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Novel targeted deregulation of c-Myc cooperates with Bcl-XL to cause plasma cell neoplasms in mice
Wan Cheung Cheung,1 Joong Su Kim,2 Michael Linden,3 Liangping Peng,2 Brian Van Ness,3 Roberto D. Polakiewicz,1 and Siegfried Janz2
1Cell Signaling Technology Inc., Beverly, Massachusetts, USA. 2Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA. 3University of Minnesota, Minneapolis, Minnesota, USA.
Address correspondence to: Siegfried Janz, Laboratory of Genetics, Center for Cancer Research, NCI, Building 37, Room 3140A, Bethesda, Maryland 20892-4256, USA. Phone: (301) 496-2202; Fax: (301) 402-1031; E-mail: sj4s@nih.gov.
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1Cell Signaling Technology Inc., Beverly, Massachusetts, USA. 2Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA. 3University of Minnesota, Minneapolis, Minnesota, USA.
Address correspondence to: Siegfried Janz, Laboratory of Genetics, Center for Cancer Research, NCI, Building 37, Room 3140A, Bethesda, Maryland 20892-4256, USA. Phone: (301) 496-2202; Fax: (301) 402-1031; E-mail: sj4s@nih.gov.
Find articles by Kim, J. in: JCI | PubMed | Google Scholar
1Cell Signaling Technology Inc., Beverly, Massachusetts, USA. 2Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA. 3University of Minnesota, Minneapolis, Minnesota, USA.
Address correspondence to: Siegfried Janz, Laboratory of Genetics, Center for Cancer Research, NCI, Building 37, Room 3140A, Bethesda, Maryland 20892-4256, USA. Phone: (301) 496-2202; Fax: (301) 402-1031; E-mail: sj4s@nih.gov.
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1Cell Signaling Technology Inc., Beverly, Massachusetts, USA. 2Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA. 3University of Minnesota, Minneapolis, Minnesota, USA.
Address correspondence to: Siegfried Janz, Laboratory of Genetics, Center for Cancer Research, NCI, Building 37, Room 3140A, Bethesda, Maryland 20892-4256, USA. Phone: (301) 496-2202; Fax: (301) 402-1031; E-mail: sj4s@nih.gov.
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1Cell Signaling Technology Inc., Beverly, Massachusetts, USA. 2Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA. 3University of Minnesota, Minneapolis, Minnesota, USA.
Address correspondence to: Siegfried Janz, Laboratory of Genetics, Center for Cancer Research, NCI, Building 37, Room 3140A, Bethesda, Maryland 20892-4256, USA. Phone: (301) 496-2202; Fax: (301) 402-1031; E-mail: sj4s@nih.gov.
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1Cell Signaling Technology Inc., Beverly, Massachusetts, USA. 2Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA. 3University of Minnesota, Minneapolis, Minnesota, USA.
Address correspondence to: Siegfried Janz, Laboratory of Genetics, Center for Cancer Research, NCI, Building 37, Room 3140A, Bethesda, Maryland 20892-4256, USA. Phone: (301) 496-2202; Fax: (301) 402-1031; E-mail: sj4s@nih.gov.
Find articles by Polakiewicz, R. in: JCI | PubMed | Google Scholar
1Cell Signaling Technology Inc., Beverly, Massachusetts, USA. 2Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA. 3University of Minnesota, Minneapolis, Minnesota, USA.
Address correspondence to: Siegfried Janz, Laboratory of Genetics, Center for Cancer Research, NCI, Building 37, Room 3140A, Bethesda, Maryland 20892-4256, USA. Phone: (301) 496-2202; Fax: (301) 402-1031; E-mail: sj4s@nih.gov.
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Published June 15, 2004 - More info
J Clin Invest. 2004;113(12):1763–1773. https://doi.org/10.1172/JCI20369.
© 2004 The American Society for Clinical Investigation
Received: October 23, 2003; Accepted: April 14, 2004
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Methods
Generation of Myc transgenics. Gene targeting (52) and cre-loxP recombination (53) were used to insert a mouse Myc gene into the mouse germ-line Igh locus (Figure 1A). The inserted Myc consisted of an intron-less cDNA clone, the noncoding first exon with the natural P1/P2 promoter, 1.5 kb of genomic 5′ flank containing the normal transcription-regulatory region, and a short stretch of 3′ untranslated region (UTR) harboring the Myc major polyadenylation site. The UTR 3′ of the polyadenylation signal, which has been shown to be dispensable in vivo (54), was not present in the construct. The Myc coding region also contained an artificial histidine tag added in frame at its 3′ end. The tag made it possible to distinguish Myc mRNA and Myc protein encoded by the inserted MycHis gene from message and protein encoded by the normal, endogenous Myc gene. MycHis was inserted in opposite transcriptional orientation in intron 1 of the Cα locus. The construction of the inserted Myc, referred to as iMycCα, the assembly of the targeting vector, and the generation of the transgenic mice are described in the Supplemental Methods section and illustrated in Supplemental Figure 1 (suplemental material available at http://www.jci.org/cgi/content/full/113/12/1763/DC1). The Myc transgenic mice were of mixed C57BL/6 ∞ 129SvJ background (Figure 1C).
