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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 cooperates with h-Ras to drive tumorigenesisPassage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification.The indicated cell lines were grown in complete media and assayed for h-Ras expression via immunoblot. Lamin B serves as a loading control.Cells were plated in soft agar and allowed to grow for a period of 3 weeks. Plates were then washed, fixed, and stained with 0.01% crystal violet. Colonies greater than 50 μm were counted and are plotted (top). Representative images of colony growth are shown for each cell line. Images were taken at 40× with insets taken at 200× magnification.Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in (A), and tumor incidence is plotted as % of tumor-free mice over time (n = 10).Indicated tumors were harvested, fixed, and stained for the proliferative marker mKi67. Three random fields from each tumor were counted for mKi67 positivity, and are plotted as a % of total cell number, n = 5 (right: representative images). Images were taken at 400× magnification.Data information: Boxes highlight area of magnified images. Error bars represent ± SEM, and statistical significance was determined using a Student's t-test. ***P < 0.001.
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fig04: Cyclin D1b cooperates with h-Ras to drive tumorigenesisPassage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification.The indicated cell lines were grown in complete media and assayed for h-Ras expression via immunoblot. Lamin B serves as a loading control.Cells were plated in soft agar and allowed to grow for a period of 3 weeks. Plates were then washed, fixed, and stained with 0.01% crystal violet. Colonies greater than 50 μm were counted and are plotted (top). Representative images of colony growth are shown for each cell line. Images were taken at 40× with insets taken at 200× magnification.Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in (A), and tumor incidence is plotted as % of tumor-free mice over time (n = 10).Indicated tumors were harvested, fixed, and stained for the proliferative marker mKi67. Three random fields from each tumor were counted for mKi67 positivity, and are plotted as a % of total cell number, n = 5 (right: representative images). Images were taken at 400× magnification.Data information: Boxes highlight area of magnified images. Error bars represent ± SEM, and statistical significance was determined using a Student's t-test. ***P < 0.001.

Mentions: Previous studies have suggested that mammary transformation and tumor development are critically dependent upon the kinase activity of cyclin D1 (Landis et al, 2006), whereby knock-in of a kinase dead allele of cyclin D1 (K112E) dramatically inhibited tumor formation within this tissue type (Landis et al, 2006). To define the ability of cyclin D1b to cooperate with oncogenes reliant on the cyclin D1/CDK4 pathway, lentivirus containing vector- or h-RAS-expressing constructs was introduced to both Ccnd1+/+ (+/+ h-Ras) and Ccnd1KI/KI (KI/KI h-Ras) lines. While no anti-proliferative effects were noted in KI/KI h-Ras lines, +/+ h-Ras lines underwent sustained cell cycle arrest. Consistent with this observation, analysis of senescence markers (β-galactosidase activity) uncovered intense positive blue staining only in the +/+ h-Ras line (+/+ vec lines were negative) (Fig4A), demonstrating that in this cell type, h-Ras induction promotes oncogene-induced senescence, consistent with previous findings (Weyemi et al, 2012; Peeper et al, 2002). Given that KI/KI h-Ras lines were refractory to this effect (Fig4B), +/+ Vec, KI/KI vec, and KI/KI h-Ras lines were assayed their ability to form colonies in soft agar, a known marker of transformation. Consistent with tumor formation in vivo (Fig3A), +/+ vec lines were unable to grow in soft agar, while KI/KI vec cells were capable of forming colonies after 4 weeks (Fig4C). Strikingly, the addition of h-Ras enhanced the ability of these cells to grow in soft agar, increasing colony number by ~fivefold. This effect was further demonstrated in vivo, where KI/KI h-Ras lines were capable of enhancing tumor formation kinetics by ~5.5-fold over KI/KI vec cells (Fig4D). Furthermore, analysis of proliferative markers within these tumors found an increase in the number of Ki67-positive cells in the KI/KI h-Ras tumors, demonstrating the enhanced proliferative capacity of these cells (Fig4E). Collectively, these data provide evidence to support the role of cyclin D1b as critical oncogenic ‘hit’, functioning to promote cellular transformation and bypass of senescence programs initiated by the further oncogenic insult.


