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a Laboratory of Molecular Immunoregulation and
b Basic Research Program, Science Applications International Corporation (SAIC)-Frederick, Inc., Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland, USA;
c Dana-Farber Harvard Cancer Center, Boston, Massachusetts, USA; and
d Invitrogen Corporation, Carlsbad, California, USA
Key Words. IFI-200 • Tumor suppressor • Myeliod • Differentiation
Correspondence: Jonathan R. Keller, Ph.D., Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Building 560, Room 12–03, Frederick, Maryland 21702–1201, USA. Telephone: 301-846-1461; Fax: 301-846-6646; e-mail: kellerj{at}ncifcrf.gov
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ABSTRACT |
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INTRODUCTION |
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p205 is a member of a family of interferon-inducible genes known as the IFI-200 family [6–8]. Other members of the IFI-200 family include murine p202, p203, p204, and the human myeloid cell differentiation antigen (MNDA), IFI-16, and AIM-2 (absent in melanoma–2). IFI-200 proteins are defined by having at least one 200-amino-acid homology region, designated as either a or b domain, that is highly conserved among family members. p203, p205, MNDA, and AIM-2 all contain one 200-amino-acid homology domain, whereas all other family members contain both a and b regions. Currently, no human counterparts exist for any of the mouse IFI-200 family members, and vice versa. However, between murine and human proteins, the highest homology exists for the p205 and MNDA polypeptides, which have 44% identity to each other. Like p205, MNDA is expressed in a lineage-specific manner in myeloid cells [9–11]. Treatment of monocytes, HL-60 cells, U-937 cells, and THP-1 cells with interferons results in robust induction of MNDA mRNA [12]. Because of this myeloid-specific expression pattern, it is likely that MNDA may have an important role in human myelopoiesis; however, its precise function is unclear and remains to be elucidated.
Despite the high homology between p205 and its murine orthologues, expression patterns of p202 and p204 vary widely. p202 is widely expressed in various tissues, and p202 mRNA can be detected at moderate levels in the lungs, kidneys, gut, and heart and at higher levels in the spleen, lymph nodes, thymus, and bone marrow [7]. p204 is expressed in myelomonocytic cells and is strongly induced in interferon (IFN)–treated fibroblasts. In comparison, p205 mRNA and protein is not detectable in IFN-treated fibroblasts [8, 13].
In contrast to the differences in their expression patterns, p202 and p204 have similar inhibitory effects on cell growth. It has been demonstrated that overexpression of p202 in NIH3T3 and AKR2B fibroblasts inhibits cell proliferation [14, 15]. Similarly, p204 expression inhibits the growth of the fibroblast cell lines NIH3T3, B6MEF, and B/cMEF in vitro [16, 17]. Furthermore, expression of p202 reduces growth and causes reversion of the transformed phenotype of prostate cancer cells [18].
Currently, no known function (antiproliferative or otherwise) has been assigned to p205. Indeed, p205 remains the most poorly characterized of the murine IFI-200 family members. However, p205 is induced in purified murine c-Kit+ Sca-1+ hematopoietic stem cells during myeloid cell differentiation, suggesting that p205 may have a role in myeloid cell development [5]. We have investigated the ability of p205 to regulate cell growth and mapped its functional domains. Based on the data in this report, we postulate that p205 has an important role in myelomonocytic cell differentiation by exerting an antiproliferative effect on myeloid cell growth.
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MATERIALS AND METHODS |
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The plasmid pEGFP-p205 was constructed by ligation of the 1.4-kb p205 cDNA HaeIII fragment to BamHI linker sequences into the BamHI site of pEGFP-C1 (Clontech). The resulting plasmid encodes enhanced green fluorescent protein (EGFP) fused at the C-terminal end with the p205 protein lacking its first 29 amino acids.
The p205 coding sequence was amplified by PCR using pBluescriptSKII-p205 as template and the following primer pair: 5'-GCAGAATTCCGCACCATGGTGAATGAATACAAGAGAATT-3' and 5'-GCAGGATAATCACTGGACAGTTGA-3'. Following amplification of the 1,302-bp DNA fragment, the PCR product was digested with a mixture of EcoRI and BamHI and ligated to pCB6+ vector (a gift from Karen Vousden, Regulation of Cell Growth Laboratory, NCI-Frederick, Frederick, MD). The correct p205 coding sequence was verified by dideoxy sequencing, and this plasmid was termed pCB6+-p205. Similarly, the pCB6+-p204 plasmid was generated by first amplifying the p205 coding sequence using PCR.