Figure 1Experimental overview of the generation of double-transgenic Myc/Bcl-XL mice. (A) Generation of Myc transgenic mice. Shown are the normal mouse Igh locus (top) and the targeted Igh locus with the inserted MycHis gene (bottom). The transcriptional orientation of Igh and MycHis is indicated by a black and a red arrow, respectively. (B) Generation of Bcl-XL transgenic mice. Depicted is a scheme of the Bcl-x transgene, which consists of the mouse 3′ κ enhancer; the promoter of the mouse variable κ gene, Vκ21; the mouse Bcl-x cDNA fused to the Flag epitope_encoding sequence; and the 3′ untranslated region of the human growth hormone (3′ hGH), a facilitator of Bcl-x expression. (C) Myc/Bcl-XL bitransgenics were on a mixed genetic background containing alleles from strains C57BL/6, 129SvJ, and FVB/N.
Generation of Bcl-XL transgenics. Injection of plasmid DNA into male pronuclei of FVB/N zygotes was used to generate the Bcl-XL transgenic mice (Figure 1B). The transgene, referred to as 3′KE-Bcl-XL, was a modification of a previously developed Bcl-x transgene developed by Tim Behrens (University of Minnesota, Minneapolis, Minnesota, USA) (31). It contains the same Bcl-xFlag gene but uses the 3′ κ enhancer and VK21 promoter (excised from plasmid K3′E.KP.LUC; ref. 55) in place of the intronic E∝ enhancer and TK promoter to drive Bcl-x expression. Prior to microinjection, plasmid 3′κE-Bcl-xL was purified by cesium chloride centrifugation and linearized by digestion with NotI/AseI. Data presented in this report were collected using animals from the founder line 1967 (56). The Bcl-XL transgenic mice were of inbred FVB/N background (Figure 1C).
Generation of double-transgenic Myc/Bcl-XL mice. Hemizygous Myc transgenic mice were crossed with hemizygous Bcl-XL transgenic mice to generate bitransgenics, which were born at the expected frequency of approximately 25%. The bitransgenic mice, henceforth referred to as Myc/Bcl-XL mice, were of mixed C57BL/6 ∞ 129SvJ ∞ FVB/N background (Figure 1C). Single-transgenic Myc and Bcl-XL F1 progeny (∼50% of offspring in the above-mentioned cross) and nontransgenic F1 progeny (∼25% of offspring) were used as controls unless otherwise noted. Breeding and maintenance of mice and all experimentation involving mice were approved under Institutional Animal Care and Use Committee Protocol 0006A56361 (University of Minnesota) and Animal Study Protocol LG-028 (National Cancer Institute [NCI]).
Histology and immunohistochemistry. Tissues were fixed overnight in 10% buffered formalin and embedded in paraffin. Deparaffinized tissue sections were stained with H&E for histological examination by light microscopy or left unstained for marker analysis by immunohistochemistry. The latter involved incubation with biotin-conjugated antibodies to mouse B220 (RA3-6B2), CD138 (281-2; both from BD Biosciences, San Jose, California, USA), and Igκ (Southern Biotechnology Associates Inc., Birmingham, Alabama, USA), or incubation with unlabeled antibodies from rabbit to mouse phosphohistone H3 (Ser10) and cleaved caspase-3 (Asp175; both from Cell Signaling Technology Inc., Beverly, Massachusetts, USA) followed by a labeled secondary, goat anti-rabbit antibody. Antibody binding in tissue sections was visualized by the VECTASTAIN ABC alkaline phosphatase kit or the ABC peroxidase kit (Vector Laboratories Inc., Burlingame, California, USA) using Vector Blue or NovaRED as substrates. Endogenous alkaline phosphatase and peroxidase activities were inhibited by levamisole and hydrogen peroxide, respectively. Tissue sections were counterstained with hematoxylin.
ELISA. Serum IgG and IgM concentrations were measured by ELISA using capture antibodies binding to both heavy chains and light chains (2 ∝g/ml; Caltag Laboratories Inc., Burlingame, California, USA). BSA (1%) was used for blocking. Serial dilutions of serum samples (1:2,000, 1:10,000, 1:50,000, 1:250,000, 1:500,000) were incubated at 4°C overnight in coated ELISA plates. Individual isotypes (IgG1, IgG2a, IgG2b, IgG3, and IgM) were detected with the help of biotinylated goat anti-mouse antibodies (Caltag Laboratories Inc.), followed by binding to alkaline phosphatase–conjugated streptavidin (Caltag Laboratories Inc.) and addition of 1 mg/ml alkaline phosphatase substrate in diethanolamine buffer. The optical densities were measured at 405 nm using a microplate reader.
