This work is consistent with the LTBP1 C-terminus adopting a flexible “knotted rope” structure, which may facilitate cell matrix interactions, and the accessibility to proteases or other factors that could contribute to TGFβ activation.Proteins from the LTBP/fibrillin family perform key structural and functional roles in connective tissues. LTBP1 forms the large latent complex with TGFβ and its propeptide LAP, and sequesters the latent growth factor to the extracellular matrix. Bioinformatics studies suggest the main structural features of the LTBP1 C-terminus are conserved through evolution. NMR studies were carried out on three overlapping C-terminal fragments of LTBP1, comprising four domains with characterised homologues, cbEGF14, TB3, EGF3 and cbEGF15, and three regions with no homology to known structures. The NMR data reveal that the four domains adopt canonical folds, but largely lack the interdomain interactions observed with homologous fibrillin domains; the exception is the EGF3-cbEGF15 domain pair which has a well-defined interdomain interface.  The human genome encodes four latent transforming growth factor-β (TGFβ) binding proteins (LTBPs). These are large extracellular proteins which contain variable numbers of epidermal growth factor-like (EGF), calcium-binding EGF (cbEGF) and “TGFβ” binding protein-like (TB) domains, but all of which conform to a similar domain architecture . SignifiLTBPs play an important role in the TGFβ signalling pathway, which is a key component of many developmental and disease processes LTBPs have been demonstrated to interact with components of the extracellular matrix including human fibrillin1 The bioinformatic analysis and NMR experiments reported here were designed to investigate the domain organisation of the fibrillin1-binding region of LTBP1 which is adjacent to the LAP-binding TB2 domain. This is the first detailed study of the domain architecture of the LTBPs and reveals a dynamic “knotted rope” model for the C-terminus of LTBP1. This may have significant implications for its interaction with fibrillin and other ECM proteins, and also help decipher the role of LTBP1 in TGFβ regulation.6 tag at the N-terminus. The EGF3-cbEGF15 protein was produced as described previously, spanning LTBP1L residues 1617–1722 6 tag was not removed from TB3-EGF3 as this construct contained additional factor Xa sensitive sites. TB3-EGF3 was purified by anion exchange FPLC with the His6 tag still present. After purification all proteins were characterised by SDS-PAGE and Coomassie staining (Prokaryotic expression constructs were generated by cloning DNA fragments into the pQE30 expression vector (Qiagen), incorporating a Hisstaining , and the1H frequencies ranging from 500 to 950 MHz. All experiments were carried out at 25°C. Data was processed using NMRpipe Experiments were carried out using home-built instruments operating at 15N labelled protein, using 3D 15N-edited TOCSY experiments to identify spin systems, and 3D 15N-edited NOESY experiments to identify the through-space connections between residues. 3D experiments for resonance assignment of cbEGF14-TB3 were collected at 500 MHz while spectra for TB3-EGF3 were collected at 600 MHz. Backbone 15N and 1HN assignments for cbEGF14-TB3 and TB3-EGF3 have been deposited in the BMRB with accession number 19322. The 15N-edited 3D data for EGF3-cbEGF15 were not of sufficient quality to allow complete backbone assignment. Therefore, assignment of the NMR spectrum of EGF3-cbEGF15 was carried out using 13C and 15N double-labelled protein as described previously The spectra of cbEGF14-TB3 and TB3-EGF3 were assigned with 2O: 5% D2O mixture. EGF3-cbEGF15 experiments were carried out at pH 5.4 with 10 mM calcium chloride and a protein concentration of 5 mM. Experiments on cbEGF14-TB3 were carried out at pH 5.9, with 20 mM calcium chloride and a protein concentration of 3 mM. TB3-EGF3 experiments were carried out at pH 6.9 with no calcium chloride or other salt present, and a protein concentration of 4 mM. Spectra for chemical shift comparisons were all collected at pH 6 with 10 mM calcium chloride.Unless otherwise stated, all NMR experiments were carried out at 25°C in a 95% H2O with 5% D2O, 5 mM calcium, at pH 5.4), and in a 3.5% PEG-hexanol liquid crystal alignment medium 2O, 10% D2O, 5 mM calcium at pH 5.4). RDC data were used to define the interdomain orientation of EGF3 and cbEGF15 as follows. The X-ray coordinates of human Notch-1 EGF12-EGF13 (PDB 2VJ3) N-15N bond relative to the principle axes of alignment tensor and the energy minimisation carried out in the homology modelling procedure was found to alter atomic positions leading to poor fits. Relative domain orientation was determined as follows. First, the RDC values for the individual domains were fitted to the X-ray coordinates of individual domains, using an in-house program, to identify a subset of residues that gave a good fit. The overall fit between experimental and calculated RDC values was assessed using the Q value, defined as Q  =  ½/RDCrmsResidual dipolar coupling (RDC) values for EGF3-cbEGF15 were obtained from IPAP spectra 15N-labelled protein samples were prepared at pH 5.9 with 20 mM calcium chloride, for cbEGF14-TB3, and at pH 5.4 with 10 mM calcium chloride, for EGF3-cbEGF15, and then freeze dried. These samples were then re-dissolved in 100% D2O (to the same volume as the sample prior to freeze drying), and a series of HSQCs collected over 24 hours to examine the loss of peak intensity.Hydrogen/deuterium exchange experiments were used to provide information about amide solvent accessibility and hydrogen bonding in various constructs. In these experiments 1H-15N Heteronuclear NOE experiments were carried out on 15N-labelled protein samples in order to examine the sub-nanosecond dynamics of specific amides. Spectra with and without 1H saturation were collected as interleaved experiments. Error bars were calculated by taking the standard deviation of random background noise and recalculating NOE ratios after adding this standard deviation to the NOE peak intensities and subtracting it from the blank peak intensities.Homology modelling was carried out using Modeller 9 v7 The LTBP1 TB2-cbEGF13 domain pair was modelled using the TB2 solution structure Alignments were generated using the ClustaW feature of the MEGA5 suite of programs LTBP and true TGFβ first emerged with the evolution of deuterostomes LTBP sequences all contain linker regions of 17 or more amino acids between the cbEGF14 and TB3 domains, and 34 or many more residues between the TB3 and EGF3 domains. These long linker regions have no clear homology to previously identified domains and are not seen between homologous TB/EGF/cbEGF domains in fibrillin.These sequences are poorly conserved between LTBP variants, suggesting that these regions may be unstructured and potentially flexible. Interdomain packing interactions between adjacent TB and cbEGF domains have been described previously and are often centred around specific glycine-aromatic motifs in the N-terminal (cb)EGF or TB domain that form non-polar interactions with the C-terminal (cb)EGF domain Homology models were generated for the four domains of LTBP1 cbEGF14-TB3-EGF3-cbEGF15 by assuming that these domains adopt the canonical domain folds seen for other (cb)EGF and TB domain structures To test the hypotheses of LTBP1 domain structure and linker flexibility suggested by bioinformatics analysis, three overlapping protein fragments spanning from the N-terminus of cbEGF14 to the C-terminus of LTBP1 were produced . These f15N labelling alone was used for the cbEGF14-TB3 and TB3-EGF3 assignments, while EGF3-cbEGF15 was assigned with 13C and 15N labelling as described previously 15N-edited NOESY and TOCSY experiments (BMRB accession 19322). TB3-EGF3 was the largest fragment assigned with 172 amino acids (including a His6 tag); 78% of the backbone amides were assigned (BMRB accession 19322). Comparison of HSQC spectra from the three LTBP1 fragments shows that residues present in more than one construct give rise to peaks with very similar chemical shifts in the relevant HSQC spectra  typical of cbEGF domains in an N-terminal position. In native LTBP1, cbEGF13 would be expected to pack against cbEGF14 leading to an increased calcium affinity in cbEGF14.The binding of Cad<∼25 µM); some peaks, corresponding to the calcium-bound form, appear and strengthen as calcium is added, while others, corresponding to the calcium-free state, weaken and disappear  exchange monitored by HSQC spectra following dissolution of the protein fragments in Ds in D2O . Three os in D2O  located 1H-15N heteronuclear NOE experiments, which report on fast-timescale backbone dynamics, were carried out on all three protein fragments . RDCs were measured for 18 and 30 residues in EGF3 and cbEGF15, respectively . The larThese NMR studies have validated structural models for the four C-terminal domains of LTBP1 and given significant insight into the overall dynamics and interdomain interactions of this region. The numerous flexible linkers identified would make many regions of the LTBP1 C-terminus unsuitable for crystallographic studies, or other structural investigations that rely on rigid body modelling. The data presented here demonstrate that the C-terminus of LTBP1 behaves like a “knotted rope” in solution, where the linkers either side of the TB3 domain act as the highly flexible “rope” allowing the TB3 domain and EGF3-cbEGF15 domain pair, , to move freely relative to each other and the rest of LTBP1. The presence of flexible linkers mean that long-range interdomain interactions, such as EGF3 interacting with cbEGF14 or other parts of the LTBP1 molecule, cannot be excluded by these studies.Bioinformatics observations support this model, and also demonstrate that the structural and dynamic properties of the LTBP C-terminus may have emerged prior to the divergence of acorn worms and the other deuterostomes over 500 million years ago Several proteins such as fibulin4 Interestingly LTBP2 and LTBP4 have even longer linkers joining their equivalents of LTBP1's TB3 and EGF3 domains . While rThe work presented here establishes for the first time the structural organisation for the C-terminus of LTBP1, a molecule of significant importance in TGFβ regulation. These results will help guide further studies to understand the complex relationship between TGFβ signalling and ECM localisation, and reveal the true relevance of LTBP1's knotted tail.Figure S1Protein characterisation. A) Protein products analysed by SDS-PAGE and Coomassie staining, under both non-reducing and reducing conditions. The cbEGF14-TB3 fragment migrated unusually far under non-reducing conditions, especially when compared with reducing conditions, this behaviour was seen reproducibly, and may reflect unusual behaviour of this fragment in the presence of SDS and electric fields. Mass spectrometry confirmed the protein was of the expected size in each case. Bi-iii) 500 MHz HSQC spectra of all three constructs, collected at pH 6. Some example peaks corresponding to the TB3 domain are highlighted with blue circles in the cbEGF14-TB3, and TB3-EGF3 spectra, while some peaks corresponding to EGF3 residues are highlighted with orange circles in the TB3-EGF3 and EGF3-cbEGF15 spectra.(TIF)Click here for additional data file.Figure S2Calcium sensitivity of the HSQC spectra of cbEGF14-TB3 and EGF3-cbEGF15. A) Overlaid 500 MHz HSQC spectra of 2 mM cbEGF14-TB3 collected with different concentrations of calcium, coloured according to the legend in the top left. The sample was prepared at pH 5.9, in 95% H2O : 5% D2O without any additional salt. Assignments for peaks showing significant shifts are highlighted. B) 750 MHz HSQC spectra of 5 mM EGF3-cbEGF15 recorded with and without 10 mM calcium at pH 5.4. The inlay in the bottom right shows the upfield shifted methyl region of 1D spectra of a 2 mM EGF3-cbEGF15 sample, collected at different calcium concentrations. The appearance of new peaks rather than shifts demonstrates slow exchange between the calcium-free and calcium-bound states of the protein (1D spectra collected in D2O at pH 7.7 and 750 MHz).(TIF)Click here for additional data file.Figure S3Slowly-exchanging amides mapped onto the homology models of cbEGF14-TB3 and EGF3-cbEGF15. Slowly-exchanging amides in cbEGF14-TB3 (A) and EGF3-cbEGF15 (B) highlighted in the protein sequence, and as blue spheres on the homology model.(TIF)Click here for additional data file.


