Search

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., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

The X-chromosome from the CBA/N mouse which carries the defective Bruton's tyrosine kinase (Btk) allele (Xxid) has been introgressively backcrossed onto the plasmacytoma (PCT) induction-susceptible BALB/cAN. Inbred BALB/c.CBA/N-xid/xid (C.CBA/N) mice raised and maintained in our conventional colony were given three 0.5 ml injections of pristane and were highly refractory to PCT induction. Only one PCT was found among 59 mice followed for > or =300 days. Twenty mice were examined at day 200 for foci of plasma cells in the oil granuloma. Ten mice had small foci of plasma cells, most of which were plasmacytotic, embedded in the inflammatory oil granuloma. In one there were multiple foci, but most of the mice had only one or two foci. F1 hybrid XxidY males derived from CBA/N females crossed to BALB/cAnPt were also resistant to PCT induction, while heterozygous and homozygous XY males were susceptible. C.CBA/N mice can develop extensive mucosal plasma cells as well as plasma cell accumulations in oil granuloma tissue, but the precursors of these plasma cells do not give rise to PCT in genetically susceptible hosts. The failure of C.CBA/N mice to develop PCT is probably due to the elimination of B cell clones that can be perpetuated by repeated exposure to thymus-independent type 2 antigens.

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.

Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, mean mutant rates were determined in the spleen of mice exposed to sustained oxidative stress and were found to be increased approximately 3-fold in plasmacytoma-susceptible BALB/c and C.D2-Idh1-Pep3 mice, but not in plasmacytoma-resistant DBA/2N mice. This finding suggests a correlation between the genetic susceptibility to inflammation-induced peritoneal plasmacytomagenesis and the phenotype of increased mutagenesis in lymphoid tissues, raising the possibility that plasmacytoma resistance genes may inhibit tumor development by minimizing oxidative mutagenesis in B cells.

Purpose: 6-[211At]-astato-MNDP is a high-LET endoradiotherapeutic drug that selectively targets to an oncogenically associated alkaline phosphatase isoenzyme expressed by certain tumors. A detailed histopathological study of the late tissue effects of its endogenous alpha-particle emissions has been carried out in a murine tumor model., Materials and Methods: Thyroid-blocked male C57BI/10 mice bearing a s.c. transplanted rectal adenocarcinoma were treated with a single i.p. injection of 10-750 kBq 6-[211At]-astato-MNDP. Cured mice (131) were studied. Detailed autopsies and histological examinations were performed on all mice. The study was concluded after 756 days., Results: Lymphoma, plasmacytoma, and intercurrent infections secondary to chronic pulmonary fibrosis were the most commonly found late manifestations of alpha-radiation exposure. Low grade B-cell non-Hodgkin's lymphoma occurred in 19 (24.7%) of 77 mice, 13-17 months after receiving 3.5-185 kBq 6-[211At]-astato-MNDP. The incidence of lymphoma alone and its latency was similar to that of the control population (23.3%). Treatment doses exceeding 200 kBq 6-[211At]-astato-MNDP, were associated with the development of soft tissue plasmacytoma in 7 (13%) of 54 mice, after 17-22 months. Generalized debilitation and nonspecific infections supervening pulmonary fibrosis significantly contributed to the late morbidity and mortality observed in mice treated with 300-750 kBq 6-[211At]-astato-MNDP. Dosimetry has afforded LD50/360 and LD50/420 estimates of 12-14 and 10-12 Cobalt-Gray equivalent (CGyE), respectively, for chronic lung damage. There was no histological evidence of chronic radiation damage to other critical healthy tissues. Normal thyroid morphology was preserved., Conclusions: Dose activities of 6-[211At]-astato-MNDP exceeding 300 kBq, were associated an increased risk of tumor induction and development of varying degrees of chronic pulmonary fibrosis implicated in the onset of terminal intercurrent infections. Within the therapeutic dose range 55-300 kBq 6-[211At]-astato-MNDP, mortality associated with the incidence of significant late radiation damage in critical normal tissues and latent carcinogenesis was less than 15%. Data from this murine model suggest that clinically relevant activities of 6-[211At]-astato-MNDP may be given without unacceptable toxicity.

