Cytosolic and Transmembrane Protein Extraction Methods of Breast and Ovarian Cancer Cells: A Comparative Study.

Efficient extraction of proteins is a great challenge for numerous downstream proteomic analyses. During the protein extraction procedure, it is critical to maintain the conformational stability, integrity, as well as higher yield of the protein. To do so, 5-different lysis buffers of Tris and HEPES have been used as the primary buffering reagents with variable compositions at different concentrations and pH using human cancer cells. In this study, different protein lysates of human breast cancer cells T47D and MDA-MB-231 and ovarian cancer cell PA-1 were subjected to run SDS-PAGE for separation of proteins based on their molecular size, followed by Coomassie blue, silver staining, and immunoblot assays to compare the extraction yield of total cytoplasmic proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the integral membrane protein, integrin ß-1. Our results revealed that Tris-based lysis buffer with 50 mM concentration, pH 7.5, is relatively the efficient and reliable protein extraction method for a wide range of MW subcellular markers, cytoplasmic GAPDH and transmembrane integrin ß-1 proteins. We anticipate that this simple and cost-effective protein extraction protocol might be extremely useful across a broad range of subcellular proteins in different biologic samples.

Expression, purification and renaturation of truncated human integrin ß1 from inclusion bodies of Escherichia coli.

Integrins are a family of transmembrane receptors and among their members, integrin ß1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin ß1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin ß1. The recombinant plasmid was transformed into Escherichiacoli BL21 (DE3) cells and after induction by isopropyl-ß-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS-PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin ß1. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn(2+) stimulation.

In vivo dedifferentiation of human epidermal cells.

Consistent with our previous study, we herein offer further evidence to demonstrate the dedifferentiation of differentiating epidermal cells into stem cells or stem cells -like in vivo. The epidermal sheets eliminated of basal cells were labeled with 6-diamidino-2-phenylindole (DAPI), and then were transplanted onto the full-thickness skin wounds nude mice. Immunohistochemical examination of the survival sheets showed that some cells were positive for both DAPI and either cytokeratins (CK19, CK14) or beta1 integrin in spinous and granular layers at day 7 after transplantation. Furthermore, there was a significant increase in the percentages of both alpha6briCDdim and alpha6briCD71bri populations in survival epidermal sheet grafts 7 d after transplantation compared with those before xenotransplantation (P<0.05), as determined by flow cytometry. The results collectively indicated that some of the differentiated cells in engrafted epidermal sheets dedifferentiated into stem cells or stem cells-like in vivo, which offer us new evidence and insights into the dedifferentiation.

Specific lectin binding to beta1 integrin and fibronectin on the apical membrane of madin-darby canine kidney cells.

Although lectins have previously been used to identify specific cell types in the kidney and various other tissues, the proteins labeled were not identified. We hypothesized that fluorescently labeled lectins could provide a useful tool for direct labeling of membrane-associated glycoproteins. Protein fractions from Madin-Darby canine kidney (MDCK) cells were exposed to a panel of 16 fluorescently labeled lectins to identify suitable lectin-protein pairs. Peanut agglutinin (PNA) selectively bound a 220-240 kDa O-linked glycoprotein with a slightly acidic isoelectric point, while Sambucus nigra agglutinin (SNA) labeled a 130 kDa glycoprotein with a highly acidic isoelectric point. Both proteins were readily labeled by lectins applied to the apical surface of living confluent cells. The proteins were isolated by lectin affinity columns and identified by mass spectrometry. Peptides from the PNA-binding protein shared molecular weight and amino acid composition with fibronectin. Fragments of the SNA-binding protein showed amino-acid identity with peptides from beta1 integrin. The identities of these proteins were validated by Western blotting. Binding of PNA to a 220 kDa protein was inhibited by an anti-fibronectin antibody, and binding of a 130 kDa protein by SNA was diminished by an anti-beta1 integrin antibody. We conclude that PNA and SNA can be used as specific markers for fibronectin and beta1 integrin, respectively, in MDCK cells.

Differences of alpha3beta1 integrin glycans from different human bladder cell lines.

