Mossaad Abdel-Ghany

Lung Endothelial Dipeptidyl Peptidase IV Promotes Adhesion and Metastasis of Rat Breast Cancer Cells via Tumor Cell Surface-associated Fibronectin

Endothelial cell adhesion molecules are partly responsible for the distinct organ distribution of cancer metastases. Dipeptidyl peptidase IV (DPP IV) expressed on rat lung capillary endothelia is shown here to be an adhesion receptor for rat breast cancer cells and to mediate lung colonization by these tumor cells. Fibronectin (FN) assembled on breast cancer cell surfaces into multiple, randomly dispersed globules from cellular and plasma FN is identified as the principal ligand for DPP IV. Ligand expression correlates quantitatively with the tumor cells’ capabilities to bind to DPP IV and to metastasize to the lungs. DPP IV/FN-mediated adhesion and metastasis are blocked when tumor cells are incubated with soluble DPP IV prior to conducting adhesion and lung colony assays. Adhesion is also blocked by anti-DPP IV monoclonal antibody 6A3 and anti-FN antiserum. However, adhesion to immobilized FN is unaffected by soluble plasma FN and, thus, can happen during hematogenous spread of cancer cells at high plasma FN concentrations. The ability of many cancer cells to capture FN molecules on their surface and to augment such deposits by FN self-association during passage in the blood suggests that DPP IV/FN binding may be a relatively common mechanism for lung metastasis.

The Breast Cancer β4 Integrin and Endothelial Human CLCA2 Mediate Lung Metastasis

Adhesion of blood-borne cancer cells to the endothelium is a critical determinant of organ-specific metastasis. Here we show that colonization of the lungs by human breast cancer cells is correlated with cell surface expression of the α6β4 integrin and adhesion to human CLCA2 (hCLCA2), a Ca2+-sensitive chloride channel protein that is expressed on the endothelial cell luminal surface of pulmonary arteries, arterioles, and venules. Tumor cell adhesion to endothelial hCLCA2 is mediated by the β4 integrin, establishing for the first time a cell-cell adhesion property for this integrin that involves an entirely new adhesion partner. This adhesion is augmented by an increased surface expression of the α6β4 integrin in breast cancer cells selected in vivo for enhanced lung colonization but abolished by the specific cleavage of the β4 integrin with matrilysin. β4 integrin/hCLCA2 adhesion-blocking antibodies directed against either of the two interacting adhesion molecules inhibit lung colonization, while overexpression of the β4 integrin in a model murine tumor cell line of modest lung colonization potential significantly increases the lung metastatic performance. Our data clearly show that the β4/hCLCA2 adhesion is critical for lung metastasis, yet expression of the β4 integrin in many benign breast tumors shows that this integrin is insufficient to bestow metastatic competence on cells that lack invasiveness and other established properties of metastatic cells.

Cloning and characterization of lung-endothelial cell adhesion molecule- 1 suggest it is an endothelial chloride channel

Lung-endothelial cell adhesion molecule-1 (Lu-ECAM-1) is an endothelial cell surface molecule that mediates adhesion of metastatic melanoma cells to lung endothelium. Here we analyze the organization of the Lu-ECAM-1 protein complex, report the sequence of Lu-ECAM-1 cDNAs, and reveal a novel function of the protein. Lu-ECAM-1 immunopurified from bovine aortic endothelial cells (BAEC) consists of tightly associated glycoproteins of 90, 38, and 32 kDa, with minor components of 130 and 120 kDa. We present evidence that all of these protein species are encoded by a single open reading frame whose initial translation product is proteolytically processed to yield the other products. Correct processing in vitro was demonstrated by transfection of the longest cDNA into human embryonic kidney 293 cells; immunoblot analysis showed that the ∼120-kDa precursor gave rise to 90- and 38-kDa products. RNA blots of BAEC mRNA detected messages in agreement with the sizes of the cDNA clones in addition to several of high molecular weight. DNA blot analysis showed that Lu-ECAM-1 is conserved throughout its length in all mammals tested, usually as a single or low copy gene. In the bovine, Lu-ECAM-1 protein is 88% identical to a calcium-dependent chloride channel described recently in tracheal epithelium, Ca-CC. Probes for Lu-ECAM-1 mRNA and protein confirmed the presence of a homolog in this tissue. We show that messages for both proteins are present in lung while only Ca-CC is present in trachea and only Lu-ECAM-1 is present in BAEC. These results suggest that endothelial cells express a chloride channel that is related to, but distinct from, that expressed in tracheal epithelium. They further suggest that an adhesion molecule can also be a chloride channel.

Molecular characteristics and functional diversity of CLCA family members.

1. In the present brief review, we describe some of the molecular and functional characteristics of a novel mammalian family of putative Ca2+‐activated chloride channels (CLCA).

Truncated Dipeptidyl Peptidase IV Is a Potent Anti-Adhesion and Anti-Metastasis Peptide for Rat Breast Cancer Cells

A novel adhesion receptor/ligand pair was shown recently to mediate lung vascular arrest and metastasis of rat breast cancer cells. The interacting adhesion molecules are endothelial dipeptidyl peptidase IV (DPP IV) and tumor cell surface-associated, polymeric fibronectin (FN). A truncated DPP IV (DPP IV(31–767): amino acids 31–767) in which the FN-binding site is preserved is shown here to mask the breast cancer cell surface-associated FN complexes, causing a dose-dependent inhibition of adhesion to endothelial DPP IV and impeding lung colony formation by approximately 80%. Since surface accumulation of FN is chiefly occurring during dissemination in the blood and since many cancer cell types have surface receptors by which they may initiate FN accumulation on their surfaces, the present anti-metastatic treatment modality may extend its efficacy farther than appreciated by this study.

