Autonomously replicating sequences (ARSs) of the yeast S. cerevisiae function as replication origins on plasmids and probably also on chromosomes. ARS function requires a copy of the ARS core consensus (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') and additional sequences 3' to the T-rich strand of the consensus. Our analysis of an ARS from chromosome III, the C2G1 ARS, suggests that ARS function depends on the presence of an exact match to the core consensus and the presence of additional near matches in the 3' flanking region. We have demonstrated that ARS function can be mediated by multiple matches to the core consensus by constructing synthetic ARS elements from oligonucleotides containing copies of the consensus sequence. We find that two copies of the core consensus are sufficient for ARS activity and that an artificial ARS as efficient as a natural chromosomal ARS can be constructed from multiple core consensus elements in a specific orientation.

Visible radiation at resonant frequencies is transduced to thermal energy by surface plasmons on gold nanoparticles. Temperature in </=10-microliter aqueous suspensions of 20-nanometer gold particles irradiated by a continuous wave <em>Ar</em>+ ion laser at 514 nm increased to a maximum equilibrium value. This value increased in proportion to incident laser power and in proportion to nanoparticle content at low concentration. Heat input to the system by nanoparticle transduction of resonant irradiation equaled heat flux outward by conduction and radiation at thermal equilibrium. The efficiency of transducing incident resonant light to heat by microvolume suspensions of gold nanoparticles was determined by applying an energy balance to obtain a microscale heat-transfer time constant from the transient temperature profile. Measured values of transduction efficiency were increased from 3.4% to 9.9% by modulating the incident continuous wave irradiation.

The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of androgen receptor (AR), and proliferation of these two cell lines is paradoxically repressed by androgen. After continuous passage in androgen-containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R2 sublines was further evaluated by examining the role of critical regulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.

There is now evidence that oestrogens and androgens can influence male and female reproductive systems. In order to accurately identify the sites of action of oestrogens and androgens, we have proceeded to the histological localization of the two oestrogen receptor (ER) subtypes, ERalpha and ERbeta, and the androgen receptor (AR) in the reproductive tissues of adult rats of both sexes. AR was detected by immunocytochemistry, while ERalpha and ERbeta were localized by both immunocytochemistry and in situ hybridization. In the pituitary gland of animals of both sexes, ERalpha was found in the majority of nuclei of secretory cells in the anterior pituitary. The intermediate and posterior lobes did not show any staining. ERbeta was not found to be expressed in any of the pituitary lobes. Using AR antibodies, nuclear staining was detected in about 50% of secretory cells of the anterior lobe, the intermediate and posterior lobes being completely unstained. In the testis, ERalpha was localized in nuclei of Leydig cells as well as in round spermatocytes and spermatids, while ERbeta could only be detected in Sertoli cell nuclei. AR immunoreactivity was found in nuclei of Sertoli, peritubular myoid and Leydig cells. In the prostate, ERbeta was observed in epithelial cells of tubulo-alveoli, while the stroma was unlabelled. ERalpha was not found to be expressed in any prostate cells. In the prostate, AR was detected in nuclei of epithelial, stromal and endothelial cells. In seminal vesicles, staining of ERalpha was found in nuclei of epithelial and stromal cells. Similar findings were observed using AR antibodies. While ERbeta mRNA could not be detected by in situ hybridization, weak staining for ERbeta was localized in epithelial cells of seminal vesicles. In the ovary, both ERalpha and ERbeta were found to be expressed. ERbeta mRNA was found in granulosa cells of growing follicles, while ERalpha was present in theca cells, interstitial gland cells and germinal epithelium. AR immunoreactivity was detected in granulosa cell nuclei in growing follicles and also in scattered interstitial cells. In the oviduct and uterus, ERalpha was observed in nuclei of epithelial cells as well as of stromal and muscle cells. Similarly, AR immunoreactivity was present in nuclei of epithelial cells, stromal and muscle cells in both the oviduct and uterus. ERbeta was not detected in the oviduct and uterus. The present findings indicate a cell-specific localization of ERalpha, ERbeta and AR in reproductive tissues in rats of both sexes. By establishing the precise sites of action of oestrogens and androgens they contribute to a better understanding of the respective role of these steroids in reproduction function.

Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors by which the prosurvival effects of androgen and AR drive prostate cancer progression are poorly defined. Here, we identify a novel feed-forward loop involving cooperative interactions between ligand-activated AR and heat-shock protein 27 (Hsp27) phospho-activation that enhance AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. Androgen-bound AR induces rapid Hsp27 phosphorylation on Ser(78) and Ser(82) residues in an AR- and p38 kinase-dependent manner. After this androgen-induced, non-nuclear phospho-activation, Hsp27 displaces Hsp90 from a complex with AR to chaperone AR into the nucleus and interact with its response elements to enhance its genomic activity. Inhibition of Hsp27 phosphorylation, or knockdown using the antisense drug OGX-427, shifted the association of AR with Hsp90 to MDM2, increased proteasome-mediated AR degradation, decreased AR transcriptional activity, and increased prostate cancer LNCaP cell apoptotic rates. OGX-427 treatment of mice bearing LNCaP xenografts transfected with an androgen-regulated, probasin-luciferase reporter construct resulted in decreased bioluminescence and serum PSA levels as pharmacodynamic readouts of AR activity, as well as AR, Hsp27, and Hsp90 protein levels in LNCaP tumor tissue. These data identify novel nongenomic mechanisms involving androgen, AR, and Hsp27 activation that cooperatively interact to regulate the genomic activity of AR and justify further investigation of Hsp27 knockdown as an AR disrupting therapeutic strategy in prostate cancer.

Hoxa-10 is an AbdominalB-like homeobox gene that is expressed in the developing genitourinary tract during embryogenesis and in the adult uterus during early pregnancy. Null mutation of Hoxa-10 in the mouse causes both male and female infertility. Defective implantation and decidualization resulting from the loss of maternal Hoxa-10 function in uterine stromal cells is the cause of female infertility. However, the mechanisms by which Hoxa-10 regulates these uterine events are unknown. We have identified two potential mechanisms for these uterine defects in Hoxa-10(-/-) mice. First, two PGE2 receptor subtypes, EP3 and EP4, are aberrantly expressed in the uterine stroma in Hoxa-10(-/-) mice, while expression of several other genes in the stroma (TIMP-2, MMP-2, ER, and PR) and epithelium (LIF, HB-EGF, Ar, and COX-1) are unaffected before implantation. Further, EP3 and EP4 are inappropriately regulated by progesterone (P4) in the absence of Hoxa-10, while PR, Hoxa-11 and c-myc, three other P4-responsive genes respond normally. These results suggest that Hoxa-10 specifically mediates P4 regulation of EP3 and EP4 in the uterine stroma. Second, since Hox genes are implicated in local cell proliferation, we also examined steroid-responsive uterine cell proliferation in Hoxa-10(-/-) mice. Stromal cell proliferation in mutant mice in response to P4 and 17beta-estradiol (E2 was significantly reduced, while epithelial cell proliferation was normal in response to E2. These results suggest that stromal cell responsiveness to P4 with respect to cell proliferation is impaired in Hoxa-10(-/-) mice, and that Hoxa-10 is involved in mediating stromal cell proliferation. Collectively, these results suggest that Hoxa-10 mutation causes specific stromal cell defects that can lead to implantation and decidualization defects apparently without perturbing epithelial cell functions.

