From www.bloodjournal.org by guest on February 2, 2015. For personal use only. bcl-2 Proto-oncogene Expression in Normal and Neoplastic Human Myeloid Cells By Domenico Delia, Antonella Aiello, Davide Soligo, Enrico Fontanella, Cecilia Melani, Francesco Pezzella, Marco A. Pierotti, and Giuseppe Della Porta The present study provides immunobiochemical and molecular data on the differentiation-linked expression of the bcl-2 proto-oncogene in normal and neoplastic myeloid cells. Using a recently developed monoclonal antibody (MoAb) t o the bcl-2 molecule, staining of normal bone marrow myeloblasts, promyelocytes, and myelocytes, but neither monocytes nor most polymorphonuclear cells, was demonstrated. By two-color flow cytometric analysis, bcl-2 was evidenced in CD33' and CD33+/CD34+myeloid cells as well as in the more primitive CD33-/CD34+ population. The leukemic cell lines HL-60, KGI, GM-1, and K562 were bcl-2 positive together with ll of 14 acute myeloid leukemias (AML) and three of three chronic myeloid leukemias (CML) in blast crises; six of seven CML were negative. Among myelodysplastic cases, augmentation of the bcl-2 positive myeloblastic compartment was found in refractory anemia with excess of blasts (RAEB) and in transformation (RAEB-t). Western blots of myeloid leukemias and control lymphocytes extracts evidenced an anti-bcl-2 immunoreactive band of the expected size (26 Kd). Moreover, the HL-60 and KGI cell lines, both positive for the bcl-2 protein, exhibited the appropriate size bcl-2 mRNA (7.5 Kb). These findings clearly indicate that the bcl-2 gene is operative in myeloid cells and that the anti-bcl-2 MoAb identifies its product and not a crossreactive epitope. Induction of HL-60 differentiation toward the monocytic and granulocytic pathways was accompanied by a marked decrease in bcl-2 mRNA and protein levels; bivariate flow cytometric analysis showed that the fraction becoming bcl-2 negative was in the G1 phase of the cell cycle. These data establish that the bcl-2 proto-oncogene is expressed on myeloid cells and their progenitors and is regulated in a differentiation-linked manner, 0 1992by The American Society of Hematology. T on the expression of bcl-2 proto-oncogene in normal and malignant myeloid cells, including a population of CD34'/ CD33- hematopoietic progenitor cells. HE GENE bcl-2 is involved in the t(14;18) chromosomal translocation present in more than 70% follicular B-cell lymphomas,',' where the fusion of the 3' nontranslated region of bcl-2 with the joining (JH) segment of Ig heavy chain gene results in a chimeric transcript encoding a normal bcl-2 product.* The inappropriately elevated mRNA levels and the lack of point mutations of the coding region3 support the view that the deregulation of the gene which leads to quantitative differences in the levels of bcl-2 protein4 is the major factor of transformation. Normal bcl-2 transcripts have been detected in activated but not in resting T- and B-cell lymphocyte^^.^ and in a variety of T-, B-, and pre-B lymphoblastoid cell lines negative for the t( 14;18) chromosomal breakpoint.'.' Moreover, immunohistochemical analysis using recently produced monoclonal antibodies (MoAbs) specific for the human bcl-2 protein7a8have shown that medullary but not cortical thymocytes and mantle zone but not germinal center B cells of normal lymph nodes are bcl-2 positive. Resting blood T and B lymphocytes also express the protein, despite the reported undetectable levels of mRNA.' Together, these findings indicate that bcl-2 is actively regulated during normal lymphopoiesis. The bcl-2 gene encodes a major nonglycosylated protein of 26 Kd lacking leader or kinase d0mains.4~~~'~ Subcellular fractionation studies have recently established that bcl-2 is an inner mitochondrial membrane protein," the first example of a proto-oncogene with such localization. At present, while the biochemical function of bcl-2 protein is unknown and the reported in vitro GTP-binding activity" has