Colchicine resistant (CHR) lines of stable phenotype have been isolated from cultured Chinese hamster (CHO) cells. Successive single-step selections for increasing resistance were performed by isolating resistant colonies at each step. Two complementary assays involving [3H] colchicine uptake by whole cells and binding of [3H] colchicine by cytoplasmic extracts were developed to test for altered permeability and altered intracellular target protein, respectively. All clones isolated appeared to have decreased permeability to the drug while their colchicine-binding ability was not reduced. The amount of reduction in colchicine uptake correlated strongly with cellular resistance. The CHR lines were also cross resistant to other drugs such as actinomycin D, vinblastine and Colcemid; furthermore, the degree of cross resistance was positively correlated with the degree of colchicine resistance. The non-ionic detergent Tween 80 potentiated the cytotoxic action of colchicine on mutant cells as well as its rate of uptake into whole cells.

Reduced permeability in CHO cells as a mechanism of resistance to colchicine.

https://www.cell.com/cell/pdf/0092-8674(85)90117-5.pdf

Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases.

Recombinant protein therapeutics have changed the face of modern medicine in the past decade, and they continue to provide innovative and effective therapies for numerous previously refractory illnesses. Today, they are used in the treatment of a variety of human diseases, ranging from cancers to infertility. These proteins are generally synthesized by large-scale cultivation of genetically engineered “host” cells, which harbor artificially transfected genes encoding for the proteins of interest. For protein therapeutics to be effective, they must be synthesized in biologically active forms, requiring proper folding and post-translational modifications. In many cases, this includes glycosylation, a type of modification where certain carbohydrate moieties are added to specific amino acid residues of the protein. Glycoproteins, as these are usually called, are generally synthesized in mammalian cells, because common microbial hosts like Escherichia coli lack the requisite machinery to synthesize appropriate glycoforms. Several rodentor human-derived cells like 3T3, CHO, BHK, HeLa and HepG2 are frequently used in biomedical research for heterologous protein expression. Despite the availability of a plenitude of cell lines, nearly 70% of all recombinant protein therapeutics produced today are made in Chinese Hamster Ovary (CHO) cells. The current annual sales for biologics produced using CHO cells alone exceed US$30 billion worldwide. The first recombinant therapeutic protein produced in mammalian cells, tissue plasminogen activator (r-tPA, Activase) synthesized using CHO cells, was approved for clinical use in 1987. This marked the beginning of an array of highly successful CHO-based therapeutics (Table), which continue to revolutionize the field of medicine to this day. The knowledge and expertise amassed over the past two decades will most certainly ensure that CHO cells continue to remain the industry's premier workhorse for therapeutic protein production, at least in the near future. In this article, we revisit the evolution of CHO cells, tracing from their origins to the development of current production cell lines, and discuss some important aspects that make them robust and versatile protein expression hosts. Unlike some of its rodent relatives (mouse and rat), the generation of genomic resources for Chinese hamster has been quite limited. Genomic and proteomic tools using these resources can potentially aid in understanding and improving recombinant protein production processes leading to a significant enhancement in the speed with which these therapeutics transition from laboratory molecules to life-saving medicines. We, therefore, devote special attention to the value and need for such information in meeting the challenges of future research and manufacturing processes.

Recombinant protein therapeutics from CHO cells : 20 years and counting

This review gives a comprehensive account of the molecular toxicology of the bicyclic peptides obtained from the poisonous mushrooms of the genus Amanita. The discussion of the biochemical events will be preceded by a consideration of the chemistry of the toxic peptides. The structural features essential for biological activities of both the amatoxins and the phallotoxins will be discussed, also including the most important analytical data. Similar consideration will be given to antamanide, a cyclic peptide, which counteracts phalloidin. In addition, the phallolysins, three cytolytic proteins from Amanita phalloides will be discussed. The report on the biological activity of the amatoxins will deal with the sensitivity of the different RNA-polymerases towards the toxins and with their action on various cell types. Consideration will also be given to systems in which alpha-amanitin was used and can be used as a molecular tool; in the past, many investigators used the inhibitor in molecular biology, genetics, and even in physiological research. As for the phallotoxins, discussion of the affinity of these toxins for actin is provied. Further discussion attempts to understand the course of intoxication by filling in the gap between the first molecular event, formation of microfilaments, and the various lesions in hepatocytes during the intoxication.

Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms.

This review will attempt to cover the present information on the multiple forms of eukaryotic DNA-dependent RNA polymerases, both at the structural and functional level. Nuclear RNA polymerases constitute a group of three large multimeric enzymes, each with a different and complex subunit structure and distinct specificity. The review will include a detailed description of their molecular structure. The current approaches to elucidate subunit function via chemical modification, phosphorylation, enzyme reconstitution, immunological studies, and mutant analysis will be described. In vitro reconstituted systems are available for the accurate transcription of cloned genes coding for rRNA, tRNA, 5 SRNA, and mRNA. These systems will be described with special attention to the cellular factors required for specific transcription. A section on future prospects will address questions concerning the significance of the complex subunit structure of the nuclear enzymes; the organization and regulation of the gene coding for RNA polymerase subunits; the obtention of mutants affected at the level of factors, or RNA polymerases; the mechanism of template recognition by factors and RNA polymerase.

Eukaryotic RNA polymerases.

Mutants of Chinese hamster ovary cells that are resistant to α-amanitin can be isolated. At least some of these mutants contain an altered form of DNA-dependent RNA polymerase II, as indicated by its resistance to α-amanitin. These results indicate that mutation to α-amanitin resistance involves a change of a structural gene.

V. L. Chan

Papers

Mammalian cells with altered forms of RNA polymerase II.