Nucleolar proteins with altered expression in leukemic cell lines.

Kaisa J. Teittinena, Pauliina Kärkkäinena, Johanna Salonenb, Gunilla Rönnholmc,Hanna Korkeamäkia, Mauno Vihinenb,

Nisse Kalkkinenc, Olli Lohia

leukemia.

Leukemia is cancer of the blood cells. It starts in the bone marrow (the soft tissue inside most bones. Bone marrow is where blood cells are made).

Normally the bone marrow makes: White blood cells (which help body fight Infection), Red blood cells (which carry oxygen to all parts of body) and Platelets (which help blood clot).

With leukemia, the bone marrow starts to make a lot of abnormal white blood cells, called leukemia cells. They don't do the work of normal white blood cells, they grow faster than normal cells, and they don't stop growing when they should.

Over time, leukemia cells can crowd out the normal blood cells. This can lead to serious problems such as anemia, bleeding, and infections. Leukemia cells can also spread to the lymph nodes or other organs and cause swelling or pain.

INTRODUCTION.

leukemia is grouped by how fast it gets worse and

what kind of white blood cell it Affects:

-Acute leukemia gets worse very fast and cause symptoms in few time.-Chronic leukemia gets worse slowly and may not cause symptoms for years.

-Lymphocytic (or lymphoblastic) leukemia affects white blood cells called lymphocytes. -Myelogenous leukemia affects white blood cells called myelocytes.

types .

Acute lymphoblastic leukemia, or ALL.

Acute myelogenous leukemia, or AML.

Chronic lymphocytic leukemia, or CLL.

Chronic myelogenous leukemia, or CML.

types .

There are more likely to get leukemia when people:

Was exposed to large amounts of radiation. Was exposed to certain chemicals at work, such as

benzene. Had some types of chemotherapy to treat another

cancer. Have Down syndrome or some other genetic

problems. Smoke.

Risk factors.

Prohibitin. Mitochondrial prohibitin complexes control cell

proliferation, cristae morphogenesis and the functional integrity of mitochondria.

TDP-43. has been shown to be involved in regulation of

pre-mRNA splicing and repression of transcription.

Nuclear proteins.

Nucleophosmin. It is essential for normal embryonic

development and, specifically, for the control of centrosome duplication and genomic stability.

Nuclear proteins.

Some Proteins can be find with an altered

expression in the nucleoli of leukemic cells (changes in nucleolar proteomes). Prohibitin (PHB) and TAR-DNA-binding protein-43 (TDP-43) were strongly expressed in the nucleoli.

Our results demonstrate that leukemic cells have differences in their nucleolar protein composition, and suggest that it may be possible to exploit these differences in identification of leukemia subtypes.

Nucleolar protein-leukemia

This study aimed to identify nucleolar proteins that are differen- tially expressed in

various cell lines derived from acute leukemias.

Objective.

MATERIALES Y METODOS.

FUNDAMENTO: aislar células (ya sean células

procariotas, eucariotas o vegetales), cultivarse en condiciones controladas y obtener poblaciones celulares homogéneas para estudiar los procesos que ocurren en las células.

¿Para que?: Se realizo el cultivo para poder estudiar en el laboratorio estas células plasmáticas.

CULTIVO.

FUNDAMENTO: una manera eficiente para

aislar y obtener nucléolos purificados sin las demás partes del núcleo.

¿para que?: para poder medir la concentración de proteínas totales en los nucléolos.

AISLAMIENTO NUCLEOLOS.

FUNDAMENTO: Separa las proteínas según su

punto isoeléctrico y peso molecular.

¿para que?: Análisis de los extractos nucleolares y sus características.

2 DIGE.

FUNDAMENTO: identificar proteínas.

¿para que?: identificar las proteínas nucleolares.

IDENTIFICACION DE PROTEINAS.

FUNDAMENTO: detectar proteínas específicas

en una muestra determinada.

¿Para que?: Detectar las proteínas: PHB y TDP-43.

WESTERN BLOT.

FUNDAMENTO: se emplean anticuerpos para

tinción de las proteínas.

¿para que?: producir la tinción de las proteínas: PHB y TDP-43.

INMUNOFLUORECENCIA.

