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Identification of PML Oncogenic Domains (PODs) in Human Megakaryocytes
Experimental Cell Research 271, 277–285 (2001) doi:10.1006/excr.2001.5377, available online at http://www.idealibrary.com on
Identification of PML Oncogenic Domains (PODs) in Human Megakaryocytes Arnaud Drouin, Alain Schmitt, Jean-Marc Masse´, Anne-Marie Cieutat, Serge Fichelson, and Elisabeth M. Cramer 1 INSERM U. 474, Institut Cochin de Ge´ne´tique Mole´culaire, Paris, France
Megakaryocytes (Mks) are unique cells in the human body in that they carry a single and polyploid nucleus. It is therefore of interest to understand their nuclear ultrastructure. PML oncogenic domains (PODs) were described in several types of eukaryotic cells using human autoantibodies which recognize nuclear antigens with a specific speckled pattern (dots) in indirect immunofluorescence (IF). Two main antigens, PML and Sp 100, usually colocalize and concentrate in these nuclear subdomains. We investigated the presence of PODs using IF and immunoelectron microscopy (IEM) in cells from megakaryocytic lineage: the HEL cell line and human cultured Mks. Antibodies against PML, Sp100, and anti-nuclear dots were used in single and double labeling. PODs were identified in HEL cells and in human Mks, and their ultrastructure was characterized. We then used IF to quantify PODs within Mks and showed that their number increased proportionally to nuclear lobularity. In summary, we report the identification of PODs in human Mks at an ultrastructural level and an increase in PODs number in parallel with Mk ploidy. We show that endomitosis not only leads to DNA increase but also to the multiplication of at least one of the associated nuclear structures. © 2001 Elsevier Science
Electron microscopic examination has previously identified distinct nuclear compartments in eukaryotic cells where specific proteins and nucleic acids may concentrate [3–5]. Some of these nuclear structures were initially described by electron microscopy [4] and further identified as autoantigenic targets in human autoimmune diseases and called “multiple nuclear dots” or “dots” [5–7]. The cell growth repressor PML (promyelocytic leukemia protein) and the transcription factor Sp100 colocalize within these nuclear dots, which have therefore also been named PML nuclear bodies, or PML oncogenic domains (PODs) [4, 8 –11]. Since Mk nucleus is a unique model for the study of polyploidy, and because the presence of PODs has not so far been demonstrated within Mks, we found it noteworthy to determine whether PODs were present within the nuclei of cells from the megakaryocytic lineage. We analyzed PML and Sp100 nuclear distribution by immunofluorescence (IF) and immunoelectron microscopy (IEM) in the HEL cell line (derived from a human erythroleukemia with Mk differentiative potential) [12] and human Mks cultured from CD34 ⫹ precursors. We also examined the evolution of the number of PODs during the endomitotic process and ploidy increase.
Key Words: nucleus; ultrastructure; megakaryocyte; endomitosis; PML oncogenic domains; human.
INTRODUCTION
Megakaryocytes (Mks), bone marrow precursors of platelets, are unique cells in humans since they become polyploid as nuclear DNA multiplies without cell division. Cell ploidy can then reach 32N to 64N without chromosome size increase or separate nuclear membrane formation. This physiological process, called endomitosis, has been recently approached by several investigators [1, 2] but remains poorly understood. 1
To whom reprint requests should be addressed at INSERUM U. 474, Maternite´ de Port-Royal, 5 e`me e´tage, Paris, France. Fax: 33 1 43 25 11 67. E-mail: [email protected].
MATERIALS AND METHODS HEL cell line and human MKs. The HEL cell line was grown and cultured as previously described [12]. Human cultured MKs were grown from circulating precursors. Blood CD34 ⫹ cells were isolated from leukapheresis samples obtained from patients undergoing autologous peripheral blood stem cell transplantation (for IEM study) and cord blood (for IF study). CD34 ⫹ cells were isolated using a magnetic cell-sorting system as previously described [13]. The cells were cultured for 13 days in a serum-free liquid medium containing thrombopoietin (TPO) (Rhu PEG-MGDF; Amgen, Thousand Oaks, CA) at a final concentration of 10 ng/mL. When CD34 ⫹ cells were isolated from cord blood, they were cultured in the presence of TPO in serum-free media, as previously described [14]. All human cells were collected after informed consent was obtained from the patients, in accordance with the institutional guidelines of the Committee on Human Investigation. Antibodies. Human autoantibodies from the serum of one patient with systemic lupus erythematosus (SLE) was kindly provided by Dr. C. Andre´ (Hoˆpital H. Mondor, Creteil, France). This anti-dots
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serum displays anti-PML and anti-Sp100 affinities in Western blot [7]. Monoclonal anti-PML antibody and polyclonal anti-Sp100 were kind gifts from Dr. H. de The´ (Centre Hayem, Hoˆpital Saint Louis, Paris, France) and have been described previously [6, 15]. Polyclonal antibodies to glycoprotein IIbIIIa (GPIIbIIIa) and to GPIb were used to identify immature Mks by IF and IEM, respectively, and were kindly given by Dr. Michael Berndt (Baker Medical Research Institute, Victoria, Australia) [16]. Polyclonal rabbit antibodies to von Willebrand factor (vWF) were purchased from Dakopatts, Denmark, and used at a concentration of 100 mg/mL. For IF, specific primary antibodies were revealed by goat antirabbit immunoglobulin (Ig) G coupled to rhodamin (GAR-TRITC) or by rabbit anti-human IgG coupled to fluorescein (RAH-FITC) (Beckton Dickinson, Mountain View, CA) at 1:40 and 1:400 dilutions, respectively. For mitotic spindle