The membrane IgM-associated heterodimer on human B cells is a newly defined B cell antigen that contains the protein product of the mb-1 gene

7/8embrane IgM (mIgM) on human B lymphocytes is noncovalently associated with a disulfide-linked dimer that contains phosphoproteins of 47 and 37 kDa. In this study, the biochemical properties and the identity of these Ag receptor-associated components have been addressed. Both subunits carry N-linked carbohydrate groups. After deglycosylation, the 47-kDa and 37-kDa proteins have similar molecular masses, of about 23 kDa, and relatively acidic but different isoelectric points. The accumulated data, together with a previously performed comparison of tryptic peptides, suggest that the two components are structurally distinct and possibly encoded by different genes. Indeed, a mAb, raised against a synthetic peptide that was made on the basis of the published carboxyl-terminal amino acid sequence of the human mb-1 gene product, specifically reacted with the 47-kDa but not the 37-kDa subunit. None of the established B cell-specific mAb characterized in the Fourth International Workshop on Leukocyte Antigens, including CD24, CD37, and CD72, detect the mIgM-linked heterodimer, which makes it a newly defined human B cell Ag.     

An immunoperoxidase study of S-100 protein in neoplastic cells in serous effusions. Use as a marker for melanoma

S-100 protein has been demonstrated on histologic sections in a number of neural and nonneural tissues, including a variety of neoplasms. Since pleural or peritoneal effusions are frequently the initial presentation of cancer, a study was undertaken to determine if S-100 protein in exfoliated cancer cells could be used as a marker for melanoma. Cells in 36 serous fluids obtained from 32 patients were retrospectively examined for S-100 protein by the peroxidase-antiperoxidase technique. All samples had been previously studied as Papanicolaou-stained cytology specimens, and 25 samples had been studied by transmission electron microscopy. All benign effusions were negative for S-100 protein. Malignant effusions were negative except for some that contained malignant melanoma cells: two of five pigmented melanomas and both cases of amelanotic melanoma. This study indicates that S-100 protein in malignant cells is a useful marker for malignant melanomas, especially the amelanotic type.     

Mesenchymal stem cells as all-round supporters in a normal and neoplastic microenvironment

ABSTRACT: Mesenchymal stem cells (MSC) represent a heterogeneous population exhibiting stem cell-like properties which are distributed almost ubiquitously among perivascular niches of various human tissues and organs. Organism requirements such as tissue damage determine interdisciplinary functions of resident MSC including self-renewal, migration and differentiation, whereby MSC support local tissue repair, angiogenesis and concomitant immunomodulating. However, growth of tumor cells and invasion also causes local tissue damage and injury which subsequently activates repair mechanisms and consequently, attracts MSC. Thereby, MSC exhibit a tissue-specific functional biodiversity which is mediated by direct cell-to-cell communication via adhesion molecule signaling and by a tightly regulated exchange of a multifactorial panel of cytokines, exosomes, and micro RNAs. Such interactions determine either tumor-promoting or tumor-inhibitory support by MSC. Moreover, fusion with necrotic/apoptotic tumor cell bodies contributes to re-program MSC into an aberrant phenotype also suggesting that tumor tissue in general represents different types of neoplastic cell populations including tumor-associated stem cell-like cells. The present work summarizes some functional characteristics and biodiversity of MSC and highlights certain controversial interactions with normal and tumorigenic cell populations, including associated modulations within the MSC microenvironment.     

DNA aneuploidy as a specific marker of neoplastic cells in FNAB of the thyroid

OBJECTIVE: To investigate whether DNA image cytometry (ICM) could be of value in the specific identification of neoplastic cells in cytologic specimens of the thyroid. STUDY DESIGN: FNABs of thyroid from 162 patients with different benign and neoplastic diseases were investigated. Nuclear DNA content in thyroid cells was measured after Feulgen staining using a TV image analysis system. Data were correlated with clinical and histologic patient follow-up. The occurrence of abnormal DNA stemlines was used as a marker for aneuploidy and thus for neoplasia. RESULTS: None of the 89 cases without tumor cells revealed DNA aneuploidy. An abnormal DNA stemline was found in 55% of histologically proven benign thyroid tumors (follicular or oncocytic adenomas) and in 59.5% of malignant tumors. The positive predictive value of DNA aneuploidy in FNABs of the thyroid for neoplasms was 100%. The negative predictive value of DNA nonaneuploidy for the prediction of tumor-free histologic or clinical follow-up was 79.4%. CONCLUSION: DNA image cytometry may be helpful in the specific identification of neoplastic follicular cells. DNA ICM on FNABs of the thyroid is an additional tool to achieve early identification of patients with nodular lesions of the thyroid that have to be operated on. DNA euploidy excludes the presence of neither malignancy nor neoplasia.     

