We previously reported that sterol regulatory element-binding protein-1 (SREBP-1) is involved in the transcriptional regulation of androgen receptor (AR) and formation of fatty acid through altered expression of fatty acid synthase (FASN). In this article, we provide a new finding that SREBP-1 induced oxidative stress in prostate cancer cells through increased production of reactive oxygen species (ROS) and expression of NADPH oxidase 5 (Nox5). We have shown that (i) expression of SREBP-1 protein is positively associated with the clinical Gleason grades in human prostate cancer; (ii) genetic overexpression or knockdown of SREBP-1 in prostate cancer cells resulted in corresponding increased or decreased AR, FASN and Nox5 expression, fatty acid and lipid droplet accumulation, and ROS generation; and (iii) SREBP-1 induces and promotes the growth, migration, invasion, and castration-resistant progression of prostate cancer cells in vitro and in vivo. Our data show a novel molecular mechanism by which SREBP-1 promotes prostate cancer growth and progression through alterations in the concerted intracellular metabolic and signaling networks involving AR, lipogenesis, and ROS in prostate cancer cells.

Growth of normal and neoplastic prostate is mediated by the androgen receptor (AR), a ligand-dependent transcription factor activated by high affinity androgen binding. The AR is highly expressed in recurrent prostate cancer cells that proliferate despite reduced circulating androgen. In this report, we show that epidermal growth factor (EGF) increases androgen-dependent AR transactivation in the recurrent prostate cancer cell line CWR-R1 through a mechanism that involves a post-transcriptional increase in the p160 coactivator transcriptional intermediary factor 2/glucocorticoid receptor interacting protein 1 (TIF2/GRIP1). Site-specific mutagenesis and selective MAPK inhibitors linked the EGF-induced increase in AR transactivation to phosphorylation of TIF2/GRIP1. EGF signaling increased the coimmunoprecipitation of TIF2 and AR. AR transactivation and its stimulation by EGF were reduced by small interfering RNA inhibition of TIF2/GRIP1 expression. The data indicate that EGF signaling through MAPK increases TIF2/GRIP1 coactivation of AR transactivation in recurrent prostate cancer.

Previous studies from our laboratory have shown anti-proliferative and pro-apoptotic effects of 3,3'-diindolylmethane (DIM) through regulation of Akt and androgen receptor (AR) in prostate cancer cells. However, the mechanism by which DIM regulates Akt and AR signaling pathways has not been fully investigated. It has been known that FOXO3a and glycogen synthase kinase-3beta (GSK-3beta), two targets of activated Akt, interact with beta-catenin, regulating cell proliferation and apoptotic cell death. More importantly, FOXO3a, GSK-3beta, and beta-catenin are all AR coregulators and regulate the activity of AR, mediating the development and progression of prostate cancers. Here, we investigated the molecular effects of B-DIM, a formulated DIM with higher bioavailability, on Akt/FOXO3a/GSK-3beta/beta-catenin/AR signaling in hormone-sensitive LNCaP and hormone-insensitive C4-2B prostate cancer cells. We found that B-DIM significantly inhibited the phosphorylation of Akt and FOXO3a and increased the phosphorylation of beta-catenin, leading to the inhibition of cell growth and induction of apoptosis. We also found that B-DIM significantly inhibited beta-catenin nuclear translocation. By electrophoretic mobility shift and chromatin immunoprecipitation assays, we found that B-DIM inhibited FOXO3a binding to the promoter of AR and promoted FOXO3a binding to the p27(KIP1) promoter, resulting in the alteration of AR and p27(KIP1) expression, the inhibition of cell proliferation, and the induction of apoptosis in both androgen-sensitive and -insensitive prostate cancer cells. These results suggest that B-DIM-induced cell growth inhibition and apoptosis induction are partly mediated through the regulation of Akt/FOXO3a/GSK-3beta/beta-catenin/AR signaling. Therefore, B-DIM could be a promising non-toxic agent for possible treatment of hormone-sensitive but most importantly hormone-refractory prostate cancers.

BACKGROUND

Eukaryotic replication origins exhibit different initiation efficiencies and activation times within S-phase. Although local chromatin structure and function influences origin activity, the exact mechanisms remain poorly understood. A key to understanding the exact features of chromatin that impinge on replication origin function is to define the precise locations of the DNA sequences that control origin function. In S. cerevisiae, Autonomously Replicating Sequences (ARSs) contain a consensus sequence (ACS) that binds the Origin Recognition Complex (ORC) and is essential for origin function. However, an ACS is not sufficient for origin function and the majority of ACS matches do not function as ORC binding sites, complicating the specific identification of these sites.