The primers 5'-GCAGAATTCCGCACCATGGTGAATGAATACAAGAGAATT-3' and 5'-GCAGGATCCTCACTTTCTAGCATT-3' were used with pBluescript-p204 as template. The 1,947-bp PCR product was also digested with a mixture of EcoRI and BamHI and ligated to pCB6+ vector, resulting in the plasmid pCB6+-p204. The correct p204 coding sequence was verified by dideoxy sequencing.
Construction of p205 Mutants
To construct deletion mutants of p205, PCR primers (29–35 bases long) were designed with an EcoRI site in both the forward and reverse primers to produce p205 (wild-type, full-length translated region of p205 with 425 amino acids) and the translated region with the following deletions:
a: The entire C-terminal sequence of p205, including the a domain, was truncated, resulting in a protein comprising amino acids 1–207 Rb:Amino acids 401–425 were truncated from the p205 sequence, resulting in a mutant without the LXCXE retinoblastoma protein (Rb)–binding site. DAPIN: 51 N-terminal residues encoding most of the IFI-200 domain in apoptosis and interferon response (DAPIN)/PYRIN motif were removed. The PCR reactions contained 0.5–1.0 µg of DNA, 200 nM dNTPs mix, 1x PCR buffer (PerkinElmer, Boston, MA), 1 µM (each) of appropriate primers, and 2.5 units of Taq polymerase (Perkin-Elmer/Cetus). The samples were incubated in a programmable thermal controller (Perkin-Elmer/Cetus) for 1 cycle of 95°C for 5 minutes; followed by 30 cycles of 95°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute; followed by a cycle of 72°C for 8 minutes. PCR products with intact single 3' adenine overhangs were ligated into pCR 2.1-TOPO (topoisomerase-I) vector and cloned into chemically competent TOP 10F' Escherichia coli cells (Invitrogen Corporation). The inserts were verified by sequencing, then removed by digestion with EcoRI and shuttled into EGFP expressing pTracer-CMV2 vector for growth inhibition studies.
p205
TSTAQA
For the construct lacking 37 amino acids (121–157) that contain the five-repeat TSTAQA(G/R) sequence, forward and reverse primers were designed to make separate N-terminal and C-terminal products, both lacking the 37-amino-acid TSTAQA(G/R) sequence. The forward primer for the N-terminal had an EcoRI site, while the reverse primer had a BamHI site. The forward primer for the C-terminal product had a BamHI site, while the reverse primer had a NotI site. Two separate PCR reactions were performed for N-terminal and C-terminal products, as described above, and the products were ligated into TOPO vector individually, as described above. After sequencing, the N-terminal product was digested with a mixture of EcoRI and BamHI and the C-terminal fragment with a mixture of BamHI and NotI.A triple ligation was then performed with the pTracer-CMV2 vector, as described above.
pS261D and pS261A To construct the site-directed mutant of p205 (Serine 261 changed to aspartate [pS261D] or alanine [pS261A]), forward and reverse primers were designed to make separate N-terminal and C-terminal PCR products. The forward primer for the N-terminal and the reverse primer for the C-terminal had EcoRI sites and were similar to the ones made for the full-length translated region of p205, while the N-terminal reverse primer and the C-terminal forward primer had two bases modified to change Serine 261 to either aspartate or alanine. Initially, two separate PCR reactions were performed for N-terminal and C-terminal products, as described above, followed by a third PCR reaction containing 0.5 µg (each) of amplified N-terminal and C-terminal products and 1 µM (each) of appropriate primers (the full-length N-terminal forward primer and C-terminal reverse primer) under the same PCR conditions as described above. The resulting PCR product was ligated into the pCR 2.1-TOPO vector, sequenced, removed by digestion with EcoRI, and cloned into pTracer-CMV2. Protein expression from all above constructs was verified by Western blot analysis using a C-terminal–specific p205 antibody (Cocalico Biologicals, Inc., Reamstown, PA); for C-terminal truncation mutants we used an N-terminal–specific p204 antibody (kindly provided by Peter Lengyel), which recognizes both p204 and p205 proteins.
Cell Culture
EML cells [4] were maintained in Iscove’s Modified Dulbecco’s medium (IMDM), 20% horse serum (GIBCO-BRL, Gaithersburg, MD), 2 mmol/L L-glutamine, and penicillin-streptomycin solution (GIBCO-BRL) at 1/100 dilution and supplemented with 8%–15% BHK/MLK conditioned media (CM), as a source of SCF, or 100 ng/mL recombinant SCF (PeproTech, Rocky Hill, NJ) (EML complete medium). To induce the differentiation of EML cells, complete media was supplemented with 30 ng/mL IL-3 (PeproTech), and 10–5 M atRA (Sigma, St. Louis). MPRO and EML-derived promyelocytic cells (EPRO) [4] were maintained in Dulbecco’s Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS) (Gemini Bio-Products, Calabasas, CA), 2 mmol/L L-glutamine, and 1/100 dilution penicillin-streptomycin, supplemented with 20 ng/mL recombinant murine GM-CSF (PeproTech) and were differentiated with addition of atRA (10–5 M).
