Phosphotyrosine Protein Research Articles

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

AbstractAbstract 3376Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures:No relevant conflicts of interest to declare.

ARAP3 inhibits peritoneal dissemination of scirrhous gastric carcinoma cells by regulating cell adhesion and invasion

During the analysis of phosphotyrosine-containing proteins in scirrhous gastric carcinoma cell lines, we observed an unusual expression of Arf-GAP with Rho-GAP domain, ankyrin repeat and PH domain 3 (ARAP3), a multimodular signaling protein that is a substrate of Src family kinases. Unlike other phosphotyrosine proteins, such as CUB domain-containing protein 1 (CDCP1) and Homo sapiens chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa), which are overexpressed and hyperphosphorylated in scirrhous gastric carcinoma cell lines, ARAP3 was underexpressed in cancerous human gastric tissues. In this study, we found that overexpression of ARAP3 in the scirrhous gastric carcinoma cell lines significantly reduced peritoneal dissemination. In vitro studies also showed that ARAP3 regulated cell attachment to the extracellular matrix, as well as invasive activities. These effects were suppressed by mutations in the Rho-GTPase-activating protein (GAP) domain or in the C-terminal two tyrosine residues that are phosphorylated by Src. Thus, the expression and phosphorylation state of ARAP3 may affect the invasiveness of cancer by modulating cell adhesion and motility. Our results suggest that ARAP3 is a unique Src substrate that suppresses peritoneal dissemination of scirrhous gastric carcinoma cells.

Mitochondrial tyrosine phosphoproteome: New insights from an up‐to‐date analysis

Tyrosine phosphorylation is a newcomer in the mitochondrial signaling and is currently emerging as an important mechanism for regulating mitochondrial processes. But to what extent? By analyzing an updated draft of the mitochondrial tyrosine phosphoproteome, the following observations can be drawn: more than a hundred mitochondrial proteins undergo tyrosine phosphorylation, phosphotyrosine proteins are distributed in each of the submitochondrial compartments, and mitochondrial tyrosine phosphorylated proteins are involved in a variety of functions as metabolism (electron transport chain, Krebs cycle, fatty acid and amino acid metabolism), solute and protein transport, mitochondrial translation machinery, quality protein assessment, oxidative stress, apoptosis, fission, and other. This large and varied collection suggests that tyrosine phosphorylation could be a widespread mechanism in modulating mitochondrial functions. Moreover the in silico model is here used to explore potential effects of tyrosine phosphorylation on selected mitochondrial proteins pointing out some future perspectives in this field.

The metalloprotease, NN-PF3 from Naja naja venom inhibits platelet aggregation primarily by affecting α2β1 integrin

NN-PF3 is a non-toxic, anticoagulant, high-molecular-mass (67.81kDa) metalloprotease from Indian cobra (Naja naja) venom. In the present study, NN-PF3 was investigated for the mechanism of inhibition of collagen-induced aggregation of human platelets. The complete inhibition of collagen-induced aggregation and partial inhibition of ADP- and epinephrine-induced aggregation has the respective IC(50) of 75 ± 5, 185 ± 10, and 232 ± 12nM, whereas no inhibition of thrombin-, arachidonic acid-, and ristocetin-induced aggregation of platelets was observed in platelet-rich plasma. Further, native NN-PF3 and EDTA-inactivated NN-PF3 inhibited collagen-induced aggregation of washed platelets with respective IC(50) of 75 ± 4 and 180 ± 6nM. The higher inhibitory effect of native NN-PF3 compared with EDTA-inactivated NN-PF3 suggests the enzymatic and non-enzymatic mechanism of inhibition. NN-PF3 pretreatment affected the collagen binding but not the fibrinogen, and fibronectin binding of washed platelets in adhesion assay suggested that the collagen receptors are affected. Western blot study using anti-integrin α2β1 mAb 6F1 suggested that NN-PF3 binds to integrin α2β1 in a primary structure-dependent manner only and is not cleaved. There was a drastic reduction in the intensity of several intracellular signaling phosphotyrosine protein bands when monoclonal anti-phosphotyrosine antibody was used, suggesting that the major activation pathway of platelets get affected, which occurs through glycoprotein VI. NN-PF3 did not bind to collagen as revealed by Western blot using anti-collagen mAb. Furthermore, neither the proteolytic cleavage of fibrinogen nor its degradation products by NN-PF3 contributed for the collagen-induced platelet aggregation inhibition.

