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Originally published In Press as doi:10.1074/jbc.M500189200 on April 29, 2005

J. Biol. Chem., Vol. 280, Issue 26, 24642-24648, July 1, 2005
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Identification and Characterization of Novel Isoforms of Human DP-1

DP-1{alpha} REGULATES THE TRANSCRIPTIONAL ACTIVITY OF E2F1 AS WELL AS CELL CYCLE PROGRESSION IN A DOMINANT-NEGATIVE MANNER*

Hironori Ishida{ddagger}, Yoshikazu Masuhiro{ddagger}, Akie Fukushima{ddagger}, Jose Guillermo Martinez Argueta{ddagger}, Noboru Yamaguchi§, Susumu Shiota{ddagger}, and Shigemasa Hanazawa{ddagger}

From the {ddagger}Division of Oral Infectious Diseases and Immunology, Faculty of Dental Science, Kyushu University, Higashiku, Fukuoka, 812-8582 and the §Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan

Received for publication, January 6, 2005 , and in revised form, April 26, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cell cycle-regulating transcription factors DP-1 and E2F form a heterodimeric complex and play a central role in cell cycle progression. Two different DP subunits (DP-1 and DP-2) exist in humans. In this study, we identified two novel DP-1 isoforms (DP-1{alpha} and DP-1{beta}) and characterized their structure and function. DP-1{alpha} is composed of 278 amino acids and lacks a portion of the C-terminal heterodimerization domain, whereas DP-1{beta} is composed of 357 amino acids with a frameshift that causes truncation of the C-terminal domain. Yeast two-hybrid and immunoprecipitation assays demonstrated that DP-1{alpha} binding to E2F1 was significantly reduced as compared with that of wild-type DP-1 or DP-1{beta}. Immunofluorescence analysis revealed that the subcellular localization of both DP-1 isoforms changed from the cytoplasm to the nucleus in HEK 293 cells cotransfected with E2F1 and wild-type DP-1 or DP-1{beta}. However, such a translocation for DP-1{alpha} was barely observed. Reverse transcription-PCR results showed that the three DP-1 isoforms are expressed ubiquitously at equal levels in several normal human tissues. We also demonstrated the expression of these isoforms at the protein level by Western blotting. Interestingly, we observed a significant decrease in transcriptional activity, a marked delay of cell cycle progression, and an inhibition of cell proliferation in DP-1{alpha}-transfected HEK 293 cells. Together, the results of the present study suggest that DP-1{alpha} is a novel isoform of DP-1 that acts as a dominant-negative regulator of cell cycle progression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The E2F family of transcription factors plays an essential role in regulating cell cycle progression (1, 2). This family consists of two subgroups, termed E2F and DP. Currently, the E2F/DP family in mammals is known to include seven E2F members (E2F1–7) and two DP members (DP-1 and DP-2) (3–10). All E2F/DP family proteins contain two highly conserved domains: the sequence-specific DNA-binding domain and the dimerization domain. E2F exhibits strong transcriptional activity when it forms a heterodimer with DP protein. E2F1 was initially identified as a cellular factor required for the transactivation of the adenovirus E2 promoter by the E1A oncoprotein (11). Subsequently, several studies (7, 12–14) have shown that this transcription factor regulates the timely expression of numerous genes (e.g. cyclin E, CDC2, cyclin A, B-Myb, E2F1, and p107) involved in cell cycle progression as well as several enzymes (e.g. DNA polymerase {alpha}, thymidine kinase, and dihydrofolate reductase) required for DNA replication.

DP-1 was first identified in 1993 as the partner of the E2F family member E2F1 (15, 16). Several studies (17–19) have shown that DP-1 is expressed at high levels in various murine and human tissues. Interestingly, a recent study (20) demonstrated that targeted inactivation of the Dp-1 locus in mice causes severe abnormalities during development of extra-embryonic tissue, which leads to embryonic lethality. This suggests an important role for DP-1 in morphogenesis. Therefore, it is of interest to identify any DP-1 isoform(s) as well as their precise functional roles. Although DP-2 has been known to have several isoforms that result from tissue-specific alternative splicing and that produce proteins that are 55, 48, and 43 kDa in size (17, 21, 22), DP-1 isoforms have not yet been identified. In this study, we explored whether there are additional isoform(s) of human DP-1. We identified herein two novel human DP-1 isoforms, termed DP-1{alpha} and DP-1{beta}, and characterized their structures and functions. We also investigated the interaction between the DP-1 isoforms and E2F1, as well as their transcriptional activity and role during cell cycle progression. We suggest that DP-1{alpha}, a novel isoform, acts as a dominant-negative regulator of cell cycle progression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of DP-1 and Its Isoforms—Full-length DP-1 and isoform cDNAs were amplified by PCR using specific primers (forward, 5'-ATGGCAAAAGATGCCGGTCTAATTG-3'; reverse, 5'-TCAGTCGTCCTCGTCATTCTCGTTG-3') and Pyrobest DNA polymerase (Takara, Otsu, Japan) from a human testis cDNA library (BD Biosciences). The PCR conditions were as follows: preincubation at 98 °C for 20 s, 30 cycles of denaturation at 98 °C for 20 s, annealing at 70 °C for 20 s, and extension at 72 °C for 3 min. The PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide, and fragments were extracted from the agarose gel using the QIAEX II kit (Qiagen, Hilden, Germany). The extracted fragments were amplified by PCR using specific primers. EcoRI and ApaI restriction enzyme sites were incorporated into the forward and reverse primers, respectively. The PCR products were electrophoresed and extracted under the same conditions. The extracted fragments were digested with EcoRI and ApaI and cloned into the pcDNA3 vector.

