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Retinal pigment epithelium (RPE) degeneration plays an important role in a group of retinal disorders such as retinal degeneration (RD) and age-related macular degeneration (AMD). The mechanism of RPE cell death is not yet fully elucidated. Ferroptosis, a novel regulated cell death pathway, participates in cancer and several neurodegenerative diseases. Glutathione peroxidase 4 (GPx-4) and ferroptosis suppressor protein 1 (FSP1) have been proposed to be two main regulators of ferroptosis in these diseases; yet, their roles in RPE degeneration remain elusive. Here, we report that both FSP1-CoQ

One hallmark of cancer cells is sustaining proliferative signaling that leads to uncontrolled cell proliferation. Both the Forkhead box (FOX) M1 transcription factor and the Epidermal Growth Factor (EGF) receptor Pathway Substrate 8 (EPS8) are known to be activated by mitogenic signaling and their levels upregulated in cancer. Well-known to regulate Rac-mediated actin remodeling at the cell cortex, EPS8 carries a nuclear localization signal but its possible nuclear role remains unclear. Here, we demonstrated interaction of FOXM1 with EPS8 in yeast two-hybrid and immunoprecipitation assays. Immunostaining revealed co-localization of the two proteins during G2/M phase of the cell cycle. EPS8 became nuclear localized when CRM1/Exportin 1-dependent nuclear export was inhibited by Leptomycin B, and a functional nuclear export signal could be identified within EPS8 using EGFP-tagging and site-directed mutagenesis. Downregulation of EPS8 using shRNAs suppressed expression of FOXM1 and the FOXM1-target CCNB1, and slowed down G2/M transition in cervical cancer cells. Chromatin immunoprecipitation analysis indicated recruitment of EPS8 to the CCNB1 and CDC25B promoters. Taken together, our findings support a novel partnering role of EPS8 with FOXM1 in the regulation of cancer cell proliferation and provides interesting insight into future design of therapeutic strategy to inhibit cancer cell proliferation.

Secreted PDZD2 (sPDZD2) is a signaling molecule generated upon proteolytic processing of the multi-PDZ-containing protein PDZD2. Previous analysis of gene-trap mice deficient in the synthesis of full-length PDZD2, but not the secreted form, revealed a role of PDZD2 in the regulation of glucose-stimulated insulin secretion. Here, using the pancreatic INS-1E β cells as in vitro model, we showed that depletion of PDZD2/sPDZD2 by RNA interference suppressed the expression of β-cell genes Ins1, Glut2 and MafA whereas treatment with recombinant sPDZD2 rescued the suppressive effect. Similar to GLP-1, sPDZD2 stimulated intracellular cAMP levels, activated β-cell gene expression in a PKA-dependent manner and induced the phosphorylation and nuclear localization of PDX1. Depletion of PDX1 inhibited the sPDZD2 insulinotropic effect, which could also be demonstrated in mouse islets. In summary, our findings are consistent with sPDZD2 serving a signaling function in regulating β-cell gene expression.

Effective retinal drug delivery remains a challenge for treating vision-threatening diseases. Encapsulated-cell therapy (ECT) can provide local drug delivery without repeated invasive injections but is plagued by unsteady performance and biosafety issues. Here, an injectable composite alginate-collagen (CAC) ECT gel with a Tet-on inducible pro-caspase 8 mechanism that acted as an orally-inducible biosafety switch was developed for safer drug delivery. The optimised gels (2 mg/ml collagen, 1.5% high molecular weight alginate and 50,000 cells/gel) could be effectively terminated in vitro (≥20 pg/ml Doxycycline) and in vivo (1 mg/ml oral Doxycycline after 48 h). Also, they displayed effective proliferation control and continuous delivery of bioactive glial-cell derived neurotrophic factor (GDNF) with no significant gel degradation in vitro and in rat vitreous. Most importantly, intravitreally injected gels demonstrated therapeutic efficacy in Royal College of Surgeons rats with degenerating retina in reducing photoreceptor apoptosis and retina function loss. Furthermore, double gel injections into the same eye yielded better outcomes without compromising gel viability. Retrieved gels showed no host-tissue attachment or cell-protrusion 6 months post-implantation. The CAC ECT system exhibited mechanical stability, good encapsulation power, cell viability support, multiplexed GDNF dosage, and compatibility with different cell types (HEK293 and ARPE-19) without immunosuppressant, making it an attractive, safe and well-controlled platform for treating various eye diseases.

Background: The tumour-node-metastasis (TNM) classification is the most widely used tool for penile cancer. However, the current system is based on few studies and has been unchanged since 2009. We determined whether a modified pathological N staging system that incorporates the laterality and number of lymph node metastases (LNMs) increases the accuracy of the results in predicting survival compared with the 7th edition of the pathological N staging system of the American Joint Committee on Cancer (AJCC) for penile cancer. Methods: The clinical and histopathologic data from 111 patients with penile cancer with LNMs were analysed. Univariate and multivariate Cox proportional hazard regression analyses were used to determine the impact of the clinical and pathological factors on disease-specific survival of these patients. The predictive accuracy was further assessed using the concordance index. Results: According to the 7th edition of the pathological N classification, the 3-year disease-specific survival (DSS) rates for patients with pN1, pN2, and pN3 disease are 89.6%, 65.9%, and 33.6%, respectively (PN1–N2=0.030, PN2–N3

// Patty C.H. Cai 1 , Lei Shi 2 , Vincent W.S. Liu 1 , Hermit W.M. Tang 1 , Iris J. Liu 1 , Thomas H.Y. Leung 1 , Karen K.L. Chan 1 , Judy W.P. Yam 3 , Kwok-Ming Yao 2 , Hextan Y.S. Ngan 1 and David W. Chan 1 1 Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, P.R.China 2 Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, P.R.China 3 Department of Pathology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, P.R.China Correspondence: Hextan YS Ngan, email: // David W Chan, email: // Keywords : TAK1, NF-κB signaling, ovarian cancer, high-grade tumor Received : May 07, 2014 Accepted : July 26, 2014 Published : July 27, 2014 Abstract Transforming growth factor (TGF)-β-activating kinase 1 (TAK1) is a serine/threonine kinase which is frequently associated with human cancer progression. However, its functional role in tumorigenesis is still controversial. Here, we report that TAK1 enhances the oncogenic capacity of ovarian cancer cells through the activation of NF-κB signaling. We found that TAK1 is frequently upregulated and significantly associated with high-grade and metastatic ovarian cancers. Mechanistic studies showed that Ser412 phosphorylation is required for TAK1 in activating NF-κB signaling and promotes aggressiveness of ovarian cancer cells. Conversely, suppression of TAK1 activity by point mutation at Ser412, RNAi mediated gene knockdown or TAK1 specific inhibitor ((5Z) -7-Oxozeaenol) remarkably impairs tumor growth and metastasis in ovarian cancer in vitro and in vivo . Our study underscores the importance of targeting TAK1 as a promising therapeutic approach to counteract the ovarian cancer progression.