Expression as well as properties of integrins are altered upon transformation. Cell adhesion regulated by integrins is modulated by glycosylation, one of the most frequent biochemical alteration associated with tumorogenesis. Characterisation of carbohydrate moieties of alpha3beta1 integrin on the cultured human bladder carcinoma (T-24, Hu456, HCV 29T) and human normal ureter and bladder epithelium (HCV 29, Hu609) cell lines was carried out after an electrophoresis and blotting, followed by immunochemical identification of alpha3 and beta1 integrin chains and analysis of their carbohydrates moieties using highly specific digoxigenin-labelled lectins. In all the studied cell lines alpha3beta1 integrin was glycosylated although in general each subunit differently. Basic structures recognized in beta1 subunit were tri- or tetraantennary complex type glycans in some cases sialylated (T-24, HCV 29, HCV 29T) and fucosylated (Hu609, HCV 29T). Positive reaction with Phaseolus vulgaris agglutinin and Datura stramonium agglutinin suggesting the presence of beta1-6 branched N-linked oligosaccharides was found in cancerous cell lines (T-24, Hu456) as well as in normal bladder epithelium cells (Hu609). High mannose type glycan was found only in beta1 subunit from Hu456 transitional cell cancer line. On the other hand alpha3 subunit was much less glycosylated except the invasive cancer cell line T-24 where high mannose as well as sialylated tri- or tetraantennary complex type glycans were detected. This observation suggests that changes in glycosylation profile attributed to invasive phenotype are rather associated with alpha3 not beta1 subunit.

Characterization of a murine monoclonal antibody specific for swine beta1 integrin.

Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160-1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM160-1A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human beta 1 integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine alpha 2 integrin. Therefore, the NaM160-1A3 antibody was directed against porcine beta 1 integrin and allowed the purification of the complex alpha 2 beta 1, also termed Very Late Antigen 2 (VLA-2). It did not recognize human beta 1 integrin.

Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells.

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.

Entamoeba histolytica contains a beta 1 integrin-like molecule similar to fibronectin receptors from eukaryotic cells.

Entamoeba histolytica trophozoites do interact with extracellular matrix components in order to invade and finally destroy tissue. An important step in this interaction involves the binding of a 140-kDa membrane protein that binds to fibronectin. The similarity of this amoebic receptor to fibronectin receptors from higher eukaryotic cells was defined by indirect immunofluorescence, western blot and immunohistochemistry, using polyclonal monospecific antibodies raised against the amoebic protein. These results suggest that lower eukaryotic cells have and use a beta 1 integrin-like molecule as well as mechanisms similar to those present in higher eukaryotic cells during interaction with extracellular matrix components.

Maturation of the liver-specific peroxisome versus laminin, collagen IV and integrin expression.

The interaction of cells with extracellular matrix components contributes to their specific differentiation. We studied hepatic peroxisomes and their changing features during embryonic development, and we immunolocalized in the same tissue the extracellular matrix components laminin and collagen IV as well as the integrin receptor subunits alpha 1, alpha 2, beta 1, and beta 4. Rat and human embryonic liver peroxisome expression were studied at the light- and electron-microscopic level by means of localizing catalase-, D-amino acid oxidase- and polyamine oxidase activities and by means of the immunocytochemistry of six peroxisomal proteins. The successive import of catalase and the peroxisomal beta-oxidation enzymes, the late appearance of the other enzymes, and the gradual increase of peroxisomal size and number to adult values occurred as asynchronous events. Although still immature, peroxisomes were recognized at every stage examined and coexisted with laminin and collagen IV in both species. The beta 1 integrin subunit was immunodetected as early as at 12.5 days in rat. It was concluded that these extracellular matrix factors may be important for the differentiation of liver parenchyma from the liverbud stage onwards. However, the stepwise maturation of the liver-specific peroxisome suggests the involvement of many other regulating factors.

Integrin expression in developing smooth muscle cells.

