Protein Tyrosine Phosphatase Gamma (PTPγ) is a Novel Leukocyte Marker Highly Expressed by CD34+ Precursors

Protein Tyrosine Phosphatase gamma (PTPγ) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPγ is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPγ mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPγ specific antibody that recognizes the protein by flow cytometry. PTPγ expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPγ was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34+ haematopoietic precursors express high PTPγ level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPγ as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation

Rapid recognition of drug-resistance/sensitivity in leukemic cells by Fourier transform infrared microspectroscopy and unsupervised hierarchical cluster analysis

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A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

Background: Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. Methods: TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results: Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions: The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions

Regulative Loop between \u3b2-catenin and Protein Tyrosine Receptor Type \u3b3 in Chronic Myeloid Leukemia

Protein tyrosine phosphatase receptor type \u3b3 (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. \u3b2-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between \u3b2-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene \u3b2-Catenin. PTPRG was found to be capable of dephosphorylating \u3b2-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of \u3b2-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and \u3b2-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and \u3b2-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells

Predictive value of tyrosine phosphatase receptor gamma for the response to treatment tyrosine kinase inhibitors in chronic myeloid leukemia patients.

Protein tyrosine phosphatase receptor gamma (PTPRG) is a member of the receptor-like family protein tyrosine phosphatases and acts as a tumor suppressor gene in different neoplasms. Recent studies reported the down-regulation of PTPRG expression levels in Chronic Myeloid Leukemia disease (CML). In addition, the BCR-ABL1 transcript level is currently a key predictive biomarker of CML response to treatment with Tyrosine Kinase Inhibitors (TKIs). The aim of this study was to employ flow cytometry to monitor the changes in the expression level of PTPRG in the white blood cells (WBCs) of CML patients at the time of diagnosis and following treatment with TKIs. WBCs from peripheral blood of 21 CML patients were extracted at diagnosis and during follow up along with seven healthy individuals. The PTPRG expression level was determined at protein and mRNA levels by both flow cytometry with monoclonal antibody (TPγ B9-2) and RT-qPCR, and BCR-ABL1 transcript by RT-qPCR, respectively. PTPRG expression was found to be lower in the neutrophils and monocytes of CML patients at time of diagnosis compared to healthy individuals. Treatment with TKIs nilotinib and Imatinib Mesylate restored the expression of PTPRG in the WBCs of CML patients to levels observed in healthy controls. Moreover, restoration levels were greatest in optimal responders and occurred earlier with nilotinib compared to imatinib. Our results support the measurement of PTPRG expression level in the WBCs of CML patients by flow cytometry as a monitoring tool for the response to treatment with TKIs in CML patients

Defective CFTR Expression and Function Are Detectable in Blood Monocytes: Development of a New Blood Test for Cystic Fibrosis

BACKGROUND: Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION: Western blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE: CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials

Sviluppo e applicazioni di nuovi strumenti biotecnologici per lo studio del ruolo fisiopatologico della protein-tirosin fosfatasi ricettoriale gamma (PTPRG)

