Induction of cyclin E-cdk2 kinase activity, E2F-dependent transcription and cell growth by Myc are genetically separable events

Abstract

The c-myc gene has been implicated in three distinct genetic programs regulating cell proliferation: control of cyclin E-cdk2 kinase activity, E2F-dependent transcription and cell growth. We have now used p27(-/-) fibroblasts to dissect these downstream signalling pathways. In these cells, activation of Myc stimulates transcription of E2F target genes, S-phase entry and cell growth without affecting cyclin E-cdk2 kinase activity. Both cyclin D2 and E2F2, potential direct target genes of Myc, are induced in p27(-/-) MycER cells. Ectopic expression of E2F2, but not of cyclin D2, induces S-phase entry, but, in contrast to Myc, does not stimulate cell growth. Our results show that stimulation of cyclin E-cdk2 kinase, of E2F-dependent transcription and of cell growth by Myc can be genetically separated from each other.

Fig. 1. Immortalization and induction of apoptosis by Myc in primary p27^+/+ and p27^–/– MEFs. (A) Number of colonies growing upon infection of either p27^–/– or p27^+/+ cells with control (‘pbabe’) or Myc-expressing virus (‘pbabe-Myc’), after selection with puromycin and incubation for 2 weeks. The graph shows a quantitation of a representative experiment. The western blot documents equal expression of Myc proteins in p27^–/– and p27^+/+ cells. (B) Growth curve of pools of p27^+/+ and p27^–/– cells recovered after infection with the indicated viruses; 5 × 10⁴ drug-resistant cells were plated at the start of the experiment. (C) Induction of apoptosis by Myc. p27^+/+ and p27^–/– MEFs, recovered after infection with the indicated viruses and selection, were plated in the absence or presence of serum; photographs were taken 48 h after plating.

Fig. 2. Induction of cell cycle progression and cell growth by Myc. (A) Western blots documenting DNA-damage-independent expression of p53 in p27^–/– and p27^+/+ 3T3 cell lines and two MycER clones, and expression of p19ARF in MycER clones. Where indicated, cells were treated with 0.5 µg/ml adriamycin for the indicated periods of time (24 h in the upper panel). (B) Percentage of cells incorporating BrdU in p27^–/– and p27^+/+ MycER cell lines. Cells were serum starved for 48 h before addition of either 200 nM 4-OHT or 10% FCS. The percentage of cells incorporating BrdU was determined 20 h later. (C) Cell cycle distribution of the indicated cell lines. Cells were starved for 48 h before re-induction. Samples were taken at the indicated time points after addition of either 200 nM 4-OHT or 10% FCS. (D) FSC profiles indicating growth of p27^–/– and p27^+/+ MycER cells after activation of Myc or addition of FCS for 24 h.

Fig. 2. Induction of cell cycle progression and cell growth by Myc. (A) Western blots documenting DNA-damage-independent expression of p53 in p27^–/– and p27^+/+ 3T3 cell lines and two MycER clones, and expression of p19ARF in MycER clones. Where indicated, cells were treated with 0.5 µg/ml adriamycin for the indicated periods of time (24 h in the upper panel). (B) Percentage of cells incorporating BrdU in p27^–/– and p27^+/+ MycER cell lines. Cells were serum starved for 48 h before addition of either 200 nM 4-OHT or 10% FCS. The percentage of cells incorporating BrdU was determined 20 h later. (C) Cell cycle distribution of the indicated cell lines. Cells were starved for 48 h before re-induction. Samples were taken at the indicated time points after addition of either 200 nM 4-OHT or 10% FCS. (D) FSC profiles indicating growth of p27^–/– and p27^+/+ MycER cells after activation of Myc or addition of FCS for 24 h.

Fig. 3. Failure of Myc to activate cyclin E-dependent kinase in p27^–/– cells. (A) Autoradiogram of cyclin E-dependent kinase assays. p27^+/+ and p27^–/– MycER cells were serum starved for 48 h and re-stimulated by addition of either 4-OHT or FCS as above. Samples were taken at the indicated time points and cyclin E-kinase activity was determined using histone H1 as substrate. (B) Quantitation of cyclin E-kinase activity (mean values from two independent experiments). (C) Western blots documenting the amount of cyclin E and p130 proteins, and cyclin E–p130 complexes in serum-starved p27^+/+ and p27^–/– MycER cells after induction of Myc. Samples were taken at the indicated time points. (D) Western blots documenting expression of cdk2 and cyclin A and quantitation of cyclin E-dependent kinase (relative to non-induced cells) in pools of p27^–/– and p27^+/+ MycER cells and in several independent clones of p27^–/– MycER cells.

Fig. 4. Activation of E2F-dependent genes by Myc is independent of p27. (A) RT–PCR documenting the expression of target genes of E2F. Total RNA was prepared from p27^–/– and p27^+/+ MycER cells after stimulation with FCS or 4-OHT, and subjected to RT–PCR analysis as detailed in Materials and methods. (B) Percentage of cyclin A-positive cells after microinjection of expression plasmids encoding the indicated proteins. Cells were serum starved for 48 h before microinjection. Four hours later, cells were re-stimulated by the addition of 4-OHT. Cyclin A expression was detected by immunofluorescence 20 h later. (C) Western blot documenting expression of cyclin A in p27^–/– MycER cells at the indicated time points after the addition of 200 nM 4-OHT to serum-starved cells. Where indicated, 25 µM roscovitine was added together with 4-OHT.

