Paradigm Shift in Gastric Cancer Prevention: Harnessing the Potential of Aristolochia olivieri Extract.

Gastric cancer, particularly adenocarcinoma, is a significant global health concern. Environmental risk factors, such as Helicobacter pylori infection and diet, play a role in its development. This study aimed to characterize the chemical composition and evaluate the in vitro antibacterial and antitumor activities of an Aristolochia olivieri Colleg. ex Boiss. Leaves' methanolic extract (AOME). Additionally, morphological changes in gastric cancer cell lines were analyzed. AOME was analyzed using HPLC-MS/MS, and its antibacterial activity against H. pylori was assessed using the broth microdilution method. MIC and MBC values were determined, and positive and negative controls were included in the evaluation. Anticancer effects were assessed through in vitro experiments using AGS, KATO-III, and SNU-1 cancer cell lines. The morphological changes were examined through SEM and TEM analyses. AOME contained several compounds, including caffeic acid, rutin, and hyperoside. The extract displayed significant antimicrobial effects against H. pylori, with consistent MIC and MBC values of 3.70 ± 0.09 mg/mL. AOME reduced cell viability in all gastric cancer cells in a dose- and time-dependent manner. Morphological analyses revealed significant ultrastructural changes in all tumor cell lines, suggesting the occurrence of cellular apoptosis. This study demonstrated that AOME possesses antimicrobial activity against H. pylori and potent antineoplastic properties in gastric cancer cell lines. AOME holds promise as a natural resource for innovative nutraceutical approaches in gastric cancer management. Further research and in vivo studies are warranted to validate its potential clinical applications.

Combined Treatment with PI3K Inhibitors BYL-719 and CAL-101 Is a Promising Antiproliferative Strategy in Human Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer arising from skeletal muscle myogenic progenitors. Recent studies have shown an important role for AKT signaling in RMS progression. Aberrant activation of the PI3K/AKT axis is one of the most frequent events occurring in human cancers and serves to disconnect the control of cell growth, survival, and metabolism from exogenous growth stimuli. In the study reported here, a panel of five compounds targeting the catalytic subunits of the four class I PI3K isoforms (p110α, BYL-719 inhibitor; p110ß, TGX-221 inhibitor; p110γ, CZC24832; p110δ, CAL-101 inhibitor) and the dual p110α/p110δ, AZD8835 inhibitor, were tested on the RMS cell lines RD, A204, and SJCRH30. Cytotoxicity, cell cycle, apoptosis, and the activation of downstream targets were analyzed. Of the individual inhibitors, BYL-719 demonstrated the most anti-tumorgenic properties. BYL-719 treatment resulted in G1/G0 phase cell cycle arrest and apoptosis. When combined with CAL-101, BYL-719 decreased cell viability and induced apoptosis in a synergistic manner, equaling or surpassing results achieved with AZD8835. In conclusion, our findings indicate that BYL-719, either alone or in combination with the p110δ inhibitor, CAL-101, could represent an efficient treatment for human rhabdomyosarcoma presenting with aberrant upregulation of the PI3K signaling pathway.

Expression of the double-stranded RNA-dependent kinase PKR influences osteosarcoma attachment independent growth, migration, and invasion.

Osteosarcoma (OS) is a rare, insidious tumor of mesenchymal origin that most often affects children, adolescents, and young adults. While the primary tumor can be controlled with chemotherapy and surgery, it is the lung metastases that are eventually fatal. Multiple studies into the initial drivers of OS development have been undertaken, but few of these have examined innate immune/inflammatory signaling. A central figure in inflammatory signaling is the innate immune/stress-activated kinase double-stranded RNA-dependent protein kinase (PKR). To characterize the role of PKR in OS, U2OS, and SaOS-2 osteosarcoma cell lines were stably transfected with wild-type or dominant-negative (DN) PKR. Overexpression of PKR enhanced colony formation in soft agar (U2OS and SaOS-2), enhanced cellular migration (U2OS), and invasive migration (SaOS-2). In contrast, overexpression of DN-PKR inhibited attachment-independent growth, migration and/or invasion. These data demonstrate a role for inflammatory signaling in OS formation and migration/invasion and suggest the status of PKR expression/activation may have prognostic value.

AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Murine thymoma viral oncogene homolog (AKT) kinases target both cytosolic and nuclear substrates for phosphorylation. Whereas the cytosolic substrates are known to be closely associated with the regulation of apoptosis and autophagy or metabolism and protein synthesis, the nuclear substrates are, for the most part, poorly understood. To better define the role of nuclear AKT, potential AKT substrates were isolated from the nuclear lysates of leukemic cell lines using a phosphorylated AKT substrate antibody and identified in tandem mass spectrometry. Among the proteins identified was adenosine deaminase acting on RNA (ADAR)1p110, the predominant nuclear isoform of the adenosine deaminase acting on double-stranded RNA. Coimmunoprecipitation studies and in vitro kinase assays revealed that AKT-1, -2, and -3 interact with both ADAR1p110 and ADAR2 and phosphorylate these RNA editases. Using site-directed mutagenesis of suspected AKT phosphorylation sites, AKT was found to primarily phosphorylate ADAR1p110 and ADAR2 on T738 and T553, respectively, and overexpression of the phosphomimic mutants ADAR1p110 (T738D) and ADAR2 (T553D) resulted in a 50-100% reduction in editase activity. Thus, activation of AKT has a direct and major impact on RNA editing.-Bavelloni, A., Focaccia, E., Piazzi, M., Raffini, M., Cesarini, V., Tomaselli, S., Orsini, A., Ratti, S., Faenza, I., Cocco, L., Gallo, A., Blalock, W. L. AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Inositide-Dependent Nuclear Signalling in Health and Disease.

Nuclear inositides have a specific subcellular distribution that is linked to specific functions; thus their regulation is fundamental both in health and disease. Emerging evidence shows that alterations in multiple inositide signalling pathways are involved in pathophysiology, not only in cancer but also in other diseases. Here, we give an overview of the main features of inositides in the cell, and we discuss their potential as new molecular therapeutic targets.

Nuclear phospholipase C isoenzyme imbalance leads to pathologies in brain, hematologic, neuromuscular, and fertility disorders.

Phosphoinositide-specific phospholipases C (PI-PLCs) are involved in signaling pathways related to critical cellular functions, such as cell cycle regulation, cell differentiation, and gene expression. Nuclear PI-PLCs have been studied as key enzymes, molecular targets, and clinical prognostic/diagnostic factors in many physiopathologic processes. Here, we summarize the main studies about nuclear PI-PLCs, specifically, the imbalance of isozymes such as PI-PLCß1 and PI-PLCζ, in cerebral, hematologic, neuromuscular, and fertility disorders. PI-PLCß1 and PI-PLCÉ£1 affect epilepsy, depression, and bipolar disorder. In the brain, PI-PLCß1 is involved in endocannabinoid neuronal excitability and is a potentially novel signature gene for subtypes of high-grade glioma. An altered quality or quantity of PI-PLCζ contributes to sperm defects that result in infertility, and PI-PLCß1 aberrant inositide signaling contributes to both hematologic and degenerative muscle diseases. Understanding the mechanisms behind PI-PLC involvement in human pathologies may help identify new strategies for personalized therapies of these conditions.

Therapeutic potential of nvp-bkm120 in human osteosarcomas cells.

