Search

Using an assay developed in this laboratory that tests the ability of live or fixed cells to bind to over 70 types of agarose beads derivatized with different molecules, we examined the surfaces of two human cancer cell lines (LS123, human colon adenocarcinoma; NCI-H446, human lung carcinoma) and one human noncancerous colon line (CCD-18Co) obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were thawed, washed in phosphate-buffered saline (PBS), fixed in 1% formaldehyde in PBS, washed again, and resuspended in distilled water. Washed, fixed cells and washed, derivatized beads were mixed in triplicate distilled water droplets and observed for cell-bead binding. Experiments were repeated several times by at least two different investigators. Of approximately 70 beads tested, beads derivatized with wheat germ agglutinin, some forms of concanavalin A, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine displayed substantially different percentages of positive and negative binding trials with the three cell lines tested. This assay provides a novel approach for the initial identification of cell surface markers that may have functional significance in cancer and developing cell systems. (Supported by NIH MBRS, NIH MARC, ONR RISE, NSF ESIE, and the Joseph Drown Foundation.)

Standard histochemical analysis of cells and tissues generally involves procedures that utilize a relatively small number of probes such as dyes, and generally requires hours or days to process. Our laboratory has developed a novel method for histochemical surveys of cell surface properties that utilizes a large number of probes (derivatized agarose beads) and takes seconds or minutes to accomplish. In this study, 4 human cell lines (CCL-255 (LS123) human colon cancer cells that are non-tumorigenic in nude mice; CRL-1459 (CCD-18CO) human colon endothelial cells that are non-malignant; CCL-220 (COLO 320DM) human colon cancer cells that are tumorigenic in nude mice; and HTB-171 (NCI H446) human lung carcinoma cells) were tested for their ability to bind to agarose beads derivatized with 51 different molecules. There were statistically significant differences in binding of the 4 cell types to all of the 51 types of beads, but 15 types of beads showed dramatic differences in binding to one or more of the 4 cell types. For example, only HTB-171 (NCI H446) bound to p-aminophenyl-beta-D-glucopyranoside-derivatized beads and only CCL-220 (COLO 320DM) bound to L-tyrosine-derivatized beads. The specificity of cell-bead binding was examined by performing assays in the presence or absence of exogenously added compounds in hapten-type of inhibition experiments. This assay, that utilizes large numbers of novel probes, may help in the development of new libraries of surface properties of specific cell types, with differing degrees of malignancy, that at this time could not be developed by using other available technologies.