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Expression of the AML-1 Oncogene Shortens the G1Phase of the Cell Cycle

2000; Elsevier BV; Volume: 275; Issue: 5 Linguagem: Inglês

10.1074/jbc.275.5.3438

1083-351X

David K. Strom, John Nip, Jennifer J. Westendorf, Bryan Linggi, Bart Lutterbach, James R. Downing, Noel Lenny, Scott W. Hiebert,

Ubiquitin and proteasome pathways

The AML-1-encoded transcription factor, AML-1B, regulates numerous hematopoietic-specific genes. Inappropriate expression of AML-1-family proteins is oncogenic in cell culture systems and in mice. To understand the oncogenic functions of AML-1, we established cell lines expressing AML-1B to examine the role of AML-1 in the cell cycle. DNA content analysis and bromodeoxyuridine pulse-chase studies indicated that entry into the S phase of the cell cycle was accelerated by up to 4 h in AML-1B-expressing 32D.3 myeloid progenitor cells as compared with control cells or cells expressing E2F-1. However, AML-1B was not able to induce continued cell cycle progression in the absence of growth factors. The DNA binding and transactivation domains of AML-1B were required for altering the cell cycle. Thus, AML-1B is the first transcription factor that affects the timing of the mammalian cell cycle. The AML-1-encoded transcription factor, AML-1B, regulates numerous hematopoietic-specific genes. Inappropriate expression of AML-1-family proteins is oncogenic in cell culture systems and in mice. To understand the oncogenic functions of AML-1, we established cell lines expressing AML-1B to examine the role of AML-1 in the cell cycle. DNA content analysis and bromodeoxyuridine pulse-chase studies indicated that entry into the S phase of the cell cycle was accelerated by up to 4 h in AML-1B-expressing 32D.3 myeloid progenitor cells as compared with control cells or cells expressing E2F-1. However, AML-1B was not able to induce continued cell cycle progression in the absence of growth factors. The DNA binding and transactivation domains of AML-1B were required for altering the cell cycle. Thus, AML-1B is the first transcription factor that affects the timing of the mammalian cell cycle. acute myeloid leukemia interleukin phosphate-buffered saline PBS containing 0.5% Tween 20 and 0.5% bovine serum albumin bromode- oxyuridine The largest form of acute myeloid leukemia-1 (AML-1),1 termed AML-1B (1.Meyers S. Lenny N. Hiebert S.W. Mol. Cell. Biol. 1995; 15: 1974-1982Crossref PubMed Scopus (351) Google Scholar) (also known as Runx1, CBFA2, or PEBP2αB1(2–4)), activates the transcription of numerous tissue specific genes, including genes encoding cytokines and cytokine receptors, T cell receptors, and myeloid-specific genes (e.g. neutrophil peptide-3 and myeloperoxidase) (5.Nuchprayoon I. Meyers S. Scott L.M. Suzow J. Hiebert S. Friedman A.D. Mol. Cell. Biol. 1994; 14: 5558-5568Crossref PubMed Google Scholar, 6.Lenny N. Westendorf J.J. Hiebert S.W. Mol. Biol. Rep. 1997; 24: 157-168Crossref PubMed Scopus (62) Google Scholar, 7.Westendorf J.J. Yamamoto C.M. Lenny N. Downing J.R. Selsted M.E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 322-333Crossref PubMed Google Scholar). When transfected alone, AML-1B activates the transcription of these genes to low levels, but it cooperatively activates transcription to high levels in concert with tissue-specific factors (e.g. C/EBPα, AP-1, ets-1, PU.1, and c-Myb) that regulate cellular proliferation and differentiation (7.Westendorf J.J. Yamamoto C.M. Lenny N. Downing J.R. Selsted M.E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 322-333Crossref PubMed Google Scholar, 8.Zhang D.E. Hetherington C.J. Meyers S. Rhoades K.L. Larson C.J. Chen H.M. Hiebert S.W. Tenen D.G. Mol. Cell. Biol. 1996; 16: 1231-1240Crossref PubMed Google Scholar, 9.Tenen D.G. Hromas R. Licht J.D. Zhang D.E. Blood. 1997; 90: 489-519Crossref PubMed Google Scholar, 10.Selvamuragan N. Chou W.-Y. Pearman A.T. Pulumati M.R. Partridge N.C. J. Biol. Chem. 1998; 273: 10647-10657Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11.Kim W.Y. Sieweke M. Ogawa E. Wee H.J. Englmeier U. Graf T. Ito Y. EMBO J. 1999; 18: 1609-1620Crossref PubMed Scopus (197) Google Scholar, 12.Mao S. Frank R.C. Zhang J. Miyazaki Y. Nimer S.D. Mol. Cell. Biol. 1999; 19: 3635-3644Crossref PubMed Scopus (112) Google Scholar). Conversely, AML-1 can repress transcription by associating with the Groucho and mSin3 co-repressors (13.Aronson B.D. Fisher A.L. Blechman K. Caudy M. Gergen J.P. Mol. Cell. Biol. 1997; 17: 5581-5587Crossref PubMed Scopus (225) Google Scholar, 14.Levanon D. Goldstein R.E. Bernstein Y. Tang H. Goldenberg D. Stifani S. Paroush Z. Groner Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11590-11595Crossref PubMed Scopus (410) Google Scholar, 15.Imai Y. Kurokawa M. Tanaka K. Friedman A.D. Ogawa S. Mitani K. Yazaki Y. Hirai H. Biochem. Biophys. Res. Commun. 