RESULTS

To identify essential origin sequences genome-wide, we utilized a tiled oligonucleotide array (NimbleGen) to map the ORC and Mcm2p binding sites at high resolution. These binding sites define a set of potential Autonomously Replicating Sequences (ARSs), which we term nimARSs. The nimARS set comprises 529 ORC and/or Mcm2p binding sites, which includes 95% of known ARSs, and experimental verification demonstrates that 94% are functional. The resolution of the analysis facilitated identification of potential ACSs (nimACSs) within 370 nimARSs. Cross-validation shows that the nimACS predictions include 58% of known ACSs, and experimental verification indicates that 82% are essential for ARS activity.

CONCLUSIONS

These findings provide the most comprehensive, accurate, and detailed mapping of ORC binding sites to date, adding to the emerging picture of the chromatin organization of the budding yeast genome.

Androgen signaling via the androgen receptor (AR) transcription factor is crucial to normal prostate homeostasis and prostate tumorigenesis. Current models of AR function are predominantly based on studies of prostate-specific antigen regulation in androgen-responsive cell lines. To expand on these in vitro paradigms, we used the mouse prostate to elucidate the mechanisms through which AR regulates another direct target, FKBP5, in vivo. FKBP5 encodes an immunophilin that has been previously implicated in glucocorticoid and progestin signaling pathways and that likely influences prostate physiology in the presence of androgens. In this work, we show that androgens directly regulate FKBP5 via an interaction between the AR and a distal enhancer located 65 kb downstream of the transcription start site in the fifth intron of the FKBP5 gene. We have found that AR selectively recruits cAMP response element-binding protein to this enhancer. These interactions, in turn, result in chromatin remodeling that affects the enhancer proper but not the FKBP5 locus as a whole. Furthermore, in contrast to prostate-specific antigen-regulatory mechanisms, we show that transactivation of the FKBP5 gene does not rely on a single looping complex to mediate communication between the distal enhancer and proximal promoter. Rather, the distal enhancer complex and basal transcription apparatus communicate indirectly with one another, implicating a regulatory mechanism that has not been previously appreciated for AR target genes.

DNA was isolated from a circular derivative of chromosome III to prepare a library of recombinant plasmids enriched in chromosome III sequences. An ordered set of recombinant plasmids and bacteriophages carrying the contiguous 210-kilobase region of chromosome III between the HML and MAT loci was identified, and a complete restriction map was prepared with BamHI and EcoRI. Using the high frequency transformation assay and extensive subcloning, 13 ARS elements were mapped in the cloned region. Comparison of the physical maps of chromosome III from three strains revealed that the chromosomes differ in the number and positions of Ty elements and also show restriction site polymorphisms. A comparison of the physical map with the genetic map shows that meiotic recombination rates vary at least tenfold along the length of the chromosome.

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR trans-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.

alpha2-Adrenergic receptors (ARs) play a key role in regulating neurotransmitter release in the central and peripheral sympathetic nervous systems. To date, three subtypes of alpha2-ARs have been cloned (alpha2A, alpha2B, and alpha2C). Here we describe the physiological consequences of disrupting the gene for the alpha2A-AR. Mice lacking functional alpha2A subtypes were compared with wild-type (WT) mice, with animals lacking the alpha2B or alpha2C subtypes, and with mice carrying a point mutation in the alpha2A-AR gene (alpha2AD79N). Deletion of the alpha2A subtype led to an increase in sympathetic activity with resting tachycardia (knockout, 581 +/- 21 min-1; WT, 395 +/- 21 min-1), depletion of cardiac tissue norepinephrine concentration (knockout, 676 +/- 31 pg/mg protein; WT, 1178 +/- 98 pg/mg protein), and down-regulation of cardiac beta-ARs (Bmax: knockout, 23 +/- 1 fmol/mg protein; WT, 31 +/- 2 fmol/mg protein). The hypotensive effect of alpha2 agonists was completely absent in alpha2A-deficient mice. Presynaptic alpha2-AR function was tested in two isolated vas deferens preparations. The nonsubtype-selective alpha2 agonist dexmedetomidine completely blocked the contractile response to electrical stimulation in vas deferens from alpha2B-AR knockout, alpha2C-AR knockout, alpha2AD79N mutant, and WT mice. The maximal inhibition of vas deferens contraction by the alpha2 agonist in alpha2A-AR knockout mice was only 42 +/- 9%. [3H]Norepinephrine release studies performed in vas deferens confirmed these findings. The results indicate that the alpha2A-AR is a major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves; however, the residual alpha2-mediated effect in the alpha2A-AR knockout mice suggests that a second alpha2 subtype (alpha2B or alpha2C) also functions as a presynaptic autoreceptor to inhibit transmitter release.

