Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. by Amir A. Jazaeri Julián Esteban ...
INTRODUCTION <ul><li>Ovarian cancer is the 5th cause of death in women, despite having undergone any treatment survival is...
Definitions: <ul><li>EVI1:  (ecotropic viral integration site 1), a proto-oncogene involved in human and murine myeloproli...
RELATION EVI1-EVI1s WITH OVARIAN CANCER <ul><li>The EVI1 is implicated in the physiopathology of ovarian cancer. </li></ul...
GENERAL OBJETIVE <ul><li>The objetive of the investigation was the function of the EVI1 and EVI1s in the development of ov...
MATERIALES Y METODOS <ul><li>MUESTRAS </li></ul><ul><ul><li>Tejidos humanos: Benignos y malignos </li></ul></ul><ul><ul><l...
<ul><li>EXTRACCION DE PROTEINAS </li></ul><ul><ul><li>Muestras congeladas pulverizadas </li></ul></ul><ul><ul><li>Extracci...
<ul><li>CULTIVO </li></ul><ul><ul><li>Cultivo de células mutadas  </li></ul></ul><ul><ul><li>(SKOV3, OV90, OVCAR3, OVCAR8,...
<ul><li>RT-PCR </li></ul><ul><li>( Reverse transcription polymerase chain reaction ) </li></ul><ul><ul><li>Amplificacion d...
<ul><li>INMUNOBLOT </li></ul><ul><ul><li>Las proteínas son separadas por electroforesis según el tamaño por la técnica SDS...
<ul><li>INMUNOFLUORESCENCIA </li></ul><ul><ul><li>Anticuerpos unidos a fluorocromos, absorben luz a longitud de onda de 20...
<ul><li>ELISA </li></ul><ul><li>(Enzyme Linked Inmunoabsorvent Assay) </li></ul><ul><ul><li>Es una tecnica de marcaje, don...
RESULTADOS <ul><li>Expresión de isoformas EVI1 en cáncer de ovario y su potencial de origen de tejidos. </li></ul><ul><ul>...
<ul><li>Efecto de la EVI1 en el crecimiento de células en el cáncer de ovario  </li></ul><ul><ul><li>Empleo de OVCAR8 con ...
<ul><li>EVI1 y quimiorresistencia en cáncer de ovario  </li></ul><ul><ul><li>Uso de serina fosforilada H2AX como indicador...
Figura 2. EVI1 mRNA expression  Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
Figura 2A Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. . (A) Semi-quantitative ...
Figura 2B Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (B) EVI1 transcripts are...
Figura 2C Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (C) Knockdown of EVI1 us...
Figura 3.  EVI1 isoform protein expression   Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovari...
Figura 3A Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (A) Using the SKOV3 ovar...
Figura 3B Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (B) Immunoblot demonstra...
Figura 3C Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (C) Western blot of a re...
Figura 4. Retroviral expression of EVI1 and EVI1s in OVCAR8 cells  Evaluation of EVI1 and EVI1s (Δ324) as potential therap...
Figura 6. Effects of EVI1 protein isoforms on ovarian cancer proliferation  Evaluation of EVI1 and EVI1s (Δ324) as potenti...
Figura 6A Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (A) Isoform selective de...
Figura 6B Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (B) Levels of basal and ...
DISCUSSION Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. No A recent investigati...
CONCLUSSIONS <ul><li>The transcripts are the key step for the overexpression of proteins, if you control the transcription...
Bibliography <ul><li>MARTINEZ SÁNCHEZ, Lina María. Biología molecular. 5. ed. Medellín: UPB. Fac. de Medicina, 2009. 313 p...
Thank you!
