The oncogenic potential of Kaposi's sarcoma-associated herpesvirus cyclin is exposed by p53 loss in vitro and in vivo  Emmy W. Verschuren, Juha Klefstrom,

Slides:



Advertisements
Similar presentations
Volume 14, Issue 2, Pages (August 2008)
Advertisements

by Matilde Murga, Oscar Fernandez-Capetillo, and Giovanna Tosato
by Ji-Long Chen, Andre Limnander, and Paul B. Rothman
PML Is a Direct p53 Target that Modulates p53 Effector Functions
Volume 116, Issue 5, Pages (March 2004)
MK-8776, a novel Chk1 inhibitor, exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis  Motofumi.
Volume 12, Issue 1, Pages (January 2013)
Effects of Betulinic Acid Alone and in Combination with Irradiation in Human Melanoma Cells  Edgar Selzer, Emilio Pimentel, Volker Wacheck, Werner Schlegel,
The Zinc Ionophore PCI-5002 Radiosensitizes Non-small Cell Lung Cancer Cells by Enhancing Autophagic Cell Death  Kwang Woon Kim, PhD, Christina K. Speirs,
Volume 19, Issue 2, Pages (February 2017)
Dissecting p53 tumor suppressor functions in vivo
Volume 2, Issue 1, Pages (July 2002)
Volume 15, Issue 1, Pages (January 2014)
Volume 4, Issue 1, Pages (July 2001)
Volume 30, Issue 1, Pages (April 2008)
Short Telomeres Limit Tumor Progression In Vivo by Inducing Senescence
Volume 44, Issue 4, Pages (November 2011)
Volume 136, Issue 4, Pages e3 (April 2009)
Volume 138, Issue 5, Pages e2 (May 2010)
Volume 1, Issue 1, Pages (February 2002)
Involvement of Fas (APO-1/CD-95) during Photodynamic-Therapy-Mediated Apoptosis in Human Epidermoid Carcinoma A431 Cells  Nihal Ahmad, Sanjay Gupta, Denise.
Cdk2 Knockout Mice Are Viable
Volume 7, Issue 6, Pages (June 2005)
Cyclin C/Cdk3 Promotes Rb-Dependent G0 Exit
Volume 22, Issue 3, Pages (March 2012)
Modeling the Therapeutic Efficacy of p53 Restoration in Tumors
A JNK-Dependent Pathway Is Required for TNFα-Induced Apoptosis
Mutations in Human ARF Exon 2 Disrupt Its Nucleolar Localization and Impair Its Ability to Block Nuclear Export of MDM2 and p53  Yanping Zhang, Yue Xiong 
Volume 17, Issue 1, Pages (January 2005)
The oncogenic potential of Kaposi's sarcoma-associated herpesvirus cyclin is exposed by p53 loss in vitro and in vivo  Emmy W. Verschuren, Juha Klefstrom,
Negative Control of p53 by Sir2α Promotes Cell Survival under Stress
AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol  Shubha Anand, Sue Penrhyn-Lowe, Ashok R Venkitaraman 
Katja Wassmann, Théodora Niault, Bernard Maro  Current Biology 
Volume 127, Issue 1, Pages (October 2006)
Thijn R Brummelkamp, René Bernards, Reuven Agami  Cancer Cell 
Linking the Rb and Polycomb Pathways
Volume 16, Issue 24, Pages (December 2006)
Oncogenic Ras-Induced Expression of Noxa and Beclin-1 Promotes Autophagic Cell Death and Limits Clonogenic Survival  Mohamed Elgendy, Clare Sheridan,
p53 stabilization is decreased upon NFκB activation
Septins Regulate Actin Organization and Cell-Cycle Arrest through Nuclear Accumulation of NCK Mediated by SOCS7  Brandon E. Kremer, Laura A. Adang, Ian.
Volume 30, Issue 2, Pages (February 2009)
Volume 4, Issue 4, Pages (October 2003)
Volume 10, Issue 3, Pages (September 2006)
Volume 50, Issue 2, Pages (April 2013)
GRM7 Regulates Embryonic Neurogenesis via CREB and YAP
Volume 3, Issue 5, Pages (May 2013)
Volume 17, Issue 2, Pages (August 1996)
Volume 15, Issue 3, Pages (September 2001)
Volume 8, Issue 3, Pages (January 1998)
Yisong Y Wan, James DeGregori  Immunity 
EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy
Volume 8, Issue 6, Pages (December 2005)
Bonnie E. Lonze, Antonella Riccio, Sonia Cohen, David D. Ginty  Neuron 
Cellular 5′-3′ mRNA Exonuclease Xrn1 Controls Double-Stranded RNA Accumulation and Anti-Viral Responses  Hannah M. Burgess, Ian Mohr  Cell Host & Microbe 
Mitochondrial p32 Is a Critical Mediator of ARF-Induced Apoptosis
DNA Damage Foci at Dysfunctional Telomeres
LAT Links the Pre-BCR to Calcium Signaling
Volume 16, Issue 2, Pages (February 2009)
Transcriptional Repression of miR-34 Family Contributes to p63-Mediated Cell Cycle Progression in Epidermal Cells  Dario Antonini, Monia T. Russo, Laura.
PUMA, a Novel Proapoptotic Gene, Is Induced by p53
Volume 17, Issue 4, Pages (April 2010)
Galectin-3 Protects Keratinocytes from UVB-Induced Apoptosis by Enhancing AKT Activation and Suppressing ERK Activation  Jun Saegusa, Daniel K. Hsu, Wei.
Volume 15, Issue 5, Pages (November 2001)
Volume 14, Issue 2, Pages (August 2008)
Volume 14, Issue 1, Pages (January 2001)
Roland Houben, Sonja Ortmann, David Schrama, Marco J
Volume 7, Issue 6, Pages (June 2014)
Inhibition of NF-κB in cancer cells converts inflammation- induced tumor growth mediated by TNFα to TRAIL-mediated tumor regression  Jun-Li Luo, Shin.
Volume 15, Issue 4, Pages (April 2016)
Yun-Gui Yang, Tomas Lindahl, Deborah E. Barnes  Cell 
Presentation transcript:

