[Cell Cycle 3:11, 1457-1462; November 2004]; ©2004 Landes Bioscience
BCCIP Functions through p53 to Regulate the Expression of p21Waf1/Cip1 Report
*Correspondence to: Zhiyuan Shen; University of New Mexico School of Medicine; Department of Molecular Genetics and Microbiology; MSC08-4660; Albuquerque, New Mexico 87131-000 USA; Tel.: 505.272.4291; Fax: 505.272.6029; Email:
[email protected] Received 08/15/04; Accepted 09/08/04
Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1213
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ACKNOWLEDGEMENTS
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BRCA2 and CDKN1A (Cip1/ p21/Waf1) Interacting Protein RNA interference short hairpin RNA
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RNAi shRNA
The BCCIP protein is a BRCA2 and CDKN1A (p21Waf1/Cip1, referred as p21 hereafter) Interacting Protein. Due to an alternative splicing, two major isoforms are expressed in human cells, BCCIPα and BCCIPβ.1 These two isoforms share identical N-terminal 258 amino acids, but each has a unique C-terminal sequence. The BCCIPα isoform (also called Tok-1α) was independently identified by two groups.2,3 Although Ono et al have shown that BCCIPα directly binds with p21 through a region identical to BCCIPβ,no interaction was originally detected between recombinant BCCIPβ and p21 proteins.3 However, we have been able to detect BCCIPβ and p21 interaction by coimmunoprecipitation of endogenous proteins.4 Ono et al have also reported that BCCIPα enhances p21 inhibition activity toward CDK2,3 and we have shown that overexpression of BCCIPβ inhibits the G1 to S cell cycle progression in HT1080 cells.4 These data suggest that BCCIP may regulate p21 functions through the direct interaction between BCCIP and p21. We have recently shown that overexpression of BCCIP increases p21 protein and mRNA levels,4 suggesting BCCIP may further regulate p21 function via the modulation of p21 expression. However, this potentially new mechanism by which BCCIP regulates p21 functions has not been completely elucidated. In this report, we show that partial shutdown of BCCIP by RNA interference (RNAi) reduces p21 level and impairs G1/S checkpoint activation in response to ionizing radiation. We further demonstrate that BCCIP regulates p21 expression through a p53-dependent mechanism. These data support the notion that BCCIP also functions through p53 to regulate p21 expression and functions.
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BCCIP, Tok-1, p21, Cip1, CDKN1A, BRCA2, p53
BCCIP
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INTRODUCTION
KEY WORDS
ABBREVIATIONS
The BCCIP protein is a BRCA2 and CDKN1A (p21Waf1/Cip1) Interacting Protein, which binds to a highly conserved domain of BRCA2, and a C-terminal domain of the CDK-inhibitor p21. We have previously reported that overexpression of BCCIP increases p21 mRNA and protein levels, and inhibits G1 to S progression. In this report, we show that a partial shutdown of BCCIP expression by RNA interference reduces p21 levels and impairs G1/S checkpoint activation in response to ionizing radiation in HT1080 cells. We further show that the regulation of p21 expression by BCCIP is dependent on p53, and BCCIP regulates p53 transcription activity. These data provide a new mechanism by which BCCIP regulates p21 functions.
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University of New Mexico School of Medicine; Department of Molecular Genetics and Microbiology; Albuquerque, New Mexico USA
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ABSTRACT
Xiangbing Meng Huimei Lu Zhiyuan Shen*
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We thank Dr. B. Vogelstein (Johns Hopkins University) for providing HCT116 (p53-/-) and the HCT116 (p53 wt) parental cells, Ms. J. Liu (University of New Mexico) for helpful discussions and critical reading of the manuscript. This research was supported by National Institute of Health NIEHS grant ES08353 and by the US Army Medical Research and Materiel Command grants DAMD17-02-1-0515 and DAMD17-03-1-0317).