Our laboratory has recently demonstrated a melatonin MT1 receptor-mediated antiproliferative signaling mechanism in androgen receptor (AR)-positive prostate epithelial cells which involves up-regulation of p27(Kip1) through dual activation of Gα(s)/protein kinase A (PKA) and Gα(q)/protein kinase C (PKC) in parallel, and down-regulation of activated AR signaling via PKC stimulation. The aim of the present investigation was to identify the transcription factor that mediates melatonin's up-regulatory effect on p27(Kip1) in LNCaP and 22Rv1 prostate cancer cells. Deletion mapping and reporter assays of the p27(Kip1) promoter revealed that the putative melatonin-responsive transcription factor binds to a 116 base-pair region of the promoter sequence, which contains a potential nuclear factor kappa B (NF-κB) binding site. When the NF-κB binding site was abolished by site-directed mutagenesis, the stimulatory effect of melatonin on p27(Kip1) promoter activity was mitigated. Notably, melatonin inhibited the DNA binding of activated NF-κB via MT1 receptor-induced PKA and PKC stimulation. Furthermore, melatonin's up-regulatory effect on p27(Kip1) transcription and consequent cell antiproliferation were abrogated by NF-κB activator but mimicked by NF-κB inhibitor. The results indicate that inhibition of constitutively active NF-κB via melatonin MT1 receptor-induced dual activation of (Gα(s)) PKA and (Gα(q)) PKC can de-repress the p27(Kip1) promoter leading to transcriptional up-regulation of p27(Kip1). MT1 receptor-mediated inhibition of activated NF-κB signaling provides a novel mechanism supporting the use of melatonin in prostate cancer chemoprevention and therapy.

BackgroundThe phosphoinositide 3 kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)/mammalian target of rapamycin (mTOR) pathway is frequently aberrantly activated in ovarian cancer and confers the chemoresistant phenotype of ovarian cancer cells. LY294002 (PI3K inhibitor) and metformin (5′-adenosine monophosphate [AMP]-activated protein kinase [AMPK] activator) are 2 drugs that were known to inhibit mTOR expression through the AKT-dependent and AKT-independent pathways, respectively. In this study, we explored the effectiveness of LY294002 and metformin in combination on inhibition of ovarian cancer cell growth.MethodsWestern blotting was used to detect the changes of PI3K/AKT/mTOR and AMPK/acetyl-CoA carboxylase (ACC) signaling activities, cell cycle control, and apoptosis. Cell growth was evaluated by cell proliferation, colony formation, and soft agar assays. Flow cytometry was used to study cell cycle distribution and cell death upon drug treatment.ResultsOur study showed that LY294002 and metformin in combination could simultaneously enhance the repression of the PI3K/AKT/mTOR pathway and the activation of the AMPK/ACC pathway. The downstream target of AKT and AMPK, mTOR, was cooperatively repressed when the drugs were used together. The cell cycle regulatory factors, p53, p27, and p21, were up-regulated. On the other hand, caspase 3 and poly (ADP-ribose) polymerase activities involved in apoptosis were also activated. Cell growth assays indicated that LY294002 and metformin could effectively inhibit ovarian cancer cell growth. Flow cytometry analysis showed that the treatment of the 2 drugs mentioned above induced cell cycle arrest at G1 phase and increased sub-G1 apoptotic cells.ConclusionThe combinational use of LY294002 and metformin can enhance inhibition of the growth and induction of the apoptosis of ovarian cancer cells. Our results may provide significant insight into the future therapeutic regimens in ovarian cancer.

Motivation:The interaction between transcription factor (TF) and transcription factor binding site (TFBS) is essential for gene regulation. Mutation in either the TF or the TFBS may weaken their interaction and thus result in abnormalities. To maintain such vital interaction, a mutation in one of the interacting partners might be compensated by a corresponding mutation in its binding partner during the course of evolution. Confirming this co-evolutionary relationship will guide us in designing protein sequences to target a specific DNA sequence or in predicting TFBS for poorly studied proteins, or even correcting and rescuing disease mutations in clinical applications. Results: Based on six, publicly available, experimentally validated TF–TFBS binding datasets for the basic Helix–Loop–Helix (bHLH) family, Homeo family, High-Mobility Group (HMG) family and Transient Receptor Potential channels (TRP) family, we showed that the evolutions of the TFs and their TFBSs are significantly correlated across eukaryotes. We further developed a mutual information-based method to identify co-evolved protein residues and DNA bases. This research sheds light on the dynamic relationship between TF and TFBS during their evolution. The same principle and strategy can be applied to co-evolutionary studies on protein–DNA interactions in other protein families. Availability: All the datasets, scripts and other related files have been made freely available at: http://jjwanglab.org/co-evo. Contact: junwen@uw.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Aberrant activation of Hedgehog (Hh) signalling has been implicated in the pathogenesis of human cancers. However, the cognate molecular mechanisms contributing to this disregulated pathway are incompletely understood. In this study, we showed that Zic2 was frequently over-expressed and associated with high-grade cervical cancer (p = 0.032), high levels of Gli1 (p < 0.001) and CyclinD1 (p < 0.001) by immunohistochemical and quantitative RT-PCR analyses. Further biochemical studies using luciferase reporter, co-immunoprecipitation, subcellular fractionation and immunofluorescence analyses demonstrated that Zic2 can physically interact with Gli1 and retain it in the nucleus, which in turn increases Gli-mediated transcriptional activity. Gain- and loss-of-function analyses of Zic2 showed that Zic2 could increase Hh signalling activity, cell proliferation and anchorage-independent growth ability in cervical cancer cells. Conversely, deletion of the zinc finger domain at C-terminus of Zic2 significantly abrogated its interaction with Gli1, the retention of Gli1 in the nucleus, effects on Hh signalling activity and oncogenic properties in cervical cancer cells. Our findings suggest that Zic2 is a positive modulator increasing Gli1 transcriptional and oncogenic activity by retaining Gli1 in the nucleus of cervical cancer cells.

Melatonin has been shown to inhibit the proliferation of malignant and transformed human prostate epithelial cells by transcriptional up-regulation of p27(Kip1) expression via MTNR1A receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel. Given that melatonin MTNR1A receptor is a G protein-coupled receptor, this study was conducted to identify the specific G proteins that mediate the antiproliferative action of melatonin on human prostate epithelial cells. In 22Rv1 and RWPE-1 cells, knockdown of either Gα(s) or Gα(q) , but not Gα(i2) expression by RNA interference, abrogated the effects of melatonin on p27(Kip1) and cell proliferation. Conversely, cellular overexpression of activated mutants of Gα(s) and Gα(q) in 22Rv1 and RWPE-1 cells mimicked the effects of melatonin on prostate epithelial cell antiproliferation by increasing p27(Kip1) expression through downstream activation of PKA and PKC in parallel. Moreover, melatonin or 2-iodomelatonin induced elevation of adenosine-3',5'-cyclic monophosphate (cAMP) in 22Rv1 and RWPE-1 cells. The effects of 2-iodomelatonin on cAMP were blocked by the nonselective MTNR1A/MTNR1B receptor antagonist luzindole but were not affected by the selective MTNR1B receptor antagonist 4-phenyl-2-propionamidotetraline (4-P-PDOT). Furthermore, knockdown of Gα(s) mitigated the stimulatory effects of 2-iodomelatonin on cAMP. Collectively, the data demonstrated, for the first time, functional coupling of MTNR1A receptor to Gα(s) in cancerous or transformed human cells expressing endogenous melatonin receptors. Our results also showed that dual activation of Gα(s) and Gα(q) proteins is involved in the signal transduction of MTNR1A receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells.