1998; 252: 582-589Crossref PubMed Scopus (101) Google Scholar, 16.Lutterbach B. Westendorf J. Linggi B. Isaac S. Seto E. Hiebert S. J. Biol. Chem. 1999; 275: 651-656Abstract Full Text Full Text PDF Scopus (223) Google Scholar). AML-1 is one of the most frequently mutated genes in human leukemia. For example, it is disrupted by the t(8;21) in AML and by the t(12;21) in childhood B-cell acute lymphoblastic leukemia (17.Miyoshi H. Kozu T. Shimizu K. Enomoto K. Maseki N. Kaneko Y. Kamada N. Ohki M. EMBO J. 1993; 12: 2715-2721Crossref PubMed Scopus (458) Google Scholar, 18.Golub T.R. Barker G.F. Bohlander S.K. Hiebert S.W. Ward D.C. Bray-Ward P. Morgan E. Raimondi S.C. Rowley J.D. Gilliland D.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4917-4921Crossref PubMed Scopus (674) Google Scholar, 19.Romana S.P. Poirel H. Leconiat M. Flexor M.A. Mauchauffe M. Jonveaux P. Macintyre E.A. Berger R. Bernard O.A. Blood. 1995; 86: 4263-4269Crossref PubMed Google Scholar). AML-1 is also targeted indirectly by the Inv (16.Lutterbach B. Westendorf J. Linggi B. Isaac S. Seto E. Hiebert S. J. Biol. Chem. 1999; 275: 651-656Abstract Full Text Full Text PDF Scopus (223) Google Scholar), which fuses CBFβ, an AML-1-interacting protein, to a smooth muscle myosin heavy chain (20.Liu P. Tarle S.A. Hajra A. Claxton D.F. Marlton P. Freedman M. Siciliano M.J. Collins F.S. Science. 1993; 261: 1041-1044Crossref PubMed Scopus (652) Google Scholar). However, the Inv (16.Lutterbach B. Westendorf J. Linggi B. Isaac S. Seto E. Hiebert S. J. Biol. Chem. 1999; 275: 651-656Abstract Full Text Full Text PDF Scopus (223) Google Scholar) fusion protein retains the ability to interact with AML-1 and inhibits expression of AML-1 target genes (21.Tanaka Y. Fujii M. Hayashi K. Chiba N. Akaishi T. Shineha R. Nishihira T. Satomi S. Ito Y. Watanabe T. Satake M. Oncogene. 1998; 17: 699-708Crossref PubMed Scopus (18) Google Scholar, 22.Kanno Y. Kanno T. Sakakura C. Bae S.C. Ito Y. Mol. Cell. Biol. 1998; 18: 4252-4261Crossref PubMed Google Scholar, 23.Lutterbach B. You H. Durst K.L. Hiebert S.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12822-12827Crossref PubMed Scopus (94) Google Scholar). Together these chromosomal translocations account for nearly one-third of all AML and one-fourth of all childhood B-cell acute lymphoblastic leukemia cases containing discernable chromosomal abnormalities (24.Rubnitz J.E. Crist W.M. Pediatrics. 1997; 100: 101-108Crossref PubMed Scopus (37) Google Scholar). Although these translocations are closely associated with acute leukemia, it is the wild-type form of AML-1 that transforms cells. Wild-type AML-1 (AML-1B) is transforming when expressed in fibroblasts, and this activity requires the C-terminal transcriptional regulatory domain (25.Kurokawa M. Tanaka T. Tanaka K. Ogawa S. Mitani K. Yazaki Y. Hirai H. Oncogene. 1996; 12: 883-892PubMed Google Scholar, 26.Tanaka T. Kurokawa M. Ueki K. Tanaka K. Imai Y. Mitani K. Okazaki K. Sagata N. Yazaki Y. Shibata Y. Kadowaki T. Hirai H. Mol. Cell. Biol. 1996; 16: 3967-3979Crossref PubMed Scopus (137) Google Scholar). Likewise, the closely related protein PEBP2αA1 (AML-3) is up-regulated by retroviral insertions that cooperate with c-Myc to induce T-cell lymphomas (27.Stewart M. Terry A. Hu M. O'Hara M. Blyth K. Baxter E. Cameron E. Onions D.E. Neil J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8646-8651Crossref PubMed Scopus (197) Google Scholar). Therefore, in fibroblasts and in mice, AML-1-family proteins are oncogenes. Thus, AML-1 has the unusual property that both the wild-type and the translocated alleles can affect cellular proliferation and differentiation pathways. Overexpression of the fusion protein encoded by the Inv (16.Lutterbach B. Westendorf J. Linggi B. Isaac S. Seto E. Hiebert S. J. Biol. Chem. 1999; 275: 651-656Abstract Full Text Full Text PDF Scopus (223) Google Scholar) inhibited cell cycle progression (28.Cao W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google Scholar). This protein can act as an AML-1 dominant repressor, suggesting a role for AML-1 in the G1 phase of the cell cycle. However, the Inv (16.Lutterbach B. Westendorf J. Linggi B. Isaac S. Seto E. Hiebert S. J. Biol. Chem. 1999; 275: 651-656Abstract Full Text Full Text PDF Scopus (223) Google Scholar) fusion protein could also interact with and regulate other factors. In addition, many proteins are required for cell cycle progression, but few factors actually promote transit through the cell cycle. Therefore, we have explored the functions of AML-1 in regulating the cell cycle. We have enforced the expression of AML-1B and found that it accelerates transit through the G1 phase, whereas transcriptionally inactive forms of AML-1 do not affect the cell cycle. This phenotype is similar to that observed upon overexpression of the D- and E-type cyclins (29.Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (980) Google Scholar, 30.Ohtsubo M. Roberts J.M. Science. 1993; 259: 1908-1912Crossref PubMed Scopus (663) Google Scholar, 31.Kato J. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11513-11517Crossref PubMed Scopus (211) Google Scholar). Thus, AML-1 is one of the first transcription factors whose enforced expression shortens the G1 phase of the cell cycle. Parental 32D.3 myeloid progenitor cells were maintained as suspension cultures in RPMI 1640 containing 10% fetal bovine serum (BioWhittaker), 2 mml-glutamine (BioWhittaker), 1% penicillin G-streptomycin (Life Technologies, Inc.), and 15 units/mL IL-3. 32D.3 cells stably transfected with AML-1B or derivatives thereof were constructed as described previously (32.Hiebert 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 (146) Google Scholar) using the sheep metallothionine promoter plasmid pMT-CB6. COS cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The designation pMT indicates a control neomycin-resistant clonal cell line that was co-selected with the AML-1B-expressing cell lines that are designated pMT-AML-1B. The numbers after each cell line name are the clone (e.g.pMT.11, control cell line number 11). Asynchronously growing cells were washed 2× in PBS and resuspended in media lacking IL-3. At the indicated times after IL-3 removal, 1 × 106 cells were centrifuged and resuspended in 1 ml of propidium iodide staining solution (0.05 mg/ml propidium iodide, 5 μg/ml DNase-free RNase A, 0.1% sodium citrate, 0.1% Triton X-100) and analyzed by flow cytometry as described (33.Strom D. Nip J. Cleveland J.L. Hiebert S.W. Cell Growth Differ. 1998; 9: 59-69PubMed Google Scholar). For bromodeoxyuridine (BrdU) analysis, asynchronously growing cells were resuspended at 0.5 × 106/ml in media containing IL-3. Cells were pulse-labeled with 10 μm BrdU (Sigma) for 30 min, washed twice to remove BrdU, and resuspended in normal growth media containing IL-3. Aliquots of 2 × 106cells were obtained at the indicated time points following the removal of BrdU. BrdU detection was performed by standard procedures (33.Strom D. Nip J. Cleveland J.L. Hiebert S.W. Cell Growth Differ. 1998; 9: 59-69PubMed Google Scholar). Briefly, cells were washed in cold PBS and fixed in cold 70% ethanol for 30 min. Cell pellets were then incubated for 30 min in 4n HCl, washed in 0.1 m borax (pH 9.1) (Sigma), followed by a PBS wash. Cells were then resuspended in 50 μl of PBS-TB (PBS containing 0.5% Tween 20 (Sigma) and 0.5% bovine serum albumin Fraction V (Sigma)), and a 1:150 dilution of an anti-BrdU antibody (BU-33) (Sigma) for 30 min at room temperature. After two washes in PBS-TB, cells were incubated with a 1:150 dilution of sheep fluorescein isothiocyanate-conjugated anti-mouse F(ab′)2(Sigma) in PBS-TB for 30 min at room temperature. Cells were again washed twice in PBS-TB, resuspended in PBS containing 5 μg/ml propidium iodide (Sigma), 1 μg/ml RNase A, and analyzed on a FACScalibur flow cytometer (Becton Dickinson). For protein analysis, cells were lysed in microextraction buffer, sonicated, and clarified as described previously (34.Hiebert S.W. Chellappan S.P. Horowitz J.M. Nevins J.R. Genes Dev. 1992; 6: 177-185Crossref PubMed Scopus (469) Google Scholar, 35.Hiebert S.W. Mol. Cell. Biol. 1993; 13: 3384-3391Crossref PubMed Scopus (130) Google Scholar). Total protein concentration of lysates was measured using the Bio-Rad protein assay reagent. Cell lysate (100 μg) was electrophoresed on a 10% polyacrylamide gel, transferred onto nitrocellulose, and probed with the indicated antibodies. Immune complexes were detected using the Super Signal detection system (Pierce) and a peroxidase-labeled goat anti-rabbit secondary antibody (Sigma). All proteins were detected using a purified rabbit polyclonal antibody specific to the runt homology domain (RHD) of AML-1. Gel mobility shift analysis and antibodies recognizing the AML family of proteins have been described (36.Meyers S. Lenny N. Sun W. Hiebert S.W. Oncogene. 