Retinoic‐acid‐induced augmentation of molecular species carrying sialosyl lewis^a antigen in colorectal‐carcinoma cell cultures

Abstract

In order to study the effects of vitamin-A metabolites on long-term carcinoma-antigen secretion, colorectal-carcinoma cells SW1116 were cultured on membrane filters in totally synthetic media with 0 to 2.6 microM retinoic acid (RA). RA altered cell division, cell size and soluble-sialosyl Le(a) (S-Le(a) secretion and S-Le(a) accumulation within cells and apical-membrane domains. Cultures treated with RA for 10-12 days grew to lower cell densities (60% of controls) and contained more protein per cell (140% of controls). RA treated cells also had 5-fold higher levels of S-Le(a) in cells and secreted 9-fold more S-Le(a) into culture media assayed per 24 hr by (ELISA) 19-9 monoclonal antibody binding. As total media S-Le(a) increased, polarity of non-lipid S-Le(a) antigen secretion increased toward the interior (apical) media. High-performance thin-layer immunobinding showed that ganglioside S-Le(a) was higher in RA-fed cells, but could not be detected in apical media of RA-fed or control cells after 24 hr. Western blots indicated that non-lipid sialosyl Lewis(a) was bound to 150- to 180-kDA molecular species principally in cells, but 210- to 300-kDa molecular species appeared in the non-lipid extract of media. Thus, the above RA alterations, monitored by 3 immunochemical techniques, include up to 9-fold stimulation of "constitutive" 150- to 300-kDa sialosyl-Lewis(a) secretion, but ganglioside Lewis(a) is sorted differently and retained by apical membranes.

Repression of the Lewis fucosyl transferase by retinoic acid increases apical sialosyl Lewisa secretion in colorectal carcinoma cultures

“…SW1116 human colorectal carcinoma cells were originally supplied by Dr. Hilary Koprowski of the Wistar Institute, and their growth characteristics and antigenic properties have been published previously [Liepkalns and Icard-Liepkalns, 1993;Liepkalns et al, 19851. Cells were seeded on Millipore (Bedford, MA) HA membranes in 7% fetal calf serum (mycoplasma and virus screened) at 200,000/cm2 in DME/F12 medium fortified with glutamine, Pen-Strep, and gentamycin (Gibco, Grand Island, NY). After 24 h all media were changed to synthetic (UG) substitute in DME/F12 plus or minus 3 pM RA.…”

Apical release of base‐labile fatty acyl groups commensurate with stimulation of glycoprotein sialosyl Lewisa secretion in colorectal carcinoma cells

“…Cells were seeded on Millipore HA membranes in 7% FCS (mycoplasma-and virus-screened) at 200,000/cm2 in DME/ F12 medium fortified with glutarnine, Pen-Strep and gentamycin (Gibco, Grand Island NY USA). After 24 hr all media were changed to synthetic (UG) substitute in DME/F12 plus or minus 3pM RA as described previously (Liepkalns and Icard-Liepkalns, 1993). Our period of conversion to high sialosyl-Lewisa-secretors was consistently 5 medium changes in 10 days.…”

“…Sialosyl Lewisa assays, protein determinations and cell counts were performed as described (Liepkalns and Icard-Liepkalns, 1993).…”

“…Several colorectal carcinoma cell lines have been found to secrete mucin or "mucin-like" glycoproteins, for example, HT-29 (Devine et al, 1991), T-84 (Roumagnac and Laboisse, 1987;McCool et aZ., 1990) and the human-derived SW 1116 (mug et nZ., Yedgar ef al., 1992;Liepkalns and Icard-Liepkalns, 1993). In a previous publication we have reported that incubation of filter-cultured carcinoma cells with retinoic acid (RA) strongly stimulates the release of sialosyl Lewis" antigens which are carried on glycoproteins in the range of 200,000 Mr (Liepkalns and Icard-Liepkalns, 1993).…”

“…Several colorectal carcinoma cell lines have been found to secrete mucin or "mucin-like" glycoproteins, for example, HT-29 (Devine et al, 1991), T-84 (Roumagnac and Laboisse, 1987;McCool et aZ., 1990) and the human-derived SW 1116 (mug et nZ., Yedgar ef al., 1992;Liepkalns and Icard-Liepkalns, 1993). In a previous publication we have reported that incubation of filter-cultured carcinoma cells with retinoic acid (RA) strongly stimulates the release of sialosyl Lewis" antigens which are carried on glycoproteins in the range of 200,000 Mr (Liepkalns and Icard-Liepkalns, 1993). The significance of this finding is underscored by recent evidence that sialosyl Lewisa and sialosyl Lewisx epitopes have been implicated as adhesion ligands that are recognized by "selectins" in endothelial-leukocyte binding assays (Varki, 1994) and may thus serve a similar function in carcinoma cells (Majuri et al, 1992).…”