EXTRACELLULAR ANNEXIN-II IS ASSOCIATED WITH DIVALENT CATION-DEPENDENTTUMOR-CELL ENDOTHELIAL-CELL ADHESION OF METASTATIC RAW117 LARGE-CELL LYMPHOMA-CELLS

Using fixed microvessel endothelial cell monolayers the molecules invo lved in the adhesion of liver-preferring murine RAW117 large cell lymp homa cells to murine liver-derived microvessel endothelial cells were identified by affinity isolation. Detergent lysates obtained from poor ly (P) or highly (Hl 0) liver-metastatic cells inhibited RAW1 17-H10 c ell adhesion to hepatic sinusoidal endothelial (HSE) cell monolayers. Allowing detergent lysates of cell surface-labeled RAW1 1 7 cells to b ind to fixed HSE cell monolayers and eluting the bound components indi cated that several tumor cell surface molecules (approximately 70, app roximately 35, approximately 32, approximately 22, and approximately 1 4 kDa) might be involved in RAW1 1 7 cell-HSE cell adhesion. The appro ximately 35 kDa component was cation dependent in its binding to targe t HSE cells. Increasing detergent concentration had no effect on bindi ng of the approximately 35 kDa component to HSE cell monolayers, where as treatment with 0.5 M NaCl resulted in its selective elution from HS E cells. Incubation of the HSE cell monolayers with detergent lysates from cell surface-labeled RAW117-H10 cells resulted in selective deple tion of the approximately 35 kDa component, suggesting that the bindin g is saturable. This divalent cation-dependent molecule is one of the major tumor cell surface components bound by several types of endothel ial cells and murine hepatocytes, whereas there was poor binding of th is component to unfixed or fixed human red blood cells. The purified, partially (approximately 40%) sequenced molecule had amino acid sequen ce identity with murine but not bovine annexin II, indicating that it was not bound from the bovine serum used to grow RAW1 1 7 cells. Using antibodies specific for annexin II flow cytometery indicated equivale nt amounts of annexin II are expressed on RAW1 1 7 cell surfaces in th e absence or presence of excess EDTA, whereas annexin I was only found in low amounts on the surfaces of RAW117 cells. Annexin II antibodies inhibited by approximately 40-50% the adhesion of RAW1 1 7 tumor cell s to live or fixed endothelial cells, and purified tumor cell surface fractions containing the approximately 35 kDa component partially inhi bited (approximately 35%) RAW117 cell-HSE cell adhesion. The data indi cate that annexin 11 is expressed on the extracellular surface of RAW1 1 7 cells, and cell surface-annexin II mediates a portion of the Ca2-dependent RAW117 cell adhesion to liver microvessel endothelial cells . (C) 1993 Wiley-Liss, Inc.