1996; 13: 303-312PubMed Google Scholar). The antisera are available from Calbiochem. Gel mobility shift analysis for E2F was performed as described (35.Hiebert S.W. Mol. Cell. Biol. 1993; 13: 3384-3391Crossref PubMed Scopus (130) Google Scholar). Immunoblot analysis for cell cycle regulatory proteins was performed with antisera purchased from Calbiochem. A 1.1-kilobase pair fragment of the murine cyclin D2 promoter was amplified from genomic DNA by polymerase chain reaction based on the published sequence (37.Jun D.Y. Kim M.K. Kim I.G. Kim Y.H. Mol. Cell. 1997; 7: 537-543Google Scholar). This fragment was cloned into pGL2 basic, which contains a polylinker sequence upstream of the firefly luciferase gene. The indicated promoter deletions were made using convenient endonuclease restriction sites. pGL2-D2 was co-transfected using the Superfect reagent (Qiagen) with pCMV5-AML-1B orpCMV5-AML-1B(L175D) into COS-7 cells, and luciferase activity was measured 40 h later. Secreted alkaline phosphatase activity expressed from the cytomegalovirus immediate early promoter was used as an internal control to correct for transfection efficiency. Regulation of the human thymidine kinase promoter was performed in the same manner but using the renilla luciferase as the reporter gene (Promega). The cyclin D1-luciferase promoter plasmid was a kind gift from Dr. Ronald Wisdom (Vanderbilt University School of Medicine) (38.Wisdom R. Johnson R.S. Moore C. EMBO J. 1999; 18: 188-197Crossref PubMed Scopus (533) Google Scholar). The observation that the Inv (16.Lutterbach B. Westendorf J. Linggi B. Isaac S. Seto E. Hiebert S. J. Biol. Chem. 1999; 275: 651-656Abstract Full Text Full Text PDF Scopus (223) Google Scholar) fusion protein inhibited cell cycle progression (28.Cao W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google Scholar) suggested that AML-1 normally functions in the G1 phase of the cell cycle. To test this possibility, we enforced the expression of AML-1B, the largest AML-1-encoded product, to determine its effects on the cell cycle. The diploid, IL-3-dependent myeloid progenitor cell line 32D.3 was used for this cell cycle analysis because these cells express relatively low levels of AML-1B. We established 32D.3 cell lines expressing AML-1B from the zinc-inducible sheep metallothionine promoter. Single cell clones expressing AML-1B were identified from three separate parental populations by immunoblot analysis using antibodies specific for AML-1 (Fig. 1). As a control for later experiments, we also selected clonal cell lines that did not express AML-1B (labeled pMT in later figures). In addition, we isolated cell lines that expressed mutant AML-1B proteins. AML-L175D contains a single amino acid change within the DNA binding domain (also known as the runt homology domain or RHD) that disrupts DNA binding (leucine 175 changed to aspartic acid, labeled L175D, Fig. 1) (39.Lenny N. Meyers S. Hiebert S.W. Oncogene. 1995; 11: 1761-1769PubMed Google Scholar), whereas AML-1a lacks the C-terminal transactivation domain (AML-1a.3, Fig. 1). Although the metallothionine promoter was used, high levels of protein expression were found in the absence of zinc. To avoid complications in the cell cycle experiments, metal induction was not used. Cell lines with higher basal expression were used for further analysis (e.g. lines 7 and 28). To compare the rates of cell cycle progression of these cell lines, DNA content analysis was performed. Enforced expression of AML-1B appeared to affect the cell cycle, as fewer cells were present in the G1 phase and more cells accumulated in S phase in AML-1B-expressing cells versus a control cell line that was established at the same time (designated pMT, Fig.2 A). The accumulation of AML-1-expressing cells in S phase could be due to a shortening of another cell cycle phase or to a lengthening of S phase. To address this issue, we determined the timing of the cell cycle phases by performing pulse-chase analysis using the nucleotide analogue BrdU. This method avoids the use of cell cycle poisons that grossly disrupt cellular morphology and physiology. A 30-min pulse of BrdU was followed over a 24-h chase period by flow cytometry using an anti-BrdU antibody to quantitate BrdU incorporation and propidium iodide staining of DNA to measure DNA content (Fig. 2, B and C). By plotting BrdU incorporation versus DNA content we were able to observe both BrdU labeled (Fig. 2 B, panel pMT.3, 0 Hour area above the horizontal line, and see arrows in Fig. 2 C) and unlabeled cells (Fig. 2 B, panel pMT.3, 0 Hour area below the horizontal line and areas highlighted