Ricin-resistant mutants of CHO cells

Abstract

Ricin is a potent cytotoxin composed of two disulfide-linked heteropeptides, an A chain and a B chain. Galactose-containing glycoconjugates at the eukaryotic cell surface bind to the B chain, leading to endocytosis of the toxin and inactivation of the 60S ribosomal subunit by the A chain. Ricin-resistant (Ric{dollar}\sp{lcub}\rm R{rcub}{dollar}) mutants of animal cells may be expected to arise from alterations in (1) carbohydrates of plasma membrane receptors for the toxin, (2) internalization of ricin or (3) components of the protein synthetic apparatus. In the course of these studies a previously isolated Ric{dollar}\sp{lcub}\rm R{rcub}{dollar} glycosylation mutant (LEC10) of chinese hamster ovary (CHO) cells was characterized genetically, and four novel Ric{dollar}\sp{lcub}\rm R{rcub}{dollar} CHO cell lines (LEC17, Lec26, Lec27 and Lec28) were shown to be ribosomal mutants.;LEC10 mutants, unlike parent CHO cells, express the glycosyltransferase UDP-GlcNAc: {dollar}\beta{dollar}1,4 Mannoside-N-acetylglucosaminyltransferase III (GlcNAc-TIII). To investigate genetic mechanisms that might account for the differential expression of GlcNAc-TIII, the frequency of LEC10 mutants and revertants in CHO cell populations untreated or mutagenized with various agents was determined. Treatment with several point mutagens and a frame-shift mutagen, but not with 5-azacytidine, resulted in an increase in the frequency of LEC10 phenotypes. Hybrid studies indicated that the LEC10 mutation acts in cis. Revertants arose at high frequency and consisted of two classes: one class gave rise to the LEC10 phenotype at high frequency ({dollar}\sim{dollar}10{dollar}\sp{lcub}-4{rcub}{dollar}), while the second class re-reverted at the spontaneous frequency for the forward mutation ({dollar}\sim{dollar}10{dollar}\sp{lcub}-7{rcub}{dollar}). The combined results suggest that a novel genetic instability at the LEC10 locus might be relevant in the regulation of expression of GlcNAc-TIII activity.;Selections for Ric{dollar}\sp{lcub}\rm R{rcub}{dollar} glycosylation mutants as well as for cells with the LEC10 phenotype following transfection of mutant genomic DNA into parent CHO cells, resulted in the isolation of four novel ricin- and abrin-resistant cell lines. Three mutants (Lec26, Lec27 and Lec28) were recessive whereas the most extensively studied mutant (LEC17) was dominant in somatic cell hybrids with parent cells. Post-mitochondrial supernatants prepared from the four mutants were more resistant than parent cell extracts to inhibition of protein synthesis by ricin A chain and abrin. Polysomes prepared from the Ric{dollar}\sp{lcub}\rm R{rcub}{dollar} line were also more resistant than those from parents to the N-glycanase activity of ricin and abrin, confirming that the alteration in the four mutants resides in the cells' ribosomes. Thus, as many as four potential gene products involved in the interaction of ricin A chain and its analogues with the eukaryotic ribosome have been identified.