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1.
Oncogene ; 32(15): 1876-87, 2013 Apr 11.
Article in English | MEDLINE | ID: mdl-22710718

ABSTRACT

Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood cancer of striated muscle characterized by the presence of the PAX3-FOXO1A or PAX7-FOXO1A chimeric oncogenic transcription factor. Identification of their targets is essential for understanding ARMS pathogenesis. To this aim, we analyzed transcriptomic data from rhabdomyosarcoma samples and found that P-cadherin expression is correlated with PAX3/7-FOXO1A presence. We then show that expression of a PAX3 dominant negative variant inhibits P-cadherin expression in ARMS cells. Using mouse models carrying modified Pax3 alleles, we demonstrate that P-cadherin is expressed in the dermomyotome and lies genetically downstream from the myogenic factor Pax3. Moreover, in vitro gel shift analysis and chromatin immunoprecipitation indicate that the P-cadherin gene is a direct transcriptional target for PAX3/7-FOXO1A. Finally, P-cadherin expression in normal myoblasts inhibits myogenesis and induces myoblast transformation, migration and invasion. Conversely, P-cadherin downregulation by small hairpin RNA decreases the transformation, migration and invasive potential of ARMS cells. P-cadherin also favors cadherin switching, which is a hallmark of metastatic progression, by controlling N- and M-cadherin expression and/or localization. Our findings demonstrate that P-cadherin is a direct PAX3-FOXO1A transcriptional target involved in ARMS aggressiveness. Therefore, P-cadherin emerges as a new and attractive target for therapeutic intervention in ARMS.


Subject(s)
Cadherins/metabolism , Forkhead Transcription Factors/metabolism , Paired Box Transcription Factors/metabolism , Rhabdomyosarcoma, Alveolar/metabolism , Animals , Base Sequence , Cadherins/genetics , Cell Movement/genetics , Cell Transformation, Neoplastic/genetics , Forkhead Box Protein O1 , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Transgenic , Neoplasm Invasiveness/genetics , PAX3 Transcription Factor , PAX7 Transcription Factor/metabolism , Paired Box Transcription Factors/genetics , RNA Interference , RNA, Small Interfering , Rhabdomyosarcoma, Alveolar/pathology , Sequence Alignment , Transcription, Genetic
2.
Cell Death Differ ; 15(8): 1221-31, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18369372

ABSTRACT

Differentiation of skeletal myoblasts into multinucleated myotubes is a multi-step process orchestrated by several signaling pathways. The Rho small G protein family plays critical roles both during myogenesis induction and myoblast fusion. We report here that in C2C12 myoblasts, expression of RhoE, an atypical member of this family, increases until the onset of myoblast fusion before resuming its basal level once fusion has occurred. We show that RhoE accumulates in elongated, aligned myoblasts prior to fusion and that its expression is also increased during injury-induced skeletal muscle regeneration. Moreover, although RhoE is not required for myogenesis induction, it is essential for myoblast elongation and alignment before fusion and for M-cadherin expression and accumulation at the cell-cell contact sites. Myoblasts lacking RhoE present with defective p190RhoGAP activation and RhoA inhibition at the onset of myoblast fusion. RhoE interacts also with the RhoA effector Rho-associated kinase (ROCK)I whose activity must be downregulated to allow myoblast fusion. Consistently, we show that pharmacological inactivation of RhoA or ROCK restores myoblast fusion in RhoE-deficient myoblasts. RhoE physiological upregulation before myoblast fusion is responsible for the decrease in RhoA and ROCKI activities, which are required for the fusion process. Therefore, we conclude that RhoE is an essential regulator of myoblast fusion.


Subject(s)
Myoblasts/metabolism , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Cell Differentiation , Cell Fusion , Cell Line , Cell Shape , Down-Regulation , GTPase-Activating Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron, Scanning , Muscle Fibers, Skeletal/cytology , Myoblasts/cytology , Myoblasts/ultrastructure , Signal Transduction , Up-Regulation
3.
J Biol Chem ; 275(46): 36457-64, 2000 Nov 17.
Article in English | MEDLINE | ID: mdl-10967094

ABSTRACT

GTPases of the Rho family control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation, and apoptosis. We report here the characterization of a new Rho member, which shares 85% and 78% amino acid similarity to TC10 and Cdc42, respectively. This GTPase, termed as TC10-like (TCL) is encoded by an unexpectedly large locus, made of five exons spanning over 85 kilobases on human chromosome 14. TCL mRNA is 2.5 kilobases long and is mainly expressed in heart. In vitro, TCL shows rapid GDP/GTP exchange and displays higher GTP dissociation and hydolysis rates than TC10. Using the yeast two-hybrid system and GST pull-down assays, we show that GTP-bound but not GDP-bound TCL protein directly interacts with Cdc42/Rac interacting binding domains, such as those found in PAK and WASP. Despite its overall similarity to TC10 and Cdc42, the constitutively active TCL mutant displays distinct morphogenic activity in REF-52 fibroblasts, producing large and dynamic F-actin-rich ruffles on the dorsal cell membrane. Interestingly, TCL morphogenic activity is blocked by dominant negative Rac1 and Cdc42 mutants, suggesting a cross-talk between these three Rho GTPases.