Murine myeloid progenitor BaF3 and 32D-C123 cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mmol/L L-glutamine, 1/100 dilution penicillin-streptomycin (GIBCO-BRL), and 30 ng/ml recombinant murine IL-3 (PeproTech). Murine NIH3T3 fibroblasts were maintained in DMEM with 10% FBS and 1/100 dilution penicillin-streptomycin (GIBCO-BRL). Human embryonic kidney 293 cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, 2 mmol/L L-glutamine, and 1/100 dilution penicillin-streptomycin (GIBCO-BRL).
Lineage-negative (Lin–) bone marrow progenitor cells were maintained in IMDM, 10% FCS, 2 mmol/L L-glutamine, and 1/100 dilution penicillin-streptomycin supplemented with 30ng/ml IL-3 and 100 ng/ml SCF. Lin– cells and all cell lines were grown at 37°C, in a 5% CO2 incubator.
Isolation of Lineage-Negative Bone Marrow Progenitors Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, 1996). Bone marrow cells (BMCs) were harvested from the femurs of 12- to 18-week-old Balb/c mice and separated based on cell density using Lymphocyte Separation Medium (Organon Technika, Durham, NC). The light density fraction was obtained after centrifugation, and cells were further purified by immunomagnetic depletion of terminally differentiated cell populations. Specifically, cells were incubated with a cocktail of antibodies directed against granulocytes, Gr-1 (RB6–8C5); B cells, B220 (RA3–6B2); T cells. CD8 (Lyt-2) and CD4 (L3T4); macrophages, CD11b (Mac-1) and Ter-119; and erythroid cells (Pharmingen, San Diego) at a concentration of 0.1 µg antibody/106 cells in media. The cells were incubated for 30 minutes at 4°C, then washed twice and resuspended in media at a density of 108 cells/ml. Magnetic beads coated with antirat immunoglobulin G (IgG; Dynal, New Hyde Park, NY) were incubated with cells at a concentration of 20 beads/cell at 4°C on a rotating platform for 45 minutes. The Lin+ cells were removed using a magnetic particle concentrator, and the resulting Lin– cell population was used in the experiments described herein.
Transfections Electroporation of BaF3, 32D-C123, 293, and Lin– cells was performed as follows. Plasmid DNA (20 µg) was electroporated into 1 x 107 cells using a BTX Electro Cell Manipulator 600 (Biotechnologies and Experimental Research, Inc., San Diego). The DNA and cells were first incubated at room temperature for 10 minutes in 0.5 ml of medium lacking serum and penicillin-streptomycin. The DNA and cell mixture was then transferred to a 4-mm electroporation cuvette, which was placed in the Electro Cell Manipulator, and a 50-msec pulse was delivered at 400 volts, 800 µF. After allowing the cells to recover for 10 minutes at room temperature, the cells were resuspended in 20 ml of medium supplemented with appropriate growth factors and incubated as described before.
Cell Sorting
Twenty-four hours after electroporation, the cells were sorted by fluorescence activated cell sorter (FACS) for GFP fluorescence. GFP+-sorted BaF3 and 293 cells transfected with pEGFP-C1 or pEGFP-p205 were resorted 1 week later and plated as single cells so that cell lines could be established. GFP+- and GFP–-sorted Baf3, 32D-C123, 293 and Lin– cells from pTr, pTr-205, pTr-204, or pTr-202 electroporations were either used for cell growth assays or lysed in 1x SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for western blot analysis.
Proliferation Assay FACS-sorted cells were seeded in triplicate (5 x 103 cells/100 µl) in growth medium supplemented with the appropriate growth factors in a 96-well plate and allowed to grow for 24, 48, or 72 hours at 37°C and 5% CO2. The cells were labeled with 1.0 µCi [3H]-thymidine for 8–12 hours before harvesting. The cells were harvested using a Skatron automated cell harvester (PerkinElmer), and the amount of [3H]-thymidine incorporated was determined using a Betaplate scintillation counter (PerkinElmer).