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MATERIALS AND METHODS
Cell Culture and Gene Transfection. HT1080 cells were cultured in αMEM (Gibco BRL, Grand Island, NY) with 10% fetal bovine serum, 20 mM glutamine, and 1% penicillin/streptomycin. HCT116 cells were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University), and were cultured in DMEM (Biowhittaker, Walkersville, MD) with 10% fetal bovine serum (Biowhitaker, Walkersville, MD) and 1% penicillin/streptomycin (Gibco BRL, Grand Island, NY). Plasmid transfection with mammalian cells was performed with the Geneporter transfection kit (Gene Therapy Systems Inc., San Diego, CA). Western Blot and p21 Protein Stability Assay. Antibodies to p53, p21, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), DB Transduction Laboratory (Lexington, KY), and Sigma (Saint Louis, Missouri) respectively. To perform Western blot, cells were lysed in a buffer (50 mM HEPES pH7.6, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40). Proteins were separated by 10% or 15% SDS-PAGE. BCCIP was detected by specific rabbit antibodies generated earlier.2 The anti-β-actin monoclonal antibody was used to confirm loading of
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Figure 1 (Right). Shutdown of BCCIP by RNAi reduces p21 expression. (A) Regions of BCCIP targeted by RNAi. Illustrated are the two major isoforms of BCCIP proteins and the approximate site for RNAi targeting. NAD, N-terminal acidic domain; ICD, internal conserved domain; CVD, C-terminal variable domain. As shown here, regions specific for BCCIPα or BCCIPβ are selected for RNAi targeting on appropriate isoform. A common region of BCCIPα and BCCIPβ are selected for RNAi targeting on both isoforms. The details of targeting nucleotide sequences and shRNA vector construction are described in Materials and Methods. (B) Reduction of p21 protein level in BCCIP-shutdown HT1080 cells. HT1080 cells were transfected with pPUR/U6 vacant vector, pPUR/U6/ shRNA-BCCIPα, pPUR/U6/shRNA-BCCIPβ, and pPUR/U6/ shRNA-BCCIPαβ (see Materials and Methods for details about these vectors). Stable clones expressing respective shRNA were selected with puromycin. Shown are the protein levels of BCCIP (top panel) and p21 (middle panel) as detected by respective antibodies. Anti β-actin blot (bottom panel) was used to confirm loading of equal amount of protein for each sample. These cell lines are designated: Control (lane 1), RNAi-BCCIPα (lane 2), RNAi-BCCIPβ (lane 3), and RNAi-BCCIPαβ (lane 4). (C) Reduction of p21 mRNA level in BCCIP-silenced HT1080 cells. p21 (top panel) and actin (bottom panel) mRNA levels were detected in the same cell lines in Figure 1B by Northern blot.
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equal quantity of proteins in each samples. To quantify the protein level on a Western blot, the GeneGenome Chemiluminescence Capture and Analysis System (SynGene, Frederick, MD) was used. To measure the degradation dynamics of p21 proteins, cells were treated with 20 µg/ml cycloheximide for 0.5–2.0 hours, and the p21 protein levels were analyzed by Western blot as specified above. Expression of shRNA from pPUR/U6 Plasmid. Specific sequences targeting BCCIP and p53 were cloned into pPUR/U6 plasmid that carries a U6 promoter for short RNA transcription, and a puromycin marker for selection in mammalian cells. The construction of pPUR/U6 vector has been reported.5 Briefly, the shRNA expression cassette reported by Sui et al.6 were transferred into pPUR vector (Clontech, Palo Alto, CA). To build vectors that express shRNA against BCCIPα, BCCIPβ, and BCCIPα/BCCIPβ, the following target sequences were cloned into pPUR/U6 respectively: 5’-GGG AAC CTT CAT GAC TGT TGG-3’, 5’-GGG AAG CAA ATG GTC TTT TGA-3’, and 5’-GGG AAG TGC TAC TTT TAC CTT-3’. As a result, the following RNAi vectors were constructed: pPUR/U6/shRNA-BCCIPα, pPUR/U6/shRNABCCIPβ, and pPUR/U6/shRNA-BCCIPαβ. These vectors direct the syntheses of gene specific shRNA in vivo, and can be Figure 2. Impaired G1/S checkpoint activation in cells with partial BCCIP shutdown. (A) transfected into mammalian cells by selection with puromycin G1/S Transition in Cells without Irradiation. Early passages of BCCIP shutdown HT1080 to stably express shRNA. In addition, pPUR/U6 vectors cell lines (shown in Fig. 1B) were used for this assay. Various time points (labeled on the expressing shRNA specific to p53 genes were constructed left) after 0.1 µg/ml