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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 promotes double-strand breaks and PARP-1 activityCcnd1+/+ and Ccnd1KI/KI cells were transfected with a pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or scramble control for 48 h (as in Figure5). Cells were then fixed and stained for markers of double-strand breaks (p-H2AX and 53BP1, 400× objective). Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci/cell (p-H2AX) or % of cells with > 2 foci/cell (53BP1). Error bars represent ± SEM.Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h and probed for expression of auto-modified PARP1 via immunoblot. Gapdh and cyclin D1b serve as controls.Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control.Indicated MAF lines were treated as in (A) and harvested 48 h post-transfection. Cells were analyzed for total PAR levels via immunoblot. cyclin D1 levels serve as siRNA validation controls.cyclin D1b expression was induced in the prostate cancer cell line LNCaP (previously described (Augello et al, 2014)) and stained for p-H2AX and 53BP1 foci as in (A) via immunofluorescence (400× objective). Total number of foci/cell is reported for LNCaP vector control and cyclin D1b-expressing isogenic pairs in biological triplicate.Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.Data information: Statistical significance was determined using a two-tailed Student's t-test.*P < 0.05 **P < 0.01, ***P < 0.001.
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fig06: Cyclin D1b expression promotes double-strand breaks and PARP-1 activityCcnd1+/+ and Ccnd1KI/KI cells were transfected with a pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or scramble control for 48 h (as in Figure5). Cells were then fixed and stained for markers of double-strand breaks (p-H2AX and 53BP1, 400× objective). Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci/cell (p-H2AX) or % of cells with > 2 foci/cell (53BP1). Error bars represent ± SEM.Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h and probed for expression of auto-modified PARP1 via immunoblot. Gapdh and cyclin D1b serve as controls.Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control.Indicated MAF lines were treated as in (A) and harvested 48 h post-transfection. Cells were analyzed for total PAR levels via immunoblot. cyclin D1 levels serve as siRNA validation controls.cyclin D1b expression was induced in the prostate cancer cell line LNCaP (previously described (Augello et al, 2014)) and stained for p-H2AX and 53BP1 foci as in (A) via immunofluorescence (400× objective). Total number of foci/cell is reported for LNCaP vector control and cyclin D1b-expressing isogenic pairs in biological triplicate.Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.Data information: Statistical significance was determined using a two-tailed Student's t-test.*P < 0.05 **P < 0.01, ***P < 0.001.

Mentions: Given the collective observations above that cyclin D1b elicits divergent effects on cellular transformation, and clinical observations that cyclin D1b expression is associated with therapeutic resistance, it was imperative to challenge the impact of therapeutic intervention and or genotoxic stress on cyclin D1b-driven tumor cells. Initially, the presence of DNA damage was assessed in Ccnd1+/+ and Ccnd1KI/KI by quantifying intrinsic p-H2AX and 53BP1 foci, established markers of DNA breaks and known metrics for genome integrity. As expected, a majority of the Ccnd1+/+ cells showed a low frequency of both p-H2AX and 53BP1 foci/cell (Fig6A). In contrast, cells expressing endogenous cyclin D1b harbored increased double-strand breaks, demonstrated by the enhanced prevalence of p-H2AX and 53BP1 foci (Fig6A, quantified right). These data were confirmed in an independently derived Ccnd1KI/KI cell line (Supplementary Fig S4A), suggesting that Ccnd1KI/KI cells maintain heightened intrinsic DNA damage. Furthermore, cyclin D1b was required to maintain markers of DNA damage, as loss of cyclin D1b expression reduced the levels of both p-H2AX and 53BP1 foci to those found in Ccnd1+/+ control cells (Fig6A, quantified right), suggesting that cyclin D1b expression is associated with markers of DNA damage.


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 promotes double-strand breaks and PARP-1 activityCcnd1+/+ and Ccnd1KI/KI cells were transfected with a pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or scramble control for 48 h (as in Figure5). Cells were then fixed and stained for markers of double-strand breaks (p-H2AX and 53BP1, 400× objective). Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci/cell (p-H2AX) or % of cells with > 2 foci/cell (53BP1). Error bars represent ± SEM.Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h and probed for expression of auto-modified PARP1 via immunoblot. Gapdh and cyclin D1b serve as controls.Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control.Indicated MAF lines were treated as in (A) and harvested 48 h post-transfection. Cells were analyzed for total PAR levels via immunoblot. cyclin D1 levels serve as siRNA validation controls.cyclin D1b expression was induced in the prostate cancer cell line LNCaP (previously described (Augello et al, 2014)) and stained for p-H2AX and 53BP1 foci as in (A) via immunofluorescence (400× objective). Total number of foci/cell is reported for LNCaP vector control and cyclin D1b-expressing isogenic pairs in biological triplicate.Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.Data information: Statistical significance was determined using a two-tailed Student's t-test.*P < 0.05 **P < 0.01, ***P < 0.001.
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fig06: Cyclin D1b expression promotes double-strand breaks and PARP-1 activityCcnd1+/+ and Ccnd1KI/KI cells were transfected with a pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or scramble control for 48 h (as in Figure5). Cells were then fixed and stained for markers of double-strand breaks (p-H2AX and 53BP1, 400× objective). Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci/cell (p-H2AX) or % of cells with > 2 foci/cell (53BP1). Error bars represent ± SEM.Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h and probed for expression of auto-modified PARP1 via immunoblot. Gapdh and cyclin D1b serve as controls.Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control.Indicated MAF lines were treated as in (A) and harvested 48 h post-transfection. Cells were analyzed for total PAR levels via immunoblot. cyclin D1 levels serve as siRNA validation controls.cyclin D1b expression was induced in the prostate cancer cell line LNCaP (previously described (Augello et al, 2014)) and stained for p-H2AX and 53BP1 foci as in (A) via immunofluorescence (400× objective). Total number of foci/cell is reported for LNCaP vector control and cyclin D1b-expressing isogenic pairs in biological triplicate.Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.Data information: Statistical significance was determined using a two-tailed Student's t-test.*P < 0.05 **P < 0.01, ***P < 0.001.
Mentions: Given the collective observations above that cyclin D1b elicits divergent effects on cellular transformation, and clinical observations that cyclin D1b expression is associated with therapeutic resistance, it was imperative to challenge the impact of therapeutic intervention and or genotoxic stress on cyclin D1b-driven tumor cells. Initially, the presence of DNA damage was assessed in Ccnd1+/+ and Ccnd1KI/KI by quantifying intrinsic p-H2AX and 53BP1 foci, established markers of DNA breaks and known metrics for genome integrity. As expected, a majority of the Ccnd1+/+ cells showed a low frequency of both p-H2AX and 53BP1 foci/cell (Fig6A). In contrast, cells expressing endogenous cyclin D1b harbored increased double-strand breaks, demonstrated by the enhanced prevalence of p-H2AX and 53BP1 foci (Fig6A, quantified right). These data were confirmed in an independently derived Ccnd1KI/KI cell line (Supplementary Fig S4A), suggesting that Ccnd1KI/KI cells maintain heightened intrinsic DNA damage. Furthermore, cyclin D1b was required to maintain markers of DNA damage, as loss of cyclin D1b expression reduced the levels of both p-H2AX and 53BP1 foci to those found in Ccnd1+/+ control cells (Fig6A, quantified right), suggesting that cyclin D1b expression is associated with markers of DNA damage.

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