Seminario biomol 2

DELATED IN BREAST CANCER1(DBC1) DEFICIENCY RESULTS IN APOPTOSIS OF

BREAST CENCER CELLS THROUGH IMPAIRED RESPONSES TO UV-INDUCED DNA DEMAGE

Wootae kim, Ja- Eun KiMSantiago Díaz

María Clara González C.Third semester –Medicine

INTRODUCTION Breast cancer is an issue that plagues women around the

world, mostly for its poor prognosis if not diagnosed early, it was found that DBC1 protein is eliminated in the cells of breast cancer, is characterized by the regulation of various processes the main this being promoter or tumor suppressor, in the same way that the SIRT1 protein, these proteins is almost always dependent on one another and always have been studied in haw,, this study was commissioned independent study DBC1 and SIRT1, in response to genotoxic stress in this case exposure to UV rays.

GENERALITIES

CANCER Poliferation of cells

Poliferation of cells

Invade tissue at a given time and invade other tissues.

Invade tissue at a given time and invade other tissues.

Damage or mutation in the DNA that could not

be repaired in time

Damage or mutation in the DNA that could not

be repaired in time

the cell couldn´t do apoptosis

the cell couldn´t do apoptosis

UV radiationUV radiation

The snuffThe snuff

PollutionPollution

DBC1 Encoded by the DBC1 gene

Encoded by the DBC1 gene

Regule various processes

Regule various processes

Transcriptionn Transcriptionn

Heterochromatin formation

Heterochromatin formation

Metabolism of the RNA splicingMetabolism of

the RNA splicing

Apoptosis and cell proliferationApoptosis and cell proliferation

Suppress several functions of SIRT1Suppress several

functions of SIRT1Suppress or promote cancer

Suppress or promote cancer

APOPTOSIS Is the programmed

cell deathIs the programmed

cell death

Intracellular or extracellular stimulus

Intracellular or extracellular stimulus

Ineffective DNA repair

Ineffective DNA repair

Is essential for

Embryonic development

Embryonic development

The development of organs and systemsThe development of organs and systems

maintenance of cellular homeostasis

maintenance of cellular homeostasis

defense against pathogens

defense against pathogens

defense against tumor development.

defense against tumor development.

DNA DAMAGE BY UV RADIATION Is a genotoxic for DNA molecule

Is a genotoxic for DNA molecule

A mutagen capable of increasing the levels of spontaneous mutation in the cell

A mutagen capable of increasing the levels of spontaneous mutation in the cell

which can be

Mutation in germs cell

Hereditary diseases in future generationsHereditary diseases in future generations

Mutation in somatic cells

Degenerative diseases and carcinogenic processes

Degenerative diseases and carcinogenic processes

GENERAL PURPOSE

This study utilized in cancer cells lacking the interaction between DBC1 and SIRT1, in order to investigate the functions of SIRT1-independent to DBC1 in response to stress degenerative

MATERIALES Y METODOS

CULTIVO DE LAS CELULAS Y LA IRRADIACION UV

1.Extracion de celulas

2.Dulbecco’s modified eagle medium + Suero salino

3. Adicion : 100 ml de penicilina G sódica 100 ml de sulfato de estreptomicina 0.25g/ml anfotericina B

4. Las células que crecieron , fueron lavadas FS

5. UV (254nm)



DBC1 Y TRANSFECCION DE siRNA

•AUGUAUUGGCCUGUAUUAG

•CAGCUUGCAUGACUACUUU

• Transfeccion se realizo con 20 nm de siRNA

•Lipofectaina RNAmax


PREPARACION DE CELULAS ENTERAS Y FRACCION DE CROMATINA

1. Lisis de celulas ( NENET)

2. 10` en hielo y 5` centrifugacion

3. Sedimento se incubo con 0.2 HCL 1N

4. 20` en hielo y centrifugar

5. Neutralización con NAOH 0.2N

6. FRACCIÓN DE CROMATINA

SOBRENADANTE ( CELL ENTERAS)


WASTERN BLOT

•Protocolo de rutina

•Anticuerpos: Anti B actina Dimero de pirimidina ciclobutano. yH2AX pH3H3 pS317 ChK1 ChK2 pS1524 BRCA1 ps428-ATR ATR pS1981-ATM


MEDIDA DE LA FORMACIÓN FOTOPRODUCTO INDUCIDA POR UV Y REPARACIÓN POR ANÁLISIS DE SLOT-BLOT1.Cell se trataron con radiacion UV ( 3J/m2)

2.Purificacion DNA genómico (DNeasy Blood and Tissue kit (Qiagen, MD, USA).

