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Biología molecular

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Biología molecular

  1. 1. THE USE OF CASPASE INHIBITORS IN PULSED-FIELD GEL ELECTROPHORESIS MAY IMPROVE THE ESTIMATION OF RADIATION-INDUCED DNA REPAIR AND APOPTOSIS Josep Balart, Gemma Pueyo, Lara I de Llobet, Marta Baro, Xavi Sole, SusannaMarin, Oriol Casanovas, Ricard Mesia, Gabriel Capella Sara Rendón V Verónica Sarassa Molecular Biology Universidad Pontificia Bolivariana
  2. 2. INTRODUCTION In this study the scientiststried to prove by the PFEG (pulsed-field gel Electrophoresis)that Caspase inhibitors would help in radiation-inducedDNA double-strand break (DSB) repair. They used tumor cells, caspase inhibitors and mice.
  3. 3. KEY WORDSAPOPTOSIS: Genetically programmed and regulated cell death. It intervenes in two ways: begins at the end third of the G1 phase to impede a damaged cell going to synthesis phase (so mutations can’t reproduce during DNA replication). The other way: it can act on the G2 phase to impede cells that haven’t reached maturity enter mitosis.
  4. 4. KEY WORDS APOPTOSIS WAYS EXTRINSIC INTRINSECCalled “death receptors” Use the protein Bcl-2 path, it establishes (gene B-cell lymphoma conections with the 2)to regulate extracellular preapoptotic factors. For space, recieving example the C citocromeproapoptotic signals from (necessary in the the outside and from activation of the 9- neighbor cells. caspase in cytosol)
  5. 5. KEY WORDS APOPTOSIS: FADD On the inside interacts with Apoptosis Fas two proteins: DaXXEXTRINSIC RECEPTORS Tradd Cell TNF: proliferation Raidd Traff Apoptosis C CitocromeINTRINSIC Bcl-2 SMAC/DIABLO
  6. 6. KEY WORDSAPOPTOSIS: FAS: surface protein of 36 kDa with a cytoplasmatic domain of “cell death”. DaXX: estimulates cell cycle and mitosis, having the contrary effect that of the apotosis. FADD: factor associated death domain. TNF: tumor necrosis factor Tradd: TNF receptor associated death domain
  7. 7. KEY WORDSAPOPTOSIS: Raidd (receptor associated interleukine death domain), activation of initiator caspases. Traf (TNF receptor associated factor) activated protein kinase and stimulate cell proliferation. Bcl-2: Regulation of the apoptosis action on the mitochondria SMAC/DIABLO: an inhibitor of caspase inhibitors Citocromo C: activates a protein complex called "apoptosome", which directly activatescaspase-9
  8. 8. KEY WORDS DNA REPARATION The double-strand breaks is a post Capacity of a cell Both strands replication repair. to identify and of the double Divides in two types:correct damage tothe DNA molecules helix are broken 1. Non-homologous that encode its end joining genome. 2. Homologous recombination.
  9. 9. KEY WORDSPULSED-FIELD GEL ELECTROPHORESIS(PRGE) It has been developed for separating large molecules of DNA, all using multiple electric fields Chromosomal DNAintegrated in agarose plugs Plugs treated with enzymes, that leaves only the naked DNA With the restriction enzymes, the plugs are cut. Then, inmersed in the wells of gel and sealed with agarose.
  10. 10. KEY WORDSBy the method PRGE (puled-field gelelectrophoresis) the apoptosis and caspaseactivation can be determinated becausethis method shows the quantity offragmented DNA. + fragmented DNA= + apoptosis,+activation of caspases - fragmented DNA = - apoptosis, + DNA repair
  11. 11. PURPOSE To demostrate if the DNAfragmentation in experiments with PFGE in induced by apoptosis using Caspase-3.
  12. 12. MATERIALES Y MÉTODOSLÍNEAS CELULARES (CULTIVOS) Se utilizaron células tumorales: la utilizada en el estudio fue la escamosa humana carcinoma de A431. Fue tomada de una colección americana de células (El carcinoma pancreático NP18). Se hizo un cultivo celular en ratones macho durante 6 a 8 semanas Los tumores se generaron mediante inyección subcutánea de un millón de células NP18 en el flanco de cada ratón.
