Abstract e appunti sulla funzione delle ciglia nella cellula (ancora da selezionare)

Tubulin and microtubules as targets for anticancer drugs

John A. Hadfield*, Sylvie Ducki, Nicholas Hirst and Alan T. McGown

Microtubules are intracellular organelles formed from the protein tubulin. These organelles have a number of essential cellular functions including chromosome segregation, the maintenance of cell shape, transport, motility, and organelle distribution. Drugs that affect the tubulin-microtubule equilibrium (taxol®, vinca alkaloids) are effective anticancer drugs. This review describes the molecular target, methods used in screening, the structures of compounds known to interact with tubulin, and the clinical use of these agents. In addition the ability of these agents to destroy tumour vasculature is described. This represents an exciting new molecular target in the design of anticancer drugs.

Riflessioni e appunti.

I tassani interferiscono con i microtubuli, i “binari” che guidano i cromosomi durante la divisione cellulare. Recentemente, è stata  identificata una proteina, chiamata PI3K-C2A, che regola i microtubuli durante la divisione cellulare. Alcuni tumori mammari possiedono bassi livelli di PI3K-C2A, e sono più aggressivi e proliferativi.Tuttavia questa perdita di PI3K-C2A rappresenta il tallone d’Achille delle cellule tumorali poiché risultano più sensibili alla terapia con i tassani.

Recettore HERC2 e carcinoma mammario:

Con l’acronimo HER2/neu viene identificato il recettore 2 per il fattore di crescita epidermico umano; HER2/neu appartiene alla famiglia delle proteine ErbB, più comunemente conosciuta come la famiglia dei recettori epidermici dei fattori di crescita. HER2 è una proteina a funzione di recettore di membrana del tipo tirosin chinasico, posizionata esternamente alla cellula (faccia esterna), coinvolta nelle vie di trasduzione del segnale che portano alla crescita e al differenziamento cellulare. Circa il 30% dei tumori della mammella si sviluppano in concomitanza a un’amplificazione del gene HER2/neu o alla sovraespressione del suo prodotto proteico, che concorre a un aggravamento della prognosi. La sua sovraespressione si verifica anche in altri tumori come: il carcinoma ovarico, il carcinoma dello stomaco, il carcinoma del colon-retto e nelle forme aggressive di carcinoma all’utero, come il carcinoma uterino endometriale sieroso. L’iperespressione del gene HER2 può essere soppressa tramite induzione di altri geni che ostacolano la produzione del recettore di HER2.

L’espressione della proteina HER2/ERBB2 viene regolata da recettori per gli estrogeni; Inoltre, l’estradiolo il quale agisce attraverso il recettore degli estrogeni, regola l’espressione di HER2/ERBB2

Nel tumore mammario si usano inibitori dei microtubuli (taxani). Questi farmaci antitumorali, i taxani, sono originariamente derivati da alberi della famiglia del tasso. Il principale ostacolo all’utilizzo dei taxani è legato al fatto che questi composti sono poco solubili in acqua e devono essere veicolati in solventi. Grazie a questo, i taxani sono divenuti tra i farmaci antitumorali più utilizzati nella clinica, risultando efficaci nel trattamento di molte neoplasie, in primo luogo del cancro della mammella. Il rovescio della medaglia è però costituito dalle azioni negative dei solventi: questi provocano frequentemente reazioni allergiche (orticaria, crisi asmatiche, spasmi alla gola, violenti dolori alla schiena) che richiedono la somministrazione preventiva di farmaci cortisonici e antistaminici. Nel 1992 un medico, Patrick Soon-Shiong e un chimico, Neil Desai, ebbero l’idea di tentare di superare questi problemi fondendo il farmaco (paclitaxel) con l’albumina, la sostanza che fisiologicamente, nel nostro organismo, è deputata a trasportare sostanze insolubili in acqua. L’albumina è la più abbondante proteina presente nel plasma e entra facilmente nelle cellule tumorali.

Published in final edited form as:
Drug Discov Today Dis Mech. 2013 December 1; 10(3-4): e135–e142. doi:10.1016/j.ddmec.2013.03.004.

Cilia and cilia-associated proteins in cancer
Tamina Seeger-Nukpezah, Joy L. Little, Victoria Serzhanova, and Erica A. Golemis
*

Program in Developmental Therapeutics, Fox Chase Cancer Center, Philadelphia, PA 19111

Abstract

The primary cilium is a well-established target in the pathogenesis of numerous developmental and chronic disorders, and more recently is attracting interest as a structure relevant to cancer. Here we discuss mechanisms by which changes in cilia can contribute to the formation and growth of tumors. We emphasize the cancer-relevance of cilia-dependent signaling pathways and proteins including mTOR, VHL, TSC, WNT, Aurora-A, NEDD9, and Hedgehog, and highlight the emerging role of ciliary dysfunction in renal cell carcinoma, medulloblastoma, and breast cancer.

www.jasn.org

Context-Dependent Regulation of Wnt Signaling through the Primary Cilium

Edwin C. Oh*and Nicholas Katsanis*†‡
*Center for Human Disease Modeling, Duke University, Durham, North Carolina; and Departments of
Cell Biology and

BRIEF REVIEW

Pediatrics, Duke University, Durham, North Carolina

ABSTRACT

The primary cilium is a highly conserved environmental sensor and modulator of fluid movement in tubular structures. The growing recognition of mutations among its many components has led to the discovery of new disorders collectively called cil- iopathies. Ciliary dysfunction disturbs a variety of signaling pathways along its basal body and axoneme that are critical for embryonic development and cell and organ homeostasis. Among the many pathways, here we discuss the emerging role of Wnt proteins in morphogenic signaling and ciliary biology during health and disease.

J Am Soc Nephrol 24: 10–18, 2013. doi: 10.1681/ASN.2012050526

Published in final edited form as:
Neurobiol Dis. 2010 May ; 38(2): 167–172. doi:10.1016/j.nbd.2009.12.022.

The role of primary cilia in neuronal function

Jeong Ho Lee and Joseph G. Gleeson*
Department of Neurosciences and Pediatrics, Howard Hughes Medical Institute, University of California, San Diego, CA, USA

Abstract

The “ciliopathies” are a newly defined group of disorders characterized by defects in the structure or function of the cellular primary cilium. Patients with these disorders display variably expressive fibrocystic renal disease, retinal blindness, polydactyly, obesity, and brain dysgenesis as well as neurocognitive impairments. Joubert syndrome is a ciliopathy defined by cerebellar vermis hypoplasia, oculomotor apraxia, intermittent hyperventilation, and mental retardation. Recent evidence suggests important roles for the primary cilium in mediating a host of extracellular signaling events such as morphogen, mitogen, homeostatic and polarity signals. Based upon the clinical features of ciliopathies and cilia mediated signaling pathways, the data support a role for the primary cilium in modulating neurogenesis, cell polarity, axonal guidance and possibly adult neuronal function.

Absence of primary cilia in cell cycle-arrested human breast cancer cells

Kentaro Nobutani1,2,3, Yohei Shimono3, Midori Yoshida1,3,4, Kiyohito Mizutani1,3, Akihiro Minami2,3, Seishi Kono5, Toru Mukohara6, Takashi Yamasaki7, Tomoo Itoh7, Shintaro Takao5, Hironobu Minami6, Takeshi Azuma2 and Yoshimi Takai1,3*

1Division of Pathogenetic Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 1-5-6 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
2Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

3Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
4Department of Oral and Maxillofacial Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

5Division of Breast Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku,
Kobe 650-0017, Japan
6Division of Medical Oncology/Hematology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
7Department of Diagnostic Pathology, Kobe University Hospital, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

Previous studies using cultured cells showed that primary cilia are present in quiescent cells, but are absent in proliferating cells. We studied here the relationship between the presence or absence of primary cilia and the cell cycle arrest of normal epithelial cells and cancer cells in the human normal breast and breast cancer tissues. In normal breast tissues, although most epithelial cells were nonproliferating as estimated by the immunofluorescence staining of the proliferation marker Ki-67, primary cilia were present only in 20–40% of the epithelial cells. In breast cancer tissues, primary cilia were not observed in any of the breast cancer cells. Furthermore, primary cilia were hardly observed in the nonproliferating cancer cells in the orthotopic and metastatic human breast cancer xenograft tumors in mice. These results indi- cate that the absence of primary cilia does not necessarily represent the proliferating phases of normal epithelial cells and cancer cells.

Published in final edited form as:
Exp Hematol. 2016 December ; 44(12): 1181–1187.e2. doi:10.1016/j.exphem.2016.08.009.

Primary Cilia are Present on Human Blood and Bone Marrow Cells and Mediate Hedgehog Signaling

Mohan Singh, Parvesh Chaudhry, and Akil A. Merchant
Department of Medicine, Division of Hematology, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles CA, 90033, USA

Abstract

Primary cilia are non-motile microtubule-based organelles that are present on the cellular membrane of all eukaryotic cells. Functional cilia are required for the response to developmental signaling pathways such as Hedgehog (Hh) and Wnt/β-catenin. Although the Hh pathway has been shown to be active in leukemia and other blood cancers, there have been no reports describing the presence of primary cilia in human blood or leukemia cells. In the present study, we show that nearly all human blood and bone marrow cells have primary cilia (97–99%). In contrast, primary cilia on acute myeloid leukemia cell lines (KG1, KG1a, K562) were less frequent (10– 36% of cells) and were often shorter and dysmorphic, with less well defined basal bodies. Finally, we show that treatment of blood cells with the Hh pathway ligand (SHh) causes translocation of SMO to the primary cilia and activation of Hh target genes, demonstrating that primary cilia in blood cells are functional and participate in Hh signaling. Loss of primary cilia on leukemia cells may have important implications for aberrant pathway activation and response to SMO inhibitors currently in clinical development.

Leptin-promoted cilia assembly is critical for normal energy balance

Yu Mi Han, … , Bonghee Lee, Min-Seon Kim
J Clin Invest. 2014;124(5):2193-2197. https://doi.org/10.1172/JCI69395.