Construction of Plasmids—Wild-type DP-1 and its isoforms were amplified by PCR with primers containing EcoRI and SalI or XhoI sites. PCR fragments were subcloned into the EcoRI-SalI site of pGEX4T-1 (Amersham Biosciences), pGBT9 (BD Biosciences), and pEGFP-C2 (BD Biosciences) or the EcoRI-XhoI site of pcDNA3-6xMyc. E2F1 was amplified as a BamHI-EcoRI fragment by PCR and subcloned into the BamHI-EcoRI site of pGEX4T-1, pGAD10 (BD Biosciences). Alternatively, E2F1 was cloned into the BglII-EcoRI site of pEYFP-C1 (BD Biosciences). p3xFLAG-CMV7.1-E2F1 was constructed by isolating the HindIII-EcoRI fragment and cloning the fragment into the HindIII-EcoRI site of p3xFLAG-CMV7.1 (Sigma). All cDNAs were generated with Pyrobest DNA polymerase (Takara) by using DNA isolated from a human testis cDNA library (BD Biosciences). All PCR products were verified by sequencing. 6xE2F-Luc and dominant-negative (dn) DP-1 have been described elsewhere (23).

Cell Culture and Transfection—Human embryonic kidney fibroblast 293 (HEK 293) and HeLa cells were grown to 30–50% confluency in 100-mm plates in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (Thermo Trace, Melbourne, Australia), 100 units/ml penicillin, and 100 µg/ml streptomycin in a 5% CO2 atmosphere. For luciferase assays, cells were transfected with the indicated plasmid DNAs by the standard calcium phosphate precipitation method. In other experiments, Polyfect (Qiagen) was used for plasmid DNA transfection.

Expression of DP-1 and Its Isoforms in Human Tissues—To detect expression of DP-1 isoforms, reverse transcription-PCR (RT-PCR)1 was performed with the Access RT-PCR system (Promega, Madison, WI). Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan) from several human cell lines. Total RNA (0.1 µg) was reverse-transcribed in reaction mixture (50 µl) with 10 pmol of the following isoform-specific primers: wild-type, {beta} (reverse1, 5'-ACGCGTCGACTCAGTCGTCCTCGTCATTCTCGTTG-3') and DP-1{alpha} (reverse2, 5'-CATTGGAGATGCGTCG-3') for 45 min at 48 °C. Three microliters of this reaction mixture were subjected to PCR after addition of Ex Taq (Takara) PCR mixture. PCR amplification was performed in a final volume of 20 µl with 20 pmol of the following gene-specific primers: wild-type, {beta} (forward1, 5'-ACTGAATTCATGGCAAAAGATGCCGGTCTAATTG-3'; reverse1, 5'-ACGCGTCGACTCAGTCGTCCTCGTCATTCTCGTTG-3') and DP-1{alpha} (forward1, 5'-ACTGAATTCATGGCAAAAGATGCCGGTCTAATTG-3'; reverse2', 5'-ACGCGTCGACTCAAATTTGTCATTGGAGATGCGTCG-3'). To detect the expression of DP-1 isoforms in human normal tissues, we used the multiple tissue cDNA panels (BD Biosciences). The PCR conditions were as follows: preincubation at 96 °C for 20 s followed by 22 cycles of denaturation at 96 °C for 20 s, annealing at 70 °C for 20 s, and extension at 72 °C for 2 min. Five microliters of the PCR products were electrophoresed on a 2% agarose gel.

Immunoprecipitations—HEK 293 cells were harvested and washed two times with PBS (–) at 24 h following transfection of FLAG and Myc tag-fused expression vectors by Polyfect (Qiagen). The transfected cells were lysed in TNE buffer (10 mM Tris-HCl (pH 7.9), 150 mM NaCl, 1 mM EDTA) containing 1% Nonidet P-40 and a protease inhibitor mixture (Roche Diagnostics). Proteins in the cell lysate were precipitated with anti-FLAG M2-Agarose (Sigma) for 1 h at 4 °C. Immunocomplexes were washed five times with TNE buffer and were then eluted by 3xFLAG peptide (Sigma). Supernatants were suspended in 2xSDS-PAGE sample buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromphenol blue, 200 mM dithiothreitol) and boiled for 2 min at 95 °C. Equal quantities of protein extracts of the samples were separated by SDS-PAGE and analyzed by Western blotting.

