Michihiko Kuwano

Chinese Hamster Cell Variants Resistant to the A Chain of Ricin Carry Altered Ribosome Function

Ricin, a toxic lectin from Ricinus communis, is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 microgram/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [125I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [125I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli

SummaryRibosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The isoelectric points of the smaller and the wild type S1 species are similar in the gel electrophoresis system of OFarrell (1975). Genetic analyses by Hfr conjugation and P1 phage transduction indicate that the mutation affecting S1 (rpsA) is located close to the serC gene [20 min on the E. coli genetic map of Bachmann et al. (1976)], with a co-transduction frequency of 61%. The most probable gene order is serC-rpsA-cmlB.

Altered Expression of Epidermal Growth Factor Receptor Gene in a Classical Multidrug‐resistant Variant of a Human Cancer Cell Line, KB

A variant clone resistant to high doses of colchicine (KB‐C1) derived from human cancer KB cell line is resistant to various anticancer agents. The KB‐C1 cells were much more resistant to epidermal growth factor and a chimeric toxin, EGF‐Pseudomottas exotoxin (PE), than the parental KB cells. KB‐C1 cells have decreased numbers of EGF‐receptors, though the affinity of the receptors is similar to that in the parental KB cells. A drug‐sensitive revertant (C1‐R2) partially recovered its EGF‐receptor activity. Northern blot analysis showed a decreased level of EGF‐receptor mRNA in KB‐C1 cells, while the multidrug‐resistance gene, mdr‐1, was expressed at very high levels in KB‐C1 cells, but not in KB or C1‐R2 cells. The drug‐resistant cells were less tumorigenic than the parental cells when injected into nude mice. A decreased expression of EGF‐receptor in these cells may be one of the pleiotropic properties of multidrug‐resistant cells and may perhaps represent the basis for their reduced tumorigenicity.