labeling, mouse monoclonal antibodies to human ␣- and -tubulin (Sigma, St. Louis, MO) were pooled and used at 1:100 dilution and revealed by goat anti-mouse IgG coupled to rhodamin (GAM-TRITC) at 1:40 dilution. For IEM, specific antibodies were revealed using IgG fractions anti-rabbit or anti-mouse coupled to 10- or 15-nm-diameter gold particles (British Biocell International, Cardiff, Wales, UK). Immunofluoresence. Nuclear protein locations were studied in the HEL cells and in human Mks in primary culture at days 6 and 13. Cultured cells were cytocentrifuged at 500 ⫻ g for 4 min onto cover slips, fixed in 4% formaldehyde in Hank balanced salt solution (HBSS; Gibco BRL, Gaithersburg, MD) for 20 min, permeabilized with 0.5% Triton X-100 in HBSS containing 0.1 mmol/L EGTA, and blocked with 0.5% bovine serum albumin in phosphate-buffered saline (PBS). Cells were incubated for 1 h in the primary antibody, washed, incubated 1 h with the appropriate secondary antibody, and washed again. Quantitative studies were performed by using triple staining; antidots serum from the patient with SLE was revealed by RAH-FITC in order to detect PODs; anti-vWF were revealed with GAR-TRITC in order to specifically identify Mks; anti–␣- and -tubulin were revealed by GAM-TRITC in order to visualize mitotic spindles; and Vectashield mounting medium with DAPI was used to visualize nuclear lobes and estimate ploidy. Cells were then observed with a Leica microscope. IEM. Cells were fixed in glutaraldehyde (1.5%) in phosphate buffer (0.1 M, pH 7.2) for 1 h at 22°C under a chemical hood, washed three times with phosphate buffer, embedded in 2.3 M sucrose in ˚ ) were cut PBS, and frozen in liquid N 2. Ultrathin cryosections (700 A at ⫺95°C (Reichert Ultracut S, Vienna, Austria) and were collected on nickel grids. Thin sections were then incubated with anti-PML, anti-Sp100, and anti-dots primary antibodies, followed by appropriate IgG fractions coupled to colloidal gold at a 1:30 dilution. Double immunolabeling was performed by using anti-GPIb in order to identify Mks as such and by using anti-dots to visualize PODs. Sections were counterstained with uranyl and lead. Observations were performed with a CM10 electron microscope (Philips, Eindoven, The Netherlands).
PODs per cell nucleus was observed (Fig. 1, inset). By using IEM, we found PML and Sp100 to be mostly located as a rim around nuclear structures, forming homogenous gray structures in ultrathin sections (Fig. 1). The mean diameter of these structures was 500 nm. Nuclear Body Identification in Human-Cultured Mks
By using IEM with anti-GPIb and anti-dots sera on Mks at days 8 and 13 of culture, double staining appeared in demarcation and plasma membranes, respectively, which indicate megakaryocytic lineage, and in intranuclear PODs (Figs. 2a, 2b). In single labeling with anti-Sp100 antibodies, specific staining could be found in the cell nucleus; consistent, albeit weaker, labeling could also be found with anti-PML antibodies. Both antigens appeared to be concentrated in small circular nuclear structures about 500 nm in diameter. They appeared as homogenous gray, electron-dense structures, occasionally surrounded by a rim of denser material (Fig. 3). Nuclear Body Quantification
RESULTS
IF studies with anti-dots autoantibodies allowed a semiquantitative evaluation of PODs. The reaction was performed in HEL cells, where a range of 7 to 12 PODs per nucleus was identified. When cord blood CD34 ⫹ progenitors are cultured with TPO, they give rise to fully mature Mks. In this system, most cells reach a maximum of 16N DNA content, and the ploidy remains inferior to the one of bone marrow Mks [14]. In order to estimate the number of PODs per Mk, and to attempt to relate this number to cell ploidy, we used triple staining. DAPI stain allowed distinction of different nuclear lobes; specific immunofluorescent staining for vWF using rhodamine allowed recognition of Mk; and anti-dots serum (revealed by fluorescein) specifically labeled PODs and allowed evaluation of their number. It appeared that the small, apparently diploid Mks contain an average of 5 PODs (Figs. 4a– 4c). A Mk with three visible lobes contains an average of 15 PODs (Figs. 4d– 4f, Figs. 5a, 5b). This number could reach an amount of 50 elements in large, mature polylobulated Mks (Figs. 5c, 5d) Thus, the PODs’ number was much higher in these large cells compared with the maximal amount previously described in diploid cells.
Nuclear Body Identification in HEL Cells
PODs Distribution during Endomitosis
Autoantibodies from the serum of the patient with SLE, when used in IF on eukaryocytic cells, give a typical speckled nuclear pattern, or dots [7]. Identification of PODs was then possible by this method on HEL cells. The staining pattern appeared mostly punctiform, associated with a low level of diffuse staining on the remaining nuclear surface. A mean of 9.5 (7 to 12)
Endomitotic events were observed. Mks could be recognized as such due to their unusually large size. In order to visualize more accurately the repartition of PODs in relationship to the mitotic spindles and the chromosomes, triple staining was performed (pooled anti–␣- and -tubulin antibodies were used in order to visualize the mitotic spindles, anti-dots were used for
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FIG. 1. EM view of HEL cells treated by autoantibodies from the serum of a patient with SLE and immunogold-specific labeling appears as a rim of particles (arrowheads) surrounding a dense core, allowing the ultrastructural identification of a POD. N, nucleus; Nu, nucleolus. Magnification, 61,000⫻. Inset: HEL cells stained with autoantibodies (anti-dots) from a patient with SLE by IF. The cell displays a typical pattern of multinuclear dots. A range of 7 to 12 PODs can be quantified in each cell nucleus.
