Download BRCA1 Modulates Xenobiotic Stress-inducible Gene

1. In quali circostanze è stata identificata?
2. Qual è il suo ruolo fisiologico?
3. Com’è coinvolta nella risposta agli xenobiotici?
4. Cosa può comportare una sua alterazione funzionale?
5. Che ruolo può avere come target farmacologico?
The BRCA1 gene was identified and cloned in 1994
based on its linkage to early onset breast cancer
and breast-ovarian cancer syndromes in women.
While inherited mutations of BRCA1 are responsible for about 40–45% of
hereditary breast cancers, these mutations account for only 2-3% of all breast
cancers, since the BRCA1 gene is rarely mutated in sporadic breast cancers.
However, BRCA1 expression is frequently reduced or absent in sporadic cancers,
suggesting a much wider role in mammary carcinogenesis. (Eliot M. Rosen et al.
2003)
The BRCA1 gene on chromosome 17q12-21 was
identified and cloned by Miki et al. (1994)
Domains associated with
specific assays or activities are
depicted. BRCA1 contains the
N-terminal RING domain,
nuclear localization signal
(NLS), and two C-terminal
BRCT domains.
(Marcelo A. Carvalho et al.
2007)
Cell cycle progression, several highly specialized
DNA repair processes, DNA damage responsive cell
cycle checkpoints, regulation of a set of specific
transcriptional pathways, and apoptosis.
The interplay between ERα and BRCA1 pathways and its impact on cell
proliferation (1) and genome stability maintenance (2). Solid arrows indicate a
direct role while the dashed arrow indicates an indirect pathway (Yanfen Hu
2009)
L’interazione con p53 testimonia il ruolo chiave nella progressione del
ciclo cellulare, per la comune espressione di oncosoppressori (p21,
p27, Bax). MEF BRCA-/- presentano inoltre una scarsissima
proliferazione a meno di un contemporaneo knockout di p53 (Zheng
et al., 2001).
BRCA1 è coinvolta in ogni
meccanismo di riparo del
DNA (HR, NHEJ, MR) in
misura diversa.
La sua presenza nelle foci di
riparo insieme a RAD51 è
indipendente da altri fattori
come H2AX o MDC1. (Roger
A. Greenberg 2008)
Chu-Xia Deng 2006
Tra le funzioni della stabilità genomica mediate da BRCA1
si nota come essa medi la nucleazione del fuso mitotico
garantendo la separazione dei nucleotidi fratelli solo
quando tutti i cromosomi sono stabilmente “agganciati”.
Il modello schematico di come ciò possa accadere è
spiegato nella figura in basso. (Chu-Xia Deng 2006)
(C – D) immunofluorescenza Dapi e anticorpi
anti-tubulina. (E – F) anticorpi fluorescenti anti
H3. (C – F) Brca1D11/D11 (D – F) Brca1wt.
BRCA1 targets the conserved C-terminal activation domain (AF-2) of ER-a, since it
blocked the ligand-dependent activation of AF-2 in a specific assay of AF-2
dependent transactivation. These findings suggest a mammary tissue-specific
function for BRCA1 (Fan et al., 1999a)
Figure 1. The ubiquitination cascade.
Figure 2. BRCA1/BARD1 ubiquitinate phosphorylated
RNAPII. DNA damage results in the phosphorylation of
RNAPII, and BRCA1/BARD1 bind to the phospho-RNAPII to
catalyze its ubiquitination.
Figure 3. BRCA1-dependent E3 ubiquitin ligase prevents
centrosomal hypertrophy. The activity of the
BRCA1/BARD1 ubiquitin ligase marks centrosomes as
post-duplicated, preventing amplification of centrosome
number, a common feature of breast tumors. This activity
of BRCA1 also regulates the activity of the centrosome in
assembling the microtubule network of the cell.
(Lea M. Starita, Jeffrey D. Parvin 2006)
In the present study, we have investigated the
effects of BRCA1 on xenobiotic stress-inducible gene
expression.