Flow cytometry and cell sorting. To determine subpopulations of lymphocytes in mouse tissues, single-cell suspensions from bone marrow, spleen, lymph nodes, and tumors were treated with FcBlock (anti–CD16/CD32, 2.4G2) and directly stained with mAb’s to mouse B220 (RA3-6B2), CD138 (281–2), CD3 (17A2), or IgM (II/41; all from BD Biosciences). These antibodies were conjugated to FITC, phycoerythrin (PE), allophycocyanin, or biotin. Isotype-specific controls demonstrated the specificity of labeling. To estimate the proliferation of normal B and plasma cells from spleen and bone marrow as well neoplastic plasma cells from plasma cell tumors, BrdU incorporation was measured in vitro according to the manufacturer’s protocol (BD Biosciences). Forty-eight hours after intraperitoneal injection of 0.5 mg BrdU, the cells were stained with allophycocyanin-labeled anti-B220 and PE-labeled anti-CD138 antibodies, fixed, permeabilized, treated with DNase, and incubated with a FITC-labeled antibody to BrdU. To analyze cell cycling in B and plasma cells, the cells were stained with 50 ∝g/ml propidium iodide (PI) in the presence of 0.1% sodium citrate and 0.1% Triton X-100. To determine rates of programmed cell death in B cells and plasma cells, the FITC-conjugated monoclonal active caspase-3 antibody apoptosis kit I from BD Biosciences was used (catalog number 550480). In all experiments, cells were analyzed on a Beckman Coulter FC 500 and sorted on an EPICS ALTRA (Beckman Coulter Inc., Fullerton, California, USA).
Cell sorting with magnetic beads (MACS). To determine proliferation and apoptosis in B splenocytes, the MACS mouse B cell sorting kit with B220 beads (Miltenyi Biotec Inc., Auburn, California, USA) was used to purify B220+ cells. Briefly, spleen cells freed of red blood cells were separated on MACS VS+ columns using magnetic beads conjugated to an antibody to mouse B220 (CD45R). Recovery of B cells from single-cell suspensions was approximately 35%. The remaining cells were either B220– (∼35%) or lost (∼30%) during cell separation. The purity of MACS-sorted B220+ splenocytes was greater than 90%, as determined by FACS analysis.
Immunoblotting. Proteins from clarified lysates of FACS-sorted homogenized cells from spleen, bone marrow, and plasma cell tumors were resolved electrophoretically in denaturing 10% SDS-PAGE gels and transferred by electroblotting to nitrocellulose membranes. Membranes were probed with rabbit anti-mouse antibodies to histidine tag (Cell Signaling Technology Inc.) and Flag tag (Sigma-Aldrich, St. Louis, Missouri, USA), or rabbit anti-mouse antibodies to cyclin D2 (sc-593), cyclin D1 (sc-717), and XBP-1 (sc-7160) from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). The positions of the MycHis and Bcl-xLFlag proteins were visualized using the Phototope-HRP detection system (Cell Signaling Technology Inc.). To confirm equal loading, the membranes were stripped and reprobed using an antibody specific for actin (CLONTECH Laboratories Inc., Palo Alto, California, USA).
Southern analysis. Genomic DNA (20 ∝g) was digested with BamHI and EcoRI, fractionated on a 0.7% agarose gel, transferred to a nylon membrane, and crosslinked under UV light. Following prehybridization (Hybrisol I; Intergen, Gaithersburg, Maryland, USA) at 42°C, the membrane was hybridized to a 1.1-kb Cκ probe labeled with 32P-CTP using a random-priming kit. The probe was generated by PCR using a primer pair that was designed by Michael Kuehl (NCI, NIH): 5′-GATGCTGCACCAACTGTATCCA-3′ and 5′-GGGGTGATCAGCTCTCAG-CTT-3′.
Paraproteins. Paraproteins (M-spikes, extragradients) were detected using the Paragon SPE electrophoresis kit (Beckman Coulter Inc.). Ig isotypes were determined by ELISA using Immulon 2 plates (DYNEX Technologies Inc., Chantilly, Virginia, USA) and isotype-specific goat anti-mouse serum labeled with HRP (Southern Biotechnology Associates Inc.). Mouse serum samples were diluted from 10–3 to 1.28 ∞ 10–5. Plates were read on a Molecular Dynamics (Sunnyvale, California, USA) microplate reader at 450 nm.
