and normal plasma cells Expression of the bcl-2 gene

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1992 79: 495-502

Expression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells M Pettersson, H Jernberg-Wiklund, LG Larsson, C Sundstrom, I Givol, Y Tsujimoto and K Nilsson

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Expression of the bcl-2 Gene in Human Multiple Myeloma Cell Lines and Normal Plasma Cells By M. Pettersson, H. Jernberg-Wiklund, L.-G. Larsson, C. Sundstrom, 1. Givol, Y . Tsujimoto, and K. Nilsson The bcl-2 gene, encoding a mitochondrial membrane protein suggested to play an important role in cell survival, is translocated into the lg loci in about 80% of human follicular lymphomas, which results in a high level of expression. This report shows that bcl-2 was expressed in eight of eight human multiple myeloma cell lines and in normal lymph node and bone marrow plasma cells. In the majority of the myeloma lines, the level of expression was comparable with that observed in Karpas 422, a follicular lymphoma cell line carrying a 14;18 translocation of the bcl-2 gene. DNA rearrangements of the bel-2 locus were evident in only one of the myeloma cell lines, U-266-1970. In this cell line, which exhibited the highest bcl-2 expression, a fourfold increased copy

number of the bcl-2 gene was estimated by Southern analysis. This amplification was lost in cells of later passages (U-266-1984), suggesting that bel-2 might possibly have played a role in the tumor development in vivo. Our results are in contrast t o previous observations in murine plasmacytoma, in which bcl-2 was shown to be silent. The results also contradict the published observation that bel-2 is not expressed at terminal stages of B-cell differentiation. It is at present unclear whether the high expression of bcl-2 in human myeloma is the result of a deregulated expression associated with the malignant phenotype or a mere reflection of the bcl-2 expression typical of normal plasma cells. 0 1992 by The American Society of Hematology.

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cells. Mature B cells, including murine plasmacytoma and human myeloma, have been reported to resemble pro-B and normal resting B cells in that the expression is downreg~lated.~.'~ In light of the suggested role of Bcl-2 in cell survival, the reported absence of bcl-2 expression in myeloma cells is surprising considering the often long lifespan of normal plasma cells. Therefore, we analyzed the bcl-2 expression in human multiple myeloma cell lines by Northern blot and immunocytochemistry and in cells of non-neoplastic lymph nodes and bone marrows by immunocytochemistry. We report here that the bcl-2 gene was highly expressed in all of the myeloma cell lines examined. The levels of mRNA corresponded to the amount of protein in the cases investigated. The Bcl-2 protein was also observed in normal lymph node mantle zone B cells and in normal lymph node and bone marrow plasma cells.

HE bcl-2 GENE WAS originally defined as the portion of chromosome 18 involved in the t( 14;18)(q32;q21) translocation occuring in most cases ( > 80%) of human follicular lymphoma. This DNA rearrangement juxtaposes the bcl-2 gene with the Ig heavy chain locus.'-3In addition, translocation of the bcl-2 gene into the Ig light chain loci has been reported in chronic lymphocytic leukemia (CLL) at a frequency of about The coding region of the gene is not disrupted by the translocations, which, however, result in a high expression of the bcl-2 gene as compared with the normal counterpart The 25-Kd Bcl-2 protein was recently suggested to be an integral inner mitochondrial membrane protein.' Although the function of the Bcl-2 protein is still essentially unknown, several investigations indicate that the protein plays an important role in cell survival. Introduction of the bcl-2 gene into murine interleukin-3 (IL-3)-dependent cell lines induces survival of these cells in the absence of IL-3. However, the cells do not proliferate, but rather persist in the Go phase of the cell cycle.'" Transgenic mice bearing a bcl-2-Ig minigene develop a polyclonal expansion of IgMIgD' B cells, with a prolonged survival as resting cells in G,."