Colony Formation and Single Cell Growth Assay For the colony-formation assay, calcium phosphate transfection of NIH3T3 cells was accomplished using previously described methods [19]. Briefly, 10 µg of plasmid DNA (pCB6+, pCB6+-p205, or pCB6+-p204) was used for transfection of 2.5 x 105 NIH3T3 cells in 60-mm tissue-culture dishes. The cells were washed 16 hours after addition of the Ca2+/DNA precipitate and then allowed to recover for an additional 24 hours. The cells were then enumerated, and 5 x 103 cells were plated in 100-mm tissue-culture dishes in DMEM supplemented with 10% FBS, 1/100 dilution penicillin-streptomycin, and 400 µg/ml Geneticin (GIBCO-BRL). The cells were allowed to grow for 2 weeks with two medium changes, and then they were washed, fixed with methanol, and stained with Giemsa (Sigma). Colonies 2 mm or greater in size were scored.
For single-cell growth assays, Lin–, GFP+ were seeded in Terasaki plates, 60 wells per plate (Nunc, Kamstrup, Denmark) at a concentration of one cell per well in 20 µl growth medium plus IL-3 and SCF, and incubated for 5 days, as described above.
Early Apoptosis Assay 32D-C123 cells were transfected with pCMS-EGFP (pCMS) and pCMS-EGFP-p205 (pCMS-205) reporter plasmids, as described above, and after 48 hours the cells were washed and resuspended in binding buffer and stained with Annexin V-PE and 7-AAD to detect apoptotic cells according to the procedures outlined by the manufacturer (Pharmingen).
Generation of p205 Antibody A 15-amino-acid sequence corresponding to the C-terminus of p205 (NH2-KVTKAGKKKEASTVQ-COOH) was synthesized and conjugated to KLH (Peptide Express, Ft. Collins, CO). The conjugated peptide was resuspended in H2O and delivered to Cocalico Biologicals) for production of anti-sera in rabbits. The anti-serum was shown to be specific for identification of p205 expression in western blot analysis using lysates from p205-, p204-, and p202-transfected cells.
Western Blotting
EML, EPRO, and MPRO nuclear and cytosolic cellular fractions were isolated as described previously [20]. Twenty-five µg of each sample were separated on 4%–12% gradient SDS-PAGE and transferred to Immobilon-P PVDF membrane (Millipore, Bedford, MA) for immunoblotting. For experiments that used cells that had been sorted for EGFP expression, equal cell numbers were lysed in 1xSDS-PAGE sample loading buffer and boiled for 10 minutes. Equal volumes were then separated on 4%–12% gradient SDS-PAGE and transferred to Immobilon-P PVDF for immunoblotting. To verify that equal protein amounts were transferred in each lane of the gel, the membranes were stained with Ponceau S (Sigma). The immunoblots were developed using anti-rabbit HRP-conjugated secondary antibody (Promega, Madison, WI) and LumiGlo HRP substrate (New England Biolabs, Beverly, MA). Chemiluminescence was detected by exposing the immunoblots to RAR film (Eastman Kodak Co., Rochester, NY) for times ranging from 5 seconds to 15 minutes. Anti-p202 and anti-p204 antibodies were the generous gifts of Peter Lengyel and have been described previously [21, 22]. Immunoblots were stripped by incubation in 200-mM glycine (pH 2.5), 0.05% Tween-20 for 2 hours at 80°C. Western blotting using anti-actin antibodies (product number A2668; Sigma) was performed to verify equal loading of the samples.
Microinjection of NIH3T3 Cells
Microinjection was performed as described previously [23]. Briefly, NIH3T3 cells were plated onto glass coverslips and grown to 95% confluence before being serum-deprived for 24–30 hours in DMEM supplemented with 0.4% FBS. The quiescent cells were microinjected (10–11 ml) with coded samples, serum-stimulated with media containing 10% FBS, incubated at 37°C for 10–12 hours, and pulsed with [3H]-thymidine (0.5 µCi/ml) for 4 hours. The cells were fixed with 3.7% glutaraldehyde (v/v PBS, pH 7.4), and autoradiography was performed for 2 days in nuclear tracking emulsion. The cells were stained with Giemsa (Sigma), scored, and photographed. This assay measured the ability of an injected factor to inhibit serum-induced entry of the G0/G1 cell into S phase. These data are corrected for background in that the DNA synthesis labeling efficiency is defined as the ratio of label uptake in microinjected cells that incorporate [3H]-thymidine into nuclei divided by label uptake in noninjected cells, near the injected area, that incorporate label into nuclei multiplied by 100.