Direct evidence that hyperglycemia, rather than concomitant increases in known risk factors, induces atherosclerosis is lacking. Most diabetic mice do not exhibit a higher degree of atherosclerosis unless the development of diabetes is associated with more severe hyperlipidemia. We hypothesized that normal mice were deficient in a gene that accelerated atherosclerosis with diabetes. The gene encoding aldose reductase (AR), an enzyme that mediates the generation of toxic products from glucose, is expressed at low levels in murine compared with human tissues. Mice in which diabetes was induced through streptozotocin (STZ) treatment, but not nondiabetic mice, expressing human AR (hAR) crossed with LDL receptor-deficient (Ldlr-/-) C57BL/6 male mice had increased aortic atherosclerosis. Diabetic hAR-expressing heterozygous LDL receptor-knockout mice (Ldlr+/-) fed a cholesterol/cholic acid-containing diet also had increased aortic lesion size. Lesion area at the aortic root was increased by STZ treatment alone but was further increased by hAR expression. Macrophages from hAR-transgenic mice expressed more scavenger receptors and had greater accumulation of modified lipoproteins than macrophages from nontransgenic mice. Expression of genes that regulate regeneration of glutathione was reduced in the hAR-expressing aortas. Thus, hAR increases atherosclerosis in diabetic mice. Inhibitors of AR or other enzymes that mediate glucose toxicity could be useful in the treatment of diabetic atherosclerosis.

The androgen receptor (AR) is a transcription factor that is pivotal for the development of prostate cancer. Here, we have identified two related histone demethylases, JMJD2A and JMJD2D, which form complexes with ligand-bound AR. We found that AR interacts through its ligand binding domain with JMJD2A and JMJD2D. On the other hand, JMJD2A utilizes its catalytic domain or C-terminus to bind to AR, and JMJD2D does so via its C-terminus. Further, overexpression of JMJD2A or D stimulates AR function and this is dependent on JMJD2 catalytic activity. Conversely, downregulation of JMJD2A, which is often overexpressed in prostate tumors, reduces basal transcription of the AR target gene, prostate-specific antigen, in LNCaP prostate cancer cells. Altogether, our data have identified a novel class of AR coactivators, whose (over)expression in prostate tumors could contribute to the constitutive activation of AR and thus to androgen-depletion independency of advanced prostate cancer cells.

With ∼30 000 deaths annually in the United States, prostate cancer (PCa) is a major oncologic disease. Here we show that the microRNAs miR-130a, miR-203 and miR-205 jointly interfere with the two major oncogenic pathways in prostate carcinoma and are downregulated in cancer tissue. Using transcriptomics we show that the microRNAs repress several gene products known to be overexpressed in this cancer. Argonaute 2 (AGO2) co-immunoprecipitation, reporter assays and western blot analysis demonstrate that the microRNAs directly target several components of the mitogen-activated protein kinase (MAPK) and androgen receptor (AR) signaling pathways, among those several AR coregulators and HRAS (Harvey rat sarcoma viral oncogene homolog), and repress signaling activity. Both pathways are central for the development of the primary tumor and in particular the progression to its incurable castration-resistant form. Reconstitution of the microRNAs in LNCaP PCa cells induce morphological changes, which resemble the effect of androgen deprivation, and jointly impair tumor cell growth by induction of apoptosis and cell cycle arrest. We therefore propose that these microRNAs jointly act as tumor suppressors in prostate carcinoma and might interfere with progression to castration resistance.

To examine the role of androgen receptor (AR) in Sertoli cells (SC), we used a SC-specific AR knockout (S-AR-/y) mouse to further evaluate the chronological changes of seminiferous tubules and the molecular mechanisms of SC androgen/AR signals on spermatogenesis. Testes morphology began changing as early as postnatal day (PD) 10.5 in wild-type (WT), but not in S-AR-/y mice. After puberty (PD 50), the SC nuclei of WT testes migrated to the basal area of the seminiferous epithelium; however, in S-AR-/y testes, SC nuclei were disarranged and dislocated. Results from electron microscopy further showed an obvious duplication of basal lamina of the seminiferous epithelium in S-AR-/y testes at PD 50 compared with WT testes. Using quantitative RT-PCR analyses, the expression of SC gene profiles were compared in PD 10.5 testes. In S-AR-/y testes, the expression levels of 1) vimentin were significantly increased and laminin alpha5 was significantly decreased in PD 10.5, which contributed to functional defects in cytoskeletons and production of the basement membrane component of SC leading to cell morphology deterioration and thus affecting the integrity of seminiferous epithelium; 2) claudin-11, occludin, and gelsolin were significantly decreased in PD 10.5, which contributed to defects in intact junctional complex formation of SC leading to impairment of the integrity of the blood-testis barrier; 3) calcium channel, voltage-dependent, P/Q-type, alpha1A subunit; tissue-type plasminogen activator; transferrin; and epidermal fatty-acid-binding protein were significantly decreased in PD 10.5, which contributed to functional defects in production and/or secretion of specific proteases, transport proteins, and paracrine factors of SC, leading to impairment of its germ cells' nursery functions.

