Search

Phosphatidylinositol (PI) metabolism represents the core of a network of signaling pathways which modulate many cellular functions including cell proliferation, cell differentiation, apoptosis, and membrane trafficking. An array of kinases, phosphatases, and lipases acts on PI creating an important number of second messengers involved in different cellular processes. Although, commonly, PI signaling was described to take place at the plasma membrane, many evidences indicated the existence of a PI cycle residing in the nuclear compartment of eukaryotic cells. The discovery of this mechanism shed new light on many nuclear functions, such as gene transcription, DNA modifications, and RNA expression. As these two PI cycles take place independently of one another, understanding how nuclear lipid signaling functions and modulates nuclear output is fundamental in the study of many cellular processes. J. Cell. Physiol. 231: 1645-1655, 2016. © 2015 Wiley Periodicals, Inc.

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer that arises from the skeletal muscle. Recent studies have identified an important role of AKT signaling in RMS progression. This suggests targeting components of the PI3K/Akt pathway may be an effective therapeutic strategy. Here, we investigated the in vitro activity of the class I PI3K inhibitors [1] in human rhabdomyosarcoma cell lines (embryonal rhabdomyosarcoma RD and A204, alveolar rhabdomyosarcoma SJCRH30). We used a panel of four compounds which specifically target PI3K isoforms including the PI3Kα-selective (p110α ) inhibitor alpelisib BYL719, currently in clinical development by Novartis Oncology, the p110β TGX-221 inhibitor, the p110γ CZC24832, the p110δ CAL-101inhibitor and the dual p110α/p110δ inhibitor AZD8835. The effects of single drugs and of several drug combinations were analyzed to assess cytotoxicity by MTT assays, cell cycle by flow cytometry , apoptosis by caspase 3/7 assay and Western blot, as well as the phosphorylation status of the pathway. BYL719 treatment resulted in G1 phase cell cycle arrest and apoptosis. BYL719 administered in combination with CAL-101, for 48 h and 72h, decreased cell viability and induced apoptosis in a marked synergistic manner. Taken together, our findings indicate that BYL719, either alone or in combination with p110δ CAL-101inhibitor, may be an efficient treatment for human rhabdomyosarcoma cells that have aberrant upregulation of the PI3K signaling pathway for their proliferation and survival., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