Background: Le Protein-Tirosin Fosfatasi (PTPs) costituiscono una nuova classe di molecole segnale implicate nello sviluppo, nella regolazione della proliferazione, nella migrazione e trasformazione cellulare. Inoltre, il non corretto funzionamento di molte PTPs contribuiscono all\u2019eziopatogenesi di diverse patologie umane. Per tale motivo negli ultimi anni numerose ricerche si sono incentrate proprio su questa famiglia di proteine e alcune di esse sono in fase di valutazione come potenziali bersagli farmacologici. In considerazione di queste premesse, il nostro studio si \ue8 focalizzato su PTPRG (Protein-Tirosin Fosfatasi Recettoriale Gamma) un membro delle Tirosin Fosfatasi \u201cclassiche\u201d, in grado di modulare la differenziazione ematopoietica in un modello di cellule staminali embrionali murine e considerato un gene oncosoppressore nel cancro del rene, del polmone, del colon, dell\u2019ovaio e della mammella. Le nostre ricerche, grazie anche all\u2019ausilio di nuovi strumenti che abbiamo sviluppato, ci hanno permesso di meglio conoscere il profilo di espressione ed il ruolo funzionale di tale enzima e di proporre un importante ruolo di PTPRG in condizioni sia fisiologiche che patologiche. Obiettivi: 1) mettere a punto o creare nuovi strumenti per lo studio di PTPRG; 2) studiare l'espressione di PTPRG in tessuti normali; 3) studiare l'espressione di PTPRG in tessuti neoplastici; 4) effettuare studi funzionali sul ruolo di PTPRG nelle neoplasie. Metodi: purificazione di cellule ematopoietiche e loro coltura, citofluorimetria, tecniche di immunofluorescenza e immunoistochimica, reverse transcription-polymerase chain reaction (PCR), real-time quantitative reverse transcriptase (RT)-PCR, e Western Blot. Risultati: Obiettivo 1. Messa a punto di nuovi strumenti di indagine: Abbiamo ottimizzato dosaggi mediante PCR quantitativa e semi-quantitativa, creato un nuovo anticorpo per lo studio di PTPRG in citofluorimetria e messo a punto le condizioni di utilizzo per tre anticorpi, che riconoscono la proteina PTPRG in altrettanti epitopi distinti per l\u2019utilizzo in indagini immunoistochimiche di tessuti inclusi in paraffina. Obiettivo 2. Espressione di PTPRG in vivo nell\u2019uomo: a) PTPRG rappresenta un nuovo marcatore di cellule dendritiche e macrofagi specializzati; b) PTPRG viene espressa ad elevati livelli da precursori circolanti CD34+, nei quali la differenziazione ne riduce l\u2019espressione; c) PTPRG viene espressa ad elevati livelli da cellule endocrine; d) PTPRG viene espressa ad elevati livelli da cellule epiteliali, inclusi numerosi endoteli; e) abbiamo inoltre raccolto evidenze sperimentali che dimostrano la presenza della sola porzione extracellulare in vivo. Obiettivo 3. PTPRG e neoplasie: a) abbiamo descritto una sostanziale diminuzione dell'espressione della proteina nei tumore dell'ovaio (21%), della mammella (56%) e del polmone (80%), mentre una positivit\ue0 citoplasmatica \ue8 stata riscontrata nel 37% dei linfomi, soprattutto quelli ad alto grado di malignit\ue0. Inoltre, PTPRG viene sovraespressa nella maggior parte degli astrocitomi ad alto grado, mentre la proteina \ue8 normalmente presente soltanto in pochi elementi neuronali e gliali; b) l\u2019espressione di PTPRG viene notevolmente e selettivamente ridotta in pazienti affetti da Leucemia Mieloide Cronica (CML) sia a livello di sangue periferico, sia nel midollo osseo, fenomeno che coinvolge anche le cellule CD34+ e viene ristabilita nei casi di remissione molecolare della malattia. Obiettivo 4. Studi funzionali: a) PTPRG nella Leucemia Mieloide Cronica (CML): l\u2019espressione di PTPRG \ue8 ridotta o assente nelle linee cellulari di CML, ove la limitata presenza correla con i livelli pi\uf9 alti di clonogenicit\ue0 e di proliferazione, mentre la sovraespressione inibisce entrambi i parametri. L\u2019espressione di una forma mutata enzimaticamente inattiva di PTPRG non altera la clonogenicit\ue0 mentre la proliferazione viene solo parzialmente inibita; b) L\u2019espressione di PTPRG risulta fortemente ridotta od assente nei leucociti di pazienti affetti da leucemia mieloide cronica. Essa viene nuovamente ripristinata in associazione con lo stato di remissione di malattia. c) l\u2019espressione di PTPRG correla con un fenotipo tolerogenico caratteristico di cellule dendritiche differenziate in vitro derivate da pazienti affetti da adenocarcinoma duttale del pancreas. Conclusioni: Sulla base di tali osservazioni possiamo proporre PTPRGcome un nuovo marcatore regolato funzionalmente nei leucociti il cui preciso ruolo in contesti fisiologici e patologici necessita di ulteriori approfondimenti. Abbiamo descritto come i livelli d'espressione di PTPRG siano particolarmente elevati nelle cellule del sistema endocrino e come varino significativamente nel contesto di determinate neoplasie, probabilmente riflettendo lo stato indifferenziato delle cellule neoplastiche e suggerendo un complesso ruolo di questa fosfatasi. I nostri dati sperimentali indicano un ruolo oncosoppressore di PTPRG e suggeriscono che la stessa possa rappresentare un potenziale bersaglio farmacologico nella leucemia Riassunto 8 mieloide cronica. Infatti abbiamo dimostrato una sua ridotta espressione nei leucociti di pazienti affetti