Brief Report Metabolism

The majority of mammalian cells have nonmotile primary cilia on their surface that act as antenna-like sensory organelles. Genetic defects that result in ciliary dysfunction are associated with obesity in humans and rodents, which suggests that functional cilia are important for controlling energy balance. Here we demonstrated that neuronal cilia lengths were selectively reduced in hypothalami of obese mice with leptin deficiency and leptin resistance. Treatment of N1 hypothalamic neuron cells with leptin stimulated cilia assembly via inhibition of the tumor suppressors PTEN and glycogen synthase kinase 3b (GSK3b). Induction of short cilia in the hypothalamus of adult mice increased food intake and decreased energy expenditure, leading to a positive energy balance. Moreover, mice with short hypothalamic cilia exhibited attenuated anorectic responses to leptin, insulin, and glucose, which indicates that leptin-induced cilia assembly is essential for sensing these satiety signals by hypothalamic neurons. These data suggest that leptin governs the sensitivity of hypothalamic neurons to metabolic signals by controlling the length of the cell’s antenna.

Published in final edited form as:
Cell Mol Life Sci. 2018 May ; 75(9): 1521–1540. doi:10.1007/s00018-017-2740-5.

Primary cilia proteins: Ciliary and extraciliary sites and functions Kiet Hua1,* and Russell J Ferland2,*

1Department of Neuroscience and Experimental Therapeutics, Albany Medical College, Albany, New York, 12208

2Department of Neurology, Albany Medical College, Albany, New York, 12208

Abstract

Primary cilia are immotile organelles known for their roles in development and cell signaling. Defects in primary cilia result in a range of disorders named ciliopathies. Because this organelle can be found singularly on almost all cell types, its importance extends to most organ systems. As such, elucidating the importance of the primary cilium has attracted researchers from all biological disciplines. As the primary cilia field expands, caution is warranted in attributing biological defects solely to the function of this organelle, since many of these “ciliary” proteins are found at other sites in cells and likely have non-ciliary functions. Indeed, many, if not all, cilia proteins have locations and functions outside the primary cilium. Extraciliary functions are known to include cell cycle regulation, cytoskeletal regulation, and trafficking. Cilia proteins have been observed in the nucleus, at the Golgi apparatus, and even in immune synapses of T-cells (interestingly, a non-ciliated cell). Given the abundance of extraciliary sites and functions, it can be difficult to definitively attribute an observed phenotype solely to defective cilia rather than to some defective extraciliary function or a combination of both. Thus, extraciliary sites and functions of cilia proteins need to be considered, as well as experimentally determined. Through such consideration, we will understand the true role of the primary cilium in disease as compared to other cellular processes’ influences in mediating disease (or through a combination of both). Here, we review a compilation of known extraciliary sites and functions of “cilia” proteins as a means to demonstrate the potential non-ciliary roles for these proteins.

The Primary Cilium as the Cell’s

Antenna: Signaling at a

Sensory Organelle

Veena Singla and Jeremy F. Reiter*

Almost every vertebrate cell has a specialized cell surface projection called a primary cilium. Although these structures were first described more than a century ago, the full scope of their functions remains poorly understood. Here, we review emerging evidence that in addition to their well-established roles in sight, smell, and mechanosensation, primary cilia are key participants in intercellular signaling. This new appreciation of primary cilia as cellular antennae that sense a wide variety of signals could help explain why ciliary defects underlie such a wide range of human disorders, including retinal degeneration, polycystic kidney disease, Bardet-Biedl syndrome, and neural tube defects.

ORIGINAL RESEARCH

Enteric Glia Mediate Neuron Death in Colitis Through Purinergic Pathways That Require Connexin-43 and Nitric Oxide

Isola A. M. Brown,1,2 Jonathon L. McClain,1 Ralph E. Watson,3 Bhavik A. Patel,5 and Brian D. Gulbransen1,4

1Department of Physiology, 2Pharmacology and Toxicology Program, 3Department of Medicine, and 4Neuroscience Program, Michigan State University, East Lansing, Michigan; 5School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom    SUMMARY

Death of enteric neurons contributes to motility dysfunction in gastrointestinal disorders. Our work provides the first evidence of glial activation as a driver of enteric neurodegneration.

BACKGROUND & AIMS: The concept of enteric glia as regulators of intestinal homeostasis is slowly gaining acceptance as a central concept in neurogastroenterology. Yet how glia contribute to intestinal disease is still poorly understood. Purines generated during inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors (P2X7R); triggering adenosine triphosphate (ATP) release via neuronal pannexin-1 channels that subse- quently recruits intracellular calcium ([Ca]i) in surround- ing enteric glia. We tested the hypothesis that the activation of enteric glia contributes to neuron death during inflammation.

METHODS: We studied neuroinflammation in vivo using the 2,4-dinitrobenzene sulfonic acid model of colitis and in situ using whole-mount preparations of human and mouse in- testine. Transgenic mice with a targeted deletion of glial connexin-43 (Cx43) [GFAP::CreERT2þ//Cx43f/f] were used to specifically disrupt glial signaling pathways. Mice deficient in inducible nitric oxide (NO) synthase (iNOS/) were used to study NO production. Protein expression and oxidative stress were measured using immunohistochemistry and in situ Caand NO imaging were used to monitor glial [Ca]i and [NO]i.

RESULTS: Purinergic activation of enteric glia drove [Ca]i responses and enteric neuron death through a Cx43-dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43-dependent ATP release was potentiated by NO.

CONCLUSIONS: Our results show that the activation of glial cells in the context of neuroinflammation kills enteric neu- rons. Mediators of inflammation that include ATP and NO activate neurotoxic pathways that converge on glial Cx43 hemichannels. The glial response to inflammatory media- tors might contribute to the development of motility disorders. (Cell Mol Gastroenterol Hepatol 2016;2:77–91; http://dx.doi.org/ 10.1016/j.jcmgh.2015.08.007)

Deregulated hedgehog pathway signaling is inhibited by the smoothened antagonist LDE225 (Sonidegib) in chronic phase chronic myeloid leukaemia

David A. Irvine1,*, Bin Zhang2,*, Ross Kinstrie1, Anuradha Tarafdar1, Heather Morrison1, Victoria L. Campbell1, Hothri A. Moka1, Yinwei Ho2, Colin Nixon4, Paul W. Manley3, Helen Wheadon1, John R. Goodlad5, Tessa L. Holyoake1, Ravi Bhatia6 & Mhairi Copland1

Targeting the Hedgehog (Hh) pathway represents a potential leukaemia stem cell (LSC)-directed therapy which may compliment tyrosine kinase inhibitors (TKIs) to eradicate LSC in chronic phase
(CP) chronic myeloid leukaemia (CML). We set out to elucidate the role of Hh signaling in CP-CML and determine if inhibition of Hh signaling, through inhibition of smoothened (SMO), was an effective strategy to target CP-CML LSC. Assessment of Hh pathway gene and protein expression demonstrated that the Hh pathway is activated in CD34
+ CP-CML stem/progenitor cells. LDE225 (Sonidegib), a

small molecule, clinically investigated SMO inhibitor, used alone and in combination with nilotinib, inhibited the Hh pathway in CD34+ CP-CML cells, reducing the number and self-renewal capacity of CML LSC in vitro. The combination had no effect on normal haemopoietic stem cells. When combined, LDE225 + nilotinib reduced CD34+ CP-CML cell engraftment in NSG mice and, upon administration

to EGFP+ /SCLtTA/TRE-BCR-ABL mice, the combination enhanced survival with reduced leukaemia development in secondary transplant recipients. In conclusion, the Hh pathway is deregulated in
CML stem and progenitor cells. We identify Hh pathway inhibition, in combination with nilotinib, as a potentially effective therapeutic strategy to improve responses in CP-CML by targeting both stem and progenitor cells.

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder characterised by massive myeloid expansion, accumulation of differentiating granulocytic precursors and terminally differentiated effector cells leading to the key clinical features at presentation of marked peripheral blood granulocytosis, basophilia, sple- nomegaly and often thrombocytosis and anaemia1. Untreated, the clinical course of CML is one of inevitable progression from a stable chronic phase (CP) lasting about 5 years from diagnosis, where there is gradual accu- mulation of leukaemic myeloid progenitors, to accelerated phase characterised by accumulation and clonal evolu- tion of increasingly primitive myeloid precursors in the blood or bone marrow (BM) before terminating in a blast crisis (BC) with rapid accumulation of immature myeloid or lymphoid precursors resembling acute leukaemia.

In optimally responding CP-CML patients, the majority have molecular evidence of persisting disease after prolonged tyrosine kinase inhibitor (TKI) therapy2,3. Furthermore, of those patients who achieve sustained molecularly undetectable leukaemia and discontinue TKI treatment the majority suffer molecular relapse4–7. One

1Paul O’Gorman Leukaemia Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 0XB, UK. 2Division of Hematopoietic Stem Cell and Leukemia Research, City of Hope National Medical Centre, Duarte, CA, USA. 3Novartis Institutes for Biomedical Research, Basel, Switzerland. 4Histology, The Beatson Institute for Cancer Research, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK. 5Department of Histology, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK. 6Division of Hematology-Oncology, University of Alabama Birmingham, Birmingham, AL, USA. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.C. (email: Mhairi.Copland@glasgow.ac.uk)

Scientific RepoRts | 6:25476 | DOI: 10.1038/srep25476

Targeting Hedgehog signaling pathway and

autophagy overcomes drug resistance of BCR-

ABL-positive chronic myeloid leukemia

Xian Zeng,1,2,y Hui Zhao,3,4,y Yubin Li,1,y Jiajun Fan,1 Yun Sun,1 Shaofei Wang,1 Ziyu Wang,1 Ping Song,1 and Dianwen Ju1,*

1Department of Biosynthesis and Key Laboratory of Smart Drug Delivery; MOE; School of Pharmacy; Fudan University; Shanghai, China; 2Bioinformatics and Drug Design Group; Department of Pharmacy; Faculty of Science; National University of Singapore; Singapore; 3Department of Pharmacology; School of Pharmacy; Fudan University; Shanghai, China; 4Department of Pharmacology; Yong Loo Lin School of Medicine; National University of Singapore; Singapore

yThese authors equally contributed to this work.
Keywords: autophagy, BCR-ABL, CML, drug resistance, hedgehog pathway

Abbreviations: ACTB, actin; b; AKT/protein kinase B, v-akt murine thymoma viral oncogene homolog; ATG, autophagy-related; Bafi A1, bafilomycin A1; BCC, basal cell carcinoma; BCR-ABL, breakpoint cluster region-ABL proto-oncogene, non-receptor tyro- sine kinase; CASP, caspase; apoptosis-related cysteine peptidase; CML, chronic myeloid leukemia; CQ, chloroquine; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; HCQ, hydroxychloroquine; Hh, Hedgehog; MAP1LC3B, microtubule- associated protein 1 light chain 3 b; MTOR, mechanistic target of rapamycin; PARP, poly (ADP-ribose) polymerase; PBMC, human peripheral blood mononuclear cell; PCR, polymerase chain reaction; RPS6KB, ribosomal protein S6 kinase, 70kDa; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TKI, tyrosine kinase inhibitor.