~'2Although deregulated bcl-2 expression appears not to be sufficient for development of fully autonomous malignancy, the Bcl-2 and Myc proteins appear to cooperate to increase the growth rate and tumorigenicity of bone marrow cells from Ep-myc transgenic mice'",'3and EpsteinBarr virus (EBV)-immortalized B cell^.'^-'^ These observations and the recent report that Bcl-2 blocks programmed cell death (apoptosis) of murine pro-B cells' suggest that Bcl-2 contributes to the development of B-cell neoplasia by inducing a prolonged lifespan and thereby increasing the probability of other genetic alterations to occur. The finding of rearrangements of the c-myc gene in high-grade lymphomas in transgenic mice, carrying a bcl-2-Ig minigene, lends support to this hypothesis." Examination of bcl-2 messenger RNA (mRNA) levels in human and murine B cells and B-cell lines, representing different stages of B-cell development, has suggested a differentiation-associated expression of the bcl-2 gene. It is not expressed in resting normal B cells, but is upregulated upon B-cell activation."." The cell line studies suggest that bcl-2 is silent in pro-B cells, but is highly expressed in pre-B Blood, Vol79, No 2 (January 15). 1992: pp 495-502

MATERIALS AND METHODS

Cell lines. All E-cell lines were maintained in RPMI 1640 (Flow Laboratories, Ayrshire, UK) containing 10% fetal calf serum (FCS; GIBCO, Grand Island, NY), glutamine, and antibiotics (100 I U h L of penicillin and 50 pg/mL of streptomycin). The U-266, U-1958, and U-1996 lines were established in our laboratory from patients with clinically aggressive multiple m y e l ~ m a .The ~~~ pheno~' typic properties of early (U-266-1970) and late (U-266-1984) passage U-266 cells were described e l ~ e w h e r e . *U-255 ~ , ~ ~ is a lymphoblastoid cell line derived from the same patient as the U-266 line.24 Karpas 422, carrying a 14;18 tran~location,~ and ~

~

~~~

From the Department of Pathology, University Hospital, Uppsala, Sweden; and The WistarInstitute, Philadelphia, PA. Submitted April 9, 1991; accepted September 23,1991. Suppotted by grants from the Swedish Cancer Society and by Grants No. CA-50551 and CA-51864 from National Institutes of Health (YT ) . Y.T. is a Leukemia Society America Scholar. Address reprint requests to Kenneth Nilsson, MD, Laboratory of Tumor Biology, Dept. of Pathology, University Hospital, Sjukhusvagen 10, S-751 85 Uppsala, Sweden. 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 1992 by The American Society of Hematology. 0006-4971I921 7902-0019$3.00/0 495

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496

U-69SZ6are both B-lymphoma cell lines. Daudi is a Burkitt’s lymphoma*’ and L363, Karpas 707, OPM 1, and OPM 2 are multiple myeloma cell line^.^"^ The human fibroblast cell line AG1523 (The Human Mutant Genetic Cell repository, Camden, NJ) was maintained in Eagle’s supplemented with 10% newborn calf serum, glutamine, and antibiotics. DNA analysis. DNA was extracted from exponentially growing cells. The cell pellet was incubated in 1% sodium dodecyl sulphate (SDS) and 100 kg/mL of proteinase K at 37°C overnight, phenol extracted, and precipitated. Purified DNA was digested with the appropriate restriction enzyme and for Southern analysis the digest was size-fractionated in 0.8% agarose gels before transfer to nitrocellulose filters. The filters were baked and prehybridized for 4 hours at 42°C in a solution containing 50% formamide, 5X Denhardt’s solution, 2X SSC (3 mol/L sodium chloride, 0.3 mol/L sodium citrate, pH 7.0), 5 mmol/L sodium phosphate, 0.1% SDS, and 200 pg/mL of salmon sperm DNA. Hybridization was performed overnight at 42°C in a similar solution except that the concentration of Denhardt’s solution was lowered to 1X. Filters were washed in 0.5% SDS and 2X SSC at 65°C and exposed to Kodak XAR film at -70°C (Eastman Kodak, Rochester, NY). RNA analysis. Total RNA was extracted from exponentially growing cells by the LiCl/Urea method.” Fifteen micrograms of RNA was denaturated in formamide, heat-denaturated for 10 minutes at 65”C, and run in an 1% agarose gel containing 2.2 mol/L formaldehyde. After electrophoresis, the RNA was transferred to nitrocellulose filters and the hybridization and washing were performed as for the DNA filters. DNA probes. Three different bcl-2 probes were used. One probe consists of a 4.5-kb Hind111 fragment and spans the major breakpoint region in the second exon of