by arrowheads in Fig. 2 C) traversing the cell cycle during the chase period. This method of analysis gives rise to a characteristic horseshoe-shaped plot (Fig. 2 B,panel pMT.3, 0 Hour). During the 30-min labeling period, all cells that were synthesizing DNA incorporated BrdU (Fig. 2 B, panel pMT.3, O Hour, + BrdU). This includes cells at the G1to S phase transition that appear to have a 2 n DNA content, and cells at the S to G2 phase transition that have a 4 n DNA content (Fig. 2 B, panel pMT.3, 0 Hour). To measure the time required for cells to traverse the G2/M and G1 phases, we measured cells re-entering the S phase from 8 h to 16 h after labeling. Cells that were labeled with BrdU and that were re-entering the S phase had divided and contained one-half the levels of BrdU of undivided cells. This difference ensured that we were observing cycling cells (note that the plots in Fig. 2, B and C use a logarithmic scale, making this 2-fold shift small). To measure cells that were clearly in S phase versus those cells that had just begun to synthesize DNA, the gate was set to quantitate the central portion of the S phase plot. This area is equivalent to the area between the unlabeled G1 and G2/M phase cells immediately after the labeling (below the horizontal line and labeled -BrdU at 0 Hour, Fig.2 B). At 8 to 9 h post-BrdU labeling, labeled cells had exited S phase and divided as judged by a reduction in fluoresence intensity, and the first cells were beginning to re-enter S phase (Fig. 2, Band C). By 10 h after labeling, only 17% of control 32D.3 cells containing BrdU were present in early S phase (Fig.2 B, pMT.3, 10 Hour). By 12 h after BrdU labeling, 30% of cells that were originally in S phase had traversed G2/M and G1 and were re-entering S phase (Fig. 2 B, pMT.3, 12 Hour). In addition, 10 h and 12 h after labeling with BrdU, most of the unlabeled cells that were initially in the G1 and G2/M phases had moved into the S phase and the G1 phase, respectively (e.g. Fig. 2 B, compare pMT.3 0 Hour and 10 Hour, and note areas marked by arrowheads in Fig. 2 C). Cells expressing E2F-1 and its co-factor DP-1 showed similar cell cycle profiles (38% S phase 12 h post-BrdU labeling; Fig.2 B, pMAM-Neo-E2F-1.1/DP-1.2, and see Hiebertet al. (32.Hiebert 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 (146) Google Scholar)). When AML-1B-expressing cells were assayed, nearly twice as many BrdU-labeled cells expressing AML-1B were re-entering S phase 10 h after labeling as control cells (34% and 30% versus 17% for control cells; Fig. 2 B compare panels pMT-AML-1B.7 and pMT-AML-1B.28 with pMT.3 at 10 h). At 12-h post-labeling, nearly 50% more AML-1B-expressing cells were in mid-S phase (45% and 54% S-phase,pMT-AML-1B.7 and pMT-AML-1B.28, respectively; Fig. 2 B). In addition, at the 12-h time point, the unlabeled AML-1B-expressing cells showed accelerated cell cycle kinetics (Fig.2 B; note also the unlabeled cells in the 9- and 13-hpanels marked with arrowheads in Fig.2 C). 12–13 h after labeling the AML-1B-expressing cells had exited the G1 phase and were in late S phase (Fig. 2,B and C) (note the reduction in the number of cells in early S phase of the unlabeled cell portions of the plots of cells expressing AML-1B as compared with control and E2F-1-expressing cell plots (Fig. 2 B, and marked with bracketsFig. 2 C)). Similar results were obtained from more than 10 independently isolated single cell clones that were selected from three different parental populations of 32D.3 cells. The percentages of cells entering S phase 10 or 11 h after BrdU labeling from three different experiments are shown in TableI. Each AML-1B-expressing cell line entered S phase faster than the control cell lines (Fig. 2 Band Table I).Table ICell cycle analysis of AML-1B overexpressing 32D.3 cellsCell linesaCell lines used were 32D.3 clones containing either vector alone (pMT) or vector containing an AML-1B insert (1B).Cells in S phasebShown are the percentage of cells in S phase at the indicated time (10 or 11 h) after BrdU-labeling.