Decreased adenylate cyclase responsiveness of transformed cells correlates with the presence of a viral transforming protein

The adenylate cyclase responsiveness of transformed fibroblastic and epithelial cell lines to forskolin, fluoride, guanine nucleotides and cholera toxin was reduced compared to their parental counterparts. This phenomenon was observed in lines transformed by either RNA or DNA tumor viruses, and in the case of polyoma virus, coincided with the expression of middle T antigen. The data suggest that decreased responsiveness of adenylate cyclase to non-hormone activators is a general consequence of viral transformation and may be related to viral regulation of protein kinase activity.     

Hedgehog signaling regulates myelination in the peripheral nervous system through primary cilia

Myelination is an essential prerequisite for the nervous system to transmit an impulse efficiently by a saltatory conduction. In the peripheral nervous system (PNS), Schwann cells (SCs) engage in myelination. However, a detailed molecular mechanism underlying myelination still remains unclear. In this study, we hypothesized that the primary cilia of SCs are the regulators of Hedgehog (Hh) signaling-mediated myelination. To confirm our hypothesis, we used mouse dorsal root ganglion (DRG)/SC co-cultures, wherein the behavior of SCs could be analyzed by maintaining the interaction of SCs with DRG neurons. Under these conditions, SCs had primary cilia, and Hh signaling molecules accumulated on the primary cilia. When the SCs were stimulated by the addition of desert hedgehog or smoothened agonist, formation of myelin segments on the DRG axons was facilitated. On the contrary, upon administration of cyclopamine, an inhibitor of Hh signaling, myelin segments became comparable to those of controls. Of note, the ratio of SCs harboring primary cilium reached the highest point during the early phase of myelination. Furthermore, the strongest effects of Hh on myelination were encountered during the same stage. These results collectively indicate that Hh signaling regulates myelin formation through primary cilia in the PNS.     

Chemoresistance in prostate cancer cells is regulated by miRNAs and Hedgehog pathway

Many prostate cancers relapse due to the generation of chemoresistance rendering first-line treatment drugs like paclitaxel (PTX) ineffective. The present study aims to determine the role of miRNAs and Hedgehog (Hh) pathway in chemoresistant prostate cancer and to evaluate the combination therapy using Hh inhibitor cyclopamine (CYA). Studies were conducted on PTX resistant DU145-TXR and PC3-TXR cell lines and clinical prostate tissues. Drug sensitivity and apoptosis assays showed significantly improved cytotoxicity with combination of PTX and CYA. To distinguish the presence of cancer stem cell like side populations (SP), Hoechst 33342 flow cytometry method was used. PTX resistant DU145 and PC3 cells, as well as human prostate cancer tissue possess a distinct SP fraction. Nearly 75% of the SP cells are in the G0/G1 phase compared to 62% for non-SP cells and have higher expression of stem cell markers as well. SP cell fraction was increased following PTX monotherapy and treatment with CYA or CYA plus PTX effectively reduced their numbers suggesting the effectiveness of combination therapy. SP fraction cells were allowed to differentiate and reanalyzed by Hoechst staining and gene expression analysis. Post differentiation, SP cells constitute 15.8% of total viable cells which decreases to 0.6% on treatment with CYA. The expression levels of P-gp efflux protein were also significantly decreased on treatment with PTX and CYA combination. MicroRNA profiling of DU145-TXR and PC3-TXR cells and prostate cancer tissue from the patients showed decreased expression of tumor suppressor miRNAs such as miR34a and miR200c. Treatment with PTX and CYA combination restored the expression of miR200c and 34a, confirming their role in modulating chemoresistance. We have shown that supplementing mitotic stabilizer drugs such as PTX with Hh-inhibitor CYA can reverse PTX chemoresistance and eliminate SP fraction in androgen independent, metastatic prostate cancer cell lines.     