The NKX3-1 gene is a homeobox gene required for prostate tumor progression, but how it functions is unclear. Here, using chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) we showed that NKX3-1 colocalizes with the androgen receptor (AR) across the prostate cancer genome. We uncovered two distinct mechanisms by which NKX3-1 controls the AR transcriptional network in prostate cancer. First, NKX3-1 and AR directly regulate each other in a feed-forward regulatory loop. Second, NKX3-1 collaborates with AR and FoxA1 to mediate genes in advanced and recurrent prostate carcinoma. NKX3-1- and AR-coregulated genes include those found in the "protein trafficking" process, which integrates oncogenic signaling pathways. Moreover, we demonstrate that NKX3-1, AR, and FoxA1 promote prostate cancer cell survival by directly upregulating RAB3B, a member of the RAB GTPase family. Finally, we show that RAB3B is overexpressed in prostate cancer patients, suggesting that RAB3B together with AR, FoxA1, and NKX3-1 are important regulators of prostate cancer progression. Collectively, our work highlights a novel hierarchical transcriptional regulatory network between NKX3-1, AR, and the RAB GTPase signaling pathway that is critical for the genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer.

Cancer cells are known to have alterations in multiple cellular signaling pathways and because of the complexities in the communication between multiple signaling networks, the treatment and the cure for most human malignancies is still an open question. Perhaps, this is the reason why specific inhibitors that target only one pathway have been typically failed in cancer treatment. However, the in vitro and in vivo studies have demonstrated that some natural products such as isoflavones, indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM), curcumin, (-)-epigallocatechin-3-gallate (EGCG), resveratrol, lycopene, etc, have inhibitory effects on human and animal cancers through targeting multiple cellular signaling pathways and thus these "natural agents" could be classified as multi-targeted agents. This is also consistent with the epidemiological studies showing that the consumption of fruits, soybean and vegetables is associated with reduced risk of several types of cancers. By regulating multiple important cellular signaling pathways including NF-kappaB, Akt, MAPK, Wnt, Notch, p53, AR, ER, etc, these natural products are known to activate cell death signals and induce apoptosis in pre-cancerous or cancer cells without affecting normal cells. Therefore, non-toxic "natural agents" harvested from the bounties of nature could be useful either alone or in combination with conventional therapeutics for the prevention of tumor progression and/or treatment of human malignancies.

Recent studies have demonstrated that the cell growth and antiapoptotic actions of androgen could be dissociated from the transcriptional activity of the receptor and were, instead, mediated by activation of a mitogen-activated protein kinase pathway. This finding suggests an important cellular function of androgen receptor (AR) outside the nucleus. In this report, we demonstrate that androgen activates phosphatidylinositol 3-kinase (PI3K) and Akt, including AKT1 and AKT2, in AR-positive cells. Androgen-induced cell growth and survival were inhibited by PI3K inhibitor and dominant-negative Akt. AR interacts with the p85alpha regulatory subunit of PI3K, and its binding affinity is increased after androgen stimulation. The sites of interaction on the two proteins were mapped to the C-terminal Src-homology 2 domain of p85alpha and N terminus of AR. Activation of PI3K/Akt by androgen was inhibited by dominant-negative Src. Neither N-terminal truncated nor proline-rich region-deleted AR mutants, which are unable to bind to p85alpha and Src, respectively, was able to mediate androgen-induced PI3K/Akt activation. AR with deletion of C-terminal region including ligand binding domain, however, retains the ability to activate PI3K/Akt upon androgen stimulation, which supports the notion that nongenomic function of androgen is mediated by its interaction with membrane receptors (1, 3, 4). These findings indicate that a triple complex between AR, p85alpha, and Src is required for androgen-stimulated PI3K/Akt activation, and that the PI3K/Akt pathway, in addition to mitogen-activated protein kinase, mediates androgen-induced cell growth and cell survival.

