Seminario BM

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, Susanna Marin, Oriol Casanovas,Ricard Mesia, Gabriel Capella.

SIMON

RESTREPO

CAMILO

RESTREPO

KEY WORDS• PFGE Pulsed Field Gel Electrophoresis.

• CASPASES Proteins that are one of the main

executors of the apoptotic process and the pro-

inflammatory process too.

• DNA REPARATION Processes by which a cell

identifies and corrects damage to the DNA

molecules that encode its genome.

Mechanisms

• APOPTOSIS Process of programmed cell

death that may occur in multicellular organisms.

INTRODUCTION

• DNA repair in several factors which can

be determined through PFGE, as are the

activity mediated by caspases called

programmed cell death, apoptosis.

• Then the objective of PFGE is determine

the magnitude of radiation induced by

these processes and determine that the

release of DNA from cells corresponds

to the intensity of the DNA

fragmentation

• In the PFGE technique, cells

are encapsulated in agarose

to form cell-plugs, thus

preventing physic damage of

the cells while facilitating their

manipulation and placement

into agarose gels where

electrophoresis will take place.

GENERAL OBJECTIVE

• Determine the

radiation

generated by PFGE

of DNA

fragmentation and

apoptosis during

repair.

MATERIALES Y METODOS

•LINEAS CELULARES

▫Células tumorales A431

Carcinoma Escamoso.

▫Células NP18 Carcinoma

Pancreático.

•MUESTRA

▫Ratones de 6 a 8

semanas de sexo

masculino.

PROCEDIMIENTO

• A cada ratón se le inyecto de forma SC NP18 en su flanco para generar el tumor.

• 10mm Extracción/extirpación 90min incubación DMEM 30min Tripsina

a 37° en agitación

ELCTROFORESIS• Separación de partículas por peso y

por cargas

PFGE• Herramienta para examinar el ADN cromosómico que es

capaz de separar fragmentos de 10 kpb hasta 10.000 kpb.

CAMPO ELECTRICO

ELONGACION Y MIGRACION

CAMPO ELECTRICO

REDISTRIBUCION

MAS DISCRIMIACION

FLUORESCENCIA

• Es la propiedad de una sustancia de emitir luz cuando es expuesta a

radiaciones

INMUNOFLUORESCENCIA

• La inmunofluorescencia se utiliza esencialmente en la detección de

autoanticuerpos y anticuerpos contra antígenos de superficie de

células y tejidos.

•DIRECTA

ANTICUERPO MARCADO ANTIGENO FLUORESCENCIA

DIRECTA

•INDIRECTA

ANTICUERPO ANTIGENO MARCADOR ANTICUERPO

INMUNOFLUORESCENCIA

INDIRECTA

Resultados

Discussion

AUTHOR PHRASE OPINION

Frisch SM,

Screaton RA.

This is the first study to describe that the

spontaneously released DNA in cell-plugs

reflects an ongoing homeless-induced

apoptosis, referred to as anoikis .

Agree

Brown DG,

Sun XM,

Cohen GM

DNA cleavage into large fragments is an early

event

observed in the apoptotic cascade before the

typical endonuclease cleavage into 180 to 200

bp can be detected as a DNA ladder .

Agree

Bonner WM et al. Our findings show that gH2AX fluorescence, a

surrogate marker of DNA-DSB.

Agree

Liotta LA, khon E. The clinical significance of resistance to

anoikis is increasingly associated with

malignant phenotypes, therapy resistance,

and poor prognosis.

Agree

CONCLUSIONS

• New techniques may improve better and reliable results

• There should be more invest on this kind of studies

• This study shed a light to develop better treatments.

• Radiation does not cure, its just a way to prevent

cancer spreads.

Mapas Conceptuales

Simón Restrepo

CAMILO RESTREPO RODRIGUEZ

Thanks