The aim of this preliminary investigation was to assess whether human peripheral blood lymphocytes which have been pre-exposed to non-ionizing radiofrequency fields exhibit an adaptive response (AR) by resisting the induction of genetic damage from subsequent exposure to ionizing radiation. Peripheral blood lymphocytes from four healthy donors were stimulated with phytohemagglutinin for 24 h and then exposed for 20 h to 1950 MHz radiofrequency fields  at an average specific absorption rate of 0.3 W/kg. At 48 h, the cells were subjected to a challenge dose (CD) of 1.0 or 1.5 Gy X-irradiation . After a 72 h total culture period, cells were collected to examine the incidence of micronuclei (MN). There was a significant decrease in the number of MN in lymphocytes exposed to RF + XR (AD + CD) as compared with those subjected to XR alone (CD). These observations thus suggested a RF-induced AR and induction of resistance to subsequent damage from XR. There was variability between the donors in RF-induced AR. The data reported in our earlier investigations also indicated a similar induction of AR in human blood lymphocytes that had been pre-exposed to RF (AD) and subsequently treated with a chemical mutagen, mitomycin C (CD). Since XR and mitomycin-C induce different kinds of lesions in cellular DNA, further studies are required to understand the mechanism(s) involved in the RF-induced adaptive response. The data from our earlier studies indicated that non-ionizing RF is also capable of inducing AR. Human blood lymphocytes obtained from several donors were stimulated with PHA for 24 h and then exposed to 900 MHz RF  for 20 h (AD). This was followed by treatment of the cells with a genotoxic dose of 100 ng/ml MMC (CD). The incidence of MN was found to be significantly decreased in the cells exposed to AD + CD as compared with those exposed to CD alone, thus indicating RF-induced AR to the subsequent exposure to MMC. The data also indicated variability among the donors in their response to AD + CD exposures: some donors exhibited AR while others did not [17]. These observations were confirmed in two subsequent investigations, which characterized the RF-induced AR further to indicate the impact of: (i) cell cycle (cells exposed in S-phase of the cell cycle exhibited RF-induced AR while those in G0 and G1 phase did not) [18], and (ii) SAR  exhibited varying degrees of RF-induced AR, with maximum decrease in MN at an average SAR of 0.3 W/kg, a lesser decrease at 0.6 W/kg SAR, and no decrease at 0.15 W/kg or 1.25 W/kg) [19]. Thus, the overall data indicated that the extent of RF-induced AR was variable and depended on the stage at which the cells were pre-exposed to RF and on the electromagnetic characteristics of RF exposure.There is well-documented scientific evidence for the existence of very low-dose ionizing radiation-induced AR in human blood lymphocytes. Such AR has been further characterized and putatively linked to DNA-repair processes and 2, corresponding to 55 mW/kg) and then subjected to gamma-radiation (CD). A significantly increased survival, significantly decreased hematopoietic tissue damage, increased expression of cell cycle-related cyclin-D1, cyclin-E, cyclin-DK4 and cyclin-DK2 genes and decreased primary DNA damage in blood leukocytes were observed in mice exposed to AD + CD as compared with mice exposed to CD alone [20–22]. The RF-induced AR was also confirmed in independent investigations using experimental mice and rats, although no details on electromagnetic dosimetry were given . Furthermore, in vitro studies on human promyelocytic leukemia HL-60 cells that were pre-exposed to 900 MHz RF (0.25 µW/kg average SAR) and then exposed to the chemotherapeutic drug doxorubicin showed increased cell proliferation, decreased apoptosis, increased mitochondrial membrane potential, decreased intracellular Ca2+ and increased Ca2+-Mg2+-ATPase activity [23]. Together these observations provide some mechanistic insights into RF-induced AR in experimental animals and in HL-60 cells.RF-induced AR was also reported in experimental mice that were pre-exposed to 900 MHz RF  did not show significant changes in PI, while those exposed to XR and MMC alone (CD) showed a significant decrease in PI. These data further suggested that RF-induced AR was not related to alteration in cell proliferation.There is a large body of information in the peer-reviewed scientific literature concerning the mechanistic aspects of low-dose ionizing-radiation-induced AR –44. So fThis work has been supported by National Institute of Nuclear Physics (INFN), section of Naples, Project ARCAICA.


Dihydrotestosterone (DHT) stimulated transactivation of the androgen response element (ARE) was significantly greater in C4-2B cells than in LNCaP cells. DHT-induced AR transactivation was coupled with higher nuclear translocation of p65-Nrf1 in C4-2B cells, as compared to LNCaP cells. Conversely, DHT stimulation suppressed total Nrf2 levels in C4-2B cells but elevated total Nrf2 levels in LNCaP cells. Interestingly, siRNA mediated silencing of Nrf1 attenuated AR transactivation while p65-Nrf1 overexpression enhanced AR transactivation. Subsequent studies showed that Nrf1 physically interacts with AR and enhances AR’s DNA-binding activity, suggesting that the p65-Nrf1 isoform is a potential AR coactivator. In contrast, Nrf2 suppressed AR-mediated transactivation by stimulating the nuclear accumulation of the p120-Nrf1 which suppressed AR transactivation. Quantitative RT-PCR studies further validated the inductive effects of p65-Nrf1 isoform on the androgen regulated genes, PSA and TMPRSS2. Therefore, our findings implicate differential roles of Nrf1 and Nrf2 in regulating AR transactivation in PCa cells. Our findings also indicate that the DHT-stimulated increase in p65-Nrf1 and the simultaneous suppression of both Nrf2 and p120-Nrf1 ultimately facilitates AR transactivation in CRPC cells.Despite androgen deprivation therapy (ADT), persistent androgen receptor (AR) signaling enables outgrowth of castration resistant prostate cancer (CRPC). In prostate cancer (PCa) cells, ADT may enhance AR activity through induction of oxidative stress. Herein, we investigated the roles of Nrf1 and Nrf2, transcription factors that regulate antioxidant gene expression, on hormone-mediated AR transactivation using a syngeneic  Prostate cancer (PCa) is the second leading cause of cancer related deaths in American men It has been suggested that residual androgen production within the tumor microenvironment contributes to persistent AR signaling Studies have shown that ADT can induce oxidative stress and reactive oxygen species (ROS) play a significant role in PCa progression to castration resistance Nrf1 and Nrf2 are master regulators of oxidative stress induced gene expression In contrast to Nrf2, the N-terminal domain (NTD) of Nrf1 (TCF11), which anchors Nrf1 to the endoplasmic reticulum (ER) membrane and the nuclear membrane, regulates Nrf1 activation and its translocation to the nucleus Several studies have implicated the importance of Nrf2 in PCa Since LNCaP and C4-2B cells are syngeneic PCa lines, our findings in these cell lines indicate that Nrf1 and Nrf2 may have significant roles in PCa progression through the manifestation of CRPC phenotype. Our findings clearly showed that the opposing functions of Nrf2 and the p65 and p120 isoforms of Nrf1 can regulate DHT-induced AR transactivation in PCa cells. Our studies further implicate novel mechanisms via which Nrf1 and Nrf2 regulation is modified in the castration resistant C4-2B cell line to facilitate the enhanced AR transactivation.LNCaP cells were obtained from American Type Culture Collection . The C4-2B cell line is a bone metastatic subline derived from LNCaP, and was a kind gift from Dr. Lelund Chung DHT was obtained from Steraloids . Nrf1 antibody was obtained from Proteintech Group . Antibodies to Nrf2, AR, and TATA Binding Protein (TBP) were obtained from Abcam . Isotype IgG (non-specific control) and all secondary anti-human antibodies were obtained from Santa Cruz . Anti-V5 antibody was obtained from Invitrogen . The AR antibody used for ChIP assays was obtained from Active Motif . The Nrf1 specific siRNA and the non-specific control (NC1) siRNA were obtained from Integrated DNA Technologies . Plasmids were obtained from the following sources: p65-Nrf1 expression vector (p65-Nrf1-V5His) was a gracious gift from Dr. Chan AR: ′5′-GGTGAGCAGAGTGCCCTATC-3 and 5′-GAAGACCTTGCAGCTTCCAC-3′; GAPDH: ′5′-TCCCATCACCATCTTCCA-3 and 5′-CATCACGCCACAGTTTCC-3′; PSA:5′-AGGCCTTCCCTGTACACCAA-3′ and 5′-GTCTTGGCCTGGTCATTTCC-3′; and TMPRSS2∶5′-GTCCCCACTGTCTACGAGGT-3′ and 5′-CAGACGACGGGGTTGGAAG-3′. Fold changes in gene expression were calculated after normalization to their corresponding GAPDH mRNA levels.After treatment, mRNA was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen). The cDNA was prepared using the High Capacity Reverse Transcription kit from Applied Biosystems . The RT-PCR primers were synthesized at Midland Certified Reagent Company  and SyBr Green Master Mix was purchased from Applied Biosystems . The following primer sequences were used for quantitative RT-PCR: Nuclear pellets for westerns were isolated using the CER-I and CER-II extraction buffers from the NE-PER Nuclear Extraction kit . Nuclei were washed with HBSS and nuclear protein was extracted with a custom nuclear protein lysis buffer . Nuclear protein was quantified using the BCA protein estimation kit from Thermo Scientific .Protein samples were boiled in 1∶1 volume of protein and loading buffer  for five minutes. Nuclear proteins (20–50 µg) were electrophoresed on Tris-HCl PAGE gels and wet transferred to nitrocellulose membranes. After blocking with 5% milk in TBST buffer (TBS with 0.05% Tween-20), membranes were hybridized with the indicated antibodies. Bands were then detected using the Lumiglo chemiluminescent substrate system . Band intensities were quantified with Image-J Software (NIH) and values were normalized to TBP protein levels in their respective samples.luc reporter plasmid alone or in combination with equal amounts of Nrf1 expression vector (p65-Nrf1-V5-His or p120-Nrf1-V5-His), or Nrf2 expression vector (pCMV6-Nrf2), or a control expression vector (pcDNA3.1). To normalize for transfection efficiency, cells were also cotransfected with the pRL-TK (renilla luciferase) vector. Western studies were also performed with the indicated expression vectors alone. In brief, cells were incubated overnight in transfection solution  in 2 ml of serum/phenol red free media. After overnight incubation, media was removed and cells were exposed to DHT  for 24 hrs in CS-FBS containing phenol red free RPMI. Cell extracts were harvested and luciferase