Inhibition of Growth by p205: A Nuclear Protein and Putative Tumor Suppressor Expressed during Myeloid Cell Differentiation
http://www.100md.com
《干细胞学杂志》
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@ncifcrf.gov
ABSTRACT
Hematopoiesis is a complex process whereby pluripotential hematopoietic stem cells differentiate into functionally distinct cell lineages. While significant progress has been made toward understanding the cellular requirements of myeloid differentiation, the molecular mechanisms that regulate this process remain poorly defined . Since pluripotential hematopoietic stem cells are found in low frequency in bone marrow and purification does not ensure homogeneity, we used a stem/progenitor cell–line model system; the EML (erythroid, myeloid, lymphoid) cell line; and differential display analysis to identify novel genes whose expression levels are altered as EML cells are induced to differentiate into the MPRO (myeloid progenitor) cell lineage . Using this analysis, we found a significant increase in the expression of one cDNA, known as p205 (also D3 or IFI-205), which encoded a previously described protein of unknown function .
p205 is a member of a family of interferon-inducible genes known as the IFI-200 family . 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 . Treatment of monocytes, HL-60 cells, U-937 cells, and THP-1 cells with interferons results in robust induction of MNDA mRNA . 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 . 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 .
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 . Similarly, p204 expression inhibits the growth of the fibroblast cell lines NIH3T3, B6MEF, and B/cMEF in vitro . Furthermore, expression of p202 reduces growth and causes reversion of the transformed phenotype of prostate cancer cells .
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 . 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.
MATERIALS AND METHODS
Sequence Analysis of p205 cDNA
Upon dideoxy sequencing of the amplified DNA fragments, it was determined that the p205 coding sequence found in MPRO and SCF/IL-3/atRA–treated EML cells differed from published sequences (GenBank accession number M74123 ) by one nucleotide . Specifically, nucleotide 1206 of p205 cDNA is guanine in published sequences and cytosine in MPRO and SCF/IL-3/atRA–treated EML RNA transcripts. Substitution of a cytosine for a guanine at position 1206 of p205 cDNA results in an amino acid substitution of serine for arginine at position 366 of the p205 polypeptide. The full-length p205 cDNA was obtained from Thomas Hamilton (Research Institute, Cleveland Clinic Foundation, Cleve-land) and contains the same coding sequence of p205 found in MPRO and SCF/IL-3/atRA–treated EML cells. It is possible that the discrepancies between the published p205 sequence and the sequence we used in this study are due to the fact that the published p205 sequence is derived from RNA transcripts from C57BL/6 mice, whereas EML and MPRO cells are derived from BDF1 mice .
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 ); 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).
Figure 1. Subcellular localization of p205 protein. (A): Nuclear (lanes 2, 4, 6, and 8) and cytosolic (lanes 1, 3, 5, and 7) fractions were prepared from EML cells (lanes 1 and 2), EML cells treated with stem cell factor (SCF), interleukin-3 (IL-3), and all-trans retinoic acid (atRA) for 72 hours (lanes 3 and 4); EPRO cells (lanes 5 and 6), and MPRO cells (lanes 7 and 8) as described in Materials and Methods. Following separation of the proteins by 4%–12% SDS-polyacrylamide gel electrophoresis and transfer to polyvinylidene diflouride membrane, the immunoblot was probed with antibodies raised against the C-terminal 15 amino acids of p205 and developed as described in Materials and Methods. (B): Murine BaF3 (panels A, B, C, and D) and human 293 cells (panels E, F, G, and H) were transfected with either pEGFP-C1 (panels A, B, E, and F) or pEGFP-p205 (panels C, D, G, and H) plasmids, and purified by fluorescence-activated cell sorter analysis until stable cell lines were developed. The cells were washed and fixed with 1% paraformaldehyde for 10 minutes, subjected to cytocentrifugation, and treated with DAPI solution (4', 6-diamino-2-phenylindole dihydrochloride) for 10 minutes, then visualized at 100x magnification. Nuclear staining of cells with DAPI is shown in panels A, C, E, and G, while EGFP is visualized in panels B, D, F, and H. Abbreviations: EGFP, enhanced green fluorescent protein; EML, erythroid, myeloid, lymphoid; EPRO, EML-derived promyelocytic cells; MPRO, myeloid progenitor.
The nuclear localization of p205 was confirmed by introducing expression plasmids encoding p205 fused to the C-terminus of EGFP into murine IL-3–dependent BaF3 cells and human 293 cells. Specifically, BaF3 and 293 cells were transfected with either pEGFP-C1 (control plasmid) or pEGFP-p205 plasmids and sorted by FACS for cells that express EGFP in order to generate stable cell lines. BaF3 and 293 cells stably transfected with pEGFP-C1 showed EGFP expression distributed throughout the cell (Fig. 1B, 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 . Other family members, p202 and p204, have been demonstrated to inhibit cell proliferation . 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.
Figure 2. Effect of p205 expression on interleukin-3–dependent progenitor cell proliferation. (A): BaF3 cells were transfected with either pTr (p205 negative lanes) or pTr-205 (p205 positive lanes) and sorted for GFP by FACS analysis 24 hours after transfection. Equal numbers (1 x 105) of GFP+- and GFP–-sorted BaF3 cells were lysed with 1x SDS-PAGE sample buffer, separated by 4%–12% gradient SDS-PAGE, and transferred to polyvinylidene diflouride membrane. The immunoblot was probed with antibodies raised against the 15 C-terminal amino acids of p205 and developed as described in Materials and Methods. The immunoblot was stripped and re-probed with anti-actin antibodies to verify equal loading and transfer of samples. These data are representative of two separate experiments. (B): Whole cell lysates were prepared from myeloid progenitor (lane 1), BMC control (lane 2), BMC cultured in 100 ng/ml M-CSF (lane 3), and 1 x 107 BaF3 cells transfected with 20 μg pCB6-205 (lane 4), and 25 μg protein was run on SDS-PAGE gel. Western blotting was performed using an anti-p205 antibody, and the same blot was then stripped and re-probed with an anti--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 -thymidine, and the amount of -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 -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.