Osteosarcoma (OS) is the most common pediatric malignant neoplasia of the skeletal system. It is characterized by a high degree of malignancy and a severe tendency to metastasize. In the past decade, many studies have provided evidence that the phosphoinositide 3-kinase (PI3K) signaling pathway is one of the most frequently altered pathways in human cancer, and has a critical role in driving tumor initiation and progression. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120, which has recently entered clinical Phase II for treatment of PI3K-dependent cancers on three OS cell lines. We observed a concentration- and time-dependent decrease of Ser473 p-Akt as well as reduced levels of Thr37/46 p-4E-BP1, an indicator of the mammalian target of rapamycin complex 1 activity. All OS cell lines used in this study responded to BKM120 treatment with an arrest of cell proliferation, an increase in cell mortality, and an increase in caspase-3 activity. MG-63 cells were the most responsive cell line, demonstrating a significant increase in sub-G1 cells, and a rapid induction of cell death. Furthermore, we demonstrate that BKM120 is more effective when used in combination with other standard chemotherapeutic drugs. Combining BKM120 with vincristine demonstrated a more synergistic effect than BKM120 with doxorubicin in all the lines. Hence, we suggest that BKM120 may be a novel therapy for the treatment of OS presenting with anomalous upregulation of the PI3K signaling pathway.

Signal Transduction in Ribosome Biogenesis: A Recipe to Avoid Disaster.

Energetically speaking, ribosome biogenesis is by far the most costly process of the cell and, therefore, must be highly regulated in order to avoid unnecessary energy expenditure. Not only must ribosomal RNA (rRNA) synthesis, ribosomal protein (RP) transcription, translation, and nuclear import, as well as ribosome assembly, be tightly controlled, these events must be coordinated with other cellular events, such as cell division and differentiation. In addition, ribosome biogenesis must respond rapidly to environmental cues mediated by internal and cell surface receptors, or stress (oxidative stress, DNA damage, amino acid depletion, etc.). This review examines some of the well-studied pathways known to control ribosome biogenesis (PI3K-AKT-mTOR, RB-p53, MYC) and how they may interact with some of the less well studied pathways (eIF2α kinase and RNA editing/splicing) in higher eukaryotes to regulate ribosome biogenesis, assembly, and protein translation in a dynamic manner.

Phosphatidylinositol 5 Phosphate (PI5P): From Behind the Scenes to the Front (Nuclear) Stage.

Phosphatidylinositol (PI)-related signaling plays a pivotal role in many cellular aspects, including survival, cell proliferation, differentiation, DNA damage, and trafficking. PI is the core of a network of proteins represented by kinases, phosphatases, and lipases which are able to add, remove or hydrolyze PI, leading to different phosphoinositide products. Among the seven known phosphoinositides, phosphatidylinositol 5 phosphate (PI5P) was the last to be discovered. PI5P presence in cells is very low compared to other PIs. However, much evidence collected throughout the years has described the role of this mono-phosphoinositide in cell cycles, stress response, T-cell activation, and chromatin remodeling. Interestingly, PI5P has been found in different cellular compartments, including the nucleus. Here, we will review the nuclear role of PI5P, describing how it is synthesized and regulated, and how changes in the levels of this rare phosphoinositide can lead to different nuclear outputs.

Phosphoinositide 3 Kinase Signaling in Human Stem Cells from Reprogramming to Differentiation: A Tale in Cytoplasmic and Nuclear Compartments.

Stem cells are undifferentiated cells that can give rise to several different cell types and can self-renew. Given their ability to differentiate into different lineages, stem cells retain huge therapeutic potential for regenerative medicine. Therefore, the understanding of the signaling pathways involved in stem cell pluripotency maintenance and differentiation has a paramount importance in order to understand these biological processes and to develop therapeutic strategies. In this review, we focus on phosphoinositide 3 kinase (PI3K) since its signaling pathway regulates many cellular processes, such as cell growth, proliferation, survival, and cellular transformation. Precisely, in human stem cells, the PI3K cascade is involved in different processes from pluripotency and induced pluripotent stem cell (iPSC) reprogramming to mesenchymal and oral mesenchymal differentiation, through different and interconnected mechanisms.

Quantitative profiling of the endonuclear glycerophospholipidome of murine embryonic fibroblasts.