not been c~nfirmed,""~ its physiologic function in blocking programmed cell death or apoptosis is well established. For example, B cells from hyperplastic lymph nodes of bcl-2-Ig transgenic mice possess in vitro a significantly longer survival than normal B cells'4; in addition, deregulated bcl-2 prolongs the survival of growth-factordeprived hematopoietic cell In this report we present biochemical and molecular data Blood, Vol79, No 5 (March I), 1992: pp 1291-1298 MATERIALS AND METHODS Cells, biopsies, and induction of differentiation. The leukemic cell lines KG1 (myeloblastic),HL-60 (promyelocytic),GM-1 (monoblastic), K562 (erythromyeloid), Raji (B lymphoblastic), and the colon carcinoma cell line HT-29, all mycoplasma free, were maintained in RPMI-1640 (Bioproducts Inc, Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (Imine Scientific, Santa Ana, CA), 2 mmol/L glutamine, 100 U/mL penicillin, and 100 Fg/mL streptomycin (RPMI-C) in a 5% CO, humidified incubator at 37°C. Bone marrow aspirates and trephine biopsies from patients with various myeloproliferative disorders were obtained for diagnosis; the latter were fixed in either 10% buffered formalin or Bouin's solution, decalcified overnight in nitric acid or in 10% Titriplex (Merck, Darmstadt, Germany), and embedded in paraffin. Blood samples containing elevated numbers of CD34-positive hematopoietic progenitor cells were obtained from nonhematologic cancer patients undergoing autologous bone marrow transplantation and receiving recombinant human granulocyte-macrophage colonystimulating factor (GM-CSF) after myeloablative chemotherapy.'8 Spleen lymphocyteswere obtained by mechanical disaggregation of uninvolved biopsies of patients with nonhematologic tumors and undergoing splenectomy for therapeutic purposes. Bone marrow, From Istituto Nazionale per Lo Studio e La Cura Dei Tumori, Milan; Istituto di Scienze Mediche, University of Milan, Italy; and John Radcliffe Hospital, Headington, w o r d , UK Submitted July IS, 1991; accepted October 22, 1991. Supported by the Associazione Italiana Ricerca Cancro. Address reprint requests to Domenico Delia, PhD, Istituto Nazionale Tumori, Via G Venezian I , 20133 Milano, Italy. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section I734 solely to indicate this fact. 0 I992 by The American Society of Hematology. 0006-4971I92 17905-0022$3.0010 1291 From www.bloodjournal.org by guest on February 2, 2015. For personal use only. From www.bloodjournal.org by guest on February 2, 2015. For personal use only. bcl-2 IN MYELOID CELLS Fig 2. Two-color flow cytometric analysis of bcl-2 protein in CD33’ and CD34’ populations. The analyses were performed on paraformaldehyde-fixed/Tritontreated cells labeled with the specified MoAbs and with isotype-specific, FITC-and PE-conjugated second antibodies. For each fluorescence histogram (log scale), the horizontaland vertical markers (set up on negative controls) separate the reactive from unreactive cells. Analysis on bone marrow (A) and on blood cells of an rhGM-CSF-treated patient (B) are shown. The size distribution (forward v 90”light scattering) of the latter sample is depicted in top left histogram together with bitmaps 1 and 2, which encompass the small- and large-size cells, respectively; in this specimen most of CD34’ cells were found in bitmap 1. 1293 -4. d. a a €9 c3 0 0 10” 10’ lo2 bcl-2 90LS CD34 lo3 CD33 blood, and spleen suspensions were enriched in mononuclear cells by centrifugation(30 minutes at 4oog) on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Erythrocyte-depleted bone marrows were prepared by treatment with an erythrocyte lysing solution (Coulter Electronics,Hialeah, FL). Granulocytic and monocytic differentiation of HL-60 was achieved by culturing the cell line in the presence of 1.2% dimethyl sulfoxide (DMSO) (Sigma Chemical Co,St Louis, MO) and 10ng/mLof 12-0-tetradecanoylphorbol-13acetate (TPA) (Sigma), respectively. DMSO was added every 72 hours. MoAbs, cell