TRANSLATIONAL RESEARCH PAPER

Autophagy 11:2, 355–372; February 2015; © 2015 Taylor & Francis Group, LLC

   

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABLC CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABLC CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABLC cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR- ABLC cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.

Molecular Pathways

Molecular Pathways: The Hedgehog Signaling Pathway in Cancer

Ross McMillan and William Matsui

Abstract

The Hedgehog (Hh) signaling pathway regulates embryonic development and may be aberrantly activated in a wide variety of human cancers. Efforts to target pathogenic Hh signaling have steadily progressed from the laboratory to the clinic, and the recent approval of the Hh pathway inhibitor vismodegib for patients with advanced basal cell carcinoma represents an important milestone. On the other hand, Hh pathway antagonists have failed to show significant clinical activity in other solid tumors. The reasons for these negative results are not precisely understood, but it is possible that the impact of Hh pathway inhibition has not been adequately measured by the clinical endpoints used thus far or that aberrancies in Hh signal transduction limits the activity of currently available pathway antagonists. Further basic and correlative studies to better understand Hh signaling in human tumors and validate putative antitumor mechanisms in the clinical setting may ultimately improve the success of Hh pathway inhibition to other tumor types. Clin Cancer Res; 18(18); 4883–8. !2012 AACR.

The FASEB Journal express article 10.1096/fj.03-0322fje. Published online September 4, 2003.

Imatinib induces mitochondria-dependent apoptosis of the Bcr-Abl positive K562 cell line and its differentiation towards the erythroid lineage

Arnaud Jacquel,* Magali Herrant,* Laurence Legros,Nathalie Belhacene,* Frederic Luciano,* Gilles Pages,Paul Hofman,and Patrick Auberger*

*INSERM U526, Physiopathologie de la Survie et de la Mort Cellulaires et Infections Virales, Equipe Labellisée par la Ligue Nationale contre le Cancer, Faculté de Médecine, Avenue de Valombrose, 06107 Nice-Cedex 2, France; EPI 0215, IFR50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice-Cedex 2, France; and UMR 6543 Centre Antoine Lacassagne, Avenue de Valombrose, 06107 Nice, France

Corresponding author: Patrick Auberger, INSERM U526, Physiopathologie de la Survie et de la Mort Cellulaires et Infections Virales Equipe Labellisée par la Ligue Nationale contre le Cancer, 06107 Nice-Cedex 2, France. E-mail: auberger@unice.fr

ABSTRACT

Imatinib has emerged as the lead compound for clinical development against chronic myeloid leukemia. Imatinib inhibits the kinase activity of Bcr-Abl, which functions by enhancing the proliferation of hematopoietic precursors and protecting them against apoptosis. Imatinib induces apoptosis of Bcr-Abl positive cells, but how the drug effectively kills these cells remains partially understood. We show here that in K562 cells imatinib i) abolished Bcr-Abl phosphorylation and activity and as a consequence Erk1/2, JNK, and AKT activation; ii) induced mitochondrial transmembrane permeability dissipation; iii) activated caspases 3, 9, and 8, demonstrating that the effect of imatinib is integrated at the mitochondrial level; and iv) triggered caspase-dependent cleavage of Bcr-Abl. Interestingly, imatinib-mediated apoptosis was accompanied by erythroid differentiation of K562 cells. Moreover, phorbol esters inhibited imatinib-induced cell death and promoted differentiation toward the megakaryocytic lineage. Finally, we determined by c-DNA array analysis that more than 20 genes were modulated by imatinib. These genes are involved in both cell death and differentiation programs, and some of them have never been reported before to be expressed or involved in erythroid differentiation. Our results demonstrate that imatinib is responsible for a major modification of the genetic program resulting in death and/or differentiation of K562 cells.

Functional properties of ion channels and transporters in tumour vascularization

Alessandra Fiorio Pla and Luca Munaron

Department of Life Sciences and Systems Biology, Center for Complex Systems in Molecular Biology and Medicine (SysBioM), Nanostructured Interfaces and Surfaces Centre of Excellence (NIS), University of Torino, Via Accademia Albertina 13, Torino 10123, Italy

Downloaded from rstb.royalsocietypublishing.org on February 3, 2014

Vascularization is crucial for solid tumour growth and invasion, providing metabolic support and sustaining metastatic dissemination. It is now accepted that ion channels and transporters play a significant role in driving the cancer growth at all stages. They may represent novel therapeutic, diag- nostic and prognostic targets for anti-cancer therapies. On the other hand, although the expression and role of ion channels and transporters in the vas- cular endothelium is well recognized and subject of recent reviews, only recently has their involvement in tumour vascularization been recognized. Here, we review the current literature on ion channels and transporters directly involved in the angiogenic process. Particular interest will be focused on tumour angiogenesis in vivo as well as in the different steps that drive this process in vitro, such as endothelial cell proliferation, migration, adhesion and tubulogenesis. Moreover, we compare the ‘transportome’ system of tumour vascular network with the physiological one.

Published in final edited form as:
Bone. 2014 September ; 66: 111–120. doi:10.1016/j.bone.2014.05.015.

Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation

Constance P. Soves1, Joshua D. Miller1, Dana L. Begun1, Russell S. Taichman2, Kurt D. Hankenson3, and Steven A. Goldstein1,*
1Orthopaedic Research Laboratories, University of Michigan, Room 2003 Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI USA 48109.

2Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, 1011 North University Ave., Ann Arbor, MI, USA 48109.

3Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine and Department of Orthopaedic Surgery, Perelman School of Medicine University of Pennsylvania, Room 145 Myrin, Kennett Square, PA 19348.

Abstract

Maintenance of bone mass and geometry is influenced by mechanical stimuli. Paradigms suggest that osteocytes embedded within the mineralized matrix and osteoblasts on the bone surfaces are the primary responders to physical forces. However, other cells within the bone marrow cavity, such as megakaryocytes (MKs), are also subject to mechanical forces. Recent studies have highlighted the potent effects of MKs on osteoblast proliferation as well as bone formation in vivo. We hypothesize that MKs are capable of responding to physical forces and that the interactions between these cells and osteoblasts can be influenced by mechanical stimulation.

In this study, we demonstrate that two MK cell lines respond to fluid shear stress in culture. Furthermore, using laser capture microdissection, we isolated MKs from histologic sections of murine tibiae that were exposed to compressive loads in vivo. C-fos, a transcription factor shown to be upregulated in response to load in various tissue types, was increased in MKs from loaded relative to non-loaded limbs at a level comparable to that of osteocytes from the same limbs. We also developed a co-culture system to address whether mechanical stimulation of MKs in culture would impact osteoblast proliferation and differentiation. The presence of MKs in co-culture, but not conditioned media, had dramatic effects on proliferation of preosteoblast MC3T3-E1 cells in culture. Our data suggests a minimal decrease in proliferation as well as an increase in mineralization capacity of osteoblasts co-cultured with MKs exposed to shear compared to co- cultures with unstimulated MKs.

Membrane Damage Effect of Continuous Wave Ultrasound on K562 Human Leukemia Cells
Pan Wang, PhD, Yixiang Li, MS, Xiaobing Wang, PhD, Ling Guo, MS, Xiaomin Su, MS, Quanhong Liu, PhD

ived December 20, 2011, from the College of Life Sciences, Shaanxi Normal University, Shaanxi, Xi’an, China. Revision requested February 1, 2012. Revised manuscript accepted for publica- tion May 16, 2012.

This work was supported by the National Natural Science Foundation of China (grants 81000999 and 10904087), the Research Fund for the Doctoral Program of Higher Education of China (grant 20100202110006), and the Natu- ral Science Foundation of Shaanxi Province, China (grant 2011JQ4012). Pan Wang an Yixiang Li contributed equally to this work.

Address correspondence to Quanhong Liu, PhD, College of Life Sciences, Shaanxi Normal University, 710062 Shaanxi, Xi’an, China.

E-mail: lshaof@snnu.edu.cn

Abbreviations

[Ca2+]i , intracellular calcium ion concentra- tion; DiBAC4(3), bis-(1,3-dibarbituric acid)- trimethine oxanol; FDA, fluorescein diacetate; Fluo 4-AM, Fluo 4-acetoxymethyl ester; HTA, 2-hydroxyterephthalate; [K+]ex , extracellu- lar potassium ion concentration; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra zolium bromide; •OH, hydroxyl radical; PI, propidium iodide; TA, terephthalic acid

Objectives—This study investigated the bioeffects of ultrasound with a frequency of 1.1 MHz on human chronic myelogenous leukemia cell line K562.

