Beta Isoforms Research Articles

Cellular neurochemical characterization and subcellular localization of phospholipase C β1 in rat brain

The present study describes a complete and detailed neuroanatomical distribution map of the phospholipase C beta1 (PLCβ1) isoform along the adult rat neuraxis, and defines the phenotype of cells expressing PLCβ1, along with its subcellular localization in cortical neurons as assessed by double-immunofluorescence staining and confocal laser scanning. Immunohistochemical labeling revealed a considerable morphological heterogeneity among PLCβ1-positive cells in the cortex, even though there was a marked predominance of pyramidal morphologies. As an exception to the general non-matching distribution of GFAP and PLCβ1, a high degree of co-expression was observed in radial glia-like processes of the spinal cord white matter. In the somatosensory cortex, the proportion of GABAergic neurons co-stained with PLCβ1 was similar (around 2/3) in layers I, II–III, IV and VI, and considerably lower in layer V (around 2/5). Double immunofluorescence against PLCβ1 and nuclear speckle markers SC-35 and NeuN/Fox3 in isolated nuclei from the rat cortex showed a high overlap of both markers with PLCβ1 within the nuclear matrix. In contrast, there was no apparent co-localization with markers of the nuclear envelope and lamina. Finally, to assess whether the subcellular expression pattern of PLCβ1 involved specifically one of the two splice variants of PLCβ1, we carried out Western blot experiments in cortical subcellular fractions. Notably, PLCβ1a/1b ratios were statistically higher in the cytoplasm than in the nuclear and plasma membrane fractions. These results provide a deeper knowledge of the cellular distribution of the PLCβ1 isoform in different cell subtypes of the rat brain, and of its presence in the neuronal nuclear compartment.

Abstract 313: The Nuclear Liver X Receptor as a Potential Regulator of Interleukin 18 Binding Protein in the Lung

Background and Objectives: Second hand smoke (SHS) is a major risk factor for development of chronic obstructive pulmonary disease (COPD), including emphysema and chronic bronchitis in non-smokers. The role of liver X receptor (LXR) in the pathogenesis of COPD/emphysema is not well understood. Both LXR and LXR were found in the lung. The alpha isoform is involved in the regulation of lipid metabolism whereas the beta isoform is involved in the regulation of inflammation. Here we investigate the effects of cigarette smoke (CS) on LXR activation as well as target gene and protein regulation in vivo and in vitro . Methods: Sprague Dawley rats were exposed to SHS for 2 months. Lung tissue and bronchoalveolar lavage fluid (BALf) cells were examined for LXR gene expression and emphysema development (measured by mean linear intercept). Rat alveolar macrophages (AM) and rat pulmonary microvascular endothelial cells (RPMVEC) were treated with cigarette smoke extract (CSE), LPS or DMHCA, a steroidal LXR agonist) in vitro and changes in LXR and its target gene and protein expression were examined. Results: LXR gene expression was impaired and the expression of the LXR target gene, ATP binding cassette transporter A1 (ABCA1), was significantly downregulated in BALf cells from SHS-exposed rat lungs and in rat AM treated with CSE. Real-time PCR revealed significant downregulation of LXR and upregulation of LXR in BALf cells after 1 month of SHS exposure. Similar changes were also observed in CSE-treated RPMVEC. There was a significant increase in the pro-inflammatory interleukin 18 (IL18) levels in BALf cells of SHS-exposed rats, whereas the levels of IL18 binding protein (IL18bp), an endogenous inhibitor of IL18, remained unchanged. Interestingly, activation of LXR led to upregulation of IL18bp in AM and peritoneal macrophages where potential LXR response element (LXRE) sites were indentified, in silico , in the promoter region of IL18bp. Additionally, emphysematous changes and foamy macrophage accumulation were observed in lungs of LXR-deficient mice. Conclusions: These findings implicate IL18bp as a new LXR target gene involved in the anti-inflammatory signaling pathway. Our data suggest that LXR agonist treatment may be beneficial in the prevention and treatment of CS-induced emphysema.