PODs, and DAPI stain was used for chromatin) on cultured Mks. In most of them, anti-dots labeling was diffuse in the cytoplasm and PODs were undetectable; or fluorescent labeling appeared disorganized, with faint clusters of fluorescence redistributed away at the cell periphery, replacing the classical speckled interphasic pattern. The clusters displayed no interaction with the asters, spindle microtubules, or chromosomes (Fig. 6). In parallel, and as controls of the reaction, interphasic diploid cells and Mks displayed evident nuclear bodies (Fig. 6c). DISCUSSION
To our knowledge, this is the first report that demonstrates the existence of PODs in the nucleus of human Mks cells. We used the HEL cell line, which is able to undergo megakaryocytic differentiation and human cultured Mks [12]. A variety of autoantibodies from a patient with SLE and antibodies directed against PODs components were utilized, followed by IF and IEM analyses. Previous studies have reported PODs in
many eukaryotic cells, including hematopoietic cells from the granulocytic and erythroid lineages [6, 17, 18]. Occasional reports have mentioned the search for PODs in Mks, but the results concerning this cell type were of secondary importance and did not lead to a firm conclusion [6, 18]. Mks are rare cells in human bone marrow, which makes their study tedious, and the large size of their nucleus makes the identification of very small compartments by optical microscopy difficult. Therefore, in our study, we used a culture system where the Mk population is predominant, combined with the highly discriminative technique of IEM. Besides euchromatin, heterochromatin, and the nucleolus, other subnuclear compartments have been identified within the eukaryotic cell nucleus [8]. Among them are nuclear bodies, which have been recognized by electron microscopy and further characterized, thanks to their specific components (i.e., PML and Sp100 [10, 11]). Initial functional approaches to the study of PODs came from acute promyelocytic leukemia (APL), a malignant hemopathy specifically associated with a t(15;
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FIG. 2. IEM analysis of PODs in cultured Mks at day 13 (mature cells). Double immunogold labeling has been performed with anti-dots human antiserum (10 nm gold) to visualize PODs and anti-GPIb rabbit antibodies (15 nm gold) to identify Mk. (a) This large cell possesses a convoluted nucleus and characteristic organelles such as demarcation membranes and secretory granules. Magnification, 8300⫻. (b) A higher magnification view shows GPIb staining in demarcation membranes (dm) and a rim of labeling a small, dense, and round structure within the nucleus corresponding to a nuclear POD (arrowheads). Magnification, 35,000⫻.
17) translocation [19]. This translocation fuses a nuclear receptor for retinoic acid (RAR-␣) with the PML gene [20 –22]. In APL, promyelocyte differentiation is blocked and abnormal promyelocytes accumulate. The PODs of APL promyelocytes are disrupted, and PML antigen is detected in the entire cell without specific location. All transretinoic acid treatment of APL promyelocytes in culture and in patients with APL induces the reconstitution of PODs, overcomes the differentiation blockage, and leads to apoptotic death of leukemic promyelocytes [15, 23–27]. The PML protein is expressed in most tissues, including hematopoietic cells [6, 17]. PML protein is mainly nuclear, associated with the nuclear matrix [28], and localized in PODs. Transcription of the PML gene is increased by interferons [3, 29], which are growth- and tumor-suppressive cytokines. In those experiments, the number of PODs is also increased. The functions of PML protein are multiple and still under investigation. PML acts as a negative cell growth factor and as a factor which promotes cell differentiation [30]. PML can improve both erythroid and myeloid differentiation [17]. Moreover, knockout animal model
PML⫺/⫺; mice display leucopenia with a decreased capacity for retinoid acid (RA)– dependent terminal granulocytic maturation [31]. Thus, PML could mediate the RA growth-suppressive and cell-differentiationpromoting effects. In addition, PML clearly interferes with cell cycle progression [17]; PML overexpression regulates cdk2 and cyclin [30]; and PML protein interacts with the eukaryotic initiation factor 4 E (eIF-4 E), which regulates the cyclin D1 mRNA nucleocytoplasmic transport, an interaction which interestingly requires intact PODs [32]. PML can also participate in the upregulation of cell cycle inhibitor p21 [31]. Finally, PML is implicated in genomic stability, as reported by Zhong et al. [33]. In contrast to the well-described role of PML protein, PODs have still elusive functions. PODs are multiprotein complexes specifically assembled by PML protein. As PML, the PODs are associated with the nuclear matrix. Numerous proteins were reported to constitute PODs. These proteins display intranuclear and nucleocytoplasmic shuttles [4, 10, 11]. Proteins which do not constitute components of PODs can be specifically targeted and accumulated at the vicinity of PODs. Accu-
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FIG. 3. IEM analysis of PODs in cultured Mk at day 13 (mature cells) detected with anti-dots serum shows various aspects of PODs. (a, b) The structure occasionally appears surrounded by a denser ring of chromatin. Magnification, (a) 48,000⫻; (b) 67,000⫻. (c, d) In all cases, the structure is distinct from the surrounding chromatin by its homogenous, moderately dense aspect, and the labeling is distributed at the periphery of the organelle. Magnification, (c) 54,500⫻; (d) 44,000⫻.
