Mitochondrial apoptosis is induced by Alkoxy phenyl-1-propanone derivatives through PP2A-mediated dephosphorylation of Bad and Foxo3A in CLL.

Protein phosphatase 2 A (PP2A) is a tumour suppressor whose strong inhibition underlies the phosphorylation-dependent, anti-apoptotic mechanisms in Chronic Lymphocytic Leukemia (CLL). Inactivation of PP2A is due to the cooperative action of the phosphorylation of Y307 of its catalytic subunit by the aberrant cytosolic pool of the Src Family Kinase Lyn and the interaction with its protein inhibitor SET, which is overexpressed in CLL. In this study, we developed a library of compounds, the most potent being the one named CC11, which restores PP2A activity by disrupting the PP2A/SET complex, thereby triggering the mitochondrial pathway of apoptosis. This process involves the recruitment of the pro-apoptotic BH3-only proteins Bad and Bim to mitochondria, the former upon direct dephosphorylation and the latter being newly expressed upon dephosphorylation and activation of its transcription factor FoxO3a. These findings highlight that PP2A antagonizes the prosurvival pathways controlled by Akt, which phosphorylates and thereby suppresses a variety of pro-apoptotic factors and tumour suppressors including Bad and FoxO3a. Furthermore, the PP2A-mediated pro-apoptotic effect of CC11 is synergistically potentiated by the abrogation of Lyn's activity. Our results show that CC11 represents a promising lead compound for a new therapeutic rationale aimed at abrogating the aberrant oncogenic signals in CLL.

Src tyrosine kinase preactivation is associated with platelet hypersensitivity in essential thrombocythemia and polycythemia vera.

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombo-hemorrhagic complications. The acquired somatic Janus kinase 2 (JAK2) V617F mutation is present in the majority of PV and ET patients. Because aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients. JAK2 was found inactive in healthy and pathological resting cells regardless of the V617F mutation. In addition, Src was inactive in all resting platelets, but in the pathological specimens it was present in a preactivated conformation as a consequence of anomalous dephosphorylation of its inhibitory phospho-Tyr527 residue, likely mediated by Src homology-2 domain-containing protein Tyr-phosphatase-2 (SHP-2), whose constitutive activity correlated with its recruitment to Src. Low thrombin concentration triggered a more rapid Src-signaling activation, higher [Ca(2+)](c) increase, and aggregation in pathological platelets compared with controls. Thrombin-induced Src activation preceded JAK2 activation, which occurred simultaneously in normal and pathological platelets. Our results indicate that a constitutive Src kinase preactivation is implicated in platelet hypersensitivity and likely involved, at least partially, in the functional abnormalities of PV and ET platelets.

Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum and translocates to Golgi in differentiating myoblasts.

The endoplasmic-reticulum chaperone Grp94 is required for the cell surface export of molecules involved in the native immune response, in mesoderm induction and muscle development, but the signals responsible for Grp94 recruitment are still obscure. Here we show for the first time that Grp94 undergoes Tyr-phosphorylation in differentiating myogenic C2C12 cells. By means of phospho-proteomic and immunoprecipitation analyses, and the use of Src-specific inhibitors we demonstrate that the Src-tyrosine-kinase Fyn becomes active early after induction of C2C12 cell differentiation, in parallel with the recruitment and the Tyr-phosphorylation of Grp94, which peaks at 6-hour differentiation. Grp94 is Tyr-phosphorylated inside the endoplasmic reticulum by a lumenal Fyn, as indicated by fluorescence and electronmicroscopy immunolocalization, co-immunoprecipitation after chemical cross-linking and by treatment of intact endoplasmic-reticulum vesicles with proteinase K. Furthermore, fractionation of cellular membrane compartments and double-immunofluorescence studies showed that Tyr-phosphorylation of Grp94 is necessary for the protein translocation from the endoplasmic reticulum to the Golgi apparatus. These results indicate that Fyn-catalyzed Tyr-phosphorylation of Grp94 is an event required to promote the chaperone export from the endoplasmic reticulum occurring in the early phase of myoblast differentiation.

Angiogenic transforming capacity of IgG purified from plasma of type 1 diabetic patients.

We previously demonstrated that plasma of type 1 diabetic patients contains antibodies complexed irreversibly with Grp94 that also display proteolytic activity. In this work, we wanted to test whether antibodies obtained from diabetic plasma may convey an inflammatory risk on vascular cells. To this aim, IgG were purified on the Protein-G column from individual plasma of eight type 1 diabetic patients, and then tested on HUVECs to measure effects on cell growth and morphologic changes at different incubation times. The purified fractions of IgG contained a significant amount of Fab/(Fab)(2), both free and in big aggregates, and anti-Grp94 antibodies, mostly irreversibly linked with, but also free of Grp94. The purified fractions of both Fab/(Fab)(2) and whole IgG stimulated the proliferation and sustained the angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) at sub-nanomolar concentrations. IgG from normal plasma neither stimulated the cell growth nor induced any differentiation of HUVECs. The maximum cell growth stimulation occurred at 6-9 hrs and associated with the strong activation of the ERK1/2 pathway, whereas angiogenic transformation was completed later when the ERK1/2 activation was silenced and cell growth stimulation significantly reduced. Neither proteolytic activity of MMP-9 nor VEGF were apparently involved in mediating the angiogenic differentiation of HUVECs that mostly correlated with an increased expression of HSP70 closely coupled with cell membrane-bound inactive species of MMP-9. Results indicate that effects displayed on HUVECs by antibodies purified from diabetic plasma are likely sustained by immune complexes with Grp94 that may thus predict an increased risk of angiogenic transformation in vivo.

Geldanamycin-induced Lyn dissociation from aberrant Hsp90-stabilized cytosolic complex is an early event in apoptotic mechanisms in B-chronic lymphocytic leukemia.

