Diacylglycerol kinase (DGK) involvement in K562 erythroleukemia cell proliferation

Nuclear phosphoinositide metabolism has been widely described as involved in many regulatory mechanisms including cell cycle and cell proliferation (1). Our recent studies demonstrated that an increase of nuclear Diacylglycerol (DAG) regulated the G2/M progression of erythroleukemia cells, K562 (2). As nuclear DAG can be synthesized by Phospholipases C (PLC) located in the nucleus, it can also be converted to Phosphatidic acid (PA) by a class of proteins called Diacylglycerol Kinases (DGK), which phosphorylate it utilizing ATP as a source of phosphate. PA levels in the nuclear compartments peak after G2/M progression, controlling cell cycle progression (1). We found that a particular DGK isoform, DGKa, is highly localized in the nuclear compartment of K562 cells. Then, we decided to investigate if this isozyme could be involved in cell proliferation of K562 cells, stimulating the exit from G2/M checkpoint through the production of PA in the nuclear compartment. Our data show that inhibition of DGK activity by two specific inhibitors, DI (R59022) and DII (R59949), blocks K562 cell proliferation. This effect is probably due to nuclear DGKa, indeed its modulation can affect cell proliferation too. Moreover, many cell cycle related proteins seem to be targeted by DGK activity. These evidences suggest a role for DGKa in the control of cell cycle progression acting on nuclear DAG levels and increase our knowledge about the importance of PI metabolism in the nuclei of eucaryotic cells

The open body: a “new” book

At the beginning of the ‘300, Mondino de’ Liuzzi, a physician from Bologna, was the first anatomist who started again the dissection of human body neglected from the III century. He hinted at the existence of the conflict between book and body, between “auctoritas” and the direct observation of the human body . The Mondino’s masterwork “Anothomia” remained the key book up to the middle of the sixth century, when Andrea Vesalio wrote “De Umani Corporis Fabrica,” in which the body (cadaver) eventually became the main player of the book . During the years, the technologic evolution led to the wrong conviction that dissection could be dismissed, albeit, still in our day, doctors in training feel the need to associate the direct experience on the cadaver with the very valuable digital means and the modern imaging technologies even in 3D. Thinking to Anatomy as an already fully well known discipline is a mistake. The most advanced methodologies for surgical access, namely the minimally invasive surgery, require the evolution of the traditional anatomical knowledge. The Human Anatomy Institute of the University of Bologna, among the first in Italy, has recognized this need. Thanks to the generosity of the people enrolled in the Body donation programme for research and teaching, our Institute allows medical students to practice dissection on cadavers, beginning as Freshman, then Sophomore, Junior and Senior. The sharing of Bologna’s experience could be the chance to think about the perspectives offered by the dissection of the corpse: a wide range of possibilities spanning from research projects to advanced training courses in collaboration with clinicians and surgeons belonging to different branches. Moreover the practice of corpse dissection is extremely important for the recruitment of young graduates in Medicine which, by means of the experience vested acting as “tutor of anatomy”, acquire interest in the field of research of morphological sciences, spanning from macroscopic up to the cellular and molecular level. Hic mors gaudet succurrere vitae: the motto, reported in dissection room of most of the Italian anatomical institutes, represents the synthesis of the experience of an ancient discipline which, nowadays , has the chance to rewrite a new chapter dedicated to modern frontiers of scientific research and medical education

IPMK and β-catenin take part in PLC-β1-dependent signaling pathway during myogenic differentiation

Phospholipase C (PLC)-β1 catalytic activity plays an essential role in the initiation of myogenic differentiation but the effectors involved in its signaling pathway are not well defined[1,2]. Here, we show that the overexpression of the Inositol Polyphosphate Multikinase (IPMK) promotes myogenic differentiation, and that IPMK targets the same cyclin D3 promoter region activated by PLC-β1. Moreover, cyclin D3 promoter activation relies upon c-jun binding to the promoter, both in response to PLC-β1 and to IPMK overexpression. Furthermore, both IPMK and PLC-β1 overexpression determines an increase in β-catenin translocation and accumulation to the nuclei of differentiating myoblasts resulting in higher MyoD activation. Therefore, our data show that PLC-β1, IPMK and β-catenin are mediators of the same signaling pathway that regulates cyclin D3 and myosin heavy chain (MYH) induction during myogenic differentiation

A Role for Nuclear Phospholipase Cβ1 in Cell Cycle Control

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLCbeta(1), which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLCbeta(1) exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLCbeta(1) subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLCbeta(1) that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLCbeta(1) signaling in G(1) progression by means of a specific target, i.e. cyclin D3/cdk4

