{"id":114659,"date":"2018-03-11T10:42:46","date_gmt":"2018-03-11T10:42:46","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/estudio-del-proceso-de-sensibilidad-colateral-en-celulas-leucemicas-murinas-y-humanas-con-fenotipo-de-resistencia-a-multiples-farmacos-mdr\/"},"modified":"2018-03-11T10:42:46","modified_gmt":"2018-03-11T10:42:46","slug":"estudio-del-proceso-de-sensibilidad-colateral-en-celulas-leucemicas-murinas-y-humanas-con-fenotipo-de-resistencia-a-multiples-farmacos-mdr","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/oncologia\/estudio-del-proceso-de-sensibilidad-colateral-en-celulas-leucemicas-murinas-y-humanas-con-fenotipo-de-resistencia-a-multiples-farmacos-mdr\/","title":{"rendered":"Estudio del proceso de sensibilidad colateral en celulas leucemicas murinas y humanas con fenotipo de resistencia a multiples farmacos (mdr)"},"content":{"rendered":"<h2>Tesis doctoral de <strong> David Cerezo Fern\u00e1ndez <\/strong><\/h2>\n<p>T\u00edtulo: estudio del proceso de sensibilidad colateral en c\u00e9lulas leuc\u00e9micas murinas y humanas con fenotipo de resistencia a m\u00faltiples f\u00e1rmacos (mdr) autor: david cerezo fern\u00e1ndez introducci\u00f3n: la adquisici\u00f3n del fenotipo de resistencia a m\u00faltiples f\u00e1rmacos (mdr) en c\u00e9lulas tumorales las convierte en resistentes a f\u00e1rmacos antineopl\u00e1sicos y es una de las principales causas del fracaso de la quimioterapia en algunos tumores. La expresi\u00f3n de glicoprote\u00edna p (mdr-1, p-gp, abcb1) contribuye a esta resistencia expulsando los f\u00e1rmacos o regulando la muerte celular programada. Por otro lado, la sobreexpresi\u00f3n de miembros anti-apopt\u00f3ticos de la familia bcl-2 observada en tumores sangu\u00edneos se ha asociado a la resistencia a quimioterapia en varios c\u00e1nceres humanos. Tambi\u00e9n es importante estudiar alteraciones en las rutas de se\u00f1alizaci\u00f3n de mapks y la secreci\u00f3n de citocinas en las c\u00e9lulas con fenotipo mdr. objetivos: el prop\u00f3sito de este trabajo fue encontrar un est\u00edmulo capaz de inducir sensibilidad colateral en c\u00e9lulas leuc\u00e9micas murinas y humanas con fenotipo mdr, y caracterizar el tipo de muerte celular inducida. El segundo prop\u00f3sito fue estudiar la contribuci\u00f3n de mdr-1, caspasas, prote\u00ednas de la familia bcl-2, mapks y citocinas al proceso de sensibilidad colateral. materiales y m\u00e9todos: para alcanzar nuestros objetivos, usamos c\u00e9lulas leuc\u00e9micas murinas l1210 y su subl\u00ednea celular resistente l1210r, as\u00ed como una l\u00ednea celular procedente de la l\u00ednea l1210 transfectada con un pl\u00e1smido que conten\u00eda el gen mdr-1 (cbmc-6). Se realiz\u00f3 western-blot para estudiar la expresi\u00f3n de prote\u00ednas, inhibici\u00f3n de mapks y caspasas y silenciamiento de arn de mdr-1, bcl-xl, bcl-2 y bax para estudiar el papel de estas prote\u00ednas. resultados: se encontr\u00f3 que las c\u00e9lulas leuc\u00e9micas resistentes con sobreexpresi\u00f3n de p-gp, pero no sus parentales sensibles, son hipersensibles a estr\u00e9s hipot\u00e9rmico produci\u00e9ndose apoptosis a temperaturas por debajo de 4\u00c2\u00bac. La transfecci\u00f3n de la l\u00ednea celular parental con un pl\u00e1smido que contiene p-gp las convierte en sensibles a estr\u00e9s hipot\u00e9rmico, demostrando la asociaci\u00f3n entre la expresi\u00f3n de p-gp y la muerte celular provocada por el fr\u00edo. Tambi\u00e9n observamos un incremento de la expresi\u00f3n basal y actividad del fragmento activo de caspasa 3 a temperatura fisiol\u00f3gica (37\u00c2\u00bac) en las c\u00e9lulas mdr. La inhibici\u00f3n de caspasa 3 rescat\u00f3 parcialmente a las c\u00e9lulas leuc\u00e9micas mdr de la apoptosis inducida por el fr\u00edo, lo que sugiere que el mecanismo de muerte celular depende de caspasa 3. Esto demuestra que la expresi\u00f3n