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FIG. 4. Human Mk in culture are triple stained. Anti-dots antibodies coupled to FITC show PODs (a, d); DAPI staining reveals nuclear shape and allows estimation of the number of nuclear lobes, which increases with ploidy (b, e). The cytoplasmic staining for vWF certifies the Mk lineage of cell (c, f). (a– c) A small Mk with a unique nuclear lobe, which is likely diploid, displays an average of five PODs. (d–f) The large polylobulated Mk display a greater number of PODs (10 to 15) than the diploid cells.
FIG. 5. Double fluorescent staining of human Mks in culture. Anti-dots antibodies coupled to FITC (a, c, e) labels the PODs, while DAPI staining (b, f) reveals nuclear shape and allows estimation of the number of nuclear lobes, which increases with ploidy. (a, b) DAPI staining reveals three lobes in this Mk, and the nucleus contains three times more PODs than a diploid cell. (c, d) Two adjacent highly polylobulated Mks (containing an average of four nuclear lobes) are identified as such by the specific red fluorescence for vWF, maximal in the paranuclear regions. Immunostaining reveals an average number of 50 PODs per cell.
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FIG. 6. Human Mk undergoing endomitosis, triple stained with fluorescent dyes. DAPI staining (a) shows an endomitosis with two ditinct groups of chromosomes. Anti-tubulin immunolabeling (b) shows spindle microtubules (green arrows), which radiate from each aster to form a spherical conformation. Anti-dots serum (c) reveals that PODs are disorganized, forming large aggregates of fluorescence (red arrows). PODs are excluded from the mitotic spindles and chromosome areas and redistribute at the periphery of the cell. As a control, an interphasic diploid cell exhibits three PODs (red arrowheads).
mulation of nascent mRNAs was also reported in PODs, as a way of stockage that may regulate mRNA maturation, mRNA nucleocytoplasmic transport, and protein expression [34, 35]. In summary, PODs could constitute “nuclear dumps,” or nuclear storage for various proteins and mRNA. PODs could then regulate the traffic, activity, and expression of various proteins. PODs may participate to all the functions of PML: cell proliferation, cell cycle regulation, and genomic stability. In addition, since PODs are present in normal promyelocytes and disorganized in leukemic promyelocytes, it would be of interest to investigate PML distribution and PODs structure in malignant Mks from patients with megakaryoblastic leukemia and from myeloproliferative syndromes. Polyploidization results from endomitotic cycles that occur in Mk as cells stop proliferating and start differentiating. DNA accumulates in Mks without cell division, and the nuclei become polylobulated [2]. The partners of cell cycle machinery have unique expressions and probably regulations during the endomitosis of Mks [2]. The evolution of nuclear structures during endomitosis is poorly understood. Thus, we have examined the number of PODs during the polyploidization of Mks. First, we have quantified PODs in HEL cells, which are mostly diploid cells. Then we followed the evolution of the number of PODs during cytoplasmic and nuclear growth of Mk (increased nuclear lobularity and polyploidization). We found that the more numerous the nuclear lobes were, the more numerous the PODs were. As the number of nuclear lobes in Mks was
reported to be roughly proportional to Mk ploidy [36], we deduce that the number of PODs is also proportional to Mk ploidy. Increased numbers of PODs were previously reported in cancerous cells but not in cells from nontumoral hyperplastic tissues [37, 38]; this observation could be explained by the fact that malignant cells are often multiploid, whereas hyperplastic normal cells are not. The increased PODs number could then just reflect multiploidy. Growth factors, erythropoietin (EPO), and granulocyte, macrophage colony stimulatory factor (GMCSF) induce PML expression and accumulation of PODs, respectively, in erythroid and granulocytic lineages cultured from human progenitors cells [17]. In the Mk lineage, TPO is the main regulator of cell development; TPO acts at each stage of the development of Mks, proliferation, endomitosis, and maturation [39]. TPO stimulates polyploidization in cultured Mks [40, 41], and TPO ⫺/⫺ mice display Mks with smaller size and ploidy [2, 3, 39, 42, 43]. We suggest that accumulation of PODs could be induced by TPO, since TPO and interferons both have EPO-like domains, and it would be of interest to confirm this hypothesis. When the number of PODs increases in the polyploid Mk, PML, and PODs, associated proteins accumulate and then can assume an efficient cell growth control during polyploidization of Mks and differentiation control of Mks, as in other hematopoietic lineages [44]. Moreover, the level of the genomic protection role assumed by PML may thus be proportional to the DNA content of polyploid Mks. In the majority of Mks undergoing endomitosis, PODs were disorganized and became undetectable, and the fluorescent labeling appeared to be redistributed. They did not appear to interact either with the chromosomes or with the mitotic spindles. Thus, we found in Mks during endomitosis the same intracellular repartition and global disappearance of PODs, as described previously in nonMks lineages [3, 45]. Finally, a better understanding of PODs’ organization, composition, and subcellular interactions in Mks could improve our understanding of the endomitotic cell cycle. The authors are grateful to Pr. Hugues de The´ (CNRS UPR 43, Centre Hayem, Hoˆpital Saint Louis, Paris, France) for providing anti-PML and anti-Sp100 antibodies and to Dr. Chantal Andre´ (Hoˆpital Henri Mondor, Cre´teil, France) for advice and for providing autoimmune serum from the patient with SLE. We are also grateful to Najet Debili for providing the Mks used for electron microscopy.
