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Forskolin-mediated G1 arrest in acute lymphoblastic leukaemia cells: phosphorylated pRB sequesters E2Fs

Institute of Medical Biochemistry, University of Oslo, PO Box 1112, Blindern, N-0317, Oslo, Norway

View larger version (21K): [in a new window]   Fig. 1. Effect of forskolin on DNA synthesis. (A) Reh cells (0.1x105 cells/ml) were treated with forskolin (100 µM) for 3 days and DNA synthesis was measured as uptake of [3H]thymidine, as described in Materials and Methods. Vertical bars indicate the s.e.m. of three experiments. (B) Reh cells (0.1x106 cells/ml), with or without forskolin (100 µM) treatment, were counted for 10 days and the cell counts are presented as fold induction of the starting culture (0.1x106 cells/ml). One representative experiment is shown. Inset shows the cell counts up to 4 days with or without forskolin (100 µM) treatment. The cell counts are presented as the mean from five separate experiments. The vertical bars represent standard error of the mean (s.e.m.). C, control; F, forskolin.

View larger version (32K): [in a new window]   Fig. 2. Forskolin mediates a transient dephosphorylation of pRB. Reh cells were treated with forskolin (100 µM) for the hours indicated. Total cell lysate (50 µg) from treated and untreated cells were subjected to immunoblot analysis as described in Materials and Methods (A), using monoclonal antibodies towards pRB. (B) Anti-RB immunoblotting of lysates from resting T-lymphocytes (lane 1), Reh-control lysate (lane 2), forskolin-treated Reh-cells for 2 hours (lane 3), PHA/ionomycin-activated T-lymphocytes for 50 hours (lane 4) and anti-Fas antibody (CH-11)-treated jurkat wt cells for 4 hours (lane 5). (C) An immunoblot with forskolin-treated Reh-lysates, as described above, was detected with the different anti-pRB-phosphorylated antibodies, as indicated in the figure. Total expression of pRB was detected on the same blot as a control for equal loading of protein in each lane. One representative experiment of five is shown.

View larger version (36K): [in a new window]   Fig. 3. Forskolin induces a transient inhibition of the cyclin-associated kinase activities. Reh cells were treated with forskolin (100 µM) for the times indicated. Total cell lysates (500 µg) were subjected to immunoprecipitation (IP) with the appropriate antibodies, and kinase assays were performed as described in Materials and Methods, using histone 1 or GST-RB as substrate. One representative experiment of three is shown. ns, nonspecific antibody used as a negative control.

View larger version (42K): [in a new window]   Fig. 4. Effect of forskolin on the expression of G1 cyclin, CDK and CKI proteins. Reh cells were treated with forskolin (100 µM) for the hours indicated. Total cell lysates (50 µg) from treated or untreated cells were subjected to immunoblot analysis, as described in Materials and Methods, using antibodies specific for (A) cyclins and CDKs and (B) CKI proteins. One representative immunoblot of three is shown.

View larger version (31K): [in a new window]   Fig. 5. Inhibitors of phosphatase 1 and 2A prevent forskolin-mediated dephosphorylation of pRB. Reh cells were treated for 4 hours with forskolin (100 µM), in the presence or absence of tautomycin (10 µM). Total cell lysate (50 µg) from treated and untreated cells were subjected to immunoblot analysis (as described in Materials and Methods), using antibodies specific for (A) pRB, or (C) cyclins as indicated. (B) For analysis of kinase activity, 500 µg of total cell lysate was subjected to immunoprecipitation (IP) with the appropriate antibodies, and kinase assays were performed, as described in Materials and Methods, using histone 1 or GST-RB as substrate. One representative experiment of three is shown.

View larger version (23K): [in a new window]   Fig. 6. Effect of forskolin on the expression of p130 and p107. Reh cells were treated with forskolin (100 µM) for the hours indicated. Total cell lysate (50 µg) from treated and untreated cells were subjected to immunoblot analysis (see Materials and Methods), using antibodies specific for p130 (upper) and p107 (lower). One representative immunoblot of three is shown.

View larger version (39K): [in a new window]   Fig. 7. Co-immunoprecipitation of pRB with E2F-1 and E2F-4 antibodies. Reh cells were treated with forskolin (100 µM) for the hours indicated. (A) Total cell lysate (500 µg) from treated or untreated cells were subjected to immunoprecipitation as described in Materials and Methods, using antibodies specific for E2F-1 or E2F-4 as indicated. The E2F-complexes were resolved on a 10% SDS-PAGE and the resulting immunoblots were detected with antibodies specific for pRB. (B) 30 µg of the same cell lysates were subjected to immunoblot analysis to detect the total amount of E2F-1 or E2F-4 as indicated. One representative experiment of three is shown.

View larger version (46K): [in a new window]   Fig. 8. Gel retardation analysis of complex formation on the E2F-binding site. Reh cells were treated with forskolin (100 µM) for the hours indicated. (A) Total extracts were prepared as described in Materials and Methods and incubated with 32P-labelled oligonucleotide probe containing the E2F-consensus binding site. (B,C) Supershift assays were performed by incubating total cell extract with the appropriate antibodies before adding the probe containing the E2F-binding site. In all three panels, the protein-DNA complexes were subjected to a native 4% polyacrylamid gel electrophoresis and visualised by autoradiography. One representative experiment of three is shown. I, II and III denote the different protein complexes bound to the E2F-probe. The asterisks indicate supershifted bands.

View larger version (14K): [in a new window]   Fig. 9. Forskolin downregulates the E2F-promoter activity. Reh cells were transfected with the pGL3TATA-6xE2F-promoter luciferase construct or with the basic pGL3TATA vector. An SV40-ßGalactosidase construct was cotransfected with the luciferase constructs as an internal control. 20 hours after transfection, the cells were treated with and without forskolin (100 µM) for (A) 2 hours, (B) 24 hours and (C) 72 hours. After forskolin treatment the cells were harvested and lysed in reporter lysis buffer. The luciferase activity was determined and normalized for transfection efficiency, with the activity of ß-galactosidase being used as a reference. The values are presented as the mean from four experiments, and the vertical bars represent the s.e.m.

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