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Proteasome-mediated regulation of the hDlg tumour suppressor protein

International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy

View larger version (22K): [in a new window]   Fig. 1. Regulation of hDlg protein by the proteasome in CaSKi cells. HPV-16-positive CaSKi cervical tumour cells were treated with proteasome inhibitors lactacystin (LC), N-CBZ-Leu-Leu-Leu-al (CBZ) and N-acetyl-Leu-Leu-norleucinal (LL), or with DMSO as control (–) for two hours before harvesting. Equal amounts of protein extracts were then separated by PAGE and analysed by western blot with -Dlg antiserum. In vitro translated Dlg (IVT Dlg) was included as a positive control and standard molecular weight protein markers (Celbio) are shown.

View larger version (34K): [in a new window]   Fig. 2. hDlg degradation by the proteasome in epithelial cancer cells. (A) HPV-16- positive CaSKi, HPV-18-positive HeLa and HPV-negative C33I cervical tumour cells plus HaCaT immortalised skin keratinocytes were treated for either two or four hours with N-CBZ-Leu-Leu-Leu-al (CBZ) proteasome inhibitor before harvesting. Stabilisation of hDlg protein was then assessed by western blot analysis. (B) Comparison of hDlg mRNA levels in CaSKi, HeLa, HaCaT and C33I cells, analysed by RT-PCR amplification (13 cycles) of hDlg mRNA and Southern blot. PhosphoImager scanning of the Southern blot gave the following counts: CaSKi=1335, HeLa=1043, HaCaT=2272, C33I=707. The control was RT-PCR and Southern blot of TBP mRNA.

View larger version (145K): [in a new window]   Fig. 3. Comparison of cellular morphologies of the different epithelial cell lines analysed. Direct microscope photographs of cultured cells growing in monolayer are shown. Magnification 60x. (A) HeLa human cervical carcinoma cells (HPV-18 positive). (B) C33-I human cervical carcinoma cells (no HPV).(C) CaSKi human cervical carcinoma cells (HPV-16 positive). (D) HaCaT immortalised human skin keratinocytes (no HPV).

View larger version (31K): [in a new window]   Fig. 4. Density-dependent protein stabilisation of hDlg in epithelial cells. HPV-16-positive CaSKi, HPV-18-positive HeLa, plus HPV-negative HaCaT and C33I cells were grown to 25% (L), 50% (M) or 75% (H) confluence and then either treated (+) or not (–) with the proteasome inhibitor CBZ for four hours prior to cell extraction. Western blot was then performed to analyse hDlg protein levels.

View larger version (83K): [in a new window]   Fig. 5. Localisation of hDlg at cell junctions in differentiated cells inhibits its proteasome degradation. HaCaT skin keratinocytes and HeLa cervical carcinoma cells were either treated or not with CBZ proteasome inhibitor as indicated. hDlg was detected by FITC immunofluorescence and laser confocal microscopy. Representative pictures from 0.8 µm Z-axial slices are shown; settings for scanning were identical.

View larger version (15K): [in a new window]   Fig. 6. Consequences of cell transformation on density-dependent stabilisation of Dlg. Primary BRK cells were grown to 25% (L), 50% (M) or 75% (H) confluence and either incubated (+) or not (–) with the proteasome inhibitor CBZ for two hours. The Dlg protein pattern was then analysed by western blot. The same experiment was also performed in parallel on a BRK cell line stably transformed with HPV-16 E7 and EJ-ras.

View larger version (21K): [in a new window]   Fig. 7. Accumulation of hyper-phosphorylated hDlg following proteasome inhibition. CaSKi and HaCaT cells were either treated or not as indicated with the proteasome inhibitor CBZ for two hours. After cell lysis, 50 µg of each protein extract were incubated at 30°C for 30 minutes, either with () or without 2000 units of protein phosphatase. After separation on SDS-PAGE, the hDlg protein pattern was visualised by western blot analysis. Migration of protein molecular weight markers is indicated.