Cell cycle control in normal and neoplastic cells.

Recent studies of cell cycle control suggest that cyclin-dependent protein kinases play a central role in the cell's commitment to a new division cycle in late G1. The regulation of these kinases in normal and neoplastic growth is becoming clear.     

Complementary DNA cloning of a novel epithelial cell marker protein, HME1, that may be down-regulated in neoplastic mammary cells.

A full-length complementary DNA clone from a normal human mammary epithelial cell (strain 184) encoding a 25-kilodalton protein, HME1, was isolated. Expression of HME1 RNA appears to be limited to epithelial cells. The HME1 sequence has extensive sequence homology with bovine 14-3-3 protein, which is an activator of tyrosine and tryptophan hydroxylase. However, the tissue distribution, arrangement of charged amino acids, and location of potential phosphorylation sites of HME1 differ from those of 14-3-3. Compared with normal mammary epithelial cells, expression of HME1 RNA was dramatically low in two cell lines derived from human mammary carcinoma that were examined, and in a line of normal mammary epithelial cells transformed by oncogenes. HME1 therefore appears to be a cellular differentiation marker that may be down-regulated during neoplastic transformation.     

B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties.

A gene, called m-mb-1, was isolated from a murine pre-B-minus T lymphocyte subtracted library. It was found expressed as mRNA at low to medium abundance in early progenitors of the B lineage, in pre-B and mature B lineage cell lines, in normal resting B lymphocytes and in polyclonally activated B cell blasts. The gene was not expressed in plasmacytomas, in cell lines of the monocyte/macrophage, the T lymphocyte or the fibroblast lineages, nor in thymus, liver, heart, kidney, lung or brain. The nucleotide sequence of the m-mb-1 gene encodes a putative membrane glycoprotein with 220 amino acids, which includes a leader sequence, a putative extracellular domain with two potential N-glycosylation sites, a transmembrane portion and a putative intracellular domain. The partial sequence of a human homologue, h-mb-1, shows nearly 90% homology in nucleotide as well as amino acid sequences to the murine form of a stretch of the putative intracytoplasmic domain. Antibodies raised against a fusion protein of m-mb-1 with protein A, affinity purified for their m-mb-1 specificity, stained pre-B and mature B cell lines on their surface, but did not stain T cell lines and fibroblasts. Antibodies raised against a stretch of 20 amino acids of the putative intracellular domain with 90% homology between the mouse and human protein did not stain the surface of any cell lines tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human mb-1 gene: complete cDNA sequence and its expression in B cells bearing membrane Ig of various isotypes.

The transmembrane protein, IgM-alpha, a product of mb-1 gene, has been shown to be specifically associated with membrane-bound IgM on the plasma membrane of B lymphocytes. Recent studies have suggested that IgM-alpha may play a role in transducing signals from the Ag receptors during the activation of B cells. A large amount of information has been obtained in the mouse system regarding IgM-alpha and other components of the newly conceived B cell Ag receptor complex. Here we report the cloning and the nucleotide sequencing of cDNA clones of human mb-1, covering the entire length of the mRNA. At the amino acid sequence level, human and murine mb-1 share a high homology in their transmembrane and intracytoplasmic segments, suggesting an important biologic function for these regions of mb-1. A major difference, mainly in the 3' untranslated part, exists between our cDNA sequence and the published partial human mb-1 cDNA sequence. It has also been observed that human mb-1 is expressed not only by B cell lines expressing membrane-bound Ig of mu and delta isotypes but also those expressing membrane-bound Ig of alpha and gamma isotypes.     

Flow cytometric detection of the mitochondrial BCL-2 protein in normal and neoplastic human lymphoid cells.