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RESULTS |
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p205 Is a Nuclear Protein
To investigate the functional role of p205 during hematopoietic development, we analyzed the cellular localization of p205 by two methods. First, cytosolic and nuclear fractions were prepared from EML cells; EML cells treated with SCF, IL-3, and atRA for 72 hours (conditions known to induce p205 mRNA [5]); and EPRO and MPRO cells. The cellular extracts were separated by reducing SDS-PAGE on a 4% to 12% gradient, transferred to PVDF membrane, and subjected to Western blot analysis using a rabbit anti-sera raised against the C-terminal of p205 (Cocalico Biologicals) (Fig. 1A
). A single band of 55 kDa was detected only in the nuclear fractions of MPRO (lane 8), EPRO (lane 6), and SCF/IL-3/atRA–treated EML cells (lane 4). This protein was not detected in either nuclear extracts isolated from untreated EML cells (lane 2) or cytosolic extracts from EML (lanes 1 and 3), EPRO (lane 5), or MPRO cells (lane 7). Western blotting performed using rabbit pre-immune serum did not detect the band in any fraction (data not shown).
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, panels B and F) as compared to the same cells stained with DAPI to visualize the nucleus (Fig 1B, panels A and E). In contrast, BaF3 and 293 cells transfected with pEGFP-p205 showed expression of the EGFP-p205 fusion protein only in the nucleus (Fig. 1B, panels D and H) as shown by DAPI staining (Fig. 1B, panels C and G). Taken together with the Western blotting data, these results demonstrate that both endogenous p205 and exogenously expressed p205 proteins are nuclear.
IL-3–Mediated Proliferation of Myeloid Cells Is Inhibited by Expression of p205
Previous studies have shown that p205 expression is closely linked to the myeloid differentiation of hematopoietic progenitor cells [5, 8]. Other family members, p202 and p204, have been demonstrated to inhibit cell proliferation [14, 16–18, 24, 25]. Therefore, we sought to ascertain whether p205 could specifically inhibit growth of the IL-3–dependent myeloid progenitor cell lines BaF3 and 32D-C123. BaF3 cells were electroporated with pTracerCMV2 (pTr) plasmids, which contain GFP, thus facilitating separation of transfected and untransfected cell populations. At least 15% to 30% of the BaF3 cells transfected with pTr or pTr-205 expressed GFP after 24 hours (data not shown). Following electroporation, the GFP-positive (GFP+) cells were separated from GFP-negative (GFP–) cells by flow cytometry. p205 was not expressed in GFP+ cells from the vector control transfection or in GFP–-sorted cells, while it was specifically expressed in GFP+ cell populations that were obtained from transfections with pTr-205, as determined by Western blot analysis (Fig. 2A). We also compared the physiological levels of endogenous p205 protein with those achieved by transient expression assays. We found that p205 is expressed at comparable levels in BaF3 cells transfected with 20 µg pTr-205 and sorted, and murine BMCs cultured for 4 days in M-CSF (Fig. 2B). In contrast, p205 is expressed at higher levels in MPRO cell lines. Therefore, it is reasonable to conclude that p205 expression levels in transient transfection assays approximate those achieved under physiological conditions.
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, expression of p205 results in a greater than 50% decrease in [3H]-thymidine incorporation in BaF3 cells, compared with the vector controls and GFP–-sorted cells. Therefore, IL-3–induced DNA synthesis of BaF3 cells is inhibited by p205 expression. The effect of p205 expression on cell proliferation was further investigated by measuring the growth of transiently transfected BaF3 cells over 3 days in liquid cultures. BaF3 cells were electroporated as described above and separated by FACS, and GFP+ cells were seeded at a density of 5 x 103 cells/100 µl of growth medium containing IL-3 in a 96-well microtiter plate. We observed that the growth of p205-expressing BaF3 cells was inhibited over a 3-day period, compared with control cells. The percentage of growth inhibition in p205-expressing cells remained fairly constant over each 24-hour period at 47%, 40%, and 46% inhibition over 24, 48, and 72 hours, respectively (data not shown). Examination of Giemsa-stained cytocentrifuge preparations showed that p205 expression does not alter the morphology of BaF3 cells (data not shown). Thus, these data demonstrate that expression of p205 inhibits IL-3–dependent BaF3 progenitor cell growth.
To ensure that the antiproliferative effects of p205 are not restricted to a single cell line, the effect of p205 expression was examined on other IL-3–dependent myeloid cell lines, including 32D-C123. 32D-C123 cells were electroporated with either a GFP vector control or a GFP vector containing p205, separated by FACS; they were then cultured in vitro and assayed for [3H]-thymidine incorporation, as described above. Similar to the effect of p205 on BaF3 cell growth, p205-expressing 32D-C123 cells showed 40%, 55%, and 55% reductions in the levels of [3H]-thymidine incorporation compared with control cells at 24, 48, and 72 hours, respectively (Fig. 2D).
