expressed in regressing tumours after androgen ablation, and also became constitutively expressed in non-regressing tumours after the first and subsequent cycles of androgen withdrawal. Staining of cytoplasm and nuclei with anti-clusterin antibody was observed in androgen-dependent tumour cells after each cycle of intermittent androgen suppression; the nuclear staining was more intense in recurrent androgen-independent cells. The anomalous nuclear localization of clusterin, an anti-cytolytic TRPM-2 encoded protein, may serve to inhibit early events in the apoptotic process and thereby foster the generation and outgrowth of androgen-independent stem cells in an androgen-depleted environment.     

Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

Bcl-2 is an anti-apoptotic oncoprotein and its protein levels are inversely correlated with prognosis in many cancers.However, the role of Bcl-2 in the progression of prostate cancer is not clear. Here we report that Bcl-2 is required for theprogression of LNCaP prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. ThemRNA and protein levels of Bcl-2 are significantly increased in androgen-independent prostate cancer cells. shRNA-medi-ated gene silencing of Bcl-2 in androgen-independent prostate cancer cells promotes UV-induced apoptosis and suppressesthe growth of prostate tumors in vivo. Growing androgen-dependent cells under androgen-deprivation conditions resultsin formation of androgen-independent colonies; and the transition from androgen-dependent to androgen-independentgrowth is blocked by ectopic expression of the Bcl-2 antagonist Bax or Bcl-2 shRNA. Thus, our results demonstratethat Bcl-2 is not only critical for the survival of androgen-independent prostate cancer cells, but is also required for theprogression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage.     

The arrangement of nucleosomes in nucleoprotein complexes from polyoma virus and SV40.

Androgen-dependent and androgen-independent (autonomous), cloned, cultured cell lines of the androgen-dependent mouse mammary adenocarcinoma, Shionogi carcinoma 115, have been established. Growth of the dependent cells requires the presence of androgen, provided they are grown in suspension culture in medium containing dextran-charcoal-treated fetal calf serum. The growth rate of autonomous cells in the presence or absence of DHT is similar to that of dependent cells grown in its presence. An agar culture method has been developed that enables the proportion of dependent and autonomous cells in mixed populations to be determined. Autonomous cells appear in dependent clones, and their frequency increases with increasing time of subculture. Dependent cells from tumors preferentially in male animals, and dependent cell cytosols contain significant amounts (approximately 300 femtomoles per mg protein) of a specific androgen-binding macromolecule. Autonomous cells formed tumors equally well in both male and female mice, and autonomous cell cytosols contain very low levels (≤ 7 femtomoles per mg protein) of the specific androgen-binding macromolecule(s). These studies delineate a system which can be used to investigate the mechanism of steroid hormone-dependent and autonomous tumor growth, and the transitions between the hormone-dependent and autonomous states.     

Effects of androgen and antiandrogen on growth, morphology and synthesis of specific proteins in mouse mammary carcinoma cells

Primary cell cultures derived from an androgen-dependent mouse mammary carcinoma, the Shionogi SC-115 tumor, display characteristic changes in growth, morphology and protein synthesis according to the presence or absence of testosterone. In the presence of testosterone, cell proliferation was increased and cells formed characteristic clones having no contact inhibition. Ultrastructural studies of cells showed close contacts of plasma membranes having little or no gap between cells. Some cells were related by bridges of extracellular matrix. Testosterone-induced synthesis of several intracellular and secreted proteins was observed after [35S]methionine-labeling of cells, SDS-PAGE and autoradiography, as well as the disappearance of a protein in androgen-treated cells. In the absence of testosterone, cells grow as a monolayer, have contact inhibition and flattened morphology. The ultrastructurally observed cell-to-cell contacts were usually less intimate, showing spaces of irregular width between cells. None of the testosterone-induced proteins were observed in the absence of hormone. The antiandrogen cyproterone acetate, which by itself was inactive, completely suppressed the androgen-induced effects on growth, morphology and specific protein synthesis. Glycosylation of membrane proteins, as measured after labeling of cells with [3H]N-acetyl-D-glucosamine, was increased by approximately 30% in the presence of testosterone. A similar observation was made in situ by autoradiography on intact cells. Finally, we found that culture medium conditioned by testosterone-treated Shionogi cells had significant mitogenic activity on L-929 mouse fibroblasts.     

Activin A regulates growth of gastro‐intestinal epithelial cells by mediating epithelial‐mesenchymal interaction

The importance of epithelial–mesenchymal interaction on the development of gastro‐intestinal (GI) organs has been repeatedly reported, but its molecular mechanism has not been fully understood though several factors including hepatocyte growth factor and endothelin‐3 have been shown to mediate it. Activins have been demonstrated to play important roles in the regulation of organogenesis in vertebrates, but their roles in the regulation of growth and differentiation of GI organs remain to be solved. In the present study, we examined expression of activins in developing rat GI tract, and found that inhibin bA encoding activin A was specifically expressed by GI mesenchymes, while inhibin bB encoding activin B was expressed by both epithelial and mesenchymal components. We then examined the effect of activin A on the growth of fetal rat GI epithelial cells in primary culture. We found that activin A inhibited the growth of forestomach and glandular stomach epithelial cells while it stimulated the growth of colonic epithelial cells. These results suggest that activin A secreted from GI mesenchymes region‐specifically regulates the growth of attaching epithelial cells. We thus conclude that activin A mediates epithelial‐mesenchymal interaction in the developing GI tract.     

Androgen-dependent fibrinolytic activity in a murine mammary carcinoma (Shionogi SC-115 cells) in vitro.

Physiological concentrations of dihydrotestosterone (DHT) can specifically induce the release of fibrinolysin from androgen-dependent mammary carcinoma (Shionogi SC-115) cells in vitro. No fibrinolytic activity was observed in cells cultured either in the absence of DHT or presence of pharmacological concentrations of estrogen. Furthermore, an autonomous tumor derived from the Shionogi SC-115 cells produced fibrinolytic activity independent of added DHT or estrogen. These observations suggest a close correlation between fibrinolytic activity of a tumor and its ability to grow in vivo.