Microbiosensor for Alzheimer’s disease diagnostics: detection of amyloid beta biomarkers

Alzheimer's disease (AD) affects about 35.6 million people worldwide, and if current trends continue with no medical advancement, one in 85 people will be affected by 2050. Thus, there is an urgent need to develop a cost-effective, easy to use, sensor platform to diagnose and study AD. The measurement of peptide amyloid beta (Aβ) found in CSF has been assessed as an avenue to diagnose and study the disease. The quantification of the ratio of Aβ1-40/42 (or Aβ ratio) has been established as a reliable test to diagnose AD through human clinical trials. Therefore, we have developed a multiplexed, implantable immunosensor to detect amyloid beta (Aβ) isoforms using triple barrel carbon fiber microelectrodes as the sensor platform. Antibodies act as the biorecognition element of the sensor and selectively capture and bind Aβ1-40 and Aβ1-42 to the electrode surface. Electrochemistry was used to measure the intrinsic oxidation signal of Aβ at 0.65 V (vs. Ag/AgCl), originating from a single tyrosine residue found at position 10 in its amino acid sequence. Using the proposed immunosensor Aβ1-40 and Aβ1-42 could be specifically detected in CSF from mice within a detection range of 20-50 nM and 20-140 nM respectively. The immunosensor enables real-time, highly sensitive detection of Aβ and opens up the possibilities for diagnostic ex vivo applications and research-based in vivo studies.

Abstract 3741: Inhibition of PI3Kα kinase by a selective small molecule inhibitor suppresses B-cell proliferation and leukemic cell growth

Abstract Background: Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellular lipid kinases that phosphorylate the 3 position hydroxyl group of the inositol ring of phosphotidylinositol. The PI3K pathway is frequently activated in human cancers and thus represents an attractive target for small molecule inhibitors. Pan-PI3K inhibitors currently in development have been associated with adverse side-effects such as insulin resistance, thus necessitating the need to develop isoform specific inhibitors of PI3K. Herein, we describe the biological and pharmacokinetic properties of RP5264, a small molecule PI3Kα inhibitors with scope to be further developed as a clinical candidate for hematological malignancies mediated by B-cells. Methods: Activity of RP5264 on individual PI3K isoforms was determined by a Homogenous Time Resolved Fluorescence assay (Millipore, Billerica, MA) with modifications. Cell based selectivity assays against β, α, or ≤ isoforms was assessed by testing the effect of the compound on PDGF, LPA, or c5a induced Akt phosphorylation in NIH-3T3 or RAW cells. Similarly, inhibition of cellular PI3Kα activity was determined in an IgM induced human B-cell proliferation as well as LPS-induced CD19 assays. Ability to arrest cell growth and induce apoptosis was also tested. Viability assays was conducted to determine the growth inhibitory effect of the compounds in leukemic cells. Pharmacokinetic behaviour of compounds in plasma after single dose oral administration was determined in female Balb/c mice. Results: RP5264 inhibited PI3Kα activity in enzyme and cell based assays with IC50 and EC50 values of 22.2 & 24.3 nM respectively. The compound displayed a high degree of selectivity over the alpha (>1000 fold), beta (>30-50 fold), and gamma (>15-50 fold) isoforms. Additionally, the compound caused a half-maximal inhibition of human whole blood CD19 cell proliferation between 100-300 nM. Treatment of PBMC with RP5264 resulted initially in a G2/M arrest followed by subsequent increase in the number of Sub G0 cells. Viability assays demonstrated that the compound caused a significant inhibition in growth as well as Akt phosphorylation of immortalized and primary leukemic cells. Further, the compound exhibited good oral absorption with favourable pharmacokinetic properties in rodents. Conclusions: Results demonstrate the PI3K delta selective nature of RP5264 along with an ability to suppress proliferation and Akt phosphorylation in cancer cells. In vitro selectivity and potency data indicate the therapeutics potential of the compounds in hematological cancers without the deleterious effects commonly associated with the Pan PI3K inhibitors. RP5264 is poised to enter clinical development in 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3741. doi:1538-7445.AM2012-3741

Preliminary proteomic analysis of peripheral skeletal muscle atrophy in chronic obstructive pulmonary disease

