Characterization, expression and functional aspects of a novel protein tyrosine phosphatase epsilon isoform.

This report describes the identification and characterization of a novel cytoplasmic isoform of human protein tyrosine phosphatase epsilon (PTPepsilon). The novel isoform, denoted cyt-PTPepsilonPD1, displays only the N-terminal catalytic, active phosphatase domain 1 (PD1) which is common in all known PTPepsilon isoforms. In addition, it contains a unique 132-residue long C-terminal end with no known motifs or homology to other characterized proteins. RNAse protection assay on isolated leucocyte subpopulations and selected cell lines demonstrated highest expression of cyt-PTPepsilonPD1 in monocytes. The mRNA-encoding cyt-PTPepsilonPD1 is detected as distinct transcript(s) by Northern blot analysis and is a result of alternative splicing. cyt-PTPepsilonPD1 shows similar cellular localization in transfected cells, both in the cytoplasm and nucleus, as has been previously described for cytoplasmic PTPepsilon isoform. Our previous data suggest that the expression of cytoplasmic PTPepsilon inhibits the mitogen-activated protein kinase cascade through the extracellular signal-regulated kinase 1 and 2 pathway. A similar functional role is also presented here for cyt-PTPepsilonPD1, supporting our previous data suggesting that the catalytic first PD of PTPepsilon is responsible for this inhibition.

[Stem cells in adults]. / Stamceller hos voksne.

BACKGROUND: We present a literature review of the plasticity observed by adult stem cells. MATERIALS AND METHODS: We have reviewed the literature regarding stem cells from adults in order to summarise their ability to generate cells of other types than those of the tissue/organ from which they were isolated. RESULTS: Adult stem cells have recently been demonstrated to terminally differentiate into cells of other tissues than those from which they were originally isolated. For example, bone marrow cells have been shown to generate liver, nerve, heart and skeletal muscle cells in addition to their well-known ability to produce blood and mesenchymal cells. INTERPRETATION: Most studies demonstrate a proof-of-principle in animal models; much more research is needed before adult stem cells can be utilised in human medicine. However, the published reports are encouraging and give reasons for a cautious optimism with regard to future clinical use.

A splice variant of human ephrin-A4 encodes a soluble molecule that is secreted by activated human B lymphocytes.

Ephrin-A4 is a ligand for the erythropoietin-producing hepatocellular (Eph) receptor family of tyrosine kinases. We have identified a secreted form of ephrin-A4, denoted ephrin-A4 (s), which is encoded by an alternatively spliced mRNA and is produced by in vivo activated B cells in tonsils. Blood B cells secrete ephrin-A4 (s) upon stimulation via the B-cell antigen receptor. A subpopulation of tonsil cells in the crypts with a dendritic cell phenotype was shown to express EphA2, an Eph receptor tyrosine kinase that was found to be capable of binding an ephrin-A4 immunoglobulin chimeric protein. We conclude that ephrin-A4 (s) may play a role in the interaction between activated B lymphocytes and dendritic cells in human tonsils. (Blood. 2000;95:221-230)

The nature of the subset of MHC class II molecules carrying the CDw78 epitopes.

A CDw78 mAb FN1 was shown to recognize DP and/or DR molecules under the conditions of Western blotting. DP molecules were specifically retarded on a column of the FN1 immunosorbent; binding of FITC-labeled FN1 to B cell lines was completely blocked by excess of mAb to DR/DP beta chains, partially by several mAb to DP and weakly by some mAb to DR. The binding of two other CDw78 mAb, FN4 and MR11, to the B cell surface was most strongly inhibited by excess of different mAb to DR. Kinetics of stable binding of the CDw78 mAb indicated that their monovalent binding is of low affinity and that the stable binding to the surface is due to bivalent binding to two spatially close MHC class II molecules. FN1-based immunosorbent effectively immunoisolated complexes of MHC class II proteins with several tetraspanin molecules from a mild detergent lysate of a B cell line. It is concluded that FN1 and most likely also the other two CDw78 mAb recognize with low affinity determinants on MHC class II molecules (DP or DR) and preferentially bind in a stable fashion to dimerized or aggregated MHC class II molecules. Such dimers or aggregates may either exist as preformed on the cell surface or may be gradually formed and stabilized by bivalent interaction with mAb. These structures may be related to the previously described 'superdimers' of MHC class II and/or 'MHC-tetraspanin complexes'. CDw78 mAb may be valuable tools targeting such aggregated fraction of MHC class II molecules which can exhibit important signaling and antigen-presenting properties.

