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CSN6 deregulation impairs genome integrity in a COP1-dependent pathway.

Choi HH, Su CH, Fang L, Zhang J, Yeung SC, Lee MH - Oncotarget (2015)

Bottom Line: In this study, we identify CSN6's biological function in regulating genome integrity.Also, COP1 overexpression leads to downregulation of p27(Kip1), thereby promoting the expression of mitotic kinase Aurora A.Overexpression of Aurora A correlates with poor survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Understanding genome integrity and DNA damage response are critical to cancer treatment. In this study, we identify CSN6's biological function in regulating genome integrity. Constitutive photomorphogenic 1 (COP1), an E3 ubiquitin ligase regulated by CSN6, is downregulated by DNA damage, but the biological consequences of this phenomenon are poorly understood. p27(Kip1) is a critical CDK inhibitor involved in cell cycle regulation, but its response to DNA damage remains unclear. Here, we report that p27(Kip1) levels are elevated after DNA damage, with concurrent reduction of COP1 levels. Mechanistic studies showed that during DNA damage response COP1's function as an E3 ligase of p27 is compromised, thereby reducing the ubiquitin-mediated degradation of p27(Kip1). Also, COP1 overexpression leads to downregulation of p27(Kip1), thereby promoting the expression of mitotic kinase Aurora A. Overexpression of Aurora A correlates with poor survival. These findings provide new insight into CSN6-COP1-p27(Kip1)-Aurora A axis in DNA damage repair and tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

CSN6 expression leads to mitotic defect and ROS production(A) Stably expressing Myc-CSN6 (U2OS/Myc-CSN6) and Vector (U2OS vector) cells were stained with DAPI (4ʹ,6-Diamidino-2-Phenylindole, Dihydrochloride) and percentages of mitotic defects as demonstrated by bigger nuclei, micro nuclei, and fused nuclei were compared. Phase-contrast images and merged images of the same microscopic fields are shown. (B) ROS production (green fluorescence) was detected by DCFDA and fluorescence microscopy in indicated cells. Phase-contrast images and merged images of the same microscopic fields are shown. (C) CSN6 overexpressing cells have increased steady-state expressions of Aurk A and γ-H2AX. Equal amounts of cell lysates were immunoblotted with indicated antibodies.
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Figure 1: CSN6 expression leads to mitotic defect and ROS production(A) Stably expressing Myc-CSN6 (U2OS/Myc-CSN6) and Vector (U2OS vector) cells were stained with DAPI (4ʹ,6-Diamidino-2-Phenylindole, Dihydrochloride) and percentages of mitotic defects as demonstrated by bigger nuclei, micro nuclei, and fused nuclei were compared. Phase-contrast images and merged images of the same microscopic fields are shown. (B) ROS production (green fluorescence) was detected by DCFDA and fluorescence microscopy in indicated cells. Phase-contrast images and merged images of the same microscopic fields are shown. (C) CSN6 overexpressing cells have increased steady-state expressions of Aurk A and γ-H2AX. Equal amounts of cell lysates were immunoblotted with indicated antibodies.

Mentions: CSN6 is overexpressed in many types of cancer. To understand the biological consequence of this deregulation, we established CSN6 stable expressing clones and checked their cell cycle regulation. We found that CSN6 expressing clones have increased numbers of cells with bigger nuclei, small nuclei and fused nuclei (Figure 1A), suggesting mitotic defects in these cells. We also found that these CSN6 expressing clones have elevated reactive oxygen species (ROS), suggesting potential DNA damage (Figure 1B). We then found that these cells have elevated Aurora kinase A and γH2AX (a surrogate marker of DNA double strand breaks) when compared with control cell line as assayed by immunostaining (Figure 1C). Further, these CSN6 expressing cells have high levels of COP1, a downstream target of CSN6, with concurrent high Aurora kinase A and γH2AX, as evidenced by immunoblotting. This observation suggests that CSN6-COP1 axis may be involved in DNA damage and mitotic defect.