To conduct a preliminary proteomic study of chronic obstructive pulmonary disease (COPD) with peripheral skeletal muscle atrophy. A total of 16 COPD patients and 8 aged-matched persons because of bone fractures were recruited in First Hospital Affiliated to Kunming Medical College from February to July in 2010. According to body mass index, fat free mass index, quadriceps femoris perimeter and quadriceps femoris active contraction, they were divided into those with muscle atrophy (group A, n = 8) and those without (group B, n = 8). There were 6 males and 2 females with an average age of (70 ± 8) years in the group A and 5 males and 3 females with an average age of (74 ± 8) years in the group B. And the control group had 6 males and 2 females with an average age of (72 ± 6) years. All samples of total quadriceps protein were separated by two-dimensional gel electrophoresis. The abnormal protein points on electrophoresis were compared by PDQuest image software. And the differential protein expression was detected. Then the corresponding peptide quality fingerprint spectrum was analyzed by mass spectrometer. Finally the differential protein points were partially detected by a search of database. The two-dimensional gel electrophoresis yielded an excellent profile of resolution and repeatability. And 12 proteins likely to cause skeletal muscle atrophy in COPD were identified. Among them, 8 proteins belonged to structural proteins (actin alpha cardiac muscle isoform CRA_c, myosin regulatory light chain 2, ventricular/cardiac muscle isoform, myoglobin isoform myosin heavy polypeptide 7 cardiac muscle beta isoform CRA_c, actin, alpha skeletal muscle, actin alpha cardiac muscle isoform CRA_b, hemoglobin alpha 1 globin chain & myosin light chain 6B) and 4 proteins were of functional proteins (chain A, crystal structure of human enolase; troponin T, slow skeletal muscle isoform alpha, carbonate anhydrase, troponin T & slow skeletal muscle isoform b). COPD patients are often accompanied with obvious peripheral skeletal muscle atrophy. It may be caused by quantitative or qualitative changes of peripheral skeletal structural and functional proteins.

DNA topoisomerase IIβ: A player in regulation of gene expression and cell differentiation

DNA topoisomerases II regulate conformational changes in DNA topology. They act on double-stranded DNA, catalyzing its relaxation, decatenation and unknotting. Vertebrate cells express two isoforms of topoisomerase II, which are similar in structure, but different in function and regulation. Whereas the alpha isoform is indispensable for proper cell replication, the functions of the beta isoform as well as reasons for its evolution in vertebrates were long unclear. Unlike topoisomerase II alpha, the beta isoform is predominantly expressed in quiescent cells and has been implicated mainly in the process of gene transcription. Recently, new discoveries point on the role of the topoisomerase II beta in regulation of cellular differentiation and tissue development. Furthermore, contemporary discoveries are raising possibilities for novel therapeutic approaches involving selective targeting of either topoisomerase II isoform in potentiating antitumor and/or reducing adverse effects of topoisomerase II poisons.

Bone morphogenetic protein receptor type Ia localization causes increased BMP2 signaling in mice exhibiting increased peak bone mass phenotype

Bone morphogenetic protein 2 (BMP2) is a growth factor that initiates osteoblast differentiation. Recent studies show that BMP2 signaling regulates bone mineral density (BMD). BMP2 interacts with BMP receptor type Ia (BMPRIa) and type II receptor leading to the activation of the Smad signaling pathway. BMPRIa must shuttle between distinct plasma membrane domains, enriched of Caveolin-1 alpha and Caveolin-1 beta isoforms, and receptor activation occurs in these domains. Yet it remains unknown whether the molecular mechanism that regulates BMP2 signaling is driving mineralization and BMD. Therefore, the B6.C3H-1-12 congenic mouse model with increased BMD and osteoblast mineralization was utilized in this study. Using the family image correlation spectroscopy, we determined if BMP2 led to a significant re-localization of BMPRIa to caveolae of the alpha/beta isoforms in bone marrow stromal cells (BMSCs) isolated from B6.C3H-1-12 mice compared to the C57BL/6J mice, which served as controls. The control, C57BL/6J mice, was selected due to only 4 Mb of chromosome 1 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Using reporter gene assays, the B6.C3H-1-12 BMSCs responded to BMP2 with increased Smad activation. Furthermore, disrupting caveolae reduced the BMP2-induced Smad signaling in BMSCs isolated from B6.C3H-1-12 and C57BL/6J. This study suggests for the first time a regulatory mechanism of BMPRIa signaling at the plasma membrane of BMSCs that (i) associated with genetic differences in the distal Chromosome 1 segment carried by the B6.C3H-1-12 congenic and (ii) contributes to increase BMD of the B6.C3H-1-12 compared to the C57BL/6J control mice.