  13. 13. MATERIALES Y MÉTODOSLÍNEAS CELULARES (CULTIVOS) Cuando los tumores alcanzaron 10 mm de tamaño, fueron eliminados, cortados en pedazos y se incubaron durante 90 min en Dubelcco’s Modified Eagle Medium (DMEM) que tiene colagenasa IV las suspensiones de células Se incubaron durante 30 minutos en tripsina A 37 grados y se pasaron por un filtro de células de 70 um
  14. 14. MATERIALES Y MÉTODOSPRGE Los sedimentos celulares obtenidos fueron mezclados con agarosa Se ajustó el número de células por tapón (a 100.000 células) Se utilizaron alícuptas (se toma parte de ese volumen). Usaron el sedimento entero para formar los tapones de células obtenidas de la desagregación del tumor.
  15. 15. MATERIALES Y MÉTODOS PRGE Fueron pasadas a Se cambió elRefrigeración tubos con medio por uno a 4°C DMEM, después de fueron irradiadas preincubación Los tapones celulares que no La reparación del fueron irradiados DNA fue fueron tratados interrumpida paralelamente poniendo el tapón con los radiados celular en hielo
  16. 16. MATERIALES Y MÉTODOS PRGELos tapones celularesfueron transferidos, para Las esquinas seser lisadas, a una rompen fácilmentesolucion buffer helada debido a laque contiene Sodio fragilidad de loslaurolil-sarkosina y tapones de agarosaproteinasa K en EDTA(conservante). (Lisis=duró 1 h) Se aseguro que el Fragmentos de mismo numero de ADN fueron células fueran resueltos por la cargadas en el PFGE gel.
  17. 17. MATERIALES Y MÉTODOS PRGECromosomas de La suma de laSaccharomyces fluorescencia dentrocervisiae y de las extensiones deSchizosaccharomy ADN fue usada paraces pombe fueron calcular y valor lasusados como diferencias entremarcadores de cada experimento.ADN El ADN adquirio intesidadLos geles fueron fluorescente y seteñidos, lavados y transformo atransiluminados. unidades de densidad optica.
  18. 18. MATERIALES Y MÉTODOS .INMUNOFLUORESCENCIAS La inmunofluorescencia (IF) es una técnica que emplea anticuerpos conjugados a fluorocromos. Los fluorocromos son moléculas que al ser excitadas con la energía de una determinada longitud de onda son capaces de emitir energía de una longitud de onda mayor Propósito: Que la determinación de fosforilación de H2AX y la activación de las caspasas 3 se da por inmunofluorescencias
  19. 19. MATERIALES Y MÉTODOSINMUNOFLUORESCENCIAS Primero unas secciones del tapón de células se pusieron en el criostato Se agregó 4% del buffer neutro de formaldehido , se lavó con 0.1% de tritón en PBS por 10 min Incubación por una hora con proteínas bloqueadoras de la solución. Las tiras fueron incubadas con anticuerpos primarios de la antifosfohistona H2AX (gen que codifica H2) seguido de incubación con los segundos anticuerpos alexa fluor 488 mas alexa fluor 594 (Tinciones) Todo en una dilución 1:500
  20. 20. MATERIALES Y MÉTODOSINMUNOFLUORESCENCIAS . Capturación de imágenes por un microscopio especial de alta resolución y tecnología Conteo de células por un software Activación de la apoptosis de las células que estaban en monocapa se examinó mediante IF específica (uso de Nuclear-PRO 3 tinte principalmente
  21. 21. MATERIALES Y MÉTODOS Elementos que bloquean INHIBIDORES DE CASPASAS los activadores de las caspasas. •Las células •Las células •Ambas tapón fueron obtenidas sustanciastratadas con un fueron tratadas fueron ihnibidor con el inhibidor incubadas por general de Caspasa-3 zvd- una hora antescaspasas, para fmk de ser impedir encapsuladas apoptosis en la célula.