the bcl-2 gene.” The second probe is specific for the minor breakpoint cluster region and includes the 2-kb fragment called pFL2.” The third probe is the pB16 probe, which contains a part of the first exon.34 The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was obtained from Dr R. Wu (Cornell University, Ithaca, NY) and was used as a quantitative mRNA standard.” The myb probe consists of an EcoRIIBgl I1 fragment from a human c-myb clone.I6The IL-6 probe used is a Taq UBan I1 cDNA fragment.37 Westem blot analysk. Cells were lysed in a buffer containing 25 mmol/L Tris, pH 8, 1% NP40, 0.5% desoxycholate, 144 mmol/L NaCl, 0.1% SDS, 5 mmol/L dithiotreitol (DTT), 1% trasylol, 10 mmol/L iodoacetamide, and 0.5 mmol/L phenylmetylsulphonyl flouride (PMSF) at 4°C. After sonication three times for 15 seconds, the lysates were cleared by centrifugation at 17,000 rpm for 30 minutes. The extract (200 pg/lane) was boiled for 4 minutes in sample buffer (0.1 mol/L Tris, pH 6.8,3% SDS, 5% p-mercaptoethanol), run on 13% SDS/polyacrylamide gel, and subsequently electrotransferred overnight in transfer buffer (25 mmoliL Tris, pH 7.4, 20 mmol/L glycine, 20% methanol, and 0.1% SDS) to a nitrocellulose filter. The filter was preblocked in 2% non-fat milk, 0.05% Tween 20 in phosphate-buffered saline (PBS) for 1 hour before overnight incubation with anti-Bcl-2 antibodies diluted 1:50.)8 Alkaline phosphatase-conjugated goat antimouse Ig (BioRad Laboratories, Richmond, CA) diluted 1:3,000 was used as a second layer. The proteins were visualized by 5-bromo-4-chloro-3indolyl phosphate (BCIP) and p-nitro blue tetrazolium (NBT, Bio-Rad). Immunocytochemkty The myeloma cells were cytocentrifuged, dried for 30 minutes, and then fixed in acetone for 10 minutes. For normal plasma cells, snap-frozen biopsies from 15 reactive lymph nodes with varying amounts of plasma cells were cryosectioned and fixed in acetone as above. In addition, smears of

aspiration biopsies from three non-neoplastic reactive lymph nodes and seven normal bone marrows were stained after acetone fixation. As positive controls, biopsies from four cases of follicular lymphoma (follicular centroblastic-centrocyticlymphoma according to the Kiel classification) were cryosectioned and fixed in acetone. After three washes in PBS, the slides were incubated for 1 hour with monoclonal anti-Bcl-2 antibodies’“ diluted 1:lO. Rabbit antimouse Ig antibodies (Dakopatts, Glostrup, Denmark) were used as a second layer. The subsequent procedures used Vectastatin peroxidase ABC reagents (Vector Laboratories Inc, Burlingame, CA) or the APAAP complex (Dakopatts). The peroxidase activity was visualized by 3-amino-9-ethylcarbazol. Counterstaining was performed with Mayer’s hematoxylin. RESULTS

High expression of bcl-2 mRNA in myeloma cells. To investigate the expression of the bcl-2 gene in multiple myeloma cells, total RNA was prepared from eight wellcharacterized myeloma cell lines and subjected to Northern blot analysis. U-698 and U-255, a B-lymphoma and a B-lymphoblastoid cell line, respectively, were also included in the analysis, as well as normal fibroblasts (AG1523) (Fig 1A). All the myeloma cell lines expressed bcl-2 mRNA and in most cases the expression was very strong, a particularly high level of transcription was found in U-266-1970. U-255 showed an expression similar to that of U-266-1984, whereas U-698 and the fibroblasts were negative. The bcl-2 gene gives rise to three different transcripts due to differential splicing and p~lyadenylation.~~ The longest of these transcripts, 8.5 kb, was the most prominent in this Northern analysis. The high level of bcl-2 mRNA in myeloma cells prompted us to compare it with the bcl-2 expression in a cell line carrying the 14;18 translocation. Total RNA was prepared from Karpas 422, having a rearranged bcl-2 gene, from the myeloma cell lines U-266-1984 and L363 and from the Burkitt’s lymphoma cell line Daudi. As determined by Northern blot analysis, a similar level of bcl-2 mRNA expression was found in Karpas 422 and the myeloma cells, whereas no transcripts could be detected in Daudi cells (Fig 1B). The bcl-2 transcripts in Karpas 422 were not of the same size as in the other cell lines. This result is most likely due to alternative 3’ ends created from the Ig heavy chain locus. The bcl-2 mRNA levels