Initiation of BMP2 signaling in domains on the plasma membrane

Bone morphogenetic protein 2 (BMP2) is a potent growth factor crucial for cell fate determination. It directs the differentiation of mesenchymal stem cells into osteoblasts, chondrocytes, adipocytes, and myocytes. Initiation of BMP2 signaling pathways occurs at the cell surface through type I and type II serine/threonine kinases housed in specific membrane domains such as caveolae enriched in the caveolin-1 beta isoform (CAV1β, caveolae) and clathrin-coated pits (CCPs). In order for BMP2 to initiate Smad signaling it must bind to its receptors on the plasma membrane resulting in the phosphorylation of the BMP type Ia receptor (BMPRIa) followed by activation of Smad signaling. The current model suggests that the canonical BMP signaling pathway, Smad, occurs in CCPs. However, several recent studies suggested Smad signaling may occur outside of CCPs. Here, we determined; (i) The location of BMP2 binding to receptors localized in caveolae, CCPs, or outside of these domains using AFM and confocal microscopy. (ii) The location of phosphorylation of BMPRIa on the plasma membrane using membrane fractionation, and (iii) the effect of down regulation of caveolae on Smad signaling. Our data indicate that BMP2 binds with highest force to BMP receptors (BMPRs) localized in caveolae. BMPRIa is phosphorylated in caveolae and the disruption of caveolae-inhibited Smad signaling in the presence of BMP2. This suggests caveolae are necessary for the initiation of Smad signaling. We propose an extension of the current model of BMP2 signaling, in which the initiation of Smad signaling is mediated by BMPRs in caveolae.

Corrigendum to “mRNA expression of alpha and beta isoforms of glucocorticoid receptor in peripheral blood mononuclear cells of patients with tuberculosis and its relation with components of the immunoendocrine response” [Brain, Behavior, and Immunity 25 (2011) 461–467

Corrigendum to ‘‘mRNA expression of alpha and beta isoforms of glucocorticoid receptor in peripheral blood mononuclear cells of patients with tuberculosis and its relation with components of the immunoendocrine response’’ [Brain, Behavior, and Immunity 25 (2011) 461–467] Luciano D’Attilio , Ernesto Trini , Bettina Bongiovanni , Griselda Didoli , Walter Gardenez , Luis J. Nannini , Adriana Giri , Oscar A. Bottasso , Maria Luisa Bay a,⇑

Differential Splicing Alters Subcellular Localization of the Alpha but not Beta Isoform of the MIER1 Transcriptional Regulator in Breast Cancer Cells

MIER1 was originally identified in a screen for novel fibroblast growth factor activated early response genes. The mier1 gene gives rise to multiple transcripts encoding protein isoforms that differ in their amino (N-) and carboxy (C-) termini. Much of the work to date has focused on the two C-terminal variants, MIER1α and β, both of which have been shown to function as transcriptional repressors. Our previous work revealed a dramatic shift in MIER1α subcellular localization from nuclear in normal breast tissue to cytoplasmic in invasive breast carcinoma, suggesting that loss of nuclear MIER1α may play a role in breast cancer development. In the present study, we investigated whether alternative splicing to include a cassette exon and produce an N–terminal variant of MIER1α affects its subcellular localization in MCF7 breast carcinoma cells. We demonstrate that this cassette exon, exon 3A, encodes a consensus leucine-rich nuclear export signal (NES). Inclusion of this exon in MIER1α to produce the MIER1-3Aα isoform altered its subcellular distribution in MCF7 cells from 81% nuclear to 2% nuclear and this change in localization was abrogated by mutation of critical leucines within the NES. Treatment with leptomycin B (LMB), an inhibitor of the nuclear export receptor CRM1, resulted in a significant increase in the percentage of cells with nuclear MIER1-3Aα, from 4% to 53%, demonstrating that cytoplasmic localization of this isoform was due to CRM1-dependent nuclear export. Inclusion of exon 3A in MIER1β to produce the N-terminal variant MIER1-3Aβ however had little effect on the nuclear targeting of this isoform. Our results demonstrate that alternative splicing to include exon 3A specifically affects the localization pattern of the α isoform.

Synthesis and structure–activity relationships of imidazo[1,2-a]pyrimidin-5(1H)-ones as a novel series of beta isoform selective phosphatidylinositol 3-kinase inhibitors

A series of PI3K-beta selective inhibitors, imidazo[1,2-a]-pyrimidin-5(1H)-ones, has been rationally designed based on the docking model of the more potent R enantiomer of TGX-221, identified by a chiral separation, in a PI3K-beta homology model. Synthesis and SAR of this novel chemotype are described. Several compounds in the series demonstrated potent growth inhibition in a PTEN-deficient breast cancer cell line MDA-MB-468 under anchorage independent conditions.

