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

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–polymerase 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.