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First published online 23 May 2006
doi: 10.1242/jcs.02989

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Upregulation of chicken p15^INK4b at senescence and in the developing brain

S.-H. Kim^1,*, J. Rowe¹, H. Fujii^2,, R. Jones¹, B. Schmierer^3,, B.-W. Kong⁴, K. Kuchler³, D. Foster⁴, D. Ish-Horowicz² and G. Peters^1,¶

¹ Molecular Oncology, Cancer Research UK London Research Institute, Lincoln's Inn Fields, London, WC2A 3PX, UK
² Developmental Genetics Laboratories, Cancer Research UK London Research Institute, Lincoln's Inn Fields, London, WC2A 3PX, UK
³ Max F. Perutz Laboratories, Department of Medical Biochemistry, Campus Vienna Biocenter, A-1030 Vienna, Austria
⁴ Department of Animal Science, University of Minnesota, St Paul, MN 55108-6014, USA

View larger version (44K): [in a new window]   Fig. 1. Characterisation of the chicken INK4b and ARF gene products. (A) CEFs (32-38 PDs) were infected with retroviruses encoding 2xHA-tagged versions of chicken ARF and INK4b, or empty vector control (Vec). Pools of drug-resistant cells were analysed for expression of HA-tagged proteins (2HA-ARF, 2HA-INK4b), p53 and p21CIP1. Actin was used as a loading control. (B) At 8 days post-infection, viable cell numbers were compared by staining the cells with Crystal Violet. The results represent the OD at 590 nm averaged from three independent cell pools. In a separate experiment, equivalent cell pools were labelled with BrdU for 1 hour and the proportion of BrdU-positive cells determined by immunohistochemistry. (C) Samples of lysates from CEFs expressing 2xHA-tagged chicken INK4b or ARF (as in panel A) were immunoblotted with polyclonal antisera raised against ARF (SK8) or INK4b (SK14) peptides. The asterisk identifies the background band detected with the SK8 antibody, as discussed in the text. (D) CEFs (36 PDs) were infected with a retrovirus encoding human E2F1, or empty vector, and analysed for expression of endogenous ARF and INK4b by immunoblotting with the SK8 and SK14 antisera respectively. The levels of p53 were also assessed using the HP64 monoclonal antibody. Actin served as loading control. The asterisk identifies the background band detected with the SK8 antibody, as discussed in the text.

View larger version (49K): [in a new window]   Fig. 2. Response of chicken INK4b and ARF to different signalling pathways. (A) CEFs (32-38 PDs) were infected with retroviruses encoding an activated form of human H-Ras (Ras), human E2F1 or SV40 T-antigen (T-Ag), with corresponding empty vector controls (Vec). Following drug selection, the cells were analysed for expression of the exogenous Ras, E2F1 and T-Ag by immunoblotting (B) Samples of total RNA were analysed for the expression of INK4b and ARF RNA by northern blotting, using probes specific for the 3'-untranslated region of each transcript. GAPDH was used to control for loading. (C) CEFs were infected with MC29 virus or MH2 virus as indicated and total RNA was prepared at 2 days post-infection. Expression of INK4b and ARF was assessed by northern blotting, and virus infection was confirmed using a probe representing the entire ALV genome.

View larger version (37K): [in a new window]   Fig. 3. TGF-ß downregulates INK4b expression in CEFs and chicken ovarian granulosa cells. (A) CEFs (35 PDs) were treated with 2 ng/ml of recombinant TGF-ß for the indicated times, and samples of total RNA were analysed for expression of INK4b. Equal loading was assessed by staining the ribosomal RNA bands with Methylene Blue. (B) Immunofluorescence detection of Smad2/3 in CEFs that were either untreated (upper panels) or treated with recombinant TGF-ß for 1 hour. (C) Cultured cGCs were transferred into medium containing 5% FCS but lacking additional growth factors for 16 hours. The cells were then treated with the indicated amounts of TGF-ß for 4 hours and samples of total RNA were analysed for expression of INK4b and ARF.