To evaluate whether the expression of p205 could affect cell growth, we examined sorted BaF3 cell proliferation in -thymidine incorporation assays after 48 hours. As shown in Figure 2C, expression of p205 results in a greater than 50% decrease in -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 -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 -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 -thymidine incorporation assays or in single-cell cultures. Lin– cells expressing p205 showed a 60% reduction in -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 -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.
Table 1. p205 expression inhibits growth of normal bone marrow progenitors
Other IFI-200 Family Members Can Inhibit BaF3 Cell Proliferation
IFI-200 family members p202 and p204, which are closely related to p205, have been shown to negatively regulate the growth of nonhematopoietic cell lines . Therefore, we wished to confirm their effects on hematopoietic progenitor cell growth with p205. BaF3 cells were electroporated with either pTr (control), pTr-204, or pTr-202, then sorted for GFP expression, and cultured in microtiter plates to measure cell proliferation by -thymidine incorporation after 24, 48, and 72 hours. A Western blot verified that p204 and p202 were expressed in GFP+-sorted BaF3 cells (Figs. 3A and 3B, 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 -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.
Figure 3. Effect of p204 and p202 on IL-3–dependent Baf3 progenitor cell proliferation. (A, B): BaF3 cells were transfected with either pTr (p204 and p202 negative lanes), pTr-204 (p204 positive lanes), or pTr-202 (p202 positive lanes) and sorted for GFP by FACS. Equal numbers of GFP+ and GFP– BaF3 cells were lysed with 1x SDS-PAGE sample buffer, separated by 4%–12% gradient SDS-PAGE, and transferred to polyvinylidene diflouride membrane. The immunoblot was probed with (A) anti-p204 and (B) anti-p202 antibodies and developed as described in Materials and Methods. The immunoblots were stripped and re-probed with anti-actin antibodies to verify equal loading and transfer of samples. These data are representative of two separate experiments. (C): BaF3 cells were transfected with either pTr (vector; solid bars), pTr-204 (p204; gray bars), or pTr-202 (p202; striped bars), then sorted for green fluorescence by FACS as described above. GFP+-sorted 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 then were pulse-labeled with -thymidine prior to harvest. These data are representative of three separate experiments. Abbreviations: FACS, fluorescence-activated cell sorter analysis; GFP, green fluorescent protein; SDS-PAGE, SDS-polyacrylamide gel electrophoresis.
Microinjection of p205-Expressing Plasmids into NIH3T3 Cells Inhibits DNA Synthesis
While high levels of p204 mRNA and protein are induced by interferon treatment of NIH3T3 fibroblasts, low levels of p205 mRNA expression have been detected in similarly treated cells . Therefore, we evaluated whether p205 exerts an antiproliferative effect in nonhematopoietic cells, including NIH3T3 fibroblasts. To test this, NIH3T3 cells were microinjected with expression vectors containing p205, and effects on DNA synthesis were measured. pcDNA (control), pcDNA-p205 antisense, and pcDNA-p205 sense vectors were each microinjected into NIH3T3 fibroblasts, and DNA synthesis was determined after pulsing cells with -thymidine and counting labeled nuclei by autoradiography. NIH3T3 fibroblasts microinjected with pcDNA-p205 (Fig. 4, panel C) have significantly fewer nuclei that incorporated -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 ). 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.
Figure 4. Microinjection of p205-expressing plasmids into NIH3T3 cells inhibits serum-induced DNA synthesis. The plasmids (A) pcDNA, (B) pcDNA-p205 anti-sense, and (C) pcDNA-p205 were each microinjected into NIH3T3 fibroblasts as described in Materials and Methods. The cells were fixed with 3.7% glutaraldehyde (v/v phosphate-buffered saline, pH 7.4) and autoradiography was performed for 2 days in nuclear tracking emulsion. The cells were stained with Giemsa and photographed at 100x magnification.
Table 2. Microinjection of p205 cDNA into quiescent NIH3T3 cells inhibits serum-induced entry of the cells into S phase
Table 3. p205 expression inhibits colony formation of NIH3T3 cells
p205 Expression Does Not Trigger Apoptosis
To evaluate whether p205 inhibits cell growth through apoptosis, 32D-C123 cells were transfected with pCMS or pCMS-205 and stained with Annexin-V-PE and 7-AAD and analyzed by flow cytometry. We found that there was no difference in the percentage of early apoptotic cells (Annexin+ and 7-AAD–) in the GFP+ gated populations from 32D-C123 cells that were transfected with vector control and vector containing p205 (2% versus 3%, respectively; Fig. 5, 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.
Figure 5. p205 has negligible effect on apoptosis. 32D-C123 cells were transfected with (A) control (pCMS) or (B) p205 (pCMS-205), expressing plasmids, and after 48 hours were stained with Annexin V-PE and 7-AAD to detect early apoptotic cells (upper right quadrants of dot plots A and B). (C):As a positive control, 32D-C123 cells were also examined for early apoptosis 24 hours after withdrawal of growth factor (Interleukin-3).
Deletional Analysis of p205
To evaluate which domain(s) of p205 were required for growth inhibition; seven p205 substitution or deletion mutants were constructed and assayed for growth inhibition properties (Fig. 6A):
Figure 6. Summary of truncation/deletion mutants. (A): The following mutants were constructed in enhanced green fluorescent protein–expressing pTr vector and transfected into Baf3 cells: (1) p205: full-length translated p205 protein, (2) p205a: p205 with C-terminal truncation, (3) p205DAPIN: p205 bearing truncation of an N-terminal domain including the DAPIN/PYRIN motif, (4) p205Rb: p205 with C-terminal Rb-binding site deleted, (5) p205TSTAQA: p205 with the 5 seven-residue TSTAQAR repeats deleted, (6) pS261A: serine at position 261 substituted for a neutral alanine residue, (7) pS261D: Putative ataxia telangiectasia mutated (ATM) kinase phosphorylation site; serine at position 261 replaced with charged aspartate residue. The protein motifs represented in the constructs above include the protein-binding DAPIN/PYRIN and MFHATVAT motifs, as well as the Rb-binding LXCXE domain. Also represented are the 5-copy TSTAQAR repeats and the nuclear localization signal. (B): GFP+-BaF3 cells were sorted 24 hours post-transfec-tion, and expression of each mutant protein was confirmed by Western blot analysis using an N–terminal-specific p205 antibody. The same blot was stripped and re-probed with -actin antibody as a loading control. Abbreviation: GFP, green fluorescent protein.
p205 constituted the full-length p205 WT control. The p205a 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.