Immunomagnetic techniques for the enrichment and detection of isolated breast carcinoma cells in bone marrow and peripheral blood.

Detection of isolated tumor cells (TC) in bone marrow (BM) from patients with breast cancer is usually accomplished by immunocytochemical (ICC) analysis of up to 2 X 10(6) mononuclear cells (MNC). However, this method is cumbersome if large numbers of BM cells (i.e. > 1 X 10(7) cells) are to be analyzed. This emphasizes the need for TC enrichment strategies. This report describes immunomagnetic separation (IMS) techniques for enrichment and detection of viable breast carcinoma cells in BM and peripheral blood (PB). The positive IMS technique was performed by incubation of MNC with 2.8 microns magnetic particles (rat antimouse IgG1 M280-Dynabeads) coated with monoclonal antibody (mAb) against epithelial surface antigens. The rosetted tumor cells were then visualized by ICC staining using alkaline phosphatase-conjugated A45-B/B3 anticytokeratin mAb (Fab). The negative IMS technique was performed by incubation of MNC with anti-CD45-coated M450-Dynabeads (4.5 microns), followed by ICC staining of the nonrosetted cells. When 1000, 100, and 10 breast carcinoma cells were mixed with 1 X 10(7) MNC, an average of 748 (n = 9), 70 (n = 10), and 7.8 TC (n = 8), respectively, were detected with the positive IMS technique. With the negative IMS technique, 648 (n = 8), 57.8 (n = 6), and 7.3 TC (n = 6), respectively, were detected. The analysis of 1 X 10(7) MNC with the IMS techniques was compared with the ICC analysis of 2 X 10(6) unseparated MNC. A mean 3.7-fold (range 1.5-6.4) to 4.2-fold (2.5-8.2) (positive IMS) and 3.1-fold (range 2.0-5.0) to 3.8-fold (2.0-6.0) (negative IMS) higher TC detection frequency was achieved after enrichment by IMS in experiments with 100 and 1000 TC/10(7) MNC. The IMS techniques were used for examination of BM samples from locally advanced breast cancer patients. A 5.3-fold mean increase (range 2.1-13.3) in the number of TC detected was obtained when the use of positive and negative IMS together was compared with the direct ICC analysis of unseparated MNC (n = 11). Enrichment of TC by IMS techniques enables us to examine large numbers of MNC from BM or PB, which can result in the detection and characterization of minimal residual disease with increased sensitivity and specificity.

CDw78--a determinant on a major histocompatibility complex class II subpopulation that can be induced to associate with the cytoskeleton.

In the present study we demonstrate that CDw78 monoclonal antibody (mAb) recognizes a distinct subpopulation of major histocompatibility complex (MHC) class II molecules. We show that the CDw78 epitope is present on less than 10% of the total number of MHC class II molecules expressed on different cells, is not linked to a single isotype, and exhibits a characteristic expression pattern in tonsils. While mAb against MHC class II (DR, DP and DQ) stained the majority of cells both in the mantle zone and in germinal centers, the CDw78 staining was more heterogeneous with the strongest reactivity and the highest number of positive cells in the mantle zone and in the light centrocyte-rich part of the germinal centers. Antibodies to this MHC class II subpopulation (e.g. FN1) induced association with the cytoskeleton and a subsequent capping in more than 90% of peripheral blood B cells. In contrast, mAb against MHC class II (DR, DP and DQ) did not induce association with the cytoskeleton and only 10-20% of B cells were induced to cap, suggesting that CDw78 defines a population of MHC class II molecules functionally different from the majority of these antigens. Scatchard plot analysis indicates that FN1 mAb is of relatively low affinity (Ka = 1.5 x 10(8) M(-1)) and monovalent Fab fragments fail to bind to the cell surface with measurable affinity. Our data seen in the context of the ability of FN1 to co-stimulate B cells with a suboptimal dose of anti-mu suggest that CDw78 mAb might recognize a functional important subpopulation of MHC class II molecules so far not described. It seems likely that this subpopulation represents dimerized or aggregated MHC class II molecules that can selectively bind this low-affinity mAb.

Purging of tumor cells from leukapheresis products: experimental and clinical aspects.