Immunoglobulin heavy chain (Igh)/Myc recombinations are a hallmark of pristane-induced mouse plasmacytomas but are also frequently found in non-tumorous tissues. Here we describe for the first time a PCR-based technique for detecting fusions between Igh mu or Igh alpha and Myc in situ. Igh/Myc recombinations were found in transplanted and primary plasmacytomas. In addition, the gut-associated lymphoid tissues of plasmacytoma-free BALB/c mice were investigated for the presence of Igh/Myc fusions. Igh/Myc rearrangements were detected in Peyer's patch follicles and in the intestinal lamina propria both in normal mice and in mice shortly after pristane treatment. The sequence analysis showed that i) three to five different Igh/Myc hybrid sequences were present in individual follicles, ii) Igh/Myc recombinations can be subjected to additional switch recombinations as shown by related sequences in neighboring cells, and iii) cells harboring these rearrangements migrate into the adjacent lamina propria. The results indicate that Peyer's patches are a hyper-recombinogenic tissue. Myc recombination-positive cells are present in at least 100-fold more frequently than expected if recombinations were random, which suggests that this kind of trans-chromosomal rearrangement may be targeted.

Interleukin (IL)-6 is known to be an essential growth factor for myeloma cells, both in vitro and in vivo. In mice, IL-6 is required for development of B cell tumors upon infection with a retrovirus expressing the myc/raf oncogenes. In the present study, we used the pristane-oil-induced plasmacytoma model, which more closely mimics tumor transformation and progression in human multiple myeloma. Also using this system, we found that IL-6-deficient BALB/c mice are protected against tumor development. Although the pristane-induced inflammatory reaction was less pronounced in IL-6-deficient mice versus their wild-type littermates, both B cell differentiation and plasma cell formation took place, and even morphological evidence of plasma cell transformation was detected, albeit at a low frequency. However, in the absence of IL-6, there were never signs of uncontrolled proliferation of either normal B lymphocytes or tumor cells, suggesting that the role of IL-6 in murine plasmacytoma and possibly also in human multiple myeloma is to ensure abnormal survival and proliferation of previously transformed tumor cells and therefore tumor development and progression.

Continuous indomethacin (INDO) administration in the drinking water (10 to 20 microg/mL) profoundly inhibited plasmacytoma (PCT) development initiated by three 0.2- or 0.5-mL intraperitoneal (i.p.) injections of pristane in hypersusceptible BALB/c.DBA/2-Idh1-Pep3 congenic mice. The most effective inhibitions were obtained with continuous INDO treatment. When treatment was delayed until 50 to 60 days after the first pristane injection, there was approximately a 50% reduction in PCT incidence. The primary action of pristane is the induction of a chronic inflammation in the peritoneal connective tissues and the formation of a microenvironment where PCTs develop. INDO, a powerful inhibitor of prostaglandin synthases (cyclooxygenases 1 and 2), did not inhibit the formation of mesenteric oil granuloma nor the appearance of cells in this chronic inflammatory tissue carrying c-myc illegitimately joined to an Ig heavy chain switch region, ie, the t(12;15) chromosomal translocation. INDO inhibited PCT induction by the i.p. implantation of 21 x 2 mm polycarbonate discs. These solid objects predominantly induce the formation of a patchy fibroplastic tissue on contacting peritoneal surfaces. These and previous data indicate that indomethacin inhibits an intermediate stage in PCT development after the arrival of cells bearing the T(12;15) translocation in the oil granuloma and before these cells acquire transplantability to a pristane-conditioned host. The biological mechanism that explains how INDO inhibits PCT development is not yet established but appears to result from decreased production of prostaglandins in chronic inflammatory tissues (oil granuloma, fibroplasia), suggesting that prostaglandins play an active role in oil and solid plastic induced PCT formation.