The p205DAPIN 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).
The p205Rb mutant lacks the putative C-terminal Rb-binding site.
The p205TSTAQA 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 (Fig. 6A). Therefore, the serine was substituted with a charged aspartic acid residue to mimic phosphorylation at this site (pS261D).
As a control, we generated a pS261A mutant, in which the Ser261 residue was substituted for a neutral alanine residue.
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 . 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.
Figure 7. Growth inhibition by the p205 mutants. Plasmid constructs described in Figure 6 A were transfected into Baf3 cells and GFP+ cells sorted 24 hours after transfection. Cell proliferation was assayed by -thymidine uptake according to the procedures described in the Materials and Methods.
DISCUSSION
The authors are grateful to Dr. Peter Lengyel for the gift of anti-p204 and anti-p202 antibodies, Dr. Santos Landolfo for the N-terminal–specific anti-p205 antibody, Dr. Thomas Hamilton for providing the pBluescript-p205 plasmid, Drs. Karen Vousden and Margaret Ashcroft for reagents and technical assistance for the calcium phosphate experiments, and Gordon Wiegand and Louise Finch for performing FACS analysis. We thank Dr. Kim Klarmann for her insightful comments and assistance in the preparation of the manuscript. We also thank Drs. Sally Spence and Joost Oppenheim for their helpful comments and critical review of this manuscript.
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.
REFERENCES
Kehrl JH. Hematopoietic lineage commitment: role of transcription factors. STEM CELLS 1995;13:223–241.
Shivdasani RA, Orkin SH. Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2. Proc Natl Acad Sci U S A 1995;92:8690–8694.
Tenen DG, Hromas R, Licht JD et al. Transcription factors, normal myeloid development, and leukemia. Blood 1997;90:489–519.
Tsai S, Bartelmez S, Sitnicka E et al. Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development. Genes Dev 1994;8:2831–2841.
Weiler SR, Gooya JM, Ortiz M et al. D3: a gene induced during myeloid cell differentiation of Lin(lo) c-Kit(+) Sca-1(+) progenitor cells. Blood 1999;93:527–536.
Dawson MJ, Trapani JA. HIN-200: a novel family of IFN-inducible nuclear proteins expressed in leukocytes. J Leukoc Biol 1996;60:310–316.
Landolfo S, Gariglio M, Gribaudo G et al. The Ifi 200 genes: an emerging family of IFN-inducible genes. Biochimie 1998;80:721–728.
Tannenbaum CS, Major J, Ohmori Y et al. A lipopolysaccharide-inducible macrophage gene (D3) is a new member of an interferon-inducible gene cluster and is selectively expressed in mononuclear phagocytes. J Leukoc Biol 1993;53:563–568.
Briggs JA, Burrus GR, Stickney BD et al. Cloning and expression of the human myeloid cell nuclear differentiation antigen: regulation by interferon alpha. J Cell Biochem 1992;49:82–92.
Briggs R, Dworkin L, Briggs J et al. Interferon alpha selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage. J Cell Biochem 1994;54:198–206.
Briggs RC, Briggs JA, Ozer J et al. The human myeloid cell nuclear differentiation antigen gene is one of at least two related interferon-inducible genes located on chromosome 1q that are expressed specifically in hematopoietic cells. Blood 1994;83:2153–2162.
Briggs RC, Kao WY, Dworkin LL et al. Regulation and specificity of MNDA expression in monocytes, macrophages, and leukemia/B lymphoma cell lines. J Cell Biochem 1994;56:559–567.
Gariglio M, De Andrea M, Lembo M et al. The murine homolog of the HIN 200 family, Ifi 204, is constitutively expressed in myeloid cells and selectively induced in the monocyte/macrophage lineage. J Leukoc Biol 1998;64:608–614.
Lembo D, Angeretti A, Benefazio S et al. Constitutive expression of the interferon-inducible protein p202 in NIH 3T3 cells affects cell cycle progression. J Biol Regul Homeost Agents 1995;9:42–46.
Choubey D, Li SJ, Datta B et al. Inhibition of E2F-mediated transcription by p202. EMBO J 1996;15:5668–5678.
Gribaudo G, Riera L, De Andrea M et al. The antiproliferative activity of the murine interferon-inducible Ifi 200 proteins depends on the presence of two 200 amino acid domains. FEBS Lett 1999;456:31–36.
Lembo M, Sacchi C, Zappador C et al. Inhibition of cell proliferation by the interferon-inducible 204 gene, a member of the Ifi 200 cluster. Oncogene 1998;16:1543–1551.
Yan DH, Wen Y, Spohn B et al. Reduced growth rate and transformation phenotype of the prostate cancer cells by an interferon-inducible protein, p202. Oncogene 1999;18:807–811.
Phillips AC, Bates S, Ryan KM et al. Induction of DNA synthesis and apoptosis are separable functions of E2F-1. Genes Dev 1997;11:1853–1863.
Lyakh L, Ghosh P, Rice NR. Expression of NFAT-family proteins in normal human T cells. Mol Cell Biol 1997;17:2475–2484.
Choubey D, Lengyel P. Interferon action: cytoplasmic and nuclear localization of the interferon-inducible 52-kD protein that is encoded by the Ifi 200 gene from the gene 200 cluster. J Interferon Res 1993;13:43–52.