Dasatinib reduces FAK phosphorylation increasing the effects of RPI-1 inhibition in a RET/PTC1-expressing cell line

BackgroundTPC-1 is a papillary thyroid carcinoma (PTC)-derived cell line that spontaneously expresses the oncogene RET/PTC1. TPC-1 treated with the RET/PTC1 inhibitor RPI-1 displayed a cytostatic and reversible inhibition of cell proliferation and a strong activation of focal adhesion kinase (FAK). As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1.ResultsDasatinib (100 nM) strongly reduced TPC-1 proliferation and induced marked changes in TPC-1 morphology. Cells appeared smaller and more contracted, with decreased cell spreading, due to the inhibition of phosphorylation of important cytoskeletal proteins (p130CAS, Crk, and paxillin) by dasatinib. The combination of RPI-1 with dasatinib demonstrated enhanced effects on cell proliferation (more than 80% reduction) and on the phosphotyrosine protein profile. In particular, RPI-1 reduced the phosphorylation of RET, MET, DCDB2, CTND1, and PLCγ, while dasatinib acted on the phosphorylation of EGFR, EPHA2, and DOK1. Moreover, dasatinib completely abrogated the phosphorylation of FAK at all tyrosine sites (Y576, Y577, Y861, Y925) with the exception of the autoactivation site (Y397). Notably, the pharmacological treatments induced an overexpression of integrin β1 (ITB1) that was correlated with a mild enhancement in phosphorylation of ERK1/2 and STAT3, known for their roles in prevention of apoptosis and in increase of proliferation and survival. A reduction in Akt, p38 and JNK1/2 activation was observed.ConclusionsAll data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects. Following the combined treatment, cell survival pathways appeared to be mediated by STAT3 and ERK activities resulting from integrin clustering and FAK autophosphorylation. EphA2 may also contribute, at least in part, to integrin and FAK activation. In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC.

Crystal structure of the human symplekin–Ssu72–CTD phosphopeptide complex

Symplekin (Pta1 in yeast) is a scaffold in the large protein complex that is required for 3′-end cleavage and polyadenylation of eukaryotic messenger RNA precursors (pre-mRNAs) 1–4, and also participates in transcription initiation and termination by RNA polymerase II (Pol II) 5,6. Symplekin mediates interactions among many different proteins in this machinery 1,2,7–9, although the molecular basis for its function is not known. Here we report the crystal structure at 2.4 Å resolution of the N-terminal domain (residues 30–340) of human symplekin (Symp-N) in a ternary complex with the Pol II C-terminal domain (CTD) Ser5 phosphatase Ssu72 7,10–17 and a CTD Ser5 phosphopeptide. The N-terminal domain of symplekin has the ARM or HEAT fold, with seven pairs of anti-parallel α-helices arranged in the shape of an arc. The structure of Ssu72 has some similarity to that of low-molecular-weight phosphotyrosine protein phosphatase 18,19, although Ssu72 has a unique active site landscape as well as extra structural features at the C-terminus that is important for interaction with symplekin. Ssu72 is bound to the concave face of symplekin, and engineered mutations in this interface can abolish interactions between the two proteins. The CTD peptide is bound in the active site of Ssu72, unexpectedly with the pSer5-Pro6 peptide bond in the cis configuration, which contrasts with all other known CTD peptide conformations 20,21. While the active site of Ssu72 is about 25 Å away from the interface with symplekin, we found that the symplekin N-terminal domain stimulates Ssu72 CTD phosphatase activity in vitro. Furthermore, the N-terminal domain of symplekin inhibits polyadenylation in vitro, but importantly only when coupled to transcription. As catalytically active Ssu72 overcomes this inhibition, our results demonstrate a role for mammalian Ssu72 in transcription-coupled pre-mRNA 3′-end processing.

Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl-mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.

Wnt5a/Ror2-induced upregulation of xPAPC requires xShcA

Ror receptor-tyrosine kinases act as Wnt-5a receptors in beta-catenin independent Wnt-signaling pathways. In Xenopus, expression of xPAPC is regulated by a Wnt-5a/Ror2 pathway, which resembles typical signaling cascades downstream of receptor-tyrosine kinases. Here, we have identified the phospho-tyrosine binding protein ShcA as an intracellular binding partner of Ror2. ShcA binds to a conserved motif in Ror2 via its SH2-domain. Wnt-5a induces clustering of Ror2 in the cell membrane and recruitment of ShcA to the Ror2 receptor complex. We further show that ShcA is co-expressed with Ror2 in developing Xenopus embryos and ShcA is required for Wnt-5a/Ror2 mediated upregulation of xPAPC, demonstrating the functional relevance of this interaction.

