Cytometry 13:502-509 (1992)
0 1992 Wiley-Liss, Inc.
Flow Cytometric Detection of the Mitochondria1 BCL-2 Protein in Normal and Neoplastic Human Lymphoid Cells' Antonella Aiello, Domenico Delia,2Maria Grazia Borrello, Daniela Biassoni, Roberto Giardini, Enrico Fontanella, Francesco Pezzella, Karen Pulford, Marco Pierotti, and Giuseppe Della Porta Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy (A.A., D.D., M.G.B., D.B., R.G., E.F., M.P., G.D.P.) and John Radcliffe Hospital, Headington, Oxford, United Kingdom (F.P., K.P.) 'Received October 3, 1991; accepted December 17, 1991
The bcl-2 proto-oncogene, rearranged and deregulated in B-cell lymphomas bearing the t(14;lB) translocation, encodes an inner mitochondria1 membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single- and multicolor flow cytometric analysis of bcl-2 protein in relation to other markers and cell cycle, based on a fixation-permeation step of cells with paraformaldehyde and Triton XlOO and the use of a bcl-2 specific monoclonal antibody (MoAb).As an application of this method, we have examined the expression of bcl-2 in normal and neoplastic lymphoid cells. We have found that > 80% of normal Tand B-cells are bcl-2 positive; following in vitro mitogen activation, the bcl-2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl-2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two-color analysis. On lym-
The bcl-2 gene is associated with t(14;18) (q32;q21) chromosomal translocation of more than 70% of B-cell lymphomas ( 3 3 . The translocation places the 3' noncoding region of the bcl-2 proto-oncogene into the JH region of the immunoglobulin heavy chain gene (5), giving rise to a chimeric and de-regulated transcript but apparently normal gene product (19). Normal bcl-2 mRNA has been found in activated Band T-lymphocytes and in lymphoblastoid cell lines lacking the t(14;18) chromosomal abnormality (4,14, 28), indicating that bcl-2 is physiologically operative in lymphopoiesis. The recent development of monoclonal antibodies
phoblastoid cell lines, the bcl-2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl-2 gene. Among fresh Bcell non-Hodgkin's lymphomas (B-NHL), most sporadic Burkitt's cases were bcl-2 negative. Of four centroblastic-centrocytic cases with rearrangements of the bcl-2 gene, only two presented elevated amounts of bcl-2 protein, indicating that the levels of bcl-2 are not diagnostic of the translocation. The flow cytometric analysis of bcl-2 protein allows study and quantification,as the single cell level and in selected cell subsets, of the expression of the bcl-2 gene and provides an important tool for assessing its role in hematopoietic cell development, proliferation, and neoplastic conversion. 0 1992 Wiley-Liss, Inc.
Key terms: Bcl-2 proto-oncogene, B-cell lymphoma, gene rearrangements, flow cytometry
(MoAbs) to bcl-2 prompted investigations into the immunohistochemical distribution of the protein in normal and neoplastic tissues (18,26,37). Results indicate that germinal centers, but not mantle zone B-cells, of normal lymph nodes are bcl-2 negative, whereas ger-
This study was financially supported by the Associazione Italiana Ricerca sul Cancro and by the C.N.R. Target Project on Biotechnology and Bioinstrumentation (contract number 89.00242.70). 'Address reprint requests to Dr. Domenico Delia, DIVISION OSA, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy.
FLOW CYTOMETRY O F BCL-2 PROTO-ONCOGENE
minal centers of most follicular lymphomas are bcl-2 positive. In addition, diffuse lymphomas lacking the t(14;18) translocation are bcl-2 + . Hence, although helpful in distinguishing reactive from neoplastic lymphoid follicles, the bcl-2 marker may not be diagnostic of lymphoma carrying the t(14;18) translocation. The bcl-2 proto-oncogene encodes a major nonglycosylated protein of 26 Kd (32,33) recently localized in the inner mitochondria1 membrane (17). Although its biochemical function remains unknown and the GTPbinding activity (16) has not been confirmed (231, several studies show that bcl-2 plays a n important role in prolonging cell survival of growth factor-deprived cells (21,35), in conferring protection from programmed cell death or apoptosis (24,34; see 36 for review), and in maintaining B-cell memory (25). In this report we describe a method for the flow cytometric analysis of bcl-2 protein using a specific monoclonal antibody and immunofluorescence techniques. The method is based on an appropriate fixation and permeabilization of the cells for the optimal interaction of the reagent with its intracellular ligand, thus allowing a rapid and quantitative measurement of bcl-2 and correlation by two-color analysis with other immunological markers and with cell cycle. We provide a number of applications of this flow cytometric approach, demonstrating its usefulness in the study of bcl-2 regulation in normal lymphoid cells as well as in B-NHLs in relation to structural abnormalities of the bcl-2 gene.
MATERIALS AND METHODS Cell Lines and Tissues The B-lymphoblastoid cell lines K-422 and SU-DHL4 positive for the t(14;18) chromosomal translocation (11,12), Raji, Namalwa, ConcF, Daudi, BJAB, and the T-lymphoblastoid cell lines Ju rk at and JM, were maintained at 37°C in a 5% C 0 2 humidified incubator in RPMI 1640 (Flow Laboratories, Irwin, UK) supplemented with 10% heat-inactivated fetal calf serum (Flow), 100 U/ml penicillin, 100 pgiml streptomycin, and 2 mmol/L glutamine (RPMI-C). Lymphoma biopsies were obtained from patients for routine histological diagnosis and classification according to updated Kiel (29) and Working Formulation (31). Peripheral blood (PB) samples were donated by healthy volunteers. Normal spleen tissues were obtained from patients undergoing splenectomy for surgical purposes. Cell suspensions from tissues were prepared by mechanical disruption and enzymatic digestion with 50 U/ml collagenase (Sigma, St Louis, MO) and 40 U/ml DNAse (Sigma). Blood and spleen mononuclear cells were isolated by density centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and washed twice in RPMI 1640 medium (Flow Laboratories, Irvine, UK). Monocytes were removed by adherence onto Petri dishes (1 h at 37°C). Spleen B-cells and blood T-cells were purified by negative immunodepletion using CD15 plus CD3 and CD15 plus CD19 specific mono-
503
clonal antibodies, respectively, and goat-antimouse-labeled magnetic microspheres (DYNAL, Oslo, Norway).
In Vitro Mitogenic Stimulation B- and T-lymphocytes were resuspended in RPMI-C and seeded (lo6cells/ml/well) in 24-well plates (Costar, Cambridge, MA). The phorbol ester TPA (Sigma) and the calcium ionophore Ionomycin (Hoechst-Calbiochem, La Jolla, CA) were used together a t the mitogenic doses of 1 ng/ml and 0.5 pg/ml, respectively (8). Aliquotes of the activated cells were harvested every 24 h and cryopreserved till the end of the culture; subsequently they were thawed out, washed, and stained and analysed.
