Abstract
PECAM-1 (CD31/EndoCAM) is an adhesion molecule in the immunoglobulin supergene family that is expressed on endothelial cells, platelets, and some hematopoietic lineage cells. In this paper, using several polyclonal and monoclonal antibodies against PECAM-1, we identified PECAM-1 molecules on human, rat, and murine solid tumor cell lines. Immunocytochemical labeling and flow cytometric analysis using either polyclonal, monoclonal, or Fab portion of the antibodies against PECAM-1 detected a distinct distribution on tumor cell surface. Immunoblotting revealed proteins ranging from 120 to 130 kDa in tumor cells derived from different species. Immunoprecipitation and subcellular fractionation studies indicated that PECAM-1 is constitutively expressed on the surface of human tumor cells (i.e. colon adenocarcinoma). The specificity of a major polyclonal anti-PECAM-1 used in the current study (i.e. SEW-3) was confirmed by the preabsorption studies. PECAM-1 molecules on tumor cells appear to bear terminal carbohydrate moieties (i.e. sialic acid residues) different from those on platelets, since neuraminidase treatment of tumor cells, unlike platelets, did not result in a mobility shift. Polymerase chain reaction (PCR) analysis of genomic DNA derived from tumor cell lines of different species revealed the presence of PECAM-1 gene in the genome. The mRNAs of PECAM-1 in tumor cells were detected by reverse transcription-PCR followed by Southern hybridization. Screening of more than 20 human, rat, and murine solid tumor cell lines indicated that PECAM-1 is widely expressed, although the level of expression varies considerably among different cell lines. The expression of PECAM-1 message in tumor cells was confirmed by Northern blotting. DNA sequencing of the PCR fragment revealed that human tumor cell PECAM-1 matches 100% to the human endothelial cell counterpart. Finally, it was demonstrated that tumor cell PECAM-1 is involved in mediating tumor cell adhesion to endothelium, as evidenced by the ability of anti-PECAM-1 antibodies to decrease the adhesion of unstimulated tumor cells to microvascular endothelial cells.
Highlights
1, we identified PECAM-1 molecules on human, rat, cell-cell and cell-matrix interactions
A large array of Ig family adhesion molecules have been is observed within a specific tumor cell type, i.e. the level implicated in tumorigenesis and cancer metastasis
Vascular Western blotting using two pAbs and a mAb (Fig. 4) demcell adhesion molecule-1 expressed on cytokine-activated onstrated that tumor cell PECAM-1 migrates in the range of endothelial cells have been demonstrated to mediate tumor 120-130 kDa, a molecular mass similar to PECAM-1 excell adhesion to thevascular monolayer via binding to VLA- pressed in platelets and endothelial cells (Muller et al, 1989; 4 (Martin-Padura et al, 1991; Taichman et al, 1991)
Summary
Antibodies-Five antibodies against PECAM-1 were used in the present studies. Polyclonal anti-PECAM SEW-3 andSEW-16 (IgG) were generated in rabbit using affinity-purified human platelet PECAM-1 as theimmunogen. Monoclonal anti-PECAM-1, mAb 1.3, (IgG1) was produced by immunizing mice with purified human platelet PECAM-1 protein (Albelda et al, 1991; Newman et al, 1992). RAEC, were derived from rat (Sprague-Dawley)aortic rings (Diglioet aL, 1989) These endothelial cells were routinely maintained in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum (FBS, Life Technologies, Inc.) and various antibiotics (50 pg/ml gentamycin, 100 pg/ ml penicillin G, and 2.5 wg/ml amphotericin B). B16 amelanotic melanoma cell line (B16a), rat W256 carcinosarcoma (W256) cell line, and Lewis lung carcinoma (3LL) cell line were obtained from the Division of Cancer Treatment, National Institutes of Health (Frederick, MD) and adapted for cell culture as described previously (Chopra et al, 1988, 1990; Grossi et al, 1988, 1989; Tang et al, 1993b). Immunofluorescent labeling was performed essentially as described previously
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