levels were determined using the dual Luciferase Reporter Assay kit . In each experiment, firefly luciferase values (from psPSA-luc) were normalized to renilla luciferase values (from pRL-TK). Nuclear protein was extracted after treatment and evaluated by western for changes in nuclear protein expression. In parallel experiments, cells were also transfected with the Nrf1 and Nrf2 expression vectors alone in order to monitor DHT-induced expression of two AR regulated genes, PSA (prostate specific antigen) and TMPRSS2 .Cells were seeded into 6-well plates overnight before transfection with Lipofectamine-2000 reagent  and the indicated vectors. For luciferase assays, cells were transfected with either the psPSA-Short interfering RNA (siRNA) transfections were carried out using Transfast reagent from Promega . Briefly, cells were incubated overnight (∼18 hrs) in transfection solution that consisted of Transfast reagent (2∶1 dilution), and 20 nM of either Nrf1 siRNA or NC1 (control) siRNA in serum and phenol red free RPMI. For plasmid cotransfections, cells were simultaneously transfected with both siRNA and plasmid at a 2∶1 dilution of Transfast reagent, as described in the luciferase assay section. After overnight transfection, media was removed and cells were exposed to the indicated treatments. Cells were then harvested after 24 hrs and luciferase assays were carried out. In parallel samples, RNA and nuclear protein was obtained to monitor gene expression by qRT-PCR and by western.2, 100 µM EDTA, 500 µM DTT, 0.625% NP-40, protease inhibitor, and phosphatase inhibitors). After washing in PBS two times, RIPA buffer , 5 mM EDTA, 1% Deoxycholate, 1% Triton X-100, 0.1% SDS, protease inhibitor, and phosphatase inhibitors) was added to nuclear pellets and pellets were sonicated to isolate nuclear protein. Protein was pre-cleared for 30 mins with protein-G/protein-A agarose beads . AR antibody  was then added to 100 µg of nuclear protein in 400 µl RIPA lysis buffer and incubated at 4°C overnight. The protein-G/protein-A agarose beads were then added to protein and incubated for 2 hrs. After incubation, beads were washed 3 times in RIPA lysis buffer and loading dye was added to the sample. Samples were then boiled for 5 mins at 95°C, loaded onto the gel, and immunoblotted (IB) with the indicated antibodies.For co-immunoprecipitation and immunoblotting (co-IP/IB) studies, LNCaP and C4-2B cells were treated with 0, 1, or 10 nM DHT for 6 hrs. Nuclear pellets were isolated using a nuclear isolation buffer . Changes in amplified band intensities were quantified.For chromatin immunoprecipitation (ChIP) assays we used two different kits, the Covaris  truChIP chromatin shearing kit with non-ionic buffer and the Active Motif  ChIP-IT High Sensitivity kit. The assays were performed according to manufacturer’s protocols, with minor modifications. Briefly, DHT treated (6 hrs) LNCaP and C4-2B cells were sheared using the Covaris truChIP kit using an E220 focused-ultrasonicator from Covaris. The chromatin samples were diluted in ChIP buffer from the Active Motif kit. Samples were then immunoprecipitated (IP) with either the Nrf1 antibody (Proteintech Group) or the AR antibody (Active Motif). The remainder of the assay was performed according to the Active Motif kit instructions. ARE specific qRT-PCR was performed on the IP DNA using primers for ARE-II sequences located within the PSA-promoter  was used for EMSA studies. The androgen response element (ARE) and the TCF11 and TCF11/MafG (Nrf1 binding) sequences TCF11∶5′-GTCATTT-3′ and 3′-AAATGAC-5′; ARE: 5′-GATCCTTGCAGAACAGCAA GTGCTAGCTG-3′ and 3′-GAACGTCTTGTCGTTCACGATCGACCTAG-5′; and TCF11/MafG: 5′-CCCAAATGACAATGCGATTGA-3′, 3′-TCAATCGCATTGTCATTTGGG-5′ (Genomatix MatInspector). Briefly, nuclear protein was extracted from cells treated with DHT for 6 hrs and binding reactions with DIG labeled ARE oligonucleotides were carried out. ARE oligos were incubated with nuclear protein (10 µg) for 30 min, after which sample loading buffer was added and samples were electrophoresed on a 5% TBE PAGE gel . Samples were then transferred onto a nylon membrane, incubated with blocking buffer, exposed to an anti-DIG antibody, washed, and developed using the ECL chemiluminescent system, as described previously. For competition studies, nuclear extracts were pre-incubated with excess (50-fold) unlabeled ARE, TCF11, or TCF11/MafG oligos for 30 min before DIG-labeled ARE oligos were added to the reaction. For experiments using antibodies, to compete for Nrf1 binding, nuclear extracts were pre-incubated with either Nrf1 antibody or with rabbit IgG (non-specific control) for 30 min before DIG-labeled ARE oligos were added to the reaction. Electrophoresis, transfer, and hybridization were carried out as described above.The 2Each treatment condition consisted of 2–4 replicates and each experiment was performed 2-5 times. Relative expression was determined by comparing treatment values to control values after normalization to loading controls. Statistical significance was evaluated by two-way ANOVA using the GraphPad Prism software. Significant changes from controls are indicated by p-values of <0.05.luc vector transfected cells  reduces AR transactivation  regulate AR function via direct interaction with nuclear AR protein, we performed AR immunoprecipitations (IP) from nuclear extracts of untreated and DHT-treated LNCaP and C4-2B cells . The IP To determine whether the Nrf1 and AR interactions in C4-2B cells can facilitate transcription factor complex formation at the ARE sequences, we performed both ChIP assays  and EMSANext, we conducted EMSA studies to further corroborate Nrf1 association with the AR transcription complex at the ARE, using a DIG labeled ARE oligonucleotide and nuclear extracts from DHT treated C4-2B cells . In all Next, we wanted to investigate the mechanism/s via which Nrf2 down- regulates AR transactivation, as shown in Despite an initial response to hormone deprivation, most PCa patients relapse to a hormone refractory state in which tumors utilize enhanced AR function to survive during ADT. Several studies have indicated that this may be the result of augmented AR transactivation in CRPC cells We present a novel mechanism in which oxidative stress-induced transcription factors are utilized by CRPC cells to increase AR function despite low hormone levels during ADT. The levels of Nrf2 and the ratio of p65-Nrf1 to p120-Nrf1 in PCa cells dictate their effects on the activity of AR. Differential regulation of the interactions that occur between these proteins and the AR transcription complex may dictate AR function in both hormone dependent LNCaP cells and hormone independent C4-2B cells. Here, we present a schematic model of the differential actions of Nrf1 and Nrf2 in regulating AR transactivation function in CRPC cells .Compared to LNCaP cells, DHT-stimulated C4-2B cells showed elevated nuclear p65-Nrf1 levels which were associated with enhanced AR transactivation in these CRPC cells. Indeed, compared to LNCaP cells, psPSA-luc transfected C4-2B cells had a significantly greater capacity for AR transactivation at a reduced DHT concentration (1nM), even though DHT induced patterns of AR nuclear localization were similar in both cell lines . DHT stiMechanistic studies suggested that p65-Nrf1 mediates its inductive effects on AR transactivation by directly interacting with the AR transcription complex  and thatIn contrast to p65-Nrf1, the suppressive effect of Nrf2 on AR transactivation was seen in both LNCaP and C4-2B cells . Nrf2 ovPrevious studies suggested multiple roles for Nrf2 in cancer progression, recurrence, and resistance Despite its potential role in regulating Nrf2 expression or its ability to regulate Nrf2 function, little is known about the role of Nrf1, or its isoforms, in regulating PCa progression One therapeutic strategy could be to target the proteosomal pathway that may regulate p65-Nrf1 processing and its subcellular localization. Chepelev et al. (2011), recently described several mechanisms by which the full length Nrf1 (p120-Nrf1) may be processed into its smaller isoforms via the 26S proteasome pathway . Indeed, p120-Nrf1 levels were found to be significantly increased in cells following inhibition of VCP expression. Interestingly, elevated VCP expression has been previously associated with poor prognosis in PCa patients 2, N4-dibenzylquinazoline-2,4-diamine (DBeQ), have been shown to potently inhibit PCa cell growth A third strategy may be to target the degradation of p120-Nrf1 directly. Steffen et al. (2010), have shown that the violin-containing protein (VCP/p97), a ubiquitin proteasome associated protein required to extract ubiquitylated proteins from membranes before their proteasomal degradation, can facilitate p120-Nrf1 degradation In summary, our current investigations demonstrate new roles for Nrf1 and Nrf2 in regulating AR signaling in PCa. A clearer understanding of the opposing relationships between the various Nrf1 isoforms (p65-Nrf1 and p120-Nrf1) and their regulation by Nrf2, may pave the way for development of novel therapies against CRPC cells. In addition, determination of the expression and localization of p65-Nrf1, p120-Nrf1, and Nrf2 may also be a useful biomarker for identifying patients with aggressive cancer that may become resistant to ADT.Figure S1Effect of p65-Nrf1 overexpression on PSA and TMPRSS2 gene expression. LNCaP cells were transfected with either control vector (pcDNA3.1) or the p65-Nrf1 expression vector (p65-Nrf1-V5-His). Cells were stimulated with DHT (10 nM) and total RNA were isolated at 24 hr to measure the expression of two AR-regulated genes, TMPRSS2 and PSA, by qRT-PCR. Data  were normalized to GAPDH mRNA levels is respective samples and fold changes in TMPRSS2 and PSA mRNA levels are shown. .(TIF)Click here for additional data file.Figure S2Effects of Nrf1 or Nrf2 on AR gene expression and AR nuclear localization. Modulatory effects of p65-Nrf1 and Nrf2 on AR gene expression and nuclear AR levels were measured in LNCaP and C4-2B cells. The AR mRNA levels were determined by qRT-PCR and nuclear AR protein was measured by western immunoblotting. Fold changes in relative AR gene expression after, (A) p65-Nrf1 overexpression in LNCaP cells, (B) Nrf1 knockdown by siRNA in C4-2B cells, or following Nrf2 overexpression in either LNCaP (E) or C4-2B cells (F) are shown. For qRT-PCR studies, all Ct values were normalized to their corresponding GAPDH levels (n = 2). Immunoblotting of nuclear AR was carried out after (C) p65-Nrf1 overexpression (pCMV-Nrf2) in LNCaP cells or (D) following Nrf1 knockdown (siRNA) in C4-2B cells. AR nuclear levels were normalized to TBP levels in each sample (n = 2).(TIF)Click here for additional data file.