Fixation protocols For the immunodetection of the intracellular bcl-2 protein, five fixation protocols were tested; in all cases a constant number of cells (lo7)was used. The paraformaldehydeitriton (PFT) protocol was performed by resuspending the cell pellet in 2 ml of 2%paraformaldehyde (freshly prepared at the concentration of 4% in distilled water) diluted, prior to use, in 2 x phosphatebuffered saline (PBS). After 10 min on ice, 100 p1 of 1% Triton XlOO (Sigma) (final concentration 0.05%)were added, and 10 min later the cells were washed twice in cold PBS. For the aceton fixation, the cells were resuspended in 2 ml of cold acetone (-20°C) while gently vortexing, placed in the freezer for 15 min, and then washed twice in PBS. The BFA fixative was made up of 0.2 mg/ml Na2HP04.2H20 pH 6.8, 1 mg/ml KH2P04 pH 6.8, 45% acetone, 9.25% formaldehyde, 47.75% distilled water. The cells were treated for 2 sec at room temperature with 250 pl of BFA, then washed twice in cold PBS plus 0.1% bovine serum albumin (BSA) (Sigma). For the fixation in ice-cold absolute methanol, the cells were resuspended for 15 min in the alcohol (added drop wise to avoid cell clumping); thereafter the cells were washed twice in PBS. Immunofluorescence and Flow Cytometry The T-cell specific MoAb UCHTl (IgG1 isotype;CD3) and the B-cell specific MoAbs LN2 (IgG1; CD74), L26 (IgG2a; unclustered) and HD37 (IgG1,CDlS) MoAbs were obtained from the Fourth International Workshop on Human Leucocyte Differentiation Antigens (10). The anti-bcl-2 MoAbs 100 and 124 (IgG1 and IgG2a isotype, respectively), raised to a synthetic peptide corresponding to amino acids 41 to 54 of the bcl-2 protein, have been previously described (26). As negative control for the immunofluorescence, normal mouse serum at a final concentration of 1:400 was employed. Before labeling, the cells were incubated for 10 min with 2% heat-inactivated human AB serum to prevent nonspecific binding of MoAbs to Fc receptors. Double-color indirect IF staining for bcl-2 and other surface antigens was carried out on microtiter plates as described (1). The fixed cells were incubated with saturating concen-
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AIELLO ET AL.
74 -
j
108
PFT
r
loo
61
10'
10
I
*
loo
10'
10
FIG. 1. Flow cytometric evaluation of anti-bcl-2 MoAb binding to cells fixed in different ways. The indirect IF assays were performed on the B-lymphoblastoid cell line Raji fixed in various ways as described in Methods. The fluorescence histograms were obtained by normaliz-
ing the MFI of negative controls to channel 36. The upper and lower numbers refer to the percentage and MFI of reactive cells, respectively. The vertical marker, positioned on negative control samples, separates the unreactive (left) from reactive (right) cells.
trations of the anti-bcl-2 MoAb and either the B-cell marker LN2 (or L26) or the T-cell marker UCHT-1, added together. After 30 min on ice, the cells were washed four times in RPMI 1640 +5% heat-inactivated fetal calf serum (FCS, Flow) + 0.02M NaN3, and incubated again (30 min on ice) with isotype-specific, Phycoerythrin (PE) and Fluorescein (F1TC)-conjugated goat antimouse antibodies (Southern Biotechnology, Birmingham, AL) used at 1:30 final dilution. After three more washes, the cells were analysed by flow cytometry using an EPICS-C instrument (Coulter Electronics, Hialeah, FL) equipped with an argon-ion laser (settings: 488 nm, 500 mw). Whenever required, cross-color interferences of two-color labelled samples were eliminated by electronic compensation.
labeled to a specific activity of l + 2 x lo9 c p d p g with 32Pby the random primer DNA procedure (13). The human probes pFLl (6) and pFL2 (7), specific for the major and minor breakpoint cluster regions of bcl-2, respectively, were kindly provided by Dr. M. Cleary, Stanford University. Immunoglobulin heavy chain joining (JH) genes were analysed by a 6.0 Kb BamHIHind111 fragment (27).
Double-color Staining for DNA and bcl-2 PFT-treated cells were first labeled by indirect IF for bcl-2 using, as secondary reagent, a FITC-conjugated antimouse antibody, then incubated (60 min on ice) with 400 pl of 1 mg/ml RNAse A (Sigma) in PBS, and subsequently stained with 100 pl of 60 pg/ml propidium iodide (PI) (Sigma) in PBS (final PI concentration 12 pg/ml). After 30 min on ice, the cells were analyzed. Southern Blot Analysis Molecular analysis were performed as described (9). Briefly, 20 pg of high molecular weight DNA, extracted from the same biopsy used for the flow cytometric studies, was digested with 3-5 unitsipg of appropriate restriction enzymes (Biolabs, Beverly, MA) ~ agarose gel electrosize-fractionated on 0 . 6 1.4% phoresis, transblotted onto Gene Screen Plus filters (NEN, Boston, MA), and hybridized with DNA probes
RESULTS IF Detection of bcl-2 Intracellular Protein The optimal immunodetection of the bcl-2 mitochondrial protein depends upon the type of fixation and permeation necessary to achieve antibody-ligand interaction. The treatment should minimize the displacement, diffusion, and structural modifications of the molecule. To identify the most sensitive methods for bcl-2 detection, aliquots of the B-lymphoblastoid cell line Raji, reported to express normal size bcl-2 mRNA (141, were fixed with either PFT,BFA, acetone, or METOH, labeled by indirect IF with the anti-bcl-2 MoAb, and examined by flow cytometry. The analysis showed (Fig. 1) that the anti-bcl-2 MoAb reacted best with PFT-fixed cells, as evidenced by the highest number of labeled cells and highest mean fluorescence intensity (MFI). By contrast, acetone and METOH fixations gave poor results, affecting particularly the intensity of reaction. The BFA led to intermediate results. These findings indicate that the PFT fixatiodpermeation procedure is the most suitable for the immunodetection of the bcl-2 intracellular molecule. It is used throughout the course of this study.
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FLOW CYTOMETRY O F BCL-2 PROTO-ONCOGENE
Table 1 Anti-Bcl-2 Reactivity with Normal Lymphocytes and Lymphoblastoid Cell Lines"
lo3.
I
a
bcl-2 Lineage B
T
Cell Soleen Biood BJAB Namalwa Conc F Daudi Raji K422 SU-DHL4 Spleen Blood JM Jurkat
%
MFI
79 96 29 98 89 86 88 89 88 85 90 88 97
124
125 88 125
140 143 133 150 148 120 119 148 139
n
-a w
cv 0
"The flow cytometric measurements were performed by normalizing, for each cell type, the MFI of the negative control (e.g.,cells labeled with normal mouse serum as primary antibody) to channel 36 to adjustments of the photomultiplier tube voltage. This was necessary because of the different levels of autofluorescence and of nonspecific binding.
Bcl-2 Protein Levels in Normal and Neoplastic Lymphoid Cells, and Correlation with bcl-2 Gene Rearrangements Quantification of bcl-2 on lymphocyte subsets was achieved by two-color IF staining for bcl-2 (PE) and either the B- or T-cell markers (FlTC). The results (Table 1) showed that 90.4 2 7.6%and 80 2 10.2% of blood and spleen B-cells, respectively, and 88.2 2 4.3% of T-cells from both tissues were bcl-2 + . The staining intensity was slightly higher in B- than T-cells (MFI difference of 9 ? 3.1 channels) (Fig. 2A). Several T- and B-lymphoblastoid cell lines, including K422 and SU-DHL-4 positive for t(14;18) chromosomal breakpoint, were examined by single-color IF for bcl-2 expression. Given the variable autofluorescence andlor nonspecific staining of each cell line, the comparisons were performed by normalizing the mean fluorescence intensity (MFI) of negative control samples to the same channel (achieved by adjustments of the PMT voltage). Whereas all cases analysed were bcl-2 positive (Table l ) , differences in the intensity of fluorescence were noticed. In particular, BJAB contained less than 30% of bcl-2 positive cells with a MFI of 88; by contrast, K422 and SU-DHL-4 showed > 85% of strongly positive cells; (MFI > 148); this intensity of staining was higher than that measured on other B-cell lines negative for the translocation. Among the T-cell lines, J M were strongly positive for bcl-2 (MFI 148). Given the differential expression of bcl-2 protein in normal lymph node B-cells (e.g., mantle zone positive, germinal centre negative), we determined whether histotype-associated differences in bcl-2 reactivity were present in nodal-derived B-NHLs. In addition, we examined the correlation between the molecular rear-
loo
101
lo2
lo3
pan-B(FITC) FIG.2. Relative levels of bcl-2 protein in normal lymphocytes and B-NHLs. The two-color indirect IF stainings were performed as specified in Methods using antimouse isotype-specific, FITC-, and PEconjugated antibodies. For each histogram, the horizontal and vertical markers were positioned using negative control samples (background staining < 48); numbers in the quadrants (64 X 64 channels) refer to the percentage (upper)and MFI (lower)of positive cells. (a)Blood (left)and spleen lymphocytes (right) are shown. (b)Top left: CBCCF lymphoma with bcl-2 gene in germ-line (lane 3 of Fig. 3); top right: CBCCF with bcl-2 gene rearranged (lane 5 of Fig. 3); bottom left: CBCCF with bcl-2 gene rearranged (lane 2 of Fig. 3); bottom right: sBL.