Regulation of migration of primary prostate epithelial cells by secreted factors from prostate stromal cells

Stromal-epithelial interactions, which regulate the migration of prostate epithelial cells, play an important role in prostate development, prostatic hyperplasia, and prostate cancer. The objective of this study was to determine how the prostate stroma stimulates the migration of primary prostate epithelial cells (PECs). In the Boyden chamber assay, PEC migration was strongly induced by the conditioned medium of primary prostate stromal cells (PSC-CM). Stimulation of PEC migration depended on the concerted action of adhesion and motility factors in the PSC-CM. Immobilized proteins from PSC-CM mediated adhesion, spreading, and head-to-tail polarization of PECs. Migration induced by immobilized PSC-CM proteins was significantly increased by hepatocyte growth factor/scatter factor (HGF/SF). Inhibition of P13-kinase or Src-family kinases, but not MEK or PLCchi, abolished migration in the Boyden chamber assay. Consistent with their concerted activity in migration assays, the combination of adhesion and motility factors was required for efficient activation of the P13-kinase/Akt pathway. HGF/SF in the PSC-CM was the principal stimulator of the P13-kinase/Akt pathway and an important mediator of PSC-CM-induced PEC migration. In conclusion, our data show that the migration of primary PECs is regulated by the P13-kinase and Src-family kinase signaling pathways and that the activation of the P13-kinase pathway requires adhesion and motility factors from the prostate stroma.     

Estrogen metabolism by primary cultures of rat ventral prostate epithelial and stromal cells

Estrogen metabolism was examined in primary cultures of rat ventral prostate epithelial and stromal cells developed from young (approximately 3 weeks old) animals. Supraphysiologic concentrations (50 nM) of tritium-labelled estradiol (E2) and estrone (E1) were incubated separately with each cell type and the metabolites formed were measured at selected time points over a 24 h period. The metabolites were analyzed using high performance liquid chromatography. Epithelial cells exhibited an equal capability to interconvert E2 and E1 thus demonstrating the presence of similar oxidative and reductive activities for 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) [0.45 and 0.40 pmol/3 h/microgram DNA respectively]. In contrast, stromal cells showed a 6-fold lower rate of oxidation of E2 to E1 (0.08 pmol/3 h/microgram DNA) but exhibited an approx 5-fold higher rate of reduction of E1 to E2 (1.81 pmol/3 h/microgram DNA). Estriol (E3) formation from either substrate was not detected in the two cell types. The results demonstrate that rat ventral prostate epithelial cells have similar capabilities to form or remove biologically active E2. In contrast, prostate stromal cells exhibited a preferential capability to form and possibly maintain high levels of biologically active E2. These findings are discussed with reference to the actions of estrogens on prostate epithelial-stromal cellular interactions.     

Effect of keratinocyte growth factor on cell viability in primary cultured human prostate cancer stromal cells

In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.     

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2‐dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog‐related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU.     

Predominance of clathrin light chain LCb correlates with the presence of a regulated secretory pathway

Two forms of clathrin light chains, LCa and LCb, are expressed in all mammalian and avian tissues that have been examined, whereas only one type is found in yeast. Regions of structural dissimilarity between LCa and LCb indicate possible functional diversity. To determine how LCa and LCb might differentially influence clathrin function, light chain expression patterns and turnover were investigated. Relative expression levels of the two light chains were determined in cells and tissues with and without a regulated secretory pathway. LCa/LCb ratios ranged from 5:1 to 0.33:1. A higher proportion of LCb was observed in cells and tissues that maintain a regulated pathway of secretion, suggesting a specialized role for the LCb light chain in this process. The ratio of light chains in assembled clathrin was found to reflect the levels of total light chains expressed in the cell, indicating no preferential incorporation into triskelions or coated vesicles. The half-lives of LCa, LCb, and clathrin heavy chain were determined to be 24, 45, and 50 h, respectively. Thus, LCa is turned over independently of the other subunits. However, the half-lives of all three subunits are sufficiently long to allow triskelions to undergo many rounds of endocytosis, minimizing the possibility that turnover contributes to regulation of clathrin function. Rather, differential levels of LCa and LCb expression may influence tissue specific clathrin regulation, as suggested by the predominance of LCb in cells maintaining a regulated secretory pathway.     