The nature of the heteroatom substitution in the nitrogen of a 3-aza-Cope system is explored.While N-propargyl isoxazolin-5-ones suffer 3-aza-Cope rearrangements at 60°C, the corresponding N-propargyl pyrazol-5-ones need a higher temperature of 180°C for the equivalent reaction. When the propargyl group is substituted by an allyl group, the temperature of the rearrangement for both type of compounds is less affected by the nature of the heteroatom present. Treatment with a base, such as ethoxide, facilitates the rearrangement, and in the case of isoxazol-5- ones other ring opening reactions take precedence, involving N–O ring cleavage of the 5-membered ring. However when base-catalysed decomposition is prevented by substituents, products arising from a room temperature aza-Cope rearrangement are isolated. A possible mechanistic pathway based on free energies derived from density functional calculations involving cyclic intermediates is proposed.The nature of the heteroatom substitution in the nitrogen of a 3-aza-Cope system leads to a remarkable difference in the energy of activation of the reaction. N-alkyl(silyl)oxy-N-allyl enamines undergo smooth 3,3-sigmatropic rearrangements to the corresponding N-alkyl(silyl)oxy imino ethers in good to excellent yields . Following the general procedure, compounds (1a) and (2a) were obtained from 3a, and 1b and 2b from 3b.General procedure: To the desired pyrazolone (1H NMR (400 MHz CDCl3) δ 7.47 – 7.34 , 7.24 – 7.15 , 4.14 , 3.49 , 2.21 . 13C NMR (100.62 MHz CDCl3) δ 165.5 (C = O), 152.7 [NC(Ph)], 130.4 , 130.0, 129.4, 129.0, 129.0, 128.7, 127.9, 126.7, 115.2, 75.4, 74.5, 39.0, 29.2. I.R. : 3279, 2109, 1651 (C = O). EIMS : 288 , 277 (100), 249 , 199 (32); 77 (45). HRMS: calcd for C19H16N2O: 288.126263; found: 288.126523.(65%), colourless crystals, mp 123–124°C (AcOEt). 1H NMR (400 MHz CDCl3) δ 7.46 – 7.43 , 7.34 – 7.22 , 4.40 , 3.86 , 2.49 .13C NMR (100.62 MHz CDCl3) δ 149.8 [N = C(Ph)], 147.5 [N = C(Ph)C(Ph) = C], 133.7 (Ar), 132.0 (Ar), 129.6 (Ar), 128.5 (Ar), 128.1 (Ar), 127.8 (Ar), 127.4 (Ar), 126.8 (Ar), 105.6 [N = C(Ph)C(Ph) = C], 77.7 (OCH2C ≡ CH), 76.7 (OCH2C ≡ CH), 61.0 (OCH2C ≡ CH), 34.6 (NCH3). I.R. : 3289, 2123. EIMS : 288 , 249 , 221 (18), 178 (5), 115 (25), 91 (20). HRMS: calcd for C19H16N2O: 288.126263; found: 288.126219.(35%), colourless oil. 1H NMR (400 MHz CDCl3) δ 7.65 , 7.52 – 7.41 , 7.33 – 7.19 , 4.03 , 2.16 . 13C NMR (100.62 MHz CDCl3) δ 164.6 (C = O) , 154.9 [NC(Ph) = C(Ph)] , 134.8 (Ar) , 130.2 (Ar), 130.1 (Ar), 129.5 (Ar), 129.2 (Ar), 129.0 (Ar), 129.0 (Ar), 128.0 (Ar), 127.0 (Ar), 126.6 (Ar), 123.5 (Ar), 116.6 [NC(Ph) = C(Ph)], 74.6 (NCH2C ≡ CH), 74.1 (NCH2C ≡ CH), 40.6 (NCH2C ≡ CH). IR : 3298, 2116, 1667 (C = O).(35%), yellowish oil. 1H NMR (400 MHz CDCl3) δ 7.91 , 7.58 – 7.30 , 4.40 , 2.36 . 13C NMR (100.62 MHz CDCl3) δ 149.5 [N = C(Ph)C(Ph) = C], 149.0 [N = C(Ph)C(Ph) = C], 138.5 (Ar), 133.3 (Ar), 131.6 (Ar), 129.9 (Ar), 128.9 (Ar), 128.5 (Ar), 128.1 (Ar), 128.0 (Ar), 127.7 (Ar), 127.1 (Ar), 126.8 (Ar), 122.8 (Ar), 107.9 [N = C(Ph)C(Ph) = C], 77.1 (OCH2C ≡ CH), 76.8 (OCH2C ≡ CH), 61.0 (OCH2C ≡ CH). IR : 3291, 2125.(35%), yellowish oil. N-substituted pyrazolinone or its O-substituted isomer dissolved in o-dichlorobenzene (ca. 0.04 – 0.05 M), are heated at 180°C until total consumption of the starting material (TLC control). After solvent evaporation the products are purified by flash chromatography [Et2O/n-hexane (1:4)].In a round bottom flask, the 1a (30 mg) for 60 min afforded 3,4-diphenyl-1-methyl-4- pyrazolin-5-one (4a)  as a colourless crystals: mp 146–147°C (Et2O). 1H NMR (400 MHz CDCl3) δ 7.56 , 7.37 – 7.26 , 5.79 , 4.90 , 4.74 , 3.45 .13C NMR (100.62 MHz CDCl3) δ 208.9, 174.5, 158.8, 136.1, 129.9, 129.2, 128.4, 128.2, 127.0, 89.1, 79.1, 60.1, 31.8. IR  : 3061, 2921, 1956 (C = C = C), 1714 (C = O), 1496.EIMS : 288 , 230 (40), 178 (20), 128 (65), 77 (100). HRMS: calcd for C19H16N2O: 288.126263; found: 288.126455.Following the general procedure, heating 1b (14 mg) for 10 min afforded 4--1,3,4-triphenylpyrazolin-5-one (4b)  as a colourless oil. 1H NMR (400 MHz CDCl3) δ 8.07 , 7.70 , 7.47 – 7.23 , 5.90 , 4.94 , 4.77 . 13C NMR (100.62 MHz CDCl3) δ 209.0 (CH = C = CH2), 172.7 (C = O), 159.1 (N = C), 138.2, 136.0, 130.2, 130.1, 129.3, 128.9, 128.4, 127.4, 127.0, 125.3, 119.1, 89.1 (CH = C = CH2), 79.5 (CH = C = CH2), 61.6 (C–CH = C = CH2). IR : 3063, 2921, 1954 (C = C = C), 1721 (C = O), 1596, 1494. EIMS : 350 , 230 (12), 178 (10), 128 (21), 91 (32), 77 (100). HRMS: calcd for C24H18N2O: 350.141913; found: 350.142194.Following the general procedure, heating 2a (20 mg) for 10 min afforded 3,4-diphenyl-1-methyl-4-pyrazolin-5-one (4a)  as a white solid with data identical with 4.4.1.Following the general procedure, heating 2b (24 mg) for 10 min afforded 4--1,3,4-triphenylpyrazolin-5-one (4b)  as a white solid with data identical with 4.4.2.Following the general procedure, heating 5a (10 mg) for 30 min afforded the pyrazolinone 6a  as a glassy solid. 1H NMR (400 MHz CDCl3) δ 7.52 , 7.38 – 7.22 , 3.47 , 3.43 , 3.05 , 1.91 . IR : 3291 (C ≡ C-H), 2923, 1956 (C ≡ C), 1713 (C = O).Following the general procedure, heating 7a (10 mg) for 10 min afforded 6a  with data identical with the previous reaction.Following the general procedure, heating 1a or its isomer 2a is dissolved in dry THF (ca. 0.07 M) (1 eq.) under argon and 18-crown-6 ether (1 eq.) is added. After total dissolution of the reactants, EtOK (0.1 eq.) is added under anhydrous conditions. The reaction is left to react at room temperature. After consumption of the starting material (TLC control) the reaction is stopped by addition of a saturated solution of NH4Cl and extraction with Et2O (2x). The product is purified by flash chromatography [AcOEt / n-hexane (1:6)].In a round bottom flask, the propargylated pyrazolinone 5a) was obtained from 1a after 16 h as a colourless oil (65%). 1H NMR (400 MHz CDCl3) δ 7.69 – 7.64 , 7.56 – 7.52 , 7.47 – 7.43 , 6.30 , 5.32 , 3.49 . IR : 3055, 2925, 1962 (C = C = C), 1659 (C = O), 1483.Following the general procedure, 3,4-diphenyl-1-methyl-2-pyrazolin-5-one (7a) was obtained from 2a after 16 h as a colourless oil (74%). 1H NMR (400 MHz CDCl3) δ 7.47 – 7.43 , 7.34 – 7.24 , 6.79 , 5.36 , 3.81 . 13C NMR (100.62 MHz CDCl3) δ 200.0 (OCH = C = CH2), 147.7, 147.5, 133.5 (Ar), 131.6, 129.6, 129.2, 128.3, 128.2, 127.9, 127.6, 126.7, 121.7 (OCH = C = CH2), 93.0 (OCH = C = CH2), 34.7 (NCH3). IR : 3059, 2940, 1972 (C = C = C), 1605, 1563.Following the general procedure, 1-methyl-3,4-diphenyl-5-pyrazole (2O / n-hexane 1:2) to yield the title compound 8a : mp 81–82°C (Et2O / n-hexane). IR : 3291 (HC≡C), 2127 (C≡C), 1746 (C = O). 1H NMR (400 MHz CDCl3) δ 2.64 , 5.06 , 7.26 – 7.56 . EIMS : 275 , 178 (34), 105 (100), 89 (60), 77 (50). HRMS calcd for C18H13NO2: 275.09463; found: 275.09405.To a solution of 3,4-diphenylisoxazolin-5-one (Section 4.2.4) , triphenylphosphine  and propargylic alcohol  in dry THF (1.5 mL) were added, dropwise, DIAD . After the addition was completed, the solvent was removed under reduced pressure and the crude purified by CC 8  and MS 4 Å, followed by boron trifluoride diethyl etherate . After 10 min 3,4-diphenylisoxazolin-5-one (Section 4.2.4)  was added and the reaction monitored by TLC  until consumption of the starting isoxazolone. After 48 h water (5 mL) was added to the reaction mixture which was extracted with Et2O (2x5 mL) to yield the cobalt complex as a red oil, which was purified by PTLC . To a solution of this red complex in acetone (2 mL) was added Et3N (0.1 mmol) and (NH4)2Ce(NO3)6  until formation of the required isoxazolinone. Water was added (10 mL), followed by extraction of the mixture with Et2O (3x5 mL) and purification by PTLC  afforded the N-substituted isoxazolinone 8b : mp 113–115°C (Et2O / n-hexane). IR : 3291 (HC≡C), 2113 (C≡C), 1746 . 1H NMR (400 MHz CDCl3) δ 1.48 , 2.31 , 7.19 – 7.50 . 13C-NMR (100.62 MHz CDCl3) δ 28.5 [NC(CH3)2], 61.3 [NC(CH3)2], 73.7 (HC≡C), 82.4 (HC≡C), 127.4, 128.2, 128.3, 128.7, 129.5, 130.6, 170.2. EIMS : 303 , 237 , 178 (35), 67 (40). HRMS: calcd for C20H17NO2: 303.12593; found: 303.12641.Following Nicholas protocol , to a st2O / n-hexane 1:1, 2) AcOEt), the solvent was removed under reduced pressure. Purification by CC  afforded the title compound 8c  as a white solid: mp 50–51°C ( Et2O / n-hexane). IR : To 3-methyl-4-phenylisoxazolin-5-one (Section 4.2.3)  was added a solution of Na (39 mg) in EtOH (1.7 mL). To this solution was added allyl bromide . The reaction mixture was magnetically stirred, under argon, while being heated under reflux (1.8 h). On completion of the reaction  Et2O / n-hexane 1:1, 2) AcOEt), the solvent was removed under reduced pressure. Purification by CC  afforded the title compound 8c  as a white solid: mp 50–51°C ( Et2O / n-hexane). IR : 1711 (C = O), 1604 (C = C).1H NMR (400 MHz CDCl3) δ 2.29 , 4.27 , 5.30 , 5.80 , 7.27 , 7.38 , 7.46 . 13C-NMR (100.62 MHz CDCl3) δ 11.8 (CH3), 53.1 (N–CH2), 103.8 [N–C(Me) = C(Ph)], 120.5 (CH2 = CH), 127.2 (Ar), 128.2 (Ar), 128.5 (Ar), 129.4 (Ar), 129.6 (CH2 = CH), 159.3 [N–C(Me) = C(Ph)], 169.5 (C = O). EIMS : 215 , 130 (21), 116 (38), 115 (46), 89 (13), 77 (13). HRMS: calcd for C13H13NO2: 215.09463; found: 215.09397.To 3-methyl-4-phenylisoxazolin-5-one (Section 4.2.3)  was added a solution of Na (39 mg) in EtOH (1.7 mL). To this solution was added allyl bromide . The reaction mixture was magnetically stirred, under argon, while being heated under reflux (1.8 h). On completion of the reaction  Et8d)  as a white solid: mp 105–106°C (Et2O / n-hexane). IR : 1720 (C = O), 1615 (C = C). 