permeabilizaiion, immunostaining procedures, and flow cytometric anabsis. The mouse MoAb to bcl-2 protein used in this study (clone 124, IgG2a isotype) has been recently described7 and proven suitable for immunostaining of cryostat and paraffinembedded tissue sections and for Western blotting analysis; it was raised against a synthetic peptide corresponding to amino acids 41 to 54 of the bcl-2 protein. MoAbs to CD33 (MY9, IgG2b i~otype)’~ and CD34 (QBEND10, IgG1)” were obtained from Coulter Immunology (Hialeah, FL) and N. Bradley (Quantum Biosystems, Cambridge, UK), respectively. The MoAbs specific for HLA-DR (MA6, IgM), CDllb (clone 44,IgGl), and CDllc (clone 3.9, IgG1)” were obtained from the Fourth International Workshop on Human Leucocyte Antigens. The fixation/permeation protocol for optimal immunodetection of bcl-2 internal molecule will be described in detail elsewhere. Briefly, a pellet of lo7 cells was resuspended in 1 mL of 2% ioo 10’ io2 lo3 HLA-DR 100 lo’ 102 103 CD33 paraformaldehyde in phosphate-buffered saline (PBS) and kept for 10 minutes at 4°C; 100 pL of 0.05% Triton XlOO (Sigma) in PBS was then added and 10 minutes later the cells were washed twice in PBS plus 1% bovine serum albumin (BSA) and used for labeling. Single- and two-color indirect immunofluorescence (IF) staining of fixed or viable cells was performed at 4°C on 96-well microtiter plates as describedz2;the cells were preincubated for 10 minutes with 1% human heat-inactivated AB-serum to prevent binding of the antibodies to Fc receptors. For the single-color IF the cells were incubated (30 minutes) with saturating doses of the specified MoAb, washed four times in RPMI-C, and incubated again (30 minutes) with a 1:30 dilution of fluorescein (FITC)- or phycoerythrin (PE)-conjugated antimouse Ig antibodies purchased from Technogenetics(Trezzano SN,Italy; cat. 3900-2) and Southern BiotechnologyAssociates Inc (Birmingham, AL; cat. 1010-09), respectively. For the two-color IF, combinations of primary MoAbs (or irrelevant mouse Igs for the negative controls) of different isotypes were simultaneously added to the cells; their binding was shown by FITC- and PE-conjugated goat antibodies specific for the mouse IgGl isotype (cat. 1070-02; 1070-09), IgG2a (cat. 1080-02; 108009), IgG2b (cat. 1090-01; 1090-09), or IgM (1020-02; 1020-09) purchased from Southern Biotechnology. Immunocytochemical staining of air-dried paraformaldehydefixed cytospin preparations was performed by the alkalineantialkaline phosphatase (APAAP)techniqueU using a commercial kit (Dakopatts, Glostrup, Denmark); three APAAP repeats Fig 1. Immunocytochemical reactivity of anti-bcl-2 MoAb with normal bone marrow cells. Cytospin preparation of Ficoll-enriched bone marrow aspirate, fixed with 2% paraformaldehyde and permeabilized with 0.001% Triton X100, immunostained by the APAAP methods and counter-stained with Gill’s Hematoxylin. In this preparation three myeloid blasts, two late erythroblasts, and a lymphocyte are intensely stained for bcl-2 whereas more mature myeloid cells are negative; some perinuclear staining is also evident (original magnification x 1.000). Fig 3. lmmunolocalization of bcl-2 protein in normal and pathologic bone marrow biopsies. Sections of paraffin-embedded bone marrow biopsies were deparaffinized and immunostained by the APAAP technique as described in Materials and Methods. (A) Normal bone marrow biopsy showing scattered lymphocytes and two aggregates of blasts (arrows) stained for bcl-2 (original magnification x 400). (B) An RAEB showing a large nodule of bcl-2 reactive cells (original magnification x 400); inset (original magnification x 25). (C) An RAEBt with clusters of bcl-2 blasts (original magnification x 400). Fig 8. Immunocytochemical reactivity of anti-bcl-2 MoAb with normal and DMSO-treated HL-60 cells. APAAP-immunostained cytospin preparations of untreated (A) and DMSO-treated (6 days) (B) cells. Note the marked reduction in bcl-2 staining after DMSO treatment; in