WM Cells Research Articles

Polymorphisms in the Hyaluronan Synthase 1 Gene May Be Predisposing Factors for Waldenstrom's Macroglobulinemia.

The hyaluronan synthase 1 gene (HAS1), which maps to chromosome location 19q13.4 encodes a plasma membrane protein, which synthesizes hyaluronan (HA), an extracellular matrix molecule. Previously, in WM patients we detected up-regulation of HAS1 transcripts and identified aberrant splice variants of this gene, termed HAS1Va, Vb, and Vc. In patients with multiple myeloma, expression of HAS1Vb either alone or in combination with HAS1 and other variants strongly correlates with poor survival (P=0.001). Our gene expression analysis of the HAS1 family members demonstrated that 76–97% of individual CD20+ IgM+ WM cells obtained from BM aspirates and from blood (PBMC) express HAS1Va or HAS1Vb aberrant splice variants, often in the absence of full length HAS1 transcripts. Aberrant splicing of HAS1 is the result of activation of cryptic splice sites, which lead to exon skipping and/or intron retention. In turn, activation of cryptic donor and acceptor splice sites of the gene can be promoted by the mutations occurring upstream of these sites and/or at the branch point of slicing. We measured the frequency of a known polymorphism in the HAS1 gene of 16 BM and 30 PB samples obtained from WM patients, in comparison with PBMC samples from 33 healthy donors. Our results indicate that in healthy individuals, the frequency of the two alleles each reached 50% perhaps reflecting stabilizing selection. The majority of healthy donors (61%) are heterozygous for the HAS1 gene polymorphism. In contrast, 92% of analyzed WM patients are homozygous for this same polymorphism. An expression analysis of HAS1 and variants in BM cells and PBMC obtained from the same group of WM patients demonstrated that only those patients who were homozygous for the HAS1 polymorphism express HAS1 and/or its variants. The few WM patients who are heterozygous for this polymorphism have no detectable expression of aberrantly spliced HAS1 variants or full length of HAS1, a phenotype identical to that of healthy donors. Thus, our observations so far suggest that polymorphisms in HAS1 may contribute to aberrations such as exon skipping and/or activation of a new cryptic splice site leading to aberrant splicing of HAS. Based on the results obtained thus far, we speculate that individuals who are homozygous for HAS1 polymorphism(s) are at enhanced risk of developing WM due to a predisposition towards aberrant HAS1 splicing and expression of HAS1 variants, with predicted oncogenic consequences. However, the study of a much larger number of patients and healthy donors is needed to confirm these speculations and to evaluate the prognostic significance of these findings.

Cytokine modulation of antigen expression in human melanoma cell lines derived from primary and metastatic tumour tissues.

The constitutive and cytokine-mediated expression of MHC class I and II antigens and intercellular adhesion molecule-1 (ICAM-1) was evaluated on eight human melanoma cell lines derived from primary and metastatic malignancies from patients (WM human melanoma series) including three pairs of related cell lines derived from the same individual. The cytokines IL-1 beta, IL-4, IL-6, TNF alpha, TGF beta 2, IFN gamma and IFN alpha were assessed for their ability to modulate the expression of cell surface antigens. MHC class I and class II antigen expression was unregulated by IFN gamma, IFN alpha and/or TNF alpha in cell lines established from primary melanoma. In contrast the cell lines derived from metastatic deposits did not show an increase in expression of MHC antigens in response to these cytokines. Both primary and metastatic WM cell lines were shown to be resistant to spontaneous natural killer cell (NK) activity, but susceptible to effector lymphocytes mediating lymphotine activated killer (LAK) cytotoxicity as a result of activation by IL-2. Although the constitutive and cytokine-induced level of expression of ICAM-1 and MHC antigens varied between paired primary and metastatic cell lines, this did not correlate with susceptibility of the cell line target to NK or LAK cytotoxicity. Whereas the IFNs, TNF alpha, TGF beta 2 and IL-1 beta differentially modulated the expression of ICAM-1 and MHC class I, treatment with IFNs (but not IL-1 beta, TNF alpha or TGF beta 2) resulted in a significant reduction in the sensitivity of the melanoma cells to NK and LAK cytotoxicity. Constitutive ICAM-1 expression was positively correlated with the ability of WM cell lines to colonise the lungs of SCID mice upon i.v. injection. The acquisition of cytokine resistance and inability to demonstrate enhanced cell surface expression may represent an important feature associated with the development of the metastatic phenotype.