of nocodazole was added, cells were collected for DNA content using the following targeting sequences: 5’-GAC TCC AGT analysis using flow cytometry. The gradual disappearance of the G1-peak represents the Gy GGT AAT CTA C-3’, resulting in pPUR/U6/ shRNA-p53. A transition of G1-phase cells into S-phase. (B) G1/S Transition in Cells Exposed to 10 of γ-rays. The same isogenic cell lines as in Figure 2A were irradiated by 10 Gy 137Cs shRNA template sequence (5’-GGT TAT GTA CAG GAA γ–ray, and subjected to the same assay as in Figure 2A. CGC A-3’) targeted against the green fluorescent protein (GFP) mRNA was cloned into pPUR/U6, resulting in pPUR/U6/shRNA-GFP. This vector and the empty pPUR/U6 were used as pCMV-Myc/BCCIPβ. These vectors were used for transient overexpression negative controls. In our study, these control vectors produce similar of Myc- tagged BCCIP in p53 transcription assays (Fig. 6). Second, results. HA-tagged BCCIP were cloned in retrovirus vector described before.2 This Overexpression of BCCIP. Three approaches were used to overexpress vector uses puromycin as the selection marker, and was mainly used to BCCIP protein. First, full-length BCCIPα and BCCIPβ were cloned in quickly establish a population of BCCIP overexpressing cells (Fig. 4). Third, pCMV-Myc (Clontech, Palo Alto, CA) to make pCMV-Myc/BCCIPα and Myc-tagged BCCIP was overexpressed using adenovirus based method (see
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Figure 3. Silencing of BCCIP does not reduce p21 protein stability. (A) Western blot of p21 in HT1080 cells with BCCIP-shutdown. p21 protein level in HT1080 cells expressing BCCIPα and BCCIPβ shRNA were detected by anti-p21 western blot at various time after incubation with 20 µg/ml cycloheximide (top panel). Anti-BCCIP antibody was used to detect the endogenous BCCIPα and BCCIPβ protein levels (middle panel). Anti β-actin antibodies (bottom panel) was used to confirm loading of equal amount of total proteins in each lane. (B) The relative p21 protein levels in BCCIPsilenced cells. The p21 protein levels in (A) were quantified and normalized to the initial amount of p21 (0 hour after cycloheximide treatment) in the control cells. (C) The p21 protein stability in BCCIP-silenced cells. The quantified p21 protein levels (obtained from Fig. 3A) were normalized to the initial level of p21 (0 hour after cycloheximide treatment) of the same cell line to illustrate the p21 protein stabilities in each cell line.
next section). These vectors were mainly used for high efficiency (~100% of the cell population) transient expression of BCCIP (Fig. 5) when puromycin selection marker was not an option. The choices of overexpression approach are specified in figure legends. Adenovirus Mediated BCCIP Expression. Myc-tagged BCCIP overexpression was established by using an adenovirus mediated gene expression system. The system for generating recombinant adenoviruses established by Vogelstein lab was used.7 Briefly, Myc-tagged BCCIPα and BCCIPβ coding regions were inserted to the pAdTracker-CMV shuttle vector at NotI
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and XbaI sites. Then recombinant adenoviral vector plasmids were generated by homologous recombination of pAdTrack-CMV shuttle vector containing inserted gene with adenoviral backbone vector pAdEasy-1 in DY329 E. coli cells. The recombinant adenoviral vector was confirmed by the appearance of 3.0 or 4.5 kb fragment digested with PacI restriction endonuclease. Adenovirus was produced in package cells Ad293 by transfecting with 5 µg of recombinant adenoviral vector DNA predigested with PacI. Adenoviruses were collected from the packaging cells ten days after transfection by repeated freezing-thawing for five times followed by centrifuging. The supernatant containing virus was further used to infect Ad293 packaging cells in order to amplify adenovirus that mediates the over-expression of BCCIP. To achieve 100% infection efficiency of the cell population, the aforementioned adenoviruses were tittered based on expression of GFP marker. Then viruses were used to infect HT1080 at the predetermined titer. Confirmation of BCCIP expression was done by Western blot three days after infection. p53 Transcription Activity Assay. A p53 reporter plasmid p53-Luc was purchased from Stratagene (La Jolla,CA). This report plasmid p53-Luc confers p53-dependent expression of luciferase. To measure the p53 transcription activity, 1 µg of