The bcl-2 proto-oncogene, rearranged and deregulated in B-cell lymphomas bearing the t(14;18) translocation, encodes an inner mitochondrial membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single- and multicolor flow cytometric analysis of bcl-2 protein in relation to other markers and cell cycle, based on a fixation-permeation step of cells with paraformaldehyde and Triton X100 and the use of a bcl-2 specific monoclonal antibody (MoAb). As an application of this method, we have examined the expression of bcl-2 in normal and neoplastic lymphoid cells. We have found that greater than 80% of normal T-and B-cells are bcl-2 positive; following in vitro mitogen activation, the bcl-2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl-2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two-color analysis. On lymphoblastoid cell lines, the bcl-2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl-2 gene. Among fresh B-cell non-Hodgkin's lymphomas (B-NHL), most sporadic Burkitt's cases were bcl-2 negative. Of four centroblastic-centrocytic cases with rearrangements of the bcl-2 gene, only two presented elevated amounts of bcl-2 protein, indicating that the levels of bcl-2 are not diagnostic of the translocation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkaline phosphatase activity as a membrane marker for activated B cells

Alkaline phosphatase activity was assayed by a microtiter assay (with p-nitrophenylphosphate used as substrate) in the plasma membrane of mouse spleen cells activated in vitro by the B cell mitogen LPS and the T cell-dependent B cell mitogen, PWM. No activity was detected in spleen cells cultured in the presence of the T cell mitogens PHA and Con A. This alkaline phosphatase activity was detected in the blast-enriched 30 to 40% fraction of discontinuous Percoll gradients in LPS-treated cultures, and the enzymatic activity assayed was susceptible to inhibition by the alkaline phosphatase inhibitors EDTA and L-phenylalanine. These data support the idea that the membrane alkaline phosphatase activity could be used as a marker for activated B cells.     

Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway.

Thioredoxin, despite its function as an intracellular disulfide reducing enzyme and its lack of a signal sequence, has been found to play some roles extracellularly. Here we show that thioredoxin is actively secreted by a variety of normal and transformed cells, including fibroblasts, airway epithelial cells, and activated B and T lymphocytes. Neither brefeldin A nor dinitrophenol, two drugs that block transport through the exocytic pathway, inhibit secretion of thioredoxin, indicating that the latter does not follow the classical ER-Golgi route. The secretory mechanism for thioredoxin shares several features with the alternative pathway described for interleukin-1 beta, such as the potentiating effect on secretion of several unrelated drugs and the sensitivity to methylamine. However, unlike interleukin-1 beta, thioredoxin is not detected in membrane-bound compartments of secreting cells. In addition, when COS7 are transfected with plasmids encoding pro-interleukin-1 beta or thioredoxin, only the latter is detectable extracellularly.     

Differentiation and the neoplastic transformation of normal cells

It is suggested that blastomogenic factors can be considered as the means isolating normal cells from each other. Carcinogenic substances caused cell functions to disturb, to inhibit or to intensify the synthesis of specific substances, to change the cell surface disrupted contacts of interactions between cells, pressing cells to become on the way of evolution, and to create heterogenic populations of cells.     

The multidrug resistance protein 1: a functionally important activation marker for murine Th1 cells

Previously, we described the expression of an energy-dependent pump in resting murine Th2 (but not resting Th1) cells which extruded the fluorescent dye Fluo-3. After stimulation with Ag and APCs, Th1 cells also expressed this pump. Furthermore, expression of the murine multidrug resistance protein 1 (mrp1) correlated with the presence of the pump. In this study, we report that Fluo-3 is indeed transported by murine mrp1 or its human ortholog MRP1, as revealed by transfection of HEK 293 cells with mrp1 or MRP1 cDNA. Like antigenic activation, IL-2 dose-dependently enhanced the Fluo-3-extruding activity in murine Th1 cells. Although TNF-alpha and IL-12 by themselves only weakly enhanced Fluo-3 extrusion, each of them did so in strong synergism with IL-2. An Ab directed against mrp1 was used to quantify the expression of mrp1 protein in T cells at the single-cell level. Like the Fluo-3 pump, mrp1 protein expression was enhanced by IL-2. Immunohistochemical studies using confocal laser microscopy indicated that mrp1 is localized mainly at the plasma membrane. In addition, protein expression of mrp1 was induced in Vbeta8+CD4+ T cells 12 h after in vivo application of Staphylococcal enterotoxin B. Finally, mrp1 was functionally relevant during the activation process of Th1 cells, because T cell activation could be suppressed by exposure of cells to the mrp1 inhibitor MK571. Thus, we present mrp1 as a novel, functionally important activation marker for Th1 cells and short-term in vivo activated CD4+ T cells, whereas its expression seems to be constitutive in Th2 cells.