Western Blotting—Cellular proteins separated by SDS-PAGE were transferred to Immobilon membrane (Nihon Millipore, Tokyo, Japan) by transfer blot (Bio-Rad Laboratories). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 1 h and washed with Tris-buffered saline containing 0.1% Tween 20 buffer. FLAG- or Myc-tagged DP-1 isoforms and E2F1 proteins were detected using mouse monoclonal anti-FLAG M2 antibody (Sigma, 1:1,000 dilution) or mouse monoclonal anti-c-Myc antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA) and peroxidase-conjugated rabbit anti-mouse IgG (1:1,000, Dako Japan, Kyoto, Japan). The ECL Plus detection system (Amersham Biosciences) was used. Hyperfilm was exposed for 15 s to 10 min. Western blot assays were performed as described previously (24).

Endogenouse DP-1 isoforms were detected using rabbit polyclonal anti-DP-1 antibody (1:1,000, sc-610, Santa Cruz Biotechnology) and alkaline phosphatase goat anti-rabbit IgG (1:1,000, Zymed Laboratories Inc., South San Francisco, CA). CDP-Star (Roche Applied Science, 1:200 dilution) in detection buffer (100 mM Tris-HCl, 100 mM NaCl) was used for detection. Hyperfilm was exposed for 4 min. Western blot assays were performed as described previously (24).

Yeast Two-hybrid Assay—pGBT9-DP-1s and pGAD10-E2F1 were transformed into the yeast strains AH109 or Y187, respectively. Transformed AH109 and Y187 were fused in synthetic dropout nutrient medium lacking tryptophan, leucine, histidine, and adenine and containing 2% glucose and 0.67% yeast nitrogen base without amino acids (Difco, Detroit, MI) overnight at 30 °C. Subsequently, cells were plated onto synthetic dropout nutrient plates containing the same components as well as 1.5% agar and X-{alpha}-gal (BD Biosciences) and cultured for 3 days at 30 °C. Yeast {alpha}-galactosidase assays were carried out in fused cells according to the manufacturer's protocol (BD Biosciences).

Immunofluorescence Microscopy—HEK 293 cells were transfected with GFP- or YFP-fused expression constructs. Transfected cells were cultured for 24 h at 37 °C in DMEM with 10% FBS, washed three times with PBS (–), and then fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Cells were washed three times with PBS (–), and coverslips were mounted on glass slides using Vectashield (Vector Laboratories, Berkochen, Germany). GFP or YFP fusion proteins were analyzed with a Zeiss LSM 510 META confocal imaging system (Carl Zeiss, Oberkochen, Germany).

Luciferase Assay—HEK 293 cells were transfected with 200 ng of reporter construct (195 ng 6xE2F-Luc reporter plasmid and 5 ng of pRL-tk-Luc internal control plasmid), 100 ng of pcDNA3-6xMyc-DP-1s, and 100 ng of p3xFLAG-CMV7.1-E2F1. The total amount of vector DNA was kept constant by balancing with the empty expression plasmid. The transfected cells were lysed in passive lysis buffer (Promega). Luciferase activities were detected with the TR717 microplate luminometer (Tropix) using the Dual luciferase reporter system (Promega). Luciferase assays were performed essentially as described previously (23, 24). All experiments were performed in triplicate.

Flow Cytometric Analysis—To analyze the cell cycle, HEK 293 cells were grown in a 10-cm dish with 10% FBS containing DMEM until the cells formed a subconfluent monolayer. Cells were then transfected with 10 µg of the indicated expression constructs by Polyfect as described previously (24). Flow cytometry was used to analyze cell cycle distribution by a modification of a described procedure (25). Briefly, after a 24-h incubation, cells were washed twice with PBS (–) and fixed for 2 h with cold 70% ethanol. After washing twice with PBS (–), cells were stained with propidium iodide (50 µg/ml containing 0.25 mg/ml RNase) for 30 min. Samples were passed through a nylon mesh (Kyoshin Riko, Tokyo, Japan) with a pore size of 40 µm and then analyzed on an EPICS XL flow cytometer (Beckman Coulter Inc.). Propidium iodide fluorescence was measured with a 620/15-nm band pass filter using linear amplification. A minimum of 5,000 events were collected per sample. Cell cycle analysis was performed using FlowJo (Tree Star, Inc., San Carlos, CA).