p53-Luc plasmid DNA was transfected into HCT116 cells. Forty eight hours after the transfection, luciferase activity was measured by a Luciferase Reporter Assay Kit (Clontech, Palo Alto, CA). In some experiments (see Figure legends for details), 1 µg vectors expressing exogenous p53 or BCCIP were cotransfected with the p53 reporter. The p53 expressing vector (pFC-p53) was purchased from Strategene (La Jolla, CA). BCCIP overexpression vectors were pCMV-Myc/BCCIPα and pCMV-Myc/BCCIPβ. Northern Blot of p21 mRNA. Total RNA was extracted by RNAqueousTM kit (Ambion, Houston, TX). Twenty µg of total RNA from each cell line was resolved on a 0.8% agarose gel containing formaldehyde and blotted to nylon membranes by capillary transfer. The membranes were hybridized to a full-length p21 cDNA labeled with 32P-dCTP by Ready-To-GoTM DNA labeling kit (Amersham Pharmacia, Piscataway, NJ). The blot was hybridized overnight at 42˚C with p21 probe in ULTRAhybTM buffer (Ambion Inc, Austin, TX), washed three times for 5–10 minutes at room temperature with 2x SSC, 0.1% SDS, and then three times for 15 minutes at 42˚C with 0.1x SSC, 0.1% SDS. The same membrane were stripped and rehybridized with β-actin probes. DNA Content Based Cell Cycle Analysis. The method originally described by Vindelov et al was adapted to stain nuclear DNA for flow cytometry analysis.8 Briefly, cells were trypsinized, washed with PBS and fixed with 70% ethanol. Then cells were pelleted, washed with PBS, and resuspended in 200 µl of citrate buffer (250 mM Sucrose, 0.05% DMSO, 40 mM Trisodium citrate, pH7.6). Nine hundred microliters of Solution A [0.003% trypsin in stock buffer (3.4 mM Trisodium citrate, 0.1% Nonidet P 40, 1.5 mM Spermine tetrahydrochloride, 0.5 mM Trizma, pH 7.6) were added and cells were incubated at room temperature for 10 minutes. Afterwards, 750 µl Solution B (0.025% Trypsin inhibitor, 0.01% Ribonuclease A in stock buffer) was added for another incubation of 10 minutes at room temperature. Then 750 µl of Solution C (0.0416% Propidium iodide, 3.3 mM Spermine tetrahydorchloride in stock buffer) was added to the cells, which were then ready for flow cytometry analysis. In each assay, 20,000 cells were collected by FACScan (Becton Dickinson, San Jose, CA) and analyzed by CellQuest software provided.
RESULTS Downregulation of BCCIP Reduces p21 Expression. Because BCCIP interacts with p21, it has been suggested that BCCIP mainly regulates p21 function via their direct interactions. However, a previous report4 has shown that overexpression of BCCIP increases p21 mRNA and protein levels, suggesting that BCCIP may also affect p21 functions through expression regulation. To confirm this, we reduced BCCIP expression in HT1080 cells by RNAi using short hairpin RNA (shRNA). Because the mRNAs of BCCIPα and BCCIPβ are products of alternative splicing,1 they share identical 5’-sequences but have unique 3’-sequences. Sequences targeted to BCCIPα, BCCIPβ, or both isoforms (Fig. 1A) were cloned into a shRNA
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expression vector,5 and transfected into HT1080 cells. After selection in puromycin, stable transfectants with about 90% reduction of BCCIPα and 50% reduction of BCCIPβ expression were obtained (Fig. 1B, top panel). These cell lines displayed relatively stable growth and retained the reduced BCCIP levels for up to 3–4 passages in the cell culture. We measured the expression of p21 protein and mRNA in these cells in their first three passages. As shown in Figures 1B (middle panel) and C, downregulation of BCCIP reduces both protein and mRNA levels of p21. These data further support the notion that BCCIP regulates p21 expression. BCCIP-Shutdown Impairs G1/S Checkpoint Activation in Response to Ionizing Radiation. Considering that overexpression of BCCIP elevates p21 protein levels and inhibits G1 to S cell cycle progression,4 and that partial shutdown of BCCIP by RNAi reduces p21 levels (Fig. 1), we predict that downregulation of BCCIP would impair G1/S checkpoint activation in response to DNA damage. We treated the cells with 0.1 µg/ml of nocodazole to block cells at M phase, so that no new G1 cells could be generated. The gradual disappearance of the G1 peak was measured by DNA-content analysis with flow cytometry. Without irradiation (Fig. 2A), G1 cells exited G1-phase by eight hours after incubation with nocodazole regardless to the BCCIP status in these cell lines. After 10 Gy of