Subject(s)
GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/metabolism , cdc42 GTP-Binding Protein/chemistry , rho GTP-Binding Proteins/chemistry , rho GTP-Binding Proteins/metabolism , Actins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Size , Cytoskeleton/metabolism , GTP Phosphohydrolases/genetics , Humans , Immunohistochemistry , Mice , Microscopy, Electron, Scanning , Molecular Sequence Data , Mutation/genetics , Organ Specificity , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Proteins/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , Rats , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Two-Hybrid System Techniques , Wiskott-Aldrich Syndrome Protein , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases , rho GTP-Binding Proteins/genetics
4.
J Biol Chem ; 275(42): 33046-52, 2000 Oct 20.
Article in English | MEDLINE | ID: mdl-10900195

ABSTRACT

In this study we show that expression of active Cdc42Hs and Rac1 GTPases, two Rho family members, leads to the reorganization of the vimentin intermediate filament (IF) network, showing a perinuclear collapse. Cdc42Hs displays a stronger effect than Rac1 as 90% versus 75% of GTPase-expressing cells show vimentin collapse. Similar vimentin IF modifications were observed when endogenous Cdc42Hs was activated by bradykinin treatment, endogenous Rac1 by platelet-derived growth factor/epidermal growth factor, or both endogenous proteins upon expression of active RhoG. This reorganization of the vimentin IF network is not associated with any significant increase in soluble vimentin. Using effector loop mutants of Cdc42Hs and Rac1, we show that the vimentin collapse is mostly independent of CRIB (Cdc42Hs or Rac-interacting binding)-mediated pathways such as JNK or PAK activation but is associated with actin reorganization. This does not result from F-actin depolymerization, because cytochalasin D treatment or Scar-WA expression have merely no effect on vimentin organization. Finally, we show that genistein treatment of Cdc42 and Rac1-expressing cells strongly reduces vimentin collapse, whereas staurosporin, wortmannin, LY-294002, R(p)-cAMP, or RII, the regulatory subunit of protein kinase A, remain ineffective. Moreover, we detected an increase in cellular tyrosine phosphorylation content after Cdc42Hs and Rac1 expression without modification of the vimentin phosphorylation status. These data indicate that Cdc42Hs and Rac1 GTPases control vimentin IF organization involving tyrosine phosphorylation events.


Subject(s)
Actins/metabolism , Intermediate Filaments/physiology , Vimentin/metabolism , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Cell Line , Embryo, Mammalian , Fibroblasts/physiology , Humans , Intermediate Filaments/ultrastructure , Phosphorylation , Phosphotyrosine/metabolism , Rats , Recombinant Proteins/metabolism , Transfection
5.
Sci Total Environ ; 233(1-3): 47-56, 1999 Aug 15.
Article in English | MEDLINE | ID: mdl-10492897

ABSTRACT

In order to characterize the estrogenic activity of chemicals, we established complementary in vitro recombinant receptor-reporter gene assays in stably transfected MCF-7 and HeLa cells. MCF-7 cells which express the endogenous estrogen receptor alpha (ER alpha) were stably transfected with only an estrogen-regulated luciferase gene. These cells enable the detection of compounds which bind to ER alpha or interfere with the induction of ER alpha mediated gene expression. Furthermore, HeLa cells, which do not express endogenous ERs, were transfected with an ER alpha or an ER beta construct together with an estrogen-regulated luciferase gene, or a chimeric GAL4-ER alpha receptor and the corresponding luciferase reporter gene. Finally, we tested these four cellular models as tools to check the estrogenic activities of several potential xenoestrogens and to detect estrogenic activity in wastewater sewage treatment effluents. In all of the models, nonylphenol mixture (NPm), 4n-nonylphenol (4nNP), 2,4'-DDE, 4,4'-DDE and wastewater sewage treatment effluent were active, while PCB mixture (Aroclor 1254), PCB 77, atrazine and lindane (gamma hexachlorocyclohexane) were inactive. Dioxin partially activates the estrogen receptor in MCF-7 cells while in HeLa-derived cell lines, it decreased the estrogenic-induced expression of luciferase.