Methods—Membrane potential changes were evaluated by flow cytometry using fluo- rescent probe bis-(1,3-dibarbituric acid)-trimethine oxanol staining. Other related changes such as potassium ion efflux and intracellular calcium ion overload were also measured. The plasma membrane integrity was monitored by flow cytometry com- bined with fluorescein diacetate and propidium iodide double fluorescent dye staining. A cell-counting assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis were used to examine the viability of K562 cells after ultrasound exposure. The acoustic cavitation activity in ultrasound fields was assessed by monitoring hydroxyl radical production.

Results—As the ultrasonic intensity increased, the hydroxyl radical produced in the medium increased, and cell membrane damage and cell viability loss were enhanced. The ultrasonic intensity at 0.64 W/cm2 did not cause substantial cell damage, whereas ultrasound exposure at 1 and 2.1 W/cm2 could induce serious cell death (14.0% and 40.7%, respectively). Moreover, ultrasound at 0.64 W/cm2 did not cause substantial membrane potential changes, whereas ultrasound exposure at 1 W/cm2 could induce depolarization, and fast hyperpolarization occurred when the ultrasonic intensity increased to 2.1 W/cm2. In addition, compared with control cells, in different ultra- sound-treated cells, the potassium ion continuously outflowed with a prolonged incu- bation time, whereas the intracellular calcium ion oscillations became more apparent.

Conclusions—The damaging effects of ultrasound on the cell membrane and cell via- bility were intensity dependent. The membrane potential changes may be due to acoustic cavitation accompanied by alterations in the balance of ions on opposite sides of the cellular membrane.

Key Words—cavitation; K562 cells; membrane integrity; membrane potential; ultrasound

Review

Targeting Hedgehog (Hh) Pathway for the Acute Myeloid Leukemia Treatment

Toshiki Terao 1,2 and Yosuke Minami 1,*

1

Department of Hematology, National Cancer Center Hospital East, Kashiwa 277-8577, Japan; terao.toshiki@kameda.jp
Division of Hematology/Oncology, Department of Internal Medicine, Kameda Medical Center, Kamogawa 296-8602, Japan

2
* Correspondence: yominami@east.ncc.go.jp; Tel.: +81-47-133-1111; Fax.: +81-71-336-502

Received: 22 February 2019; Accepted: 28 March 2019; Published: 3 April 2019

Abstract: The Hedgehog (Hh) pathway, containing the Patched (PTCH) and Smoothened (SMO) multitransmembrane proteins, is the main regulator of vertebrate embryonic development. A non-canonical Hh pathway was recently observed in numerous types of solid cancers and hematological malignancies. Although acute myeloid leukemia (AML) is a common and lethal myeloid malignancy, the chemotherapy for AML has not changed in the last three decades. The Hh pathway and other intracellular signaling pathways are important for the tumor cells’ cycle or therapeutic resistance of AML cells. In this article, we will review the current trends in Hh pathway inhibitors for treating AML.

[CANCER RESEARCH 60, 1014 –1020, February 15, 2000]

Apoptosis Induction of Human Myeloid Leukemic Cells by Ultrasound Exposure1

Hagit Ashush, Leon A. Rozenszajn,2 Michal Blass, Mira Barda-Saad, Damir Azimov, Judith Radnay, Dov Zipori, and Uri Rosenschein

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel [H. A., L. A. R., M. B., M. B-S., D. A., J. R.]; Department of Molecular Cell Biology, Weizmann Institute of Sciences, Rehovot, Israel [D. Z.]; and Tel Aviv Medical Center, Tel Aviv University, Tel Aviv, Israel [U. R.]

ABSTRACT

Therapeutic ultrasound (ULS) and the resulting cavitation process has

been shown to induce irreversible cell damage. In this study, we wanted to

further investigate the mechanism of ULS-induced cell death and to

determine whether apoptosis is involved. High intensity focused pulsed

ULS sonication at a frequency of 750 KHz was delivered to HL-60, K562,

U937, and M1/2 leukemia cell line cultures. ULS exposure used with

induction of transient cavitation in the focal area was delivered with an

intensity level of 103.7 W/cm2 and 54.6 W/cm2 spatial-peak temporal-

average intensity. As a control, ULS of lower intensity was delivered at

2
22.4 W/cm spatial-peak temporal-average intensity, presumably without

generation of cavitation. Our results indicated that DNA damage induced by ULS cavitation did not involve generation of free radicals in the culture media. Morphological alterations observed in cells after exposure to ULS included: cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation, and apoptotic body formation. Apoptotic cells were evaluated by fluorescence microscopy and detected using the termi- nal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, which identifies DNA breaks, and by the leakage of phosphatidylserine from the inner to the outer side of the membrane layer of treated cells. Some bioeffects induced on sonicated HL-60 cells, such as inhibition of cell proliferation, DNA repair, and cell-dependent apoptosis, were found to be similar to those produced by 􏰎-irradiation. Thus, much of the cell damage induced by therapeutic ULS in leukemia cells surviving ULS exposure appears to occur through an apoptotic mechanism.

Article Addendum

Linoleic acid inhibits TRP channels with intrinsic voltage sensitivity

Implications on the mechanism of linoleic acid action

Moshe Parnas, Maximilian Peters and Baruch Minke*

Departments of Physiology and the Kühne Minerva Center for Studies of Visual Transduction; Faculty of Medicine of the Hebrew University; Jerusalem, Israel

Key words: TRP channels, open channel block, divalent cations, lipids, membrane

   

Open channel block (OCB) is a process by which ions bind to the inside of a channel pore and block the flow of ions through that channel. Repulsion of the blocking ions by membrane depolarization is a known mechanism for open channel block removal. For the N-methyl-D-aspartate (NMDA) channel, this mechanism is necessary for channel activation and is involved in neuronal plasticity. Several types of Transient Receptor Potential (TRP) channels, including the Drosophila TRP and TRP-Like (TRPL) channels, also exhibit open channel block. For the Drosophila TRP and TRPL channels, removal of open channel block is necessary for the production of the physiological response to light. Recently, we have shown that lipids such as polyunsaturated fatty acids (PUFAs), represented by linoleic acid (LA), alleviate OCB under physiological conditions, from the Drosophila TRP and TRPL channels and from the mammalian NMDA channel. Here we show that OCB removal by LA is not confined to the Drosophila TRPs but also applies to mamma- lian TRPs such as the heat activated TRPV3 channel. TRPV3 shows OCB alleviation by LA, although it shares little amino acid sequence homology with the Drosophila TRPs. Strikingly, LA inhibits the heat-activated TRPV1 and the cold temperature- activated TRPM8 channels, which are intrinsic voltage sensitive channels and do not show OCB. Together, our findings further support the notion that lipids do not act as second messengers by direct binding to a specific site of the channels but rather act indirectly by affecting the channel-plasma membrane interface.

Published in final edited form as:
Bone. 2014 September ; 66: 111–120. doi:10.1016/j.bone.2014.05.015.

Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation

Constance P. Soves1, Joshua D. Miller1, Dana L. Begun1, Russell S. Taichman2, Kurt D. Hankenson3, and Steven A. Goldstein1,*
1Orthopaedic Research Laboratories, University of Michigan, Room 2003 Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI USA 48109.

2Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, 1011 North University Ave., Ann Arbor, MI, USA 48109.

3Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine and Department of Orthopaedic Surgery, Perelman School of Medicine University of Pennsylvania, Room 145 Myrin, Kennett Square, PA 19348.

Abstract

Maintenance of bone mass and geometry is influenced by mechanical stimuli. Paradigms suggest that osteocytes embedded within the mineralized matrix and osteoblasts on the bone surfaces are the primary responders to physical forces. However, other cells within the bone marrow cavity, such as megakaryocytes (MKs), are also subject to mechanical forces. Recent studies have highlighted the potent effects of MKs on osteoblast proliferation as well as bone formation in vivo. We hypothesize that MKs are capable of responding to physical forces and that the interactions between these cells and osteoblasts can be influenced by mechanical stimulation.

In this study, we demonstrate that two MK cell lines respond to fluid shear stress in culture. Furthermore, using laser capture microdissection, we isolated MKs from histologic sections of murine tibiae that were exposed to compressive loads in vivo. C-fos, a transcription factor shown to be upregulated in response to load in various tissue types, was increased in MKs from loaded relative to non-loaded limbs at a level comparable to that of osteocytes from the same limbs. We also developed a co-culture system to address whether mechanical stimulation of MKs in culture would impact osteoblast proliferation and differentiation. The presence of MKs in co-culture, but not conditioned media, had dramatic effects on proliferation of preosteoblast MC3T3-E1 cells in culture. Our data suggests a minimal decrease in proliferation as well as an increase in mineralization capacity of osteoblasts co-cultured with MKs exposed to shear compared to co- cultures with unstimulated MKs.

Effect of Rapamycin as an Inhibitor of the mTOR Cell Cycle Entry Complex on the Selective Lysis of Human Leukemia Cells Lines in Vitro Using 20 kHz Pulsed Low-Frequency Ultrasound

A Capstone Project Submitted in Partial Fulfillment of the Requirements of the Renée Crown University Honors Program at Syracuse University

Yasmine Hemida

Abstract

It has been shown that the mammalian target of rapamycin (mTOR) pathway, which regulates cell growth and proliferation, is aberrant in many hematological malignancies. Rapamycin inhibits mTOR signaling which regulates cell growth and cell cycle progression. This study sought to determine the effects of mTOR inhibitors on the cell sizes of normal and neoplastic cells and to determine ultrasonic sensitivity of normal and neoplastic cells treated with mTOR inhibitors. The effects of rapamycin (rapa), an extensively studied natural product that affects cell cycle entry by inhibiting mTORC1, and its analogs temsirolimus (tems) and everolimus (eve), were examined on leukemic cell lines (U937: Human monocytic leukemia, THP-1: Human acute monocytic lymphoma, K562: Human chronic myelogenous leukemia, and MOLT-4, Human acute lymphoblastic leukemia) in combination with 20kHz ultrasound. Each cell line was treated for 48 hours with each analog before being exposed to ultrasound. By using low frequency ultrasound of 5 pulses (0.6 seconds of ultrasound and 0.4 seconds without ultrasound) at 20 kHz and 30% amplitude (60W), we determined that as the concentrations of Rapa, Eve, and Tems (2, 20, and 50nM) increased, there is usually the most sonic damage observed in U937, MOLT-4, K562, and THP-1 cells. The most preferentially damaged cell line is K562 by all of the analogs and with all the concentrations used. In addition, larger cells were more susceptible to ultrasonic damage than were smaller cells. Further, there are no significant differences among the three analogs in the potentiation of ultrasonic damage. This preliminary therapeutic approach involving the use of ultra-sound in combination with mTOR inhibitors might eventually provide an improved approach to the treatment of leukemia and other hematological malignancies in a clinical setting.

