Alkaline phosphatase expression in human cell lines derived from primary hepatomas

Isozyme patterns of alkaline phosphatase (ALP) were electrophoretically examined in human cell lines derived from one hepatoblastoma, five hepatocellular carcinomas (HCCs) and two cholangiocellular carcinomas. Most of the cell lines tested had a liver-type ALP isozyme. In addition, an abnormal ALP isozyme, which was similar to variant ALP, was detected in one hepatoblastoma and two HCC cell lines. One HCC cell line of these variant-like ALP-positive cell lines was alpha-fetoprotein (AFP)-negative. These findings suggest that variant-like ALP may be useful for the identification of human hepatoma cell lines, especially in AFP or albumin-negative cell lines.     

Alkaline phosphatase isozymes as possible markers of differentiation in human testicular teratocarcinoma cell lines

The alkaline phosphatase (ALP) activity in eight independent cell lines derived from human testicular germ cell tumors was characterized. Seven out of eight of the lines had high ALP levels, and most of the activity in each case was of the liver/bone/kidney ALP type, as judged by thermostability, inhibition, and electrophoretic studies. Low levels of a heat stable, placental-like ALP were also present in these seven lines; only a small subpopulation of the cells of each line reacted strongly with an anti-placental ALP monoclonal antibody. The heat-stable, placental-like isozyme characteristic of these lines differed from the normal placental ALP in its inhibition profile. Thus it is possible that a subpopulation of the cells in these lines expresses a new embryonic ALP form.     

Heterogeneity of alkaline phosphatases in different HeLa lines

Alkaline phosphatase (ALP) components in 8 cell lines of HeLa were examined. Line to line heterogeneity in ALP expression was observed using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylanine inhibition, and reactivity against antiplacental ALP antiserum. Six lines contained a placentallike ALP isozyme and varying amounts of a liverlike ALP isozyme. One line contained a liverlike ALP isozyme only. One line contained a new ALP form which was clearly distinguished from the placental, liver, bone, and intestinal ALPs. Thus, derepression of the placental ALP structural locus appeared to have occurred in 6 of the 8 lines. However, where expressed, the placentallike ALP varied electrophoretically from line to line, and in only one case was the mobility identical to that of a common placental ALP phenotype. This phenotypic heterogeneity of the "derepressed' placentallike ALP contrasts markedly with the phenotypic stability of other enzymes expressed in HeLa cells.     

B cell sensitivity to natural killing: correlation with target cell stage of differentiation and state of activation

When 13 B cell lines were phenotyped with a panel of B cell, stage-specific monoclonal antibodies and ordered with respect to differentiation state; their sensitivity to natural killer (NK) cell-mediated conjugate formation and cytolysis was found to be stage dependent. Target cell lines expressing an early B cell phenotype (B1+B2-CALLA+DU-ALL1 +/-) or an intermediate phenotype (B1+B2+CALLA-DU-ALL1+) were NK resistant. Late phenotypic B cells (B1+B2-CALLA-DU-ALL1-) were highly susceptible to NK cytolysis. Individual B cell lines when induced with sodium butyrate or retinoic acid expressed altered B cell phenotype and NK susceptibility. For example, SB, an intermediate B cell line and initially NK resistant, when induced to express a later B cell phenotype became NK sensitive. BJA.B, a late B cell line and initially NK sensitive, when induced to differentiate lost most of its sensitivity to natural killing. Since loss of the B2 antigen is associated with B cell activation, we further phenotyped the B cell lines and induced B cell lines with the 4F2 and 5E9 (anti-transferrin receptor) monoclonal reagents. All cell lines tested expressed each of these antigens, but with varying intensities. While the intensity of 4F2 expression appeared to correlate well with NK sensitivity on both resting and differentiated B cell lines, the intensity of 5E9 expression did not. Peripheral blood B cells express a similar pattern of reactivity to natural killing when stimulated with pokeweed mitogen (PWM) in vitro. B cell sensitivity to NK-mediated events peaked at day 4 of incubation and correlated with the loss of the B2 antigen and acquisition of the 4F2 and 5E9 antigens; sensitivity to natural killing was diminished in the presence of the PCA-1 antigen. The expression of the NK-susceptible phenotype among B cells appears to be differentiation stage- and activation state-dependent. It is during this period of ontogeny that the NK cell may cytolytically regulate the B cell transition to a plasma cell.     