Accumulation in “normal” lungs of live tumorigenic cells during sub‐cutaneous growth of weakly and highly metastasizing tumors and their In vitro sensitivity to growth regulation by normal fibroblasts

The main purposes of the study were: (1) in vivo comparison of accumulation of live tumorigenic cells (LTC) in macroscopically normal lungs of animals bearing 6 s.c. Syrian hamster sarcomas differing in spontaneous metastasizing activity (SMA); (2) in vitro examination of the sensitivity of these cell strains to the growth-regulating signals of normal fibroblasts. Cell strains used differed in SMA from very weak (WM) to extremely high (HM). The numbers of LTC doses in "normal" lung tissue of tumor-bearing animals were determined in s.c. transplantation tests by titrating single-cell suspensions prepared from the lung tissues of 5 tumor-bearing animals, for each cell strain, every 5 days during 30 days of s.c. tumor growth, until the appearance of the first spontaneous lung metastases. The sensitivity of WM and HM cells to growth-regulating signals of normal hamster embryo fibroblasts (HEFs) was examined by in vitro co-culturing during 6 days with daily determination of 3H-TdR incorporation in the WM and HM cells grown with or without contact with HEFs. The data presented demonstrate (1) the surprisingly similar efficiency in the occupation of macroscopically normal lung tissues by live tumorigenic cells of WM and HM strains, disseminating spontaneously from the s.c. tumors; (2) the significantly lower sensitivity of HM cells, in contrast to WM, to growth inhibition by contact with HEFs and especially their marked ability to usurp the growth-stimulating signals of normal HEFs.

Analysis of leucocyte differentiation antigens in blood and bone marrow from patients with Waldenström's macroglobulinaemia.

Peripheral blood and bone marrow mononuclear cells from 10 patients with newly diagnosed Waldenström's macroglobulinaemia were analysed for the presence of leucocyte differentiation antigens using flow cytometry. Most patients had circulating intracytoplasmic-mu cells that were positive for the B-cell specific antigens B1, B2 and B4 and for the plasma cell antigen PCA-1. When such monoclonal cyto-mu + cells were purified by immune rosette depletion they were shown to express B1 to a much lesser extent than B2, B4 and PCA-1. In bone marrow samples cells with a similar phenotype, but with higher B1 expression were found in all patients. When the serum IgM paraprotein concentrations were compared to surface markers longitudinally from presentation and during the first courses of chemotherapy, a decreased serum IgM level was accompanied by a decrease in B1 cells in peripheral blood. These data demonstrate that WM cells possess a unique phenotype with coexpression of B-cell and plasma cell associated antigens.

Lipoteichoic acid from Bacillus subtilis subsp. niger WM: isolation and effects on cell wall autolysis and turnover.

Lipoteichoic acid (LTA) was extracted by means of hot aqueous phenol from Bacillus subtilis subsp. niger WM cells grown under various conditions in chemostat culture. The extracts were partially purified by nuclease treatment and gel permeation chromatography. Chemical analyses revealed a composition consistent with a polyglycerol phosphate polymer. The influence on autolysis of the LTAs thus obtained was studied with both whole cells and autolysin-containing native walls of B. subtilis subsp. niger WM. Lysis rates of phosphate-limited cells could be reduced to about 40% of the control rate by the addition of LTA, whereas lysis of cells grown under phosphate-sufficient conditions was affected to a much lesser extent. The lysis of native walls prepared from variously grown cells proved to be fairly insensitive to the addition of LTA. The effect of LTA on wall turnover was studied by following the release of radioactively labeled wall material during exponential growth. The most obvious effect of LTA was a lowered first-order rate of release of labeled wall material; calculations according to the model for cell wall turnover in Bacillus spp. formulated by De Boer et al. (W. R. De Boer, F. J. Kruyssen, and J. T. M. Wouters, J. Bacteriol. 145:50-60, 1981) revealed changes in wall geometry and not in turnover rate in the presence of LTA.

Immunological phenotype of neoplasms involving the B cell in the last step of differentiation.

The immunological phenotype of diseases involving the last step of B cell differentiation--multiple myeloma (MM, 38 patients) and Waldenström's macroglobulinaemia (WM, 12 patients)--was analysed with a panel of monoclonal antibodies (McAb) as well as conventional markers. Most of the bone marrow plasma cells (80%) from MM patients reacted with the McAb OKT10, FMC8 and FMC48. Plasma cells were consistently negative with FMC7, Leu-1 and mouse rosettes. Ia, B1 and SIg were expressed in a minority of plasma cells (less than 30%) in half of the cases. The circulating neoplastic cells from five MM patients showed a more immature phenotype, with a higher reactivity for OKIa, B1 and increased SIg and a lower expression of CIg, than bone marrow plasma cells. The malignant cells of WM patients differed from those of MM in the reactivity with FMC7, being positive in 10 out of 11 cases, and in their high expression of B1, Ia and SIg with a predominant mu+ phenotype. Mouse rosettes and Leu-1 were positive in one case; OKT10 was positive in three out of five WM patients studied. This phenotype indicates that WM cells correspond to an earlier stage of B cell differentiation than MM plasma cells. The McAb J5 was positive in three out of six MM and two out of four WM analysed. The antigenic differences observed in MM and WM patients support the notion that the cells of the neoplastic clone are able to undergo a certain degree of differentiation.