Lauren Amable

Family 18 chitinase-oligosaccharide substrate interaction: subsite preference and anomer selectivity of Serratia marcescens chitinase A.

The sizes and anomers of the products formed during the hydrolysis of chitin oligosaccharides by the Family 18 chitinase A (ChiA) from Serratia marcescens were analysed by hydrophilic interaction chromatography using a novel approach in which reactions were performed at 0 degrees C to stabilize the anomer conformations of the initial products. Crystallographic studies of the enzyme, having the structure of the complex of the ChiA E315L (Glu315-->Leu) mutant with a hexasaccharide, show that the oligosaccharide occupies subsites -4 to +2 in the substrate-binding cleft, consistent with the processing of beta-chitin by the release of disaccharide at the reducing end. Products of the hydrolysis of hexa- and penta-saccharides by wild-type ChiA, as well as by two mutants of the residues Trp275 and Phe396 important in binding the substrate at the +1 and +2 sites, show that the substrates only occupy sites -2 to +2 and that additional N -acetyl-D-glucosamines extend beyond the substrate-binding cleft at the reducing end. The subsites -3 and -4 are not used in this four-site binding mode. The explanation for these results is found in the high importance of individual binding sites for the processing of short oligosaccharides compared with the cumulative recognition and processive hydrolysis mechanism used to digest natural beta-chitin.

Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis (P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.

Structure-Activity Relationship Studies of Fostriecin, Cytostatin, and Key Analogs, with PP1, PP2A, PP5, and (β12–β13)-Chimeras (PP1/PP2A and PP5/PP2A), Provide Further Insight into the Inhibitory Actions of Fostriecin Family Inhibitors

Fostriecin and cytostatin are structurally related natural inhibitors of serine/threonine phosphatases, with promising antitumor activity. The total synthesis of these antitumor agents has enabled the production of structural analogs, which are useful to explore the biological significance of features contained in the parent compounds. Here, the inhibitory activity of fostriecin, cytostatin, and 10 key structural analogs were tested in side-by-side phosphatase assays to further characterize their inhibitory activity against PP1c (Ser/Thr protein phosphatase 1 catalytic subunit), PP2Ac (Ser/Thr protein phosphatase 2A catalytic subunit), PP5c (Ser/Thr protein phosphatase 5 catalytic subunit), and chimeras of PP1 (Ser/Thr protein phosphatase 1) and PP5 (Ser/Thr protein phosphatase 5), in which key residues predicted for inhibitor contact with PP2A (Ser/Thr protein phosphatase 2A) were introduced into PP1 and PP5 using site-directed mutagenesis. The data confirm the importance of the C9-phosphate and C11-alcohol for general inhibition and further demonstrate the importance of a predicted C3 interaction with a unique cysteine (Cys269) in the β12–β13 loop of PP2A. The data also indicate that additional features beyond the unsaturated lactone contribute to inhibitory potency and selectivity. Notably, a derivative of fostriecin lacking the entire lactone subunit demonstrated marked potency and selectivity for PP2A, while having substantially reduced and similar activity against PP1 and PP1/PP2A- PP5/PP2A-chimeras that have greatly increased sensitivity to both fostriecin and cytostatin. This suggests that other features [e.g., the (Z,Z,E)-triene] also contribute to inhibitory selectivity. When considered together with previous data, these studies suggest that, despite the high structural conservation of the catalytic site in PP1, PP2A and PP5, the development of highly selective catalytic inhibitors should be feasible.

Disruption of serine/threonine protein phosphatase 5 (PP5:PPP5c) in mice reveals a novel role for PP5 in the regulation of ultraviolet light-induced phosphorylation of serine/threonine protein kinase Chk1 (CHEK1).