Fig. 5. Activation of cyclin A–cdk2 kinase activity by Myc in p27^–/– MycER cells. (A) Autoradiogram of cdk2 kinase assays. p27^+/+ and p27^–/– MycER cells were serum starved for 48 h and re-stimulated by the addition of either 4-OHT or FCS as before. Samples were taken at the indicated time points and cdk2-kinase activity was determined using histone H1 as substrate. Under these conditions, cyclin A regulates most of the cdk2 activity. (B) Quantitation of cdk2-kinase activity (mean values from two independent experiments). (C) Western blots documenting the amount of p130, cyclin A and cdk2 in p27^–/– MycER cells after stimulation with either 4-OHT or FCS as indicated. Cellular lysates were either left untreated (–) or depleted three times with α-cdk2 antibodies (+) before loading on the gel. (D) Western blots documenting the amount of p130 and of cyclin A in p27^–/– MycER cells after stimulation with either 4-OHT or FCS as indicated. Cellular lysates were either left untreated (–) or depleted three times with α-p130 antibodies (+) before loading on the gel.

Fig. 6. Ectopic expression of cyclin D2 and of E2F2 in p27^–/– MycER cells. (A) Western blots documenting expression of cyclin D2 (left) and of E2F2 (right) in pools of p27^–/– MycER cells after infection with either control retroviruses (top), or retroviruses encoding cyclin D2 (lower left) or E2F2 (lower right). Cells were either growing exponentially or serum starved for 24 or 48 h. After 48 h, 200 nM 4-OHT was added and samples were harvested after the indicated times. (B) Western blot documenting the expression of cyclin A in p27^–/– MycER control infected cells (top), and in cells expressing cyclin D2 (middle) or E2F2 (bottom) constitutively. (C) BrdU incorporation. The indicated cells were serum starved for 48 h before addition of 4-OHT. BrdU incorporation was measured 20 h later. (D) Cell cycle distribution as determined by FACScan of the indicated p27^–/– MycER cell lines after addition of 4-OHT. (E) Top: FSC profiles of p27^–/– MycER/cyclin D2 and of p27^–/– MycER/E2F2 cells, relative to control cells, after 48 h of serum deprivation. Bottom: FSC profiles of serum-deprived p27^–/– MycER/cyclin D2 (left) and of p27^–/– MycER/E2F2 (right) cells after addition of 4-OHT, relative to non-induced control cells. Cells were deprived of serum for 48 h before addition of 4-OHT; samples were analysed after 20 h. (F) FSC profiles of cells gated for individual cell cycle phases. p27^–/– MycER/E2F2 cells (as in E) were serum starved (control) and MycER was activated by addition of 4-OHT for 20 h.

Fig. 6. Ectopic expression of cyclin D2 and of E2F2 in p27^–/– MycER cells. (A) Western blots documenting expression of cyclin D2 (left) and of E2F2 (right) in pools of p27^–/– MycER cells after infection with either control retroviruses (top), or retroviruses encoding cyclin D2 (lower left) or E2F2 (lower right). Cells were either growing exponentially or serum starved for 24 or 48 h. After 48 h, 200 nM 4-OHT was added and samples were harvested after the indicated times. (B) Western blot documenting the expression of cyclin A in p27^–/– MycER control infected cells (top), and in cells expressing cyclin D2 (middle) or E2F2 (bottom) constitutively. (C) BrdU incorporation. The indicated cells were serum starved for 48 h before addition of 4-OHT. BrdU incorporation was measured 20 h later. (D) Cell cycle distribution as determined by FACScan of the indicated p27^–/– MycER cell lines after addition of 4-OHT. (E) Top: FSC profiles of p27^–/– MycER/cyclin D2 and of p27^–/– MycER/E2F2 cells, relative to control cells, after 48 h of serum deprivation. Bottom: FSC profiles of serum-deprived p27^–/– MycER/cyclin D2 (left) and of p27^–/– MycER/E2F2 (right) cells after addition of 4-OHT, relative to non-induced control cells. Cells were deprived of serum for 48 h before addition of 4-OHT; samples were analysed after 20 h. (F) FSC profiles of cells gated for individual cell cycle phases. p27^–/– MycER/E2F2 cells (as in E) were serum starved (control) and MycER was activated by addition of 4-OHT for 20 h.

Fig. 7. Activation of E2F2 promotes cell cycle progression, but not cell growth. (A) Left: western blot documenting expression of cyclin A before and after addition of 4-OHT, in pools of p27^–/– cells infected with either a retrovirus expressing an E2F2-ER chimera (Vigo et al., 1999) or control virus. Cells were serum starved for 72 h and induced by addition of 200 nM 4-OHT for 24 h. Right: western blot documenting the expression of E2F2-ER upon retroviral infection. (B) FACScan profile showing the DNA content of serum-starved p27^–/– E2F2-ER cells before (left) and after (right) addition of 4-OHT for 24 h. (C) FSC profiles of p27^–/– E2F2-ER cells under the same conditions as (B).