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

Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.by

Amir A. Jazaeri

Julián Esteban David HincapiéMolecular Biology

III Semester MedicineUPB 2009

INTRODUCTION

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.

Evaluation of EVI1 and EVI1s (Δ324) as potential therapeutic targets in ovarian cancer.

Definitions:

EVI1: (ecotropic viral integration site 1), a proto-oncogene involved in human and murine myeloproliferative disorders and myelogenous leukemias.

EVI1s: A short form of EVI1.

CANCER: The name for diseases in which the body's cells become abnormal and divide without control. Cancer cells may invade nearby tissues.

OVARY: The female gonad, the ovary is one of a pair of reproductive glands in women.


RELATION EVI1-EVI1s WITH OVARIAN CANCER

The EVI1 is implicated in the physiopathology of ovarian cancer.

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.


GENERAL OBJETIVE

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


MATERIALES Y METODOS

MUESTRAS

Tejidos humanos: Benignos y malignos Almacenados -80ºC Tincion hematoxilina-eosina Posmenopausia


EXTRACCION DE PROTEINAS

Muestras congeladas pulverizadas Extracción DNA Extracción Proteínas Electroforesis


CULTIVO

Cultivo de células mutadas (SKOV3, OV90, OVCAR3, OVCAR8, CAOV3, ES2, IGROV1)

Cultivo de líneas celulares (HEK293T)

Inmortalización de epitelio ovárico con H118 SV40 T-antígeno


RT-PCR (Reverse transcription polymerase chain reaction)

Amplificacion del RNA a traves de la sintesis previa de su cDNA utilizando trasncriptasa inversa y seguida de PCR convencional.


INMUNOBLOT

Las proteínas son separadas por electroforesis según el tamaño por la técnica SDS-PAGE.

Las proteínas se transfieren a un filtro, que tiene anticuerpos que reaccionan con la proteína de interés.

El anticuerpo unido al filtro identifica la presencia de la proteina.


INMUNOFLUORESCENCIA

Anticuerpos unidos a fluorocromos, absorben luz a longitud de onda de 200 a 400nm y emiten luz de 400 a 700 nm.

Se usa para determinación de proteína, drogas u hormonas.

Directa: Ac proporcional a Ag presente.

Indirecta: menor Ac fluorescente mayor Ag en la muestra.


ELISA(Enzyme Linked Inmunoabsorvent Assay)

Es una tecnica de marcaje, donde el marcador es una enzima. Pueden ser:

Homogeneos: no necesitan paso de separacion Heterogeneos: requiere separacion, se divide en:

El Ac se marca (directo e indirecto) El Ag se marca, fijandose al Ac (sandwich)


RESULTADOS

Expresión de isoformas EVI1 en cáncer de ovario y su potencial de origen de tejidos.

Estas isoformas se presentaron en 17 muestras de cancer de ovario y varias lineas celulares.

Tambien se observaron expresion de estas isoformas en las fimbrias de la trompa de falopio.

Ovarios normales: no presenta proteinas relacionadas con EVI1.

Ovarios con tumores, cancerigenos y fimbrias de trompa de falopio: presentan altas cantidades de proteinas ralcionadas con EVI1.


Efecto de la EVI1 en el crecimiento de células en el cáncer de ovario

Empleo de OVCAR8 con supresion de 3q26 EVI1 y EVI1s se expreso de manera estable HEK293T:

EVI1s: no indujo crecimiento EVI1: indujo pequeña proliferacion


EVI1 y quimiorresistencia en cáncer de ovario

Uso de serina fosforilada H2AX como indicador Uso de cisplatino EVI1 no fue modificado despues del daño del DNA No hubo diferencias estadisticas entre el control de

siRNA y caidas de EVI1 con respecto a la induccion de la apoptosis.


Figura 2. EVI1 mRNA expression


Figura 2A


. (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.

Figura 2B


(B) EVI1 transcripts are also expressed in a number ofestablished 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.

Figura 2C


(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.

Figura 3. EVI1 isoform protein expression


Figura 3A


(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.

Figura 3B


(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.

Figura 3C


(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).

Figura 4. Retroviral expression of EVI1 and EVI1s in OVCAR8 cells


(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.

Figura 6. Effects of EVI1 protein isoforms on ovarian cancer proliferation


Figura 6A


(A) Isoform selective depletion of EVI1 proteins using siRNA.

Figura 6B


(B) Levels of basal and daunorubicin-induced double-stranded DNA breaks as indicated by γ-H2AX immunoblotting following EVI1 isoform knockdown.

DISCUSSION


Scientist Approach CoincidenceYes/No

Sunde JS et al. 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.

Yes

Najundan M et al.

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.

Yes

Osterberg L et al.

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.

No

Najundan M et al.

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.

Yes

CONCLUSSIONS

The transcripts are the key step for the overexpression of proteins, if you control the transcription, proliferative processes are controlled.

Perhaps the expression of genes in a tissue can be complications in other tissues, reflecting histological.

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.

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.


Bibliography

MARTINEZ SÁNCHEZ, Lina María. Biología molecular. 5. ed. Medellín: UPB. Fac. de Medicina, 2009. 313 p.

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para-laboratorio-15375-363707.html http://www.genboot.com/category/genetica/ http://www.teagasc.ie/research/reports/dairyproduction/5067/eopr-

5067.asp http://coleccion.educ.ar/coleccion/CD23/contenidos/familia/

index3.html http://www.sanidadanimal.info/cursos/inmuno2/ca093.htm


Hecho por: Julián David Hincapié

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

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