Interleukin 6 (IL-6) is a cytokine that regulates not only immune and inflammatory responses but also the growth of some tumors, including prostate carcinomas. IL-6 signals through Janus kinase, signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase and is also able to induce androgen receptor (AR)-mediated gene activation in prostate cancer, which is an important process in prostate cancer androgen-independent progression. We now show that IL-6-induced AR-mediated gene activation requires the activation of STAT3 by IL-6 in LNCaP prostate cancer cells. In particular, STAT3 associates with AR in an androgen-independent but IL-6-dependent manner. Inhibition of STAT3 rather than mitogen-activated protein kinase results in inhibition of AR-mediated gene activation in response to IL-6. These findings not only identify STAT3 as an important signaling molecule required for IL-6-signaling to induce AR-mediated gene activation in prostate carcinoma cells but also reveal the importance of activated STAT3 in human tumor development and progression.

Typically, the initial response of a prostate cancer patient to androgen ablation therapy is regression of the disease. However, the tumor will progress to an "androgen-independent" stage that results in renewed growth and spread of the cancer. Both nuclear factor-kappaB (NF-kappaB) expression and neuroendocrine differentiation predict poor prognosis, but their precise contribution to prostate cancer progression is unknown. This report shows that secretory proteins from neuroendocrine cells will activate the NF-kappaB pathway in LNCaP cells, resulting in increased levels of active androgen receptor (AR). By blocking NF-kappaB signaling in vitro, AR activation is inhibited. In addition, the continuous activation of NF-kappaB signaling in vivo by the absence of the IkappaBalpha inhibitor prevents regression of the prostate after castration by sustaining high levels of nuclear AR and maintaining differentiated function and continued proliferation of the epithelium. Furthermore, the NF-kappaB pathway was activated in the ARR(2)PB-myc-PAI (Hi-myc) mouse prostate by cross-breeding into a IkappaBalpha(+/-) haploid insufficient line. After castration, the mouse prostate cancer continued to proliferate. These results indicate that activation of NF-kappaB is sufficient to maintain androgen-independent growth of prostate and prostate cancer by regulating AR action. Thus, the NF-kappaB pathway may be a potential target for therapy against androgen-independent prostate cancer.

We evaluated the pharmacological characteristics of (R)-2-(2-aminothiazol-4-yl)-4'-{2-[(2-hydroxy-2-phenylethyl)amino]-ethyl} acetanilide (YM178). YM178 increased cyclic AMP accumulation in Chinese hamster ovary (CHO) cells expressing human beta3-adrenoceptor (AR). The half-maximal effective concentration (EC50) value was 22.4 nM. EC50 values of YM178 for human beta1- and beta2-ARs were 10,000 nM or more, respectively. The ratio of intrinsic activities of YM178 versus maximal response induced by isoproterenol (nonselective beta-AR agonist) was 0.8 for human beta3-ARs, 0.1 for human beta1-ARs, and 0.1 for human beta2-ARs. The relaxant effects of YM178 were evaluated in rats and humans bladder strips precontracted with carbachol (CCh) and compared with those of isoproterenol and 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP-12177A) (beta3-AR agonist). EC50 values of YM178 and isoproterenol in rat bladder strips precontracted with 10(-6) M CCh were 5.1 and 1.4 microM, respectively, whereas those in human bladder strips precontracted with 10(-7) M CCh were 0.78 and 0.28 microM, respectively. In in vivo study, YM178 at a dose of 3 mg/kg i.v. decreased the frequency of rhythmic bladder contraction induced by intravesical filling with saline without suppressing its amplitude in anesthetized rats. These findings suggest the suitability of YM178 as a therapeutic drug for the treatment of symptoms of overactive bladder such as urinary frequency, urgency, and urge incontinence.