irradiation (Fig. 2B), a significant amount of HT1080/control cells remain in G1 phase by 8 and 16 hours after nocodazole incubation. This suggests an activation of G1/S checkpoint in response to radiation in the HT1080/ control cells. However, in HT1080/RNAi-BCCIPα, HT1080/RNAi-BCCIPβ, and HT1080/ RNAi-BCCIPαβ cells, much less amount of cells remain in G1 phase by 16 hours after irradiation as compared to HT1080/control (Fig. 2B). This suggests an impaired G1/S checkpoint activation in the BCCIP downregulated cells. Considering that overexpression of BCCIP inhibits G1/S transition4 and that BCCIP silencing impairs G1/S checkpoint activation in response to DNA damage (Fig. 2B), our data strongly suggest a role of BCCIP in G1/S regulation. The BCCIP-Induced p21 Upregulation is Not Caused by p21 Stabilization. Having established that partial shutdown of BCCIP reduces p21 levels (Fig. 1) and impairs G1/S checkpoint activation (Fig. 2), we next investigated the mechanisms by which BCCIP regulates p21 levels. One possibility is that BCCIP upregulates p21 protein level by stabilizing p21 protein. We used two approaches to test this. First, the p21 protein stability was measured in BCCIP-silenced cells. Cycloheximide was added to block new protein synthesis, and the endogenous p21 protein level was detected by Western blot (Fig. 3A). When the relative amount of protein was quantified and normalized to the control cells before cycloheximide treatment, shutdown of BCCIPα or BCCIPβ reduces p21 by 50–60% (Fig. 3B). When p21 protein levels were normalized to the pretreatment level of the same cells (Fig. 3C), no decrease of p21 stability was observed in BCCIP silenced cells. In fact, shutdown of BCCIPβ slightly stabilized p21 protein (Fig. 3C). Second, p21 protein stability was measured in BCCIP overexpressing HT1080 cells. Consistent with our previous report4 overexpression of BCCIP increases p21 protein levels by 150–200% (Fig. 4A and B). However, it does not stabilize the p21 protein, and in fact p21 protein stability was slightly reduced in cells overexpressing BCCIP proteins (Fig. 4C). Together, these data (Figs. 3 and 4) suggest BCCIP upregulates p21 expression not through a stabilization of p21 protein. Upregulation of p21 by BCCIP is p53-Dependent in HT1080 Cells. Because p53 is a critical transcription activator for p21 expression, we tested whether upregulation of p21 expression by BCCIP is dependent on p53. The p53 expression in HT1080 cells was silenced by shRNA (Fig. 5A), and BCCIPα or BCCIPβ was overexpressed in these cells. As shown in Figure 5B), overexpression of BCCIP increases p21 expression in control HT1080 cells, but not in p53 silenced HT1080 cells. These data suggest that the upregulation of p21 expression by BCCIP is dependent on p53. Measurement of p21 reduction in response to BCCIP silencing was not informative in p53 silenced cells because the basal p21 levels in these cells were already low. BCCIP Stimulates p53 Transcription Activity. Because BCCIP regulation on p21 expression is dependent on p53, we further asked whether BCCIP regulates p53 transcription activity. We employed a p53 reporter plasmid (p53-luc) that contains 14 tandem repeats of a p53 binding sequence (Fig.
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Figure 4. Overexpression of BCCIP does not stabilize p21 protein (A) Western blot of p21 in HT1080 cells over-expressing BCCIP. HA-BCCIPα and HA-BCCIPβ were overexpressed in HT1080 cells by retrovirus mediated gene expression (see Materials and Methods). Control cell line was infected with retrovirus that carries an empty vector. At various times after 20 µg/ml cycloheximide was added to each cell line, p21 protein levels were detected by anti-p21 blot (top panel). The level of overexpressed exogenous HA-BCCIP was detected with anti-HA antibody (middle panel) and anti β-actin antibodies (bottom panel) was used to confirm loading of equal amount of total proteins in each lane. (B) The relative p21 protein levels in BCCIP overexpressed cells. The p21 protein levels in (A) were quantified and normalized to the initial amount of p21 (0 hour after cycloheximide treatment) in the control cell. (C) The p21 protein stability in BCCIP overexpressed cells. The quantified p21 protein levels (obtained from Fig. 4A) were normalized to the initial level of p21 (0 hour after cycloheximide treatment) of the same cell line to illustrate the p21 protein stabilities in each cell line.
