lewis-x-antigen and Carcinoma--Embryonal

Human CD34(+) cells, highly enriched for hematopoietic stem and progenitors, and CD15(+) cells, more terminally differentiated myeloid cells in blood, represent distinct maturation/differentiation stages. A proteomic approach was used to identify proteins differentially present in these two populations from human cord blood. Cytosolic proteins were extracted and subjected to two-dimensional gel electrophoresis followed by mass spectrometry. On average, 460 protein spots on each gel were detected; 112 and 15 proteins, respectively, were found to be differentially expressed or post-translationally modified in CD34(+) and CD15(+) cells. This suggests that CD34(+) cells have a relatively larger proteome than mature CD15(+) myeloid cells and production of many stem/progenitor cell-associated proteins ceases or is dramatically down-regulated as the CD34(+) cells undergo differentiation. Of approximately 140 protein spots, 47 different proteins were positively identified by mass spectrometry and database search; these proteins belong to several functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Multiple heat shock proteins and chaperones, as well as proteins important for intracellular trafficking, were predominantly present in CD34(+) cells. Most of the identified proteins in CD34(+) cells are expressed in germ cell tumors, as well as in embryonal carcinoma and neuroblastoma. Approximately eight novel proteins, whose functions are unknown, were identified. This study presents, for the first time, global cellular protein expression patterns in human CD34(+) and CD15(+) cells, which should help to better understand intracellular processes involved in myeloid differentiation and add insight into the functional capabilities of these distinct cell types.

Topics: Antigens, CD34; Carcinoma, Embryonal; Cytoskeleton; Electrophoresis, Gel, Two-Dimensional; Fetal Blood; Humans; Isoelectric Focusing; Lewis X Antigen; Mass Spectrometry; Myeloid Cells; Neuroblastoma; Protein Folding; Protein Processing, Post-Translational; Proteomics; RNA, Small Nuclear; Signal Transduction; Stem Cells

Retinoic acid induces P19 mouse embryonal carcinoma cells to differentiate to endoderm and increases expression of the heterotrimeric G-protein subunits Galpha12 and Galpha13. Retinoic acid was found to induce differentiation and sustained activation of c-Jun amino-terminal kinase, but not of ERK1,2 or of p38 mitogen-activated protein kinases. Much like retinoic acid, expression of constitutively active forms of Galpha12 and Galpha13 induced differentiation and constitutive activation of c-Jun amino-terminal kinase. Expression of the dominant negative form of c-Jun amino-terminal kinase 1 blocked both the activation of c-Jun amino-terminal kinase and the induction of endodermal differentiation in the presence of retinoic acid. These data implicate c-Jun amino-terminal kinase as a downstream element of activation of Galpha12 or Galpha13 obligate for retinoic acid-induced differentiation.

Topics: Animals; Anisomycin; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Embryonal; Cell Differentiation; Embryonic and Fetal Development; Enzyme Activation; GTP-Binding Proteins; JNK Mitogen-Activated Protein Kinases; Lewis X Antigen; Mice; Mitogen-Activated Protein Kinases; Tretinoin

Convertases of the subtilisin/kexin family are responsible for the biological activation of a variety of pro-proteins, pro-hormones, and pro-trophic factors, and thus can modulate various aspects of embryonic development. We investigated the expression of each convertase by Northern hybridization during cell differentiation in vitro, using the mouse embryonal carcinoma cell line P19 as a model. The neuroendocrine convertase PC2 and 7B2, its specific binding protein, are co-induced during neuronal differentiation of P19 cells with retinoic acid, whereas the other convertases are not or follow different patterns of temporal expression. The mature forms of PC2 and 7B2 proteins are detected together by immunoblotting following induction of mRNA expression, indicating that these proteins are processed early during brain development. These results demonstrate that PC2 and 7B2 gene expression and protein processing are in a close temporal association during neuronal differentiation and point to the value of the P19 cell model to study the significance and the regulation of this relationship in mammalian brain development.

Topics: Animals; Carcinoma, Embryonal; Cell Differentiation; Enzyme Induction; Furin; Gene Expression Regulation, Developmental; Lewis X Antigen; Mesoderm; Mice; Nerve Tissue Proteins; Neuroendocrine Secretory Protein 7B2; Neurofilament Proteins; Neurons; Pituitary Hormones; Proprotein Convertase 2; Proprotein Convertase 5; Proprotein Convertases; Protein Processing, Post-Translational; RNA, Messenger; Serine Endopeptidases; Subtilisins; Tretinoin; Tumor Cells, Cultured

Treatment of mouse teratocarcinoma F9 cells with all-trans-retinoic acid (RA) causes a 9-fold increase in steady-state levels of mRNA for UDP-Gal:beta-D-Gal alpha1,3-galactosyltransferase (alpha1,3GT) beginning at 36 h. Enzyme activity rises in a similar fashion, which also parallels the induction of laminin and type IV collagen. Nuclear run-on assays indicate that this increase in alpha1,3GT in RA-treated F9 cells, like that of type IV collagen, is transcriptionally regulated. Differentiation also results in increased secretion of soluble alpha1,3GT activity into the growth media. The major alpha-galactosylated glycoprotein present in the media of RA-treated F9 cells, but not of untreated cells, was identified as laminin. Differentiation of F9 cells is accompanied by an increase in alpha-galactosylation of membrane glycoproteins and a decrease in expression of the stage-specific embryonic antigen, SSEA-1 (also known as the Lewis X antigen or LeX), which has the structure Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-R. However, flow cytometric analyses with specific antibodies and lectins, following treatment of cells with alpha-galactosidase, demonstrate that differentiated cells contain LeX antigens that are masked by alpha-galactosylation. Thus, RA induces alpha1,3GT at the transcriptional level, resulting in major alterations in the surface phenotype of the cells and masking of LeX antigens.