rangement of the bcl-2 gene and the levels of the protein. The latter question was relevant to the potential diagnostic value of the anti-bcl-2 MoAb. Twenty-two B-NHLs (all expressing membrane monoclonal light chain immunoglobulin; data not shown), 2 of CLL-type (WF:A), 10 centroblastic-centrocytic follicular (CBCCF) (WF: 5B, 4C, lD), 1 centrocytic (CC) (WF:E), 2 CBCC diffuse (CBCCD) (WF:F), 1 centroblastic (CBj (WF:Gj, 2 immunoblastic (IMB) (WF:H), 4 sporadic Burkitt's lymphomas (sBLj (WF:J), were analysed. A hairy cell leukemia variant with splenic involvement (HCLv) was also included. Since most of these tumors contain variable amounts of normal infiltrating T-lymphocytes, the analysis were performed on samples labeled by two-color IF for bcl-2 and the anti-B cell marker L26 (or, in some cases, the T-cell marker CD3).
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AIELLO ET AL.
Table 2 Anti-Bcl-2 Reactivity with B-NHL" Histotypeb CLL CBCCF
cc
CBCCD CB IMB BL
No of cases 2 10 1
2 1
2 4
5
1 2 3 4 % of bcl-2
6
+
B-cells 96 1.5 97.6 ? 2 92 98.5 ? 1.5 85 90 -+ 7 24 ? 14.9
*
- 4.5 Kb
11 Kb-
"Of nine cases studied at molecular level (8 CBCC an 1CB), 4 (3 CBCCJCBCCD; WF: 1 B,IC,lD,lF) presented rearrangements of bcl-2 gene, 2 of which (2 CBCC) overexpressed the bcl-2 protein. Of the remaining cases, the sBL were mostly negative or dimly positive (one case), whereas the others presented normal levels of bcl-2 protein. bCLL: lymphoma of chronic lymphocytic leukemia type (WF:A); CBCCF: centroblastic-centrocytic follicular (WF: B,C,D); CC: centrocytic (WF:E); CBCCD: centroblastic-centrocytic diffuse (WF:F); CB: centroblastic (WF:G); IMB: immunoblastic(WF:H); sBL: sporadic Burkitt's lymphoma (WF:J).
The anti-bcl-2 MoAb reacted with a large fraction of neoplastic cells from the HCLv and the CLL, CBCCF, CBCCD, CB, and IMB histotypes (Table 2). By contrast, 3 of 4 sBL were bcl-2 unreactive; the single positive case was weakly labeled in only half of the tumor cells. Nine lymphomas were analysed by Southern blotting; all presented rearranged bands for the joining region (JH) of the Ig heavy chain gene (not shown) confirming their monoclonal B-cell nature. When tested with the bcl-2 specific probes pFLl and pFL2, four of these cases (3 CBCCF and 1 CBCCD) exhibited rearranged bands with the former probe (Fig. 3). A correlation with the flow cytometric data indicated upregulated levels of bcl-2 protein (e.g., increased MFI) in only two (2 CBCCF) of the four cases (Fig. 2b). Of the lymphomas with bcl-2 in germ-line configuration, one CBCCF presented increased levels of bcl-2 protein.
FIG.3. Southern blot analysis of bcl-2 in B-NHL. Autoradiographs of DNAs digested with PstI or Hind-I11 restriction enzymes and hybridized with the pFL-1 probe (specific for the major breakpoint cluster region of bcl-2 gene) are shown. Rearranged bands are evident in lanes 2,4, and 5. The molecular size of the germ-line bands with each enzyme are indicated. For correlations with bcl-2 protein expression refer to legend to Figure 2.
100
b
Previous studies have noted that activated but not resting T- and B-lymphocytes bear detectable amounts of bcl-2 mRNA, reaching maximal levels within 6 to 12 h after stimulation (28). Yet, immunohistochemical data indicate that the bcl-2 protein is mostly localized in nonproliferating (e.g., Ki-67 negative) lymphocytes (26). We therefore wanted to examine, in a more direct way and by bivariate flow cytometric analysis, the relationship among bcl-2 expression, cell activation, and proliferation. For this purpose, purified T- and B-cells were cultured with mitogenic doses of TPA-Ionomycin and analysed at different intervals of time for bcl-2 and DNA content. The results are illustrated in Figure 4. The lymphocytes (> 80% bcl-2+ at day 0) underwent a transient and moderate increase in bcl-2 reactivity by day 1. By
OT-cells
r9
U
n
+
0
3
W
Bcl-2 Expression in Activated Lymphocytes and Correlation with the Cell Cycle
Hind 111
Pst I
1
I
1
I
3
5
7
days of activation FIG. 4. Time-course bcl-2 protein expression in mitogen-treated lymphocytes. Purified T- and B-lymphocytes were activated with rnitogenic doses of TPA-ionomycin and assayed every 24 h for bcl-2 expression and proliferative status by single and two-color flow cytometry. The percentages of bcl-2 positive cells (overall and in the S-G2/M phase of the cell cycle) obtained from the average of two independent experiments, are reported.
day 2 the reactivity declined, but the fraction of cells in cycle increased markedly. With progression of activation, the number of bcl-2 + T-cells remained constant (70%), but the number of B-cells dropped to 39%.The fraction of proliferating cells also declined. The bcl-2 protein was evidenced, by two-color IF analysis, in all phases of the cell cycle (Fig. 5). I n ad-
FLOW CYTOMETRY O F BCL-2 PKOTO-ONCOGENE
G1 G2/M
t t
106
Q
0 R)
491
102
6
100
10'
102
bcl -2
163
PI (DNA)
FIG.5. Single- and two-color flow cytometric analysis of bcl-2 and cell cycle in activated B-cells. The time-dependent downregulation of the bcl-2 protein is evident from the single-parameter histograms (left). The bcl-2iDNA dot-plots evidence a large fraction of cycling (S-GS/M) cells positive for bcl-2 protein.
dition, most of the cells in S-G2IM were, throughout the course of activation, bcl-2 positive (Fig. 4). These results establish that, in mitogen-stimulated lymphocytes, the expression of bcl-2 is markedly downregulated in B- but not T-cells; in addition, it is not cell-cycle-phase specific.
