The use of animal models in multiple myeloma.

In myeloma, the understanding of the tissular, cellular and molecular mechanisms of the interactions between tumor plasma cells and bone cells have progressed from in vitro and in vivo studies. However none of the known animal models of myeloma reproduce exactly the human form of the disease. There are currently three types of animal models: (1) injection of pristane oil in BALB/c mice leads to intraperitoneal plasmacytomas but without bone marrow colonization and osteolysis; (2) injection of malignant plasma cell lines in immunodeficient mice SCID or NOD/SCID; the use of the SCID-hu or SCID-rab model allows the use of fresh plasma cells obtained from MM patients; (3) injection of allogeneic malignant plasma cells (5T2MM, 5T33) in the C57BL/KalwRij mouse induces bone marrow proliferation and osteolytic lesions. These cells did not grow in vitro and can be propagated by injection of plasma cells isolated from bone marrow of a mouse at end stage of the disease into young recipient mice. The 5TGM1 is a subclone of 5T33MM cells and can grow in vitro. Among the different models, the 5TMM models and SCID-hu/SCID-rab models were extensively used to test pathophysiological hypotheses and to assess anti-osteoclastic, anti-osteoblastic or anti-tumor therapies in myeloma. In the present review, we report the different types of animal models of MM and describe their interests and limitations.

Insertional hypermutation in mineral oil-induced plasmacytomas.

Unless stimulated by a chronic inflammatory agent, such as mineral oil, plasma cell tumors are rare in young BALB/c mice. This raises the questions: What do inflammatory tissues provide to promote mutagenesis? And what is the nature of mutagenesis? We determined that mineral oil-induced plasmacytomas produce large amounts of endogenous retroelements--ecotropic and polytropic murine leukemia virus and intracisternal A particles. Therefore, plasmacytoma formation might occur, in part, by de novo insertion of these retroelements, induced or helped by the inflammation. We recovered up to ten de novo insertions in a single plasmacytoma, mostly in genes with common retroviral integration sites. Additional integrations accompany tumor evolution from a solid tumor through several generations in cell culture. The high frequency of de novo integrations into cancer genes suggests that endogenous retroelements are coresponsible for plasmacytoma formation and progression in BALB/c mice.

The bisphosphonate zoledronic acid inhibits the development of plasmacytoma induced in BALB/c mice by intraperitoneal injection of pristane.

OBJECTIVES: Bisphosphonates (BPs) are mostly used in the palliative care of myeloma-associated osteolytic lesions. Recent studies have suggested that BPs may also exert direct antitumor effects on myeloma cells. We have investigated the effect of the potent bisphosphonate, zoledronic acid (ZOL), on the development of pristane (2,6,10,14-tetramethylpentadecane)-induced plasmacytoma (PCT) in six-week-old BALB/c mice. METHODS: Different groups of pristane-treated mice also received ZOL (100 microg/kg) commencing after the development of PCT or ZOL (20 microg/kg) from the first day. Control groups received pristane alone, ZOL alone (20 microg/kg), or phosphate-buffered saline. The study was terminated on day 300, and the remaining mice were autopsied and abdominal tissues were examined histologically for PCT. RESULTS AND CONCLUSIONS: Statistical analysis revealed a significant delay in PCT development in the group receiving pristane plus ZOL (20 microg/kg) from the first day compared to the groups receiving pristane alone and pristane combined with ZOL (100 microg/kg) after the appearance of PCT (Log-rank, P = 0.0001 and 0.0001; respectively). Kaplan-Meier analysis revealed a significant difference in survival between the group treated with pristane alone and the groups receiving pristane plus ZOL (20 microg/kg) from the first day or ZOL (100 microg/kg) after the appearance of PCT (Log-rank, P = 0.016 and 0.023; respectively). These results indicate a direct anti-tumor effect of ZOL in pristane-induced PCT development BALB/c mice, which may contribute to their significantly increased survival. This hypothesis should now be further investigated in clinical trials.

BCL2 accelerates inflammation-induced BALB/c plasmacytomas and promotes novel tumors with coexisting T(12;15) and T(6;15) translocations.