Hecho por: Julián David Hincapié
Upcoming SlideShare
Loading in …5
×

Evaluation of EVI1 and EVI1s (δ324) by Julian David Hincapie

627 views

Published on

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
627
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
3
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Evaluation of EVI1 and EVI1s (δ324) by Julian David Hincapie

  1. 1. Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. by Amir A. Jazaeri Julián Esteban David Hincapié Molecular Biology III Semester Medicine UPB 2009
  2. 2. INTRODUCTION <ul><li>Ovarian cancer is the 5th cause of death in women, despite having undergone any treatment survival is very low. Recently genes EVI1 - EVI1s have been associated with ovarian carcinoma, its overexpression and function in chemoresistance, which has promoted the study of these to achieve a therapeutic potential new way. </li></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  3. 3. Definitions: <ul><li>EVI1: (ecotropic viral integration site 1), a proto-oncogene involved in human and murine myeloproliferative disorders and myelogenous leukemias. </li></ul><ul><li>EVI1s: A short form of EVI1. </li></ul><ul><li>CANCER: The name for diseases in which the body's cells become abnormal and divide without control. Cancer cells may invade nearby tissues. </li></ul><ul><li>OVARY: The female gonad, the ovary is one of a pair of reproductive glands in women. </li></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  4. 4. RELATION EVI1-EVI1s WITH OVARIAN CANCER <ul><li>The EVI1 is implicated in the physiopathology of ovarian cancer. </li></ul><ul><li>High levels of transcription of EVI1 were presented in different ovarian cancer and also these high levels were associated with chemoresistance that showed the tissue. </li></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  5. 5. GENERAL OBJETIVE <ul><li>The objetive of the investigation was the function of the EVI1 and EVI1s in the development of ovarian cancer and his possible therapeutic use in the treatment of the patology </li></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  6. 6. MATERIALES Y METODOS <ul><li>MUESTRAS </li></ul><ul><ul><li>Tejidos humanos: Benignos y malignos </li></ul></ul><ul><ul><li>Almacenados -80ºC </li></ul></ul><ul><ul><li>Tincion hematoxilina-eosina </li></ul></ul><ul><ul><li>Posmenopausia </li></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  7. 7. <ul><li>EXTRACCION DE PROTEINAS </li></ul><ul><ul><li>Muestras congeladas pulverizadas </li></ul></ul><ul><ul><li>Extracción DNA </li></ul></ul><ul><ul><li>Extracción Proteínas </li></ul></ul><ul><ul><li>Electroforesis </li></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  8. 8. <ul><li>CULTIVO </li></ul><ul><ul><li>Cultivo de células mutadas </li></ul></ul><ul><ul><li>(SKOV3, OV90, OVCAR3, OVCAR8, CAOV3, ES2, IGROV1) </li></ul></ul><ul><ul><li>Cultivo de líneas celulares </li></ul></ul><ul><ul><li>(HEK293T) </li></ul></ul><ul><ul><li>Inmortalización de epitelio ovárico con H118 SV40 T-antígeno </li></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  9. 9. <ul><li>RT-PCR </li></ul><ul><li>( Reverse transcription polymerase chain reaction ) </li></ul><ul><ul><li>Amplificacion del RNA a traves de la sintesis previa de su cDNA utilizando trasncriptasa inversa y seguida de PCR convencional. </li></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  10. 10. <ul><li>INMUNOBLOT </li></ul><ul><ul><li>Las proteínas son separadas por electroforesis según el tamaño por la técnica SDS-PAGE. </li></ul></ul><ul><ul><li>Las proteínas se transfieren a un filtro, que tiene anticuerpos que reaccionan con la proteína de interés. </li></ul></ul><ul><ul><li>El anticuerpo unido al filtro identifica la presencia de la proteina. </li></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  11. 11. <ul><li>INMUNOFLUORESCENCIA </li></ul><ul><ul><li>Anticuerpos unidos a fluorocromos, absorben luz a longitud de onda de 200 a 400nm y emiten luz de 400 a 700 nm. </li></ul></ul><ul><ul><li>Se usa para determinación de proteína, drogas u hormonas. </li></ul></ul><ul><ul><li>Directa: Ac proporcional a Ag presente. </li></ul></ul><ul><ul><li>Indirecta: menor Ac fluorescente mayor Ag en la muestra. </li></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  12. 