Synthesis of a spin-labeled anti-estrogen as a dynamic motion probe for the estrogen receptor ligand binding domain

The preparation and characterization of a novel nitroxide spin probe based on a steroidal anti-estrogen is described. The probe 5 demonstrated very high binding affinity for both the alpha and beta isoforms of the estrogen receptor–ligand binding domain. EPR spectrometric studies demonstrate conformational constraints for the ligand, consistent with the nitroxyl moiety occupying a position just beyond the receptor-solvent interface.

Hypothyroidism Effects on Wild Type and Mutant Rats with Altered Cardiac Titin Expression

Thyroid hormone levels play an important role in cardiac regulation, and a state of hypothyroidism leads to dilated forms of cardiomypathy. Inappropriate thyroid levels can also impact the sarcomeric protein titin, which is responsible for maintaining passive tension and structural integrity. The effects of hypothyroidism were studied after administration of propylthiouracil (PTU) to wild type (Wt) and homozygous mutant (Hm) rats (Greaser et al J Mol Cellul Cardiol 44:983, 2008) (the latter of which express a giant titin isoform). Enzyme-linked immunosorbent assay results demonstrated that PTU lowered thyroxine levels in the treated rats. Electrophoretic cardiac myosin heavy chain analysis indicated that hypothyroidism induced a transition to predominantly beta myosin heavy chain expression, while Wt control samples continued to express an age-appropriate range of alpha and beta isoforms. PTU-treated Wt rats had a larger N2BA to N2B titin ratio, and the slower migrating N2BA was in higher proportion than the faster N2BA. PTU did not affect titin isoform expression in Hm. Because it is believed that thyroid hormone regulates titin isoform expression through the PI3K/Akt pathway, we investigated several participants by immunoblotting. The Akt PH domain was not altered by PTU administration, although expression was higher in Hm rats. Akt phospho-Ser473 did not vary between treatments, but mTOR phospho-Ser2448 demonstrated a higher phosphorylation state in Wt. Echocardiography data showed significantly decreased fractional shortening in Hm control, Hm PTU, and Wt PTU relative to Wt control. Wt control also exhibited a higher ejection fraction and a lower isovolumic relaxation time compared to the other treatments. It can be concluded that hypothyroidism has significant effects on titin isoform expression and other cardiac functional characteristics. Supported by NIH HL77196.

An Evaluation of Single Nucleotide Polymorphisms in the Human Heat Shock Protein 90 kDa Alpha and Beta Isoforms

Single nucleotide polymorphisms (SNPs) in genes coding for proteins that maintain the cytosolic aryl hydrocarbon receptor (AHR) complex may affect individual susceptibility to dioxin-like compound (DLC)-induced toxicity. The cytosolic 90 kDa heat shock proteins (HSP90s) are ubiquitous chaperone proteins that bind to and stabilize numerous client proteins, including non-ligand-bound AHR. The objective of this study was to characterize SNPs in the human cytosolic HSP90 genes (HSP90AA1 and HSP90AB1). DNA sequencing of 101 human samples detected eight and seven unique SNPs at the HSP90AA1 and HSP90AB1 loci, respectively. For HSP90AA1, two non-synonymous (L71M and E554D) and one rare early termination (Q107X) SNP were observed. One SNP (E554D) was a rare novel polymorphism located in the middle substrate binding region. All SNPs detected in the HSP90AB1 gene were synonymous. With the exception of Q107X, in silico analyses predicted all HSP90 SNPs would have very low to medium risk of affecting the regulation of alternative splicing in gene transcription or protein function. Overall, a very limited presence of SNPs with predicted functional consequence in key domains of the human HSP90 proteins was observed in this study.