Acidic stress facilitates tyrosine phosphorylation of HLJ1 to associate with actin cytoskeleton in lung cancer cells

Acidosis is a common stress in solid tumours and is also a major determinant of tumour growth, metabolism, and metastasis. During cellular stress, heat shock proteins play an important role in actin cytoskeleton stability. HLJ1, a member of the DnaJ-like heat shock protein 40, has been characterised as a tumour suppressor gene; however, the effect of acidic stress on HLJ1 is unknown. In this study, we found that the migration ability of human lung adenocarcinoma cells was significantly impaired following the increased protein level of HLJ1 under acidic culture conditions. However, HLJ1 transcriptional activity was no different in the normal and acidic culture medium. Incubation of the cells in an acidic extracellular pH (pHe 6.4) caused up-regulated tyrosine phosphorylation of HLJ1 within 2 h. We further identified the sub-cellular distribution of tyrosine phospho-HLJ1 and its tyrosine-phosphorylated sites. Most importantly, acidic stress was observed to remarkably enhance the interaction between HLJ1 and β-actin, which was a tyrosine phosphorylation-dependent association. In conclusion, our results not only validate that HLJ1 is a tyrosine phosphoprotein, but also suggest that the increased level of tyrosine phospho-HLJ1 is crucial for binding with the actin cytoskeleton, especially in acidic pHe. We propose that acidic stress increases the association between HLJ1 and β-actin to modulate migration of human lung cancer cells.

The Trypanosoma brucei Life Cycle Switch TbPTP1 Is Structurally Conserved and Dephosphorylates the Nucleolar Protein NOPP44/46

Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development. Moreover, inactivation of T. brucei protein-tyrosine phosphatase 1 (TbPTP1) triggers differentiation of bloodstream stumpy forms into tsetse procyclic forms through unknown downstream effects. Here, we link these events by showing that NOPP44/46 is a major substrate of TbPTP1. TbPTP1 substrate-trapping mutants selectively enrich NOPP44/46 from procyclic stage cell lysates, and TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. To provide insights into the mechanism of NOPP44/46 recognition, we determined the crystal structure of TbPTP1. The TbPTP1 structure, the first of a kinetoplastid protein-tyrosine phosphatase (PTP), emphasizes the conservation of the PTP fold, extending to one of the most diverged eukaryotes. The structure reveals surfaces that may mediate substrate specificity and affords a template for the design of selective inhibitors to interfere with T. brucei transmission.

Use of Cholesterol in Sperm Cryopreservation: Present Moment and Perspectives to Future

Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

Prepubertal genistein exposure affects erbB2/Akt signal and reduces rat mammary tumorigenesis

Breast cancer is the most common malignancy in women worldwide and pharmaceutical agents have therapeutic and preventive effects in breast cancer. The human epidermal growth factor receptor 2/neu is one of the most important oncogenes in human breast cancer. Prepubertal exposure to endogenous estradiol and a phytoestrogen, genistein (Gen), has been shown to reduce future breast cancer risk. Gen downregulates tyrosine kinase regulated protein expression and reduces prostate cancer. In this study, the effects of prepubertal exposure to Gen on rat mammary carcinogenesis and the erbB2/Akt signal pathway were investigated. Prepubertal female Sprague-Dawley rats were daily exposed to Gen at 125 mg/kg (Gen-1) and 500 mg/kg (Gen-5) from postnatal days 22-28. Subsequently, the rats were given a single dose of 100 mg/kg 7.12-dimethylbenz [a] anthracene on postnatal day 42 to induce mammary tumor. The mRNA expression of erbB2 and amplified in breast cancer 1 (AIB1) was detected by reverse transcription-polymerase chain reaction. The protein levels of proliferating cell nuclear antigen (PCNA), erbB2, phosphotyrosine protein, Akt, and p-Akt were detected by immunohistochemistry and Western blotting. The activity of protein tyrosine kinase (PTK) was detected by liquid scintillation counting. The percentage of rats with mammary tumors in breast cancer model (BCM), Gen-1, and Gen-5 was 71.43, 52.38, and 33.34%, respectively. The incidence of 7.12-dimethylbenz [a] anthracene-induced mammary tumors significantly decreased in Gen-5 compared with that in BCM. The mRNA levels of AIB1 and erbB2 and the protein levels of erbB2, p-Akt, and PCNA protein expression were downregulated for a long time in the mammary tumors in Gen-5 groups. The activity of PTK was also decreased for a long time. However, the total Akt protein expression did not change significantly among BCM, Gen-1, and Gen-5. Prepubertal exposure of Sprague-Dawley female rats to 500 mg/kg Gen can reduce later breast cancer risk and its protective effect is associated with persistent downregulation of the expression of erbB2, p-Akt, AIB1, and PCNA and with low PTK activity in the mammary tumor. Our results suggest that erbB2/Akt signaling plays a role in tumor formation and targeting erbB2/Akt signaling with prepubertal exposure to Gen may provide greater efficacy to the current therapies used to treat tumors.