1H NMR (400 MHz CDCl3) δ 4.06 , 5.16 , 5.25 , 5.78 , 7.18 – 7.48 . 13C NMR (100.62 MHz CDCl3) δ 54.9 (N–CH2), 105.2 [N–C(Ph) = C(Ph)], 121.0 (CH2 = CH), 127.2 (Ar), 127.7 (Ar), 128.2(Ar), 128.3(Ar), 128.7(Ar), 129.0(Ar), 129.2(Ar), 129.3(Ar), 131.0 (CH2 = CH), 162.8 [N–C(Ph)], 169.9 (C = O). EIMS : 277 , 236 (12), 192 (27), 178 (76), 133 (13), 117 (51), 89 (46), 77 (22). HRMS: calcd for C18H15NO2: 277.11028; found: 277.11037.3,4-Diphenylisoxazolin-5-one (Section 4.2.4) , using the previous protocol, afforded the title compound  in CCl4 / CHCl3 (1:3) (20 mL) under argon was added diethyl 2-(ethoxymethylene)malonate , 0.93 , 1.23 , 4.00 , 4.12 , 5.26 , 5.35 , 5.90 , 7.88 . 13C NMR (100.62 MHz CDCl3) δ -5.3 [Si(CH3)2], 14.1 (OCH2CH3), 17.8 [C(CH3)3], 25.8 [C(CH3)3], 59.9 (CH2N), 60.3 (OCH2CH3), 60.6 (OCH2CH3), 61.4 (NCH = C), 119.6 (CH2 = CH), 131.1 (CH2 = CH), 149.5 (N–CH), 166.2 (C = O). EIMS : 357 , 312 , 300 , 226 , 198 (20), 57 . HRMS: calcd for C17H31NO5Si: 357.19715; found: 357.19796.To a stirred solution of tert-butyldimethylsilyloxy)amino) methylene]malonate , at RT under nitrogen, was added SbCl5 . On completion of the reaction (10 m), , the solvent was removed under reduced pressure. Purification by CC  afforded diethyl 2-[amino) methylene]malonate (11) , and ethyl 2-allyl-5-oxo-2,5-dihydroisoxazole-4-carboxylate (8e) . Compound 11 was unstable and lactonised spontaneously on standing to the 2-allyl-isoxazolinone 8e.To a magnetically stirred solution of 2-, 80.6 (CH = C = CH2), 88.0 (CH = C = CH2), 126.9 (Ar), 127.1 (Ar), 127.7 (Ar), 128.8 (Ar), 129.2 (Ar), 129.6 (Ar), 131.6 (Ar), 134.3 (Ar), 166.6 (N = C), 175.1 (C = O), 203.0 [CH = C = C(CH3)2]. EIMS : 275 , 231 , 172 (89), 128 (99), 102 (89), 77 (82). HRMS: calcd for C18H13NO2: 275.09463; found: 275.09556.Following the general procedure, 3,4-diphenyl-4-isoxazolin-5-one (9b) was obtained by heating 8b at 60°C for 10 m, followed by CC , as a colourless solid (85%): mp 77–80 (Et2O / n-hexane). IR : 1969 (C = C = C), 1790 (C = O). 1H NMR (400 MHz CDCl3) δ 1.37 , 1.62 , 5.65 , 7.28 – 7.54 . 13C NMR (100.62 MHz CDCl3) δ 19.3 (CH3), 19.7 (CH3), 58.5 [N = C(Ph)C], 86.6 [CH = C = C(CH3)2], 102.2 (CH = C = C(CH3)2), 127.0 (Ar), 127.7 (Ar), 128.1 (Ar), 128.5 (Ar), 128.9 (Ar), 129.4 (Ar), 129.7 (Ar), 131.2 (Ar), 132.2 (Ar), 134.7 (Ar), 167.2 (N = C), 177.5 (C = O), 203.5 (CH = C = C(CH3)2). EIMS  303 , 288 , 259 , 244 , 200 , 141 , 89 , 77 .Following the general procedure, 3,4-diphenyl-4-isoxazolin-5-one (9c) was obtained by heating 8c at 180°C for 7 h, followed by CC , as a viscous oil (95%): IR : 1791 (C = O), 1643 (C = N). 1H NMR (400 MHz CDCl3) δ 1.95 , 2.85 , 3.10 , 5.25 , 5.30 , 5.60 , 7.20 – 7.44 . 13C NMR (100.62 MHz CDCl3) δ 12.4 (CH3), 36.4 (C–CH2), 59.3 (C–CH2), 121.4 (CH2 = CH), 126.2 (Ar), 127.3, 127.8, 128.9, 129.5, 129.7, 133.3 (CH2 = CH), 168.3 (C = N), 178.7 (C = O). EIMS : 215 , 116 (15), 115 (40), 77 (30). HRMS: calcd for C13H13NO2: 215.09463; found: 215.09414.Following the general procedure, 4-allyl-3-methyl-4-phenylisoxazolin-5-one (9d) was obtained by heating 8d at 180°C for 5 h, followed by CC , as a pale yellow solid (97%): mp 71–72°C. IR : 1800 , 1644 (C = N).1H NMR (400 MHz CDCl3) δ 3.03 , 3.32 , 5.25 , 5.14 , 5.54 , 7.33 – 7.52 . 13C NMR (100.62 MHz CDCl3) δ 37.5 (C–CH2), 58.8 (C–CH2), 121.8 (CH2 = CH), 126.3 (Ar), 127.2 (Ar), 127.4 (Ar), 128.9 (Ar), 129.0 (Ar), 129.4 (Ar), 129.7 (Ar), 131.7 (CH2 = CH), 134.4 (Ar), 167.1 (N = C), 169.9 (C = O). EIMS : 277 , 236 , 232 (21), 178 (49), 117 (51), 103 (35), 77 (30). HRMS: calcd for C18H15NO2: 277.11028; found: 277.10956.Following the general procedure, 4-allyl-3-methyl-3,4-phenylisoxazolin-5-one (12) was obtained by heating 8e at 180°C for 5 h, followed by CC , as a colourless oil [-1): 2266 (-C≡N), 1750 (C = O). 1H NMR (400 MHz CDCl3) δ 1.32 , 2.69 , 3.56 , 4.27 , 5.25 , 5.83 .Following the general procedure, ethyl 2-cyanopent-4-enoate (less oil  (80%): I8d  in dry 1,4-dioxane (2 mL) at RT under argon, potassium ethoxide  and 18-crown-6 ether  were added. After 10 m the reaction was found complete , the mixture was neutralized with aq. 0.5 N AcOH and the solvent removed under reduced pressure. The crude was diluted with Et2O (5 mL) and washed with distilled water (2 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Purification by CC  afforded 2-(2′-ethoxyethyl)-4,5-diphenyl-2,3-dihydro-1,3-oxazin -6-one (14)  as a yellow oil: IR : 3257 (NH), 1694 (C = O), 1600, 1585, 1553. 1H NMR (400 MHz CDCl3) δ 1.05 , 2.22 , 3.44 , 3.62 , 3.77 , 5.47 , 6.46 , 7.02 – 7.24 . 13C NMR (100.62 MHz CDCl3) δ 15.0 (OCH2CH3), 32.0 (OCH2CH2), 65.7 (OCH2CH3), 66.6 (OCH2CH2), 81.6 (N–CH–O), 103.9 (N–C = C–CO), 126.1 (Ar), 127.6 (Ar), 128.2 (Ar), 129.8 (Ar), 130.2 (Ar), 131.4 (Ar), 133.9 (Ar), 134.9 (Ar), 155.5 (N–C = C–CO), 166.3 (C = O). EIMS  323 , 250 , 178 , 77 . HRMS: calcd for C20H21NO3: 323.15214; found: 323.15254; and deoxybenzoin   as a colourless solid: mp 55–56°C (lit.: [-1): 1686 (C = O). 1H NMR (400 MHz CDCl3) δ 4.29 , 7.24 – 8.04 . After the reaction in a parallel run was completed, glacial CH3COOD (3.0 eq.) was added to the reaction followed by H2O (2 mL). The reaction mixture was diluted with Et2O (5 mL) and washed with an aqueous solution of 1M NaHCO3. The aqueous layer was extracted with Et2O (3 × 5 mL), the combined organic extracts were dried over anhydrous sodium sulphate, filtered and concentrated. Purification by CC  afforded mono-deuterated deoxybenzoin  : 1H NMR (400 MHz CDCl3) δ 4.29 , 7.23 – 8.06 .To a stirred solution of compound 23.3 mg, % as a ye8e  in dry 1,4-dioxane (1 mL) at RT under argon, was added potassium ethoxide  and 18-crown-6 ether . On completion of the reaction , the solvent was removed under reduced pressure and the crude purified by cc . N-Allyl-malonamic acid ethyl ester (13) was obtained as a white solid : mp 40–41°C (Et2O). IR (KBr) νmax (cm-1): 3299 (NH), 1740 (C = O ester), 1659 (C = O amide). 1H NMR (400 MHz CDCl3) δ 1.22 , 3.26 , 3.84 , 4.13 , 5.07 , 5.14 , 5.80 , 7.29 . 13C NMR (100.62 MHz CDCl3) δ 14.0 (COOCH2CH3), 41.3 (COCH2COOEt), 41.8 (NHCH2), 61.5 (COOCH2CH3), 116.2 (CH2 = CH), 133.7 (CH2 = CH), 165.0 (NHC = O), 169.3 (COOEt). EIMS : 171 , 56 . HRMS: calcd for C8H13NO3: 171.08954; found: 171.08894.To a stirred solution of compound 8b)  in dry 1,4-dioxane (0.25 mL), under argon, was added potassium ethoxide  and 18-crown-6 ether . On completion of the reaction (10 min) , the mixture was neutralized with aq. AcOH 0.5 N and the solvent removed under reduced pressure. The crude was diluted with Et2O (5 mL) and washed with destilled H2O (2 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Purification by CC  afforded 4--3,4-diphenylisoxazolin-5-one (9b) . After 30 min of reaction, no rearrangement product could be still detected by TLC.To a stirred solution of compound (9b (55%).Using the previous conditions with potassium ethoxide (0.1 eq.) and 18-crown-6 ether (0.1 eq.), the reaction after 5 days yielded 8b (1 eq.) in dry 14-dioxane, under argon, were added LDA  and 12-crown-4 ether (1.5 eq.). The reaction mixture was stirred for 48 h. TLC control  showed no reaction and the starting material was recovered yield .To a stirred solution of 8b (1 eq.) in dry 1,4-dioxane, under argon, were added tert-BuLi  and 12-crown-4 ether (2 eq.). The reaction mixture was stirred for 70 h. TLC control  showed no reaction and the starting material was recovered .To a stirred solution of a Web Table  is available via the HTML version of the article.The authors declare that they have no competing interest.Calculated relative energies for intermediates and transition states relating to Scheme Click here for file


Introduction.The aim was to evaluate the changes of androgen receptor (AR) expression quantitatively and to identify influence of AR on cancer related survival markers in LNCap cell line. Materials and Methods. We compared expressions of AR, heat shock protein 27 (HSP27), clusterin (CLU), glucose-related protein 78 (GRP78), and cellular FLICE-like inhibitory protein (c-FLIP) and their genes between es-LNCaP , ls-LNCaP , and si-LNCaP (AR siRNA transfected ls-LNCaP) by Western blotting and RT-PCR. Results. The expressions of AR, HSP27, CLU, GRP78, and c-FLIP were increased in ls-LNCaP compared with es-LNCaP . However, in si-LNCaP cell line, protein expressions were reversed to the level of es-LNCaP cell lines , and gene expressions on real-time PCR were also reversed to the expression level of es-LNCaP . Conclusions. This finding suggests that androgen receptor can be related to the increased expression of cancer related survival markers such as HSP27, GRP78, CLU, and c-FLIP in late stage prostate cancer, and also inhibition of AR gene can be a therapeutic target in this stage of cancer. Nearly 29% of patients with newly diagnosed cancer in United States were diagnosed with prostate cancer which is the second most common cause of cancer death  [At early stage, the prostate cancer is influenced markedly by androgen acting through the androgen receptor (AR) and, clinically, could be treated with surgical castration, radiation, or antiandrogen therapy. However, after initial response to these treatments, most androgendependent prostate cancer cells commonly progress to a highly aggressive, metastatic, androgenindependent state.Androgen