Matched pairs of human prostate stromal cells display differential tropic effects on LNCaP prostate cancer cells

Prostate stromal cells may play binary roles in the process of prostate cancer development. As the first to be encountered by infiltrating prostate cancer cells, prostate stromal cells form the first defense line against prostate cancer progression and metastasis. However, interaction between prostate cancer and stromal cells may facilitate the formation of a tumor microenvironment favoring cancer cell growth and survival. To establish an experimental system for studying the interaction between cancer and stromal cells, we isolated three matched pairs of normal and cancer-associated human prostate stromal clones. In this report, we describe the morphologic and behavioral characteristics of these cells and their effect on LNCaP prostate cancer cells in co-culture. Unlike LNCaP prostate cancer cells, the isolated prostate stromal clones are large fibroblast-like cells with a slow proliferation rate. Growth and survival of these clones are not affected by androgens. The stromal cells display high resistance to serum starvation, while cancer-associated stromal clones have differentiated survival ability. In co-culture experiments, the stromal cells protected some LNCaP prostate cancer cells from death by serum starvation, and cancer-associated stromal clones showed more protection. This work thus established a panel of valuable human prostate stromal cell lines, which could be used in co-culture to study the interaction between prostate cancer and prostate stromal cells.     

Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.     

An ultrastructural study of primary cilia,abnormal cilia and ciliary knobs from the ciliated cells of the guinea-pig trachea

Summary Single primary cilia are found in developing as well as mature ciliated cells of guinea-pig tracheal epithelium. A few biciliated cells were observed, and in a rare case one cell had developed four such processes. Primary cilia are characterized by a 9 + 0 microtubular arrangement near the base, while a transition to an 8 + 1 pattern occurs at a slightly more distal position. Spokes are lacking, and dynein arms are absent or incompletely developed. The function, if any, of primary cilia remains unknown.In the population of the motile 9 + 2 cilia atypical forms are very rare, i.e. <0.1%. Of the various abnormalities cilia with supernumary microtubules are most common. Only one atypical basal body was observed. Although some of the aberrant forms undoubtedly are non-motile, their very low number suggests that they have no practical influence on the muco-ciliary clearance.Local extrusions of the ciliary membrane, here named ciliary knobs, are believed to be fixation artefacts. It is suggested that they represent circumscribed regions of the ciliary membrane which are sensitive to changes in the environmental osmotic pressure.     

Potent agonists of the Hedgehog signaling pathway

A family of biaryl substituted 1,4-diaminocyclohexanamides of 3-chlorobenzothiophene-2-carboxylic acid is reported as picomolar modulators of Hedgehog protein function. SAR for the 1,4-diaminocyclohexane group is shown to be exquisitely sensitive to substitution on the 4-amino group, and SAR for the 3-chlorobenzothiophene group is highly specific. Preliminary SAR studies of the biaryl substituent led to a picomolar compound with in vivo activity.     

Growth and hormonal responsiveness of human endometrial stromal cells in culture

The present review describes and discusses published results on growth and hormonal responsiveness of human endometrial stromal cells in culture. The proliferative potential of serially subcultured cells, that is, the number of cell doublings before cells enter mitotic senescence and cease to divide, was unusually high in stromal cells from several endometrial specimens, a property that may reflect the unique proliferative capacity of human endometrium when compared to other adult tissues. Fluorescent visualization of microfilaments revealed distinct age-related changes in the distribution of cytoskeletal fibers. Addition of ovarian steroids to the culture medium of stromal cells resulted in significant morphologic changes. From comparative studies using different culture media it became evident that medium components remarkably influenced cell morphology during early culture periods in an irreversible manner. Cultured stromal cells yielded interesting results in experiments designed to define the role of polyamines in growth regulation. Proliferation was greatly inhibited when polyamine levels were reduced by specific inhibition of ornithine decarboxylase, the first and rate limiting enzyme in polyamine synthesis which produces putrescine by catalytic conversion from ornithine. The antiproliferative effects were reversed by addition of putrescine to the culture medium. These results clearly establish a causal link between polyamine depletion and growth deficiencies and reveal an essential function of polyamines in stromal cell proliferation. Hormonally regulated parameters in cultured stromal cells include aromatase activity, pregnancy-associated plasma protein-A, 51K secreted protein, prolactin and laminin. The hormonally regulated production of prolactin and laminin, both considered markers of decidualization, together with morphologic changes of stromal cells to decidual-like cells, strongly suggest that human endometrial stromal cells, when subjected to appropriate hormonal stimulation, are capable of differentiating into decidual cells in culture. Cultured stromal cells therefore offer a unique opportunity to examine the complex changes in gene expression associated with decidualization. In addition, in vitro decidualization may prove to be an effective diagnostic tool in certain cases of infertility. Finally, decidualization of cultured stromal cells represents a relevant end point for testing compounds of potential clinical importance, such as synthetic progestins or antifertility drugs.     

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.