da tale patologia; sulla base di queste evidenze, la sua misurazione potrebbe trovare potenziali applicazioni cliniche nella conferma della diagnosi e durante il \u201cfollow up\u201d della malattia. Abbiamo inoltre osservato una correlazione tra espressione di PTPRG e fenotipo tolerogenico in cellule dendritiche di pazienti affetti da adenocarcinoma pancreatico, suggerendo quindi un possibile rapporto tra espressione di PTPRG e acquisizione, da parte di cellule specializzate, della capacit\ue0 di modulare negativamente la risposta immunitaria.Background: Protein tyrosine phosphatases (PTPs) have emerged as a new class of signalling molecules that play important roles in the development, regulating cell proliferation, differentiation, migration and transformation. Moreover, deregulation of several PTPs contributes to the pathogenesis of human diseases. As a result, substantial research over the last decade has focused on the structure and function of PTPs, and a number of these enzymes are now being tested as potential pharmaceutical targets. Considering these assumptions, we focused on Receptor-type Tyrosine-Protein Phosphatase Gamma (PTPRG), a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPRG is known to regulate haematopoietic differentiation in a murine embryonic stem cells model and to be involved as a putative tumor suppressor gene in kidney, lung, colon, ovarian and breast cancers. Our studies, supported by the unique tools we developed, led us to recognize new features for this phosphatase, including a possible critical role in the pathogenesis of chronic myeloid leukemia. Aims: 1) set up or develop new tools for analysis of PTPRG; 2) study PTPRG expression in normal tissues; 3) study PTPRG expression in neoplasia; 4) identification of functional role/s for PTPRG. Methods: haematopoietic cells purification and culture, flow cytometry, immunostaining of cells and tissues by immunofluorescence and immunohistochemistry, reverse transcriptionpolymerase chain reaction (PCR), real-time quantitative reverse transcriptase (RT)- PCR, Western Blot analysis. Results: Aim 1. Set up new tools: We developed a quantitative PCR (QPCR) and semi-quantitative PCR assay, developed a new antibody suitable for flow cytometric detection of PTPRG, set up the conditions for immunohistochemical staining of paraffin embedded tissues by three different antibodies recognizing as many different domains of PTPRG. Aim 2. PTPRG expression in normal tissues: We demonstrated that: a) PTPRG is a new biomarker for monocytes, dendritic cells and specialized macrophages; b) PTPRG is highly expressed by CD34+ circulating precursors and is modulated during differentiation; c) PTPRG is highly expressed by endocrine cells; d) PTPRG is highly expressed by epithelial cells, including several endothelium; e) The isolated PTPRG extracellular domain is expressed in vivo . Aim 3. PTPRG expression in neoplasia: a) We demonstrated a marked loss of PTPRG immunoreactivity in subsets of ovarian (21%), breast (56%) and lung (80%) neoplasms. Conversely, cytoplasmic positivity was found in 37% of lymphomas, mainly of high-grade histotypes, while normal lymphocytes were negative. Brain tissue showed PTPRG expression in a few neuronal and glial elements while PTPRG was overexpressed in the majority of high-grade astrocytomas; b) PTPRG is specifically down modulated in Chronic Myeloid Leukemia (CML) patients in both peripheral blood and bone marrow, including in CD34+ cells, and is re-expressed following molecular remission of the disease. Aim 4. Functional studies: a) PTPRG in disease: PTPRG is down-regulated in Chronic Myeloid Leukemia (CML) cell lines where reduced expression correlates with higher clonogenicity and proliferation, while overexpression inhibits both parameters. Clonogenicity is unaffected whereas proliferation is partially inhibited by the expression of a phosphatase inactive mutant; b) PTPRG is down-regulated in leukocytes of patients affected by CML and its expression is recovered following molecular remission of the disease. c) PTPRG expression correlates with a tolerogenic phenotype in vitro differentiated dendritic cells isolated from patients affected by pancreatic cancer. Conclusions: We propose PTPRG as a new functionally regulated leukocyte marker whose precise role in normal and pathological context deserve further investigation. We describe particularly high PTPRG expression in endocrine cells and both down- and up-regulation in neoplasia, the latter possibly reflecting the undifferentiated state of the neoplastic cells, suggesting a complex role for this phosphatase in the pathogenesis of cancer in various districts. We provide experimental evidence that PTPRG might represent a new pharmacological target. Its down-regulation represent an early and critical event in the pathogenesis of CML and the measurement of transcript and protein levels might find clinical application to confirm diagnosis and for the follow up of this disease. Finally PTPRG expression was found to correlate with a tolerogenic phenotype in monocyte-derived dendritic cells obtained from patients affected by pancreatic cancer. This observation suggest for PTPRG a possible inhibitory role in the regulation of immune response