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Identification of PML Oncogenic Domains (PODs) in Human Megakaryocytes Arnaud Drouin, Alain Schmitt, Jean-Marc Masse´, Anne-Marie Cieutat, Serge Fichelson, and Elisabeth M. Cramer 1 INSERM U. 474, Institut Cochin de Ge´ne´tique Mole´culaire, Paris, France
Megakaryocytes (Mks) are unique cells in the human body in that they carry a single and polyploid nucleus. It is therefore of interest to understand their nuclear ultrastructure. PML oncogenic domains (PODs) were described in several types of eukaryotic cells using human autoantibodies which recognize nuclear antigens with a specific speckled pattern (dots) in indirect immunofluorescence (IF). Two main antigens, PML and Sp 100, usually colocalize and concentrate in these nuclear subdomains. We investigated the presence of PODs using IF and immunoelectron microscopy (IEM) in cells from megakaryocytic lineage: the HEL cell line and human cultured Mks. Antibodies against PML, Sp100, and anti-nuclear dots were used in single and double labeling. PODs were identified in HEL cells and in human Mks, and their ultrastructure was characterized. We then used IF to quantify PODs within Mks and showed that their number increased proportionally to nuclear lobularity. In summary, we report the identification of PODs in human Mks at an ultrastructural level and an increase in PODs number in parallel with Mk ploidy. We show that endomitosis not only leads to DNA increase but also to the multiplication of at least one of the associated nuclear structures. © 2001 Elsevier Science
Electron microscopic examination has previously identified distinct nuclear compartments in eukaryotic cells where specific proteins and nucleic acids may concentrate [3–5]. Some of these nuclear structures were initially described by electron microscopy [4] and further identified as autoantigenic targets in human autoimmune diseases and called “multiple nuclear dots” or “dots” [5–7]. The cell growth repressor PML (promyelocytic leukemia protein) and the transcription factor Sp100 colocalize within these nuclear dots, which have therefore also been named PML nuclear bodies, or PML oncogenic domains (PODs) [4, 8 –11]. Since Mk nucleus is a unique model for the study of polyploidy, and because the presence of PODs has not so far been demonstrated within Mks, we found it noteworthy to determine whether PODs were present within the nuclei of cells from the megakaryocytic lineage. We analyzed PML and Sp100 nuclear distribution by immunofluorescence (IF) and immunoelectron microscopy (IEM) in the HEL cell line (derived from a human erythroleukemia with Mk differentiative potential) [12] and human Mks cultured from CD34 ⫹ precursors. We also examined the evolution of the number of PODs during the endomitotic process and ploidy increase.
Key Words: nucleus; ultrastructure; megakaryocyte; endomitosis; PML oncogenic domains; human.
INTRODUCTION
Megakaryocytes (Mks), bone marrow precursors of platelets, are unique cells in humans since they become polyploid as nuclear DNA multiplies without cell division. Cell ploidy can then reach 32N to 64N without chromosome size increase or separate nuclear membrane formation. This physiological process, called endomitosis, has been recently approached by several investigators [1, 2] but remains poorly understood. 1
To whom reprint requests should be addressed at INSERUM U. 474, Maternite´ de Port-Royal, 5 e`me e´tage, Paris, France. Fax: 33 1 43 25 11 67. E-mail: [email protected].
MATERIALS AND METHODS HEL cell line and human MKs. The HEL cell line was grown and cultured as previously described [12]. Human cultured MKs were grown from circulating precursors. Blood CD34 ⫹ cells were isolated from leukapheresis samples obtained from patients undergoing autologous peripheral blood stem cell transplantation (for IEM study) and cord blood (for IF study). CD34 ⫹ cells were isolated using a magnetic cell-sorting system as previously described [13]. The cells were cultured for 13 days in a serum-free liquid medium containing thrombopoietin (TPO) (Rhu PEG-MGDF; Amgen, Thousand Oaks, CA) at a final concentration of 10 ng/mL. When CD34 ⫹ cells were isolated from cord blood, they were cultured in the presence of TPO in serum-free media, as previously described [14]. All human cells were collected after informed consent was obtained from the patients, in accordance with the institutional guidelines of the Committee on Human Investigation. Antibodies. Human autoantibodies from the serum of one patient with systemic lupus erythematosus (SLE) was kindly provided by Dr. C. Andre´ (Hoˆpital H. Mondor, Creteil, France). This anti-dots
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serum displays anti-PML and anti-Sp100 affinities in Western blot [7]. Monoclonal anti-PML antibody and polyclonal anti-Sp100 were kind gifts from Dr. H. de The´ (Centre Hayem, Hoˆpital Saint Louis, Paris, France) and have been described previously [6, 15]. Polyclonal antibodies to glycoprotein IIbIIIa (GPIIbIIIa) and to GPIb were used to identify immature Mks by IF and IEM, respectively, and were kindly given by Dr. Michael Berndt (Baker Medical Research Institute, Victoria, Australia) [16]. Polyclonal rabbit antibodies to von Willebrand factor (vWF) were purchased from Dakopatts, Denmark, and used at a concentration of 100 mg/mL. For IF, specific primary antibodies were revealed by goat antirabbit immunoglobulin (Ig) G coupled to rhodamin (GAR-TRITC) or by rabbit anti-human IgG coupled to fluorescein (RAH-FITC) (Beckton Dickinson, Mountain View, CA) at 1:40 and 1:400 dilutions, respectively. For mitotic spindle labeling, mouse monoclonal antibodies to human ␣- and -tubulin (Sigma, St. Louis, MO) were pooled