Possible co-regulation of genes associated with enhanced progression of mammary adenocarcinomas.

Tumor progression is a multistep process in which alterations in the expression of numerous gene products may give rise to highly malignant cellular variants. In the present study, we analyzed the differential expression of several genes in cellular variants of mammary adenocarcinomas with high or low malignancy potential, which originated in a common ancestor. To assess the generality of our findings, high and low malignancy variants were derived from two different mammary adenocarcinoma cell lines, namely DA3 and CSML cells. Of major importance is the fact that the differences between high- and low-malignancy variants observed in one system of mammary adenocarcinoma cells (DA3 cells) were identically reproduced in the other system of mammary adenocarcinoma cells (CSML cells). The high malignancy variants of tumors both DA3-high and CSML-high (previously called CSML-100), expressed higher levels of factors that induce monocyte migration than the low malignancy DA3-low and CSML-low (previously called CSML-0) variants. In addition, it was found that DA3-high and CSML-high cell variants expressed higher levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and matrix metalloproteinases (MMPs) than the low malignancy variants (DA3-low and CSML-low). These results suggest that MCP-1, IL-6 and MMPs potentially contribute to mammary adenocarcinoma progression and that their expression is regulated by a common pathway. The expression of MCP-1, IL-6 and MMPs in both DA3-high and CSML-high cells was up-regulated by tumor necrosis factor alpha (TNFalpha). The fact that TNFalpha exerted similar effects on the expression of these three factors in both cell systems raises the possibility of a coordinated co-regulation of tumor-promoting factors.

A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma.

The homing of hemopoietic stem cells to the bone marrow is mediated by specific interactions occurring between CXCR4, which is expressed on hemopoietic stem cells, and its ligand, stromal cell-derived factor-1 (SDF-1), a CXC chemokine secreted by bone marrow stromal cells. In the present study we evaluated the possibility that neuroblastoma cells use a mechanism similar to that used by hemopoietic stem cells to home to the bone marrow and adhere to bone marrow stromal cells. Our study suggests that CXCR4 expression may be a general characteristic of neuroblastoma cells. SH-SY5Y neuroblastoma cells express not only CXCR4, but also its ligand, SDF-1. CXCR4 expression on SH-SY5Y neuroblastoma cells is tightly regulated by tumor cell-derived SDF-1, as demonstrated by the ability of neutralizing Abs against human SDF-1alpha to up-regulate CXCR4 expression on the tumor cells. The reduction in CXCR4 expression following short term exposure to recombinant human SDF-1alpha can be recovered as a result of de novo receptor synthesis. Recombinant human SDF-1alpha induces the migration of CXCR4-expressing SH-SY5Y neuroblastoma cells in CXCR4- and heterotrimeric G protein-dependent manners. Furthermore, SH-SY5Y cells interact at multiple levels with bone marrow components, as evidenced by the fact that bone marrow-derived constituents promote SH-SY5Y cell migration, adhesion to bone marrow stromal cells, and proliferation. These results suggest that SH-SY5Y neuroblastoma cells are equipped with adequate machinery to support their homing to the bone marrow. Therefore, the ability of neuroblastoma tumors to preferentially form metastases in the bone marrow may be influenced by a set of complex CXCR4-SDF-1 interactions.

The FX enzyme is a functional component of lymphocyte activation.

Fucose is an essential constituent of selectin ligands. These molecules mediate the initial contact between extravasating leukocytes and endothelial cells. The generation of GDP-L-fucose by the FX enzyme is the final step of fucose biosynthesis. Recently, we demonstrated that outside-in signaling regulates the expression of the FX enzyme in certain cancer cells. The present study demonstrates that the polyclonal activation of T and B cells significantly up-regulated the expression of the FX enzyme and of the fucosylated selectin ligands sLe-x and CLA. Treatment of T cells with FX antisense oligonucleotides significantly decreased selectin ligand expression upon activation. We conclude that FX is regulated by outside-in signals also in lymphocytes and that this enzyme is involved in the biosynthesis of selectin ligands in such cells. We propose that FX takes part in the cascade of events leading to the extravasation of activated lymphocytes.

Differential expression of genes by tumor cells of a low or a high malignancy phenotype: the case of murine and human Ly-6 proteins.