Peripheral blood progenitor cell autografts are being used increasingly in conjunction with high-dose therapy of cancer patients, in the belief that these products have a low probability of containing tumor cells. However, recent findings demonstrate that tumor cell involvement is frequent in leukapheresis products. Although the clinical value of purging has not been clinically established by prospective randomized trials, several studies indicate that contaminating tumor cells in autografts contribute to relapse of the disease in the recipients. We describe our experimental and clinical experience in purging tumor cells from leukapheresis products. Based on our work with purging of lymphoma cells from bone marrow by the use of anti-B cell and anti-T cell antibodies and immunobeads, a purging procedure to deplete leukapheresis products of lymphoma cells has been developed. Moreover, we present data showing that breast cancer cells can be efficiently removed from leukapheresis products using antibreast cancer antibodies, either in combination with immunobeads or as immunotoxins. Our experience with enrichment of CD34 cells employing immunobeads in leukaphresis products from patients with breast cancer and lymphomas shows high purity and yield of CD34 cells. In spite of this, contaminating tumor cells can be observed, strongly suggesting that a combination of CD34 cell enrichment and a purging procedure might be warranted.

Solid-phase method for differential display of genes expressed in hematopoietic stem cells.

A solid-phase differential display method was designed to analyze differential gene expression in samples with low amounts of mRNA. The principle was based on using a biotinylated probe to capture the mRNA and priming both the first-strand synthesis and the subsequent polymerase chain reaction step. Coupling the mRNA to a solid phase during the procedure simplified the purification steps, limited sample loss and enabled rapid handling of mRNA. DNA contamination was also minimized when the mRNA was bound to a solid phase. Optimization of the differential display method was achieved by analyzing both the enzymatic conditions and the required cell amounts. The approach was used for the characterization of genes expressed in the most immature hematopoietic progenitor cells (CD34+CD38-). The majority of the differentially expressed fragments represented previously uncharacterized sequences.

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RI alpha 2 C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation.

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RI alpha, RII alpha, C alpha and C beta, barely detectable levels of RI beta mRNA, and no detectable RII beta or C gamma mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RI alpha and RII alpha, but no RI beta or RII beta. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[32P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RI beta. Immunofluorescence staining of RI alpha and RII alpha demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RI alpha, whereas the RII alpha subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RI alpha subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RII alpha subunits remains unchanged.

All-trans- and 9-cis-retinoic acid inhibit growth of normal human and murine B cell precursors.

In the present paper we demonstrate that physiologic levels (10 nM) of both all-trans- and 9-cis-retinoic acid (RA) are potent inhibitors of the growth of human as well as murine B cell precursors in vitro. Ten nanomolar concentrations of all-trans- and 9-cis-RA reduced the DNA synthesis ([3H]thymidine uptake) of human B cell precursors (CD19+ IgM-) stimulated with O-tetradecanoylphorbol-13-acetate and ionomycin by approximately 55% and 70%, respectively. Human B cell precursors stimulated with low m.w. B cell growth factor were also inhibited by RA. Ten nanomolar concentrations of either isoform of RA reduced DNA synthesis by approximately 50%. No effect of RA on differentiation to sIgM positive cells was noted. The potent growth-inhibiting effect of RA on human B cell precursors was confirmed in the murine cell system. B lymphopoiesis from murine hematopoietic precursors (Lin-B220(+)-containing cells) was induced by stimulation with IL-7. Concentrations of all-trans- and 9-cis-RA as low as 10 pM reduced the colony-forming ability of the IL-7-stimulated Lin-B220(+)-containing cells. Ten nanomolar concentrations of either isoform reduced colony formation by approximately 60%. RA was not toxic to the cells, as the inhibition of colony formation after 24 h was reversible at concentrations as high as 1 microM. The growth-inhibiting effect of RA was directly mediated, as revealed by single cell analysis of the Lin-B220(+)-containing cells. Thus, vitamin A appears to have an important role in regulation of B lymphopoiesis.

Characterization of a promoter region supporting transcription of a novel human beta-galactoside alpha-2,6-sialyltransferase transcript in HepG2 cells.

In humans, two transcripts encoding beta-galactoside alpha-2,6-sialytransferase (EC 2.4.99.1.) have previously been described. One of the transcripts is widely expressed, whereas the other is restricted to mature B-cells. In this study we demonstrate the existence of a third transcript in the hepatoma cell-line HepG2. The expression of this transcript is controlled by a promoter region which efficiently supports transcription in HepG2 cells, and which harbours putative binding sites for liver-enriched and acute phase inducible transcription factors.

All-trans retinoic acid directly inhibits granulocyte colony-stimulating factor-induced proliferation of CD34+ human hematopoietic progenitor cells.