Differential patterns of expression of glycosylphosphatidylinositol-anchored carcinoembryonic antigen and alkaline phosphatase in various cancer cell lines

The expression of glycosylphosphatidylinositol (GPI-anchored) carcinoembryonic antigen (CEA) and alkaline phosphatase (ALP) on the cell surface of various cancer cell lines and a lung diploid cell line (WI38) was investigated, with exposure of the cell lines to a cell differentiation agent (sodium butyrate) to induce cell differentiation and expression of the two tumor-associated antigens. In three colon (SW1222, SW1116, and HT-29) and stomach (MKN-45) cancer cell lines, all of which are double producers of CEA and ALP, the maximum expression of GPI-anchored CEA occurred with butyrate at a lower concentration than did that of GPI-anchored ALP. GPI-anchored ALP derived from colon (SW1222 and SW1116) and stomach (MKN-45 and MKN-1) cancer cell lines was heat-stable with and without exposure to butyrate, but GPI-anchored ALP derived from lung cancer cell lines (PC-6, PC13, PC-14, WI26VA4, and WI38VA13) showed a variety of heat stabilities, depending on cell line, butyrate exposure, and SV40 transformation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Secretion of antileucoprotease from a human lung tumor cell line

Two human tumor cell lines were analyzed for the production of human antileucoprotease (ALP). One of them, a human squamous lung carcinoma cell line (HS-24) synthesized, as confirmed by Western blot analysis, high amounts of ALP in serum-free medium. The supernatant inhibited elastase, chymotrypsin and trypsin. Northern blot analysis with an 18-mer radiolabelled oligonucleotide, derived from an ALP specific cDNA clone, revealed a specific mRNA of about 700-800 nucleotides in HS-24 tumor cells. In contrast, a secondary human lung tumor cell line (SB-3), derived from the adrenal cortex, did not synthesize ALP when assayed under identical conditions. The supernatant inhibited only trypsin and chymotrypsin.     

Down-regulation of murine collagen-induced arthritis by a T cell hybridoma

T cell hybridoma cell lines were generated by somatic cell fusion of BW 5147 myeloma cells and splenic cells from mice suppressed for collagen-induced arthritis (CIA). Two cell lines were characterized for their cell surface phenotype, antigen recognition and capacity to down-regulate the erythema and edema associated with CIA. Cell line T101N was determined to portray the cell surface phenotype Ly1+2- L3T4- Thy1+ by a direct binding assay. Cell line T104B1 was determined to express only the Thy1+ alloantigen. Panning studies, measurement of IL-2 production in vitro and the suppression of antibodies to type I and type II collagen in vivo indicate that the hybridoma cells are not isotype specific in their recognition of the polymorphic interstitial collagens. Down-regulation of the erythema and edema of CIA occurred on injection of 1 X 10(5) T101N cells but not T104B1 cells. Histology of the tarsus region of the hind paw of CIA mice 33 days after the administration of T101N cells showed contrasting histopathology compared to that of CIA mice. The joints of CIA mice given T101N cells showed aligned articular surfaces resembling normal joint structure and only residual pannus. The data indicate that collagen-specific cloned T cell lines can modulate the gross pathology and joint architecture of joints exhibiting CIA.     

Induction of alkaline phosphatase activity by exposure of human cell lines to a low-frequency electric field from apparatuses used in clinical therapies

Low‐frequency (LF) electric fields (EFs) are currently used in clinical therapies of several bone diseases to increase bone regenerative processes. To identify possible molecular mechanisms involved in these processes, we evaluated the effects on cell cultures of 1 h exposures to the signal generated by an apparatus of current clinical use (frequency 60 kHz, frequency of the modulating signal 12.5 Hz, 50% duty cycle, peak‐to‐peak voltage 24.5 V). Two different human cell lines, bone SaOS‐2 and liver HepG2, were used. Exposures significantly increased alkaline phosphatase (ALP) enzymatic activity in both cell lines. The increase was about 35% in SaOS‐2 cells and about 80% in HepG2 cells and occurred in the first 4 h after exposure and decreased to almost no change by 24 h. Since ALP represents a typical marker of bone regeneration, these results represent a first molecular evidence of biological effects from 60 kHz EF exposures. The finding of similar effects in cells derived from two different tissues more likely indicates the effective operation of the mechanism in living organisms. Bioelectromagnetics 32:113–119, 2011. © 2010 Wiley‐Liss, Inc.     