The oncogenic potential of Kaposi's sarcoma-associated herpesvirus cyclin is exposed by p53 loss in vitro and in vivo  Emmy W. Verschuren, Juha Klefstrom, Gerard I. Evan, Nic Jones  Cancer Cell  Volume 2, Issue 3, Pages 229-241 (September 2002) DOI: 10.1016/S1535-6108(02)00123-X

Figure 1 Expression of K cyclin sensitizes MEFs to death triggered by various stress signals Wt MEFs were infected for 2 subsequent days with KpBMN or empty vector and were next cultured for 48 hr in medium containing 10% FBS, 0.2% FBS, or 10% FBS supplemented with etoposide (5.0 μM), TNF (50 ng/ml), or TRAIL (200 ng/ml). Floating and adhered cells were collected and stained with Annexin V-PE and Via-Probe, followed by flow cytometric analysis, an example of which is shown in the upper panel. The mean percentages of dead cells ± SEM of three independent experiments are shown Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)

Figure 2 P19ARF and E2F1 expression are dispensable, but p53 is required for apoptosis induced by K cyclin A: K cyclin does not induce p19ARF expression in MEFs. Wt MEFs were infected with K cyclin-encoding (K) or empty virus (V). Infected cells were selected by puromycin and were grown for 72 hr in medium containing 10% FBS. p53−/− MEFs are shown as a positive control, since they contain high levels of endogenous p19ARF due to the lack of a p53-mediated negative feedback loop (Stott et al., 1998). Lysates were prepared and analyzed by immunoblotting with anti-p19ARF antibody. B: K cyclin expression is associated with elevated p53 levels. MEFs of the indicated genotype were infected with empty (V) or K cyclin-encoding pBabepuro (K) vector. Infected cells were selected by puromycin and were grown for 72 hr in medium containing 10% FBS until cell lysates were prepared. As a control, a subconfluent culture of wt MEFs was exposed to 8 GY of γ-irradiation and was grown for an additional 24 hr. Proteins were immunoblotted with CM-5 p53 or β-actin antibody. C: Protein lysates from MEFs infected as in B were analyzed by immunoblotting with anti-Flag antibody to detect tagged K cyclin or with CM-5 p53, p21Cip1, or β-actin antibody. D: Wt, E2F1−/−, ARF−/−, and p53−/− MEFs were infected for 2 subsequent days with KpBMN or empty vector, followed by growth for 48 hr in 10% FBS- or 0.2% FBS-containing medium. Cells were stained with Annexin V-PE and Via-Probe and were analyzed by flow cytometry to determine the percentage of apoptotic cells in the GFP-positive cell population. Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)

Figure 3 K cyclin-expressing MEFs show a p53-dependent growth arrest A: Growth curves of MEFs constitutively expressing K cyclin. Wt, E2F1−/−, p53−/− , or ARF−/− MEFs were transduced with empty or K cyclin-encoding pBabepuro retrovirus. Transduced cells were selected by puromycin and were grown in medium containing 10% FBS. Cells were plated at 0.5 × 105 cells per well at day 0, and cell numbers were counted 1, 3, and 5 days later. The experiment was repeated three times with essentially similar results, and the growth curves represent the average of these experiments. B: Microscopic analysis of cells at day 5 of the growth curve. Phase-contrast pictures were taken at a magnification of 10×. C: Cultures of MEFs transduced and cultured as in A were followed by time-lapse videomicroscopy (see the Supplementary Material for QuickTime movies). Pictures were taken every 10 min, and cultures were followed for 4 days. Movies were analyzed for cell divisions, and cumulative cell divisions of wt or p53−/− MEFs expressing K cyclin were plotted. D: Growth curves of p21−/− MEFs infected with empty of K cyclin-encoding pBabepuro retrovirus and analyzed as in A. Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)