In this study we examine the effects of retinoids on purified CD34+ human hematopoietic progenitor cells. All-trans retinoic acid inhibited granulocyte colony-stimulating factor (G-CSF)-induced proliferation of CD34+ cells in short-term liquid cultures in a dose-dependent fashion with maximal inhibition of 72% at a concentration of retinoic acid of 1 mumol/L. Although no significant effects were observed on granulocyte-macrophage CSF (GM-CSF)--interleukin-3--or stem cell factor (SCF)-induced proliferation, the combinations of G-CSF and each of these cytokines were all inhibited. Moreover, retinol (3 mumol/L) and chylomicron remnant retinyl esters (0.1 mumol/L) in concentrations normally found in human plasma also had inhibitory effects. Single-cell experiments showed that the effects of retinoic acid were directly mediated. Retinoids also significantly inhibited G-CSF-induced colony formation in semisolid medium, with 88% inhibition observed at a concentration of retinoic acid of 1 mumol/L. However, we did not observe any effects of retinoic acid on G-CSF-induced differentiation as assessed by morphology and flowcytometry. Similar to previous findings using total bone marrow mononuclear cells, we observed a stimulation of GM-CSF-induced colony formation after 14 days. We also observed a stimulatory effect of low doses of retinoic acid (30 nmol/L) on blast-cell colony formation on stromal cell layers. Taken together, the data indicate that vitamin A present in human plasma has inhibitory as well as stimulatory effects on myelopoiesis.

Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells.

Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte-macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.

Complement-mediated permeabilization of platelets by monoclonal antibodies to CD9: inhibition by leupeptin, and effects on the GP Ib-actin-binding protein system.

Two monoclonal antibodies to CD9 of the IgM and IgG2a categories (FN 52 and FN 99), reproducibly induced platelet alterations in platelet-rich plasma by activation of the complement system with membrane incorporation of the pore-forming C5b-9 complex. The permeabilization could be monitored by measurements of extracellular ATP and observed as a shape change followed by an increase in light transmission in the aggregometer, and was associated with formation of tiny platelet aggregates. This could be accomplished by only minor lysis observed as extracellular lactate dehydrogenase (LDH). When leupeptin was added prior to, or immediately after the antibody, a total inhibition of the platelet alterations could be obtained. When added soon after the shape change, leupeptin had little effect on the liberation of ATP. However, whereas the ability of the platelets to become agglutinated by ristocetin was lost during the complement-mediated platelet alterations, addition of leupeptin immediately after the shape change, prevented this loss. The lost ability of the permeabilized platelets to undergo ristocetin-induced agglutination is not ascribed to degradation of GP Ib as this was relatively little affected in these studies as compared to the actin-binding protein (ABP) which was profoundly degraded. This protein represents a link between GP Ib and the submembraneous cytoskeleton, and the inhibition of its degradation by leupeptin, was clearly demonstrated. Experiments with digitonin-induced permeabilization showed that leupeptin did not inhibit permeabilization as such, but it did prevent the loss of ristocetin-induced agglutination even with this inducer.

CDw75 antigen expression in human gastric carcinoma and adjacent mucosa.

BACKGROUND: The amount of sialic acid correlates with the invasiveness and metastasizing potential of several human tumors. The CDw75 epitope is a sialylated carbohydrate determinant generated by the beta-galactosyl alpha 2,6-sialyltransferase, which can be viewed as a target for identifying biologically aggressive tumors. METHODS: The authors performed an immunohistochemical study of CDw75 epitope expression in 87 cases of gastric carcinoma and adjacent mucosa and 331 metastases (329 lymph node metastases and 2 liver metastases) with the monoclonal antibody HH2. RESULTS: Normal-appearing mucosa, foci of intestinal metaplasia, and foveolar hyperplasia, adjacent to the carcinomas, were mainly nonimmunoreactive. Only a few parietal cells of the body mucosa were stained with HH2. Two of 12 cases with dysplasia showed CDw75 antigen expression in dysplastic glands. Forty-one cases (47.1%) were immunoreactive for CDw75 antigen in the primary tumors or metastases. A very close relationship was found between the expression of the antigen in primary tumors and their respective metastases. The expression of the antigen was correlated with an infiltrative growth pattern, lymphatic invasiveness, and aneuploidy. All but two immunoreactive cases had lymph node metastases or lymphatic permeation. No relationship was found between CDw75 antigen expression and the morphologic types of gastric carcinoma, amount of lymphoid infiltrate, vascular invasion, and penetration of the gastric wall. CONCLUSIONS: The authors conclude that CDw75 antigen expression can be used as a marker of malignant transformation of gastric epithelium and is a good indicator of the biologic aggressiveness of gastric carcinoma.

CDw75 antigen expression in breast lesions.