Previous studies on peritoneal plasmacytomas (PCTs) in BALB/c (C) mice suggested that the enforced expression of the death repressor BCL2 in B cells might facilitate the malignant transformation of aberrant B cells containing Myc-activating T(12;15) translocations, generating an improved model of plasmacytomagenesis. To investigate this hypothesis, we backcrossed a human BCL2 transgene onto strain C and performed a PCT induction study with pristane in the newly generated C.BCL2 congenics. In specific pathogen-free-maintained C.BCL2 mice, PCT incidence (19 of 34, 56%) was 24 times higher than in specific pathogen-free-maintained C mice (1 of 44, 2.3%), and tumor onset (113 days) was half that of conventionally maintained C mice (220 days). BCL2 transgenic PCT harbored T(12;15) translocations (12 of 12 tumors) with an unusual clustering of translocation breakpoints in the near 5' flank of Myc (4 of 5 tumors, 80%). Five tumors contained coexisting T(12;15) and T(6;15) translocations (not observed in >300 karyotyped PCTs from conventionally maintained C mice). BCL2 transgenic C57BL/6 mice exclusively developed B lymphomas (11 of 20, 55%) that also contained T(12;15) translocations (11 of 11 cases) with breakpoints in the near 5' flank of Myc (five of five tumors). We conclude that BCL2 accelerates PCT with novel Myc-activating translocations independently of environmental antigen stimulation. Accelerated plasmacytomagenesis in strain C.BCL2 may be useful for designing and testing BCL2 inhibition strategies in human plasma cell tumors overexpressing BCL2, such as Waldenström's macroglobulinemia and multiple myeloma.

The impact of p53 loss on murine plasmacytoma development.

Mouse plasmacytomas (PCTs) are characterized by c-myc-activating translocations that juxtapose c-myc on chromosome 15 onto one of the immunoglobulin loci (IgH on chromosome 12, IgK on chromosome 6, or IgA on chromosome 16). To assess the impact of p53 loss on PCT genesis, we induced PCTs in p53-deficient BALB/cRb6.15 mouse strains. We show that p53 loss accelerates tumor development and causes a shift in the typical translocation patterns. PCTs that carry variant T(6;15) translocations become as frequent as those with typical T(12;15) translocations (41.66%). In addition, in the absence of p53, the number of translocation-negative PCTs increases from less than 1% to 16.66%. It is noteworthy that neither the shortened latency periods nor the shift in translocation patterns had an impact on the incidence of PCT development. The 42.2% incidence in N3p53-/- mice is similar to the percentages recorded in groups of conventional BALB/cAn mice. The possible mechanisms underlying the accelerated tumorigenesis and the shift in translocation patterns are discussed.

Disruption of transforming growth factor beta signaling by a novel ligand-dependent mechanism.

Transforming growth factor (TGF)-beta is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-beta receptors and one or more isoforms of TGF-beta, thus the synthesis and secretion of TGF-beta as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-beta results in TGF-beta ligand-receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-beta1 in the PCT binds to intracellular TGF-beta type II receptor (TbetaRII). Disruption of the expression of TGF-beta1 by antisense TGF-beta1 mRNA restores localization of TbetaRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TbetaRII (dnTbetaRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TbetaRII. Analysis of TGF-beta receptor-activated Smad2 suggests the intracellular ligand-receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-beta-receptor complex, and define a novel mechanism for modulating the TGF-beta signaling pathway.

Mouse plasmacytoma: an experimental model of human multiple myeloma.