Curcumin, the yellow color pigment of turmeric, is produced industrially from turmeric oleoresin. The mother liquor after isolation of curcumin from oleoresin contains approximately 40% oil. The oil was extracted from the mother liquor using hexane at 60 degrees C, and the hexane extract was separated into three fractions using silica gel column chromatography. These fractions were tested for antibacterial activity by pour plate method against Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Fraction II eluted with 5% ethyl acetate in hexane was found to be most active fraction. The turmeric oil, fraction I, and fraction II were analyzed by GC and GC-MS. ar-Turmerone, turmerone, and curlone were found to be the major compounds present in these fractions along with other oxygenated compounds.

The understanding of the key role that androgens play on the normal and pathological physiology of the prostate guided the development of different therapies for the treatment of locally advanced or metastatic prostate cancer (PCa). These so-called androgen deprivation therapies include surgical or chemical castration, achieved by the administration of gonadotropin-releasing hormone analogs; inhibition of steroidogenic enzymes; and finally, blocking of the binding of androgens to their receptor (AR) by the use of antiandrogens. Despite an excellent initial response, in approximately 2 to 3 years, most of these patients will succumb to the castration resistant form of the disease. Remarkably, even in the presence of castration levels of circulating androgens, these tumors are still dependent on a functional AR, and several molecular mechanisms have been proposed to explain this phenomenon. These include: (1) gene amplification and increased expression of the AR mRNA and protein, (2) selection of mutations in the AR that confer broader ligand specificity, (3) changes in the ratios or expression between the AR and its coregulators, (4) increased expression of steroidogenic enzymes, and (5) up-regulation of cross-talk signal transduction pathways that can activate the AR in a ligand-independent manner. We will summarize how these molecular hypotheses are being tested in the clinic by the latest therapeutic modalities.

BACKGROUND

The acquisition of an androgen-independent phenotype is the most serious issue of prostate cancer treatment. Although several experimental cell models have been reported for studying androgen independence, they have limited applications related to hormone-refractory prostate cancer. To investigate the molecular mechanism of androgen-independent growth of prostate cancer, we established a useful LNCaP cell model that resembles the clinical scenario of hormone-refractory prostate cancer.

METHODS

Androgen-sensitive LNCaP parental cells were continuously maintained in a regular cell-culture medium, that is, phenol red-positive RPMI 1640 medium supplemented with 5% fetal bovine serum and 1% glutamine. Upon passage, the androgen responsiveness of those cells decreased, to a level lower than that of parental cells. We examined the growth properties and androgen responsiveness of these different LNCaP cells in vitro and in vivo. Cytogenetic characteristics and expression of androgen receptors (ARs) and prostate-specific antigen (PSA) were determined.

RESULTS

Upon continuous passage, the biological behavior of parental C-33 cells (passage number less than 33) was altered. C-81 cells (passage number higher than 81) clearly exhibited more aggressive growth and lower androgen responsiveness than C-33 and C-51 cells (passage number between 35 and 80) in vitro and in vivo. Nevertheless, all these cells expressed a similar level of functional AR protein as well as a similar genetic profile. Moreover, in a steroid-reduced culture condition, C-81 cells secreted a higher level of PSA than C-33 cells.

CONCLUSIONS

Our LNCaP cell model closely recapitulates the progression of human prostate cancer from the androgen-responsive to the hormone-refractory state under the androgen nondeprived condition. This cell model may provide the opportunity to understand the molecular mechanisms associated with the acquisition of androgen independence during human prostate cancer progression.