Finally, we evaluated whether p205 expression could inhibit the growth of normal hematopoietic progenitors. We purified progenitor cells (Lin– cells) from normal bone marrow, transfected these cells with either vector control or vector containing p205, sorted them into GFP+ and GFP– by FACS, and then compared their growth in [3H]-thymidine incorporation assays or in single-cell cultures. Lin– cells expressing p205 showed a 60% reduction in [3H]-thymidine incorporation compared with the controls (Table 1). In addition, Lin– cells transfected with the p205-containing vector showed greater than 90% reduction in colony formation in single-cell assays in response to IL-3 plus SCF. In comparison, there was no difference in the level of [3H]-thymidine incorporation, or growth in single-cell assays, of GFP–-sorted Lin– cell populations. Thus, p205 can also function to inhibit normal hematopoietic progenitor cell growth.
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, respectively), while they are not expressed in either GFP+ cells from a vector control transfection (pTr), nor are they expressed in GFP– cells. Similar to the effect of p205, BaF3 cells expressing p204 or p202 have a 40%, 55%, and 50% decrease in [3H]-thymidine incorporation at 24, 48, and 72 hours, respectively (Fig. 3C). Thus, in addition to p205, myeloid cell growth is also inhibited by p204 and p202 at comparable levels.
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, panel C) have significantly fewer nuclei that incorporated [3H]-thymidine in comparison with cells injected with empty vector (Fig. 4, panel A) or p205 antisense vector (Fig. 4, panel B), indicating that p205 expression inhibits DNA synthesis. The labeling efficiency was determined by dividing the number of labeled nuclei from microinjected cells by the number of labeled nuclei from cells that were not microinjected (Table 2). The labeling efficiency of NIH3T3 cells microinjected with pcDNA-p205 was decreased by 75%, compared with the labeling efficiency of NIH3T3 cells microinjected with control plasmids. Thus, p205 expression can also inhibit DNA synthesis in NIH3T3 fibroblasts. To evaluate whether p205 inhibits NIH3T3 cell growth, we transfected NIH3T3 cells with pCB6+ plasmid vectors that express p205 and p204 (pCB6+-p205 or pCB6+-p204) and looked at the ability of NIH3T3 to form colonies in vitro (p204 included as a positive control [16, 17]). Transfected cells were plated at equal cell densities and cultured for 2 weeks in the presence of G418. The numbers of colonies greater than 2 mm in diameter for each transfection are shown in Table 3. NIH3T3 cells transfected with either pCB6+-p205 or pCB6+-p204 had a reduced (40% of control) ability to form colonies in vitro. NIH3T3 cells transfected with pCB6+-p205 or pCB6+-p204 expressed p205 and p204 protein 24–48 hours after transfection by western blot analysis (data not shown). Thus, p205 and p204 can inhibit NIH3T3 cell growth in vitro, indicating that their antiproliferative effect is not specific to hematopoietic progenitor cells.
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, panels A and B, upper left quadrant) in contrast to significant early apoptosis (15%) occurring 24 hours after growth factor withdrawal (Fig. 5, panel C). In addition, there was little or no difference in the percentage of late apoptotic and necrotic cells (Annexin+ and 7-AAD+) in the same GFP+ cell populations (5.5% versus 7%, respectively; upper right quadrant), while there is a 16% increase in cells undergoing late apoptosis following growth factor withdrawal. Thus, expression of p205 did not induce apoptosis in 32D-C123 cells after 48 hours, demonstrating that growth inhibition by p205 is not affected via apoptotic processes.
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a mutant was constructed to determine if the growth-inhibitory property of p205 was dependent on the 200-amino-acid motif present in the C-terminus of all IFI-200 family members. DAPIN mutant has a truncation of the N-terminal region and includes a coiled-coil region called the DAPIN/PYRIN domain, which is present on all IFI-200 family members except for p202, but still possesses the basic nuclear localization sequence (NLS). Rb mutant lacks the putative C-terminal Rb-binding site. TSTAQA mutant lacks the seven-residue TSTAQAR repeat regions unique to the p204 (eight repeats) and p205 (five repeats) proteins. A putative ATM phosphorylation site was identified at Ser261 [26, 27] (Fig. 6A). Therefore, the serine was substituted with a charged aspartic acid residue to mimic phosphorylation at this site (pS261D).
Following transfection into BaF3 cells, the relative expression and size of the p205 mutants was determined by Western blotting using an N-terminal–specific anti-p205 antibody (a gift from Santos Landolfo, Turin, Italy) (Fig. 6B).