BACKGROUND AND OBJECTIVES: There is no ideal animal model for human multiple myeloma (MM). All the models resemble the human disease in some respect, but none of them fulfils all the criteria of a perfect animal model. EVIDENCE AND INFORMATION SOURCES: The pristane oil (2,6,10,12-tetramethylpentadecane)-induced mouse plasmacytoma (MPC) model is the most widely used and accepted model and has provided the most data on plasmacytomagenesis so far. This model gives the opportunity to study the role of c-myc dysregulations, the mechanisms leading to cytogenetic changes involving Ig genes, the role of chronic inflammatory factors, the role of interleukin-6 (IL-6), insulin-like growth factor-I, prostaglandins, as well as signal transduction pathways in the neoplastic process. Therapeutic agents have been successfully tested. Although MPC growth is usually restricted to the peritoneal environment, intraperitoneal injection of MPC cell suspensions can reproduce the disseminated characteristics of the human disease in recipients. The IL-6 transgene and knockout models are important tools for clarifying the role of IL-6 in the pathogenesis of MM. Transgenic mice and retroviral gene transfer facilitate the study of oncogenes in neoplastic transformation. Spontaneous development of plasmacytomas in C57BL/ KaLwRij aging mice has several advantages, mainly because the disseminated growth, the typical bone lesions and renal involvement resemble, in part, the human disease. Furthermore, this model has already proved useful in studies on the effect of bisphosphonate in the treatment of bone disease in MM. The severe combined immunodeficiency (SCID) mouse model is also very attractive. A disseminated-like disease can be reproduced in this model. Multiple osteolytic bone lesions and bone marrow involvement are generated, and conventional drugs applied in the treatment of human multiple myeloma have proven to be effective. Nevertheless, the immune system of SCID mice basically differs from that of a MM patient. PERSPECTIVES: Taken together, all these models have contributed to our understanding of MM, but demonstrate the opportuness of developing a more appropriate model of the human disease.

Inducible mutagenesis in TEPC 2372, a mouse plasmacytoma cell line that harbors the transgenic shuttle vector lambdaLIZ.

The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues.

Efficiency alleles of the Pctr1 modifier locus for plasmacytoma susceptibility.

The susceptibility of BALB/c mice to pristane-induced plasmacytomas is a complex genetic trait involving multiple loci, while DBA/2 and C57BL/6 strains are genetically resistant to the plasmacytomagenic effects of pristane. In this model system for human B-cell neoplasia, one of the BALB/c susceptibility and modifier loci, Pctr1, was mapped to a 5.7-centimorgan (cM) chromosomal region that included Cdkn2a, which encodes p16(INK4a) and p19(ARF), and the coding sequences for the BALB/c p16(INK4a) and p19(ARF) alleles were found to be polymorphic with respect to their resistant Pctr1 counterparts in DBA/2 and C57BL/6 mice (45). In the present study, alleles of Pctr1, Cdkn2a, and D4Mit15 from a resistant strain (BALB/cDAG) carrying DBA/2 chromatin were introgressively backcrossed to the susceptible BALB/c strain. The resultant C.DAG-Pctr1 Cdkn2a D4Mit15 congenic was more resistant to plasmacytomagenesis than BALB/c, thus narrowing Pctr1 to a 1.5-cM interval. Concomitantly, resistant C57BL/6 mice, from which both gene products of the Cdkn2a gene have been eliminated, developed pristane-induced plasma cell tumors over a shorter latency period than the traditionally susceptible BALB/cAn strain. Biological assays of the p16(INK4a) and p19(ARF) alleles from BALB/c and DBA/2 indicated that the BALB/c p16(INK4a) allele was less active than its DBA/2 counterpart in inducing growth arrest of mouse plasmacytoma cell lines and preventing ras-induced transformation of NIH 3T3 cells, while the two p19(ARF) alleles displayed similar potencies in both assays. We propose that the BALB/c susceptibility/modifier locus, Pctr1, is an "efficiency" allele of the p16(INK4a) gene.

Myeloma proteins that bind Hsp65 (GroEL) are polyreactive and are found in high incidence in pristine induced plasmacytomas.

The myeloma proteins produced by 44 plasmacytomas (PCTs) recently induced by pristane in BALB/cAnPt and closely related PCT susceptible congenic strains of mice were isolated chromatographically and screened against a panel of 10 protein, nucleic acid and lipid antigens. This sample was highly unusual because 82% of the proteins had IgG isotopes. Nine of the proteins bound to Hsp65 (GroEL), and all of these were polyreactative. Twenty-one of the myeloma proteins were polyreactive and bound two or more antigens in the panel, and five were monoreactive. Thus, an antigen binding activity was determined for 59% of these myeloma proteins.

Activation of insulin-like growth factor I receptor signaling pathway is critical for mouse plasma cell tumor growth.