Global phosphoproteomic effects of natural tyrosine kinase inhibitor, genistein, on signaling pathways

Genistein is a natural protein tyrosine kinase inhibitor that exerts anti-cancer effect by inducing G2/M arrest and apoptosis. However, the phosphotyrosine signaling pathways mediated by genistein are largely unknown. In this study, we combined tyrosine phosphoprotein enrichment with MS-based quantitative proteomics technology to globally identify genistein-regulated tyrosine phosphoproteins aiming to depict genistein-inhibited phosphotyrosine cascades. Our experiments resulted in the identification of 213 phosphotyrosine sites on 181 genistein-regulated proteins. Many identified phosphoproteins, including nine protein kinases, eight receptors, five protein phosphatases, seven transcriptical regulators and four signal adaptors, were novel inhibitory effectors with no previously known function in the anti-cancer mechanism of genistein. Functional analysis suggested that genistein-regulated protein tyrosine phosphorylation mainly by inhibiting the activity of tyrosine kinase EGFR, PDGFR, insulin receptor, Abl, Fgr, Itk, Fyn and Src. Core signaling molecules inhibited by genistein can be functionally categorized into the canonial Receptor-MAPK or Receptor-PI3K/AKT cascades. The method used here may be suitable for the identification of inhibitory effectors and tyrosine kinases regulated by anti-cancer drugs.

Toxicological Disruption of Signaling Homeostasis: Tyrosine Phosphatases as Targets

The protein tyrosine phosphatases (PTPs) consist of a diverse group of enzymes whose activity opposes that of the tyrosine kinases. As such, the PTPs have critical roles in maintaining signaling quiescence in resting cells and in restoring homeostasis by effecting signal termination. Interest in these enzymes has increased in recent years following the discovery that the activity of PTPs is modulated through redox mechanisms during signaling. The molecular features that enable redox regulation of PTPs during physiological signaling also render them highly susceptible to oxidative and electrophilic inactivation by a broad spectrum of structurally disparate xenobiotic compounds. The loss of PTP activity results in a profound disregulation of protein phosphotyrosine metabolism, leading to widespread and persistent activation of signaling cascades in the cell.

Identification of tyrosine phosphoproteins in signaling pathway triggered TGF-a by using functional proteomics technology

Although several groups had conducted proteomics of nasopharyngeal carcinoma (NPC), little study was involved in phosphoproteomics, oncogenic signaling, and cancer research about NPC. Analysis of phosphotyrosine proteins from transforming growth factor alpha (TGF-a) triggered phosphotyrosine proteome permitted the identification of novel downstream substrates of the epidermal growth factor receptor (EGFR). Using functional proteomics technology based on 2-DE, 2-D western blotting, and mass spectrometry, we identified and quantified the tyrosine phosphorylation levels of 16 proteins between control and TGF-a-treated CNE2 human NPC cells. Among these proteins, tyrosine phosphorylated levels of ten proteins were increased, and those of six proteins were decreased in TGF-a-treated CNE2 cells compared with control. In addition, among these identified proteins, ANXA3, KRT8, and KRT18 were validated to be novel tyrosine-phosphorylation targets of EGFR signaling by IP-western blotting and part of a complex EGFR phosphotyrosine signaling network. These novel findings will provide new insights into the complex EGFR phosphorylation signaling and may have implications in molecular cancer therapy of NPC.

Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration. Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP), antioxidant enzymes, nitric oxide synthase (NOS), 2',5'-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel. Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol), followed by 2.3-2.4-fold decrease (P < 0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity, 30% increase (P < 0.05) in catalase activity, 2.3-fold inhibition (P < 0.05) of glutathione peroxidase, 3.3-fold increase (P < 0.05) in hydroxyl radical content, and 2.3-fold decrease (P < 0.05) in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05), but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05) in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05). NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05). Activity of SOD increased 1.6 times (P < 0.05) days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05) on the last day. Activity of 2',5'-oligoadenylate synthethase increased 2.8-fold (P < 0.05) at the beginning, and 1.6-2.3-fold (P < 0.05) during ulcer recuperation, and normalized on day 5, consistent with slowing of inflammation processes. These studies show diverse changes in total tyrosine kinase activity in gastric mucosa during the recovery process. Oxidative and nitrosative stress during lesion formation might lead to the observed reduction in tyrosine phosphorylation during ulceration.

A Phase 1 Trial of TRU-016, An Anti-CD37 Small Modular Immunopharmaceutical (SMIPTM) Protein in Relapsed and Refractory CLL: Early Promising Clinical Activity.

Abstract 3424Poster Board III-312 BackgroundCD37 is a tetraspanin family member expressed predominantly on normal and transformed B-cells across a wide range of maturational stages. TRU-016 is a novel humanized anti-CD37 SMIP protein. Pre-clinical studies have demonstrated CD37 SMIP protein mediates significantly greater direct killing of CLL cells than rituximab that is dependent upon tyrosine phosphorylation changes at 65 and 50-55 kD. Tru16 also mediates greater NK cell mediated killing of CLL cells as compared to either alemtuzumab or rituximab. A phase I study with TRU-016 was initiated based upon these data. MethodsPatients with relapsed/refractory CLL or SLL who had adequate organ function and platelets > 30,000/mm3 were eligible. Seven doses and two different schedules have been studied. The planned doses range from 0.03 mg/kg to 10 mg/kg IV once a week for 4 doses. The second schedule tests 3, 6 or 10 mg/kg on days 1, 3 and 5 the first week followed by 3 weekly doses. Dose escalation and de-escalation is based on CTC AE toxicity grades. ResultsTo date, 32 patients have been treated with TRU-016. In the weekly treatment schedule: 1 patient in each of the first 3 cohorts (0.03, 0.1, and 0.3 mg/kg); 3 patients at 1 mg/kg, 4 patients at 3 mg/kg, 7 patients at 6 mg/kg, and 5 patients at 10 mg/kg. In the TIW loading dose schedule: 8 patients at 3 mg/kg and 2 patients at 6 mg/kg. Genomic data is available for 27 patients and 19 have high risk genomic features [del(17p13.1), n=10, del(11q22.3), n=7, both=2]. The maximum tolerated dose (MTD) has not been reached. 12 serious adverse events have been reported and three may have been related to study drug, including G4 neutropenia, presumed zoster and ITP. Mild (grade 1-2) infusion toxicity has been observed. Limited pharmacodynamic data suggest changes in phosphotyrosine proteins observed pre-clinically that correlated with apoptosis have been observed at 30 minutes and 4 hours into treatment on day 1. Pharmacokinetic data from cohorts 1-7 demonstrate rapid clearance in earlier cohorts with patients in cohort 7 maintaining 10μg/ml plasma concentrations during treatment. Beginning with the 0.3 mg/kg dose, evidence of biological activity has been observed: one partial response (PR) in a patient with 17p del, two patients with leukemia cutis had partial or complete clearing, and in patients with peripheral lymphocytosis the median reduction was 83% (range 13% to 98%). Improvement in cytopenias has also been observed. Enrollment to cohort 8 and 11 is complete and further up-to-date data will be presented. ConclusionsTo date, TRU-016 is a well tolerated treatment with minimal infusional toxicity and the MTD has not been reached. One PR and a median reduction of 83% in peripheral lymphocytosis have been observed. Encouraging reduction in lymph node/spleen size and improvement in normal hematopoietic function in patients with high risk genomic CLL have been observed at low doses of CD37. The protocol has been amended to explore higher saturating doses of CD37. Future single agent and combination studies of Tru16 in CLL are warranted. DisclosuresFlinn:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Stromatt:Trubion Pharmaceuticals: Employment.