%10-h time point pMT.1016 1B.538 1B.747 1B.2744 1B.284611-h time point pMT.438 pMT.1037 1B.258 1B.349 1B.663 1B.286110-h time point pMT.327 pMT.426 1B.648 1B.749 1B.3240 1B.3648 1B.5041Shown are the results of three separate BrdU-labeling experiments listed as percentage of BrdU-labeled cells in S phase at indicated times following BrdU pulse-labeling. Experiments were performed exactly as described in the legend to Fig. 2 B.a Cell lines used were 32D.3 clones containing either vector alone (pMT) or vector containing an AML-1B insert (1B).b Shown are the percentage of cells in S phase at the indicated time (10 or 11 h) after BrdU-labeling. Open table in a new tab Shown are the results of three separate BrdU-labeling experiments listed as percentage of BrdU-labeled cells in S phase at indicated times following BrdU pulse-labeling. Experiments were performed exactly as described in the legend to Fig. 2 B. The results of DNA content analysis (Fig. 2 A) and BrdU analysis of S phase suggested that AML-1B caused accelerated entry into S phase. In principle, this could be due to a shortening of the G1 or the G2/M phases. However, DNA content analysis indicated that the total number of AML 1B-expressing cells in the G2/M phase were similar to control cells, and the number of cells in the G1 phase was reduced, suggesting a shortening of the G1 but not G2/M phases (Fig.2 A). To extend this result and to directly determine whether the G2/M phase was shortened by AML-1B expression, we used BrdU pulse-chase analysis to determine the time required to traverse G2/M (Fig. 3 and TableII). This was accomplished by measuring the time required for the first BrdU-labeled cells (those cells in late S phase at the time of labeling) to traverse G2/M and enter the G1 phase. Both control G418-resistant cells (pMT) and AML-1B-expressing cells passed through the G2/M phase and began entering the G1 phase after 3–4 h (Fig. 3 and TableII). Analysis of further time points confirmed this result. We conclude that enforced expression of AML-1B does not affect G2/M but shortens the G1 phase, consistent with the ability of an AML-1 inhibitor to arrest cells in the G1 phase (28.Cao W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google Scholar).Table IIDetermination of G2/M transit time in 32D.3-overexpressing cell linesCell linesaCell lines used were 32D.3 clones containing either vector alone (pMT) or vector containing an AML-1B insert (1B).0 h3 hbPercentage of cells in the G1 phase at 3 or 4 h after BrdU labeling.4 hbPercentage of cells in the G1 phase at 3 or 4 h after BrdU labeling.%%%32D.300.55.0pMT.101.05.2pMT.300.14.51B.700.94.91B.5100.74.2Cells were assayed as described in the legend to Fig. 2 B. At the indicated times, the percentage of G1 cells labeled with BrdU was determined using the ModFit algorithm.a Cell lines used were 32D.3 clones containing either vector alone (pMT) or vector containing an AML-1B insert (1B).b Percentage of cells in the G1 phase at 3 or 4 h after BrdU labeling. Open table in a new tab Cells were assayed as described in the legend to Fig. 2 B. At the indicated times, the percentage of G1 cells labeled with BrdU was determined using the ModFit algorithm. The observation that AML-1B-expression accelerated S phase entry in myeloid progenitor cells was unexpected. Cell cycle regulatory transcription factors and oncogenes that act in the G1 phase of the cell cycle such as E2F-1 and c-Myc do not alter the timing of the cell cycle (32.Hiebert 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 (146) Google Scholar, 33.Strom D. Nip J. Cleveland J.L. Hiebert S.W. Cell Growth Differ. 1998; 9: 59-69PubMed Google Scholar, 40.Askew D.S. Ashmun R.A. Simmons B.C. Cleveland J.L. Oncogene. 1991; 6: 1915-1922PubMed Google Scholar). However, both E2F-1 and c-Myc can induce S phase progression of 32D.3 cells in the absence of IL-3 (32.Hiebert 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 (146) Google Scholar, 33.Strom D. Nip J. Cleveland J.L. Hiebert S.W. Cell Growth Differ. 1998; 9: 59-69PubMed Google Scholar, 40.Askew D.S. Ashmun R.A. Simmons B.C. Cleveland J.L. Oncogene. 1991; 6: 1915-1922PubMed Google Scholar). Therefore, we tested whether AML-1B could also induce continued cell cycle progression in the absence of cytokine. Cell lines were cultured in media lacking IL-3 for 12 h, and cell cycle progression was measured using propidium iodide staining to determine their DNA content. E2F-1-expressing cells displayed no apparent cell cycle defects in the presence of IL-3 (Fig.4). In the absence of IL-3, E2F-1 induced continued S phase entry in the absence of the cytokine (Fig. 4). By contrast, there were fewer cells expressing AML-1B in the G1 phase and more cells in S-phase in the presence of IL-3, but in the absence of IL-3 these cells accumulated in the G1 phase (Fig. 4). Thus, AML-1B requires cooperating proliferative signals from IL-3 to accelerate S phase entry and affect the cell cycle. One of the major targets for regulation of cell cycle progression is the Rb pathway (41.Nevins J.R. Leone G. DeGregori J. Jakoi L. J. Cell. Physiol. 