Sixty per cent of BALB/cAnPt mice injected intraperitoneally (i.p.) with tetramethylpentadecane (pristane) develop plasmacytomas (PCs), whereas less than 10% of BALB/cJ develop such tumours. Most other mouse strains are completely resistant. Resistance is dominant over susceptibility in F1 hybrids between BALB/cAnPt and the resistant non-BALB/c strains, suggesting that susceptibility may be due to some genetic defect. (BALB/cAnPtxBALB/cJ)F1 hybrids have a PC incidence of 36-42%. Previously, BALB/cJ has been shown to harbour at least one resistance gene (Potter et al., Genomics 1988, Vol. 2, pp. 257-262). On the assumption that BALB/cJ may contain a segregating resistance gene, we cross BALB/cJ females with pristane-pretreated BALB/cJ males that were found to be carrying PC cells intraperitoneally 5-7 months after pristane treatment. After two selective crosses, 62% of the BALB/cJ subline BALB/cM2/22 developed PC after pristane and 52% after pristane followed by Abelson virus, while unselected controls had an incidence of 11% and 0%, respectively. Moreover, six spontaneous plasmacytomas developed in untreated females of the selected colony. Five of these carried T(12; 15) (F2; D2/3) translocations. The sixth had a T(1; 10) (G; C1) translocation and an interstitial duplication of segment (C1/E3) on one chromosome 5. It may be concluded that pristane treatment is not a prerequisite for the induction of the PC associated Ig/myc translocations.

Recombinations between c-myc and immunoglobulin (Ig) sequences that typically occur in pristane-induced mouse plasmacytomas were detected in secondary lymphoid tissues from normal mice, chiefly in the gut-associated lymphoid tissue. Based on the analysis of recombination sequences as clonotypic markers, migration of c-myc recombination-positive cells was observed between Peyer's patches and into the intestine. Treatment of plasmacytoma-susceptible BALB/cAn mice with pristane induced proliferation and migration of these cells into mesenteric lymph node, spleen, and oil granuloma within 7 days. Plasmacytoma-resistant strains of mice (DBA/2N, C3H/HeJ, C57BL/6) differed in that (1) they harbored fewer clones (Ig/c-myc recombinations were detected in 33% of resistant mice versus 91% of BALB/cAn mice after pristane treatment); (2) Ig/c-myc-positive cells were rarely detected in the oil granuloma, and (3) c-myc recombined predominantly with the Ig alpha locus in BALB/cAn mice (72%), but with the Ig mu locus in DBA/2N and in C57BL/6 (67%). The results demonstrate that normal mice generate a large number of lymphocytes with aberrant c-myc in intestinal tissues without developing tumors.

The dihydrofolate reductase (DHFR) gene is a target of c-Myc in genomic instability. The induced overexpression of c-Myc in cell lines is followed by the amplification and rearrangement of the DHFR gene. Furthermore, the constitutive upregulation of c-Myc protein coincides with genomic instability of the DHFR gene in lymphoid, non-lymphoid and in tumor lines. The amplification of the DHFR gene is locus-specific and independent of species origins. We have now addressed the question whether inducible deregulation of c-Myc is followed by DHFR gene amplification in vivo. We show that the DHFR gene is a target of c-Myc-dependent neoplasia in vivo and propose a role for genomic instability during the initiation of neoplastic transformation.