Structural Analysis of Smooth Muscle Tropomyosin Alpha and Beta Isoforms

Abstract A large number of tropomyosin (Tm) isoforms function as gatekeepers of the actin filament, controlling the spatiotemporal access of actin-binding proteins to specialized actin networks. Residues ~40-80 vary significantly among Tm isoforms, but the impact of sequence variation on Tm structure and interactions with actin is poorly understood, since structural studies have focused on skeletal muscle Tm α. We describe structures of N-terminal fragments of smooth muscle Tmα and β (sm-Tmα and sm-Tmβ). The 2.0-A structure of sm-Tmα81 (81-aa) resembles that of skeletal Tmα, displaying a similar super-helical twist matching the contours of the actin filament. The 1.8-A structure of sm-Tmα98 (98-aa) unexpectedly reveals an anti-parallel coiled-coil, with the two chains staggered by only 4-aa and displaying hydrophobic core interactions similar to those of the parallel dimer. In contrast, the 2.5-A structure of sm-Tmβ98, containing Gly-Ala-Ser at the N-terminus to mimic acetylation, reveals a parallel coiled-coil. Overall, the results suggest that sequence variation among Tm isoforms has a limited effect on actin binding, but could determine its gatekeeper function. None of the structures contains coiled-coil stabilizing elements, favoring the formation of head-to-tail overlap complexes in four out of five crystallographically independent parallel dimers. These complexes show similarly arranged 4-helix bundles stabilized by hydrophobic interactions, but the extent of the overlap varies between sm-Tmβ98 and sm-Tmα 81 from 2 to 3 helical turns. The formation of overlap complexes thus appears to be an intrinsic property of the Tm coiled-coil, with the specific nature of hydrophobic contacts determining the extent of the overlap. Overall, the results suggest that sequence variation among Tm isoforms has a limited effect on actin binding, but could determine its gatekeeper function.

Mutagenesis of a Tropomyosin Isoform from Atlantic Salmon

Atlantic salmon striated muscle tropomyosins unfold over a lower temperature range than their mammalian counterparts. A single sarcomeric isoform belonging to the alpha-class exists in the fast skeletal muscle of the trunk. It shares 20 amino acids substitutions with rabbit alpha-skeletal tropomyosin but migrates closer to the beta isoform (40 substitutions) when electrophoresed in the presence of SDS. Interesting aspects of the sequence heterogeneity (versus rabbit alpha) include: a change in charge at neutrality - salmon fast skeletal tropomyosin is of greater net negative charge - the content of core residues in the carboxyl-terminal half of the molecule and glycine in the amino-terminal half. Mutation of threonine-77 in salmon to the corresponding residue in rabbit, lysine, results in an electrophoretic shift in the absence as well as the presence of SDS. The contribution of the various isomorphisms to the properties of salmon tropomyosin is under investigation using circular dichroism and limited proteolysis.

Rare Deletions at the Neurexin 3 Locus in Autism Spectrum Disorder

The three members of the human neurexin gene family, neurexin 1 (NRXN1), neurexin 2 (NRXN2), and neurexin 3 (NRXN3), encode neuronal adhesion proteins that have important roles in synapse development and function. In autism spectrum disorder (ASD), as well as in other neurodevelopmental conditions, rare exonic copy-number variants and/or point mutations have been identified in the NRXN1 and NRXN2 loci. We present clinical characterization of four index cases who have been diagnosed with ASD and who possess rare inherited or de novo microdeletions at 14q24.3-31.1, a region that overlaps exons of the alpha and/or beta isoforms of NRXN3. NRXN3 deletions were found in one father with subclinical autism and in a carrier mother and father without formal ASD diagnoses, indicating issues of penetrance and expressivity at this locus. Notwithstanding these clinical complexities, this report on ASD-affected individuals who harbor NRXN3 exonic deletions advances the understanding of the genetic etiology of autism, further enabling molecular diagnoses.

P1-07-23: Absolute Quantification of Estrogen Receptor alpha in Breast Cancer.