receptor (AR) is a 110 kDa phosphoprotein and one of the nuclear receptor superfamily of ligand activated transcription factors which elicits the biological response of androgens –5. The AAndrogen independent prostate cancer development can be explained by the following five theories. (1) the AR hypersensitivity: under chemical castrated state induced by androgen ablation treatment, more AR is produced or the AR has enhanced sensitivity to androgen. (2) The promiscuous AR hypothesis: factors other than testosterone  acts as a mutated AR agonists due to broaden specificity of AR. (3) The outlaw AR hypothesis: hormone independent prostate cancer growth or progress of AR independently through PTEN mutation and activation of AR independent pathways such as PI3K and MAPK. (4) The bypass AR hypothesis: in androgen deprivation state, other antiapoptotic signal pathways through Bcl-2 overexpression and oncogene activation induce progression to HRPC. (5) The lurker cell hypothesis: androgen independent prostate cancer cells basically exist among epithelial stem cells even at androgen dependent state. After androgen deprivation, androgen independent malignant stem cells are selected to be activated .LNCaP cell line is androgen sensitive human prostate cancer cells derived from the lymph node metastasis , 14. IgaWe focused on the factors related to the development of androgen independent prostate cancer. In previous studies using proteomic analysis, we confirmed that high passage subcultured LNCaP cells that acquired androgen independent property and the silencing of AR with small interfering RNA (siRNA) transfection resulted in the reversion of proteomic profile to level of es-LNCap cell line . The aimLNCaP cells obtained from American Type Culture Collection  were maintained in RPMI 1640 medium and made two LNCaP clones described in previous study [μL OptiMEM medium/60 mm culture dish. The transfected cells were allowed to grow for 24, 48, and 72 h at 37°C in a 5% CO2 incubator and harvested for RT-PCR and immunoblot analysis. We performed immunohistochemical staining to confirm the expressions of AR in various LNCaP cells.Doxazosin  was prepared as described in previous study . Cells w5) were mixed in a test tube with 1 mL TRIzol solution. Prepared RNA was denaturated at 65°C for 15 min in a volume of 30 μL and cooled on ice for at least 1 min. 2.0 μg of denatured RNA were then annealed by addition of reaction mixture to a total volume of 20 μL  and incubated at 42°C for 70 min. The reaction was terminated at 95°C for 5 min, chilled on ice for 5 min, and collected by brief centrifugation. To remove RNA, 1 μL of RNase H were added to each tube followed by incubation at 37°C for 20 min. 1 μL of cDNA were used for each PCR reaction. Total RNA was extracted using the TRIzol method . Cells , and 1.0 μL cDNA as template.Amplifications of cDNAs by PCR using specific primer pairs for AR were performed in 20 μL of the reverse transcriptase product for PCR in a final volume of 25 μL. Real-time PCR was performed with an SLAN real-time PCR detection system  and SYBR Green reagents . Specific primers for human GAPDH, AR, HSP27, CLU, GRP78, and c-FLIP were designed to work in the same cycling conditions . We used 1.0 Preparation of total cell lysate and the procedures for Western blot analyses were performed essentially as described previously . The antμg) from lysates of cultured LNCaP cells were subjected to SDS-PAGE and then transferred to a PVDF filter. The filters were blocked in TBS containing 5% nonfat milk powder at 4°C overnight and then incubated for 1 h with a diluted each primary antibodies . Samples with equal amounts of protein  and then fixed with ice-cold methanol for 15 min. Samples were further permeabilized with PBS containing 0.025% Triton-X detergent (1 × PBS-TX) for 10 min and blocked with 3% BSA in 1 × PBS for 30 min. Cells were reacted with primary antibodies  for 1 hour at room temperature. Cells were washed 3 times for 5 min with 1 × PBS-TX and then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies . Diaminobenzidine (DAB) was used as the chromogen and counterstaining was done with Mayers hematoxylin. Following three 5 min washes, cellscoverslips were mounted on slides with coverslips.For TUNEL assays, fixed cells were incubated with an equilibrium buffer for 5 min using the in situ apoptosis detection kit, Fluorescein , and then treated in reaction buffer with 10 units of terminal deoxynucleotidyl transferase and 1 unit of deoxyuridine triphosphate digoxigenin at 37°C for 1 hour. The reaction was terminated by adding stop/wash buffer and then washed twice with Tris buffer. Anti-digoxigenin-FITC was added and reacted at 37°C for 30 min. After washing with distilled water, nuclei were counterstained with Hoechst 33258 , and apoptosis in the cells was observed under a fluorescent microscope. Cells with green fluorescent (FITC) colored nuclei were considered apoptotic. For quantifying apoptotic cells, apoptotic and total cells were counted in 5 random fields scoring between 300 and 500 cells, and the numbers of apoptotic cells were expressed as percentages of the total cell population. Immunocytochemical staining slides and TUNEL staining slides were observed with microscope .t-test to assess statistical significance. Values of P < 0.05 were considered statistically significant.Results were analyzed using a two-tailed Student's  To explore the feasibility of siRNA transfection efficiency in knocking down AR expression in prostate cancer cells that harbor the AR gene, fluorescent oligo staining with BLOCK-iT was used and the green staining of more than 95% of cells was confirmed in siRNA transfected cells under fluorescence microscopy . We desiAmong the four experimental cell lines , we confirmed the expression level of five prostate cancer related proteins  with immunocytochemical staining . PositivThe expression of five prostate cancer related proteins  increased in ls-LNCaP compared with es-LNCaP  consistently causes prostate apoptosis and involution in first diagnosed prostate cancer. But, when prostate cancer advance further, it progresses into a more aggressive form of castration resistant prostate cancer (CRPC), refractory to all kinds of ADT. Treatment of CRPC is very difficult and not that satisfactory so far. Docetaxel based chemotherapy is one of the most effective ways of treatments –20, but Diverse pathways have been discussed regarding progression to CRPC from androgen dependent counterpart. Among them, AR is considered having one of the most important roles with the possible mechanisms of hypersensitive AR or mutation of AR gene . LNCaP cAmong the possible mechanisms of chemotherapy failure of CRPC, several antiapoptotic factors such as c-FLIP , HSP27 , clusterHsp27 suppresses apoptosis and probably has a critical role in progression to CRPC –29. It hGRP78 is a key member of the molecular chaperone heat shock protein (HSP) 70 family –33. GRP7α induced apoptosis.Clusterin acts as an antiapoptotic factor and plays an important role in resistance to chemotherapeutic drugs . When clc-FLIP is also involved in apoptosis pathway regarding Fas signal transduction . It is gOur study showed increased expression of clusterin, HSP27, GRP78, and c-FLIP in H-81 compared to L-33. mRNAs and proteins of these factors are downregulated below the levels of L-33 after AR knock-out using siRNA technique. Furthermore, the same concentration of doxazosin could induce more significant apoptosis after AR silencing. These observations suggest that Clusterin, HSP27, GRP78, and c-FLIP take part in the progression into H-81 in LNCaP model and AR is closely related to the upregulated expression and suppression of these survival factors. Our study also helps us to speculate that while prostate cancer cells become more aggressive with change of AR and overproduction of survival factors under the extremely stressful circumstances like androgen deprivation, these cells can be reverted into treatment sensitive traits when successful AR blocking causes suppression of various survival factors. It also supports the idea that therapeutic approach targeting AR can enhance the efficacy of anticancer treatment in the patients with metastatic CRPC, resisting against all forms of treatment.Suppression of gene expression using siRNA is a simplified experimental technique which can regulate functions of specific factors at the gene level. AR silencing in the gene level is essential in the study of AR block, because AR is a transcription factor related to synthesis, regulation, and secretion of various kinds of proteins. In our present study, AR silencing successfully showed over 80% efficiency. We also showed higher level of discrimination using real time RT-PCR which enables us to find more significant differences between cell groups.It can be summarized that in this LNCaP model, we observed that c-FLIP, HSP27, clusterin, and GRP78 take part in the progression into androgen insensitive status and the existence and overexpression of AR are closely related in this process. We think that these findings can be applied in the understanding of CRPC progression and treatment resistance of metastatic CRPC. New therapeutic approaches targeting AR regulation could be an effective solution against metastatic CRPC, currently incurable, and therefore various scientific efforts should be focused.