DISCUSSION The use of immunocytochemical assays based on specific monoclonal antibodies provides a powerful tool to characterize oncoprotein expression, particularly when coupled to flow cytometry (20), which allows quantitative measurements and multiparameter analysis on single cells. The aim of this study was twofold: first, to develop a sensitive immunoassay for the flow cytometric determination of bcl-2 protein in single cells, and, second, to characterize the expression of bcl-2 in normal resting and activated lymphocytes as well as in B-cell lymphomas with or without molecular rearrangements of the gene. Regarding the staining procedure, it was important to identify the conditions that permitted optimal internalization and binding of the anti-bcl-2 MoAb to its intracellular ligand, known to be mostly localized in mitochondria (17). We have shown here that, of a number fixatives, the paraformaldehyde/triton (PFT) combination provides the best specificlbackground staining ratio. A side advantage of PFT is that it affects neither
507
the antigenicity of several membrane molecules (except CD19) nor the interaction of propidium iodide with the DNA, thus enabling the multicolor analysis of bcl-2 in relation to other immunological markers and to cell cycle. We used the PFT protocol to measure the relative levels of bcl-2 protein in normal and neoplastic lymphoid cells. Analysis of normal lymphocytes have shown comparable levels of bcl-2 protein among blood and spleen T- and B-subsets, as evidenced by the MFI measurements. Elevated levels were found on some Tand B-lymphoblastoid cell lines (e.g., JM, Conc-F, Daudi), similar to those found on cell lines carrying the t(14;18) translocation (e.g., K422 and SU-DHL-4). This latter finding is in accordance with Northern blot results showing, in certain cell lines negative for t(14;18), similar amounts of bcl-2 transcripts as in cells carrying the breakpoint (14). Introduction of the bcl-2 gene into the Ig heavy chain locus in t(14;18) bearing B-cell lymphomas leads to its deregulation and markedly increased transcriptional activity (5,14). Concomitantly, the bcl-2 oncoprotein is overproduced, as evidenced by Western blot analysis (4). Therefore, we measured the relative levels of bcl-2 protein in B-NHLs to determine how correlated they were with the molecular rearrangement of the gene. In most cases, two-color IF analysis was performed simultaneously in order to compare the levels of bcl-2 on both neoplastic and normal infiltrating lymphocytes. The following conclusions can be drawn: (1)not all B-NHLs express the bcl-2 protein; indeed, all but one case of sBL were negative, in agreement with previous immunohistochemical findings (26); (2) on bcl-2 positive cases, the protein levels are similar to those of normal infiltrating cells; and (3) not all cases with rearrangements of the bcl-2 gene overexpress the protein. The latter and unexpected result deserves further explanation in view of two recent molecular findings on t(14;18) lymphomas. One, showing that the bcl-2 gene may, in contrast to earlier data (191, carry point mutations in the 5' coding region (22,30) resulting in a n abnormal protein; the other, showing that the nontranslocated bcl-2 allele may not be silent (2). We cannot exclude that any of these factors, if present in our cases, could affect the regulation of bcl-2 (e.g., mRNA stability, translation) oncoprotein levels; further molecular investigations are needed to elucidate this point. The finding that normal proliferating follicle centre cells are bcl-2 negative (26) suggests a close association between the rate of proliferation and failure to express bcl-2. Our results on mitogen-stimulated lymphocytes show that, although found a t normal levels in 75% of activated T-cells, the bcl-2 protein is markedly reduced in activated B-cells (39%). Given that the mitogen causes proliferation of T-cells but terminal differentiation of B-cells, it is conceivable that the down-regulation of bcl-2 in the latter cells is linked to this mecha-
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characterization of ten histiocytic lymphoma cell lines. Cancer 42:2379-2391, 1978. 13. Feinberg AP, Voglestein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13, 1983; addendum 137:266-267,1984. 14. Graninger WB, Set0 M, Boutain B, Goldman P, Korsmeyer SJ: Expression of Bcl-2 and Bcl-2-Ig fusion transcripts in normal and neoplastic cells. J Clin Invest 80:1512-1515, 1987. 15. Gurfinkel N, Unger T, Givol D, Mushinski J F Expression of the bcl-2 gene in mouse B lymphocytic cell lines is differentiation stage specific. Eur J Immunol 17567-570, 1987. 16. Haldar S, Beatty C, Tsujimoto Y, Croce CM: The bcl-2 gene encodes a novel G protein. Nature 342:195-198, 1989. 17. Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ: Bcl-2 is a n inner membrane protein that blocks programmed cell death. Nature 348:334-336, 1990. 18. Hockenbery DM, Zutter M, Hickey W, Naham M, Korsmeyer SJ: BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci (USA) 88:69616965,1991. ACKNOWLEDGMENTS 19. Hua C, Raffeld M, KOHS, Fast P, Bakhshi A, Cossman J: MechThe a r t w o r k for this study w a s by Mario Azzini. Thanks are anisms of bcl-2 activation in human follicular lymphomas. Oncoexpressed t o the Gruppo Italian0 Citometria (GIC) for providgene 5233-235, 1990. ing scientific encouragement. 20. Kastan MB, Stone KD, Civin C I Nuclear oncoprotein expression as a function of lineage, differentiation stage, and proliferative activity status of normal human hematopoietic cells. Blood 74: LITERATURE CITED 1517-1524, 1989. 21. McDonnell TJ, Deane N, Platt FM, Nunez G, Jaeger U, McKearn 1. Aiello A, Delia D, Fontanella E, Giardini R, Rilke F, Della Porta G: Expression of differentiation and adhesion molecules in spoJP, Korsmeyer SJ: Bcl-2-immunoglobulin transgenic mice demradic Burkitt’s lymphomas. Hematol Oncol 8:229-238, 1990. onstrate extended B cell survival and follicular lymphoproliferation. Cell 57:79-88, 1989. 2. Amakawa R, Fukuhara S, Ohno H, Matsuyama F, Kato I, Tanable s, Sideras P, Mizuta TR, Honjo T, Okuma M: Genomic orga- 22. Mikraki V, Ladanyi M, Chaganti RSK. Structural alterations in the 5’ region of the bcl-2 gene in follicular lymphomas with bclnization of IgH gene compared with the expression of bcl-2 gene in t(l4;18)-positive lymphomas. Blood 77:1970-1976, 1991. 2-mbr or bcl-2-mcr rearrangements. Genes, Chromosomes & Can3. Bakhshi A, Jensen J P , Goldman P, Wright AJ, McBride OW, cer 3:117-121, 1991. Epstein AL, Korsmeyer SJ:Cloning the chromosomal breakpoint 23. Monica K, Chen-Levy Z, Cleary ML: Small G proteins are expressed ubiquitously in lymphoid cells and do not correspond to of t(14;18) of human lymphomas: clustering around J H on chroBcl-2. Nature 346:189-191, 1990. mosome 14 and near a transcriptional unit on 18. Cell 41:89924. Nunez G, London L, Hockenbery D, Alexander M, McKearn J P , 906, 1985. Korsmeyr SJ: Deregulated bcl-2 gene expression selectively pro4. Chen-Levy Z, Nourse J, Cleary M L The Bcl-2 candidate protooncogene product is a 24-kilodalton integral membrane protein longs survival of growth factor-deprived hemopoietic cell lines. J highly expressed in lymphoid cell lines and lymphomas carrying Immunol 144:3602-3610, 1990. the t(14;18) translocation. Molec Cell Biol9:701-710, 1989. 