Polymorphisms in promoter sequences of the p15 ( INK4B ) and PTEN genes of normal Japanese individuals.

Gene promoter regions of p15(INK4B), a cyclin-dependent kinase inhibitor, and phosphatase and tensin homolog (PTEN), a dual-function protein and lipid phosphatase, interact with regulatory factors for gene transcription and methylation. Normal individuals exhibit sequence polymorphisms in these regulatory genes. We isolated genomic DNA from whole blood of healthy Japanese individuals and sequenced promoter regions of the p15 ( INK4B ) and PTEN genes. We also examined the influence of polymorphisms on promoter activity in several cell lines. We identified polymorphisms at positions -699, -394, and -242 and an insertion at position -320 in the p15 ( INK4B ) gene and a polymorphism at position -1142 in the PTEN gene. Reporter gene analysis revealed that these polymorphisms influenced transcriptional regulation in their cell lines. Our results indicate for the first time that promoter sequences of the p15 ( INK4B ) and PTEN genes differ among normal Japanese individuals and that promoter polymorphisms can influence gene transcription.

Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.

BACKGROUND/AIMS: Hepatocyte transplantation and bioartificial liver treatment are attractive alternatives to liver transplantation. The availability of well-characterized human hepatocyte lines facilitates such cell therapies. METHODS: Human hepatocytes were immortalized with a retroviral vector SSR#197 expressing catalytic subunit of human telomerase reverse transcriptase (hTERT) and enhanced green fluorescent protein (EGFP) cDNAs flanked by a pair of loxP recombination targets. Then, Tamoxifen-dependent Cre recombinase was expressed in SSR#197-immortalized hepatocytes. Cre/LoxP recombination was performed in the established cells by simple exposure to 500 nM Tamoxifen for a week. Then, the reverted population of the cells was recovered by EGFP-negative cell sorting and characterized in vitro and in vivo using a pig model of acute liver failure (ALF) induced by d-galactosamine (0.5 g/kg) injection. RESULTS: A human hepatocyte cell line 16T-3 was established. Reverted 16-T3 cells showed the increased expression of hepatic markers in association with enhanced levels of transcriptional factors. Compared to normal human hepatocytes, albumin production and lidocaine-metabolizing activities of reverted 16-T3 cells were 0.32 and 0.50-fold, respectively. Transplantation of reverted 16T-3 cells significantly prolonged the survival of ALF pigs. CONCLUSIONS: Here we demonstrate the usefulness of Cre/LoxP -mediated reversible immortalization of human hepatocytes with Tamoxifen-mediated self-recombination.

Nuclear-mitochondrial genomic profiling reveals a pattern of evolution in epithelial ovarian tumor stem cells.

Analyses of genome orthologs in cancer on the background of tumor heterogeneity, coupled with the recent identification that the tumor propagating capacity resides within a very small fraction of cells (the tumor stem cells-TSCs), has not been achieved. Here, we describe a strategy to explore genetic drift in the mitochondrial genome accompanying varying stem cell dynamics in epithelial ovarian cancer. A major and novel outcome is the identification of a specific mutant mitochondrial DNA profile associated with the TSC lineage that is drastically different from the germ line profile. This profile, however, is often camouflaged in the primary tumor, and sometimes may not be detected even after metastases, questioning the validity of whole tumor profiling towards determining individual prognosis. Continuing mutagenesis in subsets with a mutant mitochondrial genome could result in transformation through a cooperative effect with nuclear genes - a representative example in our study is a tumor suppressor gene viz. cAMP responsive element binding binding protein. This specific profile could be a critical predisposing step undertaken by a normal stem cell to overcome a tightly regulated mutation rate and DNA repair in its evolution towards tumorigenesis. Our findings suggest that varying stem cell dynamics and mutagenesis define TSC progression that may clinically translate into increasing tumor aggression with serious implications for prognosis.

Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited.

BACKGROUND: Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static "contact first" model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism. METHODS: Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. RESULTS: In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static "contact first" model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation. CONCLUSION: The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.

Genomic characterization of KIR2DL4 in families and unrelated individuals reveals extensive diversity in exon and intron sequences including a common frameshift variation occurring in several alleles.

