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• W2023897490 abstract "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-1α and DP-1β) and characterized their structure and function. DP-1α is composed of 278 amino acids and lacks a portion of the C-terminal heterodimerization domain, whereas DP-1β 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-1α binding to E2F1 was significantly reduced as compared with that of wild-type DP-1 or DP-1β. 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-1β. However, such a translocation for DP-1α 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-1α-transfected HEK 293 cells. Together, the results of the present study suggest that DP-1α is a novel isoform of DP-1 that acts as a dominant-negative regulator of cell cycle progression. 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-1α and DP-1β) and characterized their structure and function. DP-1α is composed of 278 amino acids and lacks a portion of the C-terminal heterodimerization domain, whereas DP-1β 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-1α binding to E2F1 was significantly reduced as compared with that of wild-type DP-1 or DP-1β. 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-1β. However, such a translocation for DP-1α 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-1α-transfected HEK 293 cells. Together, the results of the present study suggest that DP-1α is a novel isoform of DP-1 that acts as a dominant-negative regulator of cell cycle progression. The E2F family of transcription factors plays an essential role in regulating cell cycle progression (1Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar, 2Stevens C. La Thangue N.B. Arch. Biochem. Biophys. 2003; 412: 157-169Crossref PubMed Scopus (179) Google Scholar). 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) (3Adams M.R. Sears R. Nuckolls F. Leone G. Nevins J.R. Mol. Cell. Biol. 2000; 20: 3633-3639Crossref PubMed Scopus (111) Google Scholar, 4He S. Cook B.L. Deverman B.E. Weihe U. Zhang F. Prachand V. Zheng J. Weintraub S.J. Mol. Cell. Biol. 2000; 20: 363-371Crossref PubMed Scopus (36) Google Scholar, 5Leone G. Nuckolls F. Ishida S. Adams M. Sears R. Jakoi L. Miron A. Nevins J.R. Mol. Cell. Biol. 2000; 20: 3626-3632Crossref PubMed Scopus (149) Google Scholar, 6Dahme T. Wood J. Livingston D.M. Gaubatz S. Eur. J. Biochem. 2002; 269: 5030-5036Crossref PubMed Scopus (16) Google Scholar, 7Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 11-20Crossref PubMed Scopus (957) Google Scholar, 8Cam H. Dynlacht B.D. Cancer Cell. 2003; 4: 311-316Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 9de Bruin A. Maiti B. Jakoi L. Timmers C. Buerki R. Leone G. J. Biol. Chem. 2003; 278: 42041-42049Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Di Stefano L. Jensen M.R. Helin K. EMBO J. 2003; 22: 6289-6298Crossref PubMed Scopus (207) Google Scholar). 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 (11Kovesdi I Reichel R. Nevins J.R. Cell. 1986; 45: 219-228Abstract Full Text PDF PubMed Scopus (321) Google Scholar). Subsequently, several studies (7Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 11-20Crossref PubMed Scopus (957) Google Scholar, 12Slansky J.E. Farnham P.J. Curr. Top. Microbiol. Immunol. 1996; 208: 1-30Crossref PubMed Scopus (239) Google Scholar, 13Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar, 14Stevaux O. Dyson N.J. Curr. Opin. Cell Biol. 2002; 14: 684-691Crossref PubMed Scopus (344) Google Scholar) 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 α, 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 (15Girling R. Partridge J.F. Bandara L.R. Burden N. Totty N.F. Hsuan J.J. La Thangue N.B. Nature. 1993; 365: 468Crossref PubMed Scopus (19) Google Scholar, 16Helin K. Wu C.L. Fattaey A.R. Lees J.A. Dynlacht B.D. Ngwu C. Harlow E. Genes Dev. 1993; 7: 1850-1861Crossref PubMed Scopus (416) Google Scholar). Several studies (17Zhang Y. Chellappan S.P. Oncogene. 