A reliable method for purifying envelope-stripped nuclei from immortalized murine embryonic fibroblasts (iMEFs) was established. Quantitative profiling of the glycerophospholipids (GPLs) in envelope-free iMEF nuclei yields several conclusions. First, we find the endonuclear glycerophospholipidome differs from that of bulk membranes, and phosphatidylcholine (PtdCho) and phosphatidylethanolamine species are the most abundant endonuclear GPLs by mass. By contrast, phosphatidylinositol (PtdIns) represents a minor species. We also find only a slight enrichment of saturated versus unsaturated GPL species in iMEF endonuclear fractions. Moreover, much lower values for GPL mass were measured in the iMEF nuclear matrix than those reported for envelope-stripped IMF-32 nuclei. The collective results indicate that the nuclear matrix in these cells is a GPL-poor environment where GPL occupies only approximately 0.1% of the total nuclear matrix volume. This value suggests GPL accommodation in this compartment can be satisfied by binding to resident proteins. Finally, we find no significant role for the PtdIns/PtdCho-transfer protein, PITPα, in shuttling PtdIns into the iMEF nuclear matrix.

BMP-2 Induced Expression of PLCß1 That is a Positive Regulator of Osteoblast Differentiation.

Bone morphogenetic protein 2 (BMP-2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide-phospholipase Cß 1 (PLCß1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCß1. Here, the function of PLCß1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP-2 we assist to a remarkable increase in PLCß1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCß1 promotes osteogenic differentiation by up-regulating alkaline phosphatase (ALP). Moreover, PLCß1 is present in the nuclear compartment of these cells and overexpression of a cytosolic-PLCß1mutant (cyt-PLCß1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR-214 is a regulator of Osterix (Osx) which is an osteoblast-specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCß1 could be a potential target of miR-214 in the control of osteogenic differentiation by gain- and loss- of function experiment. The results indicated that inhibition of miR-214 in C2C12 cells significantly enhances the protein level of PLCß1 and promotes C2C12 BMP-2-induced osteogenesis by targeting PLCß1.

PI-PLCß1b affects Akt activation, cyclin E expression, and caspase cleavage, promoting cell survival in pro-B-lymphoblastic cells exposed to oxidative stress.

The phosphoinositide-dependent signal transduction pathway has been implicated in the control of a variety of biologic processes, such as the regulation of cellular metabolism and homeostasis, cell proliferation and differentiation, and apoptosis. One of the key players in the regulation of inositol lipid signaling is the phospholipase Cß1 (PI-PLCß1), that hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtIns(4,5)P2], giving rise to the second messengers inositol triphosphate and diacylglicerol. PI-PLCß1 has been associated with the regulation of several cellular functions, some of which have not yet been fully understood. In particular, it has been reported that PI-PLCß1 protects murine fibroblasts from oxidative stress-induced cell death. The mediators of oxidative stress, reactive oxygen species (ROS), have been shown to regulate major epigenetic processes, causing the silencing of tumor suppressors and enhancing the proliferation of leukemic cells under oxidative stress. Investigation of the interplay between ROS, PI-PLCß1, and their signaling mediators in leukemia might therefore reveal innovative targets of pharmacological therapy in the treatment for leukemia. In this work, we demonstrate that in pro-B-lymphoblastic cells (Ba/F3), treated with H2O2, PI-PLCß1b conferred resistance to cell death, promoting cell cycle progression and cell proliferation and influencing the expression of cyclin A and E. Interestingly, we found that, expression of PI-PLCß1b affects the activity of caspase-3, caspase-7, and of several protein kinases induced by oxidative stress. In particular, PI-PLCß1b expression completely abolished the phosphorylation of Erk1/2 MAP kinases, down-regulated phosphatase and tensin homolog (PTEN), and up-regulated the phosphorylation of Akt, thereby sustaining cellular proliferation.

PLCß1a and PLCß1b selective regulation and cyclin D3 modulation reduced by kinamycin F during k562 cell differentiation.

