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Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
Seminario molecular
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Seminario molecular

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  • 1. Manuela Parra Cardona 3rd Semester Medicine student Texto adicional Texto adicional Texto adicional texto adicional Texto adicional texto adicional Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549 Wei Zhang ( Laboratory of Medical Genetics, Harbin Medical University) Journal of experimental & clinical cancer research
  • 2. INTRODUCTION Cell cycle strictly ordered process REGULATED BY <ul><li>Cyclins and CDKs </li></ul><ul><li>negative regulators such CKIs </li></ul>Tumor suppressor CDKN2A p16INK4a p14ARF p12 Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 3. GENERAL OBJETIVE Compare the inhibitory effects of the three transcriptional variants of CDKN2A in human lung cancer cell line A549 with endogenous expression deficiency. Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 4. CANCER Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 5. http://www.rndsystems.com/DAM_public/5174.gif Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 6. TRANSCRIPTION It 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 Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 7. LUNG CANCER Occurs when cells in the lung, change to become abnormal. Types of Lung Cancer There are 2 main types of lung cancer. • Non-Small Cell Lung Cancer This 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 Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 8. CDKN2A Tumor 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 Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 9. 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 P53 P53 degradation Arrest of cell cycle Normal cell cycle Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 10. P12 <ul><li>Defects in CDKN2a gene are involved in tumor formation in a wide range of tissues. </li></ul><ul><li>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. </li></ul>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: Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 11.  
  • 12. CULTIVO CÉLULAR F12-K con FBS (fetal bovine serum) Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 13. RT-PCR Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549 RNA TRANSCRIPTAS INVERSA DNAc AGZY H446 y células pancreáticas (expresión) DNAc de P16, P14 y P12
  • 14. Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549 WESTERN BLOTTING Proteínas <ul><li>para detectar proteínas específicas </li></ul>P16INK4A
  • 15. 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 Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549
  • 16. 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. Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549 En el estudio…. Evaluaron el estadio en el cual se encontraban del ciclo celular, después de la expresión de p16, p14 y p12.
  • 17.  
  • 18. RT-PCR of A459 Expression of P16INK4a, P14ARF and p12 were confirmed. 543 bp 493 bp 372 bp P16INK4a P14ARF P12
  • 19. Inmunohistoquímica Se detecta expresión de p16, p14 y p12, en células clonadas.
  • 20. Citometria de flujo Comparación de la inhibición de crecimiento celular que cada proteína obtuvo, en el último día de conteo celular.
  • 21. 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
  • 22.  
  • 23. Agree Agree Agree Agree 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
  • 24.  
  • 25. <ul><li>Based in the results of the protein transduction into A549 cell line, protein therapy may have good results and multiple advantages over gene therapy. </li></ul><ul><li>It would be important to find the target of p12 and the reason for cell arrest. </li></ul><ul><li>suppressive activity of p16CDK4a is the largest in comparison to p14ARF and p12. </li></ul><ul><li>cell cycle arrest of the main transcriptional variants occur before DNA replication before S phase. </li></ul>
  • 26.  

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