Reduced PKC α Activity Induces Senescent Phenotype in Erythrocytes.

The molecular mechanism mediating expression of senescent cell antigen-aggregated or cleaved band 3 and externalized phosphatidylserine (PS) on the surface of aged erythrocytes and their premature expression in certain anemias is not completely elucidated. The erythrocytes with these surface modifications undergo macrophage-mediated phagocytosis. In this study, the role of protein kinase C (PKC) isoforms in the expression of these surface modifications was investigated. Inhibition of PKC α by 30 µM rottlerin (R30) and 2.3 nM Gö 6976 caused expression of both the senescent cell marker-externalized PS measured by FACS analysis and aggregated band 3 detected by western blotting. In contrast to this observation, but in keeping with literature, PKC activation by phorbol-12-myristate-13-acetate (PMA) also led to the expression of senescence markers. We explain this antithesis by demonstrating that PMA-treated cells show reduction in the activity of PKC α, thereby simulating inhibition. The reduction in PKC α activity may be attributed to the known downregulation of PMA-activated PKC α, caused by its membrane translocation and proteolysis. We demonstrate membrane translocation of PKC α in PMA-treated cells to substantiate this inference. Thus loss of PKC α activity either by inhibition or downregulation can cause surface modifications which can trigger erythrophagocytosis.

Altered melanoma cell surface glycosylation mediates organ specific adhesion and metastasis via lectin receptors on the lung vascular endothelium.

Adhesive interactions between the molecules on cancer cells and the target organ are one of the key determinants of the organ specific metastasis. In this communication we show that b1,6 branched N-oligosaccharides which are expressed in a metastasis-dependent manner on B16-melanoma metastatic cell lines, participate in the adhesion process. We demonstrate that high metastatic cells show significantly increased translocation of one of the major carriers of these oligosaccharides, lysosome associated membrane protein (LAMP1), to the cell surface. LAMP1 on high metastatic cells, carry very high levels of these oligosaccharides, which are further substituted with poly N-acetyl lactosamine (polylacNAc), resulting in the expression of high density of very high affinity ligands for galectin-3 on the cell surface. We show that galectin-3 is expressed in highest amount in the lungs as compared to other representative organs. Blocking galectin-3 by pre-incubating the frozen sections of the lungs with 100 mM lactose, substantially inhibited the adhesion of high metastatic cells, while pre-incubation with sucrose had no effect. Finally, by in situ labeling and immunoprecipitation experiment, we demonstrated that the lung vascular endothelial cells express galectin-3 constitutively on their surface. Galectin-3 on the organ endothelium could thus serve as the first anchor for the circulating cancer cells, expressing high density of very high affinity ligands on their surface, and facilitate organ specific metastasis.

Increased sialylation and defucosylation of plasma proteins are early events in the acute phase response.

Within hours of turpentine injection to stimulate the acute phase (AP) response in rats, the N-acetylneuraminic acid content of plasma proteins increases and that of fucose decreases, each by about 60%. The two changes are inversely related (r = -0.97). The NeuAc/Gal ratio increases from the normal 0.75 to 1.0 on day 2 of the AP. Whereas 50% of the isolated oligosaccharides of normal plasma proteins are retarded on immobilized Ricinus communis agglutinin, those from day 2 AP plasma fail to do so. This indicates that NeuAc caps the normally Gal-terminated chains. alpha1-Acid glycoprotein (a positive AP protein), alpha1-macroglobulin (a non-AP protein), and alpha1-inhibitor3 (a negative AP protein) also show similar alterations in NeuAc/Gal ratio and decreases in Fuc. alpha2-Macroglobulin, which arises only during the AP, does not contain significant amounts of Fuc. Sambucus nigra agglutinin (alpha2,6-linked NeuAc-specific) binds a majority of plasma proteins, and binding is increased during the AP response. Maackia amurensis lectin (alpha2,3-linked NeuAc-specific) binds only three proteins in normal plasma and three additional proteins in AP plasma. The Fuc-specific Aleuria aurantia agglutinin and Lens culinaris agglutinin each detect five proteins in normal plasma. Their binding decreases during the AP response. These results show that: (1) sialylation and defucosylation of preexisting plasma proteins occur rapidly in the AP response; (2) sialylation caps the preexisting Gal-terminating oligosaccharides; and (3) the oligosaccharides of even the non-AP and negative AP proteins are modified. These changes are distinct from the elevation in the levels of protein-bound monosaccharides and the altered concanavalin A-binding profile the oligosaccharides of AP proteins acquire in diseases.