Background: The roles of PP5 in normal biology are poorly understood. Results: To help evaluate the biological actions of PP5, a Cre/loxP-conditional mouse line was generated. Conclusion: In response to UV light, PP5 regulates the phosphorylation of Chk1 at Ser-345. Significance: Understanding the biological roles for phosphatases is critical for understanding the role of reversible phosphorylation in the control of signaling networks. PP5 is a ubiquitously expressed Ser/Thr protein phosphatase. High levels of PP5 have been observed in human cancers, and constitutive PP5 overexpression aids tumor progression in mouse models of tumor development. However, PP5 is highly conserved among species, and the roles of PP5 in normal tissues are not clear. Here, to help evaluate the biological actions of PP5, a Cre/loxP-conditional mouse line was generated. In marked contrast to the early embryonic lethality associated with the genetic disruption of other PPP family phosphatases (e.g. PP2A and PP4), intercrosses with mouse lines that ubiquitously express Cre recombinase starting early in development (e.g. MeuCre40 and ACTB-Cre) produced viable and fertile PP5-deficient mice. Phenotypic differences caused by the total disruption of PP5 were minor, suggesting that small molecule inhibitors of PP5 will not have widespread systemic toxicity. Examination of roles for PP5 in fibroblasts generated from PP5-deficient embryos (PP5−/− mouse embryonic fibroblasts) confirmed some known roles and identified new actions for PP5. PP5−/− mouse embryonic fibroblasts demonstrated increased sensitivity to UV light, hydroxyurea, and camptothecin, which are known activators of ATR (ataxia-telangiectasia and Rad3-related) kinase. Further study revealed a previously unrecognized role for PP5 downstream of ATR activation in a UV light-induced response. The genetic disruption of PP5 is associated with enhanced and prolonged phosphorylation of a single serine (Ser-345) on Chk1, increased phosphorylation of the p53 tumor suppressor protein (p53) at serine 18, and increased p53 protein levels. A comparable role for PP5 in the regulation of Chk1 phosphorylation was also observed in human cells.

Serine/threonine protein phosphatase 5 regulates glucose homeostasis in vivo and apoptosis signalling in mouse pancreatic islets and clonal MIN6 cells

Aims/hypothesisDuring the development of type 2 diabetes mellitus, beta cells are often exposed to a high glucose/hyperlipidaemic environment, in which the levels of reactive oxygen species (ROS) are elevated. In turn, ROS can trigger an apoptotic response leading to beta cell death, by activating mitogen-activated protein kinase (MAPK) signalling cascades. Here we test the hypothesis that serine/threonine protein phosphatase 5 (PP5) acts to suppress proapoptotic c-Jun N-terminal kinase (JNK) signalling in beta cells.MethodsPpp5c−/− and Ppp5c+/+ mice were subjected to intraperitoneal glucose (IPGTT) or insulin tolerance tests. Pancreatic islets from Ppp5c−/− and Ppp5c+/+ mice or MIN6 cells treated with short-interfering RNA targeting PP5 were exposed to palmitate or H2O2 to activate MAPK signalling. Changes in protein phosphorylation, mRNA expression, apoptosis and insulin secretion were detected by western blot analysis, quantitative RT-PCR or ELISA.ResultsPpp5c−/− mice weighed less and exhibited reduced fasting glycaemia and improved glucose tolerance during IPGTT, but retained normal insulin sensitivity and islet volume. Comparison of MAPK signalling in islets from Ppp5c−/− mice and MIN6 cells revealed that the lack of PP5 was associated with enhanced H2O2-induced phosphorylation of JNK and c-Jun. Cells with reduced PP5 also showed enhanced JNK phosphorylation and apoptosis after palmitate treatment. PP5 suppression in MIN6 cells correlated with hypersecretion of insulin in response to glucose.Conclusions/interpretationPP5 deficiency in mice is associated with reduced weight gain, lower fasting glycaemia, and improved glucose tolerance during IPGTT. At a molecular level, PP5 helps suppress apoptosis in beta cells by a mechanism that involves regulation of JNK phosphorylation.