Measurement of Cell Proliferation—The HEK 293 cells transfected with the indicated expression constructs were cultured for 48 h in 10% FBS containing DMEM in the presence of Geneticin (1 mg/ml, Invitrogen). Cells (1 x 104 cells) were then selected with the drug via inoculation into each well of 24-well type culture dishes and cultured in 10% FBS containing DMEM in the presence of Geneticin. Cell numbers were measured at the selected times. The results are expressed as mean ± S.D. calculated from triplicate cultures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Structure of DP-1 Isoforms—To date, two Dp genes, Dp-1 and Dp-2, have been identified. Although a previous study (22) identified a DP-2 isoform, no DP-1 isoforms have yet been isolated. Since DP-1 effectively dimerizes with E2F1 and plays an important role as a transcription factor during cell cycling (26–32), our interest was to identify any DP-1 isoforms and to characterize their gene structure and function. To this end, we isolated cDNA clones of DP-1 from a human testis cDNA library and were able to identify two novel DP-1 isoforms in addition to the original wild-type DP-1. We named these isoforms DP-1{alpha} and DP-1{beta}. As shown in Fig. 1, A and B, DNA sequence analysis indicated that DP-1{alpha} has four nucleotides deleted in the PstI site. Consequently, the DP-1{alpha} isoform is composed of 278 amino acids, as compared with the 410 amino acids that comprise full-length DP-1, and lacks a portion of the C-terminal domain required for heterodimerization with E2Fs. We also analyzed the DNA sequence of the DP-1{beta} isoform and confirmed that exon 11 is deleted and that exons 10 and 12 are spliced together. These data show that DP-1{beta} is presumably generated by alternative splicing from canonical DP-1 pre-mRNA and, consequently, is composed of 357 amino acids with a frameshift that causes truncation of the C-terminal domain.



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  		FIG. 1.
Sequence and schematic representation of DP-1 isoforms. A, diagram and structure of wild-type DP-1 (DP-1WT), DP-1{alpha}, and DP-1{beta}. Exons (open boxes), untranslated regions (solid gray boxes), and frameshift regions (hatched boxes) are shown. Wild-type DP-1 is 410 amino acids long. DP-1{alpha} and DP-1{beta} are deduced to be 278 and 357 amino acids long, respectively. DP-1{alpha} contains a four-nucleotide deletion in exon 9. DP-1{beta} exon 10 and 12 are directly joined, which makes DP-1{beta} mRNA shorter than wild-type DP-1 mRNA. B, alignment of wild-type DP-1, DP-1{alpha}, and DP-1{beta} cDNAs and their amino acid sequences. The sequences of the C-terminal regions of wild-type DP-1 (black), DP-1{alpha} (blue), and DP-1{beta} (orange) are indicated. The four deleted nucleotides of DP-1{alpha} are indicated in red. The asterisk indicates a stop codon.

 
Expression Profiles of DP-1 Isoforms in Various Human Tissues and Cell Lines—We analyzed mRNA expression of the DP-1 isoforms in a wide variety of human tissues and cell lines by RT-PCR. Expression of all three DP-1 isoforms was ubiquitously detected at the same level in all of the tissues and cell lines tested (Fig. 2, B–E). Using Western blotting, we analyzed expression of DP-1 isoforms at the protein level in HEK 293, KB, THP-1, and HL-60 cells and detected three endogenous DP-1 isoforms with molecular masses corresponding to 49 (WT), 33 (DP-1{alpha} ({alpha})), and 42 (DP-1{beta} ({beta})) kDa in all cell lines tested (Fig. 2F, lanes 1–4). Interestingly, the expression levels of DP-1 {alpha} protein appeared to be considerably higher in THP-1 and HL-60 cells (Fig. 2F, lanes 3 and 4).



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  		FIG. 2.
Expression profile of DP-1 isoforms in normal human tissues and established cell lines. A, diagram showing the positions of the specific primers used to analyze expression of DP-1 isoforms in normal human tissues and established cell lines. Also shown are RT-PCR profiles of wild-type DP-1 and DP-1{beta} expression in normal tissues (B) and cell lines (C) as well as DP-1{alpha} expression in normal tissues (D) and cell lines (E). F, endogenous DP-1 isoforms in total cell lysates were detected by Western blotting with anti-DP-1 antibody.

 
Interaction between DP-1 Isoforms and E2F1—Since DP-1 is known to interact with E2F1 via its heterodimerization domain, we used yeast two-hybrid screening and immunoprecipitation to assess the interaction between each new DP-1 isoform and E2F1. In our two-hybrid experiments, we observed that although no {alpha}-galactosidase activity was detected in AH105 yeast cotransfected with DP-1{alpha} and E2F1 expression vectors, strong {alpha}-galactosidase activity at the same level as that of wild-type DP-1 was detected when AH105 yeast was cotransfected with DP-1{beta} and E2F1 (Fig. 2A). Next, we used immunoprecipitation to confirm the results of the yeast two-hybrid experiments. As shown in Fig. 3B, no interaction was detected between DP-1{alpha} and E2F1, although DP-1{beta} appeared to interact with E2F1 as strongly as wild-type DP-1. The interaction between DP-1{alpha} and E2F1 may depend on the lack of a heterodimerization domain in this isoform. These results suggest the possibility that DP-1{alpha} may act as a dominant-negative regulator of DP-1.