correspond to the amount ofprotein. The finding that bcl-2 mRNA in most myeloma cells was expressed at a similar or even higher (U-266-1970) level than in Karpas 422 carrying the 14;18 translocation was unexpected. To confirm that the mRNA was translated into protein, a Western blot analysis was performed, using cell extracts from Karpas 422, the three myeloma lines U-2661984, U-266-1970, and OPM 2, and the Burkitt’s lymphoma cell line Daudi (with no detectable expression of bcl-2 mRNA). In accordance with the observed mRNA expression, the amount of protein was high in the U-266-1970 cells compared with Karpas 422, OPM 2, and U-266-1984, whereas no protein was detected in Daudi (Fig 2). Expression of Bcl-2 protein in individual cells. TO study the expression of the Bcl-2 protein in individual cells, we used immunocytochemistry. Cells from Karpas 422, Daudi,

From bloodjournal.hematologylibrary.org by guest on March 7, 2013. For personal use only. BCL-2 EXPRESSION IN MULTIPLE MYELOMA

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U-266-1970, and U-266-1984 were stained with monoclonal antihuman Bcl-2 antibodies. The U-266-1970 cells were strongly positive, whereas weaker staining was observed for both U-266-1984 and Karpas 422, thus confirming the results obtained from the Wcstern blot analysis. Daudi was completely negative (Fig 3). The high expression of Bcl-2 protein in U-266-1970 was observed in the majority of the cells, and was not confined to a subpopulation of strongly expressing cells, although some variation in intensity was observed. Examination of Bcl-2 protein in lymph node and bone marrow cells. The high expression of bcl-2 mRNA and protein in the myeloma cell lines could reflect a deregulated expression associated with the malignant phenotype or a feature of normal plasma cells. To investigate the bcl-2 expression in normal plasma cells, 18 reactive lymph nodes (sections and smears) were subjected to immunocytochemical analysis. Mature plasma cells in the medullary cords expressed a strong to moderate positivity that was diffuse and partly granular (Fig 4A and B). A fraction of bone marrow plasma cclls showed a strong, diffuse, and granular Bcl-2 expression (Fig 4C). Lymphocytes and granulocytic precursors weakly expressed Bcl-2, while erythroid cells and megakaryocytes were negative. In control sections of non-neoplastic lymph nodes and follicular lymphomas, Bcl-2 expression was moderate in mantle zone lymphocytes (Fig 4D) and strong in folliclular lymphoma cells (Fig 4E). Germinal center cells of the non-neoplastic lymph node were completely negative (Fig 4D). DNA analysis of the bcl-2 gene in the myeloma cell lines. Because follicular lymphomas carrying the 14;18 translocation usually have a high expression of the bcl-2 gene, we next investigated whether the high level of bcl-2 mRNA found in the myeloma cell lines was due to rearrangements of the bcl-2 gene. DNA samples were prepared from the eight myeloma cell lines and digested with EcoRI or HindIII. As a control for a normal bcl-2 gene pattern, DNA from monocytes was analyzed in parallel. bcl-2 probes from both the major and the minor breakpoint region were used for hybridization. No rearrangements were detectable in any of the myeloma cell lines (Fig 5A, B, and C). The middle band seen in the U-266-1970 digest (Fig SA) is also detected in all the other cell lines after a longer exposure. However, a different pattern was observed when DNA from the Karpas 422 cell line, carrying the translocation, was digested with HindIII and hybridized to the major breakpoint region probe (Fig 5C).Similarly, no rearrangements

18sFig 1. Expression of bcl-2 mRNA. Total RNA was extracted from exponentially growingcells. Fifteenmicrogramsper lane was fractionated on a formaldehyde-containing agarose gel and blotted to a nitrocellulose filter. Hybridizationwas performedwith a probe spanning the major breakpoint region of the bcl-2 gene. (A) The level of bcl-2 mRNA in fibroblasts (AG1523). eight myeloma cell lines, a lymphoblastoid (U-255). and a B-lymphoma (U-698) cell line. The amount of RNA was controlled by hybridizationto a GAPDH probe. (B) Comparison of bcl-2 mRNA levels in Karpas 422 cells, carrying the 14;18 translocation, the two myeloma cell lines U-266-1984 and L363, and the Burkitt‘s lymphoma cell line Daudi.

