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Lina Acevedo ForeroNatalí Carvajal Pérez
Texto adicional Texto adicional
Texto adicional texto adicional
Texto adicional texto adicional
Masatoshi Takagi, Masaki Sato, Jinhua Piao, Satoshi Miyamoto, Takeshi Isoda,
Masanobu Kitagawa, Hiroaki Honda, Shuki Mizutani.
ATM-dependent DNAATM-dependent DNAdamage-response pathwaydamage-response pathway
as a determinant in as a determinant in chronic myelogeneus leukemiachronic myelogeneus leukemia
INTRODUCTIONINTRODUCTION
LEUKEMIALEUKEMIA
• Clonal bone marrow stem cell disorder in which a proliferation of mature granulocytes (neutrophils,eosinophils and basophils) and their precursors is found.
• White blood cells
• Characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow
• The accumulation of these cells in the blood
Chronic myeloid leukemia (CML) Chronic myeloid leukemia (CML)
BCR-ABLBCR-ABL
Philadelphia chromosome, a reciprocal translocation between chromosomes 9 and 22, which results in oncogenic BCR-ABL gene
fusion
Cell proliferation activity Anti-apoptotic activity by tyrosine phosphorylation
Instability induced by BCR-ABL lead to the loss of function of p53, p16 or p14, an important cell cycle checkpoints.
verify whether the processes at each phase of the cell cycle have been completed before progression into the next phase.
BCR-ABLBCR-ABL
CHEKPOINTS
Assess DNA damage, which is detected by sensor mechanisms.
Uses a signal mechanism either to stall the cell cycle until repairs are made or, if repairs cannot be made, to target the cell for destruction. In this specific case (CML), an oncogenic stimuli enforce cell proliferation, which is enabled by DNA replication.
DNA damage
DNA damage facilitates activation of DNA damage checkpoint pathways
ATM (ataxia telangiectasia mutated)
activation
Can lead to cell cycle arrest, apoptosis to prevent the progression of oncogenic transformation
Ataxia-telangiectasia Ataxia-telangiectasia
• Autosomal recessive disorder• Characterized by cerebellar ataxia, telangiectasia,
immune defects, and a predisposition to malignancy.
the molecule responsible for A-T, plays a central role in the DNA damage-response pathway.
ATM
General ObjectiveGeneral Objective
Investigate the possible role of DNA damage check points for preventing blast crisis by crossing BCR- ABL transgenic CML mice with hemi-or homozygous Atm-knockout mice.
Materiales y MétodosMateriales y MétodosInmunohistoquímica
• Aplicado a una muestra de tejido orgánico, correctamente fijada e incluida en parafina.
• Inmunotinción que permite demostrar antígenos presentes en células o tejidos utilizando anticuerpos marcados.
• Utiliza un anticuerpo específico que es capaz de unirse a antígenos correspondientes.• Esto es visible solo si el anticuerpo esta marcado con una sustancia para que puede
localizarse el complejo antígeno - anticuerpo.• Inmunoperoxidasa se usan como marcadores enzimas capaces de poner color a
un sustrato incoloro (peroxidadsa, fosfatasa alcalna)
Parafina Inmunoperoxidasa tinción Al tejido se le añadio Vectastain
(formaldehido) elite kit (detectar)
Anticuerpos Ser 1981-fosforilado, Thr 68-fosforilado, generic ATM y ChK2
Presencia de ATM fosforilado se cuantifico usando imágenes de software
Materiales y MétodosMateriales y MétodosCélulas
Obtuvo una IL-3 produce línea celular WEHI-3B (leucémica) IL-3 Produce línea celular de Ba/F3
Células se mantuvieron en RPMI 1640 con 10% de suero fetal y 10% de WEIH-3B.
Materiales y MétodosMateriales y MétodosWestern Blot
Técnica usada para detectar proteínas específicas en una muestra determinada.
Materiales y MétodosMateriales y MétodosWestern Blot
Materiales y MétodosMateriales y MétodosRatones
BCR-ABL ratón transgénico heterocigotoATM knockout ratones heterocigotos(se cruzaron)
Se generaron y usaron 4 genotipos diferentes:-BCR-ABL ratón transgénico heterocigoto con el alelo ATM wild-Alelo ATM knockout heterocigoto -Alelo ATM knockout homocigoto -Alelo ATM raton knockout homocigoto
Análisis de genotipo ATM fue realizado por PCR genómico para distinguir el alelo ATM wild o el ATM knockout
Materiales y MétodosMateriales y MétodosCitometría de flujo
Utilización de luz láser, para el recuento y clasificación de células(Medula ósea fue lisado con una solución salina ; y las células mononucleares fueron aisladas) según: características morfológicas, presencia de biomarcadores, y en la ingeniería de proteínas.(kit H2AX)
Resultados
Resultados
Resultados
Resultados
Resultados
Resultados
Resultados
Resultados
Discussion
Author Phrase Agree / disagree
Tomasz Skorski “Several studies have directly addressed the relationshipbetween BCR-ABL and molecules involved in DNA repair”
Agree
M. Swift, D. Morrell, E. Cromartie, A.R. Chamberlin, M.H. Skolnick, D.T. Bishop
“Nevertheless, it remains controversial whether the risk of cancer development is higher inA-T carriers, and the relationship between ATM SNPs and cancersusceptibility remains uncertain”
Agree
Discussion
Author Phrase Agree / disagree
T. Stankovic, P. Weber, G. Stewart, T. Bedenham, J. Murray, P.J. Byrd, P.A. Moss,A.M. Taylor
“Thus, although ATM is known to be important for preventing oncogenicTransformation”
Agree
T. Shigeta, M. Takagi, D. Delia, L. Chessa, S. Iwata, Y. Kanke, M. Asada, M. Eguchi,S. Mizutani
“We previously reported that A-T heterozygous carrier-derivedcells are defective in apoptosis induction associated with moderatedefects in cell-cycle regulation”
Agree
Conclusions
• The DNA damage checkpoint is ATM, this is responsible for the initiation of the damage of the cells, thus affecting the bone marrow and thus generates tumorigenic cells.
• BCR-ABL expression and the level and activity of proteins involved in DNA repair, particularly the repair of DNA doublestrand breaks have high relationship with each other and pathology Chronic myelogenous leukemia (CML)
Conclusions
• Use of DNA damage checkpoint to prevent the progression of oncogenic transformation is a great advance in the control of propagation. The DNA damage-response pathways may play an important role in the clinical course of CML and its blast crisis.
• ATM failure actually accelerates the development of tumors arises when an oncogenic event in the cells. However, it can also serve as a reference for various modes of prevention within the same disease, but lack know many mechanisms of action.