Localization of DP-1 Isoforms and E2F1 in HEK 293 Cells— Next, we used confocal imaging to observe the subcellular localization of the unbound DP-1 isoforms, the E2F1 monomer, and the DP-1/E2F1 heterodimers in HEK 293 cells transfected with GFP- or YFP-fused expression vectors. When each DP-1 isoform and E2F1 was expressed in cells transfected with GFP- or YFP-fused expression vector alone, we observed that all DP-1 proteins were predominantly expressed in the cytoplasm. In contrast, E2F1 was strongly expressed in the nuclei of the cells (Fig. 4A, upper panel). In cells cotransfected with YFP-E2F1 and GFP-DP-1WT or -DP-1{beta}, we clearly observed that these DP-1 isoforms localize to the nuclei in all cell lines tested. Interestingly, we also observed that DP-1{alpha} is almost completely localized to the cytoplasm, even when both GFP-DP-1{alpha} and YFP-E2F1 are coexpressed (Fig. 4A, lower panel). These results suggest that this phenomenon is due to DP-1{alpha} lacking a heterodimerization domain.

Transcriptional Activity in HEK 293 Cells Transfected with DP-1 Isoforms—Since E2F/DP-1 is a key transcription factor involved in cell cycle regulation, we used a luciferase assay to measure the transcriptional activity of DP-1 isoforms in HEK 293 cells cotransfected with a combination of DP-1 expression vectors and a reporter luciferase construct (6xE2F-Luc). As shown in Fig. 5, marked transcriptional activity was observed in cells transfected with pcDNA3/DP-1WT and DP-1{beta}. However, when cells were transfected with pcDNA3/DP-1{alpha}, we observed a significant decrease in transcriptional activity. In addition, we observed that transcriptional activity is completely inhibited in dnDP-1-transfected cells. These results suggested that DP-1{alpha} may act as a negative regulator of the transcription factor E2F/DP-1.

DP-1{alpha} Regulates Cell Cycle Progression of HEK 293 Cells in a Dominant-negative Manner—To confirm the possibility that DP-1{alpha} may negatively regulate cell cycle progression, we investigated the effects of DP-1 isoforms on cell cycle progression using flow cytometry. As shown in Fig. 6, each DP-1 isoform was transfected into HEK 293 cells, and cell cycle progression of the transfected or control cells was analyzed 24 h later. We observed a significant increase of the G2/M cell population in wild-type DP-1-transfected cells (Fig. 6C). However, the G2/M cell population in DP-1{alpha}-transfected cells was dramatically decreased (Fig. 6D). In contrast, no significant effects were observed in DP-1{beta}-transfected cells (Fig. 6E). These results demonstrated that DP-1{alpha} regulates the cell cycle in a dominant-negative manner.



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  		FIG. 3.
Interaction between DP-1 isoforms and E2F1. A, yeast two-hybrid assays were performed as described under "Materials and Methods." {alpha}-Galactosidase activity was measured in Saccharomyces cerevisiae strain AH109 cotransformed with combinations of empty vector (pGAD10; left (L); pGBT9, 1), DP-1 isoforms (WT, 2; {alpha}, 3; {beta}, 4), and E2F1 (pGAD10; right (R)). B, HEK 293 cells were cotransfected with combinations of empty vector, DP-1 isoforms (pcDNA3-6xMyc), and E2F1 (p3xFLAG-CMV-7.1), and cell lysates were immunoprecipitated with anti-FLAG antibodies. The presence of DP-1 isoforms in the immunoprecipitates (IP) was detected by Western blotting (WB) using anti-Myc antibody. The presence of E2F1 and DP-1 isoforms in cell lysates was detected by Western blotting using anti-Myc or anti-FLAG antibody as indicated.

 



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  		FIG. 4.
Immunofluorescence analysis of the localization of DP-1 isoforms and E2F1 in HEK 293 cells. HEK 293 cells were transfected with pEGFP-C2 (A) or pYFP-C1 empty vector (B) and with E2F1-fused pYFP-C1 (C). Cells were also transfected with wild-type DP-1 (D), DP-1{alpha} (E), DP-1{beta} (F), or dnDP-1 (G) fused to pEGFP-C2. In addition, cells were cotransfected with combinations of GFP-DP-1WT- (H), DP-1{alpha}- (I), DP-1{beta}- (J), or dnDP-1- (K) fused pEGFP-C2 and YFP-E2F-1.