12. <ul><li>ELISA </li></ul><ul><li>(Enzyme Linked Inmunoabsorvent Assay) </li></ul><ul><ul><li>Es una tecnica de marcaje, donde el marcador es una enzima. Pueden ser: </li></ul></ul><ul><ul><li>Homogeneos: no necesitan paso de separacion </li></ul></ul><ul><ul><li>Heterogeneos: requiere separacion, se divide en: </li></ul></ul><ul><ul><ul><li>El Ac se marca (directo e indirecto) </li></ul></ul></ul><ul><ul><ul><li>El Ag se marca, fijandose al Ac (sandwich) </li></ul></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  13. 13. RESULTADOS <ul><li>Expresión de isoformas EVI1 en cáncer de ovario y su potencial de origen de tejidos. </li></ul><ul><ul><li>Estas isoformas se presentaron en 17 muestras de cancer de ovario y varias lineas celulares. </li></ul></ul><ul><ul><li>Tambien se observaron expresion de estas isoformas en las fimbrias de la trompa de falopio. </li></ul></ul><ul><ul><li>Ovarios normales: no presenta proteinas relacionadas con EVI1. </li></ul></ul><ul><ul><li>Ovarios con tumores, cancerigenos y fimbrias de trompa de falopio: presentan altas cantidades de proteinas ralcionadas con EVI1. </li></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  14. 14. <ul><li>Efecto de la EVI1 en el crecimiento de células en el cáncer de ovario </li></ul><ul><ul><li>Empleo de OVCAR8 con supresion de 3q26 </li></ul></ul><ul><ul><li>EVI1 y EVI1s se expreso de manera estable </li></ul></ul><ul><ul><li>HEK293T: </li></ul></ul><ul><ul><ul><li>EVI1s: no indujo crecimiento </li></ul></ul></ul><ul><ul><ul><li>EVI1: indujo pequeña proliferacion </li></ul></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  15. 15. <ul><li>EVI1 y quimiorresistencia en cáncer de ovario </li></ul><ul><ul><li>Uso de serina fosforilada H2AX como indicador </li></ul></ul><ul><ul><li>Uso de cisplatino </li></ul></ul><ul><ul><li>EVI1 no fue modificado despues del daño del DNA </li></ul></ul><ul><ul><li>No hubo diferencias estadisticas entre el control de siRNA y caidas de EVI1 con respecto a la induccion de la apoptosis. </li></ul></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  16. 16. Figura 2. EVI1 mRNA expression Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  17. 17. Figura 2A Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. . (A) Semi-quantitative RT-PCR revealed higher EVI1 and EVI1s expression in serous epithelial ovarian cancers compared to normal ovaries. Identity of EVI1and EVI1swas verified by cloning and DNA sequencing. In addition other lower expressed cDNA bands were alsoamplified and identified as EVI1 transcript variants using siRNA(panel C). Negative control experiments consisted of omission of reverse transcriptase and RNA and resulted in no amplified PCR product.
  18. 18. Figura 2B Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (B) EVI1 transcripts are also expressed in a number of established ovarian cancer cell lines aswell as the H118 immortalized ovarian surface epithelial cells. The CAOV3 and OVCAR8 ovarian cancer cell lines had no detectable EVI1mRNA.
  19. 19. Figura 2C Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (C) Knockdown of EVI1 using siRNA directed against the region spliced out in EVI1s results in degradation of EVI1 as well as the other minor transcripts.
  20. 20. Figura 3. EVI1 isoform protein expression Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  21. 21. Figura 3A Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (A) Using the SKOV3 ovarian cancer cells as a representative model, 185, 145, and 90 kDa bands corresponding to MDS-EVI1 (ME), EVI1, and EVI1s, respectively, were identified and their identities confirmed using isoform selective siRNA. Oligonucleotides A, B, C, and 03 target regions of the EVI1 open reading frame common to all isoforms and exhibited varying degrees of knockdown. SiB reproducibly provided greater than 90% knockdown of all EVI1 isoforms. Si04 targets sequences spliced out in EVI1s and hence results in isoform-selective knockdown of EVI1 and ME. Si 2Kb targets the splice junction uniquely used in EVI1s and thus selectively inhibits this isoform.
  22. 22. Figura 3B Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (B) Immunoblot demonstrating the expression of ME, EVI1, and EVI1s in 10 serous ovarian cancer samples. SKOV3 cells and OVCAR8 EVI1 null cells expressing exogenous EVI1 and EVI1s were included to facilitate isoform identification.