Lyn, a tyrosine kinase belonging to the Src family, plays a key role as a switch molecule that couples the B-cell receptor to downstream signaling. In B-CLL cells, Lyn is overexpressed, anomalously present in the cytosol, and displays a high constitutive activity, compared with normal B lymphocytes. The aim of this work was to gain insights into the molecular mechanisms underlying these aberrant properties of Lyn, which have already been demonstrated to be related to defective apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Herein, Lyn is described to be in an active conformation as integral component of an aberrant cytosolic 600-kDa multiprotein complex in B-CLL cells, associated with several proteins, such as Hsp90 through its catalytic domain, and HS1 and SHP-1L through its SH3 domain. In particular, Hsp90 appears tightly bound to cytosolic Lyn (CL), thus stabilizing the aberrant complex and converting individual transient interactions into stable ones. We also demonstrate that treatment of B-CLL cells with geldanamycin, an Hsp90 inhibitor already reported to induce cell death, is capable of dissociating the CL complex in the early phases of apoptosis and thus inactivating CL itself. These data identify the CL complex as a potential target for therapy in B-CLL.

Stable complexes formed by Grp94 with human IgG promoting angiogenic differentiation of HUVECs by a cytokine-like mechanism.

To explore the molecular mechanisms by which complexes of Grp94 with IgG, purified from the plasma of diabetic subjects, could drive an inflammatory risk in vascular cells, native Grp94 was co-incubated with human, non-immune IgG to obtain the formation of complexes that were then tested on human umbilical vein endothelial cells (HUVECs). Co-incubation of Grp94 with IgG led to the formation of stable, SDS-resistant complexes that displayed effects partly similar and partly significantly different from those of Grp94 alone. Both Grp94 alone and with IgG stimulated the cell growth and promoted angiogenesis by a mechanism of autocrine/paracrine activation of the expression of heat shock protein (HSP)90 and HSP70. However, the most striking alterations in the cell cytoskeleton, characterized by dramatic rearrangement of actin and increased formation of podosomes, were induced by Grp94 with IgG, and were mediated by the enhanced expression of HSP90. At variance with Grp94 alone, Grp94 with IgG promoted the angiogenic differentiation by activating a signaling pathway apparently independent of the intense stimulation of the ERK1/2 pathway that was instead more directly involved in mediating the proliferative effects on HUVECs. Results show unprecedented cytokine-like effects of Grp94 and a so far undisclosed capacity to bind irreversibly IgG, forming complexes that, with respect to Grp94 alone, display a more intense angiogenic transforming capacity that may predict an increased inflammatory risk in vascular cells in vivo.

Functional VEGF and VEGF receptors are expressed in human medulloblastomas.

Vascular endothelial growth factor (VEGF) is one of the key regulators of tumor neoangiogenesis. It acts through two types of high-affinity tyrosine kinase receptors (VEGF receptor-1 [VEGFR-1]/fms-related tyrosine kinase 1 [Flt-1] and VEGFR-2/kinase domain receptor [KDR]) expressed on endothelial cells. VEGFRs have also been detected on cancer cells, suggesting a possible autocrine effect of VEGF on their growth. We studied the expression of VEGF, VEGFR-1, and VEGFR-2 in human medulloblastoma cell lines (DAOY, D283Med, and D341Med) and investigated the possible autocrine mechanisms of VEGF on medulloblastoma cell proliferation. Reverse transcriptase PCR analysis showed the presence of VEGF and VEGFR mRNAs in all cell lines studied. Of the three VEGF isoforms, VEGF(121) and VEGF(189) were detected by Western blot analysis in all three medulloblastoma cell lines, whereas VEGF(165) was identified only in DAOY cells. Medulloblastoma cell lines expressed both VEGFR-1 and VEGFR-2. We also demonstrated expression of VEGF and its receptors in medulloblastoma tumor specimens. Exogenous VEGFR-2 inhibitor reduced the VEGF-dependent cell proliferation of DAOY and D283Med cells. In DAOY cells, VEGF(165) induced phosphorylation of VEGFR-2/KDR and of downstream proteins in the signal transduction pathway. These data suggest a possible autocrine role for VEGF in medulloblastoma growth. Targeting VEGF signaling may represent a new therapeutic option in the treatment of medulloblastoma.

Characterization of immune complexes of idiotypic catalytic and anti-idiotypic inhibitory antibodies in plasma of type 1 diabetic subjects.

An increase in proteolytic activity is an early common feature of diabetes, and is associated with the development of vascular complications. We performed an extensive proteomic investigation on plasma of type 1 diabetic subjects to discover why some of them apparently lacked any measurable proteolytic activity. Activity was found enclosed in immune complexes in which Fab/(Fab)(2) displayed a serine-like catalytic activity. Disaggregation of complexes by means of Protein G affinity chromatography led to the separation of free subunits of Fab, showing a specific amidolytic activity, from Fab that displayed activity on casein and remained closely complexed with whole IgG. On both types of Fab the serine catalytic site appeared to be the same, being located in close vicinity to the antigen-binding site. The distinct substrate specificity was due to the different conformation adopted by the catalytic site depending on the structure of Fab/(Fab)(2), whether in complexes or as free subunits. Catalytic Fab/(Fab)(2) originated from idiotypic antibodies developed against Grp94, identified as the primary antigen covalently complexed with Fab. Whole IgG present in immune complexes were instead mostly formed with anti-idiotypic antibodies developed against the adduct of Fab/(Fab)(2) with Grp94, and were responsible for blocking any catalytic activity. In dot-blot experiments with native Grp94, we confirmed that in any diabetic plasma circulated anti-Grp94, idiotypic, and anti-idiotypic antibodies.