particular, cells with a mort? mature chromatin pattern and nuclear indentations are bcl-2 negative whereas two blasts exhibit cytoplasmic positivity (original magnification x 1,OOO). From www.bloodjournal.org by guest on February 2, 2015. For personal use only. DELIA ET AL 1294 allowed optimal amplification of the reaction. Sections from paraffin-embedded bone marrow specimens were deparaffinized in xylene and graded ethanols, incubated with PBS containing 5 % decomplemented human A B serum, and immunostained by the APAAP method; normal mouse serum and an MoAb specific for the leukocyte common antigen CD45 (Dako) were used as negative and positive controls of the APAAP reaction, respectively. Antibcl-2 reactive cells were quantified at 400 X magnification by means of an ocular grid in more than 20 randomly selected fields (equivalent to 2 mm' of tissue). Double-color staining for bcl-2 (green) and DNA (red) was performed on cells that had been previously paraformaldehydefixedlTriton-permeabilizedand indirectly labeled for bcl-2 using an FITC-tagged secondary antibody; the cells were then incubated (30 minutes at room temperature) with 1 mglmL RNAse A (Sigma) in PBS and finally in 1 mL of 10 WglmL propidium iodide (Sigma) in PBS. Flow cytometric analysis of single- and two-color stained samples was performed on an EPICS-C instrument (Coulter Electronics, Hialeah, FL) equipped with a 5-W argon-ion laser set at 488 nm and 5 0 mW output power. Whenever required, color overlaps were eliminated by electronic compensation. Westemblot analysis. Lysates were prepared by solubilizing 10' cells in 2 mL of a 50 mmol/L Tris buffer solution (TBS) p H 7.5 containing0.5% NP40,1% antagosin. 0.001% phenylmethylsulphonyl fluoride (PMSF); after ultracentrifugation, the supernatants were recovered, boiled for 5 minutes in the presence of 5 % B-mercaptwthanol and loaded (50 FLlslot) on 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel, electrophoresed, and blotted onto BA8S nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After neutralization with 5 % BSA in TBS, the membranes were incubated (20 hours at 4°C) with the anti-bcl-2 MoAb (5 )rg/mL) or with an equal amount of normal mouse IgG (for the negative control) diluted in TBS containing 5 % BSA and 0.01% NaN3, hence washed extensively with TBS with or without 0.05% Triton XIOO, incubated for 1 hour at room temperature (RT) with '"I-labeled goat antimouse Ig (5 x 10" cpm in 20 mL) (Amersham, Amersham, UK), washed again, and exposed to autoradiographic film. Northem blots. Poly-A enriched mRNA from treated o r untreated cell lines was extracted by a kit (cat. 27-9254-01) purchased from Pharmacia LKB (Uppsala, Sweden). Northern blots were performed as described"; mRNA samples (5 kgllane) were electrophoresed in I % agaroselformaldehyde, transferred onto Duralon UV nylon membranes (Stratagene, La Jolla, CA), and autocrosslinked. Prehybridizations, hybridizations, and washes under stringent conditions were performed as described." The bcl-2 probe was a 1.5-kb Hindlll-EcoRI insert from pFLl obtained from M. Cleary.' One hundred nanograms of the fragment, radiolabeled to a specific activity of 2 to 5 x 10" cpm/pg with "P by the random primer DNA labeling procedure, was used per hybridization. Table 1. Reactivity of the Anti-bcl-2 MoAb With Myeloid Disorders No. of Cases 3 1 3 1 FAB Diagnosis RA RAEB RAEB RAEB 1 1 RAEB-t 2 RAEB-t 3 AML" AMLt CML" RAEB-t bcl-2' Cells + + ++ +++ + ++ +++ Distribution Clusters Clusters Clusters Clusters and diffuse Clusters Clusters Clusters and diffuse 11 1 3 CML CML-BC 3 ET 2 PV 6 - +++ +++ +++ ++ Diffuse Rare nodules Diffuse Clusters The studies were performed on bone marrow sections and aspirates the former were labeled by the APAAP method, the latter by immunofluorescence and examined byflow cytometry ( t 7 of 11 cases). A semiquantitative estimate of the number of bcl-2' cells was based on a comparison with normal bone marrow biopsies as follows: +, up to 