We studied the specific expression patterns and distributions of alpha1 and beta1 integrin subunits, the major cell adhesion receptors in smooth muscle, in developing smooth muscle cells from 16-, 18-, and 20-day embryonic gizzards and from 1- and 7-day post hatch chick gizzards by SDS-PAGE, immunoblotting, and immunoelectron microscopy. Antibodies raised against alpha1 and beta1 integrins isolated from avian gizzards were used as probes. Gels and blots showed that the amount of alpha1 and beta1 integrins increased as age increased, with major increases at 1 and 7 days post hatch. Image analysis of immunoelectron micrographs demonstrated that statistically significant labeling increases occurred between embryonic Days 16 and 18, between embryonic Day 20 and 1 day post hatch, and between 1 day and 7 days post hatch. Immunolabeling with both anti-alpha1 and anti-beta1 integrin was prominent at membrane-associated dense plaques (MADPs) and at filament anchoring regions at cell ends. This indicates that alpha1 and beta1 integrin expression coincides temporally with the intracellular proliferation and reorientation of myofilaments. The similarity in distribution patterns of alpha1 and beta1 integrins during development suggests that the two integrin subunits are synchronously expressed during development and do not appear sequentially. (J Histochem Cytochem 46:119-125, 1998)

Direct role of the carboxy-terminal cell-binding domain of fibronectin in neural crest cell motility.

We have analyzed the interaction of neural crest cells with fragments of fibronectin corresponding to the different spliced variants of the COOH-terminal cell-binding domain (COOH-ter CBD). We have shown that this domain can support cell adhesion and migration and that both the IIICS and HepII regions are involved in these events. The rate of locomotion is high, although undirectional, compared to that of whole fibronectin. Interactions with the COOH-ter CBD are controlled by alpha4beta1 and maybe other beta1 integrins and cell-surface proteoglycans. These receptors act cooperatively to mediate attachment, spreading, and migration on fibronectin.

[Electrophoretic characteristics of integrin++ beta1 subunit from human smooth muscles]. / Electroforeticheskoe povedenie beta1 sub''edinitsy integrina iuz gladkikh myshts cheloveka.

Total fraction of beta 1 integrin family was isolated from human smooth muscle by affinity chromatography on immobilized anti-beta 1 monoclonal antibodies. SDS-PAGE analysis and subsequent immunoblotting demonstrated that integrin samples contain unknown before high molecular mass (205 kD-nonreduced and 230 kD-reduced) material immunologically related to beta 1 integrin subunit. One dimensional peptide mapping showed that the 205 kD protein is not a novel beta 1 related integrin subunit, but a beta 1 integrin subunit dimer. Reduction of electrophoretic samples with dithiothreitol led to the removal of the major part of the beta 1 immunoreactive material from 205 kD to 130 kD region, indicating a disulfide nature of B1 integrin subunit dimer. The 230 kD protein turned out to be an only partly reduced beta 1 integrin disulfide bonded dimer. Possible in vivo existence of the disulfide bonded dimer and oligomer integrin forms is discussed.

Transforming growth factor-beta (TGF-beta) stimulates the expression of beta1 integrins and adhesion by rat mesangial cells.

Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of mesangial matrix (MM) accumulation in human and experimental glomerulonephritis. To clarify molecular mechanisms responsible for pathological MM deposition, we examined the effect of TGF-beta on the production of beta1 integrins and on adhesion function of rat mesangial cells (MC). In immunoprecipitation experiments using [35S]methionine-labeled MC, stimulation of MC with TGF-beta for 48 h resulted in an increase in the synthesis of alpha1beta1 (collagen/laminin receptor) and alpha5beta1 (fibronectin receptor) integrins accompanied by increases in the synthesis of their ligands, collagen type I (collagen I), and fibronectin. A time-dependent increase in beta1, alpha1 integrin subunit mRNA peaking 48 h after exposure to TGF-beta was shown by Northern blot analysis. After 48 h of treatment with TGF-beta, MC displayed significant increases in adhesion to fibronectin, collagen I, and laminin as compared to untreated MC. Anti-beta1 antiserum significantly inhibits MC adhesion to fibronectin, collagen I, and laminin. Anti-alpha1 subunit antibody very strongly inhibited adhesion to collagen I and laminin, but not to fibronectin. Synthetic peptides containing RGD sequences specifically blocked adhesion to fibronectin. These data suggest that TGF-beta may promote MM deposition by increasing MC synthesis of both matrix proteins and beta1 integrins which facilitate binding of these proteins to the MC surface and thus enhance their incorporation into MM.

Functional significance of CD9 association with beta 1 integrins in human epidermal keratinocytes.