 
DP-1{alpha} Inhibits Proliferation of HEK 293 Cells—Since flow cytometric analysis suggested that DP-1{alpha} is able to inhibit the proliferation of transfected cells, we next examined the proliferation kinetics of HEK 293 cells transfected with each DP-1 isoform. As shown in Fig. 7 (A and B), a marked decrease in cell proliferation was observed in DP-1{alpha}-transfected cells as compared with that of DP-1WT-transfected cells at 6 days after initiation of the cultures. A significant decrease was also observed in DP-1{beta}-transfected cells. Taken together, we believe that DP-1{alpha} plays an important role as a cell growth suppressor and is one of the important components in the regulation of E2F/DP-dependent promoters in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A recent study (20) demonstrated that knocking out Dp1 in mice results in embryonic lethality prior to 12.5 days of gestation due to a failure in the development of extra-embryonic tissues. In light of these results, it is important to explicitly define the functional role of DP-1 and DP-2 in relation to E2F during cell cycle progression and proliferation. Although DP-2 has been shown to exist as several isoforms that result from tissue-specific alternative splicing, DP-1 isoforms have not been previously identified. In this study, we described two novel DP-1 isoforms. Interestingly, DP-1{alpha} appears to regulate E2F1 in a dominant-negative manner during cell cycle progression.

We identified two novel DP-1 isoforms, DP-1{alpha} and DP-1{beta}, and found that the 381 nucleotides in the N-terminal region that encode the DNA-binding domain were identical in DP-1 and its two novel isoforms. Four nucleotides in the heterodimerization domain of DP-1{alpha} are deleted, and it is therefore only 278 amino acids long. Although we do not know how this deletion occurs, its short length suggests that it is most likely not mediated by general splicing factors. In contrast, DP-1{beta} results from the deletion of exon 11 and the subsequent splicing together of exons 10 and 12. This presumably occurs through alternative splicing from canonical DP-1 pre-mRNA. However, this isoform has a completely conserved heterodimerization domain. Together, these observations suggest that although DP-1{beta} retains the ability to form a heterodimer with E2F, heterodimer formation between DP-1{alpha} and E2F may be extremely weak.



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  		FIG. 5.
Transcriptional activity of DP-1 isoform-transfected HEK 293 cells. HEK 293 cells were cotransfected with plasmids as indicated. Cotransfected cells were cultured for 24 h, and luciferase activity was measured. The results are expressed as mean ± S.D. for the ratio of luciferase to luciferase activity in triplicate cultures. {alpha}, DP-1{alpha}; {beta}, DP-1{beta}; *, p < 0.05 versus wild-type DP-1.

 



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  		FIG. 6.
DNA histograms of DP-1 isoform-transfected HEK 293 cells. HEK 293 cells were transfected with expression vectors (A–F, 8 µg) as indicated and cultured for 24 h, washed, and fixed. Cells were then stained with propidium iodide and analyzed by flow cytometry.

 
E2F1 is known to accumulate in nuclei, whereas DP-1 is primarily distributed in the cytoplasm. Since each DP-1 isoform interacts with E2F1 in a different manner, we explored the localization of each DP-1 isoform as well as E2F1 in HEK 293 cells. Although both DP-1{beta} and wild-type DP-1 translocated to the nucleus from the cytoplasm, interestingly, we observed that DP-1{alpha} was retained primarily in the cytoplasm and that only a small portion translocated to the nucleus. This phenomenon may depend on the lack of interaction between DP-1{alpha} and E2F1. These results suggest that E2F1 transcriptional activity in combination with DP-1{alpha} is much lower than that which occurs via interactions between E2F1 and DP-1{beta} or wild-type DP-1. We therefore examined E2F1 transcriptional activity in HEK 293 cells cotransfected with reporter and each DP-1 isoform. As expected, although cotransfection of wild-type DP-1 clearly increased E2F1 transcriptional activity, the E2F1 activity in cells cotransfected with DP-1{alpha} was significantly lower than that in cells cotransfected with either wild-type DP-1 or control. Wu et al. (33) prepared DP-1 dominant-negative mutants (dnDP-1) in which the DNA-binding was deleted and showed that although cotransfection of wild-type DP-1 to C33-A cell greatly increased the transcriptional activity of E2F1, cotransfection with the dnDP-1 mutant failed to augment E2F1 activity. These researchers also demonstrated that the dnDP-1 mutant dramatically inhibits cell cycle progression. Since DP-1{alpha} is a dominant-negative isoform that occurs naturally in mammalian cells, we examined whether it is actually expressed in normal human tissues and established cell lines and whether it indeed acts as a negative regulator of cell cycle progression.