20% stained cells; + +, up to 30% stained cells; + + +, more than 30% stained cells: -,similar to normal controls. Abbreviations: RA, refractoly anemia; RAEB, refractory anemia with excess of blasts; RAEB-t, RAEB in transformation; AML, acute myeloid leukemia: CML, chronic myeloid leukemia: CML-BC, CML in blast crisis; ET, essential thrombocythemia; PV, polycythemia vera. (*); labeled cells on erythrocyte-dcpleted samples; however, on Ficoll-separatcd samples (and thus depletcd of most of the granulocytes) 78% 2 8.6% wcrc found positivc. In the latter samples the CD33+ myeloid cells were 28.5% 2 21.1% and the CD34' cells 5.3% 2 3.5%. Dual-color IF analysis showed that 89.5% 2 3.6% of the CD33' and 75.7% 2 21.2% of the CD34' fractions were bcl-2 positive (Fig 2A). Coexpression of bcl-2 and CD34 was also found in peripheral blood samples from patients trcated with recombinant human (rh) GM-CSF and presenting elevated numc RESULTS Detection of bcl-2 protein in myeloid cells. bcl-2 positive myeloid cells were found in normal bone marrows as shown by the immunostaining of cytospin preparations (Fig 1); 70% of myeloblasts, 83% of promyelocytes, 40% of myelocytes, 12.5% of metamyelocytes, 12.5% of polymorphonuclear cells, and 60% of monoblasts were bcl-2+, whereas monocytes were totally negative. Although the localization of the protein was prcvalcntly cytoplasmic, some perinuclear staining was observed. The immunofluorescence flow cytometric analysis of bcl-2 in normal bone marrows cvidcnccd 18% 2 5.3% of 26 kDFig 4. lmmunoblot analysis of bcl-2 protein in myeloid cells. Lysates from equal numbers of cells (2.5 x los) were run under reducing conditions on SDS-polyacrylamidegel electrophoresis (SDSPAGE), transblotted to nitrocellulose, reacted with the anti-bcl-2 MoAb and with '%goat antimouse, and finally exposed to autoradiographic film. Spleen lymphocytes and HT-29 cell line were included as positive and negative controls. AML is a fresh acute myeloblastic leukemia. From www.bloodjournal.org by guest on February 2, 2015. For personal use only. bcl-2 IN MYELOID CELLS 1295 A HL60 ..tu [I kb kb - 9.5 7.54.4- 2.42.4- 1.4Fig 5. bcl-2 mRNA in KG1 and HL-60 cells. Equal amounts of poly-A enriched mRNA (5 pg/lane) were size-fractionated in agarose, blotted onto nylon filter, hybridized with UP-labeledDNA probe pFLl specific for bcl-2, and exposed t o autoradiographic film with intensifying screen. Poly-A RNA from the B-lymphoblastoid cell line Raji and the colon carcinoma HT29 were included as positive and negative controls, respectively. Exposure times were 60 hours (A) and 9 days (6). 1.4- 0.3 - 0.3- cont. 2d 5d 5d TPA 2d DMSO The myeloid leukemic cell lines HL-60, KG1, GMI, and K562 were found, by IF and immunocytochemistry, positive for bcl-2; the mean fluorescence staining intensity values indicated KGl as having thc highest amount of protein per cell (not shown). Westem and Northem blot analysis. To verify that, on myeloid cells, the binding of bcl-2 MoAb was to the bcl-2 gene product rather than to a cross-reactive epitope, further studies at protein and mRNA level were performed. On Western blots of myeloid leukemia lysatcs the antibcl-2 MoAb identified a protein of identical size (26 Kd) to that of spleen lymphocyte lysates (used here as positive control) and corresponding to bcl-2 (Fig 4). The 26-Kd protein was also dcmonstrated in the early myelo/ erythroblastic cell line K562; this finding, which suggests that the bcl-2 gene may be expressed by erythroid progenitors, is supported by thc reactivity of the anti-bcl-2 MoAb bers of CD34' circulating progenitor cells; onc of these cases was CD33-, HLA-DR' (Fig 2b). Immunohistochemical staining of paraffin-embedded normal bone marrow biopsy sections evidenced 14.5% (range 10% to 16%) of anti-bcl-2 reactive nucleated elements, most of which were lymphoid cells and myeloid blasts (Fig 3A). On fresh leukemias the anti-bcl-2 MoAb rcacted with 11 of 14 AML, 1 of 7 CML, and 3 of 3 CML in