As shown above, p205 significantly affects cell proliferation as compared with the pTr control vector (41% growth inhibition, p = .005 by paired t-test analysis). The p205TSTAQA mutant had a growth-inhibitory effect comparable to wild-type, indicating that the repeat sequence was not required for growth inhibition in this assay (Fig. 7). However, the p205Rb and p205DAPIN mutants did not inhibit growth, demonstrating that the Rb-binding LXCXE motif and the DAPIN/PYRIN domain are required for p205 antiproliferative activity. Other studies have already noted that the growth-inhibitory activity of p204 depends on the presence of its two Rb-binding sites [25, 28]. Deletion of the entire a domain of p205 in the p205a mutant abolished antiproliferative activity to the greatest extent. This indicates that the a domain, which possesses a highly conserved protein-binding motif (MFHATVAT), in addition to the Rb-binding site, plays an important part in mediating p205 activity. Finally, the pS261D mutant exhibited enhanced growth-inhibitory properties, since it conferred 32% increased growth inhibition in comparison with the control pS261A mutant (p = .038) and 29% increased growth inhibition in comparison with wild-type p205 expression (p = .02). Therefore, a charged residue at position 261 enhances p205 activity, possibly identifying a mechanism by which p205 may be activated in vivo.
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DISCUSSION |
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Our studies in BaF3 and 32D-C123 cells show that transient expression of p205 results in an immediate decrease in DNA synthesis and in cell number as measured by [3H]-thymidine incorporation and single cell growth, respectively. Our results also showed that transient expression of two other IFI-200 family members, p204 and p202, in BaF3 cells also resulted in inhibition of cell growth and proliferation. Similar to the growth inhibitory effects of p205 on myeloid cells, NIH3T3 cells microinjected with p205 sense cDNA showed a 75% reduction in the labeling efficiency of cell nuclei. Furthermore, expression of p205 inhibits the growth of NIH3T3 cells at levels similar to p204, whose ability to inhibit NIH3T3 cell growth has been previously described [16, 17].
As all three murine IFI-200 family members inhibited BaF3 proliferation at similar levels, it is possible that p205, p204, and p202 have similar mechanisms of growth inhibition. Despite the fact that p204 contains an additional 200-amino-acid b domain, p205 and p204 are the most highly homologous IFI-200 family members. Therefore, it is not surprising that both proteins possess similar antiproliferative activities. However, a recent report found that transfection of a p204 construct lacking its b domain did not inhibit colony formation in NIH3T3 or B6MEF cells [16]. This result is unexpected, because full-length p205 is 84% identical to the truncated p204 protein (p204b). By focusing on differences between p205 and p204b, we may determine which amino acids of p205 are required for growth inhibition. In another report, transfection of NIH3T3 or B6MEF cells with p203, which has a single b domain, does not inhibit colony formation [16]. These data suggest that IFI-200 members with a single 200-amino-acid domain lack antiproliferative activity. However, our results show that an IFI-200 family member with only a single 200-amino-acid region can significantly and reproducibly inhibit cell proliferation in multiple cell types. In correlation with our observation, AIM2, a human p200 homologue with a single a domain like p205, has been shown to possess growth-inhibitory properties [32].
In agreement with results observed with other IFI-200 proteins, we have demonstrated that p205 is a nuclear protein. Human MNDA and IFI-16, as well as murine p204 and p203 proteins, are always found in the nucleus [22, 33–36], while p202 can be detected in the cytoplasm but will quickly translocate to the nucleus following treatment with interferons [21]. The nuclear localization of IFI-200 proteins is likely critical for interactions with target proteins. For instance, p202 has been demonstrated to bind to a wide variety of proteins involved in cell-cycle regulation and transcription including pRb [37], 53BP [38], NF-
B (p50 and p65) [39], and AP-1 (c-Fos and c-Jun) [39]. The p202 protein has also been demonstrated to inhibit E2F-mediated transcription [15, 40], as well as the transcriptional activities of AP-1 [39], NF-B [39], and the muscle proteins MyoD and myogenin [41]. Additionally, it has been shown that human IFI-16 fused to the GAL4 DNA-binding domain can function as a transcriptional repressor [42]. Furthermore, it has been demonstrated that induction of p202 and p204 results in an increase in the levels of hypophosphorylated pRb [17, 43]. This form of pRb is a potent inhibitor of E2F-mediated transcription.
Due to the promiscuous nature of binding and inhibition of transcription by IFI-200 proteins, it is tempting to speculate that they inhibit cell growth by a global inhibition of transcriptional activity. However, as it has recently been demonstrated that expression of p202 increases p21WAF1/CIP1 mRNA and protein levels [43], this is not the case. Furthermore, human MNDA binds nucleolin and nucleophosmin/NPM/B23 in addition to the zinc finger transcription factor YY1 [44–46]. The interaction of MNDA with the nucleolar YY1 protein results in enhanced binding of YY1 to DNA, possibly resulting in increased YY1 transcriptional activity [46]. Given the precedence of IFI-200 proteins affecting transcriptional activity, in addition to p205 specifically localizing to the nucleus, we hypothesize that p205 also binds to transcription factors and cell-cycle proteins, resulting in the antiproliferative effects of p205. In this regard, future experiments are planned to examine the effects of p205 expression on the cell cycle; specifically, we will investigate whether p205 can mediate its growth inhibitory activity by interacting with or modulating levels of cell-cycle regulatory proteins, and if expression of p205 affects cell-cycle progression.