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498

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Fig 2. Western blot analysis of the Bel-2 protein. The indicated cells were lysed by NP40 treatment and 200 pg extract was electrophoresed on a 13% SDS/polyacrylamide gel. The proteins were electrotransferred to a nitrocellulose filter that then was incubated with anti-Bcl-2 antibodies followed by treatment with alkaline phosphatase-conjugated goat antimouse antibodies. A protein molecular weight standard is indicated.

were seen after analysis of Hind111 or EcoRI digestion of the myeloma DNA when a probe from the 5' end of the gene was used (pB16; data not shown). This part of the gene has recently been shown to be juxtaposed to the Ig light chain locus in several cases of CLL.'" A striking feature with all three probes was that the DNA signals were stronger in U-266-1970 than in any of the other cell lines. By use of the c-myb and IL-6 genes as reference probes, densitrometical scanning of the gel shown in Fig 5A indicates a fourfold amplification of the bcl-2 gene in U-266-1970 compared with U-266-1984, U-1996, and normal monocytes (Fig 6). This amplification must cover any of the flanking regions of the gene because we were unable to

detect rearrangements in the parts of the gene normally involved in translocations. DISCUSSION

We have analyzed the expression of the bcl-2 gene in eight human myeloma cell lines. The results show that bcl-2 is expressed in all cases, with some variation in the level of expression. The majority of the cell lines expressed bcl-2 mRNA at levels comparable with that of the follicular lymphoma cell line Karpas 422, which contains a 14;18 translocation of the bcl-2 gene. The highest expression was observed in U-266-1970, with mRNA and protein levels even higher than in Karpas 422. Based on the expression of bcl-2 in a variety of B-cell tumors of human and murine origin, it has been suggested that bcl-2 expression is differentiation associated.'."' bcl-2 was shown to be expressed primarly at the pre-B stage, but was downregulated at later stages of differentiation and not expressed in several cases of murine plasmacytoma and the human myeloma cell line U-266, representing terminally differentiated B cells. The present results are obviously contradictory to the prcvious reports in that the bcl-2 is expressed at the terminal stages of B-cell differentiation. Apart from the discrepancy with previously reported absence of expression of bcl-2 in U-266," which possibly could be explained by clonal or methodologic differencies, our results suggest important differencies between human myeloma and murine plasmacytoma. The high expression of bcl-2 in the human myeloma cell lines may be a malignancy-associated feature or merely reflect the level of expression in normal plasma cells. Southern blot analysis of DNA from the myeloma cell lines, using bcl-2 probes spanning the regions most frequently involved in DNA rearrangements in follicular lymphomas and CLL, did not provide any evidence for major alterations at the DNA level as a cause of the high level of expression except in one line, the U-266-1970. In this line, a fourfold increased copy number of the bcl-2 gene was found. Because no increased number of chromosome 18 has been observed in this and other myeloma cell lines examined?x~~ll.#l the bcl-2 amplification in U-266-1970 must involve other areas of chromosome 18 than those detected with our bcl-2 probes. A slightly increased copy number of bcl-2 has previously been observed in the murine pre-B-cell line BAL-17, which does not carry a 14;18 translocation, but expresses very high amounts of bcl-2 mRNA." Surprisingly, the amplification found in U-266-1970 (an early passage of U-266) is lost in late passage cells (U-2661984), which also express about four times less bcl-2 mRNA. During the continuous cultivations, U-266 has undergone cytogenetic changes4"and has become independent of IL-6,'' a cytokine essential for growth and survival of feeder cell-dependent human myeloma cell lines. Furthermore, it has increased its growth rate and decreased its capacity for IgE secretion." These alterations are all indicative of the selection of a fast growing, immature phenotype as a consequence of the long-term in vitro culture. The parallel loss of the bcl-2 amplification may indicate that bcl-2 played some role, presumably as a

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MULTIPLE MYELOMA

Fig 3. Expression of the Bcl-2 protein in individual cells. Cells were cytocentrifuged and the slides incubated with monoclonal anti-Bcl-2 antibodies. Peroxidase-conjugated rabbit antimouse antibodies were used for the second layer. (A) U-266-1984; (6) Karpas 422; (C) U-266-1970; (D) Daudi cells.