DNA sequencing of 3′ VDJ regions. The DNA sequence downstream of rearranged variable (V), diversity (D), and joining (J) gene segments of the Igh locus was determined as described elsewhere (57). DNA was amplified by PCR using a 5′ primer for the third framework region of VHJ558 genes and a 3′ primer that annealed 400 nucleotides downstream of the JH4 gene segment. The sequences of the forward and reverse primers used for tumor clones were 5′-AGCCTG-ACATCTGAGGAC-3′ and 5′-TAGTGTGGAACATTCCTCAC-3′, respectively. PCR amplification conditions were 95°C for 0.5 minutes, 63°C for 0.5 minutes, and 72°C for 1.5 minutes for 35 cycles. The sequences of the forward and reverse primers used for plasmablast clones (controls) were 5′-TTTGAATTCCTGACATCTGAGGACTCTGC-3′ and 5′-TTTGG-ATCCCTCCACCAGACCTCTCTAGA-3′, respectively. PCR conditions were 95°C for 0.5 minutes, 60°C for 0.5 minutes, and 72°C for 1.5 minutes for 30 cycles. PCR products from tumors were sequenced directly, and PCR products from controls were sequenced after cloning into pBluescript (Stratagene, La Jolla, California, USA) at the DNA sequencing facility of Iowa State University (Ames, Iowa, USA). An internal JH4 sequencing primer, iJH4 (5′-CTCCACCAGACCTCTCTAGA-3′), was used in either case.
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Results
Features of single-transgenic Myc and Bcl-XL mice. Myc transgenic mice were generated to recreate and study the molecular and cellular consequences of the Myc-activating T(12;15) translocations that characterize BALB/c PCT (Figure 1A). Several noteworthy features of the newly developed mouse strain suggest that insertion of MycHis into Igh resulted in representative modeling of T(12;15). First is the precise reconstruction of the translocation breakpoint region on mouse der(12); i.e., the 5′-to-5′ juxtaposition of MycHis and Cα. Second is the potential for MycHis to interact with the complete set of correctly spaced Eα enhancers, designed to create the same complex interplay of promoter and enhancer interactions that govern the expression of translocated Myc in mouse PCT. Third is the insertion of MycHis in the Igh chromatin domain, which presumably subjects the gene to the same higher-order regulatory influences (e.g., those imposed by chromatin remodeling and positional effects in the interphase nucleus) that affect Myc involved in the T(12;15) exchange. Bcl-XL mice were generated as part of a program to elucidate in transgenic mice the oncogenic properties of death suppressor genes that are frequently overexpressed in human MM (Figure 1B). The Bcl-x transgene is a modification of a previously developed Bcl-x transgene (31) in which the intronic heavy-chain enhancer E∝ was substituted with the 3′ κ enhancer (56). Compared with E∝, the 3′ κ enhancer delays Bcl-XLFlag expression during B cell development to the mature B cell and plasma cell stage (58), possibly favoring plasma cell tumor formation over B lymphoma development.
Plasmacytosis and hypergammaglobulinemia in double-transgenic Myc/Bcl-XL mice. Enforced expression of Myc and Bcl-x by transcriptional control elements with peak activity in plasma cells (3′ Cα and 3′ κ enhancers) might result in expansion of plasma cells in vivo (plasmacytosis). To evaluate this possibility, four 8-week-old Myc/Bcl-XL mice (Figure 1C) were necropsied, and then lymphoid tissues were examined histologically and immunohistochemically. Four age-matched Myc transgenic mice, Bcl-XL transgenic mice, and nontransgenic littermates were used as controls. Myc/Bcl-XL transgenic mice invariably exhibited splenomegaly (Figure 2A), which was mainly caused by the massive accumulation of plasma cells in extrafollicular areas (Figure 2B). Immunostaining of tissue sections for B220 and κ light-chain expression, analogous to the sections shown in Figure 2B, demonstrated that the plasmacytosis sometimes involved nonlymphoid tissues, such as liver (Supplemental Figure 2). The increased tissue load with plasma cells was accompanied by a marked elevation of serum IgM and IgG in the Myc/Bcl-XL transgenic mice (Figure 2C). Bcl-XL transgenic mice also exhibited increases in serum Ig, but these were more moderate than in Myc/Bcl-XL transgenic mice and limited to three IgG isotypes (IgG3 was not higher in Bcl-XL mice than in Myc and normal mice). Myc transgenic mice were indistinguishable from normal mice (Figure 2C). ELISPOT analysis confirmed the ELISA results. It demonstrated a considerable increase in the number of antibody-producing cells in spleen and bone marrow of Myc/Bcl-XL transgenic mice compared with single-transgenic Myc and Bcl-XL mice and normal mice (Supplemental Figure 3a). Plasma cells obtained from tissues with plasmacytosis were not transplantable into pristane-primed BALB/c nude mice, suggesting that these cells had not yet undergone malignant transformation (Supplemental Figure 3b).