In response to aryl hydrocarbon receptor (AhR)
ligands, cytoplasmic AhR becomes activated and
then translocates to the nucleus where it forms a
complex with the aryl hydrocarbon receptor nuclear
translocator (ARNT). Subsequently, the AhR-ARNT
complex binds to the enhancer or promoter of genes
containing a xenobiotic stress-responsive element
and regulates the expression of multiple target
genes including cytochrome P450 subfamily
polypeptide 1 (CYP1A1). (Hyo Jin Kang et al. 2006)
The effect of exogenous BRCA1 on TCDD-induced
gene expression.
Subconfluent MCF-7 cells transfected with a BRCA1
expression vector or pcDNA3 (empty vector) were
incubated for 24 h and then treated with 10 nM
TCDD for an additional 24 h. Total cellular RNA was
then extracted to measure the relative amounts of
CYP1A1 (A) and CYP1B1 (B) by quantitative reverse
transcription-PCR.
The effect of exogenous BRCA1 on a XREcontaining promoter in TCDD treated cells.
Exponentially proliferating cells (MCF-7 (A),
T47D (B), and ZR-75–1 (C)) were transiently
transfected with empty vector or the BRCA1
expression vector and a p(XRE-1A1)-Luc
reporter plasmid, treated with 10 nM TCDD,
and assayed for luciferase.
D, MCF-7 cells were transfected with BRCA1
and the p(Cyp1a1)-Luc reporter (34) and
treated with 10 nM TCDD, and the promoter
activity was monitored.
The effect of other AhR ligands on the
activity of a XRE-containing promoter in
the presence of exogenous BRCA1.
A, exponentially proliferating MCF-7 cells
were transiently transfected with BRCA1
and p(Cyp1a1)-Luc reporter, incubated
with AhR ligands, 10 nM TCDD, 1 M
DF203, or 1 M 5F203, harvested, and
assayed for luciferase activity. Cells (MCF7 (B) and T-47D (C)) were transfected
with BRCA1 or p(XRE-1A1)-Luc reporter
plasmid and treated with 10 nM TCDD,
1M DF203, or 1M 5F203 for only 8 h
before luciferase activities were assayed.
TCDD (2,3,7,8-Tetrachlorodibenzodioxin)
BRCA1 ?
• Circa l’80% delle donne con mutazioni a carico di
BRCA1 o BRCA2 svilupperà il carcinoma della mammella
durante la propria vita, con elevati rischi anche per lo
sviluppo di carcinoma ovarico.
RUOLO DI BRCA NEL RECLUTAMENTO DI RAD50- RAD51 (riparazioni dei
danni al DNA da radiazioni)
Mutazioni di BRCA1 o
BRCA2 sono invece rare o
del tutto assenti nei
carcinomi mammari
sporadici
(non a carattere
ereditario).
Nonostante ciò in questo
tipo di tumori si
riscontrano comunque
alterazioni del pathway di
BRCA, dovute soprattutto
a modifiche nei suoi livelli
di espressione.
ALTERAZIONE NELL’
ESPRESSIONE DI BRCA
(perdita,totale o parziale, di
almeno una copia del gene o
inattivazione funzionale dovuta
a fenomeni epigenetici, quali la
metilazione del promotore)
Nelle donne che
presentano mutazioni
ereditarie nel gene per
BRCA.
Nelle donne che hanno
sviluppato un carcinoma
mammario o ovarico e
presentano alterazioni
nell’espressione di BRCA
L’ indotta
amplificazione della
risposta di ARNT ad
uno xenobiotico
(TCDD) ripristina i
normali livelli di
attività XRE anche in
assenza di BRCA
Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADPribose) polymerase
Helen E. Bryant, Niklas Schultz, Huw D. Thomas, Kayan M. Parker, Dan
Flower, Elena Lopez, Suzanne Kyle, Mark Meuth, Nicola J. Curtin & Thomas
Helleday. 2007
V-C8 (BRCA2 MUTANT)
V-C8+B2 (BRCA2 COMPLEMENTED)
Andamento della
crescita di una massa
tumorale in presenza di
inibitori di PARP1 per
topi difettivi di BRCA1
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