Commercial antibodies are used widely to quantify and localize the alpha1-adrenergic receptor (AR) subtypes, alpha1A, alpha1B, and alpha1D. We tested ten antibodies, from abcam and Santa Cruz, using western blot with heart and brain tissue from wild-type (WT) mice and mice with systemic knockout (KO) of one or all three subtypes. We found that none of the antibodies detected a band in WT that was absent in the appropriate KO or in the KO that was null for all alpha1-ARs (ABDKO). We conclude that the antibodies we tested are not specific for alpha1-ARs. These results raise caution with prior studies using these reagents. For now, competition radioligand binding is the only reliable approach to quantify the alpha1-AR subtype proteins. Receptor protein localization remains a challenge.

Androgen refractory cancer of the prostate (ARCaP) cells contain androgen receptor (AR) and synthesize and secrete prostate specific antigen (PSA). We isolated epithelia-like ARCaP(E) from parental ARCaP cells and induced them to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors including TGFbeta1 plus EGF, IGF-1, beta2-microglobulin (beta2-m), or a bone microenvironment. The molecular and behavioral characteristics of the resultant ARCaP(M) were characterized extensively in comparison to the parental ARCaP(E) cells. In addition to expressing mesenchymal biomarkers, ARCaP(M) gained 100% incidence of bone metastasis. ARCaP(M) cells express receptor activator of NF-kappaB ligand (RANKL), which was shown to increase tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in culture, and when metastatic to bone in vivo. We provide evidence that RANKL expression was promoted by increased cell signaling mediated by the activation of Stat3-Snail-LIV-1. RANKL expressed by ARCaP(M) cells is functional both in vitro and in vivo. The lesson we learned from the ARCaP model of EMT is that activation of a specific cell signaling pathway by soluble factors can lead to increased bone turnover, mediated by enhanced RANKL expression by tumor cells, which is implicated in the high incidence of prostate cancer bone colonization. The ARCaP EMT model is highly attractive for developing new therapeutic agents to treat prostate cancer bone metastasis.

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.

BACKGROUND

A complete set of nine ARSs was identified (the tenth ARS in this paper), mapped on chromosome VI of Saccharomyces cerevisiae, and characterized for functional elements.

RESULTS

The level of activity of all ARSs as chromosomal replication origins was determined by neutral/neutral 2D gel-electrophoresis. These origins were classified into three groups: (i) three high frequency origins used once nearly every cell cycle, (ii) four intermediate frequency origins used once in two to three cycles and (iii) two low frequency origins used in fewer than 5% of cell cycles. These variations in initiation frequency among origins of chromosome VI are present in three common laboratory wild-type strains. Each origin is initiated at a fixed time and passively replicated by incoming replication forks at a fixed time during a synchronous S phase. Replication of each arm of the chromosome starts from one major origin located one-fifth (left arm) and one-third (right arm) of the distance from the centromere, and expands sequentially in both directions. Two telomere vicinity origins are replicated last. Time of initiation and replication of the last replicating origin, Ori609, was remarkably variable from cell to cell.

CONCLUSIONS

Chromosome VI of S. cerevisiae contains nine replication origins that comprise five active replicons under normal cell growth conditions. A clear correlation was found between the efficiency of initiation and the order of replication. The timing of initiation of most origins, except for the first and last, is coincident with the time of passive replication by incoming forks from neighbouring origins.

BACKGROUND

Emerging evidence suggests that androgens and the androgen receptor (AR) are important mediators of castration-resistant prostate cancer (CRPC) progression. Increased expression of several enzymes responsible for cholesterol synthesis and conversion into downstream androgens has been documented in human CRPC tumors in comparison to primary tumors. Based on these observations it is hypothesized that cholesterol and its overall regulation within the cell are altered, thus modifying precursor levels for de novo androgen synthesis within the castrate tumoral environment.

METHODS

Tumoral steroid levels were assessed by LC-MS. Free and esterified cholesterol was quantified by LC-MS and a fluorescent assay. Gene and protein expression were assessed by RT-PCR and immunoblotting.