Progression of tumor cells toward a high malignancy phenotype and metastasis is a multi-event cascade involving inter alia alterations in the expression of various genes. The focus of our laboratory is on genes whose altered expression may lead, directly or indirectly, to an increased malignancy phenotype. The identification of such genes and the evaluation of the consequences of their altered expression is essential for attempts to halt tumor progression and prevent metastasis formation. Published work originating in our laboratory showed that members of the murine Ly-6 supergene family are involved in the progression of certain mouse tumors. The expression level of several members of this family was higher on highly malignant cells than on tumor cells expressing a lower malignancy phenotype. Sorting by flow cytometry of tumor cells to subpopulations expressing either high or low levels of Ly-6E.1 yielded correspondingly cells expressing a high or a low malignancy phenotype. The high malignancy, high Ly-6E.1-expressing cells also expressed high levels of the receptor for urokinase plasminogen activator (uPAR), whereas low malignancy, low Ly-6E.1-expressing cells also expressed low levels of uPAR. Transfection studies indicated that uPAR was causally involved in conferring a high malignancy phenotype upon tumor cells expressing high levels of Ly-6E.1. E48 is a human homologue of the murine ThB protein, a member of the Ly-6 supergene family (but distinct from the Ly-6E.1 protein mentioned above) and expressed on head and neck squamous carcinoma cells. Experiments currently in progress are aimed to find out whether E48 is involved in the progression of such cancer cells. Using the differential display technology, it was shown that ligation of E48 on tumor cells by the corresponding antibodies (serving as a surrogate for an as yet unidentified E48 ligand) upregulates an enzyme (FX) involved in the biosynthesis of GDP-L-fucose. Fucose is an essential component of certain selectin ligands.

The GPI-linked Ly-6 antigen E48 regulates expression levels of the FX enzyme and of E-selectin ligands on head and neck squamous carcinoma cells.

By differential display we demonstrated that antibody-mediated ligation of the GPI-linked protein product of E48, a newly discovered human Ly-6 gene, up-regulates the expression of the FX enzyme in 3 lines of head and neck squamous carcinoma cells. FX is responsible for the last step in the synthesis of GDP-L-fucose. The up-regulation of FX was E48 ligand-specific. 22AWT head and neck squamous carcinoma cells expressing high levels of E48 expressed significantly higher levels of FX than the E48 antisense transfected 22AWT cells (8-3 cells). The former cells also expressed higher levels of two major fucosylated glycans (the selectin ligand, Sialyl Lewis a, and VIM-2) than the E48 antisense transfectants. Conversely, transfection of cells from the 14CWT line expressing very low levels of E48 with E48 cDNA caused an up-regulated expression of FX and of the two fucosylated glycans in the 14C-CMV16 transfectants. Moreover, the expression levels of Sialyl Lewis a was significantly up-regulated on HNSCC upon ligation of E48 by anti-E48 antibodies. The functional significance of the E48-mediated up-regulation of Sialyl Lewis a was demonstrated in rolling experiments on E-selectin bearing surfaces under physiological conditions of shear flow and on tumor necrosis factor alpha-activated human umbilical venous endothelial cells. Only high E48/FX/Sialyl Lewis a expressing 14C-CMV16 cells could roll on purified E-selectin or establish E-selectin dependent rolling on the activated human umbilical venous endothelial cells. Low E48/FX/Sialyl Lewis a expressing 14CWT cells did not roll. These results show that E48 controls the expression of the FX enzyme and of certain fucosylated E-selectin ligands by HNSCC. E48 may thus function as a key regulator of the adhesiveness of these tumor cells to inflamed vessel walls expressing E-selectin.

MCP-1 expression as a potential contributor to the high malignancy phenotype of murine mammary adenocarcinoma cells.

The search for mechanisms that regulate tumor progression has motivated the authors' laboratory to establish a unique murine model system, consisting of two lines of DA3 mammary adenocarcinoma cells that were derived originally from a common ancestor but differed in their malignant potential. Studies indicated that the highly malignant phenotype manifested by one of the cell lines (termed Ly-6hi DA3 cells) was associated with high expression of the Ly-6E.1 antigen. To characterize the mechanisms controlling the high malignancy phenotype expressed by Ly-6hi DA3 cells, the study was focussed on the potential contribution of tumor-derived factors to the high malignancy phenotype expressed by these cells. To this end, the expression of CC chemokines, major chemoattractants of monocytes and T cells, by the highly malignant Ly-6hi DA3 cells as compared to the low malignancy Ly-6lo DA3 cells was evaluated. The results indicate that the highly malignant cells express higher levels of factors that induce monocyte migration than the low malignancy cells. Two CC chemokines were shown to be highly produced by Ly-6hi DA3 cells, MIP-1alpha and MCP-1, of which only the latter was shown to contribute to the high migratory activity expressed by the high malignancy Ly-6hi DA3 cells. Since MCP-1 may attract monocytes to tumor sites, these findings suggest that monocyte-derived mediators, such as growth factors or angiogenic cytokines, have pro-malignancy effects that contribute to the high malignancy phenotype expressed by Ly-6hi DA3 cells.