Choubey D, Lengyel P. Interferon action: nucleolar and nucleoplasmic localization of the interferon-inducible 72-kD protein that is encoded by the Ifi 204 gene from the gene 200 cluster. J Cell Biol 1992;116:1333–1341.
Smith MR, Duhe RJ, Liu Y et al. Microinjected cDNA encoding JAK2 protein-tyrosine kinase induces DNA synthesis in NIH 3T3 cells. FEBS Lett 1997;408:327–330.
Choubey D. P202: an interferon-inducible negative regulator of cell growth. J Biol Regul Homeost Agents 2000;14:187–192.
Hertel L, Rolle S, De Andrea M et al. The retinoblastoma protein is an essential mediator that links the interferon-inducible 204 gene to cell-cycle regulation. Oncogene 2000;19:3598–3608.
Kim ST, Lim DS, Canman CE et al. Substrate specificities and identification of putative substrates of ATM kinase family members. J Biol Chem 1999;274:37538–37543.
O’Neill T, Dwyer AJ, Ziv Y et al. Utilization of oriented peptide libraries to identify substrate motifs selected by ATM. J Biol Chem 2000;275:22719–22727.
De Andrea M, Ravotto M, Noris E et al. The interferon-inducible gene, Ifi204, acquires malignant transformation capability upon mutation at the Rb-binding sites. FEBS Lett 2002;515:51–57.
Miranda RN, Briggs RC, Shults K et al. Immunocytochemical analysis of MNDA in tissue sections and sorted normal bone marrow cells documents expression only in maturing normal and neoplastic myelomonocytic cells and a subset of normal and neoplastic B lymphocytes. Hum Pathol 1999;30:1040–1049.
Trapani JA, Browne KA, Dawson MJ et al. A novel gene constitutively expressed in human lymphoid cells is inducible with interferon-gamma in myeloid cells. Immunogenetics 1992;36:369–376.
Dawson MJ, Elwood NJ, Johnstone RW et al. The IFN-inducible nucleoprotein IFI 16 is expressed in cells of the monocyte lineage, but is rapidly and markedly down-regulated in other myeloid precursor populations. J Leukoc Biol 1998;64:546–554.
Choubey D, Walter S, Geng Y et al. Cytoplasmic localization of the interferon-inducible protein that is encoded by the AIM2 (absent in melanoma) gene from the 200-gene family. FEBS Lett 2000;474:38–42.
Goldberger A, Hnilica LS, Casey SB et al. Properties of a nuclear protein marker of human myeloid cell differentiation. J Biol Chem 1986;261:4726–4731.
Dawson MJ, Trapani JA. The interferon-inducible autoantigen, IFI 16: localization to the nucleolus and identification of a DNA-binding domain. Biochem Biophys Res Commun 1995;214:152–162.
Dawson MJ, Trapani JA. IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines. J Cell Biochem 1995;57:39–51.
Gribaudo G, Ravaglia S, Guandalini L et al. Molecular cloning and expression of an interferon-inducible protein encoded by gene 203 from the gene 200 cluster. Eur J Biochem 1997;249:258–264.
Choubey D, Lengyel P. Binding of an interferon-inducible protein (p202) to the retinoblastoma protein. J Biol Chem 1995;270:6134–6140.
Datta B, Li B, Choubey D, et al. p202, an interferon-inducible modulator of transcription, inhibits transcriptional activation by the p53 tumor suppressor protein, and a segment from the p53-binding protein 1 that binds to p202 overcomes this inhibition. J Biol Chem 1996;271:27544–27555.
Min W, Ghosh S, Lengyel P. The interferon-inducible p202 protein as a modulator of transcription: inhibition of NF-kappa B, c-Fos, and c-Jun activities. Mol Cell Biol 1996;16:359–368.
Choubey D, Gutterman JU. Inhibition of E2F-4/DP-1-stimulated transcription by p202. Oncogene 1997;15:291–301.
Datta B, Min W, Burma S et al. Increase in p202 expression during skeletal muscle differentiation: inhibition of MyoD protein expression and activity by p202. Mol Cell Biol 1998;18:1074–1083.
Johnstone RW, Kerry JA, Trapani JA. The human interferon-inducible protein, IFI 16, is a repressor of transcription. J Biol Chem 1998;273:17172–17177.
Gutterman JU, Choubey D. Retardation of cell proliferation after expression of p202 accompanies an increase in p21(WAF1/CIP1). Cell Growth Differ 1999;10:93–100.
Xie J, Briggs JA, Olson MO et al. Human myeloid cell nuclear differentiation antigen binds specifically to nucleolin. J Cell Biochem 1995;59:529–536.
Xie J, Briggs JA, Morris SW et al. MNDA binds NPM/B23 and the NPM-MLF1 chimera generated by the t(3;5) associated with myelodysplastic syndrome and acute myeloid leukemia. Exp Hematol 1997;25:1111–1117.
Xie J, Briggs JA, Briggs RC. Human hematopoietic cell specific nuclear protein MNDA interacts with the multifunctional transcription factor YY1 and stimulates YY1 DNA binding. J Cell Biochem 1998;70:489–506.
Bertin J, DiStefano PS. The PYRIN domain: a novel motif found in apoptosis and inflammation proteins. Cell Death Differ 2000;7:1273–1274.
Gozani O, Boyce M, Yoo L et al. Life and death in paradise. Nat Cell Biol 2002;4:E159–162.
Staub E, Dahl E, Rosenthal A. The DAPIN family: a novel domain links apoptotic and interferon response proteins. Trends Biochem Sci 2001;26:83–85.
Koul D, Obeyesekere NU, Gutterman JU et al. p202 self-associates through a sequence conserved among the members of the 200-family proteins. FEBS Lett 1998;438:21–24.
Lin W-C, Lin F-T, Nevins JR. Selective induction of E2F1 in response to DNA damage, mediated by ATM-dependent phosphorylation. Genes Dev 2001;15:1833–1844.