and used at 1:100 dilution and revealed by goat anti-mouse IgG coupled to rhodamin (GAM-TRITC) at 1:40 dilution. For IEM, specific antibodies were revealed using IgG fractions anti-rabbit or anti-mouse coupled to 10- or 15-nm-diameter gold particles (British Biocell International, Cardiff, Wales, UK). Immunofluoresence. Nuclear protein locations were studied in the HEL cells and in human Mks in primary culture at days 6 and 13. Cultured cells were cytocentrifuged at 500 ⫻ g for 4 min onto cover slips, fixed in 4% formaldehyde in Hank balanced salt solution (HBSS; Gibco BRL, Gaithersburg, MD) for 20 min, permeabilized with 0.5% Triton X-100 in HBSS containing 0.1 mmol/L EGTA, and blocked with 0.5% bovine serum albumin in phosphate-buffered saline (PBS). Cells were incubated for 1 h in the primary antibody, washed, incubated 1 h with the appropriate secondary antibody, and washed again. Quantitative studies were performed by using triple staining; antidots serum from the patient with SLE was revealed by RAH-FITC in order to detect PODs; anti-vWF were revealed with GAR-TRITC in order to specifically identify Mks; anti–␣- and -tubulin were revealed by GAM-TRITC in order to visualize mitotic spindles; and Vectashield mounting medium with DAPI was used to visualize nuclear lobes and estimate ploidy. Cells were then observed with a Leica microscope. IEM. Cells were fixed in glutaraldehyde (1.5%) in phosphate buffer (0.1 M, pH 7.2) for 1 h at 22°C under a chemical hood, washed three times with phosphate buffer, embedded in 2.3 M sucrose in ˚ ) were cut PBS, and frozen in liquid N 2. Ultrathin cryosections (700 A at ⫺95°C (Reichert Ultracut S, Vienna, Austria) and were collected on nickel grids. Thin sections were then incubated with anti-PML, anti-Sp100, and anti-dots primary antibodies, followed by appropriate IgG fractions coupled to colloidal gold at a 1:30 dilution. Double immunolabeling was performed by using anti-GPIb in order to identify Mks as such and by using anti-dots to visualize PODs. Sections were counterstained with uranyl and lead. Observations were performed with a CM10 electron microscope (Philips, Eindoven, The Netherlands).
PODs per cell nucleus was observed (Fig. 1, inset). By using IEM, we found PML and Sp100 to be mostly located as a rim around nuclear structures, forming homogenous gray structures in ultrathin sections (Fig. 1). The mean diameter of these structures was 500 nm. Nuclear Body Identification in Human-Cultured Mks
By using IEM with anti-GPIb and anti-dots sera on Mks at days 8 and 13 of culture, double staining appeared in demarcation and plasma membranes, respectively, which indicate megakaryocytic lineage, and in intranuclear PODs (Figs. 2a, 2b). In single labeling with anti-Sp100 antibodies, specific staining could be found in the cell nucleus; consistent, albeit weaker, labeling could also be found with anti-PML antibodies. Both antigens appeared to be concentrated in small circular nuclear structures about 500 nm in diameter. They appeared as homogenous gray, electron-dense structures, occasionally surrounded by a rim of denser material (Fig. 3). Nuclear Body Quantification
RESULTS
IF studies with anti-dots autoantibodies allowed a semiquantitative evaluation of PODs. The reaction was performed in HEL cells, where a range of 7 to 12 PODs per nucleus was identified. When cord blood CD34 ⫹ progenitors are cultured with TPO, they give rise to fully mature Mks. In this system, most cells reach a maximum of 16N DNA content, and the ploidy remains inferior to the one of bone marrow Mks [14]. In order to estimate the number of PODs per Mk, and to attempt to relate this number to cell ploidy, we used triple staining. DAPI stain allowed distinction of different nuclear lobes; specific immunofluorescent staining for vWF using rhodamine allowed recognition of Mk; and anti-dots serum (revealed by fluorescein) specifically labeled PODs and allowed evaluation of their number. It appeared that the small, apparently diploid Mks contain an average of 5 PODs (Figs. 4a– 4c). A Mk with three visible lobes contains an average of 15 PODs (Figs. 4d– 4f, Figs. 5a, 5b). This number could reach an amount of 50 elements in large, mature polylobulated Mks (Figs. 5c, 5d) Thus, the PODs’ number was much higher in these large cells compared with the maximal amount previously described in diploid cells.
Nuclear Body Identification in HEL Cells
PODs Distribution during Endomitosis
Autoantibodies from the serum of the patient with SLE, when used in IF on eukaryocytic cells, give a typical speckled nuclear pattern, or dots [7]. Identification of PODs was then possible by this method on HEL cells. The staining pattern appeared mostly punctiform, associated with a low level of diffuse staining on the remaining nuclear surface. A mean of 9.5 (7 to 12)
Endomitotic events were observed. Mks could be recognized as such due to their unusually large size. In order to visualize more accurately the repartition of PODs in relationship to the mitotic spindles and the chromosomes, triple staining was performed (pooled anti–␣- and -tubulin antibodies were used in order to visualize the mitotic spindles, anti-dots were used for
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FIG. 1. EM view of HEL cells treated by autoantibodies from the serum of a patient with SLE and immunogold-specific labeling appears as a rim of particles (arrowheads) surrounding a dense core, allowing the ultrastructural identification of a POD. N, nucleus; Nu, nucleolus. Magnification, 61,000⫻. Inset: HEL cells stained with autoantibodies (anti-dots) from a patient with SLE by IF. The cell displays a typical pattern of multinuclear dots. A range of 7 to 12 PODs can be quantified in each cell nucleus.