New monoclonal antibodies that define multiple epitopes and a human-specific marker on the interleukin 2 receptor molecules of primates

Twelve hybridoma cell lines producing monoclonal antibodies to the human interleukin 2 (IL-2) receptor (IL-2R) molecule were prepared. These antibodies were characterized by competitive antibody-binding assay and sequential immunoprecipitation assay with four known monoclonal antibodies to the human IL-2R molecule. The twelve new monoclonal antibodies were divided among the four known antibody types, the HIEI-, H-A26-, H-31-, and anti-Tac-type, and an additional new type, the H-48-type. The H-48 antibody did not compete with any other antibodies in the competitive binding assay. The binding of 125I-IL-2 to MT-2 cells and the IL-2-dependent growth of normal activated T-cells were both strongly inhibited by all the H-31- and anti-Tac-type antibodies, and partially or slightly inhibited by HIEI- and H-A26-type antibodies, but were not inhibited by the H-48 antibody. Thus, the same type of monoclonal antibodies had a similar effect on the function of IL-2R. These results suggest that epitopes for the same type of antibodies could be single identical epitopes or epitopes closely associated with each other. On the other hand, these antibodies also reacted variously with a panel of various human and simian lymphoid cell lines immortalized with human T-cell leukemia virus type-I (HTLV-I): the H-45 antibody reacted only with the human cell lines, the H-C1 and H-44 and H-47 antibodies reacted with human and ape cell lines, and the other antibodies reacted with cell lines of humans, apes and Old and New World monkeys. These differences in the reactivity of the antibodies with the primate cell lines suggest that the antigenic structure of the IL-2R molecule changed during evolutionary divergence of the primates.     

Acid lability of the mutated glucosylceramide-beta-glucosidase in a lymphoid cell line from type 2 Gaucher disease

Lymphoid cell lines from patients with infantile (type-2) and juvenile (type 3) Gaucher disease have been established by Epstein-Barr virus transformation and investigated and compared with the adult phenotype (type 1) with the view to enzymology. The enzymatic defect in glucosylceramide(GlcCer)-beta-glucosidase activity was more severe in type 2 and 3 than in type 1 cells. The mutant GlcCer-beta-glucosidase from our studied type 2 lymphoid cells was profoundly labile at pH 4.0 and 37 degrees C, whereas the residual GlcCer-beta-glucosidase from type 1 and type 3 were stable similar to the normal enzyme. In contrast to the distinct stability of the GlcCer-beta-glucosidases from the three phenotypes, the acid lability of the nonspecific membrane-bound beta-glucosidases from type 1, 2 and 3 were quite similar.     

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder

Summary Spleen cells from BALB/c mice immunized with cells derived from transitional cell carcinomas (TCC) of the human urinary bladder were fused with mouse myeloma Sp 2/0 Ag14 cells. Monoclonal antibodies from six established hybridomas were investigated for specificity in a cell ELISA and in indirect immunofluorescence against a large panel of fixed intact cells. Three of the antibodies reacted with half or more of the eight bladder tumors and with a few unrelated tumors. They did not react at all with malignant or normal cells of hematopoietic origin. A fourth antibody reacted with seven of eight bladder tumors. It also reacted weakly with a prostatic carcinoma, with five of six malignant or transformed B cell lines, and with a subpopulation of normal lymphocytes, but not with any of the other cells on the test panel. These four antibodies did not react with cells derived from normal urothelium. The results suggest that these antibodies might recognize cell-type-restricted antigens associated with malignancy. Another antibody reacted with almost all urothelium-derived cells. It also reacted with three of three melanomas but not with any other cells on the panel. The sixth antibody reacted with 32 of the 37 cells tested. The spectrum of reactivities displayed by the antibody suggested that it recognizes HLA antigens. Abbreviations used in this paper: TCC, transitional cell carcinoma of the urinary bladder; TAA, tumor-associated antigens; ELISA, enzyme-linked immunosorbent assay; NBCS, newborn calf serum; PBS, phosphate-buffered saline, pH 7.2; ALP, alkaline phosphatase; GDA, glutardialdehyde; BSA, bovine serum albumin; IF, indirect immunofluorescence; LCL, lymphoblastoid cell lines: B-lymphocytes transformed in vitro with Epstein-Barr virus     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

Subproteomics analysis of phosphorylated proteins: application to the study of B-lymphoblasts from a patient with Scott syndrome