Subject(s)
Environmental Monitoring/methods , Estrogens, Non-Steroidal/toxicity , Receptors, Estrogen/drug effects , Water Pollutants, Chemical/toxicity , Cell Line , Dichlorodiphenyl Dichloroethylene/toxicity , Dioxins/toxicity , Estrogen Receptor alpha , Estrogen Receptor beta , Genes, Reporter , HeLa Cells , Humans , Luciferases/genetics , Mitotane/analogs & derivatives , Mitotane/toxicity , Phenols/toxicity , Receptors, Estrogen/genetics , Sewage/adverse effects , Transfection
6.
Ann Vasc Surg ; 3(4): 313-7, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2597616

ABSTRACT

Carotid artery stump pressure was measured in 84 cases of carotid endarterectomy. The operations were performed in 71 patients over a period of five years. An altered neurological status during temporary occlusion of the carotid artery, assessed with the patient under local anesthesia, was the sole criterion for shunt placement. Stump pressure (SP) was significantly higher in the 69 unshunted cases (mean of 53.3 mmHg) than the 15 shunted cases (mean of 34.2 mmHg). Shunt was required in two of 41 cases (5%) with SP of greater than 50 mmHg, eight of 36 cases (22%) with SP of 25-50 mmHg, five of seven cases (71%) with SP of less than 25 mmHg. The clinical presentation, including history of prior stroke, and the presence of contralateral disease (including complete carotid occlusion), did not influence the need for a shunt. In this series, carotid artery stump pressure has greater predictive value for shunt requirement when it is greater than 50 mmHg or less than 25 mmHg. However, monitoring the neurological status of the patient in the awake state is still the most reliable method of determining shunt requirement. In our experience, this is associated with minimal morbidity and no mortality.


Subject(s)
Blood Pressure , Carotid Arteries/physiopathology , Cerebrovascular Circulation , Endarterectomy/methods , Aged , Aged, 80 and over , Carotid Arteries/surgery , Carotid Artery Diseases/physiopathology , Carotid Artery Diseases/surgery , Female , Humans , Male , Middle Aged , Neurologic Examination , Predictive Value of Tests , Prospective Studies
7.
Am J Obstet Gynecol ; 159(5): 1181-3, 1988 Nov.
Article in English | MEDLINE | ID: mdl-3056003

ABSTRACT

Management of a patient with cystic fibrosis who started her pregnancy in a state of pulmonary insufficiency, as defined by arterial blood gas values and a chest x-ray film, is described. A systematic approach to evaluation of patients with cystic fibrosis, including serial assessments of pulmonary functions, screening for diabetes, maintenance of adequate nutrition (including hyperalimentation), and cardiac function evaluation is described.


Subject(s)
Cystic Fibrosis/complications , Pregnancy Complications , Respiratory Insufficiency/etiology , Adult , Cesarean Section , Cystic Fibrosis/therapy , Female , Humans , Insulin/therapeutic use , Nutrition Disorders/etiology , Nutrition Disorders/therapy , Pregnancy , Pregnancy in Diabetics/complications , Pregnancy in Diabetics/drug therapy , Respiratory Function Tests , Respiratory Insufficiency/physiopathology
8.
Can Anaesth Soc J ; 30(3 Pt 1): 290-4, 1983 May.
Article in English | MEDLINE | ID: mdl-6336551

ABSTRACT

A previously healthy 18-year-old male, following appendectomy developed post-anaesthetic hyperthermia (42.1 degrees C) with an elevation of serum creatine kinase and activated partial thromboplastin time. Repeated arterial blood gases were normal. Cooling and anti-pyretic medication did not control the fever. In contrast, sodium dantrolene appeared effective in lowering the patient's temperature and normalizing the vital signs, both acutely and over the following three days. Subsequent muscle biopsy revealed a normal contracture response to caffeine alone or in the presence of halothane. However, the muscle had a larger than normal potentiation of evoked twitch tension in the presence of caffeine and halothane. Electrophoresis of the muscle revealed a marked increase of an unidentified low molecular weight protein. The patient's clinical course, and the results of the muscle studies, suggest that an abnormality of skeletal muscle.


Subject(s)
Anesthesia/adverse effects , Fever/diagnosis , Malignant Hyperthermia/diagnosis , Muscular Diseases/complications , Adolescent , Diagnosis, Differential , Fever/etiology , Humans , Male
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