IKK/NF-kB signaling: balancing life and death – a new approach to cancer therapy

Jun-Li Luo, … , Hideaki Kamata, Michael Karin
J Clin Invest. 2005;115(10):2625-2632. https://doi.org/10.1172/JCI26322.

Review Series

IkB kinase/NF-kB (IKK/NF-kB) signaling pathways play critical roles in a variety of physiological and pathological processes. One function of NF-kB is promotion of cell survival through induction of target genes, whose products inhibit components of the apoptotic machinery in normal and cancerous cells. NF-kB can also prevent programmed necrosis by inducing genes encoding antioxidant proteins. Regardless of mechanism, many cancer cells, of either epithelial or hematopoietic origin, use NF-kB to achieve resistance to anticancer drugs, radiation, and death cytokines. Hence, inhibition of IKK-driven NF-kB activation offers a strategy for treatment of different malignancies and can convert inflammation-induced tumor growth to inflammation-induced tumor regression.

Published in final edited form as:
Exp Neurol. 2012 September ; 237(1): 199–206. doi:10.1016/j.expneurol.2012.06.013.

A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors

Robert F. Hunt, Gabriela A. Hortopan, Anna Gillespie, and Scott C. Baraban
Epilepsy Research Laboratory, Department of Neurological Surgery, Biomedical Graduate Program, University of California San Francisco, San Francisco, CA 94143

Abstract

Febrile seizures are the most common seizure type in children under the age of five, but mechanisms underlying seizure generation in vivo remain unclear. Animal models to address this issue primarily focus on immature rodents heated indirectly using a controlled water bath or air blower. Here we describe an in vivo model of hyperthermia-induced seizures in larval zebrafish aged 3 to 7 days post-fertilization (dpf). Bath controlled changes in temperature are rapid and reversible in this model. Acute electrographic seizures following transient hyperthermia showed age-dependence, strain independence, and absence of mortality. Electrographic seizures recorded in the larval zebrafish forebrain were blocked by adding antagonists to the transient receptor potential vanilloid (TRPV4) channel or N-methyl-d-aspartate (NMDA) glutamate receptor to the bathing medium. Application of GABA, GABA re-uptake inhibitors, or TRPV1 antagonist had no effect on hyperthermic seizures. Expression of vanilloid channel and glutamate receptor mRNA was confirmed by quantitative PCR analysis at each developmental stage in larval zebrafish. Taken together, our findings suggest a role of heat-activation of TRPV4 channels and enhanced NMDA receptor-mediated glutamatergic transmission in hyperthermia-induced seizures.

An Emerging Role for Endothelial Nitric Oxide Synthase in Chronic Inflammation and Cancer

Lei Ying and Lorne J. Hofseth

Department of Basic Pharmaceutical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina

Abstract

Nitric oxide (NO) is a free radical that is involved in carcinogenesis. Recent literature indicates that endothelial NO synthase (eNOS) can modulate cancer-related events (angiogenesis, apoptosis, cell cycle, invasion, and metastasis). We review the literature linking eNOS to carcinogenesis to encourage future research assessing the role of eNOS in cancer prevention and treatment. [Cancer Res 2007;67(4):1407–10]

Environment-mediated drug resistance in Bcr/Abl-positive acute lymphoblastic leukemia

Niklas Feldhahn,1,† Anna Arutyunyan,1 sonia stoddart,1 Bin Zhang,1,‡ sabine M. schmidhuber,1,§ sun-Ju Yi,1 Yong-mi Kim,2,3 John Groffen1,3,4 and Nora heisterkamp1,3,4,*

1section of Molecular carcinogenesis; Division of hematology/Oncology and The saban Research Institute of children’s hospital; Los Angeles, cA UsA; 2Department of pediatrics; Division of hematology and Oncology; children’s hospital Los Angeles; University of southern california Keck school of Medicine; Los Angeles, cA UsA; 3Leukemia and Lymphoma program; Norris comprehensive cancer center; University of southern california; Leukemia Research program; children’s hospital Los Angeles; Los Angeles, cA UsA; 4Departments of pediatrics and pathology; Keck school of Medicine; University of southern california; Los Angeles, cA UsA

current affiliation: Laboratory of Molecular Immunology; The Rockefeller University; New York, NY UsA; Division of hematology and hematopoietic cell Transplantation; city of hope National Medical center; Duarte, cA UsA; §Laura Bassi centre of expertise; Theraped/Forschungsprogramm für Rezeptorbiochemie und Tumorstoffwechsel; Universitätsklinik für Kinder-und Jugendheilkunde; paracelsus Medizinische privatuniversität; Vienna, Austria

Keywords: stroma, Bcr/Abl, EMDR, innate immunity and cancer, Ph-positive ALL, transgenic mice

Abbreviations: ABL, abelson; ALL, acute lymphoblastic leukemia; BCR, breakpoint cluster region; EMDR, environment-mediated drug resistance; MEF, mouse embryonic fibroblast; Ph, Philadelphia chromosome

Although cure rates for acute lymphoblastic leukemia (ALL) have increased, development of resistance to drugs and patient relapse are common. The environment in which the leukemia cells are present during the drug treatment is known to provide significant survival benefit. here, we have modeled this process by culturing murine Bcr/Abl-positive acute lymphoblastic leukemia cells in the presence of stroma while treating them with a moderate dose of two unrelated drugs, the farnesyltransferase inhibitor lonafarnib and the tyrosine kinase inhibitor nilotinib. This results in an initial large reduction in cell viability of the culture and inhibition of cell proliferation. however, after a number of days, cell death ceases and the culture becomes drug-tolerant, enabling cell division to resume. Using gene expression profiling, we found that the development of drug resistance was accompanied by massive transcriptional upregulation of genes that are associated with general inflammatory responses such as the metalloproteinase MMp9. MMp9 protein levels and enzymatic activity were also increased in ALL cells that had become nilotinib-tolerant. Activation of p38, Akt and erk correlated with the development of environment-mediated drug resistance (eMDR), and inhibitors of Akt and erk in combination with nilotinib reduced the ability of the cells to develop resistance. however, inhibition of p38 promoted increased resistance to nilotinib. We conclude that development of eMDR by ALL cells involves changes in numerous intracellular pathways. Development of tolerance to drugs such as nilotinib may therefore be circumvented by simultaneous treatment with other drugs having divergent targets.

Leukemia (2001) 15, 1232–1239
2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00

www.nature.com/leu

Cell adhesion-mediated drug resistance (CAM-DR) protects the K562 chronic myelogenous leukemia cell line from apoptosis induced by BCR/ABL inhibition, cytotoxic drugs, and 􏰎-irradiation

JS Damiano, LA Hazlehurst and WS Dalton

Department of Interdisciplinary Oncology and Clinical Investigations Program, and H Lee Moffitt Cancer Center and Research Institute, University of South Florida Tampa FL, USA

   

Integrin-mediated cellular adhesion to extracellular matrix (ECM) components is an important determinant of chemothera- peutic response of human myeloma cells. Here, we demon- strate that when K562 chronic myelogenous leukemia (CML) cells are adhered to fibronectin (FN), they become resistant to apoptosis induced by the BCR/ABL inhibitors AG957 and STI- 571, as well as DNA damaging agents and 􏰎-irradiation. This phenomenon, termed cell adhesion-mediated drug resistance (CAM-DR), was induced by adhesion through the 􏰖5􏰗1 (VLA- 5) integrin. Phosphotyrosine analysis demonstrates that anti- apoptotic signaling through integrins in K562 cells is inde- pendent of the tyrosine kinases activated by BCR/ABL, with the possible exception of an unknown 80 kDa protein. Cytoprotec- tion of FN-adhered CML cells indicates that tumor–ECM interac- tions may be critical for the emergence of drug-resistant tumor populations and treatment failure in this disease. Antagonists of 􏰗1 integrin-mediated adhesion or corresponding signal transduction elements may sensitize CML cells to chemo- therapy and prevent resistance to the novel BCR/ABL kinase inhibitors being used for the treatment of this disease. Leuke- mia (2001) 15, 1232–1239.

Keywords: adhesion; integrin; fibronectin; apoptosis; drug resist- ance

The BCR-ABL/NF-κB signal transduction network: a long lasting relationship in Philadelphia positive Leukemias

Giovanna Carrà1, Davide Torti1, Sabrina Crivellaro1, Cristina Panuzzo1, Riccardo Taulli2, Daniela Cilloni1, Angelo Guerrasio1, Giuseppe Saglio1 and Alessandro Morotti1
1 Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy

2 Department of Oncology, University of Turin, Orbassano, Italy Correspondence to: Alessandro Morotti, email: alessandro.morotti@unito.it

Keywords: BCR-ABL; NF-κB; IκB-α; NFKBIA; CML
Received: April 17, 2016 Accepted: August 10, 2016 Published: August 22, 2016

ABSTRACT

The Nuclear Factor-kappa B (NF-κB) family of transcription factors plays a key role in cancer pathogenesis due to the ability to promote cellular proliferation and survival, to induce resistance to chemotherapy and to mediate invasion and metastasis. NF-κB is recruited through different mechanisms involving either canonical (RelA/p50) or non-canonical pathways (RelB/p50 or RelB/p52), which transduce the signals originated from growth-factors, cytokines, oncogenic stress and DNA damage, bacterial and viral products or other stimuli. The pharmacological inhibition of the NF-κB pathway has clearly been associated with significant clinical activity in different cancers. Almost 20 years ago, NF-κB was described as an essential modulator of BCR-ABL signaling in Chronic Myeloid Leukemia and Philadelphia- positive Acute Lymphoblastic Leukemia. This review summarizes the role of NF-κB in BCR-ABL-mediated leukemogenesis and provides new insights on the long lasting BCR-ABL/NF-κB connection.

Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation

Constance P. Soves1, Joshua D. Miller1, Dana L. Begun1, Russell S. Taichman2, Kurt D. Hankenson3, and Steven A. Goldstein1,*
1Orthopaedic Research Laboratories, University of Michigan, Room 2003 Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI USA 48109.

2Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, 1011 North University Ave., Ann Arbor, MI, USA 48109.

3Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine and Department of Orthopaedic Surgery, Perelman School of Medicine University of Pennsylvania, Room 145 Myrin, Kennett Square, PA 19348.

Abstract

Maintenance of bone mass and geometry is influenced by mechanical stimuli. Paradigms suggest that osteocytes embedded within the mineralized matrix and osteoblasts on the bone surfaces are the primary responders to physical forces. However, other cells within the bone marrow cavity, such as megakaryocytes (MKs), are also subject to mechanical forces. Recent studies have highlighted the potent effects of MKs on osteoblast proliferation as well as bone formation in vivo. We hypothesize that MKs are capable of responding to physical forces and that the interactions between these cells and osteoblasts can be influenced by mechanical stimulation.

In this study, we demonstrate that two MK cell lines respond to fluid shear stress in culture. Furthermore, using laser capture microdissection, we isolated MKs from histologic sections of murine tibiae that were exposed to compressive loads in vivo. C-fos, a transcription factor shown to be upregulated in response to load in various tissue types, was increased in MKs from loaded relative to non-loaded limbs at a level comparable to that of osteocytes from the same limbs. We also developed a co-culture system to address whether mechanical stimulation of MKs in culture would impact osteoblast proliferation and differentiation. The presence of MKs in co-culture, but not conditioned media, had dramatic effects on proliferation of preosteoblast MC3T3-E1 cells in culture. Our data suggests a minimal decrease in proliferation as well as an increase in mineralization capacity of osteoblasts co-cultured with MKs exposed to shear compared to co- cultures with unstimulated MKs.

ARTICLE
Received 16 Jul 2015 | Accepted 17 Aug 2015 | Published 24 Sep 2015
DOI: 10.1038/ncomms9388 OPEN

PTEN regulates cilia through Dishevelled

Iryna Shnitsar1, Mikhail Bashkurov1, Glenn R. Masson2, Abiodun A. Ogunjimi1, Sherly Mosessian3,
Eduardo Aguiar Cabeza
1, Calley L. Hirsch1, Daniel Trcka1, Gerald Gish1, Jing Jiao3, Hong Wu3, Rudolf Winklbauer4, Roger L. Williams2, Laurence Pelletier1,5, Jeffrey L. Wrana1,5 & Miriam Barrios-Rodiles1

Cilia are hair-like cellular protrusions important in many aspects of eukaryotic biology. For instance, motile cilia enable fluid movement over epithelial surfaces, while primary (sensory) cilia play roles in cellular signalling. The molecular events underlying cilia dynamics, and particularly their disassembly, are not well understood. Phosphatase and tensin homologue (PTEN) is an extensively studied tumour suppressor, thought to primarily act by antagonizing PI3-kinase signalling. Here we demonstrate that PTEN plays an important role in multicilia formation and cilia disassembly by controlling the phosphorylation of Dishevelled (DVL), another ciliogenesis regulator. DVL is a central component of WNT signalling that plays a role during convergent extension movements, which we show here are also regulated by PTEN. Our studies identify a novel protein substrate for PTEN that couples PTEN to regulation of cilia dynamics and WNT signalling, thus advancing our understanding of potential underlying molecular etiologies of PTEN-related pathologies.

BCR-ABL Promotes PTEN Downregulation in Chronic

Myeloid Leukemia

Cristina Panuzzo, Sabrina Crivellaro, Giovanna Carra`, Angelo Guerrasio, Giuseppe Saglio, Alessandro Morotti*
Department of Clinical and Biological Sciences, San Luigi Hospital, University of Turin, Orbassano, Turin, Italy

Abstract

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the t(9;22) translocation coding for the chimeric protein p210 BCR-ABL. The tumor suppressor PTEN plays a critical role in the pathogenesis of CML chronic phase, through non genomic loss of function mechanisms, such as protein down-regulation and impaired nuclear/cytoplasmic shuttling. Here we demonstrate that BCR-ABL promotes PTEN downregulation through a MEK dependent pathway. Furthermore, we describe a novel not recurrent N212D-PTEN point mutation found in the EM2 blast crisis cell line.

Citation: Panuzzo C, Crivellaro S, Carra` G, Guerrasio A, Saglio G, et al. (2014) BCR-ABL Promotes PTEN Downregulation in Chronic Myeloid Leukemia. PLoS ONE 9(10): e110682. doi:10.1371/journal.pone.0110682

Editor: Persio Dello Sbarba, Universita` degli Studi di Firenze, Italy
Received April 3, 2014; Accepted September 24, 2014; Published October 24, 2014

Copyright: ß 2014 Panuzzo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding: This research was supported by Giovani Ricercatori-Ricerca Finalizzata, ministero della salute 2010 to AM, and AIRC to GS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist. * Email: alessandro.morotti@unito.it

Physiological and Pathological Functions of Mechanosensitive Ion Channels

Yuanzheng Gu and Chen Gu*
Department of Neuroscience, the Ohio State University

Abstract

Rapid sensation of mechanical stimuli is often mediated by mechanosensitve ion channels. Their opening results from conformational changes induced by mechanical forces. It leads to membrane permeation of selected ions and thereby to electrical signaling. Newly identified mechanosensitive ion channels are emerging at an astonishing rate, including some that are traditionally assigned for completely different functions. In this review, we first provide a brief overview of ion channels that are known to play a role in mechanosensation. Next, we focus on three representative ones, including the transient receptor potential channel V4 (TRPV4), Kv1.1 voltage-gated potassium (Kv) channel, and Piezo channels. Their structures, biophysical properties, expression and targeting patterns, and physiological functions are highlighted. The potential role of their mechanosensation in related diseases is further discussed. In sum, mechanosensation appears to be achieved in a variety of ways by different proteins and plays a fundamental role in the function of various organs under normal and abnormal conditions.

RADIATION RESEARCH 186, 175–188 (2016) 0033-7587/16 $15.00
Ó2016 by Radiation Research Society.
All rights of reproduction in any form reserved. DOI: 10.1667/RR14410.1

Hemodynamic Flow-Induced Mechanotransduction Signaling Influences the Radiation Response of the Vascular Endothelium

Mohan Natarajan,a,1 Natarajan Aravindan,c Eugene A. Spragueb and Sumathy Mohana
Departments of
a Pathology and b Medicine, University of Texas Health Science Center, San Antonio, Texas 78229; and c Department of Radiation

Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

Natarajan, M., Aravindan, N., Sprague, E. A. and Mohan, S. Hemodynamic Flow-Induced Mechanotransduction Signal- ing Influences Radiation Response of Vascular Endothelium. Radiat. Res. 186, 175–188 (2016).

Hemodynamic shear stress is defined as the physical force exerted by the continuous flow of blood in the vascular system. Endothelial cells, which line the inner layer of blood vessels, sense this physiological force through mechanotrans- duction signaling and adapt to maintain structural and functional homeostasis. Hemodynamic flow, shear stress and mechanotransduction signaling are, therefore, an integral part of endothelial pathophysiology. Although this is a well- established concept in the cardiovascular field, it is largely dismissed in studies aimed at understanding radiation injury to the endothelium and subsequent cardiovascular complica- tions. We and others have reported on the differential response of the endothelium when the cells are under hemodynamic flow shear compared with static culture. Further, we have demonstrated significant differences in the gene expression of static versus shear-stressed irradiated cells in four key pathways, reinforcing the importance of shear stress in understanding radiation injury of the endothelium. This article further emphasizes the influence of hemodynam- ic shear stress and the associated mechanotransduction signaling on physiological functioning of the vascular endothelium and underscores its significance in understand- ing radiation injury to the vasculature and associated cardiac complications. Studies of radiation effect on endothelial biology and its implication on cardiotoxicity and vascular complications thus far have failed to highlight the significance of these factors. Factoring in these integral parts of the endothelium will enhance our understanding of the contri- bution of the endothelium to radiation biology. Without such information, the current approaches to studying radiation- induced injury to the endothelium and its consequences in health and disease are limited

Selective translocation of intracellular Smoothened

to the primary cilium in response to Hedgehog

pathway modulation

Yu Wanga,b, Zhe Zhouc, Christopher T. Walshc,1, and Andrew P. McMahona,1

aDepartment of Molecular and Cellular Biology and Harvard Stem Cell Institute, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138; bDepartment of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138; and cDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115

Contributed by Christopher T. Walsh, December 3, 2008 (sent for review October 24, 2008)

Smoothened (Smo), a 7-pass transmembrane protein, is essential for transduction of a Hedgehog (Hh) signal across the cell membrane. Smo is also the principle therapeutic target for several candidate drugs in the treatment of Hh-related diseases. Mammalian Smo translocates to the primary cilium in response to Sonic hedgehog (Shh) ligand-mediated signaling. A mechanistic understanding of Smo translocation and its interactions with drug candidates is pivotal to our understanding of Hh signaling and the design, development and application of successful drugs. We established a system in which Smo was dual-labeled with GFP and a 12-aa tag whose recognition by an enzymatic process enables the posttranslational labeling of Smo in the cell membrane within the living cell. These tools enable the simultaneous visualization of all cellular Smo, and more specifically, the cell membrane restricted subpopulation. Using this system, we demonstrate that cyclopamine, a widely used Hh antagonist, induces a cilial translocation of Smo similar to that reported for Shh ligand and several Hh agonists. In contrast, other antagonists abrogate the Shh-induced, cilial translocation of Smo. We present evidence that the majority of cilial-localized Smo originates from an intracellular source and may traffic to the primary cilium through an intraflagellar transport (IFT) pathway.