blast crisis (Table 1); no correlation between bcl-2 positivity and French-American-British (FAB) classification was found. Analysis of myelodysplastic syndromes evidcnced a significant increase in bcl-2' blasts in most RAEB and RAEB-t; in some cases clustcrs or nodules of positive cells were observed (Fig 3, B and C). The number of bcl-2' cells were also augmented in polycythemia vera but not in essential thrombocythemia. Table 2. bcl-2 Protein Modulation in Chemically Induced HL-60 Cell Maturation Davs of Treatment TPA bcl-2 O h i CD11b CDllc DMSO bcl-2 O h I CD11b CDllc Morphology' CG IN 0 1 2 3 4 5 97 118 <1 <1 55 99 90 64 52 92 91 86 29 90 89 88 23 91 81 78 30 89 89 63 97 118 <1 <1 89 113 1 1 90 116 16 1 89 120 14 4 89 118 13 7 114 9 11 1.5 2 24 10 27 10 84 6 7 8 74 110 12 7 69 107 10 5 65 112 6 4 6 29 45 33 40 28 For each specified marker, the percentages of positive cells after background subtraction are shown. In addition, the mean fluorescence intensity of bcl-2 positive cells, measured on a log scale, is indicated. *Morphologic analysis of DMSO-treatedcells was performed on May-GrBnwald/Giemsastained cytospin preparations; the percentage of cells bearing cytoplasmic granules (CG) and indented nuclei (IN) is reported. From www.bloodjournal.org by guest on February 2, 2015. For personal use only. DELIA ET AL 1296 with normal bone marrow erythroblasts (see Fig 1). Differences in the intensity of the bands indicated that each cell line presented variable amounts of bcl-2 protein that were, among the myeloid leukemias, highest in KG1, albeit lower than in spleen lymphocytes. Results of Northern blot hybridizations are shown in Fig 5. The bcl-2 specific probe pFLl detected a major mRNA transcript in HL-60 and KG1 of similar size (approximately 7.5 kb) to that found in the B-lymphoblastoid cell line Raji. Strikingly, the KG1 cell line expressed at least 50-fold more mRNA than Raji and this difference did not correspond to a similar augmentation in protein level. Altogether, the concordance between the immunobiochemical and molecular results indicates that myeloid cells express the bcl-2 proto-oncogene, whose product is recognized by the anti-bcl-2 MoAb. bcl-2 on in vitro differentiating cells. The pattern of bcl-2 staining in normal bone marrow (BM) myeloid cells is in accord with a differentiation-regulated expression of the bcl-2 gene. The bcl-2 positive promyelocytic cell line HL-60, which can be induced to differentiate along either the granulocytic or monocytic pathway when treated in vitro with specific chemicals,25provides a suitable model to study this relationship. Therefore, we have cultured the HL-60 cells in the presence of 1.25% DMSO and 10 ng/mL TPA and monitored at various intervals of time the levels of bcl-2 protein, in relation to other differentiation markers, morphology, and proliferation. The results are listed in Table 2. It can be seen that the DMSO-induced granulocytic maturation was accompanied by a progressive decrease in bcl-2 positivity, from greater than 95% in the untreated control to 65% by day 8 (Fig 6). Similar downregulation of the 26-Kd protein was confirmed by Western blot analysis (Fig 7). Parallel morphologic changes (eg, increased numbers of cytoplasmic granules and indented nuclei) as well as reduction of the proliferative rate (Fig 6) were observed. As expected, C D l l b and C D l l c became detectable on a small fraction of cells.z6 Dual-color flow cytometric analysis (Fig 6) showed that most of the bcl-2 negative cells were in the G1 phase of the cell cycle. These cells were, in addition, characterized by a granulocytic morphology (Fig 8). The TPA-triggered monocytic/macrophagic differentiation resulted in a rapid decrease in bcl-2 expression (Fig 6); within 24 hours the bcl-2 positivity dropped to 55% and by day 5 to about 30%. These changes were also observed on Western blots (Fig 7). The TPA-induced maturation was evidenced by the strong upregulation of C D l l b and C D l l c