CSN6 deregulation impairs genome integrity in a COP1-dependent pathway.

Choi HH, Su CH, Fang L, Zhang J, Yeung SC, Lee MH - Oncotarget (2015)

CSN6 expression leads to mitotic defect and ROS production(A) Stably expressing Myc-CSN6 (U2OS/Myc-CSN6) and Vector (U2OS vector) cells were stained with DAPI (4ʹ,6-Diamidino-2-Phenylindole, Dihydrochloride) and percentages of mitotic defects as demonstrated by bigger nuclei, micro nuclei, and fused nuclei were compared. Phase-contrast images and merged images of the same microscopic fields are shown. (B) ROS production (green fluorescence) was detected by DCFDA and fluorescence microscopy in indicated cells. Phase-contrast images and merged images of the same microscopic fields are shown. (C) CSN6 overexpressing cells have increased steady-state expressions of Aurk A and γ-H2AX. Equal amounts of cell lysates were immunoblotted with indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494904&req=5

Figure 1: CSN6 expression leads to mitotic defect and ROS production(A) Stably expressing Myc-CSN6 (U2OS/Myc-CSN6) and Vector (U2OS vector) cells were stained with DAPI (4ʹ,6-Diamidino-2-Phenylindole, Dihydrochloride) and percentages of mitotic defects as demonstrated by bigger nuclei, micro nuclei, and fused nuclei were compared. Phase-contrast images and merged images of the same microscopic fields are shown. (B) ROS production (green fluorescence) was detected by DCFDA and fluorescence microscopy in indicated cells. Phase-contrast images and merged images of the same microscopic fields are shown. (C) CSN6 overexpressing cells have increased steady-state expressions of Aurk A and γ-H2AX. Equal amounts of cell lysates were immunoblotted with indicated antibodies.
Mentions: CSN6 is overexpressed in many types of cancer. To understand the biological consequence of this deregulation, we established CSN6 stable expressing clones and checked their cell cycle regulation. We found that CSN6 expressing clones have increased numbers of cells with bigger nuclei, small nuclei and fused nuclei (Figure 1A), suggesting mitotic defects in these cells. We also found that these CSN6 expressing clones have elevated reactive oxygen species (ROS), suggesting potential DNA damage (Figure 1B). We then found that these cells have elevated Aurora kinase A and γH2AX (a surrogate marker of DNA double strand breaks) when compared with control cell line as assayed by immunostaining (Figure 1C). Further, these CSN6 expressing cells have high levels of COP1, a downstream target of CSN6, with concurrent high Aurora kinase A and γH2AX, as evidenced by immunoblotting. This observation suggests that CSN6-COP1 axis may be involved in DNA damage and mitotic defect.

Bottom Line: In this study, we identify CSN6's biological function in regulating genome integrity.Also, COP1 overexpression leads to downregulation of p27(Kip1), thereby promoting the expression of mitotic kinase Aurora A.Overexpression of Aurora A correlates with poor survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Understanding genome integrity and DNA damage response are critical to cancer treatment. In this study, we identify CSN6's biological function in regulating genome integrity. Constitutive photomorphogenic 1 (COP1), an E3 ubiquitin ligase regulated by CSN6, is downregulated by DNA damage, but the biological consequences of this phenomenon are poorly understood. p27(Kip1) is a critical CDK inhibitor involved in cell cycle regulation, but its response to DNA damage remains unclear. Here, we report that p27(Kip1) levels are elevated after DNA damage, with concurrent reduction of COP1 levels. Mechanistic studies showed that during DNA damage response COP1's function as an E3 ligase of p27 is compromised, thereby reducing the ubiquitin-mediated degradation of p27(Kip1). Also, COP1 overexpression leads to downregulation of p27(Kip1), thereby promoting the expression of mitotic kinase Aurora A. Overexpression of Aurora A correlates with poor survival. These findings provide new insight into CSN6-COP1-p27(Kip1)-Aurora A axis in DNA damage repair and tumorigenesis.

No MeSH data available.


Related in: MedlinePlus