BRCA1 Associated Epigenetic Regulation of p73 Mediates Effector Pathway For Chemosensitivity in Ovarian Carcinoma
BRCA1 Associated Epigenetic Regulation of p73 Mediates Effector Pathway For Chemosensitivity in Ovarian Carcinoma
BRCA1 Associated Epigenetic Regulation of p73 Mediates Effector Pathway For Chemosensitivity in Ovarian Carcinoma
TAP73 does not play any role in BRCA1 expressing cells but
knock down of TAP73 can abolish the effect of cisplatin.
MATERIAL AND METHODS
Tissue culture and reagents for study of murine model
Murine ovarian carcinoma cell lines (T1,T2,T3,TBR2,TBR5 and
TBR6).
Western blot analysis to confirm the absence of whole length
BRCA1 protein.
These lines were maintained in 10% fetal bovine serum ,100
IU/ML Penicillin and 100 microgram /ml of streptomycin.
For study of human cells
UWB 1.289 human cell lines.
1:1 RPMI 1640/MEGM supplemented with 3% fetal bovine serum.
RNA analysis ,lentiviral/retroviral production
And chromatin immunoprecipitation
QRT-PCR analysis
Cotransfection is used to generate lentiviral and retroviral stock.
Bisulphite treatment and methylation analysis
200 to 500ng of genomic DNA were used.
After confirmation of desired product methylation was quantitated in
sequence product by measuring the heights of the C and T peaks.
Patient sample acquisition and processing
Tumor specimens were obtained from from the MGH gynecological
tissue repository.
RNA/DNA was isolated from dry frozen tumor samples.
RESULTS
Cisplatin sensitivity of BRCA1 deficient ovarian
carcinoma cells
BRCA1 associated tumor lines consistently exhibited significantly
increased sensitivity to cisplatin comparison to wild type in murine
model.
In human assay showed the same result confirming the increase
platinum sensitivity of UWB 1.289 cells relative to their isogenic
BRCA1 counter part.
Ibrahim et all found that cisplatin induces apoptotic death in both
murine and human BRCA1 deficient cells ,as evidence by cleavage of
poly (ADP ribose) polymerase on western blot analysis.
Expression of P73 and its target genes correlate with
cisplatin sensitivity in BRCA1 deficient cells
After cisplatin induction Ibrahim etall observed robust induction of
the P53 regulated proapoptotic genes Noxa ,Puma and P53AIP1 in
both human and murine BRCA1 deficient cells but not in wild type.
These finding suggest a distinct cell death pathway in the absence of
BRCA1 which involve P53 family member P73.
TAP73 is necessary for cisplatin sensitivity
TAP73 knock down essentially abolished cisplatin induced Noxa
expression in the three murine BRCA1 deficient ovarian carcinoma
cells.
Knock down of TAP73 induced significant resistant in human BRCA1
deficient UWB 1.289 cells although it did not affect proliferation and
cell viability in the absence of cisplatin.
The ZEB1 transcriptional repressor of TAP73 in BRCA1
expressing cells
Ibrahim et all observed that binding of ZEB1 with TAP73 potently
repressed TAP73 mRNA expression.
This is the main reason that why the phenomenon of TAP73 exist only
in BRCA1 deficient ovarian carcinoma.