Burrus GR, Briggs JA, Briggs RC. Characterization of the human myeloid cell nuclear differentiation antigen: relationship to interferon-inducible proteins. J Cell Biochem 1992;48:190–202.(Jonathan M. Dermotta, Joh)
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@ncifcrf.gov
ABSTRACT
Hematopoiesis is a complex process whereby pluripotential hematopoietic stem cells differentiate into functionally distinct cell lineages. While significant progress has been made toward understanding the cellular requirements of myeloid differentiation, the molecular mechanisms that regulate this process remain poorly defined . Since pluripotential hematopoietic stem cells are found in low frequency in bone marrow and purification does not ensure homogeneity, we used a stem/progenitor cell–line model system; the EML (erythroid, myeloid, lymphoid) cell line; and differential display analysis to identify novel genes whose expression levels are altered as EML cells are induced to differentiate into the MPRO (myeloid progenitor) cell lineage . Using this analysis, we found a significant increase in the expression of one cDNA, known as p205 (also D3 or IFI-205), which encoded a previously described protein of unknown function .
p205 is a member of a family of interferon-inducible genes known as the IFI-200 family . 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 . Treatment of monocytes, HL-60 cells, U-937 cells, and THP-1 cells with interferons results in robust induction of MNDA mRNA . 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 . 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 .
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 . Similarly, p204 expression inhibits the growth of the fibroblast cell lines NIH3T3, B6MEF, and B/cMEF in vitro . Furthermore, expression of p202 reduces growth and causes reversion of the transformed phenotype of prostate cancer cells .
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 . 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.
MATERIALS AND METHODS
Sequence Analysis of p205 cDNA
Upon dideoxy sequencing of the amplified DNA fragments, it was determined that the p205 coding sequence found in MPRO and SCF/IL-3/atRA–treated EML cells differed from published sequences (GenBank accession number M74123 ) by one nucleotide . Specifically, nucleotide 1206 of p205 cDNA is guanine in published sequences and cytosine in MPRO and SCF/IL-3/atRA–treated EML RNA transcripts. Substitution of a cytosine for a guanine at position 1206 of p205 cDNA results in an amino acid substitution of serine for arginine at position 366 of the p205 polypeptide. The full-length p205 cDNA was obtained from Thomas Hamilton (Research Institute, Cleveland Clinic Foundation, Cleve-land) and contains the same coding sequence of p205 found in MPRO and SCF/IL-3/atRA–treated EML cells. It is possible that the discrepancies between the published p205 sequence and the sequence we used in this study are due to the fact that the published p205 sequence is derived from RNA transcripts from C57BL/6 mice, whereas EML and MPRO cells are derived from BDF1 mice .
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 ); 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).
Figure 1. Subcellular localization of p205 protein. (A): Nuclear (lanes 2, 4, 6, and 8) and cytosolic (lanes 1, 3, 5, and 7) fractions were prepared from EML cells (lanes 1 and 2), EML cells treated with stem cell factor (SCF), interleukin-3 (IL-3), and all-trans retinoic acid (atRA) for 72 hours (lanes 3 and 4); EPRO cells (lanes 5 and 6), and MPRO cells (lanes 7 and 8) as described in Materials and Methods. Following separation of the proteins by 4%–12% SDS-polyacrylamide gel electrophoresis and transfer to polyvinylidene diflouride membrane, the immunoblot was probed with antibodies raised against the C-terminal 15 amino acids of p205 and developed as described in Materials and Methods. (B): Murine BaF3 (panels A, B, C, and D) and human 293 cells (panels E, F, G, and H) were transfected with either pEGFP-C1 (panels A, B, E, and F) or pEGFP-p205 (panels C, D, G, and H) plasmids, and purified by fluorescence-activated cell sorter analysis until stable cell lines were developed. The cells were washed and fixed with 1% paraformaldehyde for 10 minutes, subjected to cytocentrifugation, and treated with DAPI solution (4', 6-diamino-2-phenylindole dihydrochloride) for 10 minutes, then visualized at 100x magnification. Nuclear staining of cells with DAPI is shown in panels A, C, E, and G, while EGFP is visualized in panels B, D, F, and H. Abbreviations: EGFP, enhanced green fluorescent protein; EML, erythroid, myeloid, lymphoid; EPRO, EML-derived promyelocytic cells; MPRO, myeloid progenitor.
The nuclear localization of p205 was confirmed by introducing expression plasmids encoding p205 fused to the C-terminus of EGFP into murine IL-3–dependent BaF3 cells and human 293 cells. Specifically, BaF3 and 293 cells were transfected with either pEGFP-C1 (control plasmid) or pEGFP-p205 plasmids and sorted by FACS for cells that express EGFP in order to generate stable cell lines. BaF3 and 293 cells stably transfected with pEGFP-C1 showed EGFP expression distributed throughout the cell (Fig. 1B, 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 . Other family members, p202 and p204, have been demonstrated to inhibit cell proliferation . 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.
Figure 2. Effect of p205 expression on interleukin-3–dependent progenitor cell proliferation. (A): BaF3 cells were transfected with either pTr (p205 negative lanes) or pTr-205 (p205 positive lanes) and sorted for GFP by FACS analysis 24 hours after transfection. Equal numbers (1 x 105) of GFP+- and GFP–-sorted BaF3 cells were lysed with 1x SDS-PAGE sample buffer, separated by 4%–12% gradient SDS-PAGE, and transferred to polyvinylidene diflouride membrane. The immunoblot was probed with antibodies raised against the 15 C-terminal amino acids of p205 and developed as described in Materials and Methods. The immunoblot was stripped and re-probed with anti-actin antibodies to verify equal loading and transfer of samples. These data are representative of two separate experiments. (B): Whole cell lysates were prepared from myeloid progenitor (lane 1), BMC control (lane 2), BMC cultured in 100 ng/ml M-CSF (lane 3), and 1 x 107 BaF3 cells transfected with 20 μg pCB6-205 (lane 4), and 25 μg protein was run on SDS-PAGE gel. Western blotting was performed using an anti-p205 antibody, and the same blot was then stripped and re-probed with an anti--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 -thymidine, and the amount of -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 -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.