1995; 10: 2085-2093PubMed Google Scholar, 18Wu C.L. Zukerberg L.R. Ngwu C. Harlow E. Lees J.A. Mol. Cell. Biol. 1995; 15: 2536-2546Crossref PubMed Scopus (220) Google Scholar, 19Gopalkrishnan R.V. Dolle P. Mattei M.G. La Thangue N.B. Kedinger C. Oncogene. 1996; 13: 2671-2680PubMed Google Scholar) have shown that DP-1 is expressed at high levels in various murine and human tissues. Interestingly, a recent study (20Kohn M.J. Bronson R.T. Harlow E. Dyson N.J. Yamasaki L. Development. 2003; 130: 1295-1305Crossref PubMed Scopus (58) Google Scholar) 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 (17Zhang Y. Chellappan S.P. Oncogene. 1995; 10: 2085-2093PubMed Google Scholar, 21Ormondroyd E. de la Luna S. La Thangue N.B. Oncogene. 1995; 11: 1437-1446PubMed Google Scholar, 22Rogers K.T. Higgins P.D. Milla M.M. Phillips R.S. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7594-7599Crossref PubMed Scopus (37) Google Scholar), 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-1α and DP-1β, 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-1α, a novel isoform, acts as a dominant-negative regulator of cell cycle progression.MATERIALS AND METHODSCloning 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 (23Yamada M. Kondo T. Ashizawa S. Takebayashi T. Higashi H. Hatakeyama M. Cytokine. 2002; 17: 91-97Crossref PubMed Scopus (3) Google Scholar).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) 1The 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). 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, β (reverse1, 5′-ACGCGTCGACTCAGTCGTCCTCGTCATTCTCGTTG-3′) and DP-1α (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, β (forward1, 5′-ACTGAATTCATGGCAAAAGATGCCGGTCTAATTG-3′; reverse1, 5′-ACGCGTCGACTCAGTCGTCCTCGTCATTCTCGTTG-3′) and DP-1α (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 (24Watanabe M. Yanagisawa J. Kitagawa H. Takeyama K. Ogawa S. Arao Y. Suzawa M. Kobayashi Y. Yano T. Yoshikawa H. Masuhiro Y. Kato S. EMBO J. 2001; 20: 1341-1352Crossref PubMed Scopus (245) Google Scholar).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 (24Watanabe M. Yanagisawa J. Kitagawa H. Takeyama K. Ogawa S. Arao Y. Suzawa M. Kobayashi Y. Yano T. Yoshikawa H. Masuhiro Y. Kato S. EMBO J. 2001; 20: 1341-1352Crossref PubMed Scopus (245) Google Scholar).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-α-gal (BD Biosciences) and cultured for 3 days at 30 °C. Yeast α-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 (23Yamada M. Kondo T. Ashizawa S. Takebayashi T. Higashi H. Hatakeyama M. Cytokine. 2002; 17: 91-97Crossref PubMed Scopus (3) Google Scholar, 24Watanabe M. Yanagisawa J. Kitagawa H. Takeyama K. Ogawa S. Arao Y. Suzawa M. Kobayashi Y. Yano T. Yoshikawa H. Masuhiro Y. Kato S. EMBO J. 2001; 20: 1341-1352Crossref PubMed Scopus (245) Google Scholar). 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 (24Watanabe M. Yanagisawa J. Kitagawa H. Takeyama K. Ogawa S. Arao Y. Suzawa M. Kobayashi Y. Yano T. Yoshikawa H. Masuhiro Y. Kato S. EMBO J. 2001; 20: 1341-1352Crossref PubMed Scopus (245) Google Scholar). Flow cytometry was used to analyze cell cycle distribution by a modification of a described procedure (25Shenker B.J. Hoffmaster R.H. Zekavat A. Yamaguchi N. Lally E.T. Demuth D.R. J. Immunol. 2001; 167: 435-441Crossref PubMed Scopus (99) Google Scholar). 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 × 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.RESULTSCloning and Structure of DP-1 Isoforms—To date, two Dp genes, Dp-1 and Dp-2, have been identified. Although a previous study (22Rogers K.T. Higgins P.D. Milla M.M. Phillips R.S. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7594-7599Crossref PubMed Scopus (37) Google Scholar) 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 (26Bandara L.R. Buck V.M. Zamanian M. Johnston L.H. La Thangue N.B. EMBO J. 1993; 12: 4317-4324Crossref PubMed Scopus (218) Google Scholar, 27Krek W. Livingston D.M. Shirodkar S. Science. 