  22. 22. RESULTADOSFIGURA 1En la figura 1 se analiza el resultado del PFGE para la línea celular trabajada (A431) encapsulada primero en agarosaLa cantidad del ADN liberado (lo midela densidad óptica dado en unidades arbitrarias= UA) y se representa en unidad de tiempo (en horas) Lo no irradiado (barras negras) y los tapones celulares de 45 Gy (barras blancas) La cuantificación empieza por debajo de la zona de compresión
  23. 23. RESULTADOSFIGURA 1En la figura 1 se muestra:1. DNA intactos sin irradiación en las diferentes horas de cultivo (U) y bajo el efecto de los Gy de irradiación en el tiempo (I).2. Los (I) muestran una zona de engrosamiento por debajo de los pozos de siembra del gen y un DNA barrido (esto es producto de la degradación) y además la extracción del DNA es menor en las células expuestas con más tiempo a más radiación.Gy: Unidad que mide la dosis absorbida de radiaciones ionizantes en un determinado material
  24. 24. RESULTADOSFIGURA 2 La activación de las caspasas y el aumento de la radiación inducida por la fluorescencia de H2AX disminuyó con el tiempo. Los tapones celulares con reparación se tiñeron con azul. La fluorescencia de la caspasa en células con ésta activada con rojo. H2AX con fluorescencia con verde.
  25. 25. RESULTADOSFIGURA 2 Para evaluar la activación de la apoptosis, midieron los niveles de caspasa 3. Se muestra que la apoptosis es independiente de la irradiación, que el mismo medio de la agarosa lo induce. Esto muestra que la agarosa participa de manera espontánea en la degradación del DNA.
  26. 26. RESULTADOSFIGURA 3 A-PRO tiñe núcleos Fluorescencia de la caspasa Cuadros combinados A pesar de la irradiación, la fragmentación nuclear y La la condensación de la cromatina asociada, la apoptosis fue apoptosis fue mínima pero junto a la activación específica de insignificante. caspasa-3.
  27. 27. RESULTADOSFIGURA 4 Los inhibidores de las caspasas bloquean la apoptosis inducida por la encapsulación en agarosa. Barras negras: fragmentación espontánea durante la incubación de células tapón (a mayor tiempo más fragmentación)
  28. 28. RESULTADOSFIGURA 4 Barras grises: inhibidores Zvad-fmk No diferencias significativas entre A431 y NP18
  29. 29. RESULTADOSFIGURA 5 La reducción de la apoptosis mejoró la estimación de la reparación del DNA en los injertos. PFGE se realizó en no irradiados (barras negras) e irradiado con 45 Gy(barras blancas) La fragmentación espontánea (barras negras) aumentó en ausencia de Zdv- fmk La radiación inducida por roturas disminuyó con el tiempo.
  30. 30. DISCUSSIONFrisch SM, Screaton RAThis is the first study to describe that thespontaneously released DNA in cell-plugsreflects an ongoing homeless-inducedapoptosis, referred to as anoikis.agree
  31. 31. CONCLUSIONSBrown DG, Sun XM, Cohen GM:when cells are embedded in agarose. DNA cleavage intolarge fragments is an early event observed in theapoptotic cascade before the typical endonucleasecleavage into 180 to 200 bp can be detected as a DNAladder.agree
  32. 32. CONCLUSIONSBonner WM, Redon CE, Dickey JS, NakamuraAJ, Sedelnikova OA, Solier S,Pommier Y:After elucidating the mechanism behind DNAdegradation, it seemed reasonable to concludethat apoptosis initiation was occurring in bothunirradiated and irradiated cell-plugs.agree
  33. 33. CONCLUSIONSLiotta LA, Kohn E:The clinical significance of resistance toanoikis is increasingly associated withmalignant phenotypes, therapyresistance, and poor prognosis.agree
  34. 34. CONCLUSIONS It´s difficult to quantifythe DNA repair, you can only know the cells that didn´t do apoptosis. Is assumed that if the cell didn´t do apoptosis its DNA was repaired.
  35. 35. CONCLUSIONS Although the agarose is the ideal medium for PFGE, it causes DNA fragmentation and apoptosis of some cells. This may have inceased the number of cells with apoptosis (but not only by DNA damage)
  36. 36. CONCLUSIONS Thisexperiment allows the generation of posterior studies about the origin of malignant neoplastic phenomenons.
  37. 37. CONCLUSIONS It promotes literature sources to generate new techniques to improve the effectiveness and efficiency of cancer treatments, where patients can get the expected results with the least damage possible.
  38. 38. Sara Rendón Villa
  39. 39. BIBLIOGRAPHY MARTINEZ SÁNCHEZ, Lina María. Biología molecular. 2. ed. Medellín: UPB. Fac. de Medicina, 2006. 208 p. Eder M, Gedik P (1979) Manual de Patología General y Anatomía Patológica. Traducción de la 30ª edición alemana. Editorial Científica- Médica, Barcelona.

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