Proteomics based approaches, which examine the expressed proteins of a tissue or cell type, complement the genome initiatives and are increasingly used to address biomedical questions. Proteins are the main functional output, and post-translational modifications such as phosphorylation are very important in determining protein function. To address this question, we developed a method for specific immunoprecipitation using anti-phosphotyrosine antibodies. This method is directly compatible with two-dimensional gel electrophoresis (2-DE). In this report data are presented on B-lymphoblasts from a patient suffering of Scott syndrome. Scott syndrome is an orphan inherited hemorrhagic disorder due to a lack of exposure of procoagulant phosphatidylserine at the exoplasmic leaflet of plasma membrane of blood cells. We hypothesized that a consequence of the mutation is to alter phosphorylation of proteins involved in signal transduction leading to breakdown in cellular signaling pathways mediating phosphatidylserine exposure. An immunoprecipitation method combined with 2-DE was applied to search for modifications in the expression of phosphorylated polypeptides related to Scott syndrome phenotype. We report here the construction of a B-lymphoblast subproteomic map comprising of polypeptides observed after immunoprecipitation using antibodies to phosphotyrosine. The polypeptides were identified either by mass fingerprinting, by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or by matching with various lymphoid cell 2-DE maps included in the Laboratoire de Biochimie des Protéines et Protéomique 2-DE database. A differential analysis was further performed to explore several hundred proteins in Scott B-lymphoblasts in comparison with control B-lymphoblasts. Then, image analysis allowed detection of variations between control and Scott syndrome phenotype lymphoblasts. Five spots were specifically found on 2-DE from Scott syndrome phenotype lymphoblasts, and four only appeared on 2-DE from control cells. Protein identification was achieved using a combination of mass fingerprinting and peptide identification using LC-MS/MS.     

Signals causing change in morphological phenotype,growth mode,and gene expression of vascular endothelial cells

Comparison of three different lines of bovine aortal endothelial cells provides a clear demonstration of reversible morphologic phenotype coincidental with change in expression and growth mode. These phenotypic forms can be externally controlled so that cells may exist either in an epithelioid contact-inhibitable state or as a fibroblastoid non-contact-inhibitable state. Clonal cell line N (normal) shows a strong tendency to maintain the epithelioid phenotype. Clonal cell line Sp (sprout) can readily and reversibly adopt the epithelioid or fibroblastoid phenotype. A factor in normal serum is responsible for maintaining the cells in the epithelioid phenotype. This factor could be a growth factor since several polypeptide growth factors are shown to drive cells from the fibroblastoid phenotype to the epithelioid phenotype within 11 hours. This growth factor-induced change is not mediated through induced DNA synthesis. Clonal cell line V (variant) normally maintains the fibroblastoid phenotype but can be directed to the epithelioid phenotype provided cells are on an appropriate collagenous matrix. Associated with these changes in morphological phenotype are depression of the expression of the pro α₂ chain of collagen type I which may be characteristic of the contact-inhibited state and of an 80,000 mol wt polypeptide synthesized only by cells in the fibroblastoid phenotype. An endothelial cell collagen ECl (mol wt 177,000) was synthesized by all cell lines regardless of phenotype whereas a suspected breakdown product EC3 (mol wt 100,000) was found only in the epithelioid phenotype. Other differences and similarities between cell lines include expression of a 135,000 mol wt glycoprotein GP (V and N), the procollagen of collagen type III (N) of fibronectin (N, V, Sp), and of the pro α₁ chain of collagen type I (Sp, V). The characteristic expression of each line and its response to signals controlling morphologic phenotype impinges on the question of whether there exist several distinct types of vascular endothelial cells with different functional potentials controlled by extracellular signals.     

Phenotypic and functional characterization of T cell clones derived from the cerebrospinal fluid of multiple sclerosis patients

We describe here T cell cultures and clones established from the cerebrospinal fluid (CSF) of three patients with multiple sclerosis (MS) and one chronic meningitis patient with pleocytosis. Most of the cultures were activated with phytohemagglutinin (PHA) before growth in mitogen-free interleukin 2 (IL 2), and were never restimulated. Some of the clones obtained have been propagated for over 1 yr and are strictly IL 2-dependent. Immunofluorescence analysis performed with various monoclonal antibodies revealed that the CSF-derived lines had the characteristics of activated T cells with a stable expression of either suppressor/cytotoxic or helper/inducer surface antigens. Most of the clones established had a predominantly suppressor phenotype (OKT8+), except for the clones derived from one MS patient, which expressed only the helper phenotype (anti-Leu-3a+). Consistent with these data, the CSF-derived cultures displayed a variety of immunoregulatory functions, such as the ability to lyse nonspecific and PHA-stimulated target cells, to produce IL 2 upon mitogenic activation, and to modulate polyclonally induced Ig responses. The availability of long-term CSF T cell cultures derived from MS patients at various disease stages might provide a useful tool in investigating the factor(s) involved in the etio-pathogenesis of the disease.     

