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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00809

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Activity of the human papillomavirus E6 PDZ-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes

¹ Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TA, United Kingdom
² International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy

View larger version (33K): [in a new window]   Fig. 1. (A) The C-terminal sequences of HPV18 E6 and two mutants, Thr156Glu and Arg153Leu, are shown, together with the class 1 PDZ-binding consensus sequence and PKA recognition motif. The activities of the wild-type and mutant proteins in Dlg degradation assays (in vitro and in vivo), and their response to PKA activity are shown (Gardiol et al., 1999; Kühne et al., 2000) (our unpublished results). (B) Expression of E6 mRNA in SVJD keratinocytes was analysed by semi-quantitative PCR using primers specific for HPV18 E6 or GAPDH. Equal volumes of the PCR reaction were separated on a 1% agarose-ethidium bromide gel. HPV18 DNA-positive cervical carcinoma cell line, HeLa, and all three E6 lines contained two amplified bands, one of 450 bps corresponding to full-length E6 and a second of 280 bps representing the spliced E6* product. The position of size markers is shown.

View larger version (117K): [in a new window]   Fig. 3. Morphology of E6 cells. Cells seeded onto culture dishes at a density of 5x105 cells/ml were allowed to adhere overnight before being photographed in a temperature-controlled stage chamber. a,a', Neo; b,b', Glu18E6; c,c', WT18E6; d,d', Leu18E6. (a-d) Light microscopy of cell cultures shows that cells expressing an active E6 PDZ-binding domain (WT18E6 and Leu18E6) were less likely to associate into organized colonies compared with control cells (Neo) or cells expressing an E6 with an inactive PDZ-binding motif (Glu18E6). Phase contrast microscopy (a'-d') of cells at higher magnification shows that E6 cells have undergone shape change and increased spreading. These effects were more prominent in WT18E6 and Leu18E6 cell lines. Bars, 100 µm (a-d) and 50 µm (a'-d').

View larger version (130K): [in a new window]   Fig. 6. Expression of adherens junction proteins in E6 cells. (A) Cells grown to low and high cell densities were stained with an anti-E-cadherin MAb. a,a', Neo cells; b,b', Glu18E6 cells; c,c', WT18E6 cells; d,d', Leu18E6 cells. The level of decrease of E-cadherin at peripheral contact sites in E6 cells correlated with the activity of the PDZ-binding motif. (B) Cells were stained with a MAb specific for ß-catenin (a, Neo cells; b, Glu18E6 cells; c, WT18E6 cells; d, Leu18E6 cells. Bar, 20 µm (A and B, a-d) and 40 µm (A, a'-d'). (C) Total extracts of cells grown to 80% confluency were analysed for protein expression levels of adherens junction proteins (E-cadherin, P-cadherin, -catenin and ß-catenin). Note that HeLa cells are E- and P-cadherin negative. Actin levels control for loading.

View larger version (70K): [in a new window]   Fig. 2. Dlg expression in E6 cell lines. (A) Western blot analysis of Dlg in cell extracts of E6 cell lines, Neo and HeLa cells. A decrease in total cell levels of Dlg correlates with expression of E6 with an active PDZ-binding motif. Position of markers of Mr (kDa) are as indicated. Actin levels control for loading. (B) Immunofluorescence microscopy of cells at high density stained with the anti-Dlg MAb 2D11. In Neo cells Dlg is recruited to the cell periphery at sites of intercellular contact. Peripheral levels of Dlg are significantly decreased in E6 cells expressing an active PDZ-binding domain (WT18E6 and Leu18E6), but are only partially reduced in cells expressing an E6 unable to degrade Dlg (Glu18E6). Bar, 20 µm.

View larger version (98K): [in a new window]   Fig. 4. Organization of F-actin cytoskeleton in E6 cells. Cells at high (a-d) and low (a'-d') cell densities were stained with rhodamineconjugated phalloidin. (a,a') Neo cells; (b,b') Glu18E6 cells; (c,c') WT18E6 cells; (d,d') Leu18E6 cells. Bar, 40 µm (a-d) and 20 µm (a'-d'). Arrowheads in d' indicate cells that lack actin filaments.

View larger version (54K): [in a new window]   Fig. 5. Intracellular levels of vimentin and cytokeratin 8. Western blots of total cell extracts from confluent cultures of Neo, E6 cells and fibroblasts were probed with a vimentin MAb or a cytokeratin 8 (CK8) MAb. The positions of markers of Mr (kDa) are shown.

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