The KIR2DL4 gene including a portion of exon 1 through exon 9 was sequenced from two families and eight cell lines from the International Histocompatibility Workshop (IHWS). Two known alleles and eight variants were detected. Overall, there were five synonymous and three non-synonymous changes when the variants were compared to the coding sequences of the most closely related known alleles plus a common frameshift change in five of the variant alleles. Alignment of the new variants with all known alleles showed that the regions encoding the extracellular region and the cytoplasmic tail were the most polymorphic. Two non-synonymous changes, P146H and L161V, occurred in an extracellular immunoglobulin-like domain. Five of the eight variants had a single adenine deletion in the exon encoding the transmembrane region, potentially resulting in a truncated protein lacking the cytoplasmic tail. The distribution of the deletion variant among many KIR2DL4 alleles may explain the high frequency of this variation in the population. Four of the eight consanguineous IHWS cell lines were found to be heterozygous for KIR2DL4 carrying two alleles that differed from one another by a few nucleotide substitutions. Analysis of intron sequences in the families revealed the nature and distribution of interspersed repeat elements which comprise 46% of the KIR2DL4 nucleotide sequence and consist of 12 elements including six SINEs (13.73% of the total length), one LINE (12.41%), and five LTR elements (19.51%). The results revealed the presence of extensive diversity in the KIR2DL4 gene. This is the first extensive report providing both exon and intron data in related individuals.

Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin.

BACKGROUND: Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. RESULTS: In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. CONCLUSION: The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

Proteomic analysis of androgen-regulated protein expression in a mouse fetal vas deferens cell line.

During sex differentiation, androgens are essential for development of the male genital tract. The Wolffian duct is an androgen-sensitive target tissue that develops into the epididymis, vas deferens, and seminal vesicle. The present study aimed to identify androgen-regulated proteins that are involved in development of Wolffian duct-derived structures. We have used male mouse embryos transgenic for temperature-sensitive simian virus 40 large tumor antigen at 18 d of gestation, to generate immortalized mouse fetal vas deferens (MFVD) parental and clonal cell lines. The MFVD parental and clonal cell lines express androgen receptor protein and show features of Wolffian duct mesenchymal cells. Clonal cell line MFVD A6 was selected for proteomic analysis and cultured in the absence or presence of androgens. Subsequently, two-dimensional gel electrophoresis was performed on total cell lysates. Differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and two androgen-regulated proteins were identified as mElfin and CArG-binding factor-A (CBF-A). CBF-A and mElfin are known to bind to cytoskeletal F-actin. Both proteins appeared to be regulated by androgens at the posttranslational level, possibly involving phosphorylation. Posttranslational modification of mElfin and CBF-A by androgens may be associated with a cytoskeletal change that is involved in androgen-regulated gene expression.

Hyposialated 185 kDa CD45RA+ molecules attain a high concentration in B lymphoma cells and in activated human B cells.

Alternate splicing of exons of the CD45 molecule generates multiple isoforms differing in their molecular weights (MWs). In B-lymphocytes the CD45RA isoform was previously shown to be expressed on glycoproteins with MWs of 220 and 205 kDa, while the CD45RO isoform was expressed on glycoproteins with MW of 180 kDa. The present study demonstrated that B cell lymphomas and activated B-cells contain CD45 molecules with a MW of 185 kDa that express the CD45RA and CD45RC specificities but neither the CD45RB nor the CD45RO specificities. 185 kDa CD45RA+ molecules were detected in B cell lymphoma B lines, in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and in tonsillar B cells, but not in normal, unstimulated peripheral blood B cells. These molecules were not detected in neoplastic and normal T cells. CD45RA+ 185 kDa molecules were present in B cells from three non-Hodgkin's patients in leukemic phase were not detected in B lymphocytes of seven of nine CLL patients tested. Trypsin treatment eliminated only 220 kDa CD45RA+ molecules but not 185 kDa CD45RA+ molecules, indicating that the 185 kDa CD45RA+ molecules are not expressed on the cell surface. Pulse-chase experiments, and studies on the effects of tunicamycin, neuraminidase and O-glycosidase, indicated that the 185 kDa molecules are partially glycosylated CD45RABC molecules that constitute precursors of the 220 kDa molecules. The high concentration of 185 kDa CD45RA+ molecules in B lymphoma cells and in activated B cells seems to reflect a high turnover of CD45RA+ molecules characteristic for these cells.