PODs, and DAPI stain was used for chromatin) on cultured Mks. In most of them, anti-dots labeling was diffuse in the cytoplasm and PODs were undetectable; or fluorescent labeling appeared disorganized, with faint clusters of fluorescence redistributed away at the cell periphery, replacing the classical speckled interphasic pattern. The clusters displayed no interaction with the asters, spindle microtubules, or chromosomes (Fig. 6). In parallel, and as controls of the reaction, interphasic diploid cells and Mks displayed evident nuclear bodies (Fig. 6c). DISCUSSION
To our knowledge, this is the first report that demonstrates the existence of PODs in the nucleus of human Mks cells. We used the HEL cell line, which is able to undergo megakaryocytic differentiation and human cultured Mks [12]. A variety of autoantibodies from a patient with SLE and antibodies directed against PODs components were utilized, followed by IF and IEM analyses. Previous studies have reported PODs in
many eukaryotic cells, including hematopoietic cells from the granulocytic and erythroid lineages [6, 17, 18]. Occasional reports have mentioned the search for PODs in Mks, but the results concerning this cell type were of secondary importance and did not lead to a firm conclusion [6, 18]. Mks are rare cells in human bone marrow, which makes their study tedious, and the large size of their nucleus makes the identification of very small compartments by optical microscopy difficult. Therefore, in our study, we used a culture system where the Mk population is predominant, combined with the highly discriminative technique of IEM. Besides euchromatin, heterochromatin, and the nucleolus, other subnuclear compartments have been identified within the eukaryotic cell nucleus [8]. Among them are nuclear bodies, which have been recognized by electron microscopy and further characterized, thanks to their specific components (i.e., PML and Sp100 [10, 11]). Initial functional approaches to the study of PODs came from acute promyelocytic leukemia (APL), a malignant hemopathy specifically associated with a t(15;
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FIG. 2. IEM analysis of PODs in cultured Mks at day 13 (mature cells). Double immunogold labeling has been performed with anti-dots human antiserum (10 nm gold) to visualize PODs and anti-GPIb rabbit antibodies (15 nm gold) to identify Mk. (a) This large cell possesses a convoluted nucleus and characteristic organelles such as demarcation membranes and secretory granules. Magnification, 8300⫻. (b) A higher magnification view shows GPIb staining in demarcation membranes (dm) and a rim of labeling a small, dense, and round structure within the nucleus corresponding to a nuclear POD (arrowheads). Magnification, 35,000⫻.
17) translocation [19]. This translocation fuses a nuclear receptor for retinoic acid (RAR-␣) with the PML gene [20 –22]. In APL, promyelocyte differentiation is blocked and abnormal promyelocytes accumulate. The PODs of APL promyelocytes are disrupted, and PML antigen is detected in the entire cell without specific location. All transretinoic acid treatment of APL promyelocytes in culture and in patients with APL induces the reconstitution of PODs, overcomes the differentiation blockage, and leads to apoptotic death of leukemic promyelocytes [15, 23–27]. The PML protein is expressed in most tissues, including hematopoietic cells [6, 17]. PML protein is mainly nuclear, associated with the nuclear matrix [28], and localized in PODs. Transcription of the PML gene is increased by interferons [3, 29], which are growth- and tumor-suppressive cytokines. In those experiments, the number of PODs is also increased. The functions of PML protein are multiple and still under investigation. PML acts as a negative cell growth factor and as a factor which promotes cell differentiation [30]. PML can improve both erythroid and myeloid differentiation [17]. Moreover, knockout animal model
PML⫺/⫺; mice display leucopenia with a decreased capacity for retinoid acid (RA)– dependent terminal granulocytic maturation [31]. Thus, PML could mediate the RA growth-suppressive and cell-differentiationpromoting effects. In addition, PML clearly interferes with cell cycle progression [17]; PML overexpression regulates cdk2 and cyclin [30]; and PML protein interacts with the eukaryotic initiation factor 4 E (eIF-4 E), which regulates the cyclin D1 mRNA nucleocytoplasmic transport, an interaction which interestingly requires intact PODs [32]. PML can also participate in the upregulation of cell cycle inhibitor p21 [31]. Finally, PML is implicated in genomic stability, as reported by Zhong et al. [33]. In contrast to the well-described role of PML protein, PODs have still elusive functions. PODs are multiprotein complexes specifically assembled by PML protein. As PML, the PODs are associated with the nuclear matrix. Numerous proteins were reported to constitute PODs. These proteins display intranuclear and nucleocytoplasmic shuttles [4, 10, 11]. Proteins which do not constitute components of PODs can be specifically targeted and accumulated at the vicinity of PODs. Accu-
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FIG. 3. IEM analysis of PODs in cultured Mk at day 13 (mature cells) detected with anti-dots serum shows various aspects of PODs. (a, b) The structure occasionally appears surrounded by a denser ring of chromatin. Magnification, (a) 48,000⫻; (b) 67,000⫻. (c, d) In all cases, the structure is distinct from the surrounding chromatin by its homogenous, moderately dense aspect, and the labeling is distributed at the periphery of the organelle. Magnification, (c) 54,500⫻; (d) 44,000⫻.