View larger version (59K): [in a new window]   Fig. 4. Upregulation of INK4b in senescent CEFs. (A) Growth curve of primary CEFs passaged continuously until they reached senescence. (B) Photomicrographs of young and senescent CEFs after staining for senescence-associated (SA)-ß-galactosidase activity. (C) Northern blot of RNA from CEFs at 27, 43 and 53 PDs, hybridised with probes for the unique 3'-untranslated regions of INK4b and ARF, or with a genomic DNA fragment that recognises both transcripts [described as probe 1 in Kim et al. (Kim et al., 2003)]. The p21CIP1 RNA was detected using a chick p21CIP1 cDNA probe and GAPDH served as a control for loading. (D) Equivalent samples of total protein from CEFs at 27, 43 and 53 PDs were analysed by immunoblotting for endogenous INK4b (SK14 antibody) and p53. Actin served as loading control.

View larger version (27K): [in a new window]   Fig. 5. Extension of CEF lifespan with SV40 T-antigen and shRNAs. (A) CEFs were infected at PD 37 with a retrovirus encoding SV40 T-antigen (filled squares) or empty vector control (open circles) and passaged until they reached senescence. (B) shRNA-mediated knockdown of INK4b, ARF and p53 in CEFs. For INK4b, the panel shows effects of two separate shRNAs (#2 and #4) as assessed by immunoblotting with the SK14 antibody. Knockdown of ARF expression was assessed by northern blotting of total RNA as described in Fig. 2. GAPDH served as a control for RNA loading. Knockdown of p53 was assessed by immunoblotting with the HP64 antibody. (C) Growth curves of CEFs infected at PD 30 with pRetroSuper encoding independent shRNAs against INK4b (filled squares), a single shRNA against ARF (open triangles) or empty vector (open circles). (D) An analogous experiment showing CEFs infected at PD 40 with pRetroSuper encoding shRNA against p53.

View larger version (27K): [in a new window]   Fig. 6. Loss of INK4b expression in immortalised chicken cells. (A) Polyadenylated RNA from the DT40 and DF1 cell lines, and from primary CEFs at 43 PDs, were hybridised with probes specific for INK4b and ARF as in Fig. 2. (B) DF1 cells were treated with 1 µM 5'-aza 2'-deoxycytidine (5'-aza C) for 4 days and assayed for expression of INK4b and ARF by semi-quantitative RT-PCR. Control CEF cells (PD 49) were exposed to solvent alone. GAPDH was used as loading control. The respective product sizes are 799 bp (INK4b), 592 bp (ARF) and 709 bp (GAPDH).

View larger version (59K): [in a new window]   Fig. 7. Chicken INK4b is expressed in post-mitotic cells in the roof-plate of rhombomere 1. (A-D) Whole-mount in situ hybridisation for INK4b in chicken embryos at stage 13 (B), stage 17 (A,C) and stage 20 (D). The expression, confined to r1, is detected at least until stage 24. (B-D) Magnifications of the hind-brain region. Anterior is at the top of the figure. (E-G) Roof-plate cells expressing INK4b are post-mitotic. Cross-section from an embryo at stage 24 was hybridised in situ with the chicken INK4b probe (red) and stained with anti-phospho-histone H3 antibody (green). INK4b expression is in the roof-plate. (F,G) Higher magnifications of a part of E, showing that roof-plate cells, expressing INK4b are not stained with anti-phospho-histone H3 antibody (p-H3); nuclei were detected with TO-PRO-3 iodide (TO-PRO). (G) The green and red channel of F. Note that there is no overlap between green and red signals. The arrowheads indicate the expression. Dorsal is up. rp, roof-plate; fp, floor-plate; r1, rhombomere 1; r3, rhombomere 3; ov, otic vesicle. Bars: A and D, 1 mm; B,C,E-G, 0.2 mm.