Characterization of a transformed rat retinal ganglion cell line.

The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.

Immortalisation of human bone marrow endothelial cells: characterisation of new cell lines.

BACKGROUND: Adhesion of haematopoietic progenitor cells (HPC) to human bone marrow endothelial cells (HBMEC) plays a key role in homing of HPC to bone marrow. Here we describe four new HBMEC cell lines that can be used to study the (specific) adhesion of HPC to HBMEC. DESIGN: HBMEC were immortalised with a retroviral construct containing the human papilloma virus 16 E6/E7 genes. Four cell lines were characterised. RESULTS: The cell lines showed their endothelial nature by the expression of von Willebrand Factor and VE-cadherin (CD144). Electron microscopic analysis revealed normal endothelial-cell characteristics, including the presence of Weibel-Palade bodies and intercellular junction structures. An extensive phenotypic analysis of the cell-lines was performed, they were found to resemble primary HBMEC. The only difference found was the absence of expression of E-selectin (CD62e) and VCAM-1 (CD106) on resting HBMEC cell lines. Upon stimulation with IL-1beta the expression of E-selectin, VCAM-1 and ICAM-1 (CD54) was upregulated. All resting cell lines bound CD34+ HPC. Adhesion was increased by addition of the phorbol ester PMA. Two cell lines showed increased binding upon IL-1beta prestimulation. Highest adhesion was observed after the combination of IL-1beta prestimulation of the endothelial cells and addition of PMA. Binding of CD34+ HPC to HBMEC was compared with the binding to human umbilical vein endothelial cell lines and to a human dermal microvascular endothelial cell line (HMEC-1). So far, we have only found relatively less binding of HPC to IL-1beta prestimulated HMEC-1 cells, which could be explained by a reduced induction of E-selectin and VCAM-1 upon IL-1beta stimulation of these cells. CONCLUSION: The immortalised HBMEC cell lines have maintained their normal phenotype for the majority of characteristics examined. The expression of E-selectin and VCAM-1, which are not constitutively expressed on the cell lines, can be induced by stimulation of the endothelial cells with IL-1beta. The cell lines have furthermore maintained their capability to bind HPC. They will therefore be useful to investigate the interactions between HPC and HBMEC involved in homing of HPC.

A subset of cells expressing SV40 large T antigen contain elevated p53 levels and have an altered cell cycle phenotype.

Cells transformed by the simian virus 40 (SV40) large T antigen (Tag) contain elevated levels of cellular p53 protein. To quantify this relationship, levels of p53 were measured in NIH 3T3 cells that expressed different concentrations of Tag. Using immunoblotting, average p53 levels were shown to increase linearly with Tag concentrations in these cell lines. Single-cell measurements were also performed using flow cytometry to measure p53 immunofluorescence. Surprisingly, the flow cytometry experiments showed that two distinct cell populations, based on p53 content, were present in cells expressing high levels of Tag. One cell population contained elevated p53 levels. A second population did not contain elevated p53, even though high concentrations of Tag were present in the cells. This latter cell population did not appear to arise because of mutations in either Tag or p53. The two cell populations also had phenotypic differences. In exponentially growing cells, Tag alters the cell cycle distribution (decreases the percentage of G1 phase cells and increases the percentages of S and G2 + M phase cells). This phenotype was maximum in the cell population containing elevated p53. A lesser phenotype was found in the cell population that did not contain elevated p53. These data show, firstly, that cells can express significant levels of Tag and not contain elevated levels of p53 and, secondly, that elevated p53 correlates with the altered cell cycle distribution produced by Tag in growing cells.

Receptor-mediated endocytosis of transthyretin by ependymoma cells.