SUMO (also called Sentrin) is a ubiquitin-like protein that plays an important role in regulating protein function and localization. It is known that several nuclear receptors are modified by SUMO; however, the effect of desumoylation in regulating nuclear receptor function has not been elucidated. Here we show that androgen receptor (AR)-mediated transcription is markedly enhanced by SENP1, a member of SUMO-specific protease family. SENP1's ability to enhance AR-dependent transcription is not mediated through desumoylation of AR, but rather through its ability to deconjugate histone deacetylase 1 (HDAC1), thereby reducing its deacetylase activity. HDAC1's repressive effect on AR-dependent transcription could be reversed by SENP1 and by deletion of its sumoylation sites. RNA interference depletion of endogenous HDAC1 also reduced SENP1's effect. Thus, SENP1 could regulate AR-dependent transcription through desumoylation of HDAC1. These studies provide insights on the potential role of desumoylation in the regulation of nuclear receptor activity.

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich "NR box" motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.

Motor neuron degeneration resulting from the aggregation of the androgen receptor with an expanded polyglutamine tract (AR-polyQ) has been linked to the development of spinal and bulbar muscular atrophy (SBMA or Kennedy disease). Here we report that adding 5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one (ASC-J9) disrupts the interaction between AR and its coregulators, and also increases cell survival by decreasing AR-polyQ nuclear aggregation and increasing AR-polyQ degradation in cultured cells. Intraperitoneal injection of ASC-J9 into AR-polyQ transgenic SBMA mice markedly improved disease symptoms, as seen by a reduction in muscular atrophy. Notably, unlike previous approaches in which surgical or chemical castration was used to reduce SBMA symptoms, ASC-J9 treatment ameliorated SBMA symptoms by decreasing AR-97Q aggregation and increasing VEGF164 expression with little change of serum testosterone. Moreover, mice treated with ASC-J9 retained normal sexual function and fertility. Collectively, our results point to a better therapeutic and preventative approach to treating SBMA, by disrupting the interaction between AR and AR coregulators.

OBJECTIVE

Emerging evidences implicate long noncoding RNAs (lncRNAs) are deregulated in cancer development. The purpose of the current study is to investigate the role of new lncRNA, named PlncRNA-1, in prostate cancer (CaP) pathogenesis.

METHODS

In this study, real-time q-PCR was used to demonstrate the expression of PlncRNA-1 in 16 pairs CaP tissues and matched normal tissues, 14 pairs CaP tissues and BPH tissues, 4 CaP cell lines, including LNCaP, LNCaP-AI, PC3, and C4-2, and 2 normal prostate epithelial cell lines RWPE-1 and PWR-1E. After PlncRNA-1 was suppressed by siRNA in LNCaP and LNCaP-AI cell lines, cell proliferation and apoptosis were assessed using CCK-8 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). After PlncRNA-1 and AR was suppressed by siRNA in LNCaP and LNCaP-AI cell lines, real-time q-PCR and Western blotting were used to measure reciprocal regulation of PlncRNA-1 and AR.

RESULTS

We showed that expression PlncRNA-1, was significantly higher in CaP cells relative to normal prostate epithelial cells, as well as higher in human CaPs compared with normal tissues and benign prostatic hyperplasia (BPH). Silencing of PlncRNA-1 significantly reduced cell proliferation and induced apoptosis in CaP cell lines LNCaP and LNCaP-AI. Mechanistically, PlncRNA-1 suppression by siRNA resulted in a decrease of androgen receptor (AR) mRNA, protein and AR downstream target. Of note, blockade of AR signaling with siRNA also resulted in a suppression of PlncRNA-1 expression in CaP cell lines.

CONCLUSIONS

Our study suggests reciprocal regulation of PlncRNA-1 and androgen receptor contribute to CaP pathogenesis and that PlncRNA-1 is a potential therapy target.