Solitary bone plasmacytoma (SBP) is a rare presentation of plasma cell dyscrasias. Radiotherapy has been considered the treatment of choice, however, most patients will develop multiple myeloma, 3 to 10 years after initial diagnosis and treatment. No innovations have been introduced in the treatment of SBP in the last 30 years. We began a prospective clinical trial to assess the efficacy and toxicity of adjuvant chemotherapy with low doses of melphalan and prednisone administered to patients with SBP after radiation therapy in an attempt to improve the disease-free survival and overall survival. Between 1982 and 1989, 53 patients with SBP were randomly assigned to be treated with either local radiotherapy with doses ranged from 4000 to 5000 cGy to achieve local control of disease (28 patients) or the same radiotherapy schedule followed by melphalan and prednisone given every 6 weeks for 3 years (25 patients). After a median follow-up of 8.9 years, disease-free survival and overall survival were improved in patients who were treated with combined therapy, 22 patients remain alive and free of disease in the combined treatment group compared to only 13 patients in the radiotherapy group (p < 0.01). Treatment was well tolerated; planned doses were administered in all cases; no delays in treatment or acute side-effects were observed during treatment. Long-term secondary toxicities including secondary neoplasms and acute leukaemia, have not been observed. We felt that the use of adjuvant chemotherapy after adequate doses of radiotherapy in patients with SBP improved duration of remission and survival without severe side-effects. However, as with other studies in SBP, the group was too small to draw definitive conclusions and more controlled clinical trials are necessary to define the role of this therapeutic approach in patients with SBP.

Intraperitoneal injection of pristane induces production of interleukin-6 (IL-6) and either plasmacytosis or plasmacytoma in mice, depending upon the genetic background. Pristane does not induce plasmacytoma in IL-6 knockout (IL-6-/-) mice, suggesting that IL-6 is required for this process. In the present study we determined whether IL-6 is also required for pristane-induced hyperplasia of normal plasma cells. Pristane was injected intraperitoneally into IL-6-/- and IL-6 wild-type (IL-6+/+) mice. Overall there were more deaths in IL-6+/+ mice (85%) than in IL-6-/- mice (40%), P = 0.024. Hyperplastic lymph node and spleen weight did not differ (P = 0.82 and P = 0.15, respectively) in IL-6-/- versus IL-6+/+ mice. Lymphocytosis with similar patterns of expression of B-cell (B220) and T-cell (Thy-1) antigens was noted in both IL-6-/- and IL-6+/+ mice. However, morphological studies, dual fluorescent staining for Syn-1 and B220 antigens (syn-1+ B220+ cells), and intracytoplasmic Ig staining revealed plasma cell hyperplasia in lymph node and spleen from IL-6+/+, but not IL-6-/-, mice. These plasma cells from IL-6+/+ mice were polyclonal and unable to induce tumour formation in severe combined immunodeficient mice. These data demonstrate that IL-6 is required for pristane-induced hyperplasia of polyclonal plasma cells in mice.

Interchromosomal recombinations between c-myc and immunoglobulin sequences can be found in preneoplastic lesions (oil granulomata) during pristane-induced plasmacytoma development in susceptible BALB/cAn mice. In this study we used a more sensitive approach, hybridization-enriched templates with nested PCR, to detect microclones with Ig alpha/c-myc recombinations in oil granulomata of susceptible and resistant mice. Recombinations were detected in as many as 73% (32/44) of plasmacytoma-susceptible BALB/cAn mice 30 days after an injection of pristane. Mice that are resistant to plasmacytoma induction can also harbor recombination-positive cells, but these are less frequent [2/20 DBA/2N, 8/20 (BALB/cAn x DBA/2N)F1, hereafter called CD2F1]. The clones in DBA/2N mice were small (< 400 cells), whereas in BALB/cAn, the oil granuloma harbored up to several thousand of these cells. We conclude that the molecular machinery for generating characteristic interchromosomal recombinations can be found in all strains of mice. Both the frequency of generating Ig alpha/c-myc recombinations and the expansion of recombination-positive cells are greater in susceptible mice than in resistant strains.