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Fig 4. Expression of Bcl-2 in lymph nodes and bone marrow. (A) Paraffin section of a reactive lymph node stained by hematoxylin-eosin. The medullary cords with mature plasma cells and lymphocytes is shown. (B) Frozen section stained with Bcl-2 antibody, showing a few positive plasma cells (center and left) and lymphocytes (left and top). The area corresponds t o the one shown in (A). (C) Bcl-2 expression in bone marrow plasma cells. (D) Frozen section of a germinal center with complete lack of Bcl-2 expression within the follicle and moderate expression in the surrounding mantle zone. (E) Frozen section of a follicular lymphoma with strong Bcl-2 expression of follicular cells and surrounding lymphocytes.

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Fig 5. Southem blot analysis of the bcl-2 gene. Fifteen micrograms of DNA from the indicated cell lines was digested with EcoRl or Hindlll and electrophoresed on an agarose gel before transfer t o a nitrocellulose filter. (A) DNA from monocytes and eight myeloma cell lines was digested with EcoRl and hybridized t o the bcl-2 probe spanning the major breakpoint region of the gene. (6) EcoRI-digested DNA from monocytes. U-266-1984. U-266-1970. U-1996, and U-1958 was hybridized t o a probe from the minor breakpoint region of the bcl-2 gene. (C) DNA from Karpas 422, carrying the 14;18 translocation, U-266-1984. U-266-1970. U-1996, and U-1958 was digested with Hindlll and hybridized t o the major breakpoint region bcl-2 probe.

survival gene, during the development of the tumor in vivo, but that such a function may be less important after progressive phenotypic changes in vitro. The difference in expression of bcl-2 in murine and human plasma cells tumors was unexpected. It is possible that the absence of bcl-2 expression in murine plasmacytoma, rather than the presence of bcl-2 in human myeloma,

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represents the abnormal, tumor-associated state. Considering the suggested role of bcl-2 in cell survival, one would expect the gene to be expressed in the long-lived normal plasma cells. Indeed, we observed Bcl-2-positive normal plasma cells in lymph nodes and bone marrows by using immunocytochemical techniques. Also plasma blasts, which develop after induction of differentiation in vitro of B-CLL cells,J2exhibit Bcl-2 positivity as studied by immunocytochemistry (Schena et al, manuscript in preparation). The results from these studies thus support that the presence of bcl-2 in human myeloma may reflect a normal feature of human plasma cells. That the bcl-2 expression in murine plasmacytoma may represent a malignancy-associated characteristic is indicated by the finding that Daudi (this report) and some other Burkitt’s lymphoma cell lines: which, like the plasmacytoma, carry a translocated c-myc gene, contain extremely low levels of bcl-2 mRNA and protein. This finding suggests the possibility that a deregulated c-myc expression, leading to sustained growth, is associated with a downregulation of bcl-2. It is, however, also possible that human myeloma and murine plasmacytoma represent different stages of B-cell differentiation and, as a consequence of this, express different levels of bci-2. The absence of bcl-2 in murine plasmacytoma would then reflect a normal feature of B cells at more immature stages of differentiation, associated with a blast morpho]ogy and a higher growth rate, and would not be malignancy associated. The studies showing a low bcl-2 expression in rapidly proliferating germinal center B cells”.43 and after mitogenic stimulation of B-CLL cells (Schena et

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Fig 6. Amplification of the bcl-2 gene in U-266-1970.The filter used in Fig 5A was rehybridized with c-myb and IL-6 probes and the intensity of the bands obtained with the three different probes was L~~~A, normal monocytes; compared by densitrometical scanning. lane 6, U-266-1984; lane C, U-266-1970; lane D, U-1996.

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al, manuscript in preparation) lend support to this hypothesis. These results may indicate an inverse relationship between bel-2 expression and proliferation in certain types of B cells, which might explain the low level of bcl-2 in mouse plasmacytoma cells.

ACKNOWLEDGMENT

we thank ~~~i~ Str6mstedt for assistance in the Western blot and immunocytochemical analysis. We also thank Dr David Mason for the generous gift of the anti-Bcl-2 antibodies.

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