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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

Temporally distinct roles for tumor suppressor pathways in cell cycle arrest and cellular senescence in Cyclin D1-driven tumor

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Hasan Zalzali, Mohamad Harajly, Lina Abdul-Latif, Nader El-Chaar, Ghassan Dbaibo, Stephen X Skapek, Raya Saab
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-28) contains supplementary material, which is available to authorized users.

Author’s contributions

HZ carried out the most of the cellular and molecular studies, and participated in drafting the manuscript. MH, LM, and NEC performed immunohistochemical studies, mouse genotyping for the reported experiments, and helped in drafting the manuscript. GD and SXS participated in the design of the study and reviewed the manuscript. RS conceived the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Cellular senescence represents a tumor suppressive response to a variety of aberrant and oncogenic insults. We have previously described a transgenic mouse model of Cyclin D1-driven senescence in pineal cells that opposes tumor progression. We now attempted to define the molecular mechanisms leading to p53 activation in this model, and to identify effectors of Cyclin D1-induced senescence.

Results

Senescence evolved over a period of weeks, with initial hyperproliferation followed by cell cycle arrest due to ROS production leading to activation of a DNA damage response and the p53 pathway. Interestingly, cell cycle exit was associated with repression of the Cyclin-dependent kinase Cdk2. This was followed days later by formation of heterochromatin foci correlating with RB protein hypophosphorylation. In the absence of the Cdk4-inhibitor p18Ink4c, cell cycle exit was delayed but most cells eventually showed a senescent phenotype. However, tumors later arose from this premalignant, largely senescent lesion. We found that the p53 pathway was intact in tumors arising in a p18Ink4c-/- background, indicating that the two genes represent distinct tumor suppressor pathways. Upon tumor progression, both p18Ink4c-/- and p53-/- tumors showed increased Cdk2 expression. Inhibition of Cdk2 in cultured pre-tumorigenic and tumor cells of both backgrounds resulted in decreased proliferation and evidence of senescence.

Conclusion

Our findings indicate that the p53 and the RB pathways play temporally distinct roles in senescence induction in Cyclin D1-expressing cells, and that Cdk2 inhibition plays a role in tumor suppression, and may be a useful therapeutic target.
Zusatzmaterial
Additional file 1: Figure S1: A) High-magnification images of senescence-associated heterochromatin foci (SAHF) marked by H3K9me3, in Irbp-Cyclin D1 (D1) versus wild-type (WT) pineal cells at P49. B) Senescence-associated beta galatosidase (SABG) staining of cultured pineal cells explanted from wild-type (WT), Irbp-Cyclin D1 (D1), and Irbp-Cyclin D1, p53 -/- (D1p53-/-) animals, as indicated. SABG staining was done after 10 days of culture. C) Top panel: BrdU-incorporation and immunofluorescence staining of wild-type (WT) and Irbp-Cyclin D1 (D1) pineal cells, as indicated, after 10 days in culture. Bottom panel: corresponding DAPI-stained nuclei. D) Right panels: Immunofluorescence staining for 8(OH)dG and pH2AX, in wild-type (WT), Irbp-Cyclin D1 (D1), and NAC-treated Irbp-Cyclin D1 explanted pineal cells, as indicated. Left panels: corresponding DAPI staining of nuclei. (TIFF 6 MB)
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Additional file 2: Figure S2: A) Representative immunohistochemical staining for Ki67 (left) and immunoflourescence for H3K9me3 (right) and corresponding DAPI (middle) staining of Irbp-Cyclin D1, p18Ink4c-/- pineal sections at the indicated ages. B) Immunostaining for Dec1 and DcR2, in Irbp-Cyclin D1, p18Ink4c-/- pineal glands at the indicated ages. C) SABG staining of cultured Irbp-Cyclin D1, p18Ink4c -/- pineal cells after 14 and 20 days in culture, as indicated. D) Western blotting for the indicated proteins in pineal glands of the indicated genotypes, at the indicated ages. (TIFF 5 MB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 6
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Authors’ original file for figure 7
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