Expression of Ly-6, a marker for highly malignant murine tumor cells, is regulated by growth conditions and stress.

Ly-6E.1 is highly expressed in murine tumor cells with a high malignancy phenotype and may serve as a marker for such a phenotype. In this study, we examined the effects of various growth conditions and stress on the expression levels of Ly-6E.1 by tumor cells. Previous preliminary results have shown that murine DA3 mammary tumor cells expressing high levels of Ly-6E.1 (Ly-6(hi)) are more highly tumorigenic than the same tumor cells expressing low levels of this membrane protein (Ly-6(lo)). In this study, we demonstrate that mice bearing Ly-6(hi) DA3 tumors have a significantly higher burden of spontaneous pulmonary metastasis than mice bearing Ly-6(lo) DA3 tumors. Furthermore, the survival time of the former mice was significantly shorter than that of the latter ones. We further show that certain other members of the Ly-6 gene family such as Ly-6C.1 and Ly-6G.1 are coregulated with Ly-6E.1. This was shown to occur with respect to both DA3 cells as well as A3 tumor cells which are of fibroblast origin. However, these 2 cells differ with respect to regulation of Sca-2 (TSA1, another member of the Ly-6 family) expression on these cells. Levels of Sca-2 on A3 cells appear to be coregulated with Ly-6E.1 (i.e., Ly-6(hi) A3 cells express high levels of Sca-2 and Ly-6(lo) A3 cells express low levels of Sca-2). These 2 Ly-6 proteins were, however, not coregulated on DA3 cells. Both Ly-6(hi) as well as Ly-6(lo) DA3 cells express equal levels of Sca-2. Levels of Thy-1, another glycosylphosphatidylinositol (GPI)-anchored protein expressed by A3 tumor cells, were equally expressed by both Ly-6(hi) and Ly-6(lo) A3 tumor cells. Levels of Ly-6 (but not those of CD44) on A3 tumor cells were upregulated on cells from dense cultures but were not influenced by the position of the cells in the cell cycle. Stress conditions such as serum starvation or heat shock upregulated the expression of Ly-6 by the 2 types of tumor cells but did not induce apoptosis in these cells. The kinetics of the stress-dependent upregulation of Ly-6 expression differed, however, between the epithelial and fibroblastic tumor cells.

Preleukemia in long-term plasmacytoma-regressor mice.

Our previous results have indicated that mice whose plasmacytoma regressed following curative melphalan chemotherapy manifested various persistent immunohematological abnormalities including immunosuppression, myeloproliferation, as well as excessive production of and response to growth factors. Mice not bearing plasmacytoma treated with an identical dose of melphalan chemotherapy did not exhibit such abnormalities. In the present study we show that plasmacytoma-regressor mice (PRM) contain preleukemic cells which do not progress to leukemia in these mice. However, adoptive transfer of splenocytes originating in PRM to preirradiated but otherwise untreated syngeneic recipients resulted in the development of overt leukemia in these recipients. The presence of leukemia in the primary recipient mice was ascertained by blood counts as well as by spleen histology. Furthermore, splenocytes from the irradiated primary recipients adoptively transferred to non-irradiated secondary recipients caused leukemia formation in 100% of the secondary recipients. Sex chromosome analysis of the leukemic cells in the irradiated primary recipients clearly showed that they originated in the PRM donors. Two leukemic lines were established from leukemias developing in the secondary recipients and both expressed surface markers of hematopoietic progenitor cells as well as markers of T cells. We suggest that PRM could serve as an animal model to investigate development of chemotherapy-related leukemia in humans.

TNFalpha and anti-Fas antibodies regulate Ly-6E.1 expression by tumor cells: a possible link between angiogenesis and Ly-6E.1.