Figure 2Plasmacytosis and hypergammaglobulinemia in tumor-free Myc/Bcl-XL transgenic mice. (A) Splenomegaly in double-transgenic Myc/Bcl-XL mice relative to age-matched single-transgenic Myc and Bcl-XL mice and nontransgenic littermate controls. (B) Massive accumulation of plasmablasts and plasma cells in extrafollicular areas of the spleen in Myc/Bcl-XL transgenics. Left: Low-power view of follicular B cells (top) and plasma cells immunostained for B220 and κ light-chain expression, respectively (original magnification, ∞4). Right: High-power view of plasmablasts and plasma cells stained with H&E (original magnification, ∞63). (C) Marked elevation of serum Ig’s in Myc/Bcl-XL transgenic mice, measured by ELISA. *Significant difference (P < 0.05) by Student’s t test.
Figure 3Plasmacytosis in Myc/Bcl-XL transgenic mice. (A) The percentage of B220–CD138+ plasma cells in the bone marrow (top row) and the spleen (bottom row) is indicated in the lower right quadrants of the depicted FACS scatter plots. The percentage of B220–CD3+ T cells in the spleen (center row) is indicated in the upper left quadrants. The red arrow denotes a population of B220+CD138+ plasmablasts in the bone marrow of Myc transgenic mice. (B) FACS-sorted B220+CD138+ cells from the bone marrow of Myc transgenic mice express B220 (top), CD138 (center), and Igκ (not shown) by immunostaining and exhibit cytological features of plasmablasts and plasma cells by H&E staining (bottom; original magnification, ∞63).
Bcl-XL promotes survival of MycHis-harboring plasmablasts. To better quantify the apparent expansion of plasma cells in Myc/Bcl-XL transgenic mice, FACS analysis of bone marrow cells and splenocytes was performed. Six 6- to 10-week-old bitransgenics were compared with six age-matched single-transgenics and normal mice. Representative examples of each strain are shown in Figure 3A. Myc/Bcl-XL transgenic mice harbored up to 63% (average 42%, minimum 19%) B220–CD138hi plasma cells in the bone marrow, a striking increase compared with the Myc transgenics, Bcl-XL transgenics, and normal mice (Figure 3A, top). Similarly, Myc/Bcl-XL spleen contained up to 57% plasma cells (average 34%, minimum 14%), whereas spleen from the Myc transgenic, Bcl-XL transgenic, and normal mice contained less than 2% plasma cells (Figure 3A, bottom). Although the relative number of B220–CD3+ T cells was reduced in spleen from Myc/Bcl-XL bitransgenics compared with the other strains (Figure 3A, center), the absolute number of T cells remained comparable (because of the splenomegaly of strain Myc/Bcl-XL). Thus, neither transgene by itself, nor the combination of both transgenes grossly interfered with T cell homeostasis in the spleen. Studies with lymph nodes and bone marrow confirmed this observation (results not shown). A special feature of the Myc transgenic mice relative to all other mice was the presence of a distinct population of B220hiCD138+ cells in the bone marrow (22.5% in the example shown in the top panel of Figure 3A). Although the nature of these cells has not been fully elucidated, they likely represent plasmablasts (Figure 3B). The presence of expanded populations of plasmablasts in the Myc mice and plasma cells in the Myc/Bcl-XL mice suggested that most plasmablasts harboring only deregulated Myc fail to undergo terminal differentiation. However, when protected by Bcl-XL, Myc transgenic plasmablasts survive and mature into plasma cells.
Increased turnover of Myc-harboring B cells is reduced by Bcl-XL. Myc’s potential to induce proliferation can be mitigated in vivo by Myc’s ability to trigger apoptosis (59). To compare proliferation and apoptosis in mature B cells of Myc/Bcl-XL bitransgenic mice with those in single-transgenic Myc and Bcl-XL mice and nontransgenic littermates, tissue sections of lymph nodes from 8-week-old mice were immunostained with antibody to phosphohistone H3 (a marker of mitosis) and cleaved caspase-3 (a marker of apoptosis). B cell proliferation in lymph nodes, predominantly in follicles and germinal centers, was most vigorous in Myc transgenic mice, followed by Myc/Bcl-XL transgenic, normal, and Bcl-XL transgenic mice, which had the lowest (Figure 4). To better evaluate the apparent increase in Myc-dependent proliferation, FACS analysis of PI-stained cells was combined with BrdU labeling in vivo. Myc-harboring B cells proliferated nearly 2.5 times faster by PI staining (Supplemental Figure 4a, left) and three times faster by PI/BrdU staining (Supplemental Figure 4a, center) than the controls. Apoptosis in B cells measured by immunostaining for cleaved caspase-3 was also highest in Myc transgenic mice, lowest in Bcl-XL transgenic mice, and intermediate in Myc/Bcl-XL and normal mice (Figure 4). TUNEL of lymph node, spleen, and bone marrow sections confirmed this result (not shown). To better assess the apparent increase in Myc-dependent apoptosis, freshly isolated Myc-harboring B cells were analyzed by FACS for activation of caspase-3. Apoptosis in these B cells was elevated approximately 4.5-fold compared with normal B cells (Supplemental Figure 4a, right). The propensity of Myc-containing B cells to undergo apoptosis was further increased (about twofold) upon activation of cells in vitro with LPS, anti-IgM, or both (Supplemental Figure 4b). Together, these findings indicated that the high turnover of B cells in the Myc transgenic mice is attenuated by Bcl-XL.
