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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

Genetic switch to cyclin D1b promotes serum independenceCcnd1+/+ and Ccnd1KI/KIMAF lines were arrested in G1, S, and G2/M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot. cyclin A2 and cyclin B1 serve as phase-specific cell cycle controls.Cells were plated in serum-proficient media and protein lysates generated from each genetic line. Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components. Lamin B serves as a control.Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates.Indicated cell lines were plated in serum-proficient (10%) or serum-deficient (1%) media and total cell number counted at 24, 48, and 72 h in biological triplicate.Ccnd1+/+ and Ccnd1KI/KI MAF lines were transfected with a validated pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or control siRNA in full serum. 48 h post-transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot. GAPDH and Lamin B serve as controls.Cells were treated as (E), and incubated in the indicated serum concentration for 48 h. Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation. Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive/total cell number.Data information: Error bars represent ± SEM, and statistical significance was determined using a two-tailed Student's t-test. **P < 0.01, ***P < 0.001.
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fig05: Genetic switch to cyclin D1b promotes serum independenceCcnd1+/+ and Ccnd1KI/KIMAF lines were arrested in G1, S, and G2/M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot. cyclin A2 and cyclin B1 serve as phase-specific cell cycle controls.Cells were plated in serum-proficient media and protein lysates generated from each genetic line. Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components. Lamin B serves as a control.Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates.Indicated cell lines were plated in serum-proficient (10%) or serum-deficient (1%) media and total cell number counted at 24, 48, and 72 h in biological triplicate.Ccnd1+/+ and Ccnd1KI/KI MAF lines were transfected with a validated pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or control siRNA in full serum. 48 h post-transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot. GAPDH and Lamin B serve as controls.Cells were treated as (E), and incubated in the indicated serum concentration for 48 h. Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation. Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive/total cell number.Data information: Error bars represent ± SEM, and statistical significance was determined using a two-tailed Student's t-test. **P < 0.01, ***P < 0.001.

Mentions: The concept that endogenously derived cyclin D1b induces transformation is significant and highlights distinctions from cyclin D1a, which requires overexpression to serve as an oncogene. Given the unexpected nature of these findings, Ccnd1KI/KI cells were used to characterize the molecular consequence of cyclin D1b expression. To examine cyclin D1b distribution/stability in the absence of a constitutively active promoter, Ccnd1+/+ and Ccnd1KI/KI cells were arrested in each phase of the cell cycle and expression of individual cyclin D1 isoforms determined. As shown in Fig5A, cyclin D1a levels peaked in G1, and diminished as a function of cell cycle progression (cyclin A2 and cyclin B1 serve as late S and G2/M phase-specific controls). Cyclin D1b expression mimicked that of cyclin D1a, suggesting that both isoforms are regulated in a similar fashion throughout the cell cycle. These data thus demonstrate that the oncogenic activity governed by cyclin D1b is independent of aberrant expression in later phases of the cell cycle.


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)

Genetic switch to cyclin D1b promotes serum independenceCcnd1+/+ and Ccnd1KI/KIMAF lines were arrested in G1, S, and G2/M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot. cyclin A2 and cyclin B1 serve as phase-specific cell cycle controls.Cells were plated in serum-proficient media and protein lysates generated from each genetic line. Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components. Lamin B serves as a control.Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates.Indicated cell lines were plated in serum-proficient (10%) or serum-deficient (1%) media and total cell number counted at 24, 48, and 72 h in biological triplicate.Ccnd1+/+ and Ccnd1KI/KI MAF lines were transfected with a validated pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or control siRNA in full serum. 48 h post-transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot. GAPDH and Lamin B serve as controls.Cells were treated as (E), and incubated in the indicated serum concentration for 48 h. Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation. Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive/total cell number.Data information: Error bars represent ± SEM, and statistical significance was determined using a two-tailed Student's t-test. **P < 0.01, ***P < 0.001.
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Related In: Results  -  Collection

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fig05: Genetic switch to cyclin D1b promotes serum independenceCcnd1+/+ and Ccnd1KI/KIMAF lines were arrested in G1, S, and G2/M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot. cyclin A2 and cyclin B1 serve as phase-specific cell cycle controls.Cells were plated in serum-proficient media and protein lysates generated from each genetic line. Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components. Lamin B serves as a control.Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates.Indicated cell lines were plated in serum-proficient (10%) or serum-deficient (1%) media and total cell number counted at 24, 48, and 72 h in biological triplicate.Ccnd1+/+ and Ccnd1KI/KI MAF lines were transfected with a validated pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or control siRNA in full serum. 48 h post-transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot. GAPDH and Lamin B serve as controls.Cells were treated as (E), and incubated in the indicated serum concentration for 48 h. Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation. Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive/total cell number.Data information: Error bars represent ± SEM, and statistical significance was determined using a two-tailed Student's t-test. **P < 0.01, ***P < 0.001.
Mentions: The concept that endogenously derived cyclin D1b induces transformation is significant and highlights distinctions from cyclin D1a, which requires overexpression to serve as an oncogene. Given the unexpected nature of these findings, Ccnd1KI/KI cells were used to characterize the molecular consequence of cyclin D1b expression. To examine cyclin D1b distribution/stability in the absence of a constitutively active promoter, Ccnd1+/+ and Ccnd1KI/KI cells were arrested in each phase of the cell cycle and expression of individual cyclin D1 isoforms determined. As shown in Fig5A, cyclin D1a levels peaked in G1, and diminished as a function of cell cycle progression (cyclin A2 and cyclin B1 serve as late S and G2/M phase-specific controls). Cyclin D1b expression mimicked that of cyclin D1a, suggesting that both isoforms are regulated in a similar fashion throughout the cell cycle. These data thus demonstrate that the oncogenic activity governed by cyclin D1b is independent of aberrant expression in later phases of the cell cycle.

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