molecules?6The majority of bcl-2 negative cells were in the G1 phase of the cell cycle. Downregulation of bcl-2 gene was detected on Northern blots (Fig 5); the very faint bands present at day 2 became undetectable by day 5, despite measurable amounts of protein. This finding may actually suggest that the protein has a long half-life. GI G2-M G1 G2-M t i DAYS 1 - 1 J/b d. , I a n 3 + I lo0 10' io2 bcl-2 lo3 DNA DNA Fig 6. Relationship between bcl-2 and cell cycle in differentiating HL-60 cells. The left and middle histograms were obtained from the flow cytometric analysis of single-color labeled samples, whereas those on the right were from the analysis of bel-2 (ordinate, green)/ DNA (abscissa, red) double-labeled samples; the horizontal window, set up on a negative control, defines the threshold between bcl-2 reactive and unreactive cells. DISCUSSION The bcl-2 proto-oncogene, rearranged and deregulated in B-cell lymphomas carrying the t(14;18) translocation, is expressed in normal and neoplastic cells of the lymphoid lineage negative for the t( 14;18) chromosomal abnormality12,5.6and the levels of transcription appear to correlate From www.bloodjournal.org by guest on February 2, 2015. For personal use only. bcl-2 IN MYELOID CELLS Fig 7. lmmunoblot analysis of bel-2 in differentiating HL-60 cells. Lysatesfromequal numbers of cells (2.5 x lov)were electrophoresedunder reducingconditions. Spleen lymphocytes and the HT-29 cell line were used as positive and negative controls, respectively. Note that both TPA- and DMSO-induced differentiation pathways are accompanied by a timedependent reduction of bcl-2 protein levels; this change occurs more rapidly with TPA. 1297 26 k D with proliferation and differentiation stage: This relationship has been recently further elucidated by immunohistochemical studies of lymphoid tissues using a newly developed MoAb to bcl-2 gene product,7.Rshowing strong levels of bcl-2 protein in many normal blood T and B lymphocytes, mantle-zone but not germinal center B cells, in medullary but not cortical thymocytes, and undetectable levels in proliferating lymphoid cells. AIthough studies regarding the involvement of bcl-2 in human hematopoietic cells have been mostly limited to the lymphoid lineage, bcl-2 is not lineage restricted; indeed, the presence of minimal amounts of transcripts in a monoblastoid cell line2.‘ and the recent immunodetection of the protein in normal myeloid cells2’ suggest that this protooncogene may also play a role in myeloid cell differentiation. In this report we have provided novel data on the expression of bcl-2 in normal and malignant cells of the myeloid lineage. We based this evidence partly on analysis using an anti-bcl-2 MoAb whose specificity for the 26-Kd protein was confirmed by Western blot of a variety of myloblastic leukemias (including a fresh case). Additionally, we have shown by Northern blot analysis the presence of normal-size bcl-2 transcripts of 7.5 Kb in HL-60 and KG1 cell lines. To perform quantitative and multiparameter flow cytometric measurements of bcl-2 on single cells, we developed an appropriate fixation/permeabilizationprocedure for the detection of the protein, localized primarily in the inner mitochondrial and, to a lesser extent, on the perinuclear membrane.” The bcl-2 protein levels among normal myeloid cells are inversely related to maturation; thus, a large fraction of myeloblastsand promyelocytesare bcl-2’ whereas metamyelocytes and polymorphonuclear cells are mostly bcl-2 negative, and monocytes totally negative. These findings have been substantiated by two-color flow cytometric analysis showing a large percentage of normal bone marrow bcl-2+cells expressing CD33, a marker for myeloid cells19as well as CD34, a marker for myeloid progenitor cells including stem cells.’” Coexpression of bcl-2 and CD34 molecules has been further documented on circulating CD34+ hematopoietic progenitor cells present in blood samples of rhGM-CSF-treated patients. In particular, the bcl-2 protein has been found on CD34+/CD33-/HLA-DR+ cells characterized by low right angle and low forward light Contr. 