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doi: 10.1242/10.1242/jcs.00161

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Loss of Rb overrides the requirement for ERK activity for cell proliferation

Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK

View larger version (66K): [in a new window]   Fig. 1. Rb null but not wild-type MEFs enter the cell cycle in the presence of the Mek inhibitor U0126. Primary MEFs were plated at a 2500 cells/13 mm coverslip. The following day cells were placed in medium containing 0.5% (v/v) fetal calf serum (FCS) for 16-20 hours to render the cells quiescent. For cell cycle re-entry MEFs were stimulated with medium containing 10% FCS (v/v) for 16-20 hours. Cells were treated with U0126 at a final concentration of 20 µM, or vehicle control (DMSO) or LY294002 at a final concentration of 20 µM, or vehicle control (ethanol; ETOH) for 60 minutes prior to the addition of serum. (A) Immunofluorescent images of cells treated with U0126 stained with antibody against BrdU and Texas-Red-X-phalloidin. (B) Histogram of percentage BrdU-positive cells that have been treated with U0126, LY294002 or a combination.

View larger version (26K): [in a new window]   Fig. 2. Loss of expression of CDKIs does not result in escape from requirements for MAPK activation. MEFs derived from p21Waf1/Cip1, p27Kip1 or p16Ink4-knockout mice were plated at a density of 2.5x104/ml. The following day the cells were placed in medium containing 0.5% (v/v) FCS for 24-48 hours. The cells were then treated with U0126 (20 µM final concentration) or vehicle control (DMSO) 60 minutes and then stimulated with 10% (v/v) FCS for 16-20 hours followed by immunofluorescence.

View larger version (36K): [in a new window]   Fig. 3. ERK activation is similar in Wt and Rb-null cells and is sensitive to inhibition by U0126. Following overnight serum deprivation cells were treated with U0126 at a final concentration of 20 µM or vehicle control for 60 minutes. The cells were then stimulated with 10% serum for the indicated times. (a,c) Endogenous ERK activity was analysed by in vitro kinase assay. Equal amounts of cell lysates were subjected to immunoprecipitation followed by kinase assay using myelin basic protein (MBP) as the substrate. (b,d) Total cell lysates were analysed by western blotting for active phospho-ERK.

View larger version (29K): [in a new window]   Fig. 4. ERK signalling is uncoupled from cell cycle progression in Rb-/- tumour cell lines. Asynchronously growing populations of tumour cells were plated at a density of 104/ml. The following day cells were treated with U0126 at a final concentration of 20 µM, or vehicle control (DMSO). Growth of cells was monitored by BrdU incorporation 48 hour post treatment followed by immunofluorescence. (A) Proliferation of tumour cell lines derived from gliomas treated with inhibitor(s): SF295 (Rb wild-type) and SF539 (Rb deficient). (B) Proliferation of tumour cell lines derived from non-small lung carcinomas treated with inhibitor(s): H1299 (Rb wild-type) and H2009 (Rb deficient). (C) Inhibition of ERK activation by U0126.

View larger version (22K): [in a new window]   Fig. 5. Inactivation of the Rb protein is blocked by U0126 and LY294002 in Wt MEFs. After plating cells were placed in growth medium containing 0.5% FCS overnight, rendering the cells quiescent. Cells were then treated with U0126 at a final concentration of 20 µM or LY294002 at a final concentration of 20 µM or vehicle control for 60 minutes followed by stimulation with 10% (v/v) FCS. An equal amount of total cell lysate was then subjected to western blotting for Rb using a pan-antibody that detects both the hypo and hyper phosphorylated forms of the protein.

View larger version (56K): [in a new window]   Fig. 6. Expression of cyclins is regulated differentially by ERK and PI 3-kinase signalling. Expression pattern of cyclins was assessed following serum starvation (-) or following 16 hour restimulation with 10% (v/v) FCS (+). For inhibitor experiments cells were first pre-treated with inhibitors for 60 minutes prior to serum stimulation. An equal amount of cell lysate was subjected to western blotting for (a) cyclin D1, (b) cyclin E expression and (c) cyclin A expression. (d) ß-Tubluin was used as marker for loading control.

View larger version (44K): [in a new window]   Fig. 7. Levels of p27Kip1 are regulated by both MEK and PI 3-kinase pathways in Wt and Rb-deficient MEFs. Cyclin-dependent kinase activity was assessed following serum starvation (-) or following 16-20 hour restimulation with serum (+). For inhibitor experiments, cells were first pre-treated with inhibitors for 60 minutes prior to serum stimulation. Complexes were immunoprecipitated from 500 µg of cell lysates and cdk activity was assayed by in vitro kinase assay. (A) Endogenous cdk4 was immunoprecipitated and assayed using a C-terminal fragment of Rb as substrate. (B) Endogenous cyclin E was immunoprecipitated and assayed using Histone H1 as substrate. (C) Expression of p27Kip1 was assessed by western blotting.

View larger version (14K): [in a new window]   Fig. 8. Asynchronous proliferation of Wt MEFs but not Rb-/- MEFs is reduced by inhibition of MEK. Asynchronously growing populations of cells were plated at a density of 104/ml. The following day cells were counted (day 1) followed by treatment with U0126 (20 µM final concentration) or vehicle control (DMSO), or LY294002 (20 µM final concentration) or vehicle control (ETOH). Growth of cells was then monitored each day for four more days (day 2-5). On each day the cells' media was changed and retreated with inhibitor(s) or vehicle control.

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