Angiogenic and poorly angiogenic tumor variants were obtained by an intraperitoneal inoculation of cells from clones of polyoma-virus transformed BALB/c 3T3 cells into syngeneic mice. The angiogenic tumor cells expressed a higher tumorigenicity phenotype and a higher capacity to produce artificial pulmonary metastases than cells from the poorly angiogenic tumors. The former cells expressed also significantly higher levels of the lymphocyte activation protein Ly-6E.1 than the former cells. The two types of cells did not differ in expression levels of CD44 and of a polyoma-virus specific membrane antigen. These results raise the possibility that the angiogenic phenotype is coregulated with Ly-6. The effect on Ly-6 expression of signal transduction through TNF receptors, functioning as pivotal regulators of angiogenesis was therefore studied. It was found that TNFalpha and more so antibodies against Fas down-regulate expression levels of Ly-6. This down-regulation seemed to be selective as expression levels of CD44 were not affected by this treatment.

Contribution of the intracellular domain of murine Fc-gamma receptor type IIB1 to its tumor-enhancing potential.

We have previously shown that Fc gamma receptor type II B1 (Fc(gamma)RIIB1), when expressed on non-lymphoid tumor cells, significantly enhanced their tumorigenic phenotype. This study elucidates the role of the intracellular domain of Fc(gamma)RIIB1 in the enhancement of the malignant phenotype of polyoma-transformed 3T3 cells. We investigated the tumorigenic potential conferred by different variants of the receptor: Fc(gamma)RIIB1, a full-length receptor (B1) whose intracellular region is encoded by exons 8, 9 and 10; Fc(gamma)RIIB2, a spliced variant (B2) whose cytoplasmic domain comprises exons 9 and 10 and lacks exon 8; and Fc(gamma)RIIB1-CT53, a deleted mutant whose cytoplasmic domain contains the fragment encoded by exon 8 alone. We have investigated various properties of cells transfected with each of the above variants: tumorigenicity in syngeneic mice, formation of colonies in soft agar, growth rate, production of soluble receptor and capping of the ligand-bound receptor. Results show that while the presence of exon 8 did not enhance growth rate in vitro or production of soluble Fc(gamma)R, it did enhance the tumorigenic phenotype of transfected cells (both in vivo and in vitro growth in soft agar). B1-expressing cells exhibited a significantly higher tumorigenic phenotype than B2 cells. The presence of exon 8 alone (CT53 mutant) conferred the transfected cells a higher tumorigenic phenotype than Fc(gamma)R-negative control cells but lower than intact B1 or B2 cells, indicating that the presence of B1-specific exon 8 is not sufficient but that the presence of an intact B1 intracellular domain is essential, for conferring the high tumorigenicity phenotype upon cells. We conclude that the capping, following ligand binding contributed by exon 8, and the function contributed by the specific localization of exons 9 and 10 in B1 cells may determine their malignant phenotype.

The murine Fc-gamma (Fc gamma) receptor type II B1 is a tumorigenicity-enhancing factor in polyoma-virus-transformed 3T3 cells.

The murine receptor for the Fc portion of IgG is a molecule expressed by cells of the immune system. This study suggests the hypothesis that Fc gamma receptor type II B I (Fc gamma RIIB I) functions as a progression-enhancing factor when expressed ectopically on non-lymphoid tumor cells. It has been shown previously that BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) do not express Fc gamma RII but acquire the expression of this receptor following an in vivo passage in syngeneic mice. The specific Fc gamma RII transcript present in tumor cells was identified in this report as Fc gamma RIIB I (BI). In order to determine whether or not the ectopically expressed Fc gamma RII plays a role in the progression of these transformed cells, PyV-transformed 3T3 cells were transfected with BI-cDNA. The BI transfected cells were tested for their ability to form local tumors in syngeneic mice, as compared to transfected cells which express the co-transfecting neomycine resistance (neores) DNA alone or together with the lacZ gene. Fc gamma RIIB I expressors exhibited a significantly higher tumorigenic phenotype than FcR-negative controls, though both types of cells exhibited the same growth curve in vitro. The ability of Fc gamma RIIB I to act as a potentially tumorgenicity-enhancing factor was also demonstrated as Fc gamma RII was expressed by tumor cells, originating from inoculated Fc gamma RIIB I-transfected cells, or from inoculation of a mixture of receptor-positive and -negative cells. B I-expressing cells dominated the tumor-cell population over non-expressors. This dominance strengthened the hypothesis that FcR plays a role in tumor progression in vivo.

Detection of immunoglobulin A anticardiolipin antibodies in cervical mucus from in vitro fertilization patients and fertile women.

