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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

Cyclin D1b expression sensitizes cells to senescence induced by therapeutic challengeCcnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right).Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).Data information: Error bars represent ± SEM, and statistical significance was determined using ANOVA (A) or a two-tailed Student's t-test (B). *P < 0.05, ***P < 0.001.
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fig07: Cyclin D1b expression sensitizes cells to senescence induced by therapeutic challengeCcnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right).Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).Data information: Error bars represent ± SEM, and statistical significance was determined using ANOVA (A) or a two-tailed Student's t-test (B). *P < 0.05, ***P < 0.001.

Mentions: The switch from cyclin D1a to cyclin D1b is observed in numerous human malignancies and is associated with poor prognosis. Despite this knowledge, delineation of mechanisms to target cyclin D1b-positive/cyclin D1b-driven tumor cells remains elusive. Building on the observations herein, it was hypothesized that therapeutically targeting DNA damage and PARP pathways individually or in combination would sensitize cells to cell cycle arrest and/or cell death. Thus, control (Ccnd1+/+) and Ccnd1KI/KI cells were treated either with the PARP inhibitor ABT-888, 5 Gy of radiation, or a combination and analyzed for cell cycle progression. As shown in Fig7A, both Ccnd1+/+ and Ccnd1KI/KI cells showed similar proliferative kinetics when unchallenged in normal growth conditions, and neither cell type was affected by ABT-888 alone. In Ccnd1+/+ cells, treatment with 5 Gy IR diminished BrdU incorporation by ~10%, which was unchanged with pre-treatment of ABT-888. Conversely, while Ccnd1KI/KI cells responded similarly to 5 Gy IR alone, combination of IR and ABT-888 dramatically inhibited BrdU incorporation (Fig7A, quantified right). The reduction in proliferative capacity was associated with altered morphology, indicative of senescence. As such, cells were treated as above and then assayed for senescence-associated β-galactosidase (β-Gal) activity. As shown, only a small fraction of cells stained positive in the control and ABT-888-treated conditions (Fig7B, quantified bottom), and IR alone increased the presence of β-Gal activity equally in both cell models. However, there was a pronounced induction of β-Gal staining after combination treatment in the Ccnd1KI/KI line, nearly double that of the Ccnd1+/+ control. Re-plating both the Ccnd1+/+ and Ccnd1KI/KI cells (which were co-treated) determined that while control cells were able to reengage the cell cycle and repopulate the dish after 96 h, Ccnd1KI/KI cells were markedly unable to do so. These data thus demonstrate that the intrinsic DNA damage and heightened PARP-1 activity seen in cyclin D1b-expressing cells may be exploited to thwart growth of this cell type via combined therapeutic challenge.


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)

Cyclin D1b expression sensitizes cells to senescence induced by therapeutic challengeCcnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right).Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).Data information: Error bars represent ± SEM, and statistical significance was determined using ANOVA (A) or a two-tailed Student's t-test (B). *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig07: Cyclin D1b expression sensitizes cells to senescence induced by therapeutic challengeCcnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right).Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).Data information: Error bars represent ± SEM, and statistical significance was determined using ANOVA (A) or a two-tailed Student's t-test (B). *P < 0.05, ***P < 0.001.
Mentions: The switch from cyclin D1a to cyclin D1b is observed in numerous human malignancies and is associated with poor prognosis. Despite this knowledge, delineation of mechanisms to target cyclin D1b-positive/cyclin D1b-driven tumor cells remains elusive. Building on the observations herein, it was hypothesized that therapeutically targeting DNA damage and PARP pathways individually or in combination would sensitize cells to cell cycle arrest and/or cell death. Thus, control (Ccnd1+/+) and Ccnd1KI/KI cells were treated either with the PARP inhibitor ABT-888, 5 Gy of radiation, or a combination and analyzed for cell cycle progression. As shown in Fig7A, both Ccnd1+/+ and Ccnd1KI/KI cells showed similar proliferative kinetics when unchallenged in normal growth conditions, and neither cell type was affected by ABT-888 alone. In Ccnd1+/+ cells, treatment with 5 Gy IR diminished BrdU incorporation by ~10%, which was unchanged with pre-treatment of ABT-888. Conversely, while Ccnd1KI/KI cells responded similarly to 5 Gy IR alone, combination of IR and ABT-888 dramatically inhibited BrdU incorporation (Fig7A, quantified right). The reduction in proliferative capacity was associated with altered morphology, indicative of senescence. As such, cells were treated as above and then assayed for senescence-associated β-galactosidase (β-Gal) activity. As shown, only a small fraction of cells stained positive in the control and ABT-888-treated conditions (Fig7B, quantified bottom), and IR alone increased the presence of β-Gal activity equally in both cell models. However, there was a pronounced induction of β-Gal staining after combination treatment in the Ccnd1KI/KI line, nearly double that of the Ccnd1+/+ control. Re-plating both the Ccnd1+/+ and Ccnd1KI/KI cells (which were co-treated) determined that while control cells were able to reengage the cell cycle and repopulate the dish after 96 h, Ccnd1KI/KI cells were markedly unable to do so. These data thus demonstrate that the intrinsic DNA damage and heightened PARP-1 activity seen in cyclin D1b-expressing cells may be exploited to thwart growth of this cell type via combined therapeutic challenge.

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