To evaluate whether the expression of p205 could affect cell growth, we examined sorted BaF3 cell proliferation in -thymidine incorporation assays after 48 hours. As shown in Figure 2C, expression of p205 results in a greater than 50% decrease in -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 -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 -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 -thymidine incorporation assays or in single-cell cultures. Lin– cells expressing p205 showed a 60% reduction in -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 -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.
Table 1. p205 expression inhibits growth of normal bone marrow progenitors
Other IFI-200 Family Members Can Inhibit BaF3 Cell Proliferation
IFI-200 family members p202 and p204, which are closely related to p205, have been shown to negatively regulate the growth of nonhematopoietic cell lines . Therefore, we wished to confirm their effects on hematopoietic progenitor cell growth with p205. BaF3 cells were electroporated with either pTr (control), pTr-204, or pTr-202, then sorted for GFP expression, and cultured in microtiter plates to measure cell proliferation by -thymidine incorporation after 24, 48, and 72 hours. A Western blot verified that p204 and p202 were expressed in GFP+-sorted BaF3 cells (Figs. 3A and 3B, 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 -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.
Figure 3. Effect of p204 and p202 on IL-3–dependent Baf3 progenitor cell proliferation. (A, B): BaF3 cells were transfected with either pTr (p204 and p202 negative lanes), pTr-204 (p204 positive lanes), or pTr-202 (p202 positive lanes) and sorted for GFP by FACS. Equal numbers of GFP+ and GFP– BaF3 cells were lysed with 1x SDS-PAGE sample buffer, separated by 4%–12% gradient SDS-PAGE, and transferred to polyvinylidene diflouride membrane. The immunoblot was probed with (A) anti-p204 and (B) anti-p202 antibodies and developed as described in Materials and Methods. The immunoblots were stripped and re-probed with anti-actin antibodies to verify equal loading and transfer of samples. These data are representative of two separate experiments. (C): BaF3 cells were transfected with either pTr (vector; solid bars), pTr-204 (p204; gray bars), or pTr-202 (p202; striped bars), then sorted for green fluorescence by FACS as described above. GFP+-sorted 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 then were pulse-labeled with -thymidine prior to harvest. These data are representative of three separate experiments. Abbreviations: FACS, fluorescence-activated cell sorter analysis; GFP, green fluorescent protein; SDS-PAGE, SDS-polyacrylamide gel electrophoresis.
Microinjection of p205-Expressing Plasmids into NIH3T3 Cells Inhibits DNA Synthesis
While high levels of p204 mRNA and protein are induced by interferon treatment of NIH3T3 fibroblasts, low levels of p205 mRNA expression have been detected in similarly treated cells . Therefore, we evaluated whether p205 exerts an antiproliferative effect in nonhematopoietic cells, including NIH3T3 fibroblasts. To test this, NIH3T3 cells were microinjected with expression vectors containing p205, and effects on DNA synthesis were measured. pcDNA (control), pcDNA-p205 antisense, and pcDNA-p205 sense vectors were each microinjected into NIH3T3 fibroblasts, and DNA synthesis was determined after pulsing cells with -thymidine and counting labeled nuclei by autoradiography. NIH3T3 fibroblasts microinjected with pcDNA-p205 (Fig. 4, panel C) have significantly fewer nuclei that incorporated -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 ). 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.
Figure 4. Microinjection of p205-expressing plasmids into NIH3T3 cells inhibits serum-induced DNA synthesis. The plasmids (A) pcDNA, (B) pcDNA-p205 anti-sense, and (C) pcDNA-p205 were each microinjected into NIH3T3 fibroblasts as described in Materials and Methods. The cells were fixed with 3.7% glutaraldehyde (v/v phosphate-buffered saline, pH 7.4) and autoradiography was performed for 2 days in nuclear tracking emulsion. The cells were stained with Giemsa and photographed at 100x magnification.
Table 2. Microinjection of p205 cDNA into quiescent NIH3T3 cells inhibits serum-induced entry of the cells into S phase
Table 3. p205 expression inhibits colony formation of NIH3T3 cells
p205 Expression Does Not Trigger Apoptosis
To evaluate whether p205 inhibits cell growth through apoptosis, 32D-C123 cells were transfected with pCMS or pCMS-205 and stained with Annexin-V-PE and 7-AAD and analyzed by flow cytometry. We found that there was no difference in the percentage of early apoptotic cells (Annexin+ and 7-AAD–) in the GFP+ gated populations from 32D-C123 cells that were transfected with vector control and vector containing p205 (2% versus 3%, respectively; Fig. 5, 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.
Figure 5. p205 has negligible effect on apoptosis. 32D-C123 cells were transfected with (A) control (pCMS) or (B) p205 (pCMS-205), expressing plasmids, and after 48 hours were stained with Annexin V-PE and 7-AAD to detect early apoptotic cells (upper right quadrants of dot plots A and B). (C):As a positive control, 32D-C123 cells were also examined for early apoptosis 24 hours after withdrawal of growth factor (Interleukin-3).
Deletional Analysis of p205
To evaluate which domain(s) of p205 were required for growth inhibition; seven p205 substitution or deletion mutants were constructed and assayed for growth inhibition properties (Fig. 6A):
Figure 6. Summary of truncation/deletion mutants. (A): The following mutants were constructed in enhanced green fluorescent protein–expressing pTr vector and transfected into Baf3 cells: (1) p205: full-length translated p205 protein, (2) p205a: p205 with C-terminal truncation, (3) p205DAPIN: p205 bearing truncation of an N-terminal domain including the DAPIN/PYRIN motif, (4) p205Rb: p205 with C-terminal Rb-binding site deleted, (5) p205TSTAQA: p205 with the 5 seven-residue TSTAQAR repeats deleted, (6) pS261A: serine at position 261 substituted for a neutral alanine residue, (7) pS261D: Putative ataxia telangiectasia mutated (ATM) kinase phosphorylation site; serine at position 261 replaced with charged aspartate residue. The protein motifs represented in the constructs above include the protein-binding DAPIN/PYRIN and MFHATVAT motifs, as well as the Rb-binding LXCXE domain. Also represented are the 5-copy TSTAQAR repeats and the nuclear localization signal. (B): GFP+-BaF3 cells were sorted 24 hours post-transfec-tion, and expression of each mutant protein was confirmed by Western blot analysis using an N–terminal-specific p205 antibody. The same blot was stripped and re-probed with -actin antibody as a loading control. Abbreviation: GFP, green fluorescent protein.
p205 constituted the full-length p205 WT control. The p205a 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.