de p-gp juega un papel importante en la supervivencia de las c\u00e9lulas mdr y es acompa\u00f1ada por un proceso de sensibilidad colateral a estr\u00e9s hipot\u00e9rmico. se observ\u00f3 que la l\u00ednea parental leuc\u00e9mica (l1210) y la l\u00ednea celular cbmc-6 presentan una alta expresi\u00f3n de la prote\u00edna anti-apopt\u00f3tica bcl-xl, mientras que la expresi\u00f3n de bcl-2 es baja. Por el contrario, en las c\u00e9lulas l1210r se observ\u00f3 una disminuci\u00f3n de la expresi\u00f3n de bcl-xl y un aumento de bcl-2. La inhibici\u00f3n de dirigida de prote\u00ednas anti-apopt\u00f3ticas confirma que la expresi\u00f3n de bcl-xl es un regulador clave de la supervivencia de las c\u00e9lulas leuc\u00e9micas. En contraste, el silenciamiento de bcl-2 muestra que la supervivencia de las c\u00e9lulas l1210r no depende del nivel de expresi\u00f3n de bcl-2. As\u00ed, nuestros datos demuestran que la supervivencia de la l\u00ednea celular parental depende de la expresi\u00f3n de bcl-xl, pero la adquisici\u00f3n del fenotipo mdr elimina esta dependencia y podr\u00eda depender de varios miembros anti-apopt\u00f3ticos de la familia bcl-2. Adem\u00e1s, los resultados obtenidos en las c\u00e9lulas cbmc-6 demuestran que p-gp tiene poca influencia sobre las prote\u00ednas de la familia bcl-2. Resultados similares se obtuvieron bajo condiciones de estr\u00e9s hipot\u00e9rmico y exposici\u00f3n al f\u00e1rmaco daunomicina. en relaci\u00f3n a la ruta de se\u00f1alizaci\u00f3n de mapks, demostramos su importancia en el desarrollo del fenotipo mdr, as\u00ed como en el desarrollo de sensibilidad colateral a estr\u00e9s hipot\u00e9rmico, demostrando que la inhibici\u00f3n de erk y jnk puede proteger, parcialmente, de la muerte celular inducida por el fr\u00edo en las c\u00e9lulas l1210r y cbmc-6. conclusiones: el proceso de sensibilidad colateral a estr\u00e9s hipot\u00e9rmico est\u00e1 asociado al fenotipo mdr en c\u00e9lulas murinas, y ocurre a trav\u00e9s de un proceso de apoptosis dependiente de caspasas. La expresi\u00f3n y funcionalidad de mdr-1 est\u00e1 asociada a la sensibilidad a hipotermia en c\u00e9lulas murinas. El desarrollo del fenotipo mdr est\u00e1 acompa\u00f1ado de cambios en la expresi\u00f3n de prote\u00ednas bcl-2, pero \u00e9stas no influyen en la sensibilidad colateral. La se\u00f1alizaci\u00f3n a trav\u00e9s de erk y jnk est\u00e1 implicada en la sensibilidad colateral observada en las c\u00e9lulas mdr. La se\u00f1alizaci\u00f3n a trav\u00e9s de citocinas podr\u00eda tener un papel importante en la respuesta a estr\u00e9s de las c\u00e9lulas mdr.  title: study about collateral sensitivity process in murine and human leukemic cells with multidrug resistance phenotype (mdr) author: david cerezo fern\u00e1ndez introduction: the acquisition of a multidrug-resistant (mdr) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of p-glycoprotein (mdr1, p-gp, abcb1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In the other hand, over-expression of anti-apoptotic bcl-2 family members has been reported in hematologic malignancies and has been associated with chemotherapy resistance in various human cancers. As these findings, it is important to study alterations in mdr cells on mapks pathway and citokines secretion. objectives: the aim of this work was to find a stimulus able to induce collateral sensitivity in murine and human leukemic mdr cells, and to characterize the type of death induced. The second aim was to study the contribution of mdr-1, caspases, family bcl-2 proteins, mapks and citokines to this collateral sensitivity process. materials and methods: to reach our objectives we used l1210 murine leukemia cells and its resistant derived cell line l1210r, as well as a transfected with a plasmid containing mdr-1 cell line obtained from l1210 (cbmc-6). It was used western-blot to study proteins expression, inhibition