Figure 4 Growth-arrested K cyclin-expressing MEFs synthesize DNA A: K cyclin-expressing cells become multinucleated. Cultures were infected and grown as in Figure 3A and were microscopically analyzed at day 5 of the growth curve. DNA was counterstained with Hoechst. Pictures were taken at a magnification of 32×. B: Wt, E2F1−/−, ARF−/−, or p53−/− MEFs were infected as described in Figure 3A and were grown for 3 days. BrdU (50 μM) was added to the medium, and the cells were grown for an additional 5 hr. Cells were collected and stained with PI and anti-BrdU-FITC conjugate and were analyzed by flow cytometry. The percentages of BrdU-positive cells are indicated. C: Cells infected as in Figure 3A were grown on coverslips for 3 days, followed by growth in medium containing BrdU for 5 hr. Cells were fixed in 70% ethanol and were stained with anti-BrdU-FITC (green) and Hoechst (blue) for DNA. Slides were microscopically analyzed, and pictures were taken at a magnification of 32×. A large, multinucleated BrdU-positive cell is indicated with an arrowhead. Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)

Figure 5 MEFs and MRC-5 cells expressing K cyclin become polyploid A: MEFs infected and grown as described in Figure 6A were grown for 3 days in 10% FBS-containing medium. Adhered cells were harvested, followed by staining of the DNA with PI and flow cytometric analysis to establish cell cycle profiles. The percentages of cells with a DNA content greater than 4N are indicated. B: MRC-5 cells transduced with empty or K cyclin-encoding retrovirus were grown, and growth curves were determined as described in Figure 4A. Cells cultured for 3 days were harvested and analyzed by flow cytometry as in A. Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)

Figure 6 Centrosome amplifications in K cyclin-expressing MEFs A: Wt or p53−/− MEFs were infected with empty or K cyclin-encoding vector, grown for 3 days on coverslips, and stained with anti-γ-tubulin antibody for centrosomes (green) and DAPI (blue) for DNA. The numbersindicate the amounts of centrosomes per cell. B: Quantitation of the centrosome analysis described in A. The pooled percentages of three experiments of around 70 cells each are shown. Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)

Figure 7 Centrosome amplification correlates with ploidy and is associated with genomic abnormalities A: Wt MEFs infected and grown as in Figure 7A were stained with anti-γ-tubulin and PI. The DNA content per nucleus was measured by quantitative immunofluorescence microscopy and was normalized to obtain an N = 2 population. Around 100 mock-infected cells or 150 K cyclin-infected cells were analyzed. The inset histogram illustrates that average numbers of centrosomes increase with DNA content. B: Example of the correlation analysis. The panel shows a field of wt MEFs infected with K cyclin that were stained with γ-tubulin (green) and PI (red). The numbers before the slash represent DNA contents, and the numbers after the slash represent centrosome counts. C: Confocal microsopic analysis of wt (first image) or p53−/− MEFs (second and third image) infected with K cyclin retrovirus and grown and stained as described in A. The pictures represent the image layer that best visualized the genomic abnormality, magnification 63× and 2× zoom. Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)

Figure 8 Eμ K cyclin cooperates with p53 loss in tumorigenesis A: A graphic representation of the transgenic cassette used to generate Eμ K cyclin transgenic founders. B: Kaplan Meier survival curves of K cyclin transgenic p53−/−, p53+/−, or p53+/+ mice and nontransgenic controls. All K cyclin/p53−/− mice developed lymphoblastic lymphomas. Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)

Figure 9 Model showing how expression of K cyclin in MEFs triggers multiple responses We speculate that cells expressing K cyclin undergo abortive cytokinesis (the mechanism of which is unknown) and adapt from the cytokinesis defect and become tetraploid. This may trigger the activation of p53 and concurrent (p21Cip1-independent) growth arrest and sensitization to apoptosis. Because K cyclin is insensitive to the action of Cdk inhibitors such as p21Cip1, growth-arrested cells lack functional G1 and G2 checkpoints. This leads to the formation of a polyploid/aneuploid population of cells with amplified centrosomes, which survives and expands in the absence of p53 to become tumorigenic. Additional apoptotic triggers like the phosphorylation and inactivation of endogenous Bcl-2 are likely to contribute to the final fate of K cyclin-expressing cells. Cancer Cell 2002 2, 229-241DOI: (10.1016/S1535-6108(02)00123-X)