ONCOGENIC KI-RAS BUT NOT ONCOGENIC HA-RAS BLOCKS INTEGRIN BETA-1-CHAIN MATURATION IN COLON EPITHELIAL-CELLS

Human colorectal tumors commonly contain mutations in Ki-ras but rarel y, if ever, in Ha-ras. The selectivity for Ki-ras mutations in this tu mor was explored using the HD6-4 colon epithelial cell line which cont ains no ras mutations, after adhesion to an extracellular matrix, HD6- 4 cells polarize into columnar goblet cells with distinct apical and b asal regions. Stable HD6-4 transfectants were made with mini-gene cons tructs of the oncogenic cellular Ki-ras4B(G12V) gene, the oncogenic Ha -ras(G12V) gene, or mini-gene constructs of wild-type Ki-ras4B as a co ntrol, Ki-ras mutations, but not Ha-ras mutations, disrupted colon epi thelial cell apicobasal polarity and adhesion to collagen I and lamini n. Three Ha-ras transfectants and three Ki-ras transfectants exhibited Ras proteins expressing the Val-12 mutation by Western blotting with pan-ras(G12V) antibody. Only wild-type Ki-ras transfectant cells and o ncogenic Ha-ras transfectant cells synthesized the mature, fully glyco sylated forms of beta 1 integrin. Instead of the mature integrin beta 1-chain, a faster migrating beta 1-chain intermediate was detected on the cell surface and in the cytoplasm of the oncogenic Ki-ras transfec tants. Expression of the oncogenic Ki-ras gene caused the altered beta 1 integrin maturation because phosphorothiolated antisense oligonucle otides to Ki-ras reduced expression of both the mutant Hi-Ras protein and the aberrant integrin beta 1-chain and increased expression of the mature integrin beta 1-chain. Altered glycosylation generated the new beta 1-integrin form since integrin core beta 1-chain proteins of the same molecular weight were yielded in Ki-ras, Ha-ras, and control tra nsfectants after removal of sugar residues with endoglycosidase F or f ollowing tunicamycin treatment to inhibit glycosylation. The selective effect of oncogenic Ki-ras on beta 1 integrin glycosylation was not d ue to selective activation of mitogen activated protein kinases becaus e both mutated Ki- and Ha-ras genes activated this pathway and increas ed cell proliferation. Since blocking the glycosylation of integrin be ta 1-chain inhibited the adherence, polarization, and subsequent diffe rentiation of colon epithelial cells, the selective effects of the onc ogenic cellular Ki-ras gene on integrin beta 1-chain glycosylation may account, at least in part, for the selection of Ki-ras mutations in h uman colon tumors.