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 cooperates with h-Ras to drive tumorigenesisPassage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification.The indicated cell lines were grown in complete media and assayed for h-Ras expression via immunoblot. Lamin B serves as a loading control.Cells were plated in soft agar and allowed to grow for a period of 3 weeks. Plates were then washed, fixed, and stained with 0.01% crystal violet. Colonies greater than 50 μm were counted and are plotted (top). Representative images of colony growth are shown for each cell line. Images were taken at 40× with insets taken at 200× magnification.Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in (A), and tumor incidence is plotted as % of tumor-free mice over time (n = 10).Indicated tumors were harvested, fixed, and stained for the proliferative marker mKi67. Three random fields from each tumor were counted for mKi67 positivity, and are plotted as a % of total cell number, n = 5 (right: representative images). Images were taken at 400× magnification.Data information: Boxes highlight area of magnified images. Error bars represent ± SEM, and statistical significance was determined using a Student's t-test. ***P < 0.001.
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Related In: Results  -  Collection

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fig04: Cyclin D1b cooperates with h-Ras to drive tumorigenesisPassage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification.The indicated cell lines were grown in complete media and assayed for h-Ras expression via immunoblot. Lamin B serves as a loading control.Cells were plated in soft agar and allowed to grow for a period of 3 weeks. Plates were then washed, fixed, and stained with 0.01% crystal violet. Colonies greater than 50 μm were counted and are plotted (top). Representative images of colony growth are shown for each cell line. Images were taken at 40× with insets taken at 200× magnification.Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in (A), and tumor incidence is plotted as % of tumor-free mice over time (n = 10).Indicated tumors were harvested, fixed, and stained for the proliferative marker mKi67. Three random fields from each tumor were counted for mKi67 positivity, and are plotted as a % of total cell number, n = 5 (right: representative images). Images were taken at 400× magnification.Data information: Boxes highlight area of magnified images. Error bars represent ± SEM, and statistical significance was determined using a Student's t-test. ***P < 0.001.
Mentions: Previous studies have suggested that mammary transformation and tumor development are critically dependent upon the kinase activity of cyclin D1 (Landis et al, 2006), whereby knock-in of a kinase dead allele of cyclin D1 (K112E) dramatically inhibited tumor formation within this tissue type (Landis et al, 2006). To define the ability of cyclin D1b to cooperate with oncogenes reliant on the cyclin D1/CDK4 pathway, lentivirus containing vector- or h-RAS-expressing constructs was introduced to both Ccnd1+/+ (+/+ h-Ras) and Ccnd1KI/KI (KI/KI h-Ras) lines. While no anti-proliferative effects were noted in KI/KI h-Ras lines, +/+ h-Ras lines underwent sustained cell cycle arrest. Consistent with this observation, analysis of senescence markers (β-galactosidase activity) uncovered intense positive blue staining only in the +/+ h-Ras line (+/+ vec lines were negative) (Fig4A), demonstrating that in this cell type, h-Ras induction promotes oncogene-induced senescence, consistent with previous findings (Weyemi et al, 2012; Peeper et al, 2002). Given that KI/KI h-Ras lines were refractory to this effect (Fig4B), +/+ Vec, KI/KI vec, and KI/KI h-Ras lines were assayed their ability to form colonies in soft agar, a known marker of transformation. Consistent with tumor formation in vivo (Fig3A), +/+ vec lines were unable to grow in soft agar, while KI/KI vec cells were capable of forming colonies after 4 weeks (Fig4C). Strikingly, the addition of h-Ras enhanced the ability of these cells to grow in soft agar, increasing colony number by ~fivefold. This effect was further demonstrated in vivo, where KI/KI h-Ras lines were capable of enhancing tumor formation kinetics by ~5.5-fold over KI/KI vec cells (Fig4D). Furthermore, analysis of proliferative markers within these tumors found an increase in the number of Ki67-positive cells in the KI/KI h-Ras tumors, demonstrating the enhanced proliferative capacity of these cells (Fig4E). Collectively, these data provide evidence to support the role of cyclin D1b as critical oncogenic ‘hit’, functioning to promote cellular transformation and bypass of senescence programs initiated by the further oncogenic insult.

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