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  		FIG. 7.
Growth kinetics of DP-1 isoform-transfected HEK293 cells. A, HEK 293 cells were transfected with or without each DP-1 isoform and cultured in DMEM containing 10% FBS and Geneticin at 1 mg/ml. Cell numbers were measured at the selected times. B, the cell number at day 6 in panel A is expressed as a percentage of maximum. The results are expressed as mean ± S.D. in triplicate cultures. {alpha}, DP-1{alpha}; {beta}, DP-1{beta}; *, p < 0.05 versus wild-type DP-1.

 
RT-PCR analysis of normal human tissues and cell lines showed that the three DP-1 isoforms are ubiquitously expressed. Although our Western blot analysis showed that wild-type DP-1 and DP-1{beta} are ubiquitously expressed at the protein level in all cell lines tested, we found that DP-1{alpha} protein is expressed at very high levels in THP-1 and HL-60 cells. These observations suggested that DP-1{alpha} type expression may be controlled at post-transcriptional, translational, and/or post-translational stages. It will therefore be very important to characterize the differential expression profiles of these isoforms at the protein level in many different cell types.

E2F/DP-1 plays an important role in cell cycle progression from the G1 to the S phase in many cell types. Exogenous expression of a dnDP-1 mutant with no ability to bind DNA and form a heterodimer has been reported to block G1 progression in human osteosarcoma cells (33). In light of this, we surmised that DP-1{alpha} also regulates cell cycle progression in a dominant-negative manner in cells overexpressing DP-1{alpha} and E2F1. As expected, although we observed that DP-1{alpha} overexpression clearly suppresses cell cycle progression in HEK 293 cells, the level of suppression was weaker than that observed with dnDP-1, which lacks the DNA-binding domain. This difference may be due to the fact that dnDP-1 can effectively move from the cytoplasm into the nucleus by forming a heterodimer with endogenous E2F1. DP-1{alpha} overexpression markedly inhibited proliferation of HEK 293 cells as compared with wild-type DP-1, although DP-1{beta} also appears to have significant regulatory activity. These results strongly suggest that DP-1{alpha} acts as a dominant-negative protein that negatively regulates the cell cycle. Therefore, it will be of interest to explore the functional role of this isoform during cell cycle progression.

In conclusion, we have identified and characterized two novel DP-1 isoforms. Interestingly, we demonstrated that DP-1{alpha} regulates E2F1 transcriptional activity as well as cell cycle progression in a dominant-negative manner. These findings suggest the possibility that this isoform acts as a regulatory molecule in cell differentiation and cancer cell growth.


   FOOTNOTES
 
* This work was supported by the OSAKA Cancer Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Applied Biological Sciences, College of Bioresource Science, Nihon University, Kameino, Fujisawa-City, Kanagawa 292-8510, Japan. Tel.: 81-466-84-3701. E-mail: hanazawa{at}brs.nihon-u.ac.jp.

1 The abbreviations used are: RT, reverse transcription; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; FBS, fetal bovine serum; GFP, green fluorescent protein; YFP, yellow fluorescent protein; dn, dominant-negative; Luc, luciferase; WT, wild type; PBS (–), PBS (Mg2+- and Ca2+-free). Back