Membrane rafts are sphingolipid- and cholesterol-rich microdomains that contain dynamic arrangements of signaling proteins. Notably, various components of the ubiquitous cAMP/Protein Kinase A (PKA) pathway, including β-adrenergic receptors (β-ARs), G proteins, and adenylyl cyclases (ACs), have been shown to localize differentially between membrane rafts and non-raft regions of the plasma membrane. As PKA participates in regulating diverse fundamental cellular functions, a number of which require membrane rafts, it is important to understand how PKA activity is specifically regulated in these membrane microdomains. To this end, we developed an improved FRET-based PKA activity biosensor, and targeted it to both membrane raft and non-raft regions of the plasma membrane to examine PKA activity dynamics in different plasma membrane microdomains. Disruption of membrane rafts via cholesterol depletion was shown to enhance β-AR stimulated PKA activity at the plasma membrane, suggesting that membrane rafts play a negative role in β-AR stimulated PKA activation. Furthermore, we found that membrane rafts possess higher basal PKA activity in the resting state compared to non-raft regions, which depends on the integrity of membrane rafts and proper localization of PKA. This study shows that membrane rafts play an important role in regulating the activity of PKA at the plasma membrane, and demonstrates the ability of live-cell FRET-based assays to reveal dynamic differences amongst plasma membrane microdomains, laying a foundation for further dissection of membrane regulated signal transduction.

OBJECTIVE

To analyze the long-term survival and the pattern and timing of excess mortality in patients with early-stage Hodgkin's disease.

METHODS

Between 1969 and 1997, 1,080 patients age 50 or younger were treated for clinical stage IA to IIB Hodgkin's disease. Overall survival was determined, and prognostic factors were assessed. Relative risk and absolute excess risk (AR) of mortality were calculated for the entire cohort and by prognostic groups (on the basis of B symptoms, mediastinal status, and number of sites, modified from the European Organization for Research and Treatment of Cancer).

RESULTS

The median follow-up was 12 years. The 15- and 20-year Kaplan-Meier survival estimates were 84% and 78%, respectively. Cox proportional hazards models showed that number of involved sites (P =.006), mediastinal status (P =.02), and histology (P =.02) were independent predictors of death from all causes. The AR of mortality in patients with a favorable prognosis increased over time, whereas for those with an unfavorable prognosis, the AR peaked in the first 5 years, predominantly from Hodgkin's disease. The relative risk of mortality from all causes, causes other than Hodgkin's disease, second tumors, and cardiac disease remained significantly elevated more than 20 years after treatment.

CONCLUSIONS

Patients treated for early-stage Hodgkin's disease have a sustained excess mortality risk despite good control of the disease. Treatment reduction efforts in patients with early-stage, favorable-prognosis disease should continue, but for patients with an unfavorable prognosis, modified treatment may not be advisable. The excess mortality noted beyond two decades underscores the importance of long-term follow-up care in patients treated for Hodgkin's disease.

The power of genetic association analyses is often compromised by missing genotypic data which contributes to lack of significant findings, e.g., in in silico replication studies. One solution is to impute untyped SNPs from typed flanking markers, based on known linkage disequilibrium (LD) relationships. Several imputation methods are available and their usefulness in association studies has been demonstrated, but factors affecting their relative performance in accuracy have not been systematically investigated. Therefore, we investigated and compared the performance of five popular genotype imputation methods, MACH, IMPUTE, fastPHASE, PLINK and Beagle, to assess and compare the effects of factors that affect imputation accuracy rates (ARs). Our results showed that a stronger LD and a lower MAF for an untyped marker produced better ARs for all the five methods. We also observed that a greater number of haplotypes in the reference sample resulted in higher ARs for MACH, IMPUTE, PLINK and Beagle, but had little influence on the ARs for fastPHASE. In general, MACH and IMPUTE produced similar results and these two methods consistently outperformed fastPHASE, PLINK and Beagle. Our study is helpful in guiding application of imputation methods in association analyses when genotype data are missing.

Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity.

Chromosomal DNA of Schizosaccharomyces pombe contains sequences with properties analogous to ARS elements of Saccharomyces cerevisiae. Following Sau3A fragmentation of the S. pombe genome we have recovered a number of such fragments in an M13-based shuttle vector, suitable for subsequent sequence analysis. The complete nucleotide sequence has been obtained for eight ARS+ inserts derived from the Sau3A cloning and for the ARS present in pFL20 isolated previously by Losson and Lacroute (Cell, 32, 371-377, 1983). The Sau3A clones are single fragments between 0.8 and 1.8 kb. No ARS+ clones smaller than this were recovered even though the average size Sau3A fragment in S. pombe is approximately 200-300 bp. The sequence analysis revealed that all clones are AT-rich (69-75% A + T residues), and all contain a particularly AT-rich 11 bp core element represented by the consensus sequence 5' (A/T)PuTT-TATTTA(A/T) 3'. Deletion mapping indicates that the consensus in all cases is in the vicinity of a functional ARS domain. However precise excision of the consensus by in vitro mutagenesis has little effect on ARS activity as judged by the transformation assay. We argue that the association of the consensus with the ARS domain occurs too reproducibly to be explained by chance alone. We suggest that although it may not be essential for the extrachromosomal maintenance of plasmids in S. pombe, the consensus does have a function in situ in the chromosome and thus is always present as a cryptic sequence in the isolated ARS element.