1993; 262: 1557-1560Crossref PubMed Scopus (219) Google Scholar, 28Hiebert S.W. Packham G. Strom D.K. Haffner R. Oren M. Zambetti G. Cleveland J.L. Mol. Cell. Biol. 1995; 15: 6864-6874Crossref PubMed Scopus (144) Google Scholar, 29Yamasaki L. Biochim. Biophys. Acta. 1999; 1423: 9-15PubMed Google Scholar, 30Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar, 31DeGregori J. Biochim. Biophys. Acta. 2002; 1602: 131-150PubMed Google Scholar, 32Hitchens M.R. Robbins P.D. Apoptosis. 2003; 8: 461-468Crossref PubMed Scopus (40) Google Scholar), 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-1α and DP-1β. As shown in Fig. 1, A and B, DNA sequence analysis indicated that DP-1α has four nucleotides deleted in the PstI site. Consequently, the DP-1α 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-1β isoform and confirmed that exon 11 is deleted and that exons 10 and 12 are spliced together. These data show that DP-1β 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.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-1α (α)), and 42 (DP-1β (β)) kDa in all cell lines tested (Fig. 2F, lanes 1–4). Interestingly, the expression levels of DP-1 α protein appeared to be considerably higher in THP-1 and HL-60 cells (Fig. 2F, lanes 3 and 4).Fig. 2Expression 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-1β expression in normal tissues (B) and cell lines (C) as well as DP-1α 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 α-galactosidase activity was detected in AH105 yeast cotransfected with DP-1α and E2F1 expression vectors, strong α-galactosidase activity at the same level as that of wild-type DP-1 was detected when AH105 yeast was cotransfected with DP-1β 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-1α and E2F1, although DP-1β appeared to interact with E2F1 as strongly as wild-type DP-1. The interaction between DP-1α and E2F1 may depend on the lack of a heterodimerization domain in this isoform. These results suggest the possibility that DP-1α may act as a dominant-negative regulator of DP-1.Fig. 3Interaction between DP-1 isoforms and E2F1. A, yeast two-hybrid assays were performed as described under “Materials and Methods.” α-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; α, 3; β, 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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-1β, we clearly observed that these DP-1 isoforms localize to the nuclei in all cell lines tested. Interestingly, we also observed that DP-1α is almost completely localized to the cytoplasm, even when both GFP-DP-1α and YFP-E2F1 are coexpressed (Fig. 4A, lower panel). These results suggest that this phenomenon is due to DP-1α lacking a heterodimerization domain.Fig. 4Immunofluorescence 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-1α (E), DP-1β (F), or dnDP-1 (G) fused to pEGFP-C2. In addition, cells were cotransfected with combinations of GFP-DP-1WT- (H), DP-1α- (I), DP-1β- (J), or dnDP-1- (K) fused pEGFP-C2 and YFP-E2F-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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-1β. However, when cells were transfected with pcDNA3/DP-1α, 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-1α may act as a negative regulator of the transcription factor E2F/DP-1.Fig. 5Transcriptional 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. α, DP-1α; β, DP-1β; *, p < 0.05 versus wild-type DP-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DP-1α Regulates Cell Cycle Progression of HEK 293 Cells in a Dominant-negative Manner—To confirm the possibility that DP-1α 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-1α-transfected cells was dramatically decreased (Fig. 6D). In contrast, no significant effects were observed in DP-1β-transfected cells (Fig. 6E). These results demonstrated that DP-1α regulates the cell cycle in a dominant-negative manner.Fig. 6DNA 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DP-1α Inhibits Proliferation of HEK 293 Cells—Since flow cytometric analysis suggested that DP-1α is able to inhibit the proliferation of transfected cells, we next examined the proliferation kinetics of H" @default.
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