Identification of tyrosine-phosphorylated proteins associated with metastasis and functional analysis of FER in human hepatocellular carcinoma cells

Background-Aberrant activity of tyrosine-phosphorylated proteins is commonly associated with HCC metastasis. Cell signaling events driven by these proteins are implicated in numerous processes that alter cancer cell behavior. Exploring the activities and signaling pathways of these proteins in HCC metastasis may help in identifying new candidate molecules for HCC-targeted therapy.Methods-Hep3B (a nonmetastatic HCC cell line) and MHCC97H (a highly metastatic HCC cell line) were used in this study, and the tyrosine-phosphorylated proteins expressed in these cell lines were profiled by a phosphoproteomics technique based on LC-MS/MS. Protein-protein interaction and functional clustering analyses were performed to determine the activities of the identified proteins and the signaling pathways closely related to HCC metastasis.Results-In both cell lines, a total of 247 phosphotyrosine (pTyr) proteins containing 281 pTyr sites were identified without any stimulation. The involvement of almost 30% of these in liver or liver cancer has not been reported previously. Biological process clustering analysis indicated that pTyr proteins involved in cell motility, migration, protein autophosphorylation, cell-cell communication, and antiapoptosis functions were overexpressed during metastasis. Pathway clustering analysis revealed that signaling pathways such as those involved in EGFR signaling, cytokine- and chemokine-mediated signal transduction, and the PI3K and JAK-STAT cascades were significantly activated during HCC metastasis. Moreover, noncanonical regulation of the JNK cascade might also provide new targets for HCC metastasis. After comparing the pTyr proteins that were differentially expressed during HCC cell metastasis, we selected FER, a nonreceptor tyrosine kinase, and validated its role in terms of both expression and function. The data confirmed that FER might play a critical role in the invasion and metastasis of HCC.Conclusion-The identification of pTyr proteins and signaling pathways associated with HCC metastasis could provide useful information for selecting new molecular intervention targets. Moreover, FER might serve as a novel drug target in future HCC therapy.

The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa

Before a spermatozoon can fertilize an oocyte it must undergo a cascade of biochemical and physiological changes that facilitate its binding and penetration into the oocyte. Epididymal protease inhibitor (eppin) has been found to play a critical role in male fertility through an immunological approach. In this study, we used an anti-eppin antibody to clarify the effect of eppin on human sperm functions during fertilization. Immunofluorescence studies were performed on ejaculated human spermatozoa in uncapacitated, capacitated and ionophore-treated states. Human spermatozoa were incubated in the presence or absence of anti-eppin antibody under capacitating conditions and with A23187. The effects of the antibody were evaluated on sperm motility, protein phosphotyrosine content and free intracellular calcium. Immunofluorescence results demonstrated that eppin is located on the acrosome and tail. After the acrosome reaction eppin is found on the equatorial segment and tail. We found that blocking eppin with antibodies significantly inhibited the human sperm acrosome reaction induced by A23187 in a dose-dependent manner. Finally, fluo-3 analysis demonstrated that the A23187-induced elevation of sperm intracellular calcium concentration was markedly reduced after incubation with anti-eppin antibody. However, the tyrosine phosphorylation of sperm proteins did not change. These results demonstrate that eppin can modulate intracellular calcium concentrations and subsequently affect the calcium ionophore A23187-induced acrosome reaction.

High sequence homology between protein tyrosine acid phosphatase from boar seminal vesicles and human prostatic acid phosphatase.

Boar seminal vesicle protein tyrosine acid phosphatase (PTAP) and human prostatic acid phosphatase (PAP) show high affinity for protein phosphotyrosine residues. The physico-chemical and kinetic properties of the boar and human enzymes are different. The main objective of this study was to establish the nucleotide sequence of cDNA encoding boar PTAP and compare it with that of human PAP cDNA. Also, the amino-acid sequence of boar PTAP was compared with the sequence of human PAP. PTAP was isolated from boar seminal vesicle fluid and sequenced. cDNA to boar seminal vesicle RNA was synthesized, amplified by PCR, cloned in E. coli and sequenced. The obtained N-terminal amino-acid sequence of boar PTAP showed 92% identity with the N-terminal amino-acid sequence of human PAP. The determined sequence of a 354 bp nucleotide fragment (GenBank accession number: GQ184596) showed 90% identity with the corresponding sequence of human PAP. On the basis of this sequence a 118 amino acid fragment of boar PTAP was predicted. This fragment showed 89% identity with the corresponding fragment of human PAP and had a similar hydropathy profile. The compared sequences differ in terms of their isoelectric points and amino-acid composition. This may explain the differences in substrate specificity and inhibitor resistance of boar PTAP and human PAP.