Figure 4Proliferation and apoptosis in lymph node follicles of 8-week-old Myc, Myc/Bcl-XL, and Bcl-XL mice compared with normal nontransgenic littermates. (A) Sections of a peripheral lymph node containing two follicles. Follicular B cells undergoing proliferation or apoptosis are immunostained (brown spots) for phosphohistone H3 or activated caspase-3, respectively (original magnification, ∞40). The images are ordered from top to bottom according to the apparent turnover of follicular B cells, which was highest in the Myc mice and lowest in the Bcl-XL mice. (B) The result of the microscopic enumeration of proliferating B cells (black bars) and dying B cells (gray bars) in lymph node follicles. Three lymph nodes of each mouse strain were evaluated to determine mean values and SDs of the mean.
Myc/Bcl-XL CD138+ cells actively proliferate. Normal plasma cells are end-stage B cells that have lost the ability to proliferate. To evaluate whether plasma cells of Myc/Bcl-XL transgenic mice adhered to this rule, immunostaining of tissue sections for phosphorylated histone H3 was combined with immunostaining for syndecan-1 (CD138, a marker for plasmablasts and plasma cells) or B220 (CD45, a pan–B cell marker). Doubly stained spleen sections from 8-week-old mice clearly showed that CD138+ cells participated in cell cycling (Supplemental Figure 5a, left). The same result was obtained with lymph node and bone marrow sections (not shown), and when BrdU incorporation in vivo was used as the indicator of proliferation instead of phosphorylated histone H3 (not shown). Double staining of spleen sections (Supplemental Figure 5a, right) or lymph node and bone marrow sections (not shown) for cleaved caspase-3 and CD138 indicated that Myc/Bcl-XL plasmablasts and plasma cells underwent apoptosis. This observation was confirmed with TUNEL of lymph node, spleen, and bone marrow sections (data not shown). Of importance, the semiquantitative comparison of the extent of proliferation and apoptosis in serial tissue sections of double-transgenic mice, such as those shown in Supplemental Figure 5b, indicated that although there was ongoing apoptosis in Myc/Bcl-XL CD138+ cells, the balance was tipped in favor of proliferation. This resulted in an enormous expansion of plasma cells, which was unique to the Myc/Bcl-XL mice and not observed in single-transgenic and control mice. These findings suggested that the interaction of Myc and Bcl-XL results in an expanded pool of actively proliferating CD138+ cells (most likely plasmablasts) in the double-transgenic mice.
Figure 5Plasma cell neoplasms in Myc/Bcl-XL transgenic mice. (A) Bcl-XL transgenic mice (n = 22), nontransgenic littermates (n = 10, not shown), and C57BL/6 mice (n = 18) remained tumor free. Three of four tumors that developed in Myc transgenic mice (n = 43) were diffuse large B cell lymphomas containing abundant neoplastic centroblasts (inset, H&E; original magnification, _63). (B) Typical Myc/Bcl-XL plasma cell tumor stained with H&E (original magnification, _63). (C) Myc/Bcl-XL plasma cell neoplasm immunostained for CD138 (brown; original magnification, _40). The inset shows a FACS profile of the vigorously proliferating propidium iodine_stained tumor cells. (D) Western analysis of five Myc/Bcl-XL plasma cell tumors for expression of cyclin D2 (Myc target), XBP-1 (plasma cell transcription factor), and actin (loading control, top panel). Cyclin D1/D2 expression in Myc/Bcl-XL plasma cell tumors (lanes 1_4) compared with “premalignant” plasmablasts from tumor-free Myc/Bcl-XL mice (lane 5, bottom panel). (E) Western analysis of MycHis and Bcl-XLFlag expression in flow-sorted Myc/Bcl-XL tumor cells (lanes 5_6) compared with cells from spleen (lanes 1_4) and bone marrow (lanes 7_8) of tumor-free Myc/Bcl-XL mice. Bcl-XL occurs in two alternative splice forms, with and without transmembrane domain. Myc and Bcl-XL were detected with antibodies against the histidine and Flag epitopes. (F) Southern analysis for clonotypic Igκ rearrangements in PCNs (lanes 2_4, 6, and 7), spleen (SPL) with plasma cell hyperplasia (PCH; lane 1), thymus (lane 5), and peripheral blood leukocytes (PBL; lane 8) from three Myc/Bcl-XL mice compared with liver and spleen from nontransgenic littermates. ALN, axillary LN; ILN, inguinal LN; MLN, mesenteric LN; GL, germline.