Transthyretin (TTR) is involved in the transport of thyroxine (T4) and retinol-binding protein (RBP) in cerebrospinal fluid (CSF) and serum. TTR is secreted in the CSF by the epithelial cells of choroid plexus. The binding of [(125)I]TTR to cultured ependymoma cells which form the brain cerebrospinal barrier, was studied to determine whether these cells carry receptor(s) for TTR. TTR was bound by ependymoma cells in a time-dependent manner reaching equilibrium within 2 h. Scatchard analysis was consistent with a single class of high-affinity binding sites with a K(d) of approximately 18 nM. Saturable high-affinity binding of human TTR has previously been described in rat primary hepatocytes and human renal adenocarcinoma, neuroblastoma, hepatoma and astrocytoma cells, and also transformed lung cells. Endocytosis of fluorescent or biotinylated TTR was observed in ependymoma cells in cytoplasmic vesicles but TTR did not colocalize with clathrin in endocytic coated vesicles. Endocytosis of TTR was inhibited by high sucrose concentration (0.45 M). Finally, ligand blotting and chemical-linking experiments revealed the presence of a approximately 100 kDa putative TTR receptor on the ependymoma cell membrane. Receptor binding of TTR provides a potential mechanism for the delivery of T4 within the central nervous system.

Modulation of G-protein linked cAMP accumulation in immortalized murine cortical astrocytes by retroviral infection.

The present study characterized beta-adrenergic receptors (beta-AR) in a clonal cell line (C1) immortalized from cerebral cortical astroglial cells of FVB/N mice. We also determined whether the wild type Moloney murine leukemia virus (wt-MoMuLV) and one of its neuropathogenic mutants, ts1-MoMuLV, modulated the beta-AR system in these cells. We observed that C1 cells possess a functional beta-AR system coupled to cAMP accumulation and capable of normal agonist-induced regulation (desensitization). Significant increases were observed in forskolin stimulated cAMP accumulation in C1 cells infected by wt MoMuLV and by ts1-MoMuLV. In contrast, the cAMP response to beta-AR stimulated by isoproterenol was relatively spared after viral exposure.

Enzymatic amplification staining for flow cytometric analysis of cell surface molecules.

BACKGROUND: Flow cytometric analysis is a powerful technique for the single cell assessment of cell surface expression of selected molecules. The major deficiency of flow cytometry has been its relative insensitivity. Only molecules expressed in abundance have been readily observed. METHODS: We have developed an enzymatic amplification procedure for the analysis of cell surface molecules by flow cytometry. Transformed and nontransformed cells expressing MHC class I, CD5, CD3, CD4, CD6, CD7, CD34, CD45, MHC class II, Fas ligand, and phosphatidylserine were assessed. RESULTS: Our enzymatic amplification technology increased the fluorescence signal between 10 and 100-fold for all surface molecules tested. CONCLUSIONS: Enzymatic amplification staining produces a significant enhancement in the resolving power of flow cytometric analysis of cell surface molecules. Using this technique, we have been able to detect the presence of molecules that could not be observed by the standard procedure.

Characterization of immortalized human umbilical and iliac vein endothelial cell lines after transfection with SV40 large T-antigen.

Most in vitro studies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3 and EVLC2 derived from HUVEC, and EVLK1 and EVLK2 derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM-1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but aSMC-actin was far less than in vascular SMC. Antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI-1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression.

BAP37 and Prohibitin are specifically recognized by an SV40 T antigen antibody.

We have identified two cellular proteins that are specifically immunoprecipitated by an anti-SV40 T antigen monoclonal antibody. This antibody, PAb419, recognizes an epitope contained within a region of T antigen which we have recently demonstrated is required for the initiation of immortalization by SV40 T antigen, but is not essential for maintenance of the immortal state. The two proteins were identified as BAP37 and Prohibitin. Recent results suggest Prohibitin may enhance the transcriptional inactivation of E2F by the retinoblastoma family of pocket proteins (pRb, p107, p130). BAP37 and Prohibitin are specifically recognized by PAb419 and PAb210, another anti-SV40 T antigen monoclonal antibody, which has an overlapping epitope, but not by other anti-SV40 T antigen monoclonal antibodies, demonstrating the specificity of the interaction.

Forced MyHCIIB expression following targeted genetic manipulation of conditionally immortalized muscle precursor cells.