25. Nunez G, Hockenbery D, McDonnel TJ, Sorensen CM, Korsmeyer 5. Cleary ML, Smith SD, Sklar J : Cloning and structural analysis of SJ: Bcl-2 maintains B cell memory. Nature 353:71-73, 1991. cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript 26. Pezzella F, Tse AGD, Cordell JL, Pulford KAF, Gatter KC, Mason resulting from the t(14;18) translocation. Cell 47:19-28, 1986. DY: Expression of the bcl-2 oncogene protein is not specific for the 14;18 chromosomal translocation. Am J Pathol 137:225-232, 6. Cleary ML, Sklar J : Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a 1990. breakpoint cluster region near a transcriptionally active locus on 27. Ravetch JV, Siebenlist U, Waldman T, Leder P. Structure of the chromosome 18. Proc Natl Acad Sci USA 82:7449-7453, 1985. immunoglobulin u locus: Characterization of embryonic and re7. Cleary ML, Galili N, Sklar J . Detection of a second t(14;18) breakarranged D and J genes. Cell 27:583-591, 1981. point cluster region in human follicular lymphomas. J Exp Med 28. Reed JC, Tsujimoto Y, Alpers JA, Croce CM, Nowell PC: Regulation of bcl-2 proto-oncogene expression during normal human 164:315-320, 1986. lymphocyte proliferation. Science 236: 1295-1299, 1987. 8. Delia D, Greaves M, Villa S, De Braud F. Characterization of the response of human thymocytes and blood lymphocytes to the 29. Stansfeld AG, Diebold J , Kapanci Y, Kelenyi G, Lennert K, Misynergistic mitogenicity of 12-0-tetradecanoylphorbol-13-acetate oduszewska 0, Noel H, Rilke F, Sundstrom C, Van Unnik J , (TPA)-ionomycin. Eur J Immunol 14:720-724, 1984. Wright DH: Updated Kiel classification for lymphomas. Lancet 9. Delia D, Borrello MG, Berti E, Pierotti MA, Biassoni D, Gianotti 1:292-293, 1988. R, Alessi E, Rizzetti MG, Caputo R, Della Porta G: Clonal immu- 30. Tanaka S, Louie D, Kant J , Reed JC: Frequent incidence of sonoglobulin gene rearrangements and normal T-cell receptor bcl-2 matic point mutations in translocated bcl-2 oncogenes of nonHodgkin’s Lymphomas. Blood 79229-237, 1992. and c-myc genes in primary cutaneous B-cell lymphomas. Cancer Res 494901-4905, 1989. 31. The non-Hodgkin’s lymphoma pathologic classification project: 10. Dorken B, Moller P, Pezzutto A, Shwartz-Albiez R, Moldenhauer National Cancer Institute Sponsored Study of Classification of G B-cell antigens: Section report. In: Leucocyte Typing 1V. W Non-Hodgkin’s lymphomas. Summary and description of a workKnapp et a1 (eds). Oxford University Press, Oxford, 1989, pp 15ing formulation for Clinical Usage. Cancer 49:2112-2135, 1982. 32. 32. Tsujimoto Y, Croce CM: Analysis of the structure, transcripts, 11. Dyer MJS, Fischer P, Nacheva E, Labastide W, Karpas A: A new and protein products of bcl-2, the gene involved in human follichuman B-cell non-Hodgkin’s lymphoma cell line (Karpas 422) ular lymphoma. Proc Natl Acad Sci USA 835214-5218, 1986. exhibiting both t(14;18) and t(4;11) chromosomal translocations. 33. Tsujimoto Y, Ikegaki N, Croce CM: Characterization of the proBlood 75709-714, 1990. tein product of bcl-2, the gene involved in follicular lymphomas. 12. Epstein AL, Levy R, Kim H, Henle W, Henle G, Kaplan HS: Oncogene 2:3-7, 1987. Biology of the human malignant lymphomas. IV. Functional 34. Tsujimoto Y: Stress resistance conferred by high level of bcl-
nism rather than to proliferation. This interpretation is supported by the finding in the mouse that the bcl-2 homologous gene is transcriptionally silent in the very late stages of B-cell differentiation (15), and by our observation t ha t activated T-cells, expanded in vitro for more than 40 days, still contain about 75% of bcl2 + reactive cells (not shown). In conclusion, we have described a sensitive method for the single- and multiparametric analysis of bcl-2 protein by flow cytometry. This approach can be readily applied to study the expression of bcl-2 in normal and neoplastic lymphoid cells in relation to activation, proliferation, and structural abnormalities of the bcl-2 gene.
FLOW CYTOMETRY OF BCL-2 PROTO-ONCOGENE 2a protein in human B lymphoblastoid cell. Oncogene 4: 1331-1336, 1989. 35. Vaux DL, Cory S , Adams S: Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335440-442,1988.
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36. Williams GT: Programmed cell death: apoptosis and oncogenesis. Cell, 651097-1098, 1991. 37. Zutter M, Hockenbery D, Silverman GA, Korsmeyer SJ: Immunolocalization of the Bcl-2 protein within hematopoietic neoplasms. Blood 78:1062-1068, 1991.
0 1992 Wiley-Liss, Inc.
Flow Cytometric Detection of the Mitochondria1 BCL-2 Protein in Normal and Neoplastic Human Lymphoid Cells' Antonella Aiello, Domenico Delia,2Maria Grazia Borrello, Daniela Biassoni, Roberto Giardini, Enrico Fontanella, Francesco Pezzella, Karen Pulford, Marco Pierotti, and Giuseppe Della Porta Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy (A.A., D.D., M.G.B., D.B., R.G., E.F., M.P., G.D.P.) and John Radcliffe Hospital, Headington, Oxford, United Kingdom (F.P., K.P.) 'Received October 3, 1991; accepted December 17, 1991
The bcl-2 proto-oncogene, rearranged and deregulated in B-cell lymphomas bearing the t(14;lB) translocation, encodes an inner mitochondria1 membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single- and multicolor flow cytometric analysis of bcl-2 protein in relation to other markers and cell cycle, based on a fixation-permeation step of cells with paraformaldehyde and Triton XlOO and the use of a bcl-2 specific monoclonal antibody (MoAb).As an application of this method, we have examined the expression of bcl-2 in normal and neoplastic lymphoid cells. We have found that > 80% of normal Tand B-cells are bcl-2 positive; following in vitro mitogen activation, the bcl-2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl-2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two-color analysis. On lym-
The bcl-2 gene is associated with t(14;18) (q32;q21) chromosomal translocation of more than 70% of B-cell lymphomas ( 3 3 . The translocation places the 3' noncoding region of the bcl-2 proto-oncogene into the JH region of the immunoglobulin heavy chain gene (5), giving rise to a chimeric and de-regulated transcript but apparently normal gene product (19). Normal bcl-2 mRNA has been found in activated Band T-lymphocytes and in lymphoblastoid cell lines lacking the t(14;18) chromosomal abnormality (4,14, 28), indicating that bcl-2 is physiologically operative in lymphopoiesis. The recent development of monoclonal antibodies
phoblastoid cell lines, the bcl-2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl-2 gene. Among fresh Bcell non-Hodgkin's lymphomas (B-NHL), most sporadic Burkitt's cases were bcl-2 negative. Of four centroblastic-centrocytic cases with rearrangements of the bcl-2 gene, only two presented elevated amounts of bcl-2 protein, indicating that the levels of bcl-2 are not diagnostic of the translocation. The flow cytometric analysis of bcl-2 protein allows study and quantification,as the single cell level and in selected cell subsets, of the expression of the bcl-2 gene and provides an important tool for assessing its role in hematopoietic cell development, proliferation, and neoplastic conversion. 0 1992 Wiley-Liss, Inc.
Key terms: Bcl-2 proto-oncogene, B-cell lymphoma, gene rearrangements, flow cytometry
(MoAbs) to bcl-2 prompted investigations into the immunohistochemical distribution of the protein in normal and neoplastic tissues (18,26,37). Results indicate that germinal centers, but not mantle zone B-cells, of normal lymph nodes are bcl-2 negative, whereas ger-
This study was financially supported by the Associazione Italiana Ricerca sul Cancro and by the C.N.R. Target Project on Biotechnology and Bioinstrumentation (contract number 89.00242.70). 'Address reprint requests to Dr. Domenico Delia, DIVISION OSA, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy.