1997; 173: 233-236Crossref PubMed Scopus (176) Google Scholar). To determine whether this pathway was affected either directly or indirectly by AML-1B expression, we measured the levels of cyclin-dependent kinase-2 (cdk2), cdk4, cyclin D2, and E2F. Cell lysates were prepared from control and AML-1B-expressing cells, and antibodies to each of these proteins were used to probe immunoblots to determine their levels. Each cell line expressing AML-1B contained more cdk2, slightly elevated cdk4, and substantially more cyclin D2 (Fig.5 A). Consistent with this observation, the levels of histone H1 kinase activity present in cdk2 immunoprecipitates was 2–3-fold higher in the AML-1B-expressing cells (data not shown). Electrophoretic mobility shift analysis indicated that AML-1B-expressing cells also contained more overall E2F DNA binding activity relative to control cells, with most of this increase corresponding to the “free” or active form of E2F (Fig.5 B). This was in contrast to Sp1 DNA binding activity that did not change upon AML-1B expression (Sp1, Fig. 5 B). The shortening of the G1 phase and the higher levels of cyclin D2 suggested that AML-1 might activate key cell cycle regulatory genes that affect cell cycle progression. Therefore, we scanned the promoters for the G1 cyclins and found several AML-1 binding sites in the cyclin D2 promoter. To determine whether AML-1 could activate the cyclin D2 promoter, we used this promoter linked to a luciferase reporter gene in transient assays. In COS-7 cells, the cyclin D2 promoter was activated by wild-type AML-1B but not by an AML-1B containing a point mutation that ablates DNA binding (L175D, changing residue 175 from leucine to aspartic acid, Fig.6 A). Expression from the internal control promoter was unaffected by AML-1B (data not shown, but used to correct for transfection efficiency). Promoter deletion analysis indicated that 5′ promoter sequences containing several potential and known AML-1 binding sites were required for full AML-1B-dependent transactivation, whereas a perfect AML-1 binding site 3′ to the first transcriptional start site was not sufficient for full activation (Fig. 6 B). The caveat in this analysis is that cyclin D2 is regulated during the cell cycle. Thus, the activation observed with AML-1B could be indirect if AML-1B was stimulating cell cycle progression. Therefore, we compared the ability of AML-1B to activate the cyclin D2 promoter with other cell cycle regulated promoters. Although the cyclin D2 promoter was activated to a greater extent than the cyclin D1 or human thymidine kinase promoter (which are not known to contain AML-1 binding sites), these promoters were also activated by AML-1B (Fig. 6 C). Once again, the internal control was unaffected by AML 1B (data not shown). Because we could not determine whether AML-1B directly activated cell cycle regulatory promoters, we probed the mechanism of AML-1B cell cycle control by asking whether expression of AML-1a, which lacks the C-terminal transactivation domain (3.Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6859-6863Crossref PubMed Scopus (563) Google Scholar, 42.Zeng C. van Wijnen A.J. Stein J.L. Meyers S. Sun W. Shopland L. Lawrence J.B. Penman S. Lian J.B. Stein G.S. Hiebert S.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6746-6751Crossref PubMed Scopus (228) Google Scholar), or AML-1B(L175D) could affect cell cycle progression. The clonal 32D.3 cell lines expressing AML-1a or AML-1B(L175D) (Fig. 1 and7 A) were compared with control and AML-1B-expressing cells using BrdU pulse-chase analysis (Fig.7 B). Once again, the AML-1B-expressing cells entered S phase with accelerated kinetics as compared with G418-resistant control cells. Cells expressing either AML-1a or AML-1B(L175D) did not show gross alterations in the timing of S-phase entry, indicating that both the C-terminal domain and DNA binding activity are required for AML-1B cell cycle functions. We conclude that AML-1B shortens the G1 phase by regulating the expression of downstream target genes. AML-1 is unusual in that it is an oncogene, yet it is disrupted by chromosomal translocations in acute leukemia. We observed that enforced expression of AML-1B induced accelerated S phase entry (Figs. 2 and 7and Table I). Cultures of cells expressing AML-1B had fewer cells in the G1 phase and more cells in S phase (Fig. 2). The time needed to traverse G2/M remained constant in these cells (Fig. 3, Table II), which is consistent with expression of AML-1B accelerating transit through the G1 phase of the cell cycle. Thus, AML-1B is one of the first transcription factors described that