Stage-specific differentiation markers were used to evaluate the cellular composition and the origin of nonimmortalized (PrEC) and immortalized (PZ-HPV7, CA-HPV10, RWPE-1, and 957E/hTERT) human prostate cell lines. These studies documented that immortalized and nonimmortalized prostate epithelial cells established and maintained in low (i.e., <300 micromol/L) Ca(2+) serum-free defined (SFD) medium were all derived from normal nonmalignant prostate tissues and contain CD133(+)/ABCG2(+)/alpha(2)beta(1)(Hi)/p63(-)/PSCA(-)/AR(-)/PSA(-) prostate stem cells. In these cultures, prostate stem cells are able to self-renew and generate two distinct cell lineages: the minor proliferatively quiescent neuroendocrine lineage and the major transit-amplifying cell lineage. Subsequently, CD133(-)/ABCG2(-)/alpha(2)beta(1)(Hi)/p63(+)/PSCA(-)/AR(-)/PSA(-) transit-amplifying cells proliferate frequently and eventually mature into proliferatively quiescent CD133(-)/ABCG2(-)/alpha(2)beta(1)(Lo)/p63(-)/PSCA(+)/AR(-)/PSA(-) intermediate cells. Such proliferatively quiescent intermediate cells, however, do not complete their full maturation into CD133(-)/ABCG2(-)/alpha(2)beta(1)(Lo)/p63(-)/PSCA(-)/AR(+)/PSA(+) luminal-secretory cells in low Ca(2+) SFD medium. Addition of universal type I IFN and synthetic androgen (R1881) to culture medium resulted in up-regulation of androgen receptor protein expression. However, it failed to induce full differentiation of intermediate cells into AR(+)/PSA(+) luminal-secretory cells. Our results indicate that such inability of prostate epithelial cells to complete their differentiation is due to continuous expression of Notch-1 receptor and its downstream effector, Hey-1 protein, which actively suppresses differentiation via its ability to transcriptionally repress a series of genes, including the GATA family of transcription factors.

The yeast ARS binding factor 1 (ABF1)--where ARS is an autonomously replicating sequence--and repressor/activator protein 1 (RAP1) have been implicated in DNA replication, transcriptional activation, and transcriptional silencing. The ABF1 gene was cloned and sequenced and shown to be essential for viability. The predicted amino acid sequence contains a novel sequence motif related to the zinc finger, and the ABF1 protein requires zinc and unmodified cysteine residues for sequence-specific DNA binding. Interestingly, ABF1 is extensively related to its counterpart, RAP1, and both proteins share a region of similarity with SAN1, a suppressor of certain SIR4 mutations, suggesting that this region may be involved in mediating SIR function at the silent mating type loci.

Origins of DNA replication in Schizosaccharomyces pombe lack a specific consensus sequence analogous to the Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, raising the question of how they are recognized by the replication machinery. Because all well characterized S. pombe origins are located in intergenic regions, we analyzed the sequence properties and biological activity of such regions. The AT content of intergenes is very high ( approximately 70%), and runs of A's or T's occur with a significantly greater frequency than expected. Additionally, the two DNA strands in intergenes display compositional asymmetry that strongly correlates with the direction of transcription of flanking genes. Importantly, the sequence properties of known S. pombe origins of DNA replication are similar to those of intergenes in general. In functional studies, we assayed the in vivo origin activity of 26 intergenes in a 68-kb region of S. pombe chromosome 2. We also assayed the origin activity of sets of randomly chosen intergenes with the same length or AT content. Our data demonstrate that at least half of intergenes have potential origin activity and that the relative ability of an intergene to function as an origin is governed primarily by AT content and length. We propose a stochastic model for initiation of DNA replication in the fission yeast. In this model, the number of AT tracts in a given sequence is the major determinant of its probability of binding SpORC and serving as a replication origin. A similar model may explain some features of origins of DNA replication in metazoans.