Production of sound waves by bacterial cells and the response of bacterial cells to sound

Michio Matsuhashi,* Alla N. Pankrushina,3 Satoshi Takeuchi,3 Hideyuki Ohshima, Housaku Miyoi, Katsura Endoh, Ken Murayama, Hiroshi Watanabe, Shigeo Endo, Mikio Tobi, Yoshihiro Mano, Masao Hyodo, Torakichi Kobayashi,1 Tomohiko Kaneko,3 Sugio Otani,2
Susumu Yoshimura,
3 Akira Harata,4,** and Tsuguo Sawada4

Department of Biological Science and Technology, 1Department of Information and Communication Technology, and 2Department of Material Science and Technology, Tokai University, Numazu 410–0321, Japan
3Yoshimura π-Electron Project, ERATO, Japan Science and Technology Corporation, c/o Matsushita Research Institute Tokyo, Inc., Higashimita, Tama-ku, Kawasaki 214–0033, Japan
4Department of Industrial Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113–0033, Japan

(Received October 27, 1997; Accepted February 18, 1998)

Bacterial cells enhance the proliferation of neighboring cells under stress conditions by emitting a physical signal. Continuous single sine sound waves produced by a speaker at frequencies of 6–10, 18–22, and 28–38 kHz promoted colony formation by Bacillus carboniphilus under non-per- missive stress conditions of high KCl concentration and high temperature. Furthermore, sound waves emitted from cells of Bacillus subtilis at frequencies between 8 and 43 kHz with broad peaks at approximately 8.5, 19, 29, and 37 kHz were detected using a sensitive microphone system. The similarity between the frequency of the sound produced by B. subtilis and the frequencies that in- duced a response in B. carboniphilus and the previously observed growth-promoting effect of B. subtilis cells upon B. carboniphilus through iron barriers, suggest that the detected sound waves function as a growth-regulatory signal between cells.

Hindawi Publishing Corporation BioMed Research International
Volume 2014, Article ID 273932, 6 pages http://dx.doi.org/10.1155/2014/273932

Research Article

Fetus Sound Stimulation: Cilia Memristor Effect of Signal Transduction

Svetlana Jankovic-Raznatovic,1,2 Svetlana Dragojevic-Dikic,1,2 Snezana Rakic,1,2 Branka Nikolic,1,2 Snezana Plesinac,2,3 Lidija Tasic,1,2 Zivko Perisic,1,2 Mirjana Sovilj,4,5 Tatjana Adamovic,4,5 and Djuro Koruga6

1 Department of Obstetrics and Gynecology “Narodni front”, Kraljice Natalije Street 62, 11000 Belgrade, Serbia
2 Belgrade University Medical School, Doktora Subotica Street 8, 11000 Belgrade, Serbia
3 Institute for Obstetrics and Gynecology, Clinical Center of Serbia, Majke Jevrosime 8, 11000 Belgrade, Serbia
4 Institute for Experimental Phonetics and Speech Pathology, Gospodar Jovanova Street 35, 11000 Belgrade, Serbia 5 Life Activities Advancement Center, Gospodar Jovanova Street 35, 11000 Belgrade, Serbia

6 Biomedical engineering, Faculty of Mechanical Engineering, University of Belgrade, Kraljice Marije Street 8, 11000 Belgrade, Serbia

Correspondence should be addressed to Svetlana Jankovic-Raznatovic; svetlana.jr@gmail.com
Received 24 November 2013; Revised 16 January 2014; Accepted 19 January 2014; Published 26 February 2014 Academic Editor: Irma Virant-Klun

Copyright © 2014 Svetlana Jankovic-Raznatovic et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. This experimental study evaluates fetal middle cerebral artery (MCA) circulation after the defined prenatal acoustical stimulation (PAS) and the role of cilia in hearing and memory and could explain signal transduction and memory according to cilia optical-acoustical properties. Methods. PAS was performed twice on 119 no-risk term pregnancies. We analyzed fetal MCA circulation before, after first and second PAS. Results. Analysis of the Pulsatility index basic (PIB) and before PAS and Pulsatility index reactive after the first PAS (PIR 1) shows high statistical difference, representing high influence on the brain circulation. Analysis of PIB and Pulsatility index reactive after the second PAS (PIR 2) shows no statistical difference. Cilia as nanoscale structure possess magnetic flux linkage that depends on the amount of charge that has passed between two-terminal variable resistors of cilia. Microtubule resistance, as a function of the current through and voltage across the structure, leads to appearance of cilia memory with the “memristor” property. Conclusion. Acoustical and optical cilia properties play crucial role in hearing and memory processes. We suggest that fetuses are getting used to sound, developing a kind of memory patterns, considering acoustical and electromagnetically waves and involving cilia and microtubules and try to explain signal transduction.

Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine Volume 2016, Article ID 6849473, 7 pages http://dx.doi.org/10.1155/2016/6849473

Research Article

Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture

Nathalia R. Lestard and Marcia A. M. Capella

Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil

Correspondence should be addressed to Marcia A. M. Capella; mcapella@biof.ufrj.br Received 20 January 2016; Accepted 19 June 2016
Academic Editor: Annarita Stringaro

Copyright © 2016 N. R. Lestard and M. A. M. Capella. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 10, Issue of March 8, pp. 8076–8082, 2002

© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional Cooperation among Ras, STAT5, and Phosphatidylinositol 3-Kinase Is Required for Full Oncogenic Activities of BCR/ABL in K562 Cells*

Printed in U.S.A.

Received for publication, December 3, 2001 Published, JBC Papers in Press, January 4, 2002, DOI 10.1074/jbc.M111501200

Junko Sonoyama‡, Itaru Matsumura‡, Sachiko Ezoe‡, Yusuke Satoh‡, Xian Zhang‡, Yoshihisa Kataoka‡, Emi Takai‡, Masao Mizuki‡, Takashi Machii‡, Hiroshi Wakao§, and Yuzuru Kanakura‡

From the Department of Hematology/Oncology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 and the §Helix Research Institute, 1532-3 Yana Kisarazu-shi, Chiba 292-0812, Japan

BCR/ABL tyrosine kinase generated from the chromo- somal translocation t(9;22) causes chronic myelogenous leukemia and acute lymphoblastic leukemia. To exam- ine the roles of BCR/ABL-activated individual signaling molecules and their cooperation in leukemogenesis, we inducibly expressed a dominant negative (DN) form of Ras, phosphatidylinositol 3-kinase, and STAT5 alone or in combination in p210 BCR/ABL-positive K562 cells. The inducibly expressed DN Ras (N17), STAT5 (694F), and DN phosphatidylinositol 3-kinase (􏰠p85) inhibited the growth by 90, 55, and 40%, respectively. During the growth inhibition, the expression of cyclin D2 and cyclin D3 was suppressed by N17, 694F, or 􏰠p85; that of cyclin E by N17; and that of cyclin A by 􏰠p85. In addition, N17 induced apoptosis in a small proportion of K562, whereas 694F and 􏰠p85 were hardly effective. In con- trast, coexpression of two DN mutants in any combina- tions induced severe apoptosis. During these cultures, the expression of Bcl-2 was suppressed by N17, 694F, or 􏰠p85, and that of Bcl-XL by N17. Furthermore, although K562 was resistant to interferon-􏰖- and dexamethasone- induced apoptosis, disruption of one pathway by N17, 694F, or 􏰠p85 sensitized K562 to these reagents. These results suggested that cooperation among these mole- cules is required for full leukemogenic activities of BCR/ABL.

RESEARCH PAPER
A role for transient receptor potential vanilloid 4 in

tonicity-induced neurogenic inflammationbph_590 1161..1173
N Vergnolle
1,2,3, N Cenac1,2,3, C Altier4, L Cellars3, K Chapman3, GW Zamponi4, S Materazzi5,

R Nassini5, W Liedtke6, F Cattaruzza7, EF Grady7, P Geppetti8 and NW Bunnett7

1INSERM, U563, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France, 2Université Toulouse III Paul Sabatier, Toulouse, France, 3Department of Pharmacology and Therapeutics and 4Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada, 5Department of Experimental and Clinical Medicine, Via Fossato di Mortara 19, Ferrara, Italy, 6Departments of Medicine and Neurobiology, Duke University Medical Center 2900, Durham, NC, USA, 7Departments of Surgery and Physiology, University of California, San Francisco, CA, USA, and 8Clinical Pharmacology Unit, Department of Critical Care Medicine and Surgery, University of Florence, Florence, Italy

Background and purpose: Changes in extracellular fluid osmolarity, which occur after tissue damage and disease, cause inflammation and maintain chronic inflammatory states by unknown mechanisms. Here, we investigated whether the osmosensitive channel, transient receptor potential vanilloid 4 (TRPV4), mediates inflammation to hypotonic stimuli by a neurogenic mechanism.

Experimental approach: TRPV4 was localized in dorsal root ganglia (DRG) by immunofluorescence. The effects of TRPV4 agonists on release of pro-inflammatory neuropeptides from peripheral tissues and on inflammation were examined.
Key results: Immunoreactive TRPV4 was detected in DRG neurones innervating the mouse hindpaw, where it was co-expressed in some neurones with CGRP and substance P, mediators of neurogenic inflammation. Hypotonic solutions and 4a-phorbol 12,13-didecanoate, which activate TRPV4, stimulated neuropeptide release in urinary bladder and airways, sites of neurogenic inflammation. Intraplantar injection of hypotonic solutions and 4a-phorbol 12,13-didecanoate caused oedema and granulocyte recruitment. These effects were inhibited by a desensitizing dose of the neurotoxin capsaicin, antagonists of CGRP and substance P receptors, and TRPV4 gene knockdown or deletion. In contrast, antagonism of neuropeptide receptors and disruption of TRPV4 did not prevent this oedema. TRPV4 gene knockdown or deletion also markedly reduced oedema and granulocyte infiltration induced by intraplantar injection of formalin.