Calcineurin Regulates Homologous Desensitization of Natriuretic Peptide Receptor-A and Inhibits ANP-Induced Testosterone Production in MA-10 Cells

Receptor desensitization is a ubiquitous regulatory mechanism that defines the activatable pool of receptors, and thus, the ability of cells to respond to environmental stimuli. In recent years, the molecular mechanisms controlling the desensitization of a variety of receptors have been established. However, little is known about the molecular mechanisms that underlie desensitization of natriuretic peptide receptors, including natriuretic peptide receptor-A (NPR-A). Here we report that calcineurin (protein phosphatase 2B, PP2B, PPP3C) regulates homologous desensitization of NPR-A in murine Leydig tumor (MA-10) cells. We demonstrate that both pharmacological inhibition of calcineurin activity and siRNA-mediated suppression of calcineurin expression potentiate atrial natriuretic peptide (ANP)-induced cGMP synthesis. Treatment of MA-10 cells with inhibitors of other phosphoprotein phosphatases had little or no effect on ANP-induced cGMP accumulation. In addition, overexpression of calcineurin blunts ANP-induced cGMP synthesis. We also present data indicating that the inhibition of calcineurin potentiates ANP-induced testosterone production. To better understand the contribution of calcineurin in the regulation of NPR-A activity, we examined the kinetics of ANP-induced cGMP signals. We observed transient ANP-induced cGMP signals, even in the presence of phosphodiesterase inhibitors. Inhibition of both calcineurin and phosphodiesterase dramatically slowed the decay in the response. These observations are consistent with a model in which calcineurin mediated dephosphorylation and desensitization of NPR-A is associated with significant inhibition of cGMP synthesis. PDE activity hydrolyzes cGMP, thus lowering intracellular cGMP toward the basal level. Taken together, these data suggest that calcineurin plays a previously unrecognized role in the desensitization of NPR-A and, thereby, inhibits ANP-mediated increases in testosterone production.

Abstract 3873: Single cell cisplatin measurements by ICP-MS

Cisplatin, carboplatin, and oxaliplatin are the most widely used class of cancer chemotherapy drugs in the Western world. Many cancers are initially sensitive to platinum treatment but drug resistance frequently occurs. Cellular uptake of cisplatin is related to tumor burden, thus lower intracellular cisplatin levels are associated with a decreased tumor response. Traditional methods to assess cisplatin uptake in cells involves digesting a cell population and measuring the total platinum content. This approach does not reflect the distribution and individual cellular variation of cisplatin uptake. Here, we present a new method, single cell inductively coupled plasma-mass spectrometry (SC-ICP-MS) to quantitate the platinum concentration within individual cells. Experiments were performed using the A2780 cisplatin-sensitive and the corresponding cisplatin-resistant A2780-CP70 ovarian cancer cell lines. Time course experiments were performed to measure the change of cisplatin uptake over time. Serum starvation experiments were also performed to examine if differences in cisplatin uptake were dependent on the cell cycle. SC-ICP-MS was performed by injecting cell suspensions and analyzing the platinum 195 isotope. Individual cellular cisplatin levels were collected and a histogram representing the cell population was generated using the instrument software (Syngistix Nano Application). Other metals, such as zinc, iron, and copper, were analyzed to measure the variation between cell lines after treatment. We observed a heterogeneous distribution of cisplatin concentrations within the sample reflecting that cisplatin uptake differs from cell to cell. Serum starvation affected the uptake of cisplatin within cells, changing the cisplatin distribution curve. SC-ICP-MS was also effective in measuring other metals within individual cells and differences were observed between the A2780 and A2780-CP70 cell lines. In conclusion, single cell ICP-MS analysis allows for the quantitation of cisplatin within individual cells. The heterogeneous distribution of cisplatin uptake more closely reflects what occurs within tumor cells. SC-ICP-MS allows for the development of strategies to increase cisplatin uptake, translating to better clinical responses. Citation Format: Lauren Amable, Stan Smith, Chady Stephan. Single cell cisplatin measurements by ICP-MS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3873.