This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO Plasmodium falciparumAE1 is a major membrane protein in erythrocytes  that mediates anion exchange and participates in control of the erythrocyte shape 12E8 was ∼1,500 and ∼500 kDa, respectively. Despite its heterogeneity, octyl-polyoxyethylene solubilized AE1 formed two-dimensional (2D) arrays (unit cell dimensions: a = b = ∼11 nm) in the presence of dimyristoyl phosphatidylcholine Since AE1 is a highly abundant membrane protein with a million copies in each red cell and pure plasma membrane can be readily obtained from enucleated red cells, it should be one of the very few membrane proteins that can be directly purified from natural membranes. However, the high heterogeneity of natural AE1 because of various covalent modifications, the presence of different oligomeric forms and complexes with other erythrocyte proteins has made it very difficult to purify in a highly homogeneous form for structural studies. In addition, treatment with chaotropic agents and non-physiological alkaline pH used in the separation of accessory proteins from AE1 can significantly change its native structure Additional attempts to determine the structure of AE1 have involved separately studying the membrane and cytoplasmic domains. The membrane domain generated by trypsin digestion of purified human AE1 was reconstituted into 2D sheets with lipids In the present study, we obtained biochemically homogeneous, native full-length AE1 dimers by using a non-denaturing purification method. By single particle EM reconstruction we show that the AE1 dimer has an elongated structure consisting of a double-humped cytoplasmic domain and an oval-shaped membrane domain tethered by two linkers. Image classification revealed groups of AE1 dimers with different tilt orientations of cytoplasmic domain relative to the membrane domain suggesting flexibility of the two linkers that connect the cytoplasmic domain to the anchored membrane domain. This linker’s flexibility points to a novel pivot mechanism by which AE1 is involved in regulating membrane elasticity, a critical determinant of shape changes necessary for the red cells to transit through capillaries whose dimensions are much smaller than that of the cell.Bovine AE1 is not glycosylated The heterogeneity of natural human AE1 has prevented its purification in a highly homogeneous form required for structural studies. Given that there is significantly lesser heterogeneity of bovine AE1 The ghost suspension from the previous step was pelleted at 30,000 g for 40 min. The pellet was mixed (1∶20) with 100 mM Tris-HCl, pH 7.5, containing complete protease inhibitors cocktail (Roche) and 2% dodecyl maltoside . After 2 h incubation, the suspension was centrifuged at 18,000 g for 40 min. The supernatant was loaded onto a 3×5 cm column of DE-52 (Whatman) equilibrated with 50 mM Tris-HCl, containing 0.03% DDM. Proteins were eluted from the column with a linear 0–0.5 M NaCl gradient in the same buffer. AE1 was eluted with 0.3–0.5 M NaCl. The fractions containing AE1 were collected and concentrated to ∼10 mg/ml protein concentration using a Centricon 50 centrifuge filter device (Millipore). The concentrated sample was further subjected to size-exclusion chromatography to obtain the dimeric form of AE1. Approximately 0.3 ml of the concentrated AE1 solution was loaded on a Superose 6™ 10/300 column  equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.03% DDM and 0.15 M NaCl. The fraction containing the AE1 dimer was immediately used for EM grids preparation.SDS-PAGE was performed using pre-fabricated 7.5% polyacrylamide gels from Bio-Rad Freshly prepared bovine AE1 dimers were negatively stained with 0.8% uranyl formate on grids coated with carbon film. Micrographs were then recorded on a 4k×4k CCD camera at 70,000× magnification using Leginon For OTR images, the grid was tilted at two orthogonal angles (−45° and +45°) to acquire a pair of micrographs for each specimen area of interest, one micrograph at each tilt angle. In total, 358 sets of tilt pairs were collected.For 2D image analysis and 3D structure refinement, 1275 micrographs were collected without tilting the grid. In total, 174,197 particles were extracted from the best 669 micrographs. These micrographs have defocus values ranging from −1.0 to −1.8 µm, as determined by CTFIND For OTR, we obtained two image data sets for each specimen area: a −45° tilt set and a +45° tilt set. Particles in the −45° tilt set were used for classification and particles in the +45° tilt set were used for 3D reconstructions. The following procedure was used to obtain reliable 3D maps without using initial models, thus eliminating possible model bias.ApTiltPicker.py program in Appion refine2d.py program in EMAN to generate 100 class averages. These class averages were then used as references to align the same particles with SPIDER CA S command) in SPIDER.Corresponding particles from tilt pairs were picked out automatically by the i.e., same view), but their corresponding particles in the +45° tilt set represent different views, which we called ortho-views of the class. Ortho-views of the same class have different orientation parameters, which were calculated based on the 90° tilt angle difference and the in-plane rotation parameters using a custom script called combine_ang.bat. From the ortho-views associated with each class, we obtained one 3D OTR map. Since we had 100 classes in the −45° tilt particles, we obtained a total of 100 OTR maps from the +45° tilt particles. These maps were improved iteratively by using center parameters refined by aligning ortho-views against their projections computed from the 3D OTR maps.All particles in each class of the −45° tilt set have the same feature , which corresponds to the orientation of the associated class average in the −45° tilt particle set. We visually compared this projection and the class average for feature consistency and selected the best 25 OTR maps. These 25 ORT maps then aligned using the eotest program in EMAN. The 3D model was visualized with the UCSF Chimera The averaged 3D OTR map was used as the starting model to align the 174,197 particles using the projection matching method in EMAN. A 3D map was obtained by combining the aligned particles and iteratively refined. The angular interval between projections was gradually decreased from 15° to 5° during the course of refinement. The map was corrected for the effect of contrast transfer function with phase flipping and imposed with 2-fold symmetry. The resolution of 3D reconstruction was estimated by FSC calculated with the To assess the flexibility of cytoplasmic domain, 16,061 front-view particles and 7,644 side-view particles were selected from 174,197 particles based on the result of projection matching. Both the front-view particles and side-view particles are then classified into subgroups.AE1 in the erythrocyte membrane exists either in a free state or as a multi-proteitn complex with other erythrocyte proteins. Previous investigators extracted both pools of AE1, and used alkaline and chaotropic agents to strip off accessory proteins that have been shown to significantly change the native structure of AE1 EM micrograph of negatively stained bovine AE1 particles revealed complexes of different shapes possibly representing AE1 dimers viewed at different orientations . The claTo reconstruct an initial 3D map of AE1 in the absence of a starting model, we used the orthogonal tilt reconstruction (OTR) approach We obtained an improved reconstruction by combining 174,197 particles of untilted samples by projection matching using the above averaged OTR map as a starting model . Since tThe single-particle reconstruction of the full-length dimeric AE1 has an elongated shape (15×9×6.5 nm) with a small and a larger structure . The smaOur antibodies against different parts in the cytoplasmic and membrane domain of bovine AE1 did not work well on immunoelectron microscopy. Therefore we used the published 2.6 Å resolution crystal structure of the human AE1 cytoplasmic domain to localize the cytoplasmic domain in our single-particle reconstruction. Although the crystal structure of the cytoplasmic domain was solved at pH 4.8, site-directed spin labeling studies in combination with conventional electron paramagnetic resonance and double electron resonance spectroscopy performed at neutral pH demonstrated that the structure of the cytoplasmic domain (residues 55–356) is indistinguishable from the crystal structure determined at pH 4.8 The dimeric crystal structure of the cytoplasmic domain filtered to 2.4 nm resolution resembles the small end of the EM reconstruction, in the overall size and in having a double-humped shape . The ribOur single particle reconstruction of full-length AE1 clearly shows that the cytoplasmic and membrane domains are connected through two well separated linker densities . Each liTo estimate the level of rigidity of the linkers between the cytoplasmic and membrane domains, we selected the front-view and side-view particles  AE1 tetramer bound to the membrane cytoskeleton via ankyrin, and (2) AE1 dimer linked to the membrane cytoskeleton via adducin. Recent AE1 diffusion measurements in the erythrocyte membrane estimated their near equal relative abundance Movie S1Animated view of a series of front-view class averages showing tilting motions of the AE1 cytoplasmic domain relative to the membrane domain.(GIF)Click here for additional data file.


