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BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells.

Woolery KT, Mohamed M, Linger RJ, Dobrinski KP, Roman J, Kruk PA - Biomed Res Int (2015)

Bottom Line: We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β.In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β.Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Cell Biology, University of South Florida, Tampa, FL 33612, USA.

ABSTRACT
Familial history remains the strongest risk factor for developing ovarian cancer (OC) and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE) cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (-1800) to (-900) region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

No MeSH data available.


Related in: MedlinePlus

IL-1β protein levels are increased in 185delAG BRCA1 mutation carriers. (a) Normal NFH and FH BRCA1 185delAG IOSE cells were analyzed for precursor (pro-) and cleaved IL-1β protein expression via western blot. Blots were then stripped and probed for β-actin as a loading control. Values represent relative densitometry. (b) Cells were plated in triplicate at similar densities and conditioned media were collected as described. IL-1β ELISA activity assay was performed in triplicate and the results are expressed as the mean ± standard error. Symbol (∗) denotes statistical significance at the 0.04 confidence level.
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fig1: IL-1β protein levels are increased in 185delAG BRCA1 mutation carriers. (a) Normal NFH and FH BRCA1 185delAG IOSE cells were analyzed for precursor (pro-) and cleaved IL-1β protein expression via western blot. Blots were then stripped and probed for β-actin as a loading control. Values represent relative densitometry. (b) Cells were plated in triplicate at similar densities and conditioned media were collected as described. IL-1β ELISA activity assay was performed in triplicate and the results are expressed as the mean ± standard error. Symbol (∗) denotes statistical significance at the 0.04 confidence level.

Mentions: To determine the relationship between the BRCA1 185delAG mutation and protein levels of IL-1β, we compared IL-1β protein levels in human OSE cell lines that endogenously carry this mutation to those with wild-type BRCA1. Both FH IOSE 3261-77 and 1816-686 cell lines with confirmed 185delAG mutation had 7-fold and ≥13-fold higher active IL-1β protein levels, respectively, as measured by western blot than the NFH cell line (IOSE-121) (Figure 1(a)). Likewise, 3261-77 and 1816-686 cell lines had 2- and 10-fold, respectively, higher levels of secreted IL-1β protein in their CM as measured by ELISA than the IOSE-121 cell line (Figure 1(b)).


BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells.

Woolery KT, Mohamed M, Linger RJ, Dobrinski KP, Roman J, Kruk PA - Biomed Res Int (2015)

IL-1β protein levels are increased in 185delAG BRCA1 mutation carriers. (a) Normal NFH and FH BRCA1 185delAG IOSE cells were analyzed for precursor (pro-) and cleaved IL-1β protein expression via western blot. Blots were then stripped and probed for β-actin as a loading control. Values represent relative densitometry. (b) Cells were plated in triplicate at similar densities and conditioned media were collected as described. IL-1β ELISA activity assay was performed in triplicate and the results are expressed as the mean ± standard error. Symbol (∗) denotes statistical significance at the 0.04 confidence level.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556869&req=5

fig1: IL-1β protein levels are increased in 185delAG BRCA1 mutation carriers. (a) Normal NFH and FH BRCA1 185delAG IOSE cells were analyzed for precursor (pro-) and cleaved IL-1β protein expression via western blot. Blots were then stripped and probed for β-actin as a loading control. Values represent relative densitometry. (b) Cells were plated in triplicate at similar densities and conditioned media were collected as described. IL-1β ELISA activity assay was performed in triplicate and the results are expressed as the mean ± standard error. Symbol (∗) denotes statistical significance at the 0.04 confidence level.
Mentions: To determine the relationship between the BRCA1 185delAG mutation and protein levels of IL-1β, we compared IL-1β protein levels in human OSE cell lines that endogenously carry this mutation to those with wild-type BRCA1. Both FH IOSE 3261-77 and 1816-686 cell lines with confirmed 185delAG mutation had 7-fold and ≥13-fold higher active IL-1β protein levels, respectively, as measured by western blot than the NFH cell line (IOSE-121) (Figure 1(a)). Likewise, 3261-77 and 1816-686 cell lines had 2- and 10-fold, respectively, higher levels of secreted IL-1β protein in their CM as measured by ELISA than the IOSE-121 cell line (Figure 1(b)).

Bottom Line: We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β.In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β.Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Cell Biology, University of South Florida, Tampa, FL 33612, USA.

ABSTRACT
Familial history remains the strongest risk factor for developing ovarian cancer (OC) and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE) cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (-1800) to (-900) region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

No MeSH data available.


Related in: MedlinePlus