Here we report that both PLCß1a and PLCß1b are relevant regulators of erythropoiesis in that kinamycin F, a potent inducer of γ-globin production in K562 cells, caused a selectively reduction of both PLCß1 isozymes even though the results point out that the effect of the drug is mainly directed toward the expression of the PLCß1a isoform. We have identified a different role for the two isozymes as regulators of K562 differentiation process induced by kinamycin F. The overexpression of PLCß1b induced an increase in γ-globin expression even in the absence of kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCß1 signaling pathway. Namely the amplification of the expression of the PLCß1a, but not of PLCß1b, is able to maintain high levels of expression of cyclin D3 even after treatment with kinamycin F. This could be due to their different distribution in the cell compartments since the amount of PLCß1b is mainly present in the nucleus in respect to PLCß1a. Our data indicate that the amplification of PLCß1a expression, following treatment with kinamycin F, confers a real advantage to K562 cells viability and protects cells themselves from apoptosis.

Prohibitin 2: At a communications crossroads.

Prohibitins (PHBs) are a highly conserved class of proteins first discovered as inhibitors of cellular proliferation. Since then PHBs have been found to have a significant role in transcription, nuclear signaling, mitochondrial structural integrity, cell division, and cellular membrane metabolism, placing these proteins among the key regulators of pathologies such as cancer, neuromuscular degeneration, and other metabolic diseases. The human genome encodes two PHB proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2), which function not only as a heterodimeric complex, but also independently. While many previous reviews have focused on the better characterized prohibitin, PHB1, this review focuses on PHB2 and new data concerning its cellular functions both in complex with PHB1 and independent of PHB1.

Prohibitin 2 represents a novel nuclear AKT substrate during all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells.

The AKT/PKB kinase is essential for cell survival, proliferation, and differentiation; however, aberrant AKT activation leads to the aggressiveness and drug resistance of many human neoplasias. In the human acute promyelocytic leukemia cell line NB4, nuclear AKT activity increases during all-trans retinoic acid (ATRA)-mediated differentiation. As nuclear AKT activity is associated with differentiation, we sought to identify the nuclear substrates of AKT that were phosphorylated after ATRA treatment. A proteomics-based search for nuclear substrates of AKT in ATRA-treated NB4 cells was undertaken by using 2D-electrophoresis/mass spectrometry (MS) in combination with an anti-AKT phospho-substrate antibody. Western blot analysis, an in vitro kinase assay, and/or site-directed mutagenesis were performed to further characterize the MS findings. MS analysis revealed prohibitin (PHB)-2, a multifunctional protein involved in cell cycle progression and the suppression of oxidative stress, to be a putative nuclear substrate of AKT. Follow-up studies confirmed that AKT phosphorylates PHB2 on Ser-91 and that forced expression of the PHB2(S91A) mutant results in a rapid loss of viability and apoptotic cell death. Activation of nuclear AKT during ATRA-mediated differentiation results in the phosphorylation of several proteins, including PHB2, which may serve to coordinate nuclear-mitochondrial events during differentiation.

Phosphoinositide-specific phospholipase C ß 1b (PI-PLCß1b) interactome: affinity purification-mass spectrometry analysis of PI-PLCß1b with nuclear protein.

Two isoforms of inositide-dependent phospholipase C ß1 (PI-PLCß1) are generated by alternative splicing (PLCß1a and PLCß1b). Both isoforms are present within the nucleus, but in contrast to PLCß1a, the vast majority of PLCß1b is nuclear. In mouse erythroid leukemia cells, PI-PLCß1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLCß1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLCß1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLCß1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule.

Identification of the PKR nuclear interactome reveals roles in ribosome biogenesis, mRNA processing and cell division.

