Isolation and Characterization of Chinese Hamster Ovary Cell Variants Deficient in the Expression of Fibronectin Receptor

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing ∼20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, β subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR β chain as well as enhanced amounts of a pre-β moiety in the variants. α chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of α chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR α chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of α chain mRNA leading to a deficit of mature FnR and consequent alterations in ce