3. Desnaturalizacion DNA

4.DNA descubierto a una membrana nitrocelulosa.,se humedecio con buffer(SSC) SLOT BLOT

5. 80 grados 2h.


ENSAYOS PARA LA G2 / M PUESTO DE CONTROL DE DAÑO EN EL ADN

•Punto de control de la mitosis G2 Forsforiliacion: histona H3 Ser10 (pH 3)

•2h después de la irradiación UV , las células se tiñeron con anticuerpo anti-pH 3

•Las células positivas se analizaron por fluorescencia


TINCIÓN HOECHST

1. Celulas fijadas con formaldehido 10´

2. Se permeabilizaron con Triton X-100 5´

3. Se tiñeron Hoechst 33342 5´

4. Al microscopio de fluorescencia, NO cuerpos apoptoticos.


INFECCION POR RETROVIRUS

•pBabePuro-SFP

•Las células fueron infectadas con el retrovirus en presencia de 8 mg / ml de polibreno y luego transfectadas con siRNA DBC1.

RESULTADOS

Resultados

Discussion

A.M. Trauernicht Beyond the present study, the role of DBC1 in promoting cell survival has been investigated in other cellular contexts. Knock- down of DBC1 enhances cell death of estrogen receptor-positive breast cancer cells in an estrogen-independent manner

Yes he supports this study

R. Sundararajan In contrast to its survival function, DBC1 may act as a death promoter. Upon treatment with tumor necrosis factor (TNF)-a, DBC1 is cleaved in a caspase-dependent manner and its C-terminal fragment stimulates apoptosis through mitochondrial dysregulation

He doesnt support this study

J. Yuan, K. LuoL. Zannini

Furthermore, in response to DNA damage, ATM/ATR-mediated phospho rylation of DBC1 at Thr454 enhances its interaction with SIRT1 and then this interaction leads to greater inhibition of the deacetyla se activity of SIRT1. In addition, the association between DBC1 and SIRT1 maintain s the acetylation of cell cycle regulators such as p53 and FOXO, which facilitate the cell cycle arrest and apoptosis

They dont support this study

A.M. Trauernicht In addition, DBC1 is transcriptional ly upregulated by the acquisition of endocrine hormone resistance and the absence of DBC1 intensi- fies apoptosis in such estrogen- resistant cell lines

Yes they support the study

Personal conclussions

We can conclude with the present study and the information that was given, supression of DBC 1 in a radiotherapy against cancer could be a usefull method to increse the efectiveness of the treatment

personal conclussions

Thus DBC 1 seems to be implied in reparation of GG dimers that are a product of exposure to UV light, then we can conclude that if we can modulate the activity of this protein in certain ocassions then we can use it to prevent dermic cancers that are induced by UV exposure

personal conclussions

Another conclusion its that in certain ocasions DBC 1 can be a cancer promoter, for example in breast cancer or by the promotion of methastasis in esophagical cancer, so the risk of doing some treatment that involves this protein in this moment is very hight, thats why it need further investigation

Personal conclussions

We can conclude that if we take all the functions of DBC 1, then we can see that depending of the way that a patient is going to be treat it can be an ally or an enemy. For example for quimiotheraphy DBC 1 could be an ally by reducing the activity of SIRT 1 and increasing the genotoxic stress, but if we use radiotherapy then it becomes an enemy.

Concept maps

conclusiones

GRACIAS !

Seminario biomol 2

Technology

Transcript of Seminario biomol 2