Deletional analysis of the p205 protein has identified several essential functional domains. First, deletion of the N-terminus bearing the DAPIN/PYRIN motif abolishes p205 inhibitory activity. The DAPIN domain (also referred to as the PYRIN, PAAD, or CARD domain) is a coiled-coil motif that is found on all p200 proteins, with the exception of p202, and is also implicated in mediating protein–protein interactions in the larger PYRIN family of proteins, which has been associated in the regulation of apoptotic and inflammatory pathways [47–49].
Also, deletion of the C-terminal Rb-binding site impairs p205 antiproliferative activity, as does deletion of the 200-amino-acid a domain. These findings support previous studies that implicate the Rb-binding sites of p204 in mediating its growth-inhibitory activity [25, 28], as well as studies which show that a highly conserved MFHATVAT motif (present on the p200 domain of all family members) is required to mediate homodimerization in p202 [50] and protein–protein interaction with 53BP, a protein that binds to and enhances p53 transcriptional activity [38]. Thus, it is reasonable to speculate that the C-terminal amino acid region may be essential for p205 activity in promoting the interaction of p205 with other key transcriptional regulatory elements such as Rb and 53BP.
We have also implicated a putative ATM phosphorylation site in p205 in contributing to its antiproliferative activity. A p205 mutant in which a charged aspartate residue was substituted for serine in the ATM site, to mimic a phosphorylation event, showed enhanced inhibitory activity in comparison to a p205 mutant with a neutral alanine residue at the ATM site. While the possible mechanism of ATM-dependent activation is not yet known, it is plausible that phosphorylation of p205 at this site serves to enhance protein stability, as has already been observed for ATM-dependent phosphorylation of E2F-1 in response to DNA damage [51]. Presumably, ATM may phosphorylate p205 during cell-cycle checkpoint signaling and thus promote p205-induced cell-cycle arrest.
In summary, the functional domains of p205 were identified as the N-terminal DAPIN/PYRIN motif and the conserved 200-amino-acid a domain. Within the a domain, we show that deletion of the Rb-binding site alone can abolish p205 antiproliferative activity. Deletion of these motifs resulted in loss of function of the p205 protein, therefore indicating that protein–protein interaction appears to be an essential means by which p205 mediates its antiproliferative effect, presumably by binding to and modulating the activities of key transcription factors that regulate cell growth and proliferation. Furthermore, p205 may be the substrate for ATM phosphorylation; this is suggested by the presence of an ATM phosphorylation site on p205, which, when charged, confers enhanced growth-inhibitory characteristics to p205. Thus, it remains to be investigated whether p205 could have a role in ATM checkpoint signaling in contributing to cell-cycle arrest. Like MNDA [9, 10, 29, 52], p205 is strongly expressed as hematopoietic progenitor cells undergo myeloid lineage commitment. In order for cells to differentiate, it is necessary for growth arrest to occur. We propose that during myelomonocytic cell differentiation, p205 is induced and contributes to cell growth arrest, thus allowing progenitor cells to withdraw from the cell cycle and differentiate.
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This project was funded in whole or in part by the National Cancer Institute, National Institutes of Health, under contract no. N01-CO-12400.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial, products, or organizations imply endorsement by the U.S. government.
The publisher or recipient acknowledges right of the U.S. government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.
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-actin antibody for loading control. (C): BaF3 cells were transfected with either pTr or pTr-205, sorted for green fluorescence by FACS as described above, and GFP+- (solid bars) and GFP–-sorted cells (stippled bars) were seeded in triplicate (5 x 103/100 µl) in a 96-well plate in 100-µl medium in the presence of IL-3 and allowed to grow for 24 hours. Prior to harvest, the cells were pulse-labeled with [3H]-thymidine, and the amount of [3H]-thymidine was determined as described in Materials and Methods. These data are representative of three independent experiments. (D): 32D-C123 cells were transfected with either pTr vector (grey bars) or pTr-205 (black bars) and sorted by FACS, and GFP+ cells were seeded in triplicate (5 x 103/100 µl) in a 96-well plate. The cells were allowed to grow for 24, 48, or 72 hours and were then pulse-labeled with [3H]-thymidine prior to harvest. These data are representative of three separate experiments. Abbreviations: BMC, bone marrow cell; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; SDS-PAGE, SDS-polyacrylamide gel electrophoresis.
