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First published online 12 February 2003
doi: 10.1242/jcs.00331

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Telomere-based proliferative lifespan barriers in Werner-syndrome fibroblasts involve both p53-dependent and p53-independent mechanisms

Terence Davis1, Sim K. Singhrao1, Fiona S. Wyllie1, Michele F. Haughton1, Paul J. Smith1, Marie Wiltshire1, David Wynford-Thomas1, Christopher J. Jones1, Richard G. A. Faragher2 and David Kipling1,*

1 Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
2 School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton, BN2 4GJ, UK



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  		Fig. 2. Immunoblot analysis of AG05229 and HCA2 fibroblasts with or without the expression of HPV16 E6. Lysates were prepared from cycling cells (young), senescent cells (M1), cycling E6-infected cells (CyE6) and E6-infected cells at Mint (Mint) for both AG05229 and HCA2. Expression levels were compared for various cell-cycle components as indicated. Approximately equal amounts of cell lysate protein (23 µg top, 19 µg middle and 11 µg bottom panels, respectively) were loaded per lane as verified by India-ink staining; the HCA2 cells at CyE6 lane in the bottom panel is slightly overloaded. Molecular mass markers (M) are shown in kDa. The hyperphosphorylated forms of pRb are denoted by the small arrows.

 


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  		Fig. 1. (A-H) Immunocytochemical analysis of AG05229 cells. (A-D) Cycling cells. (E-H) Cells at M1. The assays are: PC, phase contrast; p21, immunocytochemical detection of p21Waf1; SAßgal, histochemical assessment of SAß-gal activity; TUNEL, assay for apoptosis. All cells except PC are counterstained with haematoxylin. Cell-based assays were done in triplicate. (I,J) FACS analysis histograms of cell number versus FL2-H for AG05229 and HCA2 cells at M1 (pulse height is a measure of DNA content). White bar, 100 µm (A,E); black bar, 100 µm (B-D,F-H).

 


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  		Fig. 3. Extension of cellular lifespan of Werner-syndrome fibroblasts following abrogation of p53 function by HPV E6. (A) Growth curve of total cell number versus time for E6-infected AG05229 clone 6. (B) Comparison of growth levels achieved for control (C-1 to C-6) and E6-infected (E6-1 to E6-11) AG05229 clones. E6-p30 is a pooled sample of 30 G418-resistant colonies.

 


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  		Fig. 4. Immunocytochemical analysis of E6-expressing AG05229 cells. (A-D) Cycling E6-infected cells. (E-H) E6-infected cells at Mint. The assays are: PC, phase contrast; p21, immunocytochemical detection of p21Waf1; SAßgal, histochemical assessment of SAß-gal activity; TUNEL, assay for apoptosis. Arrows indicate multinucleate cells (F) and apoptotic cells (H). All cells except PC are counterstained with haematoxylin. Cell-based assays were done in triplicate. White bar, 100 µm (A,E); black bar, 100 µm (B-D,F-H).

 


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  		Fig. 5. Microinjection of control IgG or anti-p53 antibodies into senescent AG05229 cells. The injected cells are marked by Texas Red, nuclei are detected by DAPI staining and BrdU incorporation is marked by FITC. The images are merged in the bottom panels. The DO-1-injected cells show two nuclei incorporating BrdU. Bar, 100 µm.

 


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  		Fig. 6. Growth characteristics of 5229.E6p30 cells that escape Mint. (A) Growth kinetics. (B-D) Phase contrast images showing the cells at 250 days (B), 270 days (C) and 350 days (D) post-infection. Arrows indicate mitotic cells (B,C) and apoptotic-like cells (D). Bar, 100 µm.

 


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  		Fig. 7. Growth characteristics of 5229.E6 cells infected with hTERT. (A) Growth curve of population doublings achieved against time: •, E6.hTERT clone 2; {circ}, E6.hTERT clone 3; {blacksquare}, growth of the control clones (only clone 2 shown for clarity). (B) The same data as represented by a histogram. C-1 to C-3 are the control clones and hTERT-1 and hTERT-2 are the hTERT-infected clones. (C) Phase-contrast image showing the morphology of the E6.hTERT cells. Bar, 100 µm.

 

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© The Company of Biologists Ltd 2003