Rapid development of plasma cell tumors in Myc/Bcl-XL transgenic mice. To evaluate whether the sustained proliferation of Myc/Bcl-XL plasma cells leads to the development of plasma cell neoplasms, 11 double-transgenic mice were monitored for tumor incidence and survival (Figure 5A). Unlike Bcl-XL transgenic mice (n = 22) and normal mice (n = 18), which remained tumor free by 380 days of age, Myc/Bcl-XL mice exhibited a drastically reduced survival. This was caused by malignant plasma cell tumors (Figure 5B) that developed rapidly (mean onset 135 days) and with full penetrance (incidence 100%). Myc transgenic mice (n = 43) also developed neoplasms, but tumor development took a long time (mean onset 330 days), occurred with low penetrance (incidence 9.3%), and resulted in B cell lymphomas rather than plasma cell tumors: diffuse large B cell lymphoma in three of four cases (Figure 5A, inset) and unclassified B lymphoma in one case (not shown). The weak tumor phenotype of the Myc mice suggested that although the MycHis transgene reproduced the requisite molecular changes that initiate neoplastic B cell and plasma cell development in mice, MycHis’s true oncogenic potential in vivo was tempered in the absence of Bcl-XL.
Features of Myc/Bcl-XL plasma cell tumors. Plasma cell neoplasms of Myc/Bcl-XL transgenic mice were CD138+ by immunohistochemistry (Figure 5C), proliferated vigorously by flow cytometry of PI-stained tumor cells (Figure 5C, inset), and expressed transcription factors typical of plasma cells, such as XBP-1 and Blimp-1 message (RT-PCR results not shown) and XBP-1 protein (Figure 5D, top). Immunoblotting showed that Myc/Bcl-XL tumor cells expressed Myc and Bcl-XL at higher levels (Figure 5E, lanes 5 and 6) than plasma cells from tumor-free Myc/Bcl-XL mice (lanes 1 and 8). Flow-sorted tumor cells had a mean MycHis/actin ratio of 2.75 and thus contained 1.8-fold and 2.6-fold higher MycHis protein levels than flow-sorted plasma cells from tumor-free spleen (lane 1) and bone marrow (lane 8), respectively. Consistent with the high Myc level, cyclin D2, a validated Myc target (60), and cyclin D1 were overexpressed in the tumors (Figure 5D, bottom, lanes 1–4) relative to B220+CD138+ plasmablasts from tumor-free Myc/Bcl-XL mice (control, lane 5). Myc/Bcl-XL tumors were readily transplantable upon transfer of fewer than 106 tumor cells into pristane-primed BALB/c nude mice, with tumor take occurring in less than 2 weeks in two of two cases (not shown). Continuous cell lines were readily derived from two additional cases of primary plasma cell neoplasia. Together, these findings indicated that Myc/Bcl-XL transgenic mice are predisposed to plasma cell neoplasms that exhibit high levels of MycHis and Bcl-XLFlag and that have completed malignant transformation.
Origin and distribution of plasma cell tumors. The presentation of Myc/Bcl-XL transgenic mice with plasma cell tumors in multiple tissues raised the question of whether the tumors originated from a single precursor with the potential for early, widespread dissemination (monocentric origin), or from multiple precursors resulting in the outgrowth of tumors with different molecular features (multicentric origin). To investigate this, genomic tumor DNA was analyzed by Southern hybridization for clonotypic Igκ rearrangements (Figure 5F). Three different states of tumor dissemination were observed, often coexisting in the same mouse. Some tissues, such as the spleen of mouse no. 1, a borderline case of plasma cell hyper- and neoplasia, harbored multiple clones of aberrant plasma cells (lane 1), reminiscent of the clonal diversity observed in early stages of peritoneal plasmacytomagenesis in BALB/c mice (61). Other tissues, such as the mesenteric lymph node of mouse no. 3 (lane 7), contained one dominant tumor clone (yellow asterisk) that had essentially replaced the normal tissue, as reflected by the loss of the κ germ-line fragment. A third category of tissues, e.g., the lymph nodes in mouse no. 2 (lanes 2–4), harbored different tumor cell clones with distinct κ rearrangements (red asterisks). The detection of an additional cell clone in the peripheral blood leukocyte sample of the same mouse (lane 8, blue asterisk) indicated the emergence of a leukemic clone that had infiltrated the spleen (lane 6) but had not yet reached the thymus (lane 5). These findings reflected a considerable variability in the progression of Myc/Bcl-XL plasma cell tumors. Some tumors remained confined to lymphoid tissues, where they evolved in mono- or multicentric fashion, whereas other tumors acquired the ability for systemic dissemination, leading to full-blown plasma cell leukemia in some cases (not shown).