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FIG. 4. Human Mk in culture are triple stained. Anti-dots antibodies coupled to FITC show PODs (a, d); DAPI staining reveals nuclear shape and allows estimation of the number of nuclear lobes, which increases with ploidy (b, e). The cytoplasmic staining for vWF certifies the Mk lineage of cell (c, f). (a– c) A small Mk with a unique nuclear lobe, which is likely diploid, displays an average of five PODs. (d–f) The large polylobulated Mk display a greater number of PODs (10 to 15) than the diploid cells.
FIG. 5. Double fluorescent staining of human Mks in culture. Anti-dots antibodies coupled to FITC (a, c, e) labels the PODs, while DAPI staining (b, f) reveals nuclear shape and allows estimation of the number of nuclear lobes, which increases with ploidy. (a, b) DAPI staining reveals three lobes in this Mk, and the nucleus contains three times more PODs than a diploid cell. (c, d) Two adjacent highly polylobulated Mks (containing an average of four nuclear lobes) are identified as such by the specific red fluorescence for vWF, maximal in the paranuclear regions. Immunostaining reveals an average number of 50 PODs per cell.
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FIG. 6. Human Mk undergoing endomitosis, triple stained with fluorescent dyes. DAPI staining (a) shows an endomitosis with two ditinct groups of chromosomes. Anti-tubulin immunolabeling (b) shows spindle microtubules (green arrows), which radiate from each aster to form a spherical conformation. Anti-dots serum (c) reveals that PODs are disorganized, forming large aggregates of fluorescence (red arrows). PODs are excluded from the mitotic spindles and chromosome areas and redistribute at the periphery of the cell. As a control, an interphasic diploid cell exhibits three PODs (red arrowheads).
mulation of nascent mRNAs was also reported in PODs, as a way of stockage that may regulate mRNA maturation, mRNA nucleocytoplasmic transport, and protein expression [34, 35]. In summary, PODs could constitute “nuclear dumps,” or nuclear storage for various proteins and mRNA. PODs could then regulate the traffic, activity, and expression of various proteins. PODs may participate to all the functions of PML: cell proliferation, cell cycle regulation, and genomic stability. In addition, since PODs are present in normal promyelocytes and disorganized in leukemic promyelocytes, it would be of interest to investigate PML distribution and PODs structure in malignant Mks from patients with megakaryoblastic leukemia and from myeloproliferative syndromes. Polyploidization results from endomitotic cycles that occur in Mk as cells stop proliferating and start differentiating. DNA accumulates in Mks without cell division, and the nuclei become polylobulated [2]. The partners of cell cycle machinery have unique expressions and probably regulations during the endomitosis of Mks [2]. The evolution of nuclear structures during endomitosis is poorly understood. Thus, we have examined the number of PODs during the polyploidization of Mks. First, we have quantified PODs in HEL cells, which are mostly diploid cells. Then we followed the evolution of the number of PODs during cytoplasmic and nuclear growth of Mk (increased nuclear lobularity and polyploidization). We found that the more numerous the nuclear lobes were, the more numerous the PODs were. As the number of nuclear lobes in Mks was
reported to be roughly proportional to Mk ploidy [36], we deduce that the number of PODs is also proportional to Mk ploidy. Increased numbers of PODs were previously reported in cancerous cells but not in cells from nontumoral hyperplastic tissues [37, 38]; this observation could be explained by the fact that malignant cells are often multiploid, whereas hyperplastic normal cells are not. The increased PODs number could then just reflect multiploidy. Growth factors, erythropoietin (EPO), and granulocyte, macrophage colony stimulatory factor (GMCSF) induce PML expression and accumulation of PODs, respectively, in erythroid and granulocytic lineages cultured from human progenitors cells [17]. In the Mk lineage, TPO is the main regulator of cell development; TPO acts at each stage of the development of Mks, proliferation, endomitosis, and maturation [39]. TPO stimulates polyploidization in cultured Mks [40, 41], and TPO ⫺/⫺ mice display Mks with smaller size and ploidy [2, 3, 39, 42, 43]. We suggest that accumulation of PODs could be induced by TPO, since TPO and interferons both have EPO-like domains, and it would be of interest to confirm this hypothesis. When the number of PODs increases in the polyploid Mk, PML, and PODs, associated proteins accumulate and then can assume an efficient cell growth control during polyploidization of Mks and differentiation control of Mks, as in other hematopoietic lineages [44]. Moreover, the level of the genomic protection role assumed by PML may thus be proportional to the DNA content of polyploid Mks. In the majority of Mks undergoing endomitosis, PODs were disorganized and became undetectable, and the fluorescent labeling appeared to be redistributed. They did not appear to interact either with the chromosomes or with the mitotic spindles. Thus, we found in Mks during endomitosis the same intracellular repartition and global disappearance of PODs, as described previously in nonMks lineages [3, 45]. Finally, a better understanding of PODs’ organization, composition, and subcellular interactions in Mks could improve our understanding of the endomitotic cell cycle. The authors are grateful to Pr. Hugues de The´ (CNRS UPR 43, Centre Hayem, Hoˆpital Saint Louis, Paris, France) for providing anti-PML and anti-Sp100 antibodies and to Dr. Chantal Andre´ (Hoˆpital Henri Mondor, Cre´teil, France) for advice and for providing autoimmune serum from the patient with SLE. We are also grateful to Najet Debili for providing the Mks used for electron microscopy.
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