DEVELOPMENT and APPLICATIONS of NOVEL BIOTECHNOLOGICAL TOOLS to INVESTIGATE the PHYSIOPATHOLOGICAL ROLE of RECEPTOR-TYPE TYROSINE PROTEIN PHOSPHATASE GAMMA (PTPRG)

Background: Le Protein-Tirosin Fosfatasi (PTPs) costituiscono una nuova classe di molecole segnale implicate nello sviluppo, nella regolazione della proliferazione, nella migrazione e trasformazione cellulare. Inoltre, il non corretto funzionamento di molte PTPs contribuiscono all\u2019eziopatogenesi di diverse patologie nell\u2019ambito umano. Per tale motivo negli ultimi anni numerose ricerche si sono incentrate proprio su questa famiglia di proteine e alcune di esse ora sono in fase di valutazione come potenziali bersagli farmacologici. In considerazione di queste premesse, il nostro studio si \ue8 focalizzato su PTPRG (Protein- Tirosin Fosfatasi Recettoriale Gamma) un membro delle Tirosin Fosfatasi \u201cclassiche\u201d. E\u2019 noto che PTPRG regoli la differenziazione ematopoietica in un modello di cellule staminali embrionali murine e che sia coinvolta come possibile gene oncosopressore nel cancro del rene, del polmone, del colon, dell\u2019ovaio e del seno. Le nostre ricerche, grazie anche all\u2019ausilio di nuovi strumenti che abbiamo creato e messo a punto per lo studio di questa proteina, ci hanno permesso di proporre un importante ruolo di PTPRG in condizioni sia fisiologiche che patologiche. Obiettivi: 1) mettere a punto o creare nuovi strumenti per lo studio di PTPRG; 2) studiare l'espressione di PTPRG in tessuti normali; 3) studiare l'espressione di PTPRG in tessuti neoplastici; 4) effettuare studi funzionali sul ruolo di PTPRG nelle neoplasie. Metodi : Purificazione di cellule ematopoietiche e loro coltura, citofluorimetria, tecniche di immunofluorescenza e immunoistochimica, PCR, real-time PCR e Western Blot. Risultati: Obiettivo 1: Abbiamo messo a punto saggi di PCR quantitativa e semi-quantitativa, creato un nuovo anticorpo per lo studio di PTPRG in citofluorimetria, messo a punto le condizioni per tre anticorpi che riconoscono la proteina PTPRG in diversi domini nell\u2019ambito di studi di immunoistochimica di tessuti in paraffina. Obiettivo 2: Espressione nei tessuti normali: a) PTPRG \ue8 un nuovo marcatore di cellule dendritiche e macrofagi specializzati e viene espressa ad elevati livelli da b) precursori circolanti CD34+ ; c) cellule endocrine, d) cellule endoteliali ed epiteliali. e) Abbiamo raccolto evidenze sperimentali che dimostrano il rilascio della porzione extracellulare in forma solubile. Obiettivo 3: PTPRG e neoplasie: a) abbiamo descritto una sostanziale diminuzione dell'espressione della proteina nei tumore dell'ovaio (21%), della mammella (56%) e del polmone (80%), mentre una positivit\ue0 citoplasmatica \ue8 stata riscontrata nel 37% dei linfomi, soprattutto quelli ad alto grado di malignit\ue0. Inoltre, PTPRG viene sovraespressa nella maggior parte degli astrocitomi ad alto grado, mentre la proteina \ue8 normalmente presente soltanto in pochi elementi neuronali e gliali. b) l\u2019espressione di PTPRG viene notevolmente e selettivamente ridotta pazienti affetti da leucemia mieloide cronica (CML) sia a livello di sangue periferico, sia nel midollo osseo, fenomeno che coinvolge anche le cellule CD34+ e viene ristabilita nei casi di remissione molecolare della malattia. Obiettivo 4: Studi funzionali: a) PTPRG nella CML: l\u2019espressione di PTPRG \ue8 ridotta o assente nelle linee cellulari di leucemia mieloide cronica, ove la ridotta espressione correla con livelli pi\uf9 alti di clonogenicit\ue0 e di proliferazione, mentre la sovra-espressione inibisce entrambi i parametri. Mentre l\u2019espressione di una forma mutata inattiva di PTPRG non altera la clonogenicit\ue0, la proliferazione \ue8 parzialmente inibita da essa; b) l\u2019espressione di PTPRG \ue8 ridotta durante la differenziazione ematopoietica delle cellule CD34+; c) l\u2019espressione di PTPRG correla con un fenotipo tolerogenico in cellule denritiche differenziate in vitro da soggetti umani. Conclusioni: Sulla base di tali osservazioni possiamo proporre PTPRG\uf020 come un nuovo marcatore regolato funzionalmente nei leucociti il cui preciso ruolo in contesti fisiologici e patologici necessita di ulteriori approfondimenti. Abbiamo