Bone marrow involvement of plasma cell tumors. Myc/Bcl-XL plasma cell tumors demonstrated proclivity to bone marrow involvement, generating coalescent, wall-to-wall tumor masses in several mice with terminal neoplasia (Figure 6A). Histological examination of bone sections from mice with less advanced tumors routinely revealed multifocal lesions of aberrant, pleomorphic plasma cells adjacent to diminished or dissolved osseous trabeculae or located in resorption pits at the inner surface of the corticalis (Figure 6B). Some bone sections contained large sheets of neoplastic plasma cells in soft tissues surrounding the bones, indicating that the tumor had penetrated the corticalis at an obscure, nearby site. Whole-body radiographs showed osteolytic lesions and putative pathological fractures in long bones of three mice (Figure 6C). Serum and peritoneal lavage fluid of tumor-bearing mice often contained monoclonal Ig spikes (extragradients, M components) that were readily detectable by protein electrophoresis (Figure 6D). Two of three tumors from mice without evidence for M-spikes in lavage samples (Figure 6D, lanes 3, 5, and 7) did not express heavy chain (PCN-3) or light chain (PCN-7) by Western analysis (result not shown), possibly because Ig genes were deleted because of genomic instability in the tumors. To determine whether Myc/Bcl-XL plasma cell tumors exhibit evidence for hypermutation of expressed Ig genes, the 344-bp 3′ JH4 region just downstream of the rearranged VDJ gene was sequenced (Figure 6E, top). Ten tumor-derived clones with unique VH rearrangements were assessed for frequency and type of somatic mutations, and then compared with ten analogous clones from plasmablasts of tumor-free Myc/Bcl-XL mice (controls). Fifteen mutations were identified in the tumors, and four mutations were found in the plasmablasts (P = 0.022, χ2 analysis; Figure 6E, center left). The corresponding mutation frequencies in the tumors and plasmablasts were 4.36 ∞ 10–3 per bp and 1.16 ∞ 10–3 per bp, respectively (Figure 6E, center right). All mutations except one (a 1-bp deletion) were base substitutions (Figure 6E, bottom). Three mutations in the tumors occurred at WA dinucleotides (W = A or T), a preferred target of the VDJ hypermutation machinery (Supplemental Figure 6). Although these findings are too preliminary for firm conclusions, they suggest that Myc/Bcl-XL plasma cell tumors have germinal center experience.
Figure 6Myc/Bcl-XL PCNs infiltrate the bone marrow, produce monoclonal Ig, and cause osteolytic lesions. (A) Bone marrow infiltration with Igκ-producing neoplastic plasma cells (original magnification, _63). (B) Palisades of neoplastic plasma cells (brown) destroying the luminal face of a femur’s corticalis (IgG immunostaining; original magnification, _40). Two bone resorption lacunae are denoted by arrows. (C) Radiographs of large osteolytic lesion with apparent pathological fracture (1, left humerus), osteolytic lesion without fracture (2, right femur), and hairline fracture without visible osteolytic lesion (3, left forearm). (D) Protein electropherogram of serum and peritoneal lavage samples containing M-spikes (red arrows) isotyped using ELISA (bottom). (E) Frequency and type of mutations in the 3′ JH4 region of rearranged variable (V) genes. Shown at the top is a scheme of the mouse Igh locus. PCR primers J558 and JH4 were used to amplify rearranged VDJ genes and linked 3′JH4 sequences. Sequencing primers iJH4-5′ and iJH4-3′ were used to detect mutations in the 344-bp 3′ JH4 region. Shown in the center are bar diagrams of the number of mutations in the 3′ JH4 region in PCNs and plasmablasts from tumor-free Myc/Bcl-XL mice (PB, left panel). The corresponding mutation frequencies (mutations/3440 bp) are plotted to the right. Shown at the bottom are types and occurrences of base substitution mutations in the 3′ JH4 region of rearranged VH genes in PCN and PB. The tumor sample also contained a deletion, δT. The location of the mutation in the 3′ JH4 regions is depicted in Supplemental Figure 6.
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