2 4 6 3 6 9 TPA DMSO scattering properties; these features are consistent with a progenitor of most of the colony-formingcells in long-term marrow cultures.”-m However, further work is needed to determine whether the hematopoietic stem cell is bcl-2’. In view of the fact that bcl-2 protein confers stress resistance to heat shock, ethanol, methotrexate and serum deprivation,I6 and survival,"^" it is attractive to hypothesize that bcl-2 on early hematopoietic progenitor cells, eg, CD33-/ CD34’ (?stem cells), favors their long-lived capacity and confers resistance to various external insults. Overall, the pattern of bcl-2 distribution in normal bone marrow myeloid cells appears concordant with the in vitro findings on HL-60, whose differentiation toward the granulocytic and monocytic pathways results in a downregulation of bcl-2 transcription and protein levels. Correlations with cell growth and morphology have shown that the bcl-2 negative cells are mostly in G1 phase of the cell cycle and present features of mature cells. We have assessed the expression of bcl-2 molecule on fresh myeloproliferative disorders; given that the anti-bcl-2 MoAb reacts with paraffin-embedded tissues, the study was extended to bone marrow biopsies. The results have shown that greater than 70% of AML are bcl-2 positive, whereas most CML are bcl-2 negative. Increased numbers of bcl-2 positive blasts have been found in myelodysplastic syndromes. No clear-cut correlation between FAB classification and bcl-2 positivity was observed. Overall, it appears that the expression of bcl-2 in myeloid disorders reflects that found in their normal cell counterparts. The expression of bcl-2 in myeloid leukemias raises the question of its potential prognostic value. In fact, because many anticancer agents also operate through the activation of programmed cell death3’and bcl-2 levels are positively associated with resistance to apoptosis,I6 the possibility exists that bcl-2 positive leukemias are less responsive to chemotherapy than those that are negative. This speculation is now open to investigation. In conclusion, the data presented here provide the basis for future studies on the role of bcl-2 in myeloid cell commitment, maturation, and survival. ACKNOWLEDGMENT The artwork was by Mario M i n i . We thank Prof A.M. Gianni for providing blood specimens of rhGM-CSF treated patients, and Prof D.Y. Mason for helpful advice and critical suggestions. From www.bloodjournal.org by guest on February 2, 2015. For personal use only. DELIA ET AL 1298 REFERENCES 1. Bakhshi A, Jensen JP, Goldman P, Wright AJ, McBride OW, Epstein AL, Korsmeyer SJ: Cloning the chromosomal breakpoint of t(14;18) of human lymphomas: Clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 412399, 1985 2. Cleary ML, Smith SD, Sklar J: Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulintranscript resulting from the t(14;18) translocation.Cell 47:19,1986 3. Hua C, Raffeld M, KO HS, Fast P, Bakhshi A, Cossman J: Mechanisms of bcl-2 activation in human follicular lymphomas. Oncogene 5:233,1990 4. Chen-Levy Z, Nourse J, Cleary M L The Bcl-2 candidate proto-oncogene product is a 24-kilodalton integral membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation. Mol Cell Biol9:701,1989 5. Reed JC, Tsujimoto Y, Alpers JA, Croce CM, Nowell P C Regulation of bcl-2 proto-oncogene expression during normal human lymphocyte proliferation. 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For personal use only. 1992 79: 1291-1298 bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells D Delia, A Aiello, D Soligo, E Fontanella, C Melani, F Pezzella, MA Pierotti and G Della Porta Updated information and services can be found at: http://www.bloodjournal.org/content/79/5/1291.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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