OBJECTIVE: To identify and quantify antiphospholipid autoantibodies of the immunoglobulin (Ig) A isotype in cervical mucus obtained from IVF patients and fertile controls. DESIGN: The study was performed prospectively. Blood and cervical mucus samples were obtained from patients undergoing IVF treatment (n = 27) at the time of expected E2 peak, before administering hCG. Control samples were taken from fertile women (n = 16) around the time of ovulation during a spontaneous nonstimulated menstrual cycle. Anticardiolipin activity was tested using ELISA. SETTING: Infertility and IVF unit of an academic tertiary referral medical center and university-based basic research laboratory. RESULTS: Forty-eight percent (13/27) of the IVF patients and 43.8% (7/16) of the fertile controls exhibited anticardiolipin IgA activity in aspirated cervical mucus. The mean activity measured for the positive cases was similar in both groups. This activity was higher than that measured in peripheral blood of the women studied. No difference was noted between infertile patients undergoing IVF treatment and fertile women in this respect. CONCLUSIONS: In this preliminary work, we demonstrated for the first time anticardiolipin IgA activity in human cervical mucus. These observations have to be substantiated by larger scaled studies to assess their possible clinical significance.

Microenvironmental factors regulate Ly-6 A/E expression on PyV-transformed BALB/c 3T3 cells.

In previous studies non-lymphoid murine tumor cells were sorted by flow cytometry, into 2 subpopulations. The one expressed high levels of the T-cell activation protein Ly-6 A/E and the other low levels of this protein. High Ly-6 A/E expression was associated with very high tumorigenicity and metastatic phenotypes. Cells expressing low levels of this protein expressed a significantly reduced malignancy phenotype as compared to unsorted tumor populations. In view of its direct (or indirect) involvement in tumor progression we studied, in the present work, the regulation by microenvironmental factors of Ly-6 A/E expression on A3C polyoma-virus transformed cells. Ligation of membrane Ly-6 A/E by the corresponding monoclonal antibodies resulted in up-regulated expression of this protein. Similar results were obtained by exposing A3C cells to interferon-alpha. In contrast, exposing tumor cells to tumor necrosis factor-alpha or to the extracellular matrix protein laminin resulted in a down-regulation of Ly-6 A/E expression on these cells. These results provide an additional insight into the role microenvironmental factors might play in tumor progression.

An association between high Ly-6A/E expression on tumor cells and a highly malignant phenotype.

Murine Ly-6 is a molecule expressed by various cells, including several types of hematopoietic cells such as pluripotent stem cells, and activated T cells. Ly-6 is also expressed on tumor cells originating from a variety of tissues. Preliminary observations suggested that the expression of Ly-6A/E is up-regulated on highly tumorigenic variants of polyoma-virus(PyV)-transformed BALB/c 3T3 cells as compared with weakly tumorigenic variants. On the basis of these observations, we sorted PyV-transformed A3C cells or DA3 mammary adenocarcinoma cells into stable sub-populations expressing high or low levels of membrane or mRNA Ly-6A/E. In vivo studies indicated that the high-Ly-6A/E-expressing cells in both tumor systems expressed a considerably more malignant phenotype (higher efficiency in local tumor production as well as in lung colonization) than low-Ly-6A/E expressors. Since the high-Ly-6A/E expressors did not exhibit any growth advantage in vitro over low Ly-6A/E expressors, we concluded that interactions of the former cells with micro-environmental factors operating in vivo (e.g., Ly-6A/E ligands) conferred upon these cells a highly malignant phenotype. Apart from the difference in Ly-6A/E expression, no other phenotypic characteristics distinguished highly from weakly malignant tumor cells. Similarly to T cells, where antibodies to Ly-6 transduce (or co-transduce) a proliferative signal, antibodies to Ly-6A/E were found to transduce a mitogenic signal to high-Ly-6A/E-expressing tumor cells but not to low-Ly-6A/E expressors. Taken together, these results show that Ly-6A/E expression is directly or indirectly associated in vivo with a highly malignant phenotype of 2 types of non-lymphoid murine tumors.

A human monoclonal IgA autoantibody--185/12--behaves like an autoimmune antiphospholipid antibody.