Conclusions and implications: Activation of TRPV4 stimulates neuropeptide release from afferent nerves and induces neu- rogenic inflammation. This mechanism may mediate the generation and maintenance of inflammation after injury and during diseases, in which there are changes in extracellular osmolarity. Antagonism of TRPV4 may offer a therapeutic approach for inflammatory hyperalgesia and chronic inflammation.

British Journal of Pharmacology (2010) 159, 1161–1173; doi:10.1111/j.1476-5381.2009.00590.x; published online 5 February 2010

Behavioral/Systems/Cognitive

A Transient Receptor Potential Vanilloid 4-Dependent Mechanism of Hyperalgesia Is Engaged by Concerted Action of Inflammatory Mediators

Nicole Alessandri-Haber, Olayinka A. Dina, Elizabeth K. Joseph, David Reichling, and Jon D. Levine

Department of Oral and Maxillofacial Surgery and Division of Neurosciences, University of California, San Francisco, San Francisco, California 94143-0440

The transient receptor potential vanilloid 4 (TRPV4) is a primary afferent transducer that plays a crucial role in neuropathic hyperalgesia for osmotic and mechanical stimuli, as well as in inflammatory mediator-induced hyperalgesia for osmotic stimuli. In view of the clinical importance of mechanical hyperalgesia in inflammatory states, the present study investigated the role of TRPV4 in mechanical hyperal- gesia induced by inflammatory mediators and the second-messenger pathways involved. Intradermal injection of either the inflammo- gen carrageenan or a soup of inflammatory mediators enhanced the nocifensive paw-withdrawal reflex elicited by hypotonic or mechan- ical stimuli in rat. Spinal administration of TRPV4 antisense oligodeoxynucleotide blocked the enhancement without altering baseline nociceptive threshold. Similarly, in TRPV4􏰑/􏰑 knock-out mice, inflammatory soup failed to induce any significant mechanical or os- motic hyperalgesia. In vitro investigation showed that inflammatory mediators engage the TRPV4-mediated mechanism of sensitization by direct action on dissociated primary afferent neurons. Additional behavioral observations suggested that multiple mediators are necessary to achieve sufficient activation of the cAMP pathway to engage the TRPV4-dependent mechanism of hyperalgesia. In addition, direct activation of protein kinase A or protein kinase C 􏰧, two pathways that mediate inflammation-induced mechanical hyperalgesia, also induced hyperalgesia for both hypotonic and mechanical stimuli that was decreased by TRPV4 antisense and absent in TRPV4􏰑/􏰑 mice. We conclude that TRPV4 plays a crucial role in the mechanical hyperalgesia that is generated by the concerted action of inflamma- tory mediators present in inflamed tissues.

Radiation sensitization of tumor cells induced by shear stress: The roles of integrins and FAK

Chi-Wen Luo a, Chia-Ching Wu b, Hui-Ju Ch’ang a,c,

a NationalInstituteofCancerResearch,NationalHealthResearchInstitutes,Tainan,Taiwan
b DepartmentofCellBiologyandAnatomy,CollegeofMedicine,NationalChengKungUniversity,Tainan,Taiwan c DepartmentofRadiationOncology,NationalChengKungU

Recent studies revealed that the interstitial fluid flow in and around tumor tissue not only played an important role in delivering anticancer agents, but also affected the microenvironment, mostly hypoxia, in modulating tumor radio-sensitivity. The current study investigated the hypoxia-independent mechanisms of flow-induced shear stress in sensitizing tumors to radiation.

Colon cancer cells were seeded onto glass slides pre-coated with fibronectin. A parallel-plate flow chamber system was used to impose fluid shear stress. Cell proliferation, apoptosis and colony assays were measured after shear stress and/or radiation. Cell cycle analysis and immunoblots of cell adhesion signal molecules were evaluated. The effect of shear stress was reversed by modulating integrin β1 or FAK.

Shear stress of 12 dyne/cm2 for 24 h, but not 3 h, enhanced the radiation induced cytotoxicity to colon cancer cells. Protein expression of FAK was significantly down-regulated but not transcriptionally suppressed. By mod- ulating integrin β1 and FAK expression, we demonstrated that shear stress enhanced tumor radiosensitivity by regulating integrin β1/FAK/Akt as well as integrin β1/FAK/cortactin pathways. Shear stress in combination with radiation might regulate integrins signaling by recruiting and activating caspases 3/8 for FAK cleavage followed by ubiquitin-mediated proteasomal degradation.

Shear stress enhanced the radiation toxicity to colon cancer cells through suppression of integrin signaling and protein degradation of FAK. The results of our study provide a strong rationale for cancer treatment that combines between radiation and strategy in modulating tumor interstitial fluid flow.

Role of TRP ion channels in cancer and tumorigenesis George Shapovalov1,2 & Abigael Ritaine1,2 & Roman Skryma1,2 & Natalia Prevarskaya1,2

Received: 1 September 2015 / Accepted: 3 September 2015 / Published online: 3 February 2016 # Springer-Verlag Berlin Heidelberg 2016

                   

Abstract Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has cap- tured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca2+ distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca2+ homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

Tiffany C. Ho, Natalie A. Horn, Tuan Huynh, Lucy Kelava, and Jeffry B. Lansman* Department of Cellular and Molecular Pharmacology; School of Medicine; University of California, San Francisco; San Francisco, CA USA

Keywords: mechanosensitive channel, skeletal muscle, muscular dystrophy, TRPV4, calcium

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave kon = 13 x 106 M-1s-1 and koff = 1609 sec-1 with KD = ~124 μM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a KD = ~49 μM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.

ARTICLE

DOI: 10.1038/s41467-018-06753-6 OPEN

Structural insights on TRPV5 gating by endogenous modulators

Taylor E.T. Hughes 1, Ruth A. Pumroy 1, Aysenur Torun Yazici 2, Marina A. Kasimova3, Edwin C. Fluck 1, Kevin W. Huynh4, Amrita Samanta1, Sudheer K. Molugu1, Z. Hong Zhou4, Vincenzo Carnevale 3,
Tibor Rohacs
2 & Vera Y. Moiseenkova-Bell 1

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidyli- nositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron micro- scopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrange- ments in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery.

Apoptosis Induction of Human Myeloid Leukemic Cells by Ultrasound Exposure1

Hagit Ashush, Leon A. Rozenszajn,2 Michal Blass, Mira Barda-Saad, Damir Azimov, Judith Radnay, Dov Zipori, and Uri Rosenschein

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel [H. A., L. A. R., M. B., M. B-S., D. A., J. R.]; Department of Molecular Cell Biology, Weizmann Institute of Sciences, Rehovot, Israel [D. Z.]; and Tel Aviv Medical Center, Tel Aviv University, Tel Aviv, Israel [U. R.]

ABSTRACT

Therapeutic ultrasound (ULS) and the resulting cavitation process has

been shown to induce irreversible cell damage. In this study, we wanted to

further investigate the mechanism of ULS-induced cell death and to

determine whether apoptosis is involved. High intensity focused pulsed

ULS sonication at a frequency of 750 KHz was delivered to HL-60, K562,

U937, and M1/2 leukemia cell line cultures. ULS exposure used with

induction of transient cavitation in the focal area was delivered with an

intensity level of 103.7 W/cm2 and 54.6 W/cm2 spatial-peak temporal-

average intensity. As a control, ULS of lower intensity was delivered at

2
22.4 W/cm spatial-peak temporal-average intensity, presumably without

generation of cavitation. Our results indicated that DNA damage induced by ULS cavitation did not involve generation of free radicals in the culture media. Morphological alterations observed in cells after exposure to ULS included: cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation, and apoptotic body formation. Apoptotic cells were evaluated by fluorescence microscopy and detected using the termi- nal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, which identifies DNA breaks, and by the leakage of phosphatidylserine from the inner to the outer side of the membrane layer of treated cells. Some bioeffects induced on sonicated HL-60 cells, such as inhibition of cell proliferation, DNA repair, and cell-dependent apoptosis, were found to be similar to those produced by 􏰎-irradiation. Thus, much of the cell damage induced by therapeutic ULS in leukemia cells surviving ULS exposure appears to occur through an apoptotic mechanism.

Primary Cilia and the Cell Cycle
Olga V. Plotnikova
1,2, Elena N. Pugacheva3, and Erica A. Golemis1,*

1 Program in Molecular and Translational Medicine, Fox Chase Cancer Center, Philadelphia, PA 19111

2 Department of Molecular Biology and Medical Biotechnology, Russian State Medical University, Moscow, Russia

3 Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506

Abstract

Cilia are microtubule-based structures that protrude from the cell surface, and function as sensors for mechanical and chemical environmental cues that regulate cellular differentiation or division. In metazoans, ciliary signaling is important both during organismal development and in the homeostasis controls of adult tissues, with receptors for the Hedgehog, PDGF, Wnt, and other signaling cascades arrayed and active along the ciliary membrane. In normal cells, cilia are dynamically regulated during cell cycle progression: present in G0 and G1 cells, and usually in S/G2 cells, but almost invariably resorbed before mitotic entry, to re-appear post-cytokinesis. This periodic resorption and reassembly of cilia, specified by interaction with the intrinsic cell cycle machinery, influences the susceptibility of cells to the influence of extrinsic signals with cilia-associated receptors. Pathogenic conditions of mammals associated with loss of or defects in ciliary integrity include a number of developmental disorders, cystic syndromes in adults, and some cancers. With the continuing expansion of the list of human diseases associated with ciliary abnormalities, the identification of the cellular mechanisms regulating ciliary growth and disassembly has become a topic of intense research interest. Although these mechanisms are far from being understood, a number of recent studies have begun to identify key regulatory factors that may begin to offer insight into disease pathogenesis and treatment. In this chapter we will discuss the current state of knowledge regarding cell cycle control of ciliary dynamics, and provide general methods that can be applied to investigate cell cycle-dependent ciliary growth and disassembly.

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