Topics: Animals; Bucladesine; Carbohydrate Conformation; Carbohydrate Sequence; Carcinoma, Embryonal; Cell Differentiation; Cell Nucleus; Flow Cytometry; Galactosyltransferases; Gene Expression Regulation, Neoplastic; Glycosylation; Kinetics; Laminin; Lewis X Antigen; Membrane Glycoproteins; Mice; Molecular Sequence Data; Raffinose; RNA, Messenger; Teratocarcinoma; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The oligosaccharide structure carried by embryoglycan, Lex hapten, as well as E-cadherin, has been described to mediate Ca(2+)-dependent cell-cell adhesions in embryonal carcinoma cells. To examine the contribution of these two systems to intercellular adhesion, we analyzed aggregation properties of previously isolated embryoglycan-defective mutants of P19 embryonal carcinoma cells. Our data indicate that the absence of Lex and embryoglycan has no effect on homotypic cell aggregation. Pretreatment of the cells with E-cadherin-specific antibody reduced homotypic aggregation of both parental and mutant cells, suggesting that E-cadherin plays a major role in this system. When parental cells were mixed with mutant cells, the aggregates contained either parental or mutant cells; no heterotypic aggregation was observed. The absence of mixed aggregates formed between parental and Lex-or embryoglycan-negative mutant P19 cells suggests that carbohydrates are involved in cadherin-mediated cell sorting.

Topics: Animals; Antibodies, Monoclonal; Cadherins; Calcium; Carbohydrate Sequence; Carcinoma, Embryonal; Cell Adhesion; Cell Aggregation; Cells, Cultured; Clone Cells; Edetic Acid; Flow Cytometry; Immunoglobulin M; Kinetics; Lewis X Antigen; Mice; Mice, Inbred C3H; Molecular Sequence Data; Mutagenesis; Polysaccharides; Trisaccharides

cDNAs of alpha-1,3-fucosyltransferase as well as alpha-1,3/4-fucosyltransferase were placed under the control of a beta-actin promoter and cytomegalovirus enhancer and were introduced into L cells. The transfected cells expressing Le(x) antigen showed increased cell substratum adhesion as compared to the antigen-negative cells, when they were cultured for 2 to 4 h in Dulbecco-modified minimum essential medium containing 0.05% bovine serum albumin. The increased cell substratum adhesion was completely inhibited by cycloheximide and anti-integrin antiserum, and partly by an RGD peptide and EGTA. These findings indicate that Le(x) structure promotes cell adhesion to substratum-bound material secreted by cells, and that the increased adhesion is mediated by integrin. Western blotting experiments have revealed an 85 kDa protein and a 50-60 kDa protein as carriers of Le(x) antigen in transfected cells. The latter is likely to be basigin, which is a member of the immunoglobulin superfamily and is considered to be an integrin-associated protein. We hypothesize that fucosylation of basigin enhances integrin-mediated cell substratum adhesion.

Topics: Animals; Carbohydrate Sequence; Carcinoma, Embryonal; Cell Adhesion; Cycloheximide; DNA, Complementary; Edetic Acid; Fucosyltransferases; Humans; L Cells; Lewis X Antigen; Mice; Molecular Sequence Data; Oligopeptides; Recombinant Proteins; Substrate Specificity; Transfection; Tumor Cells, Cultured

The integrin superfamily of heterodimeric transmembrane adhesion receptors mediates many cell-cell and cell-matrix interactions whose functions are believed to be critical for normal morphogenesis and differentiation. By eliminating the beta 1 integrin gene through homologous recombination, we have assessed the role of the beta 1 integrin family in the F9 embryonal carcinoma model for endodermal differentiation. F9 cells were unexpectedly found to maintain three copies of the beta 1 gene and complete elimination required three sequential rounds of targeting to generate triple knockout lines (beta 1 TKO). Elimination of the beta 1 integrin family of adhesion receptors from F9 cells resulted in reduced adhesion to fibronectin, laminin and collagen, but strongly enhanced adhesion to vitronectin. The absence of beta 1 integrins did not promote significant compensatory upregulation of either beta 3 or beta 5 subunits, both of which are known to act as vitronectin receptors when associated with alpha v. The loss of beta 1 integrins severely affected morphological differentiation when the beta 1-deficient cells were induced to differentiate to either parietal or visceral endoderm. Parietal endoderm derived from beta 1-deficient cells retained a rounded morphology and migrated poorly on both fibronectin and vitronectin. Visceral endoderm derived from beta 1-deficient cells were also unable to form a normal, confluent epithelial monolayer; instead, a non-contiguous layer containing clumps of disorganized cells was observed. However, loss of beta 1 integrins did not interfere with induction by differentiating agents of tissue-specific gene products for either visceral or parietal endoderm. These results suggest that beta 1 integrins mediate morphological differentiation (migration and epithelial formation) but not tissue-specific gene expression in induced F9 cells, and that these two processes are not necessarily linked in this system.

Topics: alpha-Fetoproteins; Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Embryonal; Cell Adhesion; Cell Differentiation; Cell Movement; Collagen; DNA Primers; Fibronectins; Gene Expression; Glycoproteins; In Vitro Techniques; Integrins; Laminin; Lewis X Antigen; Mice; Molecular Sequence Data; Morphogenesis; Mutagenesis, Insertional; Oligopeptides; RNA, Messenger; Tumor Cells, Cultured; Vitronectin