descritto come i livelli d'espressione di PTPRG siano particolarmente elevati nelle cellule endocrine e come varino significativamente nel contesto di determinate neoplasie, probabilmente riflettendo lo stato indifferenziato delle cellule neoplastiche e suggerendo un complesso ruolo di questa fosfatasi. Questi risultati indicano PTPRG come un possibile bersaglio farmacologico, la cui ridotta espressione rappresenta un evento critico coinvolto nella patogenesi della leucemia mieloide cronica. La sua misurazione potrebbe trovare applicazioni cliniche nella conferma della diagnosi e durante il \u201cfollow up\u201d della malattia.Background: Protein tyrosine phosphatases (PTPs) have emerged as a new class of signalling molecules that play important roles in the development, regulating cell proliferation, differentiation, migration and transformation. Moreover, deregulation of several PTPs contributes to the pathogenesis of human diseases. As a result, substantial research over the last decade has focused on the structure and function of PTPs, and a number of these enzymes are now being tested as potential pharmaceutical targets. Considering these assumptions, we focused on Protein Tyrosine Phosphatase gamma (PTPRG), a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPRG is known to regulate haematopoietic differentiation in a murine embryonic stem cells model and to be involved as a putative tumor suppressor gene in kidney, lung, colon, ovarian and breast cancers. Our studies, supported by the unique tools we developed, lead us to recognize new features for this phosphatase including a critical role in the pathogenesis of chronic myeloid leukemia. Aims: 1) set up or develop new tools for analysis of PTPRG; 2) study PTPRG expression in normal tissue, 3) study PTPRG expression in neoplasia, 4) identification of the functional role of PTPRG Methods: Haematopoietic cells purification and culture, Flow Cytometry, Immunostaining of cells and tissues (Immunofluorescence and immunohistochemistry), Reverse transcription\u2013polymerase chain reaction (PCR), Real-time quantitative reverse transcriptase (RT)-PCR, Western Blot analysis Results: Aim 1: We developed a QPCR assay, developed a new antibody suitable for flow cytometric detection of PTPRG, set up the conditions for immunohistochemical staining of paraffin embedded tissues for three different antibodies. Aim 2: Expression in normal tissue: a) PTPRG: is a new biomarker for monocytes, dendritic cells and specialized macrophages; b) PTPRG is highly expressed by CD34+ circulating precursors, c) PTPRG is highly expressed by endocrine cells and is probably cleaved in vivo, d) PTPRG is highly expressed by endothelium and epithelial cells. Aim 3: PTPRG in neoplasia: a ) We demonstrated a marked loss of PTPRG immunoreactivity in subsets of ovarian (21%), breast (56%) and lung (80%) neoplasms. Conversely, cytoplasmic positivity was found in 37% of lymphomas, mainly of high grade histotypes, while normal lymphocytes were negative. Brain tissue showed PTPRG expression in a few neuronal and glial elements and PTPRG was overexpressed in the majority of high-grade astrocytomas. b) PTPRG is specifically down modulated in CML patients in both peripheral blood and bone marrow, including in CD34+ cells, and is re-expressed following molecular remission of the disease Aim 4: functional studies: a) PTPRG in CML: PTPRG is down-regulated in chronic myeloid leukemia (CML) cell lines where reduced expression correlates with higher clonogenicity and proliferation, while overexpression inhibits both parameters. Clonogenicity is unaffected while proliferation is partially inhibited by the expression of a phosphatase inactive mutant; b) PTPRG is modulated during haemopoietic differentiation of CD34+ cells; c ) PTPRG expression correlates with a tolerogenic phenotype in dendritic cells Conclusions: We propose PTPRG as a new functionally regulated leukocyte marker whose precise role in normal and pathological context deserve further investigation. We described particularly high PTPRG expression in endocrine cells and both down and up-regulation in neoplasia, the latter possibly reflecting the undifferentiated state of the neoplastic cells, suggesting a complex role for this phosphatase