The voltage-gated sodium channel NaV1.8 is expressed exclusively in nociceptive sensory neurons and plays an important role in pain pathways. NaV1.8 cannot be functionally expressed in non-neuronal cells even in the presence of β-subunits. We have previously identified Pdzd2, a multi PDZ-domain protein, as a potential interactor for NaV1.8. Here we report that Pdzd2 binds directly to the intracellular loops of NaV1.8 and NaV1.7. The endogenous NaV1.8 current in sensory neurons is inhibited by antisense- and siRNA-mediated downregulation of Pdzd2. However, no marked change in pain behaviours is observed in Pdzd2-decificent mice. This may be due to compensatory upregulation of p11, another regulatory factor for NaV1.8, in dorsal root ganglia of Pdzd2-deficient mice. These findings reveal that Pdzd2 and p11 play collaborative roles in regulation of NaV1.8 expression in sensory neurons. © 2009 Elsevier Inc. All rights reserved., link_to_OA_fulltext

We recently reported the isolation and characterization of a population of pancreatic progenitor cells (PPCs) from early trimester human fetal pancreata. The PPCs, being the forerunners of adult pancreatic cell lineages, were amenable to growth and differentiation into insulin-secreting islet-like cell clusters (ICCs) upon stimulation by adequate morphogens. Of note, a novel morphogenic factor, PDZ-domain containing-2 (PDZD2) and its secreted form (sPDZD2) were ubiquitously expressed in the PPCs. Our goals for this study were to evaluate the potential role of sPDZD2 in stimulating PPC differentiation and to establish the optimal concentration for such stimulation. We found that 10(-9)M sPDZD2 promoted PPC differentiation, as evidenced by the upregulation of the pancreatic endocrine markers (PDX-1, NGN3, NEURO-D, ISL-1, NKX 2.2, NKX 6.1) and INSULIN mRNA. Inhibited endogenous production of sPDZD2 suppressed expression of these factors. Secreted PDZD2 treatment significantly elevated the C-peptide content of the ICCs and increased the basal rate of insulin secretion. However, they remained unresponsive to glucose stimulation, reflected by a minimal increase in GLUT-2 and GLUCOKINASE mRNA expression. Interestingly, sPDZD2 treatment induced increased expression of the L-type voltage-gated calcium channel (Ca(v)1.2) in the ICCs, triggering calcium ion influx under KCl stimulation and conferring an ability to secrete insulin in response to KCl. Pancreatic progenitor cells from 10- and 13-week fetal pancreata showed peak expression of endogenous sPDZD2, implying that sPDZD2 has a specific role in islet development during the first trimester. In conclusion, our data suggest that sPDZD2 promotes functional maturation of human fetal PPC-derived ICCs, thus enhancing its transplanting potentials.

Early-trimester human fetal pancreas is a promising potential source of pancreatic progenitor cells. However, the ethical controversy associated with the source of these cells, and technical difficulties associated with their differentiation into insulin-producing cells have limited both their availability and utility. This study aimed to characterize a population of pancreatic progenitor cells (PPCs) isolated from human fetus and describe the effects of a novel factor, PDZ-domain containing-2 (PDZD2), and its secreted form (sPDZD2), on PPC proliferation and differentiation. In particular, we examined and characterized the expression of several stem cell (nestin, ABCG2, c-kit), growth and differentiation markers (GLP-1R, c-met, erbB1), and PDZD2 in PPCs by RT-PCR, Western blot, and immunocytochemistry. We also examined the effects of sPDZD2 on PPC proliferation and differentiation by examining BrdU incorporation, MTT, cell number, and real-time PCR as well as ELISA. PPCs were isolated, cultured and characterized from human fetal pancreas. PDZD2 and sPDZD2 were detected at high levels in both human fetal pancreas and in PPCs. sPDZD2 acted as a potent mitogen on PPCs, and inhibited the differentiation of PPC-derived islet-like cell-clusters (ICCs), evidenced by the downregulation of Isl-1, Pdx-1, and insulin mRNA levels. sPDZD2 treatment also reduced levels of C-peptide in ICCs. These results show that a novel pancreatic developmental factor, PDZD2, is sufficient to promote the proliferation of human fetal PPCs while limiting differentiation of ICCs into islet/endocrine cells. Findings from this study will contribute to the development of improved methods for islet transplantation therapy in the treatment of diabetes.

The Forkhead Box M1 (FOXM1) transcription factor plays a crucial role in regulating expression of cell cycle genes which are essentially involved in cell proliferation, differentiation and transformation. Recent studies have reported that aberrant expression of FOXM1 in a variety of human cancers is associated with their aggressive behaviour. However, the functional significance of FOXM1 in human cervical cancer is not known. We have shown that FOXM1 was significantly over-expressed in cervical squamous cell carcinoma (SCC) compared to normal cervical epithelium immunohistochemically (p < 0.001). In addition, intratumoural FOXM1 positivity was increased in cervical intraepithelial neoplasia (CIN) and carcinoma, compared with that in normal epithelium, indicating that FOXM1 is involved in tumour progression. Indeed, this is supported by clinicopathological analysis that the over-expression of FOXM1 was significantly associated with tumour late stage (p = 0.012) and cell proliferation marker, Ki67 (p < 0.001). Functionally, enforced expression of FOXM1c in FOXM1-deficient cervical cancer cells (C33A) remarkably enhanced cell proliferation and anchorage-independent growth ability. Conversely, depletion of FOXM1 by RNA interference in FOXM1-over-expressing cervical cancer cells (SiHa) caused significant inhibition on cell proliferation and anchorage-independent growth ability on soft agar. This inhibitory phenomenon was associated with the reduced expressions of cyclin B1, cyclinD1 and cdc25B but increased expression of p27Kip1 and p21Cip1. Our findings suggest a role for FOXM1 in the development and pathogenesis of human cervical SCC. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Human embryonic stem cells (hESCs) exhibit unique cell cycle structure, self-renewal and pluripotency. The Forkhead box transcription factor M1 (FOXM1) is critically required for the maintenance of pluripotency in mouse embryonic stem cells and mouse embryonal carcinoma cells, but its role in hESCs remains unclear. Here, we show that FOXM1 expression was enriched in undifferentiated hESCs and was regulated in a cell cycle-dependent manner with peak levels detected at the G2/M phase. Expression of FOXM1 did not correlate with OCT4 and NANOG during in vitro differentiation of hESCs. Importantly, knockdown of FOXM1 expression led to aberrant cell cycle distribution with impairment in mitotic progression but showed no profound effect on the undifferentiated state. Interestingly, FOXM1 depletion sensitized hESCs to oxidative stress. Moreover, genome-wide analysis of FOXM1 targets by ChIP-seq identified genes important for M phase including CCNB1 and CDK1, which were subsequently confirmed by ChIP and RNA interference analyses. Further peak set comparison against a differentiating hESC line and a cancer cell line revealed a substantial difference in the genomic binding profile of FOXM1 in hESCs. Taken together, our findings provide the first evidence to support FOXM1 as an important regulator of cell cycle progression and defense against oxidative stress in hESCs.