A human monoclonal anticardiolipin autoantibody (ACA) of the IgA-k isotype, designated 185/12, is described. The antibody was prepared from peripheral B cells, obtained from a patient with a history of habitual abortion, by immortalization with Epstein-Barr virus (EBV). The antibody displays a strong binding activity to cardiolipin and phosphatidyl L-serine, but not to phosphatidylcholine, phosphatidylinositol, ssDNA and dsDNA. It binds to cardiolipin in a concentration-related and saturable manner (Kd = 3.0 x 10(-8) M). This reaction is dependent upon the presence of bovine serum, and is fully inhibited by cardiolipin vesicles. The 185/12 antibody exhibits different binding patterns to the solid-phase bound cardiolipin-serum complex and to its individual components (cardiolipin and bovine serum). The Bmax of 185/12 binding to the complex (0.968 OD units) is higher than the sum of the Bmax values calculated for each one of the complex components (0.352 + 0.179 = 0.531 OD units). Bovine serum as well as purified beta 2-glycoprotein I (beta 2-GPI) in suspension inhibit the binding of 185/12 to the complex. 185/12 binding capacity increases in direct relation to the rising concentration of beta 2-GPI. Collectively, these data may be interpreted to suggest that 185/12 antibody, which is an IgA isotype, exhibits characteristics usually attributed only to antiphospholipid autoantibodies (APA) of the IgG isotype, that are associated with the clinical spectrum of APA syndrome (APA-S). It is, therefore, possible that autoantibodies of the IgA isotype could play a pathogenic role, which may be different from that of the IgG isotype, in the development of autoimmune phenomena.

Lung colonization by and heparanase activity in in vitro transformed 3T3 cells rendered highly tumorigenic by an in vivo passage.

Cloned BALB/c 3T3 cells transformed in vitro with polyoma virus and maintained in culture (C cells) were compared with respect to their ability to form experimental metastases, with cells from the same clones that were passaged subcutaneously in vivo (CTC cells), thereby gaining a high tumorigenicity phenotype. No correlation was found between a high subcutaneous tumorigenicity potential or the progression state of these cells, and their capacity to form experimental metastases. Both cell types were also tested for their ability to release heparan sulfate degradation products from a naturally produced, sulfate-labeled extracellular matrix (ECM). Whereas the in vitro maintained C cells did not express an endo-beta-D-glucuronidase (heparanase) activity, some of the in vivo passaged CTC cells expressed such an activity. No strict correlation was found, however, between heparanase activity and the ability of CTC cells from individual tumors to form experimental metastases. However, A9 CTC 220 cells which produced a large number of lung metastases also expressed a high heparanase activity. Both heparanase and lung colonization by A9 CTC 220 cells were inhibited to a large extent by heparin, suggesting the involvement of heparanase in the extravasation of these highly metastatic cells. A9 CTC 220 cells were found to release basic fibroblast growth factor (bFGF) from ECM. This release was partially inhibited by carrageenan lambda which also completely inhibited the heparanase activity expressed by these cells. The in vivo acquisition of heparanase activity was not a result of cell fusion between heparanase expressing host-derived cells and heparanase-negative transformed cells. This conclusion was based on the fact that both the in vitro maintained, heparanase-negative as well as the in vitro passaged, heparanase-positive cells exhibited a similar membrane and molecular market profile.

Myeloproliferation in long-term plasmacytoma-regressor mice.

MOPC-315 plasmacytoma-bearing BALB/c mice were treated with high doses of melphalan, causing a permanent and complete regression of the tumor. In the present study we analyzed plasmacytoma-regressor mice (PRM) 3-6 months after plasmacytoma regression. A second group of otherwise untreated normal mice was treated with melphalan (M--control group). A third group of mice remained untreated and served as an age- and sex-matched control group. PRM were cachectic and had an increased mortality rate compared to the M and the C control groups. Histopathological examination indicated that the spleen of PRM showed pronounced abnormalities, primarily in the red pulp. These abnormalities consisted of extramedullary hematopoiesis and myeloid-granulocytic hyperplasia. Spleens of M mice showed similar abnormalities but to a much lesser extent. Flow cytometric analysis of cellular surface markers of PRM splenocytes indicated a high number of large MAC-I- and GR-I-positive cells compared to splenocytes of M or C controls. These large cells also expressed Fc tau receptors (Fc tau RII), stained positively with non-specific esterase and adhered to plastic dishes; a certain percentage expressed MAC-2 and MAC-3 antigens. A quantitative suppression of CD4+ T cells and of B cells was also shown. Circulating levels of TNF were higher in PRM than in M or C mice. The capacity of splenocytes from PRM to secrete factors that stimulated CFU-GM colony formation in soft agar by bone-marrow cells from normal mice was significantly up-regulated compared to that of splenocytes from M or C mice.(ABSTRACT TRUNCATED AT 250 WORDS)