FLOW CYTOMETRY O F BCL-2 PROTO-ONCOGENE
minal centers of most follicular lymphomas are bcl-2 positive. In addition, diffuse lymphomas lacking the t(14;18) translocation are bcl-2 + . Hence, although helpful in distinguishing reactive from neoplastic lymphoid follicles, the bcl-2 marker may not be diagnostic of lymphoma carrying the t(14;18) translocation. The bcl-2 proto-oncogene encodes a major nonglycosylated protein of 26 Kd (32,33) recently localized in the inner mitochondria1 membrane (17). Although its biochemical function remains unknown and the GTPbinding activity (16) has not been confirmed (231, several studies show that bcl-2 plays a n important role in prolonging cell survival of growth factor-deprived cells (21,35), in conferring protection from programmed cell death or apoptosis (24,34; see 36 for review), and in maintaining B-cell memory (25). In this report we describe a method for the flow cytometric analysis of bcl-2 protein using a specific monoclonal antibody and immunofluorescence techniques. The method is based on an appropriate fixation and permeabilization of the cells for the optimal interaction of the reagent with its intracellular ligand, thus allowing a rapid and quantitative measurement of bcl-2 and correlation by two-color analysis with other immunological markers and with cell cycle. We provide a number of applications of this flow cytometric approach, demonstrating its usefulness in the study of bcl-2 regulation in normal lymphoid cells as well as in B-NHLs in relation to structural abnormalities of the bcl-2 gene.
MATERIALS AND METHODS Cell Lines and Tissues The B-lymphoblastoid cell lines K-422 and SU-DHL4 positive for the t(14;18) chromosomal translocation (11,12), Raji, Namalwa, ConcF, Daudi, BJAB, and the T-lymphoblastoid cell lines Ju rk at and JM, were maintained at 37°C in a 5% C 0 2 humidified incubator in RPMI 1640 (Flow Laboratories, Irwin, UK) supplemented with 10% heat-inactivated fetal calf serum (Flow), 100 U/ml penicillin, 100 pgiml streptomycin, and 2 mmol/L glutamine (RPMI-C). Lymphoma biopsies were obtained from patients for routine histological diagnosis and classification according to updated Kiel (29) and Working Formulation (31). Peripheral blood (PB) samples were donated by healthy volunteers. Normal spleen tissues were obtained from patients undergoing splenectomy for surgical purposes. Cell suspensions from tissues were prepared by mechanical disruption and enzymatic digestion with 50 U/ml collagenase (Sigma, St Louis, MO) and 40 U/ml DNAse (Sigma). Blood and spleen mononuclear cells were isolated by density centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and washed twice in RPMI 1640 medium (Flow Laboratories, Irvine, UK). Monocytes were removed by adherence onto Petri dishes (1 h at 37°C). Spleen B-cells and blood T-cells were purified by negative immunodepletion using CD15 plus CD3 and CD15 plus CD19 specific mono-
503
clonal antibodies, respectively, and goat-antimouse-labeled magnetic microspheres (DYNAL, Oslo, Norway).
In Vitro Mitogenic Stimulation B- and T-lymphocytes were resuspended in RPMI-C and seeded (lo6cells/ml/well) in 24-well plates (Costar, Cambridge, MA). The phorbol ester TPA (Sigma) and the calcium ionophore Ionomycin (Hoechst-Calbiochem, La Jolla, CA) were used together a t the mitogenic doses of 1 ng/ml and 0.5 pg/ml, respectively (8). Aliquotes of the activated cells were harvested every 24 h and cryopreserved till the end of the culture; subsequently they were thawed out, washed, and stained and analysed.
Fixation protocols For the immunodetection of the intracellular bcl-2 protein, five fixation protocols were tested; in all cases a constant number of cells (lo7)was used. The paraformaldehydeitriton (PFT) protocol was performed by resuspending the cell pellet in 2 ml of 2%paraformaldehyde (freshly prepared at the concentration of 4% in distilled water) diluted, prior to use, in 2 x phosphatebuffered saline (PBS). After 10 min on ice, 100 p1 of 1% Triton XlOO (Sigma) (final concentration 0.05%)were added, and 10 min later the cells were washed twice in cold PBS. For the aceton fixation, the cells were resuspended in 2 ml of cold acetone (-20°C) while gently vortexing, placed in the freezer for 15 min, and then washed twice in PBS. The BFA fixative was made up of 0.2 mg/ml Na2HP04.2H20 pH 6.8, 1 mg/ml KH2P04 pH 6.8, 45% acetone, 9.25% formaldehyde, 47.75% distilled water. The cells were treated for 2 sec at room temperature with 250 pl of BFA, then washed twice in cold PBS plus 0.1% bovine serum albumin (BSA) (Sigma). For the fixation in ice-cold absolute methanol, the cells were resuspended for 15 min in the alcohol (added drop wise to avoid cell clumping); thereafter the cells were washed twice in PBS. Immunofluorescence and Flow Cytometry The T-cell specific MoAb UCHTl (IgG1 isotype;CD3) and the B-cell specific MoAbs LN2 (IgG1; CD74), L26 (IgG2a; unclustered) and HD37 (IgG1,CDlS) MoAbs were obtained from the Fourth International Workshop on Human Leucocyte Differentiation Antigens (10). The anti-bcl-2 MoAbs 100 and 124 (IgG1 and IgG2a isotype, respectively), raised to a synthetic peptide corresponding to amino acids 41 to 54 of the bcl-2 protein, have been previously described (26). As negative control for the immunofluorescence, normal mouse serum at a final concentration of 1:400 was employed. Before labeling, the cells were incubated for 10 min with 2% heat-inactivated human AB serum to prevent nonspecific binding of MoAbs to Fc receptors. Double-color indirect IF staining for bcl-2 and other surface antigens was carried out on microtiter plates as described (1). The fixed cells were incubated with saturating concen-
504
AIELLO ET AL.
74 -
j
108
PFT
r
loo
61
10'
10
I
*
loo
10'
10
FIG. 1. Flow cytometric evaluation of anti-bcl-2 MoAb binding to cells fixed in different ways. The indirect IF assays were performed on the B-lymphoblastoid cell line Raji fixed in various ways as described in Methods. The fluorescence histograms were obtained by normaliz-
ing the MFI of negative controls to channel 36. The upper and lower numbers refer to the percentage and MFI of reactive cells, respectively. The vertical marker, positioned on negative control samples, separates the unreactive (left) from reactive (right) cells.
trations of the anti-bcl-2 MoAb and either the B-cell marker LN2 (or L26) or the T-cell marker UCHT-1, added together. After 30 min on ice, the cells were washed four times in RPMI 1640 +5% heat-inactivated fetal calf serum (FCS, Flow) + 0.02M NaN3, and incubated again (30 min on ice) with isotype-specific, Phycoerythrin (PE) and Fluorescein (F1TC)-conjugated goat antimouse antibodies (Southern Biotechnology, Birmingham, AL) used at 1:30 final dilution. After three more washes, the cells were analysed by flow cytometry using an EPICS-C instrument (Coulter Electronics, Hialeah, FL) equipped with an argon-ion laser (settings: 488 nm, 500 mw). Whenever required, cross-color interferences of two-color labelled samples were eliminated by electronic compensation.
labeled to a specific activity of l + 2 x lo9 c p d p g with 32Pby the random primer DNA procedure (13). The human probes pFLl (6) and pFL2 (7), specific for the major and minor breakpoint cluster regions of bcl-2, respectively, were kindly provided by Dr. M. Cleary, Stanford University. Immunoglobulin heavy chain joining (JH) genes were analysed by a 6.0 Kb BamHIHind111 fragment (27).