  23. 23. Figura 3C Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (C) Western blot of a representative set of serous ovarian cancers (C), normal postmenopausal ovary (O), fallopian tube fimbria (F), and benign ovarian neoplasms (B).
  24. 24. Figura 4. Retroviral expression of EVI1 and EVI1s in OVCAR8 cells Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (A) RT-PCR demonstrating the expression of Evi1 and EVI1s mRNA in OVCAR 8 infected cells compared to uninfected OVCAR8 and SKOV3 cells. (B) Expression of recombinant EVI1 and EVI1s in OVCAR 8 cells.
  25. 25. Figura 6. Effects of EVI1 protein isoforms on ovarian cancer proliferation Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  26. 26. Figura 6A Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (A) Isoform selective depletion of EVI1 proteins using siRNA.
  27. 27. Figura 6B Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. (B) Levels of basal and daunorubicin-induced double-stranded DNA breaks as indicated by γ -H2AX immunoblotting following EVI1 isoform knockdown.
  28. 28. DISCUSSION Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer. No A recent investigation implicated 3q26 copy number alterations and possibly EVI1 expression in ovarian cancer chemoresistance. It is noteworthy that in this study only EVI1 mRNA expression was evaluated using primers that do not differentiated between various EVI1 and ME transcripts. Osterberg L et al. Yes Potential oncogenic action of SnoN/SkiL, a coregulator of SMAD/TGF beta signaling that maps to 3q26.2 has been recently reported by the same group of investigators that published one of the reports on EVI1 in ovarian cancer. Najundan M et al. Yes ME and EVI1 forced expression increased the growth of an immortalized ovarian surface epithelial cell line. Interestingly however, they also observed no effects on growth of OVCAR8 ovarian cancer cells. Najundan M et al. Yes Reported a positive correlation between EVI1 immunohistochemical staining and mRNA levels, though they did not present any immublotting data and their methods precluded differentiating between ME, EVI1, and EVI1s transcripts. Sunde JS et al. Coincidence Yes/No Approach Scientist
  29. 29. CONCLUSSIONS <ul><li>The transcripts are the key step for the overexpression of proteins, if you control the transcription, proliferative processes are controlled. </li></ul><ul><li>Perhaps the expression of genes in a tissue can be complications in other tissues, reflecting histological. </li></ul><ul><li>The presence of proteins in a tissue, does not necessarily mean that the presence of these occurred due to the overexpression of a specific gene. </li></ul><ul><li>The involvement of several oncogenes in the same locus in malignancy should be treated simultaneously, in order to find specific results on the expression of proteins of the same chromosome. </li></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  30. 30. Bibliography <ul><li>MARTINEZ SÁNCHEZ, Lina María. Biología molecular. 5. ed. Medellín: UPB. Fac. de Medicina, 2009. 313 p. </li></ul><ul><li>Images: </li></ul><ul><li>http://es.123rf. com / photo _857384_resumen-mujeres-desnudas-cuerpo--3d- escena.html </li></ul><ul><li>http://mundocomocajacristal.blogspot.com/2010_04_01_archive.html </li></ul><ul><li>http://www.mifigura.com.mx/abdominoplastia.htm </li></ul><ul><li>http://rosalindelsiefranklin.blogspot.com/ </li></ul><ul><li>http://www.desdechinandega.com/salud/la-menopausia.html </li></ul><ul><li>http://www.directindustry.es/prod/consort/unidad-de-electroforesis-para-laboratorio-15375-363707.html </li></ul><ul><li>http://www.genboot.com/category/genetica/ </li></ul><ul><li>http://www.teagasc.ie/research/reports/dairyproduction/5067/eopr-5067.asp </li></ul><ul><li>http://coleccion.educ.ar/coleccion/CD23/contenidos/familia/index3.html </li></ul><ul><li>http://www.sanidadanimal.info/cursos/inmuno2/ca093.htm </li></ul>Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.
  31. 31. Thank you!
  32. 32. Hecho por: Julián David Hincapié

×