6A). As shown in (Fig. 6B), p53-dependent luciferase reporter expression was significantly lower in p53 deficient HCT116 (p53-/-) cells than that in wild type HCT116 (p53 wt) cells, and exogenous expression of p53 in the HCT116 (p53-/-) cells restores the expression of reporter gene. These results validate this reporter for an accurate measurement of the p53 transcription activity. We then cotransfected BCCIP expression vectors with the p53 reporter plasmid into p53 wild type and deficient HCT116 cells, and measured the p53 transcription activity. As shown in Figure 6C), overexpression of BCCIP significantly increases the p53-reporter expression in p53 wt cells, but not in p53-/- cells. Together, the data (Figs. 5 and 6) suggest that BCCIP upregulates p21 expression through the modulation of p53 transcription activity.
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Figure 5. The upregulation of p21 expression by BCCIP is dependent on p53. (A) Establishment of a p53 silenced HT1080 cells. The pPUR/U6/ shRNA-p53 vector was transfected into HT1080 cells, and stable shRNA expression cells were selected by puromycin. The endogenous p53 expression was silenced by shRNA targeted at p53 in HT1080 cells, resulting in HT1080/RNAi-p53 cells. The control cells were transfected with the pPUR/U6/shRNA-GFP vector (see Materials and Methods). The p53 levels in these HT1080 cells were measured by anti-p53 blot (top panel), and equal loading was shown by β-actin antibody (bottom panel). (B) Overexpression of BCCIPα or BCCIPβ does not induce p21 in p53 silenced HT1080 cells. Since puromycin marker has been used to select stable cells with p53 silencing, the retroviral based BCCIP overexpression (that uses puromycin marker) can no longer be used. Thus, exogenous Myc-tagged BCCIPα or BCCIPβ were over-expressed in the p53 silenced cells by the adenovirus mediated BCCIP expression to achieve 100% infection of the whole cell population (see Materials and Methods for details). The p21, p53, and Myc-BCCIP protein levels were detected by immuno-blot with appropriate antibodies. Anti β-actin antibodies (bottom panel) was used to confirm loading of equal amount of total proteins in each lane.
DISCUSSION
Figure 6. Expression of BCCIP stimulates p53 transcription activity. (A) Diagram of the p53-Luc reporter construct. The promoter region of p53-Luc (Stratagene, La Jolla, CA) contains 14 tandem repeats of a p53 binding sequence. Luciferase reporter gene expression can be activated by p53 binding to the promoter. (B) Expression of p53-dependent reporter gene in p53 wild type and p53-/- HCT116 cells. One µg of p53-Luc plasmid DNA was transfected into HCT116 (p53 wt) and HCT116 (p53-/-) cells. To confirm that the expression of the luciferase reporter gene was dependent on p53, exogenous p53 was expressed in HCT116 (p53-/-) by cotransfection of 1 µg pFC-p53 with 1 µg p53-Luc reporter in p53-/- HCT116 cells. (C) Expression of BCCIPα and BCCIPβ induces the p53-dependent reporter gene expression in p53 wild type HCT116 cells but not in p53 deficient cells. One microgram of pCMV-Myc/BCCIPα and pCMV-Myc/BCCIPβ overexpressing BCCIPα and BCCIPβ were cotransfected with 1 µg p53-luc reporter gene in p53 wild type and p53-/- HCT116 cells. One µg of pCMV-Myc empty vector cotransfected with p53-luc was used as the negative control. Luciferase activities were measured 48 hours after transfection.