shortens the length of the G1 phase of the mammalian cell cycle. Furthermore, this work points toward a cell cycle mechanism for the ability of AML-1 to transform cells. The ability of AML-1B to alter the cell cycle led us to examine the promoters of known components of the G1 phase cell cycle control machinery to determine whether any of these genes could be direct targets of AML-1B. We found perfect AML-1B binding sites in the p21Waf1/Cip1 cyclin-dependent kinase inhibitor promoter (16.Lutterbach B. Westendorf J. Linggi B. Isaac S. Seto E. Hiebert S. J. Biol. Chem. 1999; 275: 651-656Abstract Full Text Full Text PDF Scopus (223) Google Scholar, 43.Lutterbach B. Westendorf J.J. Linggi B. Patten A. Mariko M. Davie J. Huynh K. Bardwell V. Lavinisky R. Glass C. Rosenfeld M.G. Seto E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 7176-7184Crossref PubMed Scopus (370) Google Scholar) and the cyclin D2 promoter but not in the E2F1, cdk4, or cdk2 promoters. In AML-1B-expressing cells, endogenous p21Waf1/Cip1 protein levels were slightly elevated (approximately 2–3-fold, data not shown). Recent work indicates that p21Waf1/Cip1 can both inhibit cdk activity and promote the assembly of cyclin:cyclin-dependent kinase complexes (44.Cheng M. Olivier P. Diehl J.A. Fero M. Roussel M.F. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: 1571-1583Crossref PubMed Scopus (974) Google Scholar). Both the p21Waf1/Cip1 and cyclin D2 promoters can be regulated by AML-1B (Fig. 6, data not shown, and Refs. 16.Lutterbach B. Westendorf J. Linggi B. Isaac S. Seto E. Hiebert S. J. Biol. Chem. 1999; 275: 651-656Abstract Full Text Full Text PDF Scopus (223) Google Scholar and 43.Lutterbach B. Westendorf J.J. Linggi B. Patten A. Mariko M. Davie J. Huynh K. Bardwell V. Lavinisky R. Glass C. Rosenfeld M.G. Seto E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 7176-7184Crossref PubMed Scopus (370) Google Scholar), consistent with the requirement of AML-1B transcriptional activation for the cell cycle phenotype (Fig. 7). However, because AML-1B accelerates the cell cycle, we could not definitively determine whether up-regulation of these genes is directly responsible for the shortening of the G1 phase or whether the levels of these proteins (or the activation of their promoters) are elevated because the cells are cycling faster. Initially, we observed faster growth of the AML-1B-expressing cells, suggesting that these cells had not compensated for the acceleration of the G1 phase and were cycling faster. However, as these lines were maintained in culture, this phenotype was lost. It is likely that these cells eventually compensated for the shortening of G1 by lengthening the S phase, as has been observed for cells overexpressing the G1 cyclins (29.Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (980) Google Scholar, 30.Ohtsubo M. Roberts J.M. Science. 1993; 259: 1908-1912Crossref PubMed Scopus (663) Google Scholar). We also found that the cell culture conditions were critical for revealing the AML-1B-cell cycle phenotype. For instance, at high cell densities, the shortening of G1 was lost (data not shown), suggesting density-dependent effects. The mechanisms behind these phenotypes will require further investigation. Transcription factors such as E2F-1 and c-Myc are capable of affecting cellular proliferation, but enforced expression of these factors did not affect the timing of the 32D.3 cell cycle (32.Hiebert 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 (146) Google Scholar, 40.Askew D.S. Ashmun R.A. Simmons B.C. Cleveland J.L. Oncogene. 1991; 6: 1915-1922PubMed Google Scholar). Therefore, the phenotype of enforced AML-1B expression is more similar to that of overexpression of the G1 cyclins, accelerating the G1 phase without affecting the entry to or exit from quiescence (29.Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (980) Google Scholar, 30.Ohtsubo M. Roberts J.M. Science. 1993; 259: 1908-1912Crossref PubMed Scopus (663) Google Scholar, 31.Kato J. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11513-11517Crossref PubMed Scopus (211) Google Scholar). Although stimulation of cyclin D2 or p21Waf1/Cip1 expression may contribute to AML-1B-mediated G1 phase control, these genes are unlikely to be the only mediators of this phenotype. We are actively searching for other cell cycle control genes that may contribute to AML-1-cell cycle control. We thank Dana King and Yue Hou for technical assistance and the Vanderbilt Cancer Center DNA sequencing facility and Randy Fenrick for insightful discussions and critical evaluation of data.