Our previous investigation demonstrated that CDK11p58 repressed androgen receptor (AR) transcriptional activity and was involved in the negative regulation of AR function.CDK11p58 expression was examined in the prostate cancer tissues and adjacent tissues by IHC and qRT-PCR. Cell apoptosis was detected by flow cytometry. The metastasis of cancer cells was evaluated by the Transwell Assay. Finally we further investigated the underlying molecular mechanisms by examining expression levels of relevant proteins using western blot analysis.CDK11p58 were low in prostate cancer tissues compared with its adjacent noncancerous tissues. CDK11p58 promoted the prostate cancer cell apoptosis and inhibited its metastasis in a kinase dependent way. And finally CDK11p58 could inhibit the metastasis of AR positive prostate cancer cells through inhibition of integrin β3 and MMP2.We found that both RNA and protein expression of CDK11p58 is an anti-metastasis gene product in prostate cancer.These data indicate that CDK11 Cyclin D3 and CDK11p58 were involved in the regulation of AR-mediated transactivation. Cyclin D3/CDK11p58 holoenzyme kinase complex repress AR function through phosphorylating AR at Ser-308[p58 was autophosphorylated at Thr-370. Thr-370 is responsible for the autophosphorylation, dimerization, and kinase activity of CDK11p58[CDK11, which is encoded by two highly homologous p34cdc2-related genes, Cdc2L1 and Cdc2L2[ormation. Our stu Ser-308. Also, wp58 was involved in the negative regulation of AR, we speculated that CDK11p58 might be involved in the regulation of prostate cancer metastasis.Dysregulated AR signaling is implicated in several types of tumor, including carcinomas of the prostate, breast, liver and bladder. Abnormap58 in prostate cancers. Our study indicated that expression of CDK11p58 was decreased in the prostate cancer and CDK11p58 was involved in the negative regulation of prostate cancers.In the current study, we further examined the expression in the prostate cancer tissues and investigated the biological functions of CDK11p58 is located on human chromosome 1p36.33, a region frequently mutated in various cancers. To investigate the potential role of CDK11p58 in prostate carcinogenesis, we examined the expression of CDK11p58 protein in prostate cancer tissues and adjacent non-cancerous tissues. By western blot assay, we found that CDK11p58 was higher in normal prostate tissues than in cancer tissues  versus normal tissue (14/20) . Compared with the control, T370A, D224N, wild type CDK11p58 inhibited the proliferation of prostate cancer cells. The kinase constantly activitied mutant T370D exhibited even more inhibition effect than others  were localized mainly in nucleus. CDK11p58 was co-localized in the nucleus with AR (yellow). However, the control vector pcDNA3.0 (red) was not localized in nucleus and showed no colocalize with AR , one of the most common cancers in elderly men, is increasing annually in the world. CDK11okinesis. Our pre/CDK11p58. FurtherAndrogens and AR are indispensable for the development, regulation, and maintenance of male phenotype and reproductive physiology,6. The Ap58 could inhibit the transcription activity of AR, we speculated CDK11p58 could inhibit the metastasis of prostate cancer through AR signaling. First, we examined the expression of CDK11p58 in prostate cancer tissues to find that the expression of CDK11p58 was decreased in prostate cancer tissues compared with paired, normal epithelium, with decreased staining intensity and a lower proportion of positively stained cells in prostate cancer tissue. A similar trend in the expression of Cyclin D3, a partner protein of CDK11p58, was also observed.As CDK11p58 could inhibit the proliferation and promote the apoptosis of prostate cancer cells. To our surprise, as a Ser/Thr kinase, the proliferation inhibition was not fully kinase dependent. But the apoptosis promotion was dependent on it kinase activity.As we reported before, CDK11p58 indeed inhibited the migration and invasion of AR + LNCap cells, but not AR- PC3 cell lines. These data demonstrated that CDK11p58 could inhibit the metastasis of prostate cancer cells through the AR signaling pathway. And also it was kinase dependent. Then we examined the interaction between CDK11p58 and AR. The data showed that T370A and T370D mutants were both capable of interacting with AR. The interaction was not totally dependent on its kinase activity. However, in our previous study, the other kinase dead mutant D224N failed to interact with AR. It suggested that the aspartic acid D224 of CDK11p58 was necessary for the interaction with AR not just because of its kinase activity. Maybe, the mutant D224N has changed the CDK11p58 protein configuration and finally influenced the interaction with AR. The exact mechanism needs further investigation. This result indicated both CDK11p58 and T370A could interact with AR, but only the kinase activated CDK11p58 could inhibit the metastasis of AR positive prostate cancer cells. Western Blot assay showed that metastasis-related genes integrin β3 and MMP2, but not MMP3 was inhibited by CDK11p58 in a dose and kinase dependent manner.As we speculated, CDK11p58 in prostate cancer tissue led to the dysfunction of cell apoptosis and metastasis of cancer. CDK11p58 inhibit the metastasis of AR positive prostate cancer cells through inhibition of integrin β3 and MMP2 in a kinase dependent manner. These data indicate that CDK11p58 is an anti-metastasis gene product in prostate cancer. Taken together, we demonstrate a new role for CDK11p58 as an anti-metastasis gene in prostate cancer.Abnormal expression of CDK11Fetal bovine serum (FBS), Dulbecco modified Eagle medium (DMEM), 1640 and LipofectAMINE reagen were purchased from Invitrogen. The anti-cyclin D3 monoclonal antibody, the mouse and rabbit secondary antibody were purchased from Cell Signaling. The anti-CDK11 and anti-AR polyclonal antibody were purchased from Santa Cruz Biotechnology. HRP-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse IgG secondary antibodies were also purchased from Santa Cruz Biotechnology. The anti-MMP2 polyclonal antibody was purchased from Bioworld. The anti-MMP3 polyclonal antibody was purchased from Epitomics Company. The qRT-PCR assay system was purchased from Tiangen Company.2. Transfection for immunoprecipitation was performed in 100-mm dishes with 8 ug total plasmids.The PC3 and LNCap cell lines were obtained from cell bank of our lab. Cells were grown using 1640 medium with 10% FBS, 100 units/ml penicillin, and 100 ug/ml streptomycin at 37°C and 5% COExpression levels of CDK11 in postoperative paraffin-embedded tumor specimens from prostate cancer patients were detected with immunohistochemistry (IHC). The concentrations of antibody were as follows: anti-CDK11, 1:100 dilution; Nuclear expression of both CDK11 is defined as positive. The detailed staining procedures strictly followed the supplier’s recommendation. Negative controls were obtained by incubating parallel slides with the primary antibodies omitted. In addition, sections with confirmed positive staining from each run served as positive controls. Immunostaining of the whole slide area was evaluated by two independent pathologists who remained unaware of tumor characteristics and other staining results.μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF). Detergent insoluble materials were removed by centrifugation. Whole tissue lysates were incubated with 2 μg relevant antibody at 4°C for 2 h. Pre-equilibrated protein G-agarose beads were added and collected by centrifugation after incubation overnight and then gently washed three times with the lysis buffer. The bound proteins were eluted and analyzed using Western blots. An antibody to GAPDH was used to ensure equivalent loading.Prostate cancer cells were first lysed with ultrasoud in 1 ml of coimmunoprecipitation (CoIP) buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2, 60 mMβ-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF, 0.1 mM benzamide, 10 4 cells/ml) was added to the inside of the inserts. Assays performed at 37°C, 5% CO2 were 24 h for transfected cells. After incubation, noninvading cells were removed from the upper surface of the membrane using cotton-tipped swabs. The cells on the lower surface of the membrane were stained with Crystal violet. Cells were counted in the central field of triplicate membranes.Cells invasion was assayed using BD biocoat growth factor reduced (GFR) matrigel invasion chambers . LNCap cell suspension -conjugated goat anti rabbit secondary antibody and rhodamine-conjugated goat anti mouse for 1 h at room temperature. Cells were washed three times with PBS, stained for Hoechst 33258 (50 μg/ml) solution, respectively, in dark chamber. The coverslips were washed as described above, inverted, mounted on slides, and examined in a Zeiss or Leica TCS SP5 confocal microscope.The experimental data were expressed as the mean ± standard deviation, and the statistical significance between different groups was determined using t-tests. All statistical tests were two sided and P values less than 0.05 were considered significant.The authors declare that they have no competing interests.XZ conceived and designed the study. YC performed the experiments. LW, XX analyzed the data. PW and YW contributed reagents, materials, and analysis tools. YC wrote the paper. All authors read and approved the final manuscript.