Abstract Estrogen receptor alpha (ER) is the first biomarker to have been clinically validated as a predictor of cancer therapy response. Measurements of tumor ER expression were based on radiolabeled ligand binding to receptor present in tumor lysates. Despite substantial tissue requirement, lack of specificity for ER isoforms (alpha versus beta) or receptor integrity, quantitative determination of tumor ER content (fmol/mg total protein) was possible. Introduction of anti-ER antibodies later permitted immunohistochemical (***lHC) evaluation of ER expression. IHC determination of ER status in newly diagnosed breast cancer is now a standard of care. While these IHC assays have been shown to be as predictive of endocrine responsiveness as ligand binding assays, they remain semi-quantitative at best reporting tumor ER status either categorically (e.g. + or -) or as a numeric score which is subjective and lacks a linear relationship with endocrine responsiveness. Thus the lack of precision for quantifying ER as a predictive biomarker is one of the most important unresolved issues in breast cancer. We are working to develop a proteomic liquid chromatography-mass spectrometry (LC-MS) assay to help resolve this issue. Samples included recombinant ER (rER), immunoprecipitated (IP) rER, and IP ER from MCF7 cells. ER was digested with trypsin, lyophilised and solubilised in 5 femto-mol/microliter (100 μl) heavy peptide internal standard mix. ER peptides were resolved by LC (100 μl/minute) and detected by selected reaction monitoring MS. The area under the total ion chromatogram for each peptide were used to quantify the amount of analyte present in each sample as a single point reference to the signal of the heavy peptide spike. An 11 point calibration curve (0.1-1000 fmol on column (o/c)) of light peptides with each point in the curve spiked with 100 fmol heavy peptide was also produced to determine assay characteristics such as limits of detection (LOD), limits of quantification (LOQ), linearity, accuracy and precision. Three ER peptides were selected for quantification as they gave the greatest LOD, LOQ, linearity as well as reasonable intra- and inter-assay precision following multiple digestions of rER (intra = 3 digestions in 1 day; inter = 9 digestions over 3 weeks). Following IP of ER from four replicate MCF7 cell lysates (1mg/ml total protein) and measurement of ion intensities of the three ER peptides the mean concentration of ER was calculated to be 52 fmol (S.D of 7.5 fmol; n=4) per mg of total cell lysate after normalising for IP efficiency. We continue to develop the method to improve sensitivity and normalise for variability in IP and digestion. With the inclusion of reference peptides to known ER phosphorylation sites we are also in the process of quantifying ER phosphorylation. We aim to accurately determine ER concentration and phosphorylation status in tumor lysates and assess how these correlate with responsiveness to antiestrogen therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-23.

Relaxin and gonadal steroid receptors in uterosacral ligaments of women with and without pelvic organ prolapse

This study evaluates the expression of estrogen receptor isoforms alpha (ERα) and beta (ERβ), progesterone receptor (PR), and relaxin receptor isoforms 1 and 2 (LGR7, LGR8) in uterosacral ligament (USL) tissue of women with pelvic organ prolapse and controls. Tissue samples of USL from women with and without pelvic organ prolapse (POP) were subjected to immunohistochemistry against ERα, ERβ, PR, and LGR7 proteins. The respective mRNA expression as well as of LGR8 was assessed by quantitative real-time polymerase chain reaction. The cellular distribution of the receptor proteins was different due to cell types, independent of POP: ERα and PR were found in smooth muscle cells, but not in endothelial cells, whereas ERβ was found in endothelial cells, but not in connective tissue. ERα, ERβ, PR, and LGR7 mRNAs could be detected in all patients of both groups. ERα mRNA expression was significantly and ERβ mRNA borderline significantly higher in USL of patients with POP: ERα: p < 0.001, ERβ: p = 0.057. Enhanced effects of estrogen via altered mRNA expression patterns of ERα and ERβ--but not those of progesterone--may exist in USL of patients affected by POP. A local effect of relaxin needs to be further clarified because of this first report of prevalent ligamental expression of LGR7.

Overexpression of the VSSC‐associated CAM, beta‐2, enhances LNCaP cell metastasis associated behavior

Prostate cancer (PCa) is the second-leading cause of cancer death in American men. This is due largely to the "silent" nature of the disease until it has progressed to a highly metastatic and castrate resistant state. Voltage sensitive sodium channels (VSSCs) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two β subunits. The β-subunits modulate surface expression and gating kinetics of the channels but also have inherent cell adhesion molecule (CAM) functions. We hypothesize that PCa cells use VSSC β-subunits as CAMs during PCa progression and metastasis. We overexpressed the beta-2 isoform as a C-terminal fusion protein with enhanced cyan fluorescence protein (ECFP) in the weakly metastatic LNCaP cells. The effect of beta-2 overexpression on cell morphology was examined using confocal microscopy while metastasis-associated behavior was tested by performing several in vitro metastatic functional assays and in vivo subcutaneous tumor studies. We found that cells overexpressing beta-2 (2BECFP) converted to a bipolar fibroblastic morphology. 2BECFP cells were more adhesive than control (ECFP) to vitronectin (twofold) and Matrigel® (1.3-fold), more invasive through Matrigel® (3.6-fold in 72 hr), and had enhanced migration (2.1-fold in 96 hr) independent of proliferation in wound-healing assays. In contrast, 2BECFP cells have a reduced tumor-take and tumor volume in vivo even though the overexpression of beta-2 was maintained. Functional overexpression of VSSC β-subunits in PCa may be one mechanism leading to increased metastatic behavior while decreasing the ability to form localized tumor masses.