C2C12 is an immortalized mouse myoblast cell line. The cells readily proliferate in high-serum conditions, and differentiate and fuse in low-serum conditions. While this cell line is a very useful tool to study aspects of myogenesis, metabolism and muscle biology, however, treatment of C2C12 cells with bone morphogenic protein (BMPs) induces cells to differentiate into osteoblasts. Osteoblast differentiation is controlled by diversified signaling proteins and transcription factors, essentially BMP-2, Osterix (Osx/Sp7) and Runx2, finally associating with the expression of late osteoblast marker genes, like ALPL and Bglap. These peculiarities make C2C12 progenitor cells a skillful prototype to investigate the molecular mechanism that control cell destiny specification and terminal differentiation. In the current investigation, we took improvement of the differentiation peculiarities of the mouse C2C12 cell line to analyze whether changes in PLCbeta1 expression and its nuclear localization might regulate or affect their terminal osteogenic differentiation. We demonstrated that overexpression of PLCβ1 enhances the osteogenic differentiation of C2C12 elicited by BMP-2 as demonstrated by the presence of osteoblast marker genes expression. In the present study we also showed that miR-214 suppressed osteogenic differentiation through the regulation of nuclear PLCβ1 by targeting Osterix., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Acute myeloid leukemia (AML) is the most common type of leukemia in adults in which leukemic myeloid derived cells replace normal blood cells leading to a loss in systemic function. Once initiated the disease develops rapidly and is typically fatal within weeks or months if left untreated. AML is a complex disease and although, the exact causes of the development of AML are unknown, risk factors include age, pre-leukemic diseases such as myelodysplastic syndrome, exposure to chemicals and radiation and genetics. The mainstay treatment is still chemotherapy together with stem cell replacement therapy and while life expectancy has increased slowly, the 5 year survival rates range between 12 and 70% with relapse rates as high as 70% depending on the subtype (canceruk). These statistics illustrate the urgent requirement for the development of novel targeted therapeutics. Phospholipases C (PLC) are critical intracellular signaling enzymes that control a wide range of cellular functions including proliferation and apoptosis that have been implicated in myelodysplastic diseases and in leukemia (Faenza et al., 2013; Shah et al.). Importantly they constitute a highly druggable family of enzymes distinct from other well established drug development targets such as protein kinases. Using the human leukemic cell line THP1, we carried out a small targeted RNAi screen to establish a role of all known PLCs in cell growth, differentiation and maintenance of the transformed phenotype. We discovered that silencing of PLCb1 or PLCH2 resulted in a strong growth arrest. PLCb1 knockdown also initiated apoptosis and attenuated growth of THP1 cells in semisolid culture, which is known to reflect the ability of cells to induce leukemia in vivo. Accordingly, we found that knockdown of PLCb1 strongly attenuated THP1-mediated development of leukemia in mice. These growth inhibitory effects of PLCb1 knockdown were extended to a mouse model of human leukaemia induced by the MLL-AF9 translocation and to human primary leukemia cells. Of direct importance to the consideration for drug development we observed that PLCb1 knockdown selectively attenuated the growth of primary human AML cells, without effecting cell growth and differentiation of normal CD34+ hematopoietic stem and progenitor cells from healthy donors. We therefore propose PLCb1 as a novel candidate for a therapeutic target in AML., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Protein kinase C α has been reported to regulate cell cycle in several cell lines. Most of the reports describe a role for PKC α in G1/S transition but little is known about its possible involvement in G2/M progression. Our studies on the effects of PKC inhibitors, PKC α silencing and overexpression demonstrated a novel and positive role for PKC α in cyclin B1 regulation in human erythroleukemia cell line, K562. On the other hand, using PKC inhibitors and a PKC α inactive mutant, we could report that PKC α activity was not necessary for cyclin B1 regulation. Moreover, immunoprecipitation and immunocytochemistry experiments showed that these two proteins could physically interact each other and enter into the nuclei during G2/M progression. In order to better understand this mechanism, we investigated how PKC α could be attracted into the nuclei. We found a high increase of nuclear DAG during the G2/M phase. Then, using PMA and PLC inhibitors, we showed that PKC α translocation was due to the increase in nuclear DAG. Surprisingly, we saw the same effect on cyclin B1. Finally, in order to discover which PLC was involved, we silenced the nuclear localized PLCβ1 founding a decrease in PKC α and cyclin B1 nuclear amount. Taken together, our data demonstrate the existence of a novel DAG dependent mechanism linking PKC α and cyclin B1 which can regulate their entry into the nuclei during the G2/M phase of cell cycle., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Phospholipase C β1 (PLCβ1) is known to play an important role in cell proliferation. Previous studies reported aninvolvement of PLCβ1 in G0-G1/S transition and G2/M progression in Friend murine erythroleukemia cells (FELC). However,little has been found about its role in human models. Here, we used K562 cell line as human homologous of FELC inorder to investigate the possible key regulatory role of PLCβ1 during cell proliferation of this humancell line. Our studies on the effects of the overexpression of both these isoforms showed a specific and positive connection between cyclinD3 and PLCβ1 in K562 cells, which led to a prolonged S phase of the cell cycle and a delay in cell proliferation. In order to shed light on this mechanism, we decided to study the possible involvement of protein kinases C (PKC), known to be direct targets of PLC signaling and important regulators of cell proliferation. Our data showed a peculiar decrease of PKCα levels in cells overexpressing PLCβ1. Moreover, when we silenced PKCα, by RNAi technique, in order to mimic the effects of PLCβ1, we caused the same upregulation of cyclin D3 levels and the same decrease of cell proliferation found in PLCβ1-overexpressing cells. The key features emerging from our studies in K562 cells is that PLCβ1 targets cyclin D3, likely through a PKCα-mediated-pathway, and that, as a downstream effect of its activity, K562 cells undergo an accumulation in the S phase of the cell cycle., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013