The p205DAPIN 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).
The p205Rb mutant lacks the putative C-terminal Rb-binding site.
The p205TSTAQA 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 (Fig. 6A). Therefore, the serine was substituted with a charged aspartic acid residue to mimic phosphorylation at this site (pS261D).
As a control, we generated a pS261A mutant, in which the Ser261 residue was substituted for a neutral alanine residue.
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 . 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.
Figure 7. Growth inhibition by the p205 mutants. Plasmid constructs described in Figure 6 A were transfected into Baf3 cells and GFP+ cells sorted 24 hours after transfection. Cell proliferation was assayed by -thymidine uptake according to the procedures described in the Materials and Methods.
DISCUSSION
The authors are grateful to Dr. Peter Lengyel for the gift of anti-p204 and anti-p202 antibodies, Dr. Santos Landolfo for the N-terminal–specific anti-p205 antibody, Dr. Thomas Hamilton for providing the pBluescript-p205 plasmid, Drs. Karen Vousden and Margaret Ashcroft for reagents and technical assistance for the calcium phosphate experiments, and Gordon Wiegand and Louise Finch for performing FACS analysis. We thank Dr. Kim Klarmann for her insightful comments and assistance in the preparation of the manuscript. We also thank Drs. Sally Spence and Joost Oppenheim for their helpful comments and critical review of this manuscript.
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.
REFERENCES
Kehrl JH. Hematopoietic lineage commitment: role of transcription factors. STEM CELLS 1995;13:223–241.
Shivdasani RA, Orkin SH. Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2. Proc Natl Acad Sci U S A 1995;92:8690–8694.
Tenen DG, Hromas R, Licht JD et al. Transcription factors, normal myeloid development, and leukemia. Blood 1997;90:489–519.
Tsai S, Bartelmez S, Sitnicka E et al. Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development. Genes Dev 1994;8:2831–2841.
Weiler SR, Gooya JM, Ortiz M et al. D3: a gene induced during myeloid cell differentiation of Lin(lo) c-Kit(+) Sca-1(+) progenitor cells. Blood 1999;93:527–536.
Dawson MJ, Trapani JA. HIN-200: a novel family of IFN-inducible nuclear proteins expressed in leukocytes. J Leukoc Biol 1996;60:310–316.
Landolfo S, Gariglio M, Gribaudo G et al. The Ifi 200 genes: an emerging family of IFN-inducible genes. Biochimie 1998;80:721–728.
Tannenbaum CS, Major J, Ohmori Y et al. A lipopolysaccharide-inducible macrophage gene (D3) is a new member of an interferon-inducible gene cluster and is selectively expressed in mononuclear phagocytes. J Leukoc Biol 1993;53:563–568.
Briggs JA, Burrus GR, Stickney BD et al. Cloning and expression of the human myeloid cell nuclear differentiation antigen: regulation by interferon alpha. J Cell Biochem 1992;49:82–92.
Briggs R, Dworkin L, Briggs J et al. Interferon alpha selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage. J Cell Biochem 1994;54:198–206.
Briggs RC, Briggs JA, Ozer J et al. The human myeloid cell nuclear differentiation antigen gene is one of at least two related interferon-inducible genes located on chromosome 1q that are expressed specifically in hematopoietic cells. Blood 1994;83:2153–2162.
Briggs RC, Kao WY, Dworkin LL et al. Regulation and specificity of MNDA expression in monocytes, macrophages, and leukemia/B lymphoma cell lines. J Cell Biochem 1994;56:559–567.
Gariglio M, De Andrea M, Lembo M et al. The murine homolog of the HIN 200 family, Ifi 204, is constitutively expressed in myeloid cells and selectively induced in the monocyte/macrophage lineage. J Leukoc Biol 1998;64:608–614.
Lembo D, Angeretti A, Benefazio S et al. Constitutive expression of the interferon-inducible protein p202 in NIH 3T3 cells affects cell cycle progression. J Biol Regul Homeost Agents 1995;9:42–46.
Choubey D, Li SJ, Datta B et al. Inhibition of E2F-mediated transcription by p202. EMBO J 1996;15:5668–5678.
Gribaudo G, Riera L, De Andrea M et al. The antiproliferative activity of the murine interferon-inducible Ifi 200 proteins depends on the presence of two 200 amino acid domains. FEBS Lett 1999;456:31–36.
Lembo M, Sacchi C, Zappador C et al. Inhibition of cell proliferation by the interferon-inducible 204 gene, a member of the Ifi 200 cluster. Oncogene 1998;16:1543–1551.
Yan DH, Wen Y, Spohn B et al. Reduced growth rate and transformation phenotype of the prostate cancer cells by an interferon-inducible protein, p202. Oncogene 1999;18:807–811.
Phillips AC, Bates S, Ryan KM et al. Induction of DNA synthesis and apoptosis are separable functions of E2F-1. Genes Dev 1997;11:1853–1863.
Lyakh L, Ghosh P, Rice NR. Expression of NFAT-family proteins in normal human T cells. Mol Cell Biol 1997;17:2475–2484.
Choubey D, Lengyel P. Interferon action: cytoplasmic and nuclear localization of the interferon-inducible 52-kD protein that is encoded by the Ifi 200 gene from the gene 200 cluster. J Interferon Res 1993;13:43–52.
Choubey D, Lengyel P. Interferon action: nucleolar and nucleoplasmic localization of the interferon-inducible 72-kD protein that is encoded by the Ifi 204 gene from the gene 200 cluster. J Cell Biol 1992;116:1333–1341.