The intraperitoneal administration of pristane (2,6,10,14-tetramethylpentadecane) induces peritoneal plasmacytomas in genetically susceptible BALB/c mice. The purpose of this study was to estimate the disposition of an amount of intraperitoneally injected pristane that would conventionally be used in a tumor induction protocol. The distribution of 3H-labeled pristane in various tissues was monitored by liquid scintillation counting at different times after injection. The data show that pristane is present in the blood and detectable in all tested tissues during an observation period of one to 64 days. The levels of pristane fluctuate in some tissues such as lymph node and bone marrow but show a clear tendency to accumulate in others such as liver, spleen and kidney. Evidence is also presented for the in vivo metabolism of pristane based on the observed urinary excretion of tritium and on the high levels of radioactivity in the gall bladder fluid. It is concluded that intraperitoneally administered pristane is distributed throughout the mouse and is stored in tissues in sufficient amounts to allow interactions with the cells residing there.

A single injection of pristane was given i.p. to plasmacytoma-susceptible BALB/cAn mice. At intervals up to 6 mo thereafter, immunofluorescence labeling of intranuclear terminal deoxynucleotidyl transferase (TdT), cell surface B220 glycoprotein, cytoplasmic mu-chains of IgM (c mu), and surface mu-chains (s mu), together with mitotic arrest techniques, were used to quantitate the in vivo population dynamics of precursor B cells in the bone marrow. TdT-expressing pro-B cells (TdT+B220-, TdT+B220+), before the expression of mu-chains, showed sustained increases in both population size and the number of cells flowing through mitosis per unit time. In contrast, populations of pre-B cells (c mu + s mu -) and B cells (s mu +) were consistently depressed for long periods of time, including the phase of plasmacytoma formation. Precursor B cells in DBA/2 mice, a plasmacytoma-resistant strain, showed similar responses to pristane treatment. The results demonstrate that a single injection of pristane, which greatly increases the demand for macrophage activity in the peritoneal space, causes sustained distant alterations in B cell lymphopoiesis in the bone marrow; specifically, a prolonged increased proliferation of pro-B cells coupled with a depression and a exaggerated loss of pre-B cells and B cells. The protracted stress on B cell lymphopoiesis may be a predisposing factor in the subsequent development of c-myc-activating chromosomal rearrangements that play a critical role in plasmacytomagenesis.

Pristane (2,6,10,14-tetramethylpentadecane) is a C19-isoalkane that promotes the development of plasmacytomas in genetically susceptible BALB/c mice. Similarities between the effects of pristane and protein kinase C (PKC)-activating phorbol esters suggested that the tumor promoting activity of pristane might involve the activation of PKC. Here we show that up to 5 mol% of pristane can be homogeneously incorporated into phosphatidylcholine/phosphatidylserine bilayers. Membrane-incorporated pristane partially activated PKC and increased phorbol ester binding to the bilayer by more than 50%. Pristane (50 microM) delivered as an inclusion complex with beta-cyclodextrin to promyelocytic HL-60 leukemia cells induced a partial long-term translocation of PKC to the cell membrane. This was accompanied by differentiation of HL-60 cells into macrophage-like cells. It is concluded that activation of PKC may comprise an important aspect of the tumor promoting potential of pristane.

Genetic rearrangements activating the proto-oncogene c-myc comprise a mandatory oncogenic step in plasma cell tumor development in BALB/cAnPt mice. In the majority of plasmacytomas, c-myc activating rearrangements take the form of reciprocal chromosomal translocations t(12;15) that juxtapose c-myc to the immunoglobulin heavy chain alpha locus (IgH alpha) in particular the switch alpha region (S alpha). The genetic basis for the prevalence of S alpha/c-myc recombinations in BALB/cAnPt plasmacytomas is not known but may be related to a hypothetical regional genomic instability of the c-myc and IgH alpha loci in BALB/cAnPt mice. We wished to test whether the genomic instability of both loci might be revealed by the diversity of genetic recombinations that can be observed in IgH alpha and c-myc. We employed PCR methods to detect new recombinations of c-myc and IgH alpha in the preneoplastic stage of plasma cell tumor development and found that c-myc can be joined to more genes or genomic regions than known before. This is indicative but does not formally prove a particular genomic instability of c-myc and IgH alpha in BALB/cAnPt B cells. Since defective DNA repair provides a mechanistic explanation for genomic instability, we measured the efficiency of repair in IgH alpha and c-myc using an assay that quantitates the removal of UV-induced pyrimidine dimers within specific genomic regions. We used plasmacytoma XRPC 24 as a model system and found that both IgH alpha and c-myc were poorly repaired, whereas c-abl, a proto-oncogene not related to conventional pristane-induced plasmacytoma-genesis, was efficiently repaired.