RESULTS

Herein, using a prostate cancer xenograft mouse model it is demonstrated by Western blot analysis that proteins responsible for cholesterol regulation (LDL-r, SR-B1, HMG-CoA reductase, ACAT1,2, ABCA1) are altered during disease progression to increase influx and synthesis of cholesterol as well as free cholesterol formation from cholesteryl ester stores. In turn this can provide increased amounts of precursor for intratumoral steroidogenesis after castration. Androgens- testosterone and dihydrotestosterone- coincidently increase at CRPC to physiologically relevant levels leading to the induction of AR expression and PSA production. Furthermore, cellular cholesterol homeostasis is maintained by increased cholesterol efflux at CRPC so that excess free cholesterol does not cause toxicity to the tumor cells.

CONCLUSIONS

Cellular cholesterol regulation processes are altered during progression to CRPC. Free cholesterol from increased biosynthesis or uptake is likely a precursor for intratumoral de novo androgen synthesis.

Molecular chaperones mediate multiple aspects of steroid receptor function, but the physiological importance of most receptor-associated cochaperones has not been determined. To help fill this gap, we targeted for disruption the mouse gene for the 52-kDa FK506 binding protein, FKBP52, a 90-kDa heat shock protein (Hsp90)-binding immunophilin found in steroid receptor complexes. A mouse line lacking FKBP52 (52KO) was generated and characterized. Male 52KO mice have several defects in reproductive tissues consistent with androgen insensitivity; among these defects are ambiguous external genitalia and dysgenic prostate. FKBP52 and androgen receptor (AR) are coexpressed in prostate epithelial cells of wild-type mice. However, FKBP52 and AR are similarly coexpressed in testis even though testis morphology and spermatogenesis in 52KO males are usually normal. Molecular studies confirm that FKBP52 is a component of AR complexes, and cellular studies in yeast and human cell models demonstrate that FKBP52 can enhance AR-mediated transactivation. AR enhancement requires FKBP52 peptidylprolyl isomerase activity as well as Hsp90-binding ability, and enhancement probably relates to an affect of FKBP52 on AR-folding pathways. In the presence of FKBP52, but not other cochaperones, the function of a minimally active AR point mutant can be dramatically restored. We conclude that FKBP52 is an AR folding factor that has critically important physiological roles in some male reproductive tissues.

The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.

BACKGROUND

alpha(1)-adrenergic receptors (alpha(1)ARs) regulate blood pressure, regional vascular resistance, and venous capacitance; the exact subtype (alpha(1a), alpha(1b), alpha(1 d)) mediating these effects is unknown and varies with species studied. In order to understand mechanisms underlying cardiovascular responses to acute stress and chronic catecholamine exposure (as seen with aging), we tested two hypotheses: (1) human alpha(1)AR subtype expression differs with vascular bed, and (2) age influences human vascular alpha(1)AR subtype expression.

RESULTS

Five hundred vessels from 384 patients were examined for alpha(1)AR subtype distribution at mRNA and protein levels (RNase protection assays, ligand binding, contraction assays). Overall vessel alpha(1)AR density is 16+/-2.3fmol/mg total protein. alpha(1a)AR predominates in arteries at mRNA (P<0.001) and protein (P<0.05) levels; all 3 subtypes are present in veins. Furthermore, alpha(1)AR mRNA subtype expression varies with vessel bed (alpha(1a) higher in splanchnic versus central arteries, P<0.05); competition analysis (selected vessels) and functional assays demonstrate alpha(1a) and alpha(1b)-mediated mammary artery contraction. Overall alpha(1)AR expression doubles with age (<55 versus>> or = 65 years) in mammary artery (no change in saphenous vein), accompanied by increased alpha(1b>>alpha(1a) expression (P< = 0.001).

CONCLUSIONS

Human vascular alpha(1)AR subtype distribution differs from animal models, varies with vessel bed, correlates with contraction in mammary artery, and is modulated by aging. These findings provide potential novel targets for therapeutic intervention in many clinical settings.