Immortalized cell lines derived from mice lacking both type I and type II IFN receptors unify some functions of immature and mature dendritic cells

Cells with dendritic morphology obtained from several organs of mice lacking both type I and II IFN receptors were immortalized by a retrovirus and analysed for their phenotype and for their function to induce cognate immune responses in vitro and in vivo. Two cell lines called AG101 (skin) and AG116 (brain) were cloned and analysed in more detail. They constitutively expressed the cell surface markers CD45, CD11b, MHC class II, F4/80, N418, B7-2 and ICAM1 but were CD8- and B220-negative. Cells from both lines were capable of taking up ovalbumin (OVA). The processed protein was presented to the OVA-specific T cell hybridoma BO97.105 which responded specifically with the production of IL-2. AG101 and AG116 cells were able to induce a mixed lymphocyte reaction as shown by a 50-fold increase of IL-2 production over background. Naive T cells were stimulated by antigen-primed AG101 and AG116, resulting in a T cell proliferation which was 20-30 times over background, and in IL-2 production it was 10 times the background. The capacity of AG101 or AG116 cells to prime naive T cells was directly compared with freshly isolated and cultured cutaneous dendritic cells (DC) from 129 Sv/Ev mice (wtDC). After cognate T cell interaction, IL-6 (20-100-fold) and IL-12 p40 (100-1000-fold) were similarly up-regulated in either AG101, AG116 or mature wtDC. To analyse the capacity of the immortalized DC to induce antibodies in vivo, cell line AG116 was permanently infected with Borna disease virus (BDV) which is unable to replicate in adult mice. One hundred and twenty-nine Sv/Ev mice injected with different cell numbers of AG116 carrying BDV (but not control cells) produced antibodies against the viral BDVp40 and BDVp24 protein. Therefore, the cell lines AG101 and AG116 appear to unify some functions of immature and mature DC. They are able to pick up antigen and process it. In the absence of externally added cytokines, the antigen presented on AG101 or AG116 cells drives T cells with an efficiency similar to mature DC. The cloned cell lines may prove to be useful to study both immune response and replication of infectious agents in the absence of functional interferon receptors.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site--WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily alpha-fucosylated complex type chains with alpha2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells.     

Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites.     

Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency affecting B lymphocytes, T lymphocytes, and platelets. Previous studies on lymphocytes from WAS patients have revealed that leu-kosialin (CD43), a cell-surface glycoprotein bearing approximately 90 O-linked oligosaccharide chains, shows an aberrant electrophoretic mobility. To determine whether this finding reflects a different pattern of O-linked glycosylation in WAS cells, we have compared healthy individuals and WAS patients with respect to glycosyltransferase activities in T lymphocytes, platelets, and Epstein-Barr virus (EBV)-immortalized B cell lines. Stimulation of peripheral T cells from normal individuals in vitro with anti-CD3 antibodies and interleukin-2 was associated with a 3-fold increase in UDP-GlcNAc:Gal beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) from 0.8 to 2.2 nmol/mg/h. In contrast, peripheral T lymphocytes from WAS patients showed an inversion of this phenotype with high core 2 GlcNAc-T activity in unstimulated cells (2.3 nmol/mg/h) and a 2-3-fold decrease in activity following stimulation. Core 2 GlcNAc-T activity was also three times higher in platelets from WAS patients than in normal platelets. Glycosyltransferase activities were measured in immortalized B cell lines established from WAS and normal subjects by infection with EBV. Core 2 GlcNAc-T was less than 0.4 nmol/mg/h in WAS EBV-B cell lines compared to 2.4 nmol/mg/h in EBV-B cell lines from healthy individuals, In contrast, CMP-SA:SA alpha 2-3Gal beta 1-3GalNAc-R (where SA represents sialyl (sialic acid to GalNAc) alpha 6-sialyltransferase II activity was 2.0 nmol/mg/h in the WAS EBV-B cell and less than .01 nmol/mg/h in EBV-B cell lines derived from normal subjects. Eleven other glycosyltransferase activities were measured and found to be similar in EBV-B cell lines from WAS and normal individuals. Polylactosamine sequences were much reduced in the O-linked oligosaccharides of CD43 from WAS EBV-B cells consistent with decreased core 2 GlcNAc-T activity and expression of core 1 oligosaccharides in the cells. In conclusion, B cells, T cells, and platelets in WAS patients show abnormal expression of two developmentally regulated glycosyltransferases, consistent with the idea that the WAS immunodeficiency is due to a failure of normal lymphocyte maturation.