Phosphoinositide-dependent signaling in cancer: A focus on phospholipase C isozymes

Phosphoinositides (PI) form just a minor portion of the total phospholipid content in cells but are significantly involved in cancer development and progression. In several cancer types, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] play significant roles in regulating survival, proliferation, invasion, and growth of cancer cells. Phosphoinositide-specific phospholipase C (PLC) catalyze the generation of the essential second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (InsP3) by hydrolyzing PtdIns(4,5)P2. DAG and InsP3 regulate Protein Kinase C (PKC) activation and the release of calcium ions (Ca2+) into the cytosol, respectively. This event leads to the control of several important biological processes implicated in cancer. PLCs have been extensively studied in cancer but their regulatory roles in the oncogenic process are not fully understood. This review aims to provide up-to-date knowledge on the involvement of PLCs in cancer. We focus specifically on PLC\u3b2, PLC\u3b3, PLC\u3b4, and PLC\u3c9 isoforms due to the numerous evidence of their involvement in various cancer types

Anatomy of the optic canal and its clinical role

The optic canal is a short funnel-like structure extending from the optic foramen to the orbital apex, where the optic nerve (ON) and the ophthalmic artery (OA) pass through. The relationship between these two structures has been already well reported, in particular in the majority of cases the artery runs within the optic sheath below the ON, which is surrounded be arachnoidal membrane in this part. [1]. However, few anatomical variants have been reported, such as the origin of the OA from the intracavernous tract of the internal carotid artery [1]. In this case, it is possible for the OA not to pass thorough the optic canal, but in the superior orbital fissure [1]. For its course and for the normal location of the OA it could be difficult to analyze the anatomy of the content of the optic canal from the classic transcranial dissection [2,3]. Therefore, we performed the opening of the ventral surface of the optic canal in 6 cadavers (12 pairs of optic canals), adopting an endonasal route, performed with the auxilium of the endoscopic visualization technology. Our dissections clearly show the relationship between OA and ON in the optic canal. This knowledge is of particular importance for tumors invading the optic canal, such as tubercular sellae meningiomas, which can be safely approached through this endoscopic endonasal route, avoiding the risk of injury of OA within the optic canal [2.3

The study of human anatomy represents the first fundamental step of an academic and clinical career: lesson learned from an experimental training program in Italy

The study of human anatomy represents the starting point of the medical background. A good scholar of human anatomy summarises the theory and the practice on cadavers, in that the absence of one of the two impinges on a comprehensive knowl- edge of the human body, i.e. the road map for every physicians. We herby report the experience at the Department of Biomedical Sciences, Chair of Human Anatomy of the Alma Mater Studiorum University of Bologna, where exchanging programs with several International Academic Institutions worldwide have been established over the last dec- ade with the aim of providing a comprehensive knowledge in human anatomy, through both frontal lessons and dissection activities on cadavers. Students participating to these trainings were afterwards enrolled as “tutors” in the teaching laboratories for stu- dents of the 1st and 2nd year of the Medical School, sharing their knowledge with younger students and improving their teaching skills as well. These tutorial activities have been carried out even after the achievement of the Medical Degree, allowing an efficient interaction between the clinical knowledge of the young medical doctors and the teaching purposes of the anatomy courses. Finally, the International environment of the exchange programs represented the basis for further research and clinical ex- periences carried out by the tutors themselves, dramatically influencing the first steps of their careers. But the main issue of this presentation is the fact that in Italy and namely at the Department of Biomedical Sciences, Chair of Human Anatomy of Bolo- gna University, the gross anatomy in dissecting room is no longer a dream, but a solid reality that offers the possibility to undergraduate, postgraduate students, residents and specialists to renew constantly the knowledge of human anatomy, just carrying out dissections on cadavers

Epigenetic regulation of nuclear PLCbeta1 and Cyclin D3 during Azacitidine treatment

The Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by alterations of the hematopoietic stem cells that lead to anemia, neutropenia, bleeding problems and infections. The evidence of a clinical correlation between the presence of a monoallelic gene deletion of Phospholipase Cβ1 (PLCβ1) and the progression of MDS to Acute Myeloid Leukemia (AML) opened new perspectives of research and treatments. Patients affected by MDS with a higher risk of AML evolution have a reduction in the expression of the nuclear PLCβ1, which is also epigenetically relevant in MDS. This strengthens the importance of PLCβ1 localization. In fact, PLCβ1 is a molecular target for hypomethylating agents, such Azacitidine (AZA)(1). High-risk MDS patients that respond to the drug showed an increased expression of nuclear PLCβ1 and its downstream target Cyclin D3 (CCND3), an induction of normal myeloid differentiation, and a better prognosis. Stemming from these data, our goal was to analyze the correlation between CCND3, PLCβ1 and AZA treatment. Firstly, we treated two different cellular lines, AML HL60 and histiocytic lymphoma U937, with AZA 5μM (Ec50 for HL60 cells) for 24 hours. Then, we used Real-Time PCR and Western blot to quantify both gene and protein expression. Moreover, we showed that CCND3 promoter has one CpG island. For this reason, it is possible that AZA could directly affect both PLCβ1 and CCND3 promoters. Therefore, we studied PLCβ1 binding to CCND3 promoter by chromatin immunoprecipitation (CHIP), before and after AZA treatment. Our results evidenced that the recruitment of PLCβ1 to CCND3 promoter is specifically increased after AZA treatment, leading to suppose that PLCβ1 could have a pivotal role in MDS with either a direct or indirect effect on cell cycle, proliferation and differentiation. These complicate relations need future deepening in order to demonstrate how PLCβ1 binding actually regulates CCND3 expression and how much this expression depends on CCND3 direct promoter demethylation and PLCβ1 control

Hypo and retrotympanum: the importance of anatomical variants

The hypo- and retrotympanum host a variety of crucial anatomical structures1, characterized by high variability, which are poorly been described. The aim of our study is to describe and classify the anatomical variants of the hypo- and retrotympanum by the means of transcanal endoscopy2. We hypothesize that the retro- and hypotympanum are subject to more anatomical variability than actually thought. Moreover, the configuration as bridge variants and variably shaped sinus interconnects the different subregions. A total of 125 middle ears (83 cadaveric dissections) were explored by the means of 3mm straight and angled scopes. The variants were documented photographically and tabularized. The bony crests ponticulus, subiculum and finiculus1 were most frequently represented as ridges. The ponticulus showed the highest variability with 38% ridge, 35% bridge and 27% incomplete presentation. The subiculum was bridge - shaped only in 8% of the cases, while the finiculus in 17%. The sinus tympani had a normal shape in 66% of the cases. A subcochlear canaliculus was observed in 50%. The retro- and hypotympanum were classified respectively to the present bony crests and sinus in chambers type I to IV. In our opinion, the retro- and hypotympanum have to be considered as a tightly coherent region of the middle ear. For this purpose, we propose a straightforward classification, according to the presence of the different bony crests and sinus forming the different chambers of the retro- and hypotympanum. The introduced classification may also serve as intraoperative assessment, to be aware of the different anatomical subregions. The hidden areas of the retro- and hypotampanum are difficult to access and therefore represent a region of risk for residual cholesteatomatous disease after surgical treatment. The extension below a bridge bony crest or into a deep sinus demands thorough exploration; therefore, exact anatomical knowledge and an effective technique to visualize the whole middle ear are required

Phospholipase C Beta1 (PI-PLCbeta1)/Cyclin D3/protein Kinase C (PKC) Alpha Signaling Modulation During Iron-Induced Oxidative Stress in Myelodysplastic Syndromes (MDS)

MDS are characterized by anemia and transfusion requirements. Transfused patients frequently show iron overload that negatively affects hematopoiesis. Iron chelation therapy can be effective in these MDS cases, but the molecular consequences of this treatment need to be further investigated. That is why we studied the molecular features of iron effect and Deferasirox therapy on PI-PLCbeta1 inositide signaling, using hematopoietic cells and MDS samples. At baseline, MDS patients showing a positive response after iron chelation therapy displayed higher levels of PI-PLCbeta1/Cyclin D3/PKCalpha expression. During treatment, these responder patients, as well as hematopoietic cells treated with FeCl(3)and Deferasirox, showed a specific reduction of PI-PLCbeta1/Cyclin D3/PKCalpha expression, indicating that this signaling pathway is targeted by Deferasirox. The treatment was also able to specifically decrease the production of ROS. This effect correlated with a reduction of IL-1A and IL-2, as well as Akt/mTOR phosphorylation. In contrast, cells exposed only to FeCl(3)and cells from MDS patients refractory to Deferasirox showed a specific increase of ROS and PI-PLCbeta1/Cyclin D3/PKCalpha expression. All in all, our data show that PI-PLCbeta1 signaling is a target for iron-induced oxidative stress and suggest that baseline PI-PLCbeta1 quantification could predict iron chelation therapy response in MDS