Alterations in cell programming associated with neoplastic transformation may involve widespread changes in patterns of DNA methylation. Increased expression of IAP elements in plasmacytomas compared with LPS-stimulated normal B-cells is accompanied by extensive hypomethylation of IAP sequences (Mietz and Kuff 1990), subsets of which are revealed with the LS2, LS3 and T1 probes. Multiple common LS- and PC-specific IAP loci are hypomethylated in established plasmacytomas, showing that hypomethylation does not occur entirely randomly. Many of the same IAP loci are hypomethylated in primary plasmacytomas induced by two different methods, as soon as recognizable tumor tissue can be isolated. In primary tumors hypomethylation frequently appears to occur in DNA flanking the IAP elements. In the established tumors the hypomethylated sites occur primarily in the IAP LTR, suggesting that for these loci hypomethylation begins in the flanking DNA and is extended into the IAP LTRs during progression of the tumors. The newly hypomethylated IAP LTRs in primary plasmacytomas (as compared to normal B cells) may provide a set of reporter genes for chromosomal regions that are characteristically hypomethylated in these transformed cells and that may contain cellular genes whose activation is related to the transformation process.

Recombinations between c-myc and immunoglobulin loci are a hallmark of Burkitt's lymphomas and mouse plasmacytomas. Analyzing the fine structure of these illegitimate rearrangements has revealed differences between the recombinations in these two tumors. Recombinations are nearly reciprocal in Burkitt's lymphomas, whereas in most BALB/c plasmacytomas large stretches of c-myc sequences have been deleted. The recombinations detected during preneoplastic development of plasmacytomas, in contrast, have most of the c-myc sequences retained on either one of the translocated chromosomes. We conclude that initial recombination structures are subjected to secondary changes in mouse plasmacytomas. In Burkitt's lymphomas the primary recombination sequence is preserved in tumor cells.

Silicone gels injected intraperitoneally into strains of mice related to BALB/c develop plasmacytomas in approximately the same numbers and with similar phenotypes as previously obtained with pristane. Silicone gels produce few side effects and are well tolerated for long periods. Silicone gels contain several components that are potentially biologically active: residual vinyl groups and platinum. Microscopic and histological evidence suggests the silicone gel is degraded over a long period of time. Preliminary studies with long chain liquid dimethylpolysiloxanes with viscosities of 1000 cSt and 12,500 cSt have not produced plasmacytomas as yet. The plasmacytomagenic action of the gel appears to be due to the release of liquids from the gel matrix.

The translocation of c-myc on chromosome (chr.) 15 to an immunoglobulin heavy-chain switch region on chr. 12 is the critical oncogenic step in pristane-induced plasmacytoma (PCT) development in BALB/cAnPt mice. Applying a recently developed PCR method, we have been able to detect the most commonly occurring illegitimate recombinations between alpha-chain switch region (S alpha) and c-myc in preneoplastic B cells residing in mesenteric oil granuloma (OG) tissues 7-30 days postpristane. In this study, we compare the nucleotide sequences at the S alpha/c-myc breaksites on both the c-myc-activating chr. 12+ and the reciprocal chr. 15- from eight transplanted PCTs, seven primary PCTs, and five OGs that contained six B-cell clones. These junction sequences revealed a remarkable diversity of S alpha/c-myc recombinations. In nine cases--four PCTs and five B-cell clones--nearly precise reciprocal exchanges with a loss of only 3-35 bp in c-myc were found. Large deletions in c-myc that removed 369-878 bp were observed in seven PCTs but not in early B cells. Duplications of c-myc ranging from 103 to 229 bp were also restricted to PCTs and noticed in four cases. Clonally related but different reciprocal recombinations, 38 bp apart on chr. 12+ and 15 bp apart on chr. 15-, were isolated from two different specimens of the same OG tissue from a BALB/c mouse 30 days postpristane. A second OG from another 30-day mouse yielded four recombinational fragments--two clonally related chr. 12(+)-specific fragments and two chr. 15(-)-specific fragments--one of which carried a 143-bp insertion of a microsatellite at the breaksite. We suggest that the initial recombinational break-point regions between S alpha and c-myc in plasmacytoma precursor cells at the time of immunoglobulin heavy-chain switching are intrinsically labile and characterized by a persisting instability of c-myc, which can result in large secondary deletions of c-myc.