Although the role of BCCIP in G1/S checkpoint control has been suggested by the observation that BCCIPα (Tok-1α) enhances the inhibitory activity of p21 toward Cdk2,3 no direct evidence has been documented previously. Our data show that a partial shutdown of BCCIP expression reduces p21 expression and impairs G1/S checkpoint activation in HT1080 cells in response to ionizing radiation. We further demonstrate that BCCIP regulates p21 expression by a p53-dependent mechanism. These data firmly support a critical role of BCCIP in G1/S cell cycle regulation. Based on data reported here and those published,3,4 we propose that BCCIP may regulate p21 functions through at least two mechanisms: via inhibition of p21 suppression activity toward Cdk23 that is related to the direct interaction between BCCIP and p21; and via a p53-dependent regulation of p21 expression. A third potential mechanism is through the regulation of intracellular distribution of p21, because the yeast homologue of human BCCIP, Bcp1, is recently implicated in protein trafficking between the nucleus and cytoplasm.9 The role of BCCIP on p21 intracellular distribution in human cells remains to be investigated.
The stimulation of p53 transcription activity by BCCIP is unlikely specific for p21 expression, as BCCIP also regulates the expression of another p53 target MDM2 (Meng et al, unpublished data). However, this does not exclude the possibility that BCCIP has a more direct role in regulating p21 than other downstream targets of p53, because BCCIP also interacts with p21 and works additively with p21 to inhibit the kinase activity of Cdk2.3,4 The very similar effects of BCCIPα and BCCIPβ downregulation on p21 expression and G1/S checkpoint response (Figs. 1 and 2) suggest that both isoforms are capable of mediating these functions in human cells. However, there is only one BCCIP homolog in Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, and mouse. The C-terminus of human BCCIPα (aa259-322) has no homology with BCCIP homologs in other organisms, but the C-terminus of human BCCIPβ (aa258-314) shows varying degrees of homology with all known BCCIP homologs. Thus, BCCIPβ appears to be the most conserved isoform.
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It is noteworthy to point out that the down regulation of p21 expression and subsequent impairment of G1/S checkpoint were observed within the first three passages of cell culture and these cells displayed only partial BCCIP shutdown (~90% for BCCIPα, and ~50% for BCCIPβ). After 3–4 passages, these cells displayed certain forms of genomic instability and the BCCIP protein expression in growing cells reverts to approximately the normal level (Meng et al., unpublished data) thus were not suitable for cell cycle analysis. Furthermore, we have attempted additional strategies but failed to establish stable HT1080 cell lines with further BCCIP shutdown due to growth retardation for clones with severe BCCIP down regulation (data not shown). Interestingly, the S. cerevisiae BCCIP homolog, Bcp1 (ORF number YDR361C), is essential for viability (the Saccharomyces Genome Database at http://www.yeastgenome.org). Shutdown of C. elegans 2H343 gene (the ortholog of human BCCIPβ) leads to embryo lethality.10 These suggest that BCCIP is an essential gene for cell viability and genomic stability. In conclusion, our data suggest that BCCIP functions through p53 to regulate p21 expression, and that this regulatory mechanism plays a critical role in the cell cycle control. References 1. Meng X, Liu J, Shen Z. Genomic structure of the human BCCIP gene and its expression in cancer. Gene 2003; 302:139-46. 2. Liu J, Yuan Y, Huan J, Shen Z. Inhibition of breast and brain cancer cell growth by BCCIPα, an evolutionarily conserved nuclear protein that interacts with BRCA2. Oncogene 2001; 20:336-345. 3. Ono T, Kitaura H, Ugai H, Murata T, Yokoyama KK, Iguchi-Ariga SM, Ariga H. TOK-1, a novel p21Cip1-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2 kinase. J Biol Chem 2000; 275:31145-54. 4. Meng X, Liu J, Shen Z. Inhibition of G1 to S cell cycle progression by BCCIPb. Cell Cycle 2004; 3:343-57. 5. Meng X, Yuan Y, Maestas A, Shen Z. Recovery from DNA damage-induced G2 arrest requires actin-binding protein filamin-A/Actin-binding protein 280. J Biol Chem 2004; 279:6098-105. 6. Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99:5515-20. 7. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998; 95:2509-14. 8. Vindelov LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983; 3:323-7. 9. Audhya A, Emr SD. Regulation of PI4,5P2 synthesis by nuclear-cytoplasmic shuttling of the Mss4 lipid kinase. EMBO J 2003; 22:4223-36. 10. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 2003; 421:231-7.
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