Double-color Staining for DNA and bcl-2 PFT-treated cells were first labeled by indirect IF for bcl-2 using, as secondary reagent, a FITC-conjugated antimouse antibody, then incubated (60 min on ice) with 400 pl of 1 mg/ml RNAse A (Sigma) in PBS, and subsequently stained with 100 pl of 60 pg/ml propidium iodide (PI) (Sigma) in PBS (final PI concentration 12 pg/ml). After 30 min on ice, the cells were analyzed. Southern Blot Analysis Molecular analysis were performed as described (9). Briefly, 20 pg of high molecular weight DNA, extracted from the same biopsy used for the flow cytometric studies, was digested with 3-5 unitsipg of appropriate restriction enzymes (Biolabs, Beverly, MA) ~ agarose gel electrosize-fractionated on 0 . 6 1.4% phoresis, transblotted onto Gene Screen Plus filters (NEN, Boston, MA), and hybridized with DNA probes
RESULTS IF Detection of bcl-2 Intracellular Protein The optimal immunodetection of the bcl-2 mitochondrial protein depends upon the type of fixation and permeation necessary to achieve antibody-ligand interaction. The treatment should minimize the displacement, diffusion, and structural modifications of the molecule. To identify the most sensitive methods for bcl-2 detection, aliquots of the B-lymphoblastoid cell line Raji, reported to express normal size bcl-2 mRNA (141, were fixed with either PFT,BFA, acetone, or METOH, labeled by indirect IF with the anti-bcl-2 MoAb, and examined by flow cytometry. The analysis showed (Fig. 1) that the anti-bcl-2 MoAb reacted best with PFT-fixed cells, as evidenced by the highest number of labeled cells and highest mean fluorescence intensity (MFI). By contrast, acetone and METOH fixations gave poor results, affecting particularly the intensity of reaction. The BFA led to intermediate results. These findings indicate that the PFT fixatiodpermeation procedure is the most suitable for the immunodetection of the bcl-2 intracellular molecule. It is used throughout the course of this study.
505
FLOW CYTOMETRY O F BCL-2 PROTO-ONCOGENE
Table 1 Anti-Bcl-2 Reactivity with Normal Lymphocytes and Lymphoblastoid Cell Lines"
lo3.
I
a
bcl-2 Lineage B
T
Cell Soleen Biood BJAB Namalwa Conc F Daudi Raji K422 SU-DHL4 Spleen Blood JM Jurkat
%
MFI
79 96 29 98 89 86 88 89 88 85 90 88 97
124
125 88 125
140 143 133 150 148 120 119 148 139
n
-a w
cv 0
"The flow cytometric measurements were performed by normalizing, for each cell type, the MFI of the negative control (e.g.,cells labeled with normal mouse serum as primary antibody) to channel 36 to adjustments of the photomultiplier tube voltage. This was necessary because of the different levels of autofluorescence and of nonspecific binding.
Bcl-2 Protein Levels in Normal and Neoplastic Lymphoid Cells, and Correlation with bcl-2 Gene Rearrangements Quantification of bcl-2 on lymphocyte subsets was achieved by two-color IF staining for bcl-2 (PE) and either the B- or T-cell markers (FlTC). The results (Table 1) showed that 90.4 2 7.6%and 80 2 10.2% of blood and spleen B-cells, respectively, and 88.2 2 4.3% of T-cells from both tissues were bcl-2 + . The staining intensity was slightly higher in B- than T-cells (MFI difference of 9 ? 3.1 channels) (Fig. 2A). Several T- and B-lymphoblastoid cell lines, including K422 and SU-DHL-4 positive for t(14;18) chromosomal breakpoint, were examined by single-color IF for bcl-2 expression. Given the variable autofluorescence andlor nonspecific staining of each cell line, the comparisons were performed by normalizing the mean fluorescence intensity (MFI) of negative control samples to the same channel (achieved by adjustments of the PMT voltage). Whereas all cases analysed were bcl-2 positive (Table l ) , differences in the intensity of fluorescence were noticed. In particular, BJAB contained less than 30% of bcl-2 positive cells with a MFI of 88; by contrast, K422 and SU-DHL-4 showed > 85% of strongly positive cells; (MFI > 148); this intensity of staining was higher than that measured on other B-cell lines negative for the translocation. Among the T-cell lines, J M were strongly positive for bcl-2 (MFI 148). Given the differential expression of bcl-2 protein in normal lymph node B-cells (e.g., mantle zone positive, germinal centre negative), we determined whether histotype-associated differences in bcl-2 reactivity were present in nodal-derived B-NHLs. In addition, we examined the correlation between the molecular rear-
loo
101
lo2
lo3
pan-B(FITC) FIG.2. Relative levels of bcl-2 protein in normal lymphocytes and B-NHLs. The two-color indirect IF stainings were performed as specified in Methods using antimouse isotype-specific, FITC-, and PEconjugated antibodies. For each histogram, the horizontal and vertical markers were positioned using negative control samples (background staining < 48); numbers in the quadrants (64 X 64 channels) refer to the percentage (upper)and MFI (lower)of positive cells. (a)Blood (left)and spleen lymphocytes (right) are shown. (b)Top left: CBCCF lymphoma with bcl-2 gene in germ-line (lane 3 of Fig. 3); top right: CBCCF with bcl-2 gene rearranged (lane 5 of Fig. 3); bottom left: CBCCF with bcl-2 gene rearranged (lane 2 of Fig. 3); bottom right: sBL.
rangement of the bcl-2 gene and the levels of the protein. The latter question was relevant to the potential diagnostic value of the anti-bcl-2 MoAb. Twenty-two B-NHLs (all expressing membrane monoclonal light chain immunoglobulin; data not shown), 2 of CLL-type (WF:A), 10 centroblastic-centrocytic follicular (CBCCF) (WF: 5B, 4C, lD), 1 centrocytic (CC) (WF:E), 2 CBCC diffuse (CBCCD) (WF:F), 1 centroblastic (CBj (WF:Gj, 2 immunoblastic (IMB) (WF:H), 4 sporadic Burkitt's lymphomas (sBLj (WF:J), were analysed. A hairy cell leukemia variant with splenic involvement (HCLv) was also included. Since most of these tumors contain variable amounts of normal infiltrating T-lymphocytes, the analysis were performed on samples labeled by two-color IF for bcl-2 and the anti-B cell marker L26 (or, in some cases, the T-cell marker CD3).
506
AIELLO ET AL.
Table 2 Anti-Bcl-2 Reactivity with B-NHL" Histotypeb CLL CBCCF
cc
CBCCD CB IMB BL
No of cases 2 10 1
2 1
2 4
5
1 2 3 4 % of bcl-2
6
+
B-cells 96 1.5 97.6 ? 2 92 98.5 ? 1.5 85 90 -+ 7 24 ? 14.9
*
- 4.5 Kb
11 Kb-
"Of nine cases studied at molecular level (8 CBCC an 1CB), 4 (3 CBCCJCBCCD; WF: 1 B,IC,lD,lF) presented rearrangements of bcl-2 gene, 2 of which (2 CBCC) overexpressed the bcl-2 protein. Of the remaining cases, the sBL were mostly negative or dimly positive (one case), whereas the others presented normal levels of bcl-2 protein. bCLL: lymphoma of chronic lymphocytic leukemia type (WF:A); CBCCF: centroblastic-centrocytic follicular (WF: B,C,D); CC: centrocytic (WF:E); CBCCD: centroblastic-centrocytic diffuse (WF:F); CB: centroblastic (WF:G); IMB: immunoblastic(WF:H); sBL: sporadic Burkitt's lymphoma (WF:J).
The anti-bcl-2 MoAb reacted with a large fraction of neoplastic cells from the HCLv and the CLL, CBCCF, CBCCD, CB, and IMB histotypes (Table 2). By contrast, 3 of 4 sBL were bcl-2 unreactive; the single positive case was weakly labeled in only half of the tumor cells. Nine lymphomas were analysed by Southern blotting; all presented rearranged bands for the joining region (JH) of the Ig heavy chain gene (not shown) confirming their monoclonal B-cell nature. When tested with the bcl-2 specific probes pFLl and pFL2, four of these cases (3 CBCCF and 1 CBCCD) exhibited rearranged bands with the former probe (Fig. 3). A correlation with the flow cytometric data indicated upregulated levels of bcl-2 protein (e.g., increased MFI) in only two (2 CBCCF) of the four cases (Fig. 2b). Of the lymphomas with bcl-2 in germ-line configuration, one CBCCF presented increased levels of bcl-2 protein.