A possible role of the PDZ domain-containing protein 2 (PDZD2) in prostate tumorigenesis has been suggested. Besides, PDZD2 is posttranslationally cleaved by a caspasedependent mechanism to form a secreted PDZ domaincontaining protein 2 (sPDZD2) with unknown functions in humans. In this study, we demonstrate the endogenous expression of PDZD2 and secretion of sPDZD2 in cancerous DU145, PC-3, 22Rv1, LNCaP, and immortalized RWPE-1 prostate epithelial cells. Inhibition of endogenous sPDZD2 production and secretion by DU145, PC-3, 22Rv1, and RWPE-1 cells via the caspase-3 inhibitor Z-DEVD-FMK resulted in increased cell proliferation, which was abrogated by treatment with exogenous recombinant sPDZD2. Whereas sPDZD2induced antiproliferation in DU145, PC-3, and 22Rv1 cells, it induced apoptosis in LNCaP cells. The data suggest that endogenous sPDZD2, produced by caspase-3-mediated cleavage from PDZD2, may function as a novel autocrine growth suppressor for human prostate cancer cells. The antiproliferative effect of sPDZD2 was apparently mediated through slowing the entry of DU145, PC-3, and 22Rv1 cells into the S phase of the cell cycle. In DU145 cells, this can be attributed to stimulated p53 and p21 CIP1/WAF1 expression by sPDZD2. On the otherhand,theapoptoticeffectofsPDZD2onLNCaPcellswas apparently mediated via p53-independent Bad stimulation. Together our results indicate the presence of p53-dependent and p53-independent PDZD2/sPDZD2 autocrine growth suppressive signaling pathways in human prostate cancer cells and suggest a novel therapeutic approach of harnessing the latent tumor-suppressive potential of an endogenous autocrine signaling protein like sPDZD2 to inhibit prostate cancer growth. (Endocrinology 147: 5023–5033, 2006)

Transcriptional coactivators are essential mediators of signal amplification in the regulation of gene expression in response to hormones and extracellular signals. We previously identified Bridge-1 (PSMD9) as a PDZ-domain coregulator that augments insulin gene transcription via interactions with the basic helix-loop-helix transcription factors E12 and E47, and that increases transcriptional activation by the homeodomain transcription factor PDX-1. In these studies, we find that transcriptional activation by Bridge-1 can be regulated via interactions with the histone acetyltransferase and nuclear receptor coactivator p300. In transfection assays, transcriptional activation by Bridge-1 is increased by the inhibition of endogenous histone deacetylase activity with trichostatin A, indicating that the transcriptional activation function of Bridge-1 can be regulated by histone modifications. The exogenous expression of p300 enhances the transcriptional activation by Bridge-1 in a dose-dependent manner. In contrast, the sequestration of p300 by the overexpression of the adenoviral protein E1A, but not by an E1A mutant protein that is unable to interact with p300, suppresses the transcriptional activation by Bridge-1. We demonstrate that p300 and Bridge-1 proteins interact in immunopre-cipitation and glutathione-S-transferase (GST) pull-down assays. Bridge-1 interacts directly with multiple regions within p300 that encompass C/H1 or C/H2 cysteine- and histidine-rich protein interaction domains and the histone acetyltransferase domain. Deletion or point mutagenesis of the Bridge-1 PDZ domain substantially reduces transcriptional activation by Bridge-1 and interrupts interactions with p300. We propose that p300 interactions with Bridge-1 can augment the transcriptional activation of regulatory target genes by Bridge-1.

We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 5'-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by in vitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region -415/+54 containing Ethylene Responsive Element ERE(-355/-348) conferred threefold ethylene-induction of GUS expression, while -827/+54 which also contains ERE(-683/-676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-luc constructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at -795/-787 and -785/-777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other than Arabidopsis and that functional EEs and EREs are present in the 5'-flanking region of a gene encoding cysteine proteinase.

VCY2 is a testis-specific protein that locates in a frequently deleted azoospermia factor c region on chromosome Yq. Although its genomic structure has been characterized, the function of VCY2 is still unknown. To gain insight regarding the likely function of VCY2, we investigated the proteins that interact with VCY2 using the yeast two-hybrid system. We identified a novel VCY2 interaction partner, named VCY2IP-1, that encodes an open reading frame of 1059 amino acids. The amino acid sequence of VCY2IP-1 shows 59.3% and 41.9% homology to two human microtubule-associated proteins (MAPs), MAP1B and MAP1A, respectively. VCY2IP-1 has an extensive homology to the N-terminus and C-terminus regions of MAP1B and MAP1A, placing it within a large family of MAPs. We mapped VCY2IP-1 to chromosome 19p13.11. The VCY2IP-1 gene spans 15 kilobases (kb) and consists of seven exons. Northern blot analysis identified a single, intense band of approximately 3.2-kb VCY2IP-1 transcript, predominantly expressed in human testis. In situ hybridization of human testicular sections showed the localization of VCY2IP-1 transcripts in germ cells, and reverse transcription-polymerase chain reaction analysis demonstrated the presence of VCY2 and VCY2IP-1 transcripts in human ejaculated spermatozoa. Our expression data support the involvement of VCY2 and VCY2IP-1 in spermatogenesis. Based on the high homology of VCY2IP-1 with MAPs, we propose the involvement of VCY2 in the cytoskeletal network via interaction with VCY2IP-1.

We have previously shown that SmCP, the gene encoding Solanum melongena cysteine proteinase, is expressed during developmental events associated with programmed cell death (PCD) suggesting its involvement in protein degradation during these events (Xu and Chye, Plant Journal 17 (1999) 321–327). Here, we investigated the regulation of SmCP expression and showed that it is ethylene-inducible and is under circadian control. This circadian rhythm is entrained by light/dark (LD) cycling with peak expression in the late light period, as opposed to that in early light for rbcS, suggesting that protein degradation and photosynthesis are temporally separated by circadian control. Northern blot analysis shows that the pattern of ethylene induction of SmCP is consistent with our previous observation of its significantly increased expression at leaf senescence and fruit ripening when endogenous ethylene is abundant. To further understand SmCP regulation, we have cloned the SmCP promoter and identified a G-box (CACGTG) at −85/−80 by DNase I footprinting analysis of the −221/+17 region. Its specific interaction with nuclear proteins in S. melongena leaves and fruits was confirmed by competitive electrophoretic mobility shift assays using oligonucleotides containing the G-box and mutant derivatives. G-box binding activity was stronger in senescent than young fruits. In circadian-regulated leaves, stronger binding activity coincided with peak circadian expression of SmCP. This correlation between binding activity and expression suggests that G-box binding factors enhance SmCP transcription and that the G-box likely plays a role in circadian regulation of genes affected by LD cycling.