RESULTADOS

Leucemogénesis nucleolar

Fundamento: Aproximación proteómica para buscar proteínas cuya expresión varía entre diferentes tipos de células

leucémicas.

• Aislamiento nucleolar

Se realizó con base en varias líneas celulares reportadas en previos estudios. Marcador nucleolar: nucleofosmina/B23.

• 2-D DIGE análisis

Facilitó el análisis de la composición de proteínas nucleolares entre:AML y ALL (OCI-AML3 vs. CCRF-CEM)pre B-ALL y pre T-ALL (MHH-CALL3 vs. CCRF-CEM)

RESULTADOS

Niveles de intensidad de cada uno de las manchas de proteínas, las diferencias en los niveles expresión entrediversas líneas de células leucémicas, se compararon usando de software DeCyderTM.

36 AML/ALL29 preB/preT

RESULTADOS

RESULTADOS

Para facilitar el lugar recogiendo los geles 2-DE fueron post-teñidas con plata.Por último, las manchas de las proteínas seleccionadas fueron digeridas "en gel" y luego sometidas anano LC-de masas en tándem análisis de espectrometría

RESULTADOS

MASCOTA MS / MS programa de Búsqueda de Ion.

RESULTADOS

Expresión nucleolar de PHB

Western blot análisis en los extractos nucleolares de laslíneas de células leucémicas. Igualdad de cargas de las muestras.

Expresión nucleolar de TDP-43 y su región terminal N (TDP-43-N)

Western blot análisis. Igualdad de cargas de las muestras.

Análisis densiométrico de las intensidades de las bandas en el panel A normalizado a los niveles de CCRF-CEM.

RESULTADOS

2.9 y 1.8 a 2.7

Tinción de inmunofluorescencia de PHB en los preparativos nucleolares de CCRF-CEMy FH-CALL-3 células.

Tinción de inmunofluorescencia de TDP-43 en los preparativos nucleolar de CCRF-CEM y las células MHH-CALL3.

DAPI, 4,6-diamidino-2-phenylindole.

RESULTADOS

DISCUSSION

Author Affirmation Disagree Agree

Ayala YM et al

“Interestingly, both anti-tumorigenic and pro-tumorigenicfunctions have been reported for PHB, depending on its subcellular localization”

X

Theiss AL et al

“Loss of TDP-43 in human cells causes genomic instability and increased apoptosis”

X

Ayala YM et al

“Hyperphosphorylated, ubiquitinated,and cleaved TDP-43 was identified as the main component ofcytoplasmic inclusions in two neurodegenerative diseases, frontotemporallobar degeneration (FTLD) and amyotrophic lateralsclerosis (ALS)”

X

Sasaki S et al

“Recently, an immunoelectronmicroscopicalanalysis revealed that TDP-43 localized specificallyto the nucleoli in motor neurons”

X

CONCLUSIONS

• Given the great variety of proteins that can be expressed in cells affected by both: acute myeloid leukemia and acute lymphoblastic, you can set different lines of study to do a trace of these proteins that this article says, and thus create nucleolar protein marker as possible candidates for studies in the field of leukemia research.

• The use of different techniques applied to the field of molecular biology are a source of esencial information that not only provides valuable information to detect and classify any disease, but provides guidelines and directs us to a focus of study to finally understand why are some diseases that threaten human life.

CONCLUSIONS

CONCLUSIONS

• The therapeutic and treatment of diseases that have always prevailed in the human species, is developed from studies for extrapolating the findings in the laboratory to the clinic, so we are not only making an approach to the understanding of diseases through simple tests of low price, but we can use these findings to give an effective treatment for diseases like leukemia, through genetic manipulation, for example, or enzyme induction to accelerate processes or otherwise inhibit and other methods well documented.

CONCLUSIONS

• The understanding of the various molecular processes such as replication, transcription and translation of DNA, and familiarity to the specific terms of molecular biology allow us comprendre advances and studies that are performed daily, around the world; to enrich and thus create a feedback. Likewise, we can expose and capture our unknowns in studies by ourselves and an example are the proteins implicated in the pathology of leukemia.

MAPA CONCEPTUAL

MAPA CONCEPTUAL

MAPA CONCEPTUAL

Mapa conceptual.

Gracias…