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Overexpression of the E2 ubiquitin-conjugating enzyme UbcH10 causes chromosome missegregation and tumor formation.

van Ree JH, Jeganathan KB, Malureanu L, van Deursen JM - J. Cell Biol. (2010)

Bottom Line: The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome.UbcH10 is overexpressed in many human cancer types and is associated with tumor progression.In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

ABSTRACT
The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.

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Generation of UbcH10 transgenic mice. (A) Overview of the generation of UbcH10 transgenic mice. Arrows indicate the direction of transcription. IRES, internal ribosomal entry site. (B) Western blot analysis of lysates from transgenic and control MEFs for endogenous (Endo) UbcH10 and exogenous (Exo) HA-UbcH10. Note that endogenous UbcH10 levels increase in transgenic MEFs. Actin was used as loading control. (C–E) Western blots of splenocyte (C), lung (D), and skin (E) extracts from mice of the indicated genotypes probed for UbcH10 and HA. Tissues were collected from 3-mo-old mice.
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fig1: Generation of UbcH10 transgenic mice. (A) Overview of the generation of UbcH10 transgenic mice. Arrows indicate the direction of transcription. IRES, internal ribosomal entry site. (B) Western blot analysis of lysates from transgenic and control MEFs for endogenous (Endo) UbcH10 and exogenous (Exo) HA-UbcH10. Note that endogenous UbcH10 levels increase in transgenic MEFs. Actin was used as loading control. (C–E) Western blots of splenocyte (C), lung (D), and skin (E) extracts from mice of the indicated genotypes probed for UbcH10 and HA. Tissues were collected from 3-mo-old mice.

Mentions: Because UbcH10 is overexpressed in a broad range of human cancer types, we sought to generate transgenic mice that overexpressed UbcH10 in a wide variety of tissues and organs. We used a transgenic vector containing the CAGGS promoter, which consists of the cytomegalovirus immediate enhancer and the chicken β-actin promoter (Novak et al., 2000). The CAGGS promoter drives a floxed β-geo-stop cassette consisting of a β-galactosidase and neomycin-resistance fusion gene and three polyadenylation sites (Fig. 1 A). Downstream of this cassette, we cloned the coding sequence for the murine UbcH10 protein, which we provided with a carboxy-terminal HA epitope tag sequence. The CAGGS promoter would express HA-UbcH10 only after Cre-mediated excision of the β-geo-stop cassette. EGFP was coexpressed from an internal ribosomal entry site to serve as a reporter for HA-UbcH10 expression.


Overexpression of the E2 ubiquitin-conjugating enzyme UbcH10 causes chromosome missegregation and tumor formation.

van Ree JH, Jeganathan KB, Malureanu L, van Deursen JM - J. Cell Biol. (2010)

Generation of UbcH10 transgenic mice. (A) Overview of the generation of UbcH10 transgenic mice. Arrows indicate the direction of transcription. IRES, internal ribosomal entry site. (B) Western blot analysis of lysates from transgenic and control MEFs for endogenous (Endo) UbcH10 and exogenous (Exo) HA-UbcH10. Note that endogenous UbcH10 levels increase in transgenic MEFs. Actin was used as loading control. (C–E) Western blots of splenocyte (C), lung (D), and skin (E) extracts from mice of the indicated genotypes probed for UbcH10 and HA. Tissues were collected from 3-mo-old mice.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812857&req=5

fig1: Generation of UbcH10 transgenic mice. (A) Overview of the generation of UbcH10 transgenic mice. Arrows indicate the direction of transcription. IRES, internal ribosomal entry site. (B) Western blot analysis of lysates from transgenic and control MEFs for endogenous (Endo) UbcH10 and exogenous (Exo) HA-UbcH10. Note that endogenous UbcH10 levels increase in transgenic MEFs. Actin was used as loading control. (C–E) Western blots of splenocyte (C), lung (D), and skin (E) extracts from mice of the indicated genotypes probed for UbcH10 and HA. Tissues were collected from 3-mo-old mice.
Mentions: Because UbcH10 is overexpressed in a broad range of human cancer types, we sought to generate transgenic mice that overexpressed UbcH10 in a wide variety of tissues and organs. We used a transgenic vector containing the CAGGS promoter, which consists of the cytomegalovirus immediate enhancer and the chicken β-actin promoter (Novak et al., 2000). The CAGGS promoter drives a floxed β-geo-stop cassette consisting of a β-galactosidase and neomycin-resistance fusion gene and three polyadenylation sites (Fig. 1 A). Downstream of this cassette, we cloned the coding sequence for the murine UbcH10 protein, which we provided with a carboxy-terminal HA epitope tag sequence. The CAGGS promoter would express HA-UbcH10 only after Cre-mediated excision of the β-geo-stop cassette. EGFP was coexpressed from an internal ribosomal entry site to serve as a reporter for HA-UbcH10 expression.

Bottom Line: The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome.UbcH10 is overexpressed in many human cancer types and is associated with tumor progression.In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

ABSTRACT
The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.

Show MeSH
Related in: MedlinePlus