FIG.3. Southern blot analysis of bcl-2 in B-NHL. Autoradiographs of DNAs digested with PstI or Hind-I11 restriction enzymes and hybridized with the pFL-1 probe (specific for the major breakpoint cluster region of bcl-2 gene) are shown. Rearranged bands are evident in lanes 2,4, and 5. The molecular size of the germ-line bands with each enzyme are indicated. For correlations with bcl-2 protein expression refer to legend to Figure 2.
100
b
Previous studies have noted that activated but not resting T- and B-lymphocytes bear detectable amounts of bcl-2 mRNA, reaching maximal levels within 6 to 12 h after stimulation (28). Yet, immunohistochemical data indicate that the bcl-2 protein is mostly localized in nonproliferating (e.g., Ki-67 negative) lymphocytes (26). We therefore wanted to examine, in a more direct way and by bivariate flow cytometric analysis, the relationship among bcl-2 expression, cell activation, and proliferation. For this purpose, purified T- and B-cells were cultured with mitogenic doses of TPA-Ionomycin and analysed at different intervals of time for bcl-2 and DNA content. The results are illustrated in Figure 4. The lymphocytes (> 80% bcl-2+ at day 0) underwent a transient and moderate increase in bcl-2 reactivity by day 1. By
OT-cells
r9
U
n
+
0
3
W
Bcl-2 Expression in Activated Lymphocytes and Correlation with the Cell Cycle
Hind 111
Pst I
1
I
1
I
3
5
7
days of activation FIG. 4. Time-course bcl-2 protein expression in mitogen-treated lymphocytes. Purified T- and B-lymphocytes were activated with rnitogenic doses of TPA-ionomycin and assayed every 24 h for bcl-2 expression and proliferative status by single and two-color flow cytometry. The percentages of bcl-2 positive cells (overall and in the S-G2/M phase of the cell cycle) obtained from the average of two independent experiments, are reported.
day 2 the reactivity declined, but the fraction of cells in cycle increased markedly. With progression of activation, the number of bcl-2 + T-cells remained constant (70%), but the number of B-cells dropped to 39%.The fraction of proliferating cells also declined. The bcl-2 protein was evidenced, by two-color IF analysis, in all phases of the cell cycle (Fig. 5). I n ad-
FLOW CYTOMETRY O F BCL-2 PKOTO-ONCOGENE
G1 G2/M
t t
106
Q
0 R)
491
102
6
100
10'
102
bcl -2
163
PI (DNA)
FIG.5. Single- and two-color flow cytometric analysis of bcl-2 and cell cycle in activated B-cells. The time-dependent downregulation of the bcl-2 protein is evident from the single-parameter histograms (left). The bcl-2iDNA dot-plots evidence a large fraction of cycling (S-GS/M) cells positive for bcl-2 protein.
dition, most of the cells in S-G2IM were, throughout the course of activation, bcl-2 positive (Fig. 4). These results establish that, in mitogen-stimulated lymphocytes, the expression of bcl-2 is markedly downregulated in B- but not T-cells; in addition, it is not cell-cycle-phase specific.
DISCUSSION The use of immunocytochemical assays based on specific monoclonal antibodies provides a powerful tool to characterize oncoprotein expression, particularly when coupled to flow cytometry (20), which allows quantitative measurements and multiparameter analysis on single cells. The aim of this study was twofold: first, to develop a sensitive immunoassay for the flow cytometric determination of bcl-2 protein in single cells, and, second, to characterize the expression of bcl-2 in normal resting and activated lymphocytes as well as in B-cell lymphomas with or without molecular rearrangements of the gene. Regarding the staining procedure, it was important to identify the conditions that permitted optimal internalization and binding of the anti-bcl-2 MoAb to its intracellular ligand, known to be mostly localized in mitochondria (17). We have shown here that, of a number fixatives, the paraformaldehyde/triton (PFT) combination provides the best specificlbackground staining ratio. A side advantage of PFT is that it affects neither
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the antigenicity of several membrane molecules (except CD19) nor the interaction of propidium iodide with the DNA, thus enabling the multicolor analysis of bcl-2 in relation to other immunological markers and to cell cycle. We used the PFT protocol to measure the relative levels of bcl-2 protein in normal and neoplastic lymphoid cells. Analysis of normal lymphocytes have shown comparable levels of bcl-2 protein among blood and spleen T- and B-subsets, as evidenced by the MFI measurements. Elevated levels were found on some Tand B-lymphoblastoid cell lines (e.g., JM, Conc-F, Daudi), similar to those found on cell lines carrying the t(14;18) translocation (e.g., K422 and SU-DHL-4). This latter finding is in accordance with Northern blot results showing, in certain cell lines negative for t(14;18), similar amounts of bcl-2 transcripts as in cells carrying the breakpoint (14). Introduction of the bcl-2 gene into the Ig heavy chain locus in t(14;18) bearing B-cell lymphomas leads to its deregulation and markedly increased transcriptional activity (5,14). Concomitantly, the bcl-2 oncoprotein is overproduced, as evidenced by Western blot analysis (4). Therefore, we measured the relative levels of bcl-2 protein in B-NHLs to determine how correlated they were with the molecular rearrangement of the gene. In most cases, two-color IF analysis was performed simultaneously in order to compare the levels of bcl-2 on both neoplastic and normal infiltrating lymphocytes. The following conclusions can be drawn: (1)not all B-NHLs express the bcl-2 protein; indeed, all but one case of sBL were negative, in agreement with previous immunohistochemical findings (26); (2) on bcl-2 positive cases, the protein levels are similar to those of normal infiltrating cells; and (3) not all cases with rearrangements of the bcl-2 gene overexpress the protein. The latter and unexpected result deserves further explanation in view of two recent molecular findings on t(14;18) lymphomas. One, showing that the bcl-2 gene may, in contrast to earlier data (191, carry point mutations in the 5' coding region (22,30) resulting in a n abnormal protein; the other, showing that the nontranslocated bcl-2 allele may not be silent (2). We cannot exclude that any of these factors, if present in our cases, could affect the regulation of bcl-2 (e.g., mRNA stability, translation) oncoprotein levels; further molecular investigations are needed to elucidate this point. The finding that normal proliferating follicle centre cells are bcl-2 negative (26) suggests a close association between the rate of proliferation and failure to express bcl-2. Our results on mitogen-stimulated lymphocytes show that, although found a t normal levels in 75% of activated T-cells, the bcl-2 protein is markedly reduced in activated B-cells (39%). Given that the mitogen causes proliferation of T-cells but terminal differentiation of B-cells, it is conceivable that the down-regulation of bcl-2 in the latter cells is linked to this mecha-
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Hockenbery DM, Zutter M, Hickey W, Naham M, Korsmeyer SJ: BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci (USA) 88:69616965,1991. ACKNOWLEDGMENTS 19. Hua C, Raffeld M, KOHS, Fast P, Bakhshi A, Cossman J: MechThe a r t w o r k for this study w a s by Mario Azzini. Thanks are anisms of bcl-2 activation in human follicular lymphomas. Oncoexpressed t o the Gruppo Italian0 Citometria (GIC) for providgene 5233-235, 1990. ing scientific encouragement. 20. Kastan MB, Stone KD, Civin C I Nuclear oncoprotein expression as a function of lineage, differentiation stage, and proliferative activity status of normal human hematopoietic cells. Blood 74: LITERATURE CITED 1517-1524, 1989. 21. McDonnell TJ, Deane N, Platt FM, Nunez G, Jaeger U, McKearn 1. 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