Smith MR, Duhe RJ, Liu Y et al. Microinjected cDNA encoding JAK2 protein-tyrosine kinase induces DNA synthesis in NIH 3T3 cells. FEBS Lett 1997;408:327–330.
Choubey D. P202: an interferon-inducible negative regulator of cell growth. J Biol Regul Homeost Agents 2000;14:187–192.
Hertel L, Rolle S, De Andrea M et al. The retinoblastoma protein is an essential mediator that links the interferon-inducible 204 gene to cell-cycle regulation. Oncogene 2000;19:3598–3608.
Kim ST, Lim DS, Canman CE et al. Substrate specificities and identification of putative substrates of ATM kinase family members. J Biol Chem 1999;274:37538–37543.
O’Neill T, Dwyer AJ, Ziv Y et al. Utilization of oriented peptide libraries to identify substrate motifs selected by ATM. J Biol Chem 2000;275:22719–22727.
De Andrea M, Ravotto M, Noris E et al. The interferon-inducible gene, Ifi204, acquires malignant transformation capability upon mutation at the Rb-binding sites. FEBS Lett 2002;515:51–57.
Miranda RN, Briggs RC, Shults K et al. Immunocytochemical analysis of MNDA in tissue sections and sorted normal bone marrow cells documents expression only in maturing normal and neoplastic myelomonocytic cells and a subset of normal and neoplastic B lymphocytes. Hum Pathol 1999;30:1040–1049.
Trapani JA, Browne KA, Dawson MJ et al. A novel gene constitutively expressed in human lymphoid cells is inducible with interferon-gamma in myeloid cells. Immunogenetics 1992;36:369–376.
Dawson MJ, Elwood NJ, Johnstone RW et al. The IFN-inducible nucleoprotein IFI 16 is expressed in cells of the monocyte lineage, but is rapidly and markedly down-regulated in other myeloid precursor populations. J Leukoc Biol 1998;64:546–554.
Choubey D, Walter S, Geng Y et al. Cytoplasmic localization of the interferon-inducible protein that is encoded by the AIM2 (absent in melanoma) gene from the 200-gene family. FEBS Lett 2000;474:38–42.
Goldberger A, Hnilica LS, Casey SB et al. Properties of a nuclear protein marker of human myeloid cell differentiation. J Biol Chem 1986;261:4726–4731.
Dawson MJ, Trapani JA. The interferon-inducible autoantigen, IFI 16: localization to the nucleolus and identification of a DNA-binding domain. Biochem Biophys Res Commun 1995;214:152–162.
Dawson MJ, Trapani JA. IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines. J Cell Biochem 1995;57:39–51.
Gribaudo G, Ravaglia S, Guandalini L et al. Molecular cloning and expression of an interferon-inducible protein encoded by gene 203 from the gene 200 cluster. Eur J Biochem 1997;249:258–264.
Choubey D, Lengyel P. Binding of an interferon-inducible protein (p202) to the retinoblastoma protein. J Biol Chem 1995;270:6134–6140.
Datta B, Li B, Choubey D, et al. p202, an interferon-inducible modulator of transcription, inhibits transcriptional activation by the p53 tumor suppressor protein, and a segment from the p53-binding protein 1 that binds to p202 overcomes this inhibition. J Biol Chem 1996;271:27544–27555.
Min W, Ghosh S, Lengyel P. The interferon-inducible p202 protein as a modulator of transcription: inhibition of NF-kappa B, c-Fos, and c-Jun activities. Mol Cell Biol 1996;16:359–368.
Choubey D, Gutterman JU. Inhibition of E2F-4/DP-1-stimulated transcription by p202. Oncogene 1997;15:291–301.
Datta B, Min W, Burma S et al. Increase in p202 expression during skeletal muscle differentiation: inhibition of MyoD protein expression and activity by p202. Mol Cell Biol 1998;18:1074–1083.
Johnstone RW, Kerry JA, Trapani JA. The human interferon-inducible protein, IFI 16, is a repressor of transcription. J Biol Chem 1998;273:17172–17177.
Gutterman JU, Choubey D. Retardation of cell proliferation after expression of p202 accompanies an increase in p21(WAF1/CIP1). Cell Growth Differ 1999;10:93–100.
Xie J, Briggs JA, Olson MO et al. Human myeloid cell nuclear differentiation antigen binds specifically to nucleolin. J Cell Biochem 1995;59:529–536.
Xie J, Briggs JA, Morris SW et al. MNDA binds NPM/B23 and the NPM-MLF1 chimera generated by the t(3;5) associated with myelodysplastic syndrome and acute myeloid leukemia. Exp Hematol 1997;25:1111–1117.
Xie J, Briggs JA, Briggs RC. Human hematopoietic cell specific nuclear protein MNDA interacts with the multifunctional transcription factor YY1 and stimulates YY1 DNA binding. J Cell Biochem 1998;70:489–506.
Bertin J, DiStefano PS. The PYRIN domain: a novel motif found in apoptosis and inflammation proteins. Cell Death Differ 2000;7:1273–1274.
Gozani O, Boyce M, Yoo L et al. Life and death in paradise. Nat Cell Biol 2002;4:E159–162.
Staub E, Dahl E, Rosenthal A. The DAPIN family: a novel domain links apoptotic and interferon response proteins. Trends Biochem Sci 2001;26:83–85.
Koul D, Obeyesekere NU, Gutterman JU et al. p202 self-associates through a sequence conserved among the members of the 200-family proteins. FEBS Lett 1998;438:21–24.
Lin W-C, Lin F-T, Nevins JR. Selective induction of E2F1 in response to DNA damage, mediated by ATM-dependent phosphorylation. Genes Dev 2001;15:1833–1844.
Burrus GR, Briggs JA, Briggs RC. Characterization of the human myeloid cell nuclear differentiation antigen: relationship to interferon-inducible proteins. J Cell Biochem 1992;48:190–202.(Jonathan M. Dermotta, Joh)