The ability to carry out gene targeting in somatic stem cells while maintaining their stem cell characteristics would have important implications for gene therapy and for the analysis of gene function. Using mouse myoblasts, we have explored this possibility by attempting to alter the promoter of a myosin heavy chain gene (MyHCIIB) characteristic of physiologically "fast" muscle so as to force its unscheduled expression in physiologically "slow" muscle fibers. Conditionally immortalized muscle precursor cells were transfected with a gene targeting construct designed to replace the MyHCIIB promoter with that for the carbonic anhydrase III gene (CAIII), which is highly expressed in slow muscle. A potentially targeted clone was isolated and differentiated in culture to form myotubes which expressed MyHCIIB. Cells from the same clone were injected into both slow and fast muscle of host mice, where they contributed to fiber formation. In slow muscle, the fibers derived from this clone did not express MyHCIIB; this may reflect an instability of the targeted MyHCIIB locus and/or a failure of the hybrid promoter to function in slow fibers in vivo. Nonetheless, we have demonstrated that a "promoter knock-in" gene targeting procedure can be used to generate unique MyHCIIB-expressing myotubes in culture and that conditionally immortalized myoblasts can be subjected to extensive passaging and genetic manipulation without losing their ability to form fibers in culture and in vivo.

Expression of neurofilament L-promoter green-fluorescent protein constructs in immortalized Schwann cell-neuron coculture.

The neurofilament L/68 protein (NF-L/68) gene is expressed in the immature Schwann cell phenotype but suppressed after myelin-formation. We have investigated conditions which regulate the activity of the NF-L/68 promoter in green fluorescent protein reporter constructs expressed in the immortal rat Schwann cell strain SCL4.1/F7 in coculture with neurons. Constructs expressed in a plasmid vector containing both the full-length promoter and the 3' proximal 107 bp sequence which includes the cyclic AMP response element (CRE), were active in SCL4.1/F7 cells, but were suppressed as the cells underwent spontaneous growth-arrest. Interaction of SCL4.1/F7 with axons accelerated downregulation of expression from both constructs, however expression of the full-length promoter continued in some cells until the onset of myelin-formation. Expression of the NFL/68 construct recommenced when demyelination was induced in culture by exposure to human sera from patients with paraproteinemic gammopathy. We have demonstrated a method to study the regulation of gene expression patterns in single Schwann cells interacting with neurons and shown that different promoter regions may be controlled by axon-related and -unrelated factors.

Characterization of cell surface lectin-binding patterns of human airway epithelium.

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo- and 16HBE14o- bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.

Aryl-hydrocarbon receptor is an inhibitory regulator of lipid synthesis and of commitment to adipogenesis.

The aryl-hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In mouse embryo fibroblasts, TCDD activates expression of multiple genes, including CYP1B1, the predominant cytochrome P450 expressed in these cells. Here, we analyze constitutive functions of the AhR in primary mouse embryo fibroblasts (MEFs) and spontaneously immortalized MEF cell lines derived from wild-type (WT) C57BL/6 mice and also from congenic mice with a targeted disruption of the AhR gene (AhR-/-). After multiple passages, primary MEFs exhibit spontaneous differentiation, growth cessation and senescence. Eventually, colonies of immortalized MEFs arise to provide clonal lines. The senescent phase occurs much earlier for AhR-/- MEFs, while immortalization is substantially delayed. Comparison of AhR-/- and WT MEFs also indicates that constitutive AhR activity is required for basal expression of CYP1B1 and suppresses lipogenesis in subconfluent cultures. Primary WT and AhR-/- MEFs and the corresponding lines undergo adipogenesis when treated at confluence with the appropriate hormonal inducers. Addition of TCDD before or concurrent with hormonal induction suppressed PPAR gamma mRNA and adipogenesis, as measured by lipid accumulation, glycerol phosphate dehydrogenase activity and stearoyl CoA desaturase type 1 mRNA expression. This effect of TCDD treatment was absent in AhR-/- MEFs, establishing the role of AhR in hormone-induced adipogenesis. Such hormonal activation of confluent MEFs and preadipocytes results in a limited proliferative expansion followed by irreversible growth arrest. TCDD-treated MEFs undergo the mitotic expansion but fail to exit the cell cycle. In AhR-/- MEFs, there is no such effect of TCDD. These findings implicate the AhR as a constitutive inhibitor of triglyceride synthesis, and as an early regulator of adipocyte differentiation. AhR interference with cell-cycle arrest in differentiation may be linked to the increased rate of senescence.