AMP-activated protein kinase (AMPK) is a key energy sensor that is involved in regulating cell metabolism. Our previous study revealed that the subunits of the heterotimeric AMPK enzyme are diversely expressed during ovarian cancer progression. However, the impact of the variable expression of these AMPK subunits in ovarian cancer oncogenesis remains obscure. Here, we provide evidence to show that reduced expression of the AMPK-beta1 subunit during tumor progression is associated with the increased oncogenic capacity of advanced ovarian cancer cells. Immunohistochemical analysis revealed that AMPK-beta1 levels were reduced in advanced-stage (P = 0.008), high-grade (P = 0.013) and metastatic ovarian cancers (P = 0.008). Intriguingly, down-regulation of AMPK-beta1 was progressively reduced from tumor stages 1 to 3 of ovarian cancer. Functionally, enforced expression of AMPK-beta1 inhibited ovarian-cancer-cell proliferation, anchorage-independent cell growth, cell migration and invasion. Conversely, depletion of AMPK-beta1 by siRNA enhanced the oncogenic capacities of ovarian cancer cells, suggesting that the loss of AMPK-beta1 favors the aggressiveness of ovarian cancer. Mechanistically, enforced expression of AMPK-beta1 increased AMPK activity, which, in turn, induced cell-cycle arrest via inhibition of AKT/ERK signaling activity as well as impaired cell migration/invasion through the suppression of JNK signaling in ovarian cancer cells. Taken together, these findings suggest that the reduced expression of AMPK-beta1 confers lower AMPK activity, which enhances the oncogenic capacity of advanced-stage ovarian cancer., published_or_final_version

Melatonin inhibited thymidine incorporation into human choriocarcinoma JEG-3 cells at physiological and pharmacological concentrations in the present study. Gene expression of MT2 receptor, but not that of mt1 receptor, was detected in JEG-3 cells by reverse transcription-polymerase chain reaction (RT-PCR). The gene expression profile of the two human melatonin receptor subtypes in JEG-3 cells was identical to that previously reported for JAr cells, whose proliferation had also been shown to be similarly inhibited by physiological and pharmacological concentrations of melatonin. In contrast, melatonin had no effect on thymidine incorporation into 3A-Sub-E cells (a transformed trophoblast cell line), in which gene expression of both receptor subtypes could not be detected. The data suggest that in human placental trophoblasts, a correlation may exist between MT2 receptor gene expression and the direct anti-proliferative action of melatonin. Although melatonin has been reported to induce G1/S delay in cell cycle progression of JAr cells, no significant changes in the percentages of JEG-3 cells in different cell cycle phases upon melatonin treatment was recorded by flow cytometric analysis. This indicates that G1/S transition delay is probably not an important cellular mechanism in the direct anti-proliferative action of melatonin on human JEG-3 cells in vitro. Furthermore, melatonin inhibited the growth of both JAr and JEG-3 xenograft tumors in athymic nude mice, and prolonged the survival of those animals that developed choriocarcinoma. While the number of apoptotic tumor cells was not increased by melatonin, the pineal hormone induced significant decreases in the numbers of JAr and JEG-3 cells expressing proliferating cell nuclear antigen (PCNA) and cyclin A in the tumors. Taking into account both the in vitro and in vivo findings, it is likely that the inhibitory effect of melatonin on choriocarcinoma JAr and JEG-3 cell proliferation in vivo is largely a direct action of the hormone on the tumor cells.

The forkhead box (FOX) M1 transcription factor is required to maintain the proliferation of cancer cells. Two transcriptionally active isoforms of FOXM1, FOXM1b and FOXM1c, have been identified, but their functional differences remain unclear. FOXM1c is distinguished from FOXM1b by an extra exon (exon Va) that contains an ERK1/2 target sequence. Based on a literature search and quantitative PCR analysis, we concluded that FOXM1b is the predominant isoform that is overexpressed in cancers. The further characterization of FOXM1b and FOXM1c revealed two interesting differences. First, FOXM1b exhibited a higher transforming ability than FOXM1c in a soft agar assay. Second, the transactivating activity of FOXM1c, but not that of FOXM1b, was sensitive to activation by RAF/MEK/MAPK signaling. Importantly, the MEK1 activation of FOXM1c was associated with proteolytic processing to generate short forms that might represent constitutively active forms missing the N-terminal inhibitory domain; in contrast, the proteolytic processing of FOXM1b did not require MEK1 activation. Our findings suggest that FOXM1b is functionally more active. These results provide novel insights into the regulation of FOXM1 activity and its role in tumorigenesis.

OBJECTIVE: Suppression of Kinesin-1 by antisense oligonucleotides, or overexpression of dominant-negative acting kinesin heavy chain, has been reported to affect the sustained phase of glucose-stimulated insulin secretion in β-cells in vitro. In this study, we examined the in vivo physiological role of Kinesin-1 in β-cell development and function. RESEARCH DESIGN AND METHODS: A Cre-LoxP strategy was used to generate conditional knockout mice in which the Kif5b gene is specifically inactivated in pancreatic β-cells. Physiological and histological analyses were carried out in Kif5b knockout mice as well as littermate controls. RESULTS: Mice with β-cell specific deletion of Kif5b (Kif5b fl/-: RIP2-Cre) displayed significantly retarded growth as well as slight hyperglycemia in both nonfasting and 16-h fasting conditions compared with control littermates. In addition, Kif5b fl/-: RIP2-Cre mice displayed significant glucose intolerance, which was not due to insulin resistance but was related to an insulin secretory defect in response to glucose challenge. These defects of β-cell function in mutant mice were not coupled with observable changes in islet morphology, islet cell composition, or β-cell size. However, compared with controls, pancreas of Kif5b fl/-: RIP2-Cre mice exhibited both reduced islet size and increased islet number, concomitant with an increased insulin vesicle density in β-cells. CONCLUSIONS: In addition to being essential for maintaining glucose homeostasis and regulating β-cell function, Kif5b may be involved in β-cell development by regulating β-cell proliferation and insulin vesicle synthesis. © 2011 by the American Diabetes Association., published_or_final_version