Plasma cell neoplasia in humans generally occurs as multiple myeloma, an incurable form of cancer. Tumors with marked similarity can be induced in mice by a variety of agents, including chemicals, silicone, and oncogene-containing retroviruses, suggesting the use of murine tumors as an informative model to study plasma cell disease. Herein, we have focused on the role of insulin-like growth factor I receptor (IGF-IR) signaling in the development of plasma cell disease. The insulin receptor substrate 2/phosphatidylinositol 3'-kinase/p70S6K pathway was found to be either constitutively or IGF-I-dependently activated in all plasma cell tumors. Biological relevance was demonstrated in that plasma cell lines with up-regulated IGF-IR expression levels exhibited mitogenic responses to IGF-I. More importantly, expression of a dominant-negative mutant of IGF-IR in these lines strongly suppressed tumorigenesis in vivo. Taken together, these results demonstrate that up-regulation and activation of IGF-IR and the downstream signaling pathway involving insulin receptor substrate 2, phosphatidylinositol 3'-kinase, and p70S6K may play an important role in the development of a broad spectrum of plasma cell tumors.

Norepinephrine-mediated inhibition of antitumor cytotoxic T lymphocyte generation involves a beta-adrenergic receptor mechanism and decreased TNF-alpha gene expression.

We have previously shown that norepinephrine (NE) inhibits the in vitro generation of anti-MOPC-315 CTL activity by spleen cells from BALB/c mice rejecting a large MOPC-315 tumor as a consequence of low-dose melphalan (l -phenylalanine mustard (l -PAM)) treatment (l -PAM TuB spleen cells). Since TNF-alpha plays a key role in the generation of antitumor CTL activity in this system, we determined whether NE mediates this inhibition through inhibition of TNF-alpha production. Here, we show that NE inhibits the production of TNF-alpha protein and mRNA by l -PAM TuB spleen cells stimulated in vitro with mitomycin C-treated tumor cells. Flow cytometric analysis of intracellular expression of TNF-alpha revealed substantial NE-mediated decreases in the percentages of TNF-alpha+ cells among CD4+ and CD8+ T cells and F4/80+ activated macrophages. NE inhibition of CTL generation was largely overcome by addition of TNF-alpha to the stimulation cultures. When the beta-adrenergic antagonist propranolol was added to the stimulation cultures of l -PAM TuB spleen cells at a concentration that prevented NE-induced cAMP elevation, the NE-mediated decrease in TNF-alpha mRNA and NE-mediated inhibition of CTL generation were reversed. Collectively, these results suggest that NE inhibits antitumor CTL generation, at least in part, by inhibiting TNF-alpha synthesis through a mechanism(s) involving beta-adrenergic receptor signaling.

Elevated mutant frequencies in lymphoid tissues persist throughout plasmacytoma development in BALB/c.lambdaLIZ mice.

Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, the mean mutant frequencies in the target gene, lacI, were found to be significantly increased in lymphoid tissues of congenic BALB/c.lambdaLIZ N5 mice in the terminal stage of a plasmacytoma induction experiment, 213-280 days after the first i.p. injection of the plasmacytomagenic agent pristane (2,6,10,14-tetramethylpentadecane). In plasmacytoma-bearing mice (n = 7), mutant frequencies in the spleens and mesenteric lymph nodes were elevated 2.46-fold and 5.35-fold, respectively, when compared with age-matched controls. In plasmacytoma-negative mice (n = 11), mutant frequencies were increased 2.30-fold (spleens) and 3.48-fold (mesenteric nodes). These results, interpreted in conjunction with our previous findings (K. Felix et al., Cancer Res., 58: 1616-1619, 1998) of approximately 3-fold elevations in pristane-induced splenic mutagenesis on day 42 postpristane, indicate that increased mutant levels in lymphoid tissues persist throughout plasmacytomagenesis in genetically susceptible BALB/c mice.

Inhibition of pristane-induced peritoneal plasmacytoma formation.

While the mechanism of how Indo inhibits PCTGEN is not established, Several hypothetical explanations provide new potential experimental approaches. Indo may block production of cytokines such as Il-6 in accessory cells that are critical for B-cell growth, viability and maturation, or it may directly target B cells via PPAR-gamma receptors. The latter mode of action is described in other cell types but not yet defined in B cells.