Seminario molecular

Manuela Parra Cardona

3rd Semester

Medicine student

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Comparison of the inhibitory effects of threetranscriptional variants of CDKN2A in human

lungcancer cell line A549

Wei Zhang(Laboratory of Medical Genetics, Harbin Medical University)

Journal of experimental & clinical cancer research

INTRODUCTION

Cell cyclestrictly ordered

processREGULATED BY

•Cyclins and CDKs•negative regulators such CKIs

Tumor suppressor CDKN2A

p16INK4a p14ARF p12

Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549

GENERAL OBJETIVE

Compare the inhibitory effects of the three transcriptional variants of CDKN2A in human lung cancer cell line A549 with endogenous expression deficiency.


CANCER


http://www.rndsystems.com/DAM_public/5174.gif


TRANSCRIPTIONIt is the molecule that carries genetic information (from DNA) to the cytoplasm for the production of proteins.

RNA POLYMERASE

http://www.google.com.co/imgres?imgurl=http://www.genome.gov/Images/EdKit/bio2c_large.gif&imgrefurl=http://www.genome.gov/EdKit/bio2c.html


LUNG CANCER

Occurs when cells in the lung, change to become abnormal.

Types of Lung CancerThere are 2 main types of lung cancer.

• Non-Small Cell Lung CancerThis is the most common type of lung cancer.

• Small Cell Lung Cancer

http://www.google.com.co/imgres?imgurl=http://www.newhopeblog.com/Lung%2520cancer.jpg


CDKN2ATumor suppressor gene, that encodes proteins whit tumor suppressor activity P16INK4a, P14ARF and P12.

It is a specific cell cycle regulator of G1 stage, that inhibits the CDK4 and CDK6 thereby inhibits the phosphorylation or pRB.

P16INK4a


P14ARF

regulates tumor protein 53 (p53) activity by inhibiting MDM2, a ubiquitin ligase that otherwise targets p53 for degradation by proteasome.

DNA damage and other signs of stress can cause the release of p53 and increase its concentration, allowing to perform its role as a transcription factor.

P14ARF

MDM2

P53P53 degradatio

n

Arrest of cell cycle

Normal cell cycle


P12

•Defects in CDKN2a gene are involved in tumor formation in a wide range of tissues.•Studies examined non-small cell lung cancer (NSCLC) cell lines and found that the inactivation rate of p16INK4a and p14ARF was 84% and 55% respectively.

pRB-independent growth suppression by p12 was reported in pancreatic cells, but the tumor suppressive and cell-cycle effects of this protein are as yet unclear.

Relay with cancer development:


CULTIVO CÉLULAR

F12-K con FBS (fetal bovine serum)


RT-PCR

RNA TRANSCRIPTAS

INVERSA DNAc

AGZY H446 y células

pancreáticas (expresión)

DNAc de P16, P14 y

P12


WESTERN BLOTTING Proteínas

•para detectar proteínas específicas P16INK4A


INMUNOHISTOQUÍMICA

permiten la identificación, sobre muestras tisulares, la localización de una molécula de interés. Mediante anticuerpos marcados y diseñados para reconocer dicha molécula.

Evaluar la expresión de P!6, P14 y P12


CITOMETRIA DE FLUJO

Es el análisis de las características de células, Donde las células pasan alineadas una a una frente a un haz láser mediante un flujo continuo.


En el estudio….

Evaluaron el estadio en el cual se encontraban del ciclo celular, después de la expresión de p16, p14 y p12.

RT-PCR of A459

P16INK4a P14ARF P12

Expression of P16INK4a, P14ARF and p12 were confirmed.

543 bp493 bp

372 bp

Inmunohistoquímica

Se detecta expresión de p16, p14 y p12, en células clonadas.

Citometria de flujo

Comparación de la inhibición de crecimiento celular que cada proteína obtuvo, en el último día de conteo celular.

En el estudio …

Antes del IPTG

Después de IPTG y centrifugación

Abundante en sobrenadante

Solo sobrenadante (sin precipitado bacteria)

Se muestra proceso de purificación de proteínas con Imidazol.

Proteína blanco (p16) a los 100mM

de imidazol

mejor purificación de la proteína recombinante

del interés

Author What he said Check

Bai-qiu W, Cheng-hui

“Previous research in our laboratory also demonstrated that introduction of p16INK4a neither suppresses growth nor decreases colony formation rates by Anip973 and AGZY83-a cells expressing endogenous wild-type p16INK4a.”

Yi-zhao C, Rui-xiang X

“Transient p16INK4a transfection induces apoptosis whereas stable transfection markedly suppresses cell growth and cloning Efficiency”

Robertson KD, Jones PA

“p12 cannot bind cyclin-dependent kinase CDK4 and acts in a pRb- independent manner”

Harbour JW, Worley L, Sun J, Yan Y, Wang XT

The administration of protein therapeutic agents has proved to be feasible and effective both in vitro and in vivo

Agree

Agree

Agree

Agree

• Based in the results of the protein transduction into A549 cell line, protein therapy may have good results and multiple advantages over gene therapy.

• It would be important to find the target of p12 and the reason for cell arrest.

• suppressive activity of p16CDK4a is the largest in comparison to p14ARF and p12.

• cell cycle arrest of the main transcriptional variants occur before DNA replication before S phase.

Education

Seminario molecular