Melatonin has been shown to inhibit the proliferation of malignant and transformed human prostate epithelial cells by transcriptional up-regulation of p27(Kip1) expression via MTNR1A receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel. Given that melatonin MTNR1A receptor is a G protein-coupled receptor, this study was conducted to identify the specific G proteins that mediate the antiproliferative action of melatonin on human prostate epithelial cells. In 22Rv1 and RWPE-1 cells, knockdown of either Gα(s) or Gα(q) , but not Gα(i2) expression by RNA interference, abrogated the effects of melatonin on p27(Kip1) and cell proliferation. Conversely, cellular overexpression of activated mutants of Gα(s) and Gα(q) in 22Rv1 and RWPE-1 cells mimicked the effects of melatonin on prostate epithelial cell antiproliferation by increasing p27(Kip1) expression through downstream activation of PKA and PKC in parallel. Moreover, melatonin or 2-iodomelatonin induced elevation of adenosine-3',5'-cyclic monophosphate (cAMP) in 22Rv1 and RWPE-1 cells. The effects of 2-iodomelatonin on cAMP were blocked by the nonselective MTNR1A/MTNR1B receptor antagonist luzindole but were not affected by the selective MTNR1B receptor antagonist 4-phenyl-2-propionamidotetraline (4-P-PDOT). Furthermore, knockdown of Gα(s) mitigated the stimulatory effects of 2-iodomelatonin on cAMP. Collectively, the data demonstrated, for the first time, functional coupling of MTNR1A receptor to Gα(s) in cancerous or transformed human cells expressing endogenous melatonin receptors. Our results also showed that dual activation of Gα(s) and Gα(q) proteins is involved in the signal transduction of MTNR1A receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells.

The proliferation-associated transcription factor FOXM1 is essential for cell cycle progression into mitosis. Using synchronized human fibroblasts we detected, by immunostaining, that FOXM1 is localized predominantly in the cytoplasm in cells at late-G1 and S phases. Nuclear translocation occurs just before progression into the G2/M phase of the cell cycle and requires activity of the Raf/MEK/MAPK signaling pathway. Using pharmacological modulators, we demonstrated that activity of the Raf/MEK/MAPK pathway is both necessary and sufficient for the nuclear translocation of FOXM1. Consistent with FoxM1c being the major isoform expressed in proliferating fibroblasts, constitutively active MEK1 enhances the transactivating effect of FOXM1c, but not FOXM1b, on the cyclin B1 promoter in transient reporter assays. Here, we describe in detail the methods involved in generating these findings, which support the notion that FOXM1 is an effector of Raf/MEK/MAPK signaling in G2/M regulation.

The proliferation-associated transcription factor FOXM1 is essential for cell cycle progression into mitosis. Using synchronized human fibroblasts we detected, by immunostaining, that FOXM1 is localized predominantly in the cytoplasm in cells at late-G1 and S phases. Nuclear translocation occurs just before progression into the G2/M phase of the cell cycle and requires activity of the Raf/MEK/MAPK signaling pathway. Using pharmacological modulators, we demonstrated that activity of the Raf/MEK/MAPK pathway is both necessary and sufficient for the nuclear translocation of FOXM1. Consistent with FoxM1c being the major isoform expressed in proliferating fibroblasts, constitutively active MEK1 enhances the transactivating effect of FOXM1c, but not FOXM1b, on the cyclin B1 promoter in transient reporter assays. Here, we describe in detail the methods involved in generating these findings, which support the notion that FOXM1 is an effector of Raf/MEK/MAPK signaling in G2/M regulation.

Expression of the multi-PDZ protein Pdzd2 (PDZ domain-containing protein 2) is enriched in pancreatic islet beta cells, but not in exocrine or alpha cells, suggesting a role for Pdzd2 in the regulation of pancreatic beta-cell function. To explore the in vivo function of Pdzd2, Pdzd2-deficient mice were generated. Homozygous Pdzd2 mutant mice were viable and their gross morphology appeared normal. Interestingly, Pdzd2-deficient mice showed enhanced glucose tolerance in intraperitoneal glucose tolerance tests and their plasma insulin levels indicated increased basal insulin secretion after fasting. Moreover, insulin release from mutant pancreatic islets was found to be twofold higher than from normal islets. To verify the functional defect in vitro, Pdzd2 was depleted in INS-1E cells using two siRNA duplexes. Pdzd2-depleted INS-1E cells also displayed increased insulin secretion at low concentrations of glucose. Our results provide the first evidence that Pdzd2 is required for normal regulation of basal insulin secretion.

Background:Loss of growth inhibitory response to transforming growth factor-Β (TGF-Β) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-Β/Smads signalling cascade contribute to the TGF-Β resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-Β/Smads signalling in ovarian cancer.Methods:FOXG1 and p21 WAF1/CIP1 expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21 WAF1/CIP1 transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells.Results:Quantitative RT-PCR and western blot analyses showed that FOXG1 was upregulated and inversely associated with the expression levels of p21 WAF1/CIP1 in ovarian cancer. The overexpression of FOXG1 was significantly correlated with high-grade ovarian cancer (P0.025). Immunohistochemical analysis on ovarian cancer tissue array was further evidenced that FOXG1 was highly expressed and significantly correlated with high-grade ovarian cancer (P0.048). Functionally, enforced expression of FOXG1 selectively blocked the TGF-Β-induced p21 WAF1/CIP1 expressions and increased cell proliferation in ovarian cancer cells. Conversely, FOXG1 knockdown resulted in a 20-26% decrease in cell proliferation together with 16-33% increase in p21 WAF1/CIP1 expression. Notably, FOXG1 was able to inhibit the p21 WAF1/CIP1 promoter activity in a p53-independent manner by transient reporter assays.ConclusionOur results suggest that FOXG1 acts as an oncoprotein inhibiting TGF-Β-mediated anti-proliferative responses in ovarian cancer cells through suppressing p21 WAF1/CIP1 transcription. © 2009 Cancer Research UK All rights reserved., published_or_final_version

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

Circannual variation in the human serum levels of prostate-specific antigen, a growth marker of the prostate gland, has been reported recently. The present study was conducted to investigate the role of the photoperiodic hormone melatonin (MLT) and its membrane receptors in the modulation of human prostate growth. Expression of MT(1) and MT(2) receptors was detected in benign human prostatic epithelial tissues and RWPE-1 cells. MLT and 2-iodomelatonin inhibited RWPE-1 cell proliferation and up-regulated p27(Kip1) gene and protein expression in the cells. The effects of MLT were blocked by the nonselective MT(1)/MT(2) receptor antagonist luzindole, but were not affected by the selective MT(2) receptor antagonist 4-phenyl-2-propionamidotetraline. Of note, the antiproliferative action of MLT on benign prostate epithelial RWPE-1 cells was effected via increased p27(Kip1) gene transcription through MT(1) receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel, a signaling process which has previously been demonstrated in 22Rv1 prostate cancer cells. Taken together, the demonstration of the MT(1)/PKA+PKC/p27(Kip1) antiproliferative pathway in benign and malignant prostate epithelial cell lines indicated the potential importance of this MLT receptor-mediated signaling mechanism in growth regulation of the human prostate gland in health and disease. Collectively, our data support the hypothesis that MLT may function as a negative mitogenic hormonal regulator of human prostate epithelial cell growth.

The RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) pathway plays a pivotal role in various cellular responses, including cellular growth, differentiation, survival and motility. Constitutive activation of the ERK pathway has been linked to the development and progression of human cancers. Here, we reported that mitogen-activated protein kinase phosphatase (MKP)-3, a negative regulator of ERK1/2, lost its expression particularly in the protein level, was significantly correlated with high ERK1/2 activity in primary human ovarian cancer cells using quantitative reverse transcription-polymerase chain reaction and western blot analyses. Intriguingly, the loss of MKP3 protein was associated with ubiquitination/proteosome degradation mediated by high intracellular reactive oxygen species (ROS) accumulation such as hydrogen peroxide in ovarian cancer cells. Functionally, short hairpin RNA knock down of endogenous MKP3 resulted in increased ERK1/2 activity, cell proliferation rate, anchorage-independent growth ability and resistance to cisplatin in ovarian cancer cells. Conversely, enforced expression of MKP3 in MKP3-deficient ovarian cancer cells significantly reduced ERK1/2 activity and inhibited cell proliferation, anchorage-independent growth ability and tumor development in nude mice. Furthermore, the enforced expression of MKP3 succeeded to sensitize ovarian cancer cells to cisplatin-induced apoptosis in vitro and in vivo. These results suggest a molecular mechanism by which the accumulation of ROS during ovarian cancer progression may cause the degradation of MKP3, which in turn leads to aberrant ERK1/2 activation and contributes to tumorigenicity and chemoresistance of human ovarian cancer cells.

Tumor suppressive actions of the autocrine human secreted PDZ domain-containing protein 2 (sPDZD2) have been reported, but the mechanisms remain enigmatic. Here, we showed that sPDZD2 induced senescence of prostate cancer DU145 cells, quiescence of breast cancer MCF-7 and liver cancer Hep-G2 cells, via transcriptional activation of mutant or wild-type p53. Furthermore, sPDZD2 sensitized mutant p53-positive DU145 cells and wild-type p53-positive MCF-7 cells to apoptosis induction through genotoxic stress imposed by sub-lethal concentration of hydrogen peroxide. Together, our findings suggest a potential autocrine pathway of p53 activation by transcriptional regulation, and a new approach to reactivate p53 for cancer therapy.

The Forkhead box transcription factor FoxM1 is expressed in proliferating cells. When it was depleted in mice and cell lines, cell cycle defects and chromosomal instability resulted. Premature senescence was observed in embryonic fibroblasts derived from FoxM1 knock-out mice, but the underlying cause has remained unclear. To investigate whether FoxM1 can protect cells against stress-induced premature senescence, we established NIH3T3 lines with doxycycline-inducible overexpression of FoxM1c. Treatment of these lines with sublethal doses (20 and 100 μM) of H 2O 2 induced senescence with senescence-associated β-galactosidase expression and elevated levels of p53 and p21. Induction of FoxM1c expression markedly suppressed senescence and expression of p53 and p21. Consistent with down-regulation of the p19 Arf-p53 pathway, p19 Arf levels decreased while expression of the Polycomb group protein Bmi-1 was induced. That Bmi-1 is a downstream target of FoxM1c was further supported by the dose-dependent induction of Bmi-1 by FoxM1c at both the protein and mRNA levels, and FoxM1 and Bmi-1 reached maximal levels in cells at the G 2/M phase. Depletion of FoxM1 by RNA interference decreased Bmi-1 expression. Using Bmi-1 promoter reporters with wild-type and mutated c-Myc binding sites and short hairpin RNAs targeting c-Myc, we further demonstrated that FoxM1c activated Bmi-1 expression via c-Myc, which was recently reported to be regulated by FoxM1c. Our results reveal a functional link between FoxM1c, c-Myc, and Bmi-1, which are major regulators of tumorigenesis. This link has important implications for the regulation of cell proliferation and senescence by FoxM1 and Bmi-1. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_subscribed_fulltext

Mammalian cell culture technology has been used for decades in mass production of therapeutic proteins. However, unrestricted cell proliferation usually results in low-protein productivity. Controlled proliferation technologies such as metabolism intervention and genetic manipulation are therefore applied to enhance the productivity. Nevertheless, these strategies induced growth arrest with reduced viability and increased apoptosis. In this study, we report a new controlled proliferation technology by encapsulating human embryonic kidney (HEK) 293 cells over-expressing glial-derived neurotrophic factor (GDNF) in 3D collagen microspheres for extended culture. We investigated the viability, proliferation, cell cycle and GDNF productivity of HEK293 cells in microspheres as compared to monolayer culture. This system provides a physiologically relevant tissue-like environment for cells to grow and exerts proliferation control throughout the culture period without compromising the viability. A significant increase in the production rate of GDNF was found in the 3D microsphere system comparing with the monolayer culture. GDNF productivity was also significantly affected by the initial cell number and the serum concentration. The secreted GDNF was still bioactive as it induced neurite extension in PC12 cells. In summary, the 3D collagen microsphere system presents a cost-effective controlled growth technology for protein production in pharmaceutical manufacturing.

There is an unmet clinical demand for safe and effective pharmaceuticals/nutraceuticals for prostate cancer prevention and hormone-refractory prostate cancer treatment. Previous laboratory and human studies of our laboratory demonstrated an association between the antiproliferative action of melatonin and melatonin MT(1) receptor expression in prostate cancer. The aim of this study was to determine, using a pharmacological approach, the signaling mechanisms of melatonin in hormone-refractory 22Rv1 human prostate cancer cell antiproliferation. Both immunoreactive MT(1) and MT(2) subtypes of G protein-coupled melatonin receptor were expressed in 22Rv1 cells. Melatonin inhibited, concentration dependently, cell proliferation, upregulated p27(Kip1) gene transcription and protein expression, and downregulated activated androgen signaling in 22Rv1 cells. While the effects of melatonin were mimicked by 2-iodomelatonin, a high-affinity nonselective MT(1) and MT(2) receptor agonist, melatonin effects were blocked by luzindole, a nonselective MT(1) and MT(2) receptor antagonist, but were unaffected by 4-phenyl-2-propionamidotetraline, a selective MT(2) receptor antagonist. Importantly, we discovered that the antiproliferative effect of melatonin exerted via MT(1) receptor on p27(Kip1) gene and protein upregulation is mediated by a novel signaling mechanism involving co-activation of protein kinase C (PKC) and PKA in parallel. Moreover, we also showed that a melatonin/MT(1)/PKC mechanism is involved in melatonin-induced downregulation of activated androgen signal transduction in 22Rv1 cells. Taken together with the known molecular mechanisms of prostate cancer progression and transition to androgen independence, our data provide strong support for melatonin to be a promising small-molecule useful for prostate cancer primary prevention and secondary prevention of the development and progression of hormone refractoriness.