Is the Fischer 344/CRJ rat a protein-knock-out model for dipeptidyl peptidase IV-mediated lung metastasis of breast cancer?

Fischer 344/CRJ rats harbor a G633R substitution in dipeptidyl peptidase IV (DPP IV) that leads to retention and degradation of the mutant protein in the endoplasmic reticulum (Tsuji E, Misumi Y, Fujiwara T et al. Biochemistry 1992; 31 (47): 11921–7 [1]). However, when these rats were used as a ‘protein knock-out’ model in further evaluating the previously established role of DPP IV in metastasis, lung colonization of the highly metastatic MTF7 rat breast cancer cell line was reduced by only 33% relative to normal Fischer 344 rats. To examine whether lung endothelia leak expression of mutant DPP IV and whether mutant DPP IV exhibits the same adhesion qualities as wild type DPP IV, detailed immunohistochemical, biochemical, transfection, and FACS analyses were performed to assess the surface expression of mutant DPP IV on lung endothelia and transfected HEK293 cells and adhesion assay to compare the adhesion qualities of wild-type and mutant DPP IV. Both endothelial and transfected HEK293 cells expressed mutant, enzymatically inactive DPP IV on their surfaces, albeit at greatly reduced levels when compared to expression of wild type DPP IV. Purified mutant DPP IV had identical adhesion qualities for lung-metastatic MTF7 cells as wild type DPP IV, and competitive inhibition of MTF7 lung colonization by truncated DPP IV confirmed involvement of mutant DPP IV in lung metastasis of Fischer 344/CRJ rats. Although metastasis appears to be mediated by several, often parallel mechanisms involving multiple tumor and host factors, these data indicate that altered expression of a single component can drastically change the outcome of metastatic disease.

Isolation of Highly Purified and Stable Preparations of c-src and v-src Kinase

We describe here a simple method of purification of src kinase from insect cells infected with a baculovirus expressing c-src kinase or v-src kinase. After two affinity columns prepared with random sy

Glucose stimulation of protein acylation in the pancreatic β-cell

AIMS To determine whether protein acylation plays a role in the effects of glucose on the insulin secreting β-cell. MAIN METHODS The measurement of (3)H-palmitate incorporation into protein in the INS 832/13 cell that has a robust and well-characterized biphasic insulin secretory response to stimulation with glucose. KEY FINDINGS Stimulating the cells with glucose increased the incorporation of (3)H-palmitic acid into protein by up to 90%. Similarly, 2-aminobicyclo [2.2.1] heptane-2-carboxylic acid (BCH) the non-metabolizable analog of leucine that mimics the stimulatory effect of glucose on insulin secretion also increased the incorporation of (3)H-palmitic acid into protein. Treatment of cell lysates with hydroxylamine substantially reduced the incorporation indicating that most of the incorporation was due to enzymatic palmitoylation of proteins. Cerulenin, a classical inhibitor of protein acylation also substantially reduced the incorporation. Using PAGE and autoradiography a glucose-induced increase in protein palmitoylation and specific glucose-induced increases in the palmitoylation of proteins of 30, 44, 48 and 76kD were identified. SIGNIFICANCE The data suggest that protein acylation plays multiple roles in β-cell function.

A membrane-bound human placental protein kinase activated by endogenous polypeptides

A protein kinase (PPdPK) was purified from plasma membranes of human placenta. Phosphorylation of casein , but not of phosvitin or lactalbumin, by [γ-32P]ATP in the presence of PPdPK was stimulated about 10-fold by naturally occurring polypeptides prepared from avariety of sources similar to the procedure of Roberts et al. (Proc. Natl. Acad. Sci. U.S.A.77, 3494–3498, 1980). The amino acid phos-phorylated on casein was serine. According to gel exclusion chromatography the mol.wt, of PPdPK was 95 000. In autoradiograms, following polyacrylamide-gel electrophoresis, the autophosphorylation of PPdPK was greatly enhanced by the polypeptide activators.

CLCA adhesion in site-specific cancer metastasis

Publisher Summary This chapter discusses the calcium-activated chloride channels (CLCA) adhesion in site-specific cancer metastasis. The discovery and cloning of CLCA proteins is made possible by the isolation and purification of a protein called “lung endothelial cell adhesion molecule-1” (Lu-ECAM- 1) and the eventual cloning of this molecule. Lu-ECAM-1 was identified during a comprehensive search for molecular principles involved in organ preference of metastasis. The molecules mediate vascular arrest of blood-borne cancer cells through the tumor cell β4 integrin. Preliminary data indicate that upon β4/CLCA ligation, a slew of signaling cascades are activated within the interacting cell pair that appears to promote survival and initial metastatic growth. Some effects of such signaling appear not to involve chloride channel activity, whereas others appear to depend upon chloride channel activity. The ion channels with adhesion functions seem to indicate that in some channels ligands may serve such a gating function, whereas in others the adhesion quality appears to be independent of channel function. The adhesion property of CLCA proteins poses an intriguing novel concept in site-specific cancer metastasis and an opportunity to study the putative gating functions of the β4 integrin and the cellular effects of signaling cascades that are activated and maintained by the β4/CLCA ligation.