Characterization of new monoclonal antibodies specific for the extracellular domain of PTPRG, a candidate tumour suppressor gene

No abstract availabl

Identification of protein tyrosine phosphatase receptor gamma extracellular domain (sPTPRG) as a natural soluble protein in plasma.

PTPRG is a widely expressed protein tyrosine phosphatase present in various isoforms. Peptides from its extracellular domain have been detected in plasma by proteomic techniques. We aim at characterizing the plasmatic PTPRG (sPTPRG) form and to identify its source.The expression of sPTPRG was evaluated in human plasma and murine plasma and tissues by immunoprecipitation and Western blotting. The polypeptides identified have an apparent Mr of about 120 kDa (major band) and 90 kDa (minor band) respectively. Full length PTPRG was identified in the 100.000×g pelleted plasma fraction, suggesting that it was present associated to cell-derived vesicles (exosomes). The release of sPTPRG by HepG2 human hepatocellular carcinoma cell line was induced by ethanol and sensitive to metalloproteinase and not to Furin inhibitors. Finally, increased levels of the plasmatic ∼120 kDa isoform were associated with the occurrence of liver damage.These results demonstrate that sPTPRG represent a novel candidate protein biomarker in plasma whose increased expression is associated to hepatocyte damage. This observation could open a new avenue of investigation in this challenging field