of mapks and caspases and rnam silencing of mdr-1, bcl-xl, bcl-2 and bax family to study the role of this proteins. results: it was found that resistant leukemic cells with p-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4\u00c2\u00bac. Transfection of parental cells with a p-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between p-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37\u00c2\u00bac) in mdr cells. Treatment with a caspase-3 inhibitor partially rescues mdr leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that p-gp expression plays a role in mdr cell survival, and is accompanied by a collateral sensitivity to cold stress.     we demonstrate that parental leukemic (l1210) and cbmc-6 cells display high constitutive expression of anti-apoptotic bcl-xl protein, while bcl-2 protein expression was very low. By contrast, leukemic cells l1210r display a decrease of bcl-xl and up-regulation of bcl-2 protein expression levels. Furthermore, targeted inhibition of individual anti-apoptotic proteins by rna silencing confirms that bcl-xl expression is a key regulator of leukemic cells survival. In contrast, the silencing of bcl-2 shows that l1210r survival doesn&apos;t depend on bcl-2 expression level. Together, our data demonstrate that parental leukemic cells survival  are largely dependent on bcl-xl protein expression, but acquisition of mdr phenotype by such cells abrogate such survival dependence and suggests that resistant cells became probably dependent on more than one anti-apoptotic protein for survival at physiological conditions. Additionally, the results obtained with cbmc-6 cells demonstrate that p-gp exert slight influence in the expression level of bcl-2 family members. Similar results were obtained under cold stress and drug exposure. related to mapks signaling pathway, we have addressed it importance in mdr phenotype development, as well as in hypothermia collateral sensitivity, showing that erk and jnk inhibition can protect, partially, against stress-cold induced-death in l1210r and cbmc-6 cell lines. conclusions: collateral sensitivity to cold stress is associated to mdr phenotype in murine cells, and it occurs by a caspase-dependent apoptosis process in murine cells. Expression and functionality of mdr-1 is strongly associated to hypothermia sensitivity in murine cells. Mdr phenotype development is accompanied by changes in bcl-2 family proteins expression, but it is not shown an association with collateral sensitivity. Erk and jnk signaling pathways are implicated in collateral sensitivity process observed in mdr cells. Citokines signaling could play an important role on mdr cells stress response.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Estudio del proceso de sensibilidad colateral en celulas leucemicas murinas y humanas con fenotipo de resistencia a multiples farmacos (mdr)<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Estudio del proceso de sensibilidad colateral en celulas leucemicas murinas y humanas con fenotipo de resistencia a multiples farmacos (mdr) <\/li>\n<li><strong>Autor:<\/strong>\u00a0 David Cerezo Fern\u00e1ndez <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Murcia<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 19\/09\/2013<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>orozco Santiago Martin<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: manuel Canovas diaz <\/li>\n<li>M\u00aa pilar Garc\u00eda morales (vocal)<\/li>\n<li>Mar\u00eda  isabel Mart\u00ednez-lacaci fortuny (vocal)<\/li>\n<li>Antonio Parrado gonz\u00e1lez (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de David Cerezo Fern\u00e1ndez T\u00edtulo: estudio del proceso de sensibilidad colateral en c\u00e9lulas leuc\u00e9micas murinas y humanas con [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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