In this study, we have identified the Forkhead transcription factor FoxM1 as a physiological regulator of estrogen receptor alpha (ERalpha) expression in breast carcinoma cells. Our survey of a panel of 16 different breast cell lines showed a good correlation (13/16) between FoxM1 expression and expression of ERalpha at both protein and mRNA levels. We have also demonstrated that ectopic expression of FoxM1 in two different estrogen receptor-positive breast cancer cell lines, MCF-7 and ZR-75-30, led to up-regulation of ERalpha expression at protein and transcript levels. Furthermore, treatment of MCF-7 cells with the MEK inhibitor U0126, which blocks ERK1/2-dependent activation of FoxM1, also repressed ERalpha expression. Consistent with this, silencing of FoxM1 expression in MCF-7 cells using small interfering RNA resulted in the almost complete abrogation of ERalpha expression. We also went on to show that FoxM1 can activate the transcriptional activity of human ERalpha promoter primarily through two closely located Forkhead response elements located at the proximal region of the ERalpha promoter. Chromatin immunoprecipitation and biotinylated oligonucleotide pulldown assays have allowed us to confirm these Forkhead response elements as important for FoxM1 binding. Further co-immunoprecipitation experiments showed that FoxO3a and FoxM1 interact in vivo. Together with the chromatin immunoprecipitation and biotinylated oligonucleotide pulldown data, the co-immunoprecipitation results also suggest the possibility that FoxM1 and FoxO3a cooperate to regulate ERalpha gene transcription.

PDZD2 (PDZ domain containing 2) is a multi-PDZ protein expressed in pancreas and many other tissues. PDZD2 shows extensive homology to pro-interleukin-16 (pro-IL-16) and is localized mainly to the endoplasmic reticulum. We have recently demonstrated that PDZD2, like pro-IL-16, is proteolytically cleaved at its C-terminus to generate a secreted protein, sPDZD2 (for secreted PDZD2). To understand the possible functional role of PDZD2 in pancreas, we investigated the cellular distribution of PDZD2 in adult pancreas using an antiserum that recognizes both the full-length and secreted forms of PDZD2. Immunohistochemical analysis revealed a strong expression of PDZD2 in pancreatic islet beta cells but not alpha cells. Consistent with the beta-cell-enriched expression of PDZD2, immunoblot analysis indicated expression of both full-length PDZD2 and sPDZD2 in the insulinoma cell line INS-1E. A recombinant sPDZD2 protein was synthesized for study of its functional effect on INS-1E cells. In culture media with limiting serum, co-incubation with sPDZD2 stimulated the proliferation of INS-1E cells. The mitogenic effect of sPDZD2 was concentration-dependent, and was associated with a slight inhibition of the insulin promoter activity at high sPDZD2 concentrations. As a potential mitogen of beta-like cells, sPDZD2 may be useful for the optimization of beta-cell growth and differentiation in vitro.

The forkhead box (FOX) transcription factor FOXM1 is ubiquitously expressed in proliferating cells. FOXM1 expression peaks at the G2/M phase of the cell cycle and its functional deficiency in mice leads to defects in mitosis. To investigate the role of FOXM1 in the cell cycle, we used synchronized hTERT-BJ1 fibroblasts to examine the cell cycle-dependent regulation of FOXM1 function. We observed that FOXM1 is localized mainly in the cytoplasm in cells at late-G1 and S phases. Nuclear translocation occurs just before entry into the G2/M phase and is associated with phosphorylation of FOXM1. Consistent with the dependency of FOXM1 function on mitogenic signals, nuclear translocation of FOXM1 requires activity of the Raf/MEK/MAPK signaling pathway and is enhanced by the MAPK activator aurintricarboxylic acid. This activating effect was suppressed by the MEK1/2 inhibitor U0126. In transient reporter assays, constitutively active MEK1 enhances the transactivating effect of FOXM1c, but not FOXM1b, on the cyclin B1 promoter. RT-PCR analysis confirmed that different cell lines and tissues predominantly express the FOXM1c transcript. Mutations of two ERK1/2 target sequences within FOXM1c completely abolish the MEK1 enhancing effect, suggesting a direct link between Raf/MEK/MAPK signaling and FOXM1 function. Importantly, inhibition of Raf/MEK/MAPK signaling by U0126 led to suppression of FOXM1 target gene expression and delayed progression through G2/M, verifying the functional relevance of FOXM1 activation by MEK1. In summary, we provide the first evidence that Raf/MEK/MAPK signaling exerts its G2/M regulatory effect via FOXM1c.

We have previously shown that SmCP, the gene encoding Solanum melongena cysteine proteinase, is expressed during developmental events associated with programmed cell death (PCD) suggesting its involvement in protein degradation during these events (Xu and Chye, Plant Journal 17 (1999) 321-327). Here, we investigated the regulation of SmCP expression and showed that it is ethylene-inducible and is under circadian control. This circadian rhythm is entrained by light/dark (LD) cycling with peak expression in the late light period, as opposed to that in early light for rbcS, suggesting that protein degradation and photosynthesis are temporally separated by circadian control. Northern blot analysis shows that the pattern of ethylene induction of SmCP is consistent with our previous observation of its significantly increased expression at leaf senescence and fruit ripening when endogenous ethylene is abundant. To further understand SmCP regulation, we have cloned the SmCP promoter and identified a G-box (CACGTG) at -85/-80 by DNase I footprinting analysis of the -221/+17 region. Its specific interaction with nuclear proteins in S. melongena leaves and fruits was confirmed by competitive electrophoretic mobility shift assays using oligonucleotides containing the G-box and mutant derivatives. G-box binding activity was stronger in senescent than young fruits. In circadian-regulated leaves, stronger binding activity coincided with peak circadian expression of SmCP. This correlation between binding activity and expression suggests that G-box binding factors enhance SmCP transcription and that the G-box likely plays a role in circadian regulation of genes affected by LD cycling.

PDZD2 (PDZ-domain-containing 2; also known as PAPIN, AIPC and PIN1) is a ubiquitously expressed multi-PDZ-domain protein. We have shown that PDZD2, which shows extensive homology to pro-interleukin-16 (pro-IL-16), is localized mainly to the endoplasmic reticulum (ER). Pro-IL-16 is cleaved in a caspase-3-dependent mechanism to generate the secreted cytokine IL-16. The abundant expression of PDZD2 in the ER, and its sequence similarity to pro-IL-16, suggests that similar post-translational processing of PDZD2 may occur. Indeed, western blotting and mass spectrometry analysis of conditioned medium from cells transfected with epitope-tagged PDZD2 show that there is secretion of a PDZD2 peptide of approximately 37 kDa (sPDZD2, for secreted PDZD2) that contains two PDZ domains. Expression of PDZD2 was detected in several tissues. Furthermore, sPDZD2 secretion is suppressed by the mutation of a sequence that shows similarity to caspase recognition motifs or by treatment with a caspase inhibitor. In summary, PDZD2 is the first reported multi-PDZ protein that is processed by proteolytic cleavage to generate a secreted peptide containing two PDZ domains., published_or_final_version