Alterations on the cell surface of the oligosaccharide portion of glycoproteins and glycolipids are thought to play a role in tumorigenesis. Sialyltransferase catalyzes the incorporation of sialic acid to the carbohydrate group of glycoconjugates. Sialyltransferase has been found elevated in different tumour tissues and in the serum of cancer patients. In the present study we have examined the expression of the beta-galactosyl alpha 2,6-sialyltransferase requiring epitope CDw75, with the monoclonal antibody HH2. 142 breast lesions were included. 21% of the carcinomas in situ and 35% of the invasive carcinomas showed a diffuse cytoplasmic staining. Seven cases of invasive carcinomas also showed a distinct membrane immunoreactivity. We found no correlation between reactivity for CDw75 in malignant lesions and their metastatic potential. Only five out of 11 primary tumours with metastases expressed CDw75 in the primary tumour. In the benign lesions, there was a positive reaction in proliferating lesions, e.g. intraductal papillomas (2 out of 3 cases) and in epithelial proliferations in fibrocystic disease (10 out of 14 cases). None of the four fibroadenomas and phyllodes tumours and only one out of 22 cases of normal breast tissues showed immunoreactivity for HH2. In the malignant lesions, CDw75 was more frequently expressed in the carcinomas of high malignancy grade. The high frequency of immunoreactivity among the benign breast lesions can be indicative of activation of the epithelial cells.

Cell-specific expression of human beta-galactoside alpha 2,6-sialyltransferase transcripts differing in the 5' untranslated region.

In humans, two cDNAs have been isolated encoding beta-galactoside alpha 2,6-sialyltransferase, differing only in part of the 5' untranslated region. Primer extension data show that the two cDNAs are near full-length clones. RNase protection analysis of different cell types showed that the transcript corresponding to the alpha 2,6-sialyltransferase cDNA isolated from a B-cell library resided only in mature B cells. In contrast, the transcript corresponding to the alpha 2,6-sialyltransferase cDNA isolated from a placenta library was found in all cells tested. Our results also indicate the existence of a third alpha 2,6-sialyltransferase transcript in the hepatoma cell line HepG2. Mature B cells were found to express high amounts of alpha 2,6-sialyltransferase mRNA, compared to other cell types tested, as shown by Northern blot analysis. Moreover there was an increased expression of beta-galactoside alpha 2,6-sialyltransferase mRNA in activated B cells compared to resting B cells. In vitro transcription and translation of the cDNAs resulted in a protein of 45 kDa, but the transcripts were translated with different efficiency, suggesting a role for the 5' untranslated region in regulation of translation. We have also made an alpha 2,6-sialyltransferase construct lacking the specific 5' regions of the two cDNAs. A transcript generated from this construct was translated more efficiently in vitro than the two alpha 2,6-sialyltransferase cDNAs.

Human Golgi beta-galactoside alpha-2,6-sialyltransferase generates a group of sialylated B lymphocyte differentiation antigens.

The role of the human beta-galactoside alpha-2,6-sialyltransferase (hu alpha-2,6-ST) in the generation of B cell surface antigens was investigated by selecting subclones of COS cells (monkey kidney epithelial cells) constitutively expressing a transfected cDNA which encodes the hu alpha-2,6-ST (COS alpha-2,6-ST cells). Expression of hu alpha-2,6-ST in COS cells was sufficient to generate sialylated cell surface epitopes on different glycosylated antigens recognized by monoclonal antibodies to CDw75, CD76, and the unclustered monoclonal antibodies HB-4 and EBU-65. These epitopes were sensitive to sialidase treatment and are likely to contain terminal alpha-2,6-linked sialic acid residues. A novel antiserum raised against bacterially expressed hu alpha-2,6-ST fusion protein was used to localize the sialyltransferase in two cell lines with high expression of either endogenous (B cell line JOK-1) or recombinant (COS alpha-2,6-ST cells) hu alpha-2,6-ST. In both cell lines, the enzyme was detected only intracellularly in the juxtanuclear region and not on the cell surface. In contrast, CDw75, formerly proposed to be identical with an alpha-2,6-ectosialyltransferase, was strongly expressed on the cell surface. The different expression patterns show that neither the CDw75 antigen nor any of the other sialylated antigens analyzed is identical with the hu alpha-2,6-ST. Furthermore, the presence of a surface-expressed alpha-2,6-ST appears unlikely in these cell lines. We propose that CDw75, CD76, HB-4, and EBU-65 represent a unique group of B cell differentiation antigens the production of which requires the enzymatic activity of alpha-2,6-ST.