A 42-year-old Japanese man with chronic active Epstein-Barr virus (EBV) infection initially responded to treatment with interleukin-2 (IL-2). Six months later he developed thrombosis in the hepatic veins, and Budd-Chiari syndrome associated with severe hepatic damage was diagnosed. He also developed a solitary EBV-positive plasmacytoma in the right femur. Since these rare complications occurred after long-term IL-2 therapy, the possibility that long-term IL-2 therapy might cause Budd-Chiari syndrome and liver damage as well as EBV-associated plasmacytoma is discussed.

Background: Plasmacytomas can be induced in high frequency in susceptible strains of mice by the intraperitoneal introduction of plastics or paraffin oils, including the chemically defined oil pristane (2,6,10,14-tetramethylpentadecane). These materials persist in the peritoneal cavity, where they induce chronic inflammation during the long periods before plasmacytomas develop. Such plasmacytomas appear to arise from B cells carrying chromosomal translocations that affect c-myc transcription., Purpose: Because silicone gels are in widespread medical use and share many of the characteristics of other materials known to be inducers of plasmacytomas, we wished to determine their capacity to induce plasmacytomas in mice., Methods: In a series of parallel experiments, corn oil, pristane, silicone oil (dimethylpolysiloxane), or silicone gel from commercially obtained mammary implants was injected intraperitoneally into plasmacytoma-susceptible BALB/cAnPt-A and congenic BALB/cAnPt.DBA/2-Idh1-Pep3 mice, as well as into plasmacytoma-resistant C57BL/6N, C3H/HeJ, DBA/2N, and (BALB/c x DBA/2)F1 mice. Mice were examined at least once every 2 weeks for signs of abdominal tumor or weight loss and screened every 4-6 weeks for peritoneal plasmacytoma cells by peritoneal lavage. Tissues were examined by histologic and immunohistochemical techniques. Metaphase chromosome spreads were made from ascitic plasmacytomas without Colcemid treatment, and metaphase plates were G-banded according to standard techniques. The t(12;15) or t(6;15) translocation chromosomes were identified under the microscope in at least five metaphase plates of high banding quality. Mice were autopsied 125-400 days after the injection of test material. Gas chromatography and mass spectrometry were utilized to determine the composition of the silicone oil and silicone gel used in the injections., Results: The silicone gels tested induced plasmacytomas in BALB/cAnPt-A and BALB/cAnPt.DBA/2-Idh1-Pep3 mice. Neither corn oil used as a control nor 1000-centistoke or 12,500-centistoke dimethylpolysiloxane induced plasmacytomas in these mice. The plasmacytomas were transplantable in syngeneic hosts. Cytogenetic studies of 41 silicone-induced plasmacytomas showed that 30 had t(12;15) translocations, eight had t(6;15) translocations, and three had no translocations., Conclusions: The silicone gels used in mammary implants, which contain a complex mixture of different siloxanes, induced peritoneal plasmacytomas in genetically susceptible mice. Silicone gels provide new chemically defined materials that are effective inducers of plasmacytomas in BALB/cAnPt-A and BALB/cAnPt.DBA/2-Idh1-Pep3 mice. Further studies will be required to determine which of the components of these gels are the active materials.