CD9 is a member of the tetraspan (TM4) family of proteins and is abundantly expressed in the epidermis. As CD9 forms complexes with beta 1 integrins and the integrins are known to regulate keratinocyte behaviour, we investigated CD9 expression and function in human epidermal keratinocytes. CD9 was present in all the living layers of the epidermis, whereas the beta 1 integrins were largely confined to the basal layer; the same relative distribution was found in stratified cultures of keratinocytes. There was extensive co-localisation of CD9 and beta 1 integrins on microvilli and at cell-cell borders of basal keratinocytes; however, in contrast to the integrins, CD9 was not found in focal adhesions. CD9 was detected in beta 1 integrin immunoprecipitates and also in immunoprecipitates of CD44 and syndecan, but not of cadherins. CD9 was associated with alpha 3 beta 1 but not alpha 5 beta 1; small amounts of CD9 also co-immunoprecipitated with antibodies to alpha 2 beta 1 and alpha 6 beta 4. Antibodies to CD9 did not affect the proportion of keratinocytes that adhered to laminin 1, type IV collagen and fibronectin, but did inhibit motility of keratinocytes on tissue culture plastic. Like antibodies to the beta 1 integrin subunit, anti-CD9 inhibited suspension-induced terminal differentiation. These results suggest that CD9 may play a role in regulating keartinocyte motility and differentiation.

Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355-425) of the common beta 1 chain.

The very late activation antigens (VLA) or beta 1 integrins mediate cell attachment to different extracellular matrix proteins and intercellular adhesions. The ligand binding activity of these adhesion receptors is not constitutive and can be regulated by temperature, presence of extracellular divalent cations, stimulatory monoclonal antibodies (mAbs), and cellular activation. We have generated three novel mAbs, HUTS-4, HUTS-7, and HUTS-21, recognizing specific epitopes on the common beta 1 subunit (CD29) of VLA integrins whose expression correlates with the ligand binding activity of these heterodimeric glycoproteins. This correlation has been demonstrated for several integrin heterodimers in different cell systems using a variety of extracellular and intracellular stimuli for integrin activation. Thus, the presence of micromolar concentrations of extracellular Mn2+, preincubation with the activating anti-beta 1 mAb TS2/16, and cell treatment with phorbol esters or calcium ionophores, induced the expression of the HUTS beta 1 epitopes on T lymphoblasts. Using a panel of human-mouse beta 1 chimeric molecules, we have mapped these epitopes to the 355-425 sequence of the beta 1 polypeptide. This segment represents therefore a novel regulatory region of beta 1 that is exposed upon integrin activation. Interestingly, binding of HUTS mAbs to partially activated VLA integrins results in maximal activation of these adhesion receptors and enhancement of cell adhesion to beta 1 integrin ligands collagen, laminin, and fibronectin.

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

Immunopathogenesis of vernal keratoconjunctivitis.

We have analyzed the in situ distribution of immune cells in the conjunctival biopsy specimens obtained from patients with active vernal keratoconjunctivitis (VKC). We used immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies. Our data point to a complex immunopathogenesis of the disease. Distinct components involved in IgE-mediated immune mechanisms, as well as humoral and cell mediated immune mechanisms were detected in the conjunctival tissues. In addition, we investigated the presence and distribution of adhesion molecules. In the normal conjunctiva, intercellular adhesion molecule-1 (ICAM-1) was expressed only on the vascular endothelium, lymphocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) on epithelial and stromal mononuclear cells, and very late activation antigen-4 (VLA-4) on a few stromal mononuclear cells. Endothelial leukocyte adhesion molecule-1 (ELAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression was not detected. In VKC a marked increase of all these antigens was observed. Strong ICAM-1 expression was induced on the basal epithelial cells, and vascular endothelium. Furthermore, about 30% of the stromal mononuclear cells expressed ICAM-1. LFA-1 and ICAM-3 were expressed on the majority of infiltrating mononuclear cells. VLA-4 expression was noted on about 25% of the stromal mononuclear cells. ELAM-1 and VCAM-1 were induced on the vascular endothelial cells. Our results suggests that increased expression of adhesion molecules in VKC promotes the recruitment of inflammatory cells through blood vessels and the cell interaction between lymphocytes and antigen presenting cells, among lymphocytes, as well as between lymphocytes and epithelial cells.