The double-strand RNA-dependent protein kinase, PKR, plays a central role in inflammatory/chronic stress-mediated pathologies such as cancer, diabetes, and neuro/muscular degenerative diseases. Although a significant amount of research has been conducted to elucidate the role of PKR signaling in the cytosol, only recently has attention been paid to the role of PKR in the nuclear compartment. Previously our group reported that phosphorylated forms of PKR are present in the nucleus of acute leukemic cell lines, representing a reservoir of active kinase that responds to stress. Using the CCRF-CEM acute T-cell leukemia cell line, a PKR-specific inhibitor, co-immunoprecipitation and a proteomics approach, which included affinity purified mass spectrometry analysis (AP/MS), we identified the proteins present in active and inactive PKR nuclear complexes. Of the proteins identified in the PKR complexes, sixty-nine (69) were specific to the active complex, while thirty-eight (38) were specific to the inactive complex. An additional thirteen (13) proteins associated specifically with both complexes. The majority of the proteins identified are involved in, ribosome biogenesis, RNA splicing, mRNA stability, gene expression, cell cycle, or chromatin organization, including several with known significance to normal hematopoiesis and/or hematological disease. In agreement with the AP/MS data, basal- or over-expression of PKR under normal growth conditions favored cell proliferation in the tested cell lines, whereas pharmacological inhibition of PKR or shRNA-mediated knock-down did not. PKR was also found to influence the isoform and the level of expression of the proto-oncogene MYC.

Nuclear PLCs affect insulin secretion by targeting PPARγ in pancreatic ß cells.

Type 2 diabetes is a heterogeneous disorder caused by concomitant impairment of insulin secretion by pancreatic ß cells and of insulin action in peripheral target tissues. Studies with inhibitors and agonists established a role for PLC in the regulation of insulin secretion but did not distinguish between effects due to nuclear or cytoplasmic PLC signaling pathways that act in a distinct fashion. We report that in MIN6 ß cells, PLCß1 localized in both nucleus and cytoplasm, PLCδ4 in the nucleus, and PLCγ1 in the cytoplasm. By silencing each isoform, we observed that they all affected glucose-induced insulin release both at basal and high glucose concentrations. To elucidate the molecular basis of PLC regulation, we focused on peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor transcription factor that regulates genes critical to ß-cell maintenance and functions. Silencing of PLCß1 and PLCδ4 resulted in a decrease in the PPARγ mRNA level. By means of a PPARγ-promoter-luciferase assay, the decrease could be attributed to a PLC action on the PPARγ-promoter region. The effect was specifically observed on silencing of the nuclear and not the cytoplasmic PLC. These findings highlight a novel pathway by which nuclear PLCs affect insulin secretion and identify PPARγ as a novel molecular target of nuclear PLCs.

A role for PLCß1 in myotonic dystrophies type 1 and 2.

Phosphoinositide-phospholipase C ß1 (PLCß1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. We previously demonstrated that nuclear PLCß1 activates the cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLCß1 is essential for cyclin D3 promoter activation and gene transcription, through c-jun/AP1. Myotonic dystrophy (DM) is the most prevalent form of muscular dystrophy in adults. DM type 1 (DM1) and type 2 (DM2) are dominantly inherited multisystem disorders. DM1 is triggered by the pathological expansion of a (CTG)(n) triplet repeat in the gene coding for DMPK, the dystrophia myotonica-protein kinase, whereas a (CCTG)(n) tetranucleotide repeat expansion in the ZNF9 gene, encoding a CCHC-type zinc finger protein, causes DM2. We found that, unlike in normal myotubes, the level of expression of PLCß1 in DM1 and DM2 cells was already elevated in proliferating cells. Treatment with insulin induced a dramatic decrease in the amount of PLCß1. During differentiation, cyclin D3 and myogenin were elevated in normal myotubes, whereas differentiating DM1 and DM2 cells did not increase these proteins. Forced expression of PLCß1 in DM1 and DM2 cells increased the expression of differentiation markers, myogenin and cyclin D3, and enhanced fusion of DM myoblasts. These results highlight again that PLCß1 expression is a key player in myoblast differentiation, functioning as a positive regulator in the correction of delayed differentiation of skeletal muscle in DM human myoblasts.