Abstract LB-223: A specific Isoform of Gli1 binds the promoter region of c-fos in A2780-CP70 human ovarian cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The transcription factor Activator Protein 1 (AP1), is an important regulator in cisplatin resistance. AP1 is comprised of the two proteins, c-jun and c-fos. The transcription factor Gli1, a member of the hedgehog signaling pathway, is also an important factor in cisplatin resistance and transcriptionally regulated c-jun. The c-jun interface with Gli1 is mediated by a 130 kDa isoform of Gli1. We have investigated whether Gli1 may regulate c-fos by selectively knocking down Gli1 expression using anti-Gli1 shRNA or by treating cells with a Hedgehog pathway inhibitor, cyclopamine. A2780-CP70 cells were treated with an IC50 dose of anti-Gli1 shRNA and the changes in RNA and protein levels were analyzed over a 72 hour time course. No changes in the expression of c-jun or c-fos RNA transcripts were observed versus control. Western blot analyses showed Gli1 protein expression was reduced at 24 hours and undetectable at 48 and 72 hours. Sonic hedgehog (Shh) protein levels were dramatically reduced by 24 hours, low but measureable at 48 hours, and undetectable at 72 hours. Indian hedgehog (Ihh) expression increased at 24 hours and remained stable for the remainder of the 72 hour time course. Treatment of A2780-CP70 cells with an IC50 dose of cyclopamine resulted in a different intracellular response than treatment with Gli1 shRNA. Cyclopamine specifically targets the cell membrane receptor Smoothened. Experiments were performed analyzing RNA and protein expression over a 72 hour time period after treatment with cyclopamine. C-jun and c-fos RNA transcript levels were higher than control 6 hours after treatment and remained high for the duration of the 72 hour experiment. Western blot analyses after cyclopamine treatment showed Gli1 protein expression gradually decreasing during the 72 hours, but were never fully depleted. Expression of Shh increased over 72 hours, while Ihh levels remained stable. The identical Gli-Binding-Site (GBS) in the c-jun promoter was found in the promoter of c-fos. We investigated whether the Gli1 isoform that binds c-jun, also binds c-fos. Simultaneous Western and Southwestern blots show only one of the known Gli1 isoform binds the promoter of c-fos. Further studies with Chromatin Immunoprecipitation (ChIP) assays confirmed the 130 kDa isoform of Gli1 binds the GBS of the c-fos promoter. We conclude that the 130 kDa Gli1 isoform binds both components of AP1, c-jun and c-fos, and modulates the cellular resistance to cisplatin. Citation Format: Lauren Amable, Kenji Kudo, Elaine Gavin, Jason Fain, Eddie Reed. A specific Isoform of Gli1 binds the promoter region of c-fos in A2780-CP70 human ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-223. doi:10.1158/1538-7445.AM2013-LB-223

Abstract 834: Inhibition of Gli1 results in altered c-jun activation, inhibition of cisplatin-induced up-regulation of ERCC1, XPD, and XRCC1, and inhibition of platinum-DNA adduct repair

