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First published online 12 April 2005
doi: 10.1242/jcs.02307

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p130/p107/p105Rb-dependent transcriptional repression during DNA-damage-induced cell-cycle exit at G2

Mark W. Jackson^1,*, Mukesh K. Agarwal^1,*, Jinbo Yang¹, Patrick Bruss¹, Takeshi Uchiumi², Munna L. Agarwal¹, George R. Stark¹ and William R. Taylor,^1,,

¹ Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA
² Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan

View larger version (23K): [in a new window]   Fig. 1. Cell-cycle distribution of MEFs. Cells were treated for 12 or 24 hours with adriamycin (345 nM) and fixed. The DNA content of propidium iodide-stained cells was determined by FACS. (A) Flow cytometry profiles of MEFs treated with adriamycin. (B) Percentage of MEFs in different phases of the cell cycle. Standard errors are shown.

View larger version (86K): [in a new window]   Fig. 2. Analysis of gene expression. Total RNA was analyzed by northern blot. Transfers were stripped and probed for the expression of gapdh to assess loading. Results from two representative experiments are shown. (A) Gene expression in wild-type and p130/p107-null MEFs. MEFs were treated with adriamycin (345 nM) and northern blots were analyzed with the probes indicated. (B) Repression by p53. The effect of p53 was determined using (10)1 Val5 cells with a temperature-sensitive p53. p53-null MEFs were used as a control.

View larger version (66K): [in a new window]   Fig. 3. The role of the RB family in the repression of plk1. MEFs were exposed to adriamycin (ADR, 345 nM) and total RNA was analyzed by northern blot. (A) plk1 mRNA expression in wild-type and p130/p107-null MEFs. To indicate loading, the gel is shown stained with ethidium bromide before transfer. (B) plk1 mRNA expression in cells lacking all three RB-family proteins (triple-knockout, TKO cells). The transfer was stripped and probed for the expression of gapdh to assess loading.

View larger version (24K): [in a new window]   Fig. 4. Cell-cycle distribution of cells lacking RB proteins in response to DNA damage. Primary MEFs were treated with etoposide or adriamycin, and cell-cycle distributions were determined using FACS. (A) Effect of etoposide on wild-type (WT) and p130/p107/p105RB-null (triple-knockout, TKO) MEFs. Histograms of DNA content are shown. (B and C) Effect of etoposide and adriamycin on G2 arrest. The percentage of cells with a 4N content of DNA in treated samples is shown relative to the percentage in untreated wild-type or TKO MEFs. In (C), the cells were exposed to adriamycin for 72 hours. Results from two independent experiments are shown.

View larger version (66K): [in a new window]   Fig. 5. Analysis of PLK1 expression in response to p53 signaling. The levels of PLK1 protein and mRNA were measured using western and northern blots, respectively. To assess loading, northern and western blots were stripped and reprobed for the expression of gapdh (northern) or actin (western). (A) Expression of plk1 and p21/waf1 mRNAs in response to the overexpression of p53. TR9-7 cells containing a tetracycline-regulated p53 were incubated in the presence (p53 off) or absence (p53 on) of tetracycline. (B) The effects of p53 induced by DNA damage or high levels of p14ARF. NARF2 cells containing IPTG-inducible p14ARF were incubated in the presence of IPTG for 24 hours or in the presence of adriamycin (345 nM) for 24 hours without adding IPTG. (C) The role of p53 and p21/WAF1 in the downregulation of the PLK1 protein in response to DNA damage. HT1080 cells with (GSE) and without (WT) the dominant-negative p53 fragment GSE56 were analyzed, as well as HCT116 cells in which p21/waf1 was deleted and the corresponding parental cells. The cells were exposed to adriamycin (345 nM), and the total protein was extracted and analyzed by western blot.

View larger version (32K): [in a new window]   Fig. 6. Requirements for PLK1 downregulation in NARF2 cells. The expression levels of the indicated proteins were determined using western blot with extracts of NARF2 cells infected with viruses encoding the indicated genes. LXSN, pBabe-Puro and LV serve as controls. The NARF2 populations were exposed to IPTG for 48 hours and analyzed using antibodies to PLK1, p53, p21/WAF1, p14ARF and actin.

View larger version (34K): [in a new window]   Fig. 7. FACS analysis of KI67 and DNA content in HT1080-derived cells. Flow cytometry was used to measure KI67 expression and DNA content simultaneously. (A) Representative FACScans of TR9-7 cells. TR9-7 cells were incubated in the presence or absence of tetracycline for 72 hours to induce p53. Cells were fixed and incubated with antibodies to KI67, followed by a secondary antibody labeled with FITC. Cells were also stained with propidium iodide for analysis of DNA content. Cells with 2N or 4N DNA content showing positive staining for KI67 are shown. Controls for each treatment were treated identically, except that the antibody to KI67 was not added. (B) Quantitation of the percentage of cells that are positive for KI67. TR9-7 cells were treated as described in (A). WT and GSE cells were left untreated or exposed to etoposide (10 µM) or adriamycin (345 nM) for 72 hours. KI67 and DNA content were measured as described in (A). The percentage of cells with 2N or 4N DNA content showing positive staining for KI67 is shown. The results represent two independent experiments.

View larger version (17K): [in a new window]   Fig. 8. Model of transcriptional repression and G2 arrest. A pathway involving ATM, ATR, CHK1 and CHK2 inactivates CDC25C in response to DNA damage, leaving the CDK1/Cyclin B1 complex inactive. p53 also helps to inactivate CDK1 by inducing p21/WAF1, GADD45 and 14-3-3. Cells blocked by these pathways are in G2, with high levels of all the proteins needed for mitosis. The block can be overcome by simple re-activation of CDK1. p53 also utilizes p21/WAF1 to block the phosphorylation of RB-family proteins by CDKs other than CDK1. Hypophosphorylated RB bound to E2F then downregulates the levels of proteins needed for mitosis. The similarity of this effect to the changes in gene expression that occur during G0 suggests that the p53/RB-dependent pathway drives cells out of the cell cycle from G2.