   ACKNOWLEDGMENTS
 
We thank Dr. Hatakeyama for the kind of gift of the E2F-driven reporter plasmid and the dnDP-1 expression plasmid.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dyson, N. (1998) Genes Dev. 12, 2245–2262[Free Full Text]
  2. Stevens, C., and La Thangue, N. B. (2003) Arch. Biochem. Biophys. 412, 157–169[CrossRef][Medline] [Order article via Infotrieve]
  3. Adams, M. R., Sears, R., Nuckolls, F., Leone, G., and Nevins, J. R. (2000) Mol. Cell. Biol. 20, 3633–3639[Abstract/Free Full Text]
  4. He, S., Cook, B. L., Deverman, B. E., Weihe, U., Zhang, F., Prachand, V., Zheng, J., and Weintraub, S. J. (2000) Mol. Cell. Biol. 20, 363–371[Abstract/Free Full Text]
  5. Leone, G., Nuckolls, F., Ishida, S., Adams, M., Sears, R., Jakoi, L., Miron, A., and Nevins, J. R. (2000) Mol. Cell. Biol. 20, 3626–3632[Abstract/Free Full Text]
  6. Dahme, T., Wood, J., Livingston, D. M., and Gaubatz, S. (2002) Eur. J. Biochem. 269, 5030–5036[Medline] [Order article via Infotrieve]
  7. Trimarchi, J. M., and Lees, J. A. (2002) Nat. Rev. Mol. Cell. Biol. 3, 11–20[CrossRef][Medline] [Order article via Infotrieve]
  8. Cam, H., and Dynlacht, B. D. (2003) Cancer Cell 4, 311–316[CrossRef][Medline] [Order article via Infotrieve]
  9. de Bruin, A., Maiti, B., Jakoi, L., Timmers, C., Buerki, R., and Leone, G. (2003) J. Biol. Chem. 278, 42041–42049[Abstract/Free Full Text]
  10. Di Stefano, L., Jensen, M. R., and Helin, K. (2003) EMBO J. 22, 6289–6298[CrossRef][Medline] [Order article via Infotrieve]
  11. Kovesdi, I, Reichel, R., and Nevins, J. R. (1986) Cell 45, 219–228[CrossRef][Medline] [Order article via Infotrieve]
  12. Slansky, J. E., and Farnham, P. J. (1996) Curr. Top. Microbiol. Immunol. 208, 1–30[Medline] [Order article via Infotrieve]
  13. Muller, H., and Helin, K. (2000) Biochim. Biophys. Acta 1470, M1–M12[Medline] [Order article via Infotrieve]
  14. Stevaux, O., and Dyson, N. J. (2002) Curr. Opin. Cell Biol. 14, 684–691[CrossRef][Medline] [Order article via Infotrieve]
  15. Girling, R., Partridge, J. F., Bandara, L. R., Burden, N., Totty, N. F., Hsuan, J. J., and La Thangue, N. B. (1993) Nature 365, 468[Medline] [Order article via Infotrieve]
  16. Helin, K., Wu, C. L., Fattaey, A. R., Lees, J. A., Dynlacht, B. D., Ngwu, C., and Harlow, E. (1993) Genes Dev. 7, 1850–1861[Abstract/Free Full Text]
  17. Zhang, Y., and Chellappan, S. P. (1995) Oncogene 10, 2085–2093[Medline] [Order article via Infotrieve]
  18. Wu, C. L., Zukerberg, L. R., Ngwu, C., Harlow, E., and Lees, J. A. (1995) Mol. Cell. Biol. 15, 2536–2546[Abstract]
  19. Gopalkrishnan, R. V., Dolle, P., Mattei, M. G., La Thangue, N. B., and Kedinger, C. (1996) Oncogene 13, 2671–2680[Medline] [Order article via Infotrieve]
  20. Kohn, M. J., Bronson, R. T., Harlow, E., Dyson, N. J., and Yamasaki, L. (2003) Development 130, 1295–1305[Abstract/Free Full Text]
  21. Ormondroyd, E., de la Luna, S., and La Thangue, N. B. (1995) Oncogene 11, 1437–1446[Medline] [Order article via Infotrieve]
  22. Rogers, K. T., Higgins, P. D., Milla, M. M., Phillips, R. S., and Horowitz, J. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7594–7599[Abstract/Free Full Text]
  23. Yamada, M., Kondo, T., Ashizawa, S., Takebayashi, T., Higashi, H., and Hatakeyama, M. (2002) Cytokine 17, 91–97[Medline] [Order article via Infotrieve]
  24. Watanabe, M., Yanagisawa, J., Kitagawa, H., Takeyama, K., Ogawa, S., Arao, Y., Suzawa, M., Kobayashi, Y., Yano, T., Yoshikawa, H., Masuhiro, Y., and Kato, S. (2001) EMBO J. 20, 1341–1352[CrossRef][Medline] [Order article via Infotrieve]
  25. Shenker, B. J., Hoffmaster, R. H., Zekavat, A., Yamaguchi, N., Lally, E. T., and Demuth, D. R. (2001) J. Immunol. 167, 435–441[Abstract/Free Full Text]
  26. Bandara, L. R., Buck, V. M., Zamanian, M., Johnston, L. H., and La Thangue, N. B. (1993) EMBO J. 12, 4317–4324[Medline] [Order article via Infotrieve]
  27. Krek, W., Livingston, D. M., and Shirodkar, S. (1993) Science 262, 1557–1560[Abstract/Free Full Text]
  28. Hiebert, S. W., Packham, G., Strom, D. K., Haffner, R., Oren, M., Zambetti, G., and Cleveland, J. L. (1995) Mol. Cell. Biol. 15, 6864–6874[Abstract]
  29. Yamasaki, L. (1999) Biochim. Biophys. Acta 1423, 9–15
  30. Muller, H., Bracken, A. P., Vernell, R., Moroni, M. C., Christians, F., Grassilli, E., Prosperini, E., Vigo, E., Oliner, J. D., and Helin, K. (2001) Genes Dev. 15, 267–285[Abstract/Free Full Text]
  31. DeGregori, J. (2002) Biochim. Biophys. Acta 1602, 131–150[Medline] [Order article via Infotrieve]
  32. Hitchens, M. R., and Robbins, P. D. (2003) Apoptosis 8, 461–468[CrossRef][Medline] [Order article via Infotrieve]
  33. Wu, C. L., Classon, M., Dyson, N., and Harlow, E. (1996) Mol. Cell. Biol. 16, 3698–3706[Abstract]

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