The transcription of ERCC1 and other nucleotide excision repair genes is strongly influenced by c-jun. C-jun is transcriptionally regulated by Gli proteins of the Hedgehog pathway. We therefore studied the possible relationships between Gli1, c-jun, and the upregulation of ERCC1, XPD, and XRCC1 in cisplatin-resistant human ovarian cancer cells. We studied the paired human ovarian cancer cell lines A2780 and A2780-CP70. We used a shRNA construct that specifically degrades Gli1 message; which has been previously published by one of the co-authors (Shevde-Samant et al., 2009). Genes we assessed for mRNA and/or protein levels included: c-jun, ERCC1, XPD, XRCC1, Gli1, Gli2, SHH, IHH, GAPDH, and β-tubulin. Platinum-DNA adduct repair was assessed by atomic absorbance spectrometry with Zeeman background correction. Use of the anti-Gli1 shRNA in cisplatin-resistant cells, resulted in a block of the cells’ ability to up- regulate genes in response to cisplatin treatment, including: c-jun, ERCC1, XPD, and XRCC1. This block in upregulation of c-jun was concurrent with a change in the phosphorylation pattern of the c-jun protein, shifting that pattern from a Ser63/73 dominant pattern, to a Thr91/93 dominant pattern. A2780-CP70 cells were treated at their cisplatin IC50, and DNA repair was assessed after pretreatment with anti-Gli1 shRNA or scrambled shRNA control. Control cells repaired 78% of platinum-DNA adducts at 12 hours; compared to 33% repair in cells pretreated with anti-Gli1 shRNA; a 2.4 fold difference. Pretreatment of A2780-CP70 cells with anti-Gli1 shRNA resulted in supra-additive cell killing with cisplatin; shifting the cisplatin IC50 from 30 µM to 5 µM. Pretreatment of these cells with cyclopamine, did not shift the cisplatin IC50. We conclude that the transcriptional protein Gli1 is important in the upregulation of these three DNA repair genes in human ovarian cancer cells. And that Gli1 thereby strongly influences platinum-DNA adduct repair, and cellular sensitivity to cisplatin. This Gli1 role has c-jun as an intermediate in the pathway. 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 834. doi:1538-7445.AM2012-834

Abstract 5613: A specific isoform of Gli1 binds the Gli-binding site of the c-jun and c-fos promoters

Gli1 participates in the transcriptional regulation of c-jun, which in turn, participates in the transcriptional regulation of genes in nucleotide excision repair. The c-jun promoter and the c-fos promoter have identical Gli-binding-sites that bind Gli1. C-jun and C-fos form the transcriptionally active heterodimer Activator Protein 1, AP1. AP1, is the positive transcriptional regulator for ERCC1, and other DNA repair proteins. Gli1 is a transcription factor in the Hedgehog pathway, and there are five known isoforms of the Gli1 protein that exist in human cells. We have investigated whether there is a specific isoform of Gli1 that binds to the transcriptional regulatory sequences of c-jun and of c-fos. Detailed studies were performed in cisplatin-resistant A2780-CP70 human ovarian cancer cells. EMSA studies demonstrated the presence of a Gli protein in these cells which bind to the Gli-binding-site, as well as the consensus Gli-binding sequence. Supershift EMSA assays show that Gli1 binds the Gli binding sites of c-jun and of c-fos. Southwestern blot analyses of protein lysates from A2780-CP70 cells demonstrated that only one of the five known Gli1 protein isoforms, the 130 kDa, bind the Gli-specific binding site in the promoter of c-jun and c-fos. No Gli2 protein binds this specific binding site in the c-jun promoter, in these cells. To further confirm the 130 kDa Gli1 isoform was responsible for binding the c-jun promoter, the full-length Gli1 with a C-terminal myc tag was transfected into cells, and the protein products were assessed by immunoprecipitation and Southwestern blot analysis. The transfected full length Gli1-myc generated a 130 kDa protein that binds the Gli1-specific binding site in the promoter of c-jun. The presence of this 130 kDa Gli1 isoform was also documented in: six additional human ovarian cancer cell lines, and ten clinical ovarian tissue samples. Seven were ovarian cancer tissue and three were non-cancer ovarian tissues. The 130 kDa Gli1 isoform was present in all specimens examined, but protein levels were six-fold higher in malignant tissues. We conclude that the 130 kDa isoform of Gli1 bind the Gli-binding site in the promoters of c-jun and c-fos. Therefore, Gli1 may be the transcriptional regulator of c-jun and c-fos, and thereby regulates the transcription of ERCC1 and genes of nucleotide excision repair. 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 5613. doi:1538-7445.AM2012-5613