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Consequence of the tumor-associated conversion to cyclin D1b.

Augello MA, Berman-Booty LD, Carr R, Yoshida A, Dean JL, Schiewer MJ, Feng FY, Tomlins SA, Gao E, Koch WJ, Benovic JL, Diehl JA, Knudsen KE - EMBO Mol Med (2015)

Bottom Line: Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo.Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo.Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA.

No MeSH data available.


Related in: MedlinePlus

Humanization of the Ccnd1 exon 4/5 locus results in exclusive production of cyclin D1bRepresentative schematic of the targeting construct generated to humanize the Ccnd1 exon 4/5 genomic locus to produce cyclin D1b.Top: Schematic of primer pairs designed to discriminate between wild-type and knock-in alleles. Bottom: Representative genotyping of Ccnd1+/+, Ccnd1+/KI, and Ccnd1KI/KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct.PCR analysis of transcript b expression in organs harvested from Ccnd1+/+ and Ccnd1KI/KI mice, demonstrating production of transcript b specifically in KI mice. Gapdh serves as a control (NTC, Non-template control).Immunoblot from parallel samples in (C) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4. Lamin B serves as a control. Arrow indicates the cyclin D1b band.
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fig01: Humanization of the Ccnd1 exon 4/5 locus results in exclusive production of cyclin D1bRepresentative schematic of the targeting construct generated to humanize the Ccnd1 exon 4/5 genomic locus to produce cyclin D1b.Top: Schematic of primer pairs designed to discriminate between wild-type and knock-in alleles. Bottom: Representative genotyping of Ccnd1+/+, Ccnd1+/KI, and Ccnd1KI/KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct.PCR analysis of transcript b expression in organs harvested from Ccnd1+/+ and Ccnd1KI/KI mice, demonstrating production of transcript b specifically in KI mice. Gapdh serves as a control (NTC, Non-template control).Immunoblot from parallel samples in (C) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4. Lamin B serves as a control. Arrow indicates the cyclin D1b band.

Mentions: To develop robust genetic systems of cyclin D1b production under the endogenous promoter, a gene-targeting construct was generated wherein all C-terminal-encoding components of the murine Ccnd1 gene were replaced with the C-terminal sequences responsible for human cyclin D1b production. As shown in Fig1A, this was accomplished by replacing murine exon 4, intron 4, exon 5, and 3′ UTR with human exon 4 and intron 4 encoding sequences. The use of human exon 4/intron 4 and removal of murine exon 5/3′ UTR were necessary to both eliminate the possibility of full-length transcript a production (encoding cyclin D1a), and to foster production of transcript b, encoding the unique C-terminus harbored by cyclin D1b. Furthermore, this strategy preserves upstream splicing events of the Ccnd1 transcript, which more accurately reflects the biochemical conditions responsible for cyclin D1b production.


Consequence of the tumor-associated conversion to cyclin D1b.

Augello MA, Berman-Booty LD, Carr R, Yoshida A, Dean JL, Schiewer MJ, Feng FY, Tomlins SA, Gao E, Koch WJ, Benovic JL, Diehl JA, Knudsen KE - EMBO Mol Med (2015)

Humanization of the Ccnd1 exon 4/5 locus results in exclusive production of cyclin D1bRepresentative schematic of the targeting construct generated to humanize the Ccnd1 exon 4/5 genomic locus to produce cyclin D1b.Top: Schematic of primer pairs designed to discriminate between wild-type and knock-in alleles. Bottom: Representative genotyping of Ccnd1+/+, Ccnd1+/KI, and Ccnd1KI/KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct.PCR analysis of transcript b expression in organs harvested from Ccnd1+/+ and Ccnd1KI/KI mice, demonstrating production of transcript b specifically in KI mice. Gapdh serves as a control (NTC, Non-template control).Immunoblot from parallel samples in (C) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4. Lamin B serves as a control. Arrow indicates the cyclin D1b band.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492821&req=5

fig01: Humanization of the Ccnd1 exon 4/5 locus results in exclusive production of cyclin D1bRepresentative schematic of the targeting construct generated to humanize the Ccnd1 exon 4/5 genomic locus to produce cyclin D1b.Top: Schematic of primer pairs designed to discriminate between wild-type and knock-in alleles. Bottom: Representative genotyping of Ccnd1+/+, Ccnd1+/KI, and Ccnd1KI/KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct.PCR analysis of transcript b expression in organs harvested from Ccnd1+/+ and Ccnd1KI/KI mice, demonstrating production of transcript b specifically in KI mice. Gapdh serves as a control (NTC, Non-template control).Immunoblot from parallel samples in (C) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4. Lamin B serves as a control. Arrow indicates the cyclin D1b band.
Mentions: To develop robust genetic systems of cyclin D1b production under the endogenous promoter, a gene-targeting construct was generated wherein all C-terminal-encoding components of the murine Ccnd1 gene were replaced with the C-terminal sequences responsible for human cyclin D1b production. As shown in Fig1A, this was accomplished by replacing murine exon 4, intron 4, exon 5, and 3′ UTR with human exon 4 and intron 4 encoding sequences. The use of human exon 4/intron 4 and removal of murine exon 5/3′ UTR were necessary to both eliminate the possibility of full-length transcript a production (encoding cyclin D1a), and to foster production of transcript b, encoding the unique C-terminus harbored by cyclin D1b. Furthermore, this strategy preserves upstream splicing events of the Ccnd1 transcript, which more accurately reflects the biochemical conditions responsible for cyclin D1b production.

Bottom Line: Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo.Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo.Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA.

No MeSH data available.


Related in: MedlinePlus