Stable alterations at the cell membrane of Chinese hamster ovary cells resistant to the cytotoxicity of phytohemagglutinin

122

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGA9 mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAS) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4

Section: Methodsmentioning

confidence: 74%

Spontaneous fusion in vivo between normal host and tumor cells: possible contribution to tumor progression and metastasis studied with a lectin-resistant mutant tumor.

Kerbel¹,

Lagarde²,

Dennis³

et al. 1983

122

“…If a cell line exhibits altered resistance to at least two lectins of different carbohydrate-binding specificity, it is highly likely to be a glycosylation mutant (27,29). Complementation analysis with selected members of the 10 recessive LecR CHO complementation groups (25,27,31) will subsequently determine whether the glycosylation mutation is novel.…”

mentioning

confidence: 99%

Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

Stanley¹

1985

Membrane mutants of animal cells have been isolated by several laboratories, using a variety of selection protocols. The majority are lectin receptor mutants arising from altered glycosylation of membrane molecules. They have been obtained by selection for resistance to cytotoxic plant lectins or by alternative protocols designed, in many cases, to isolate different classes of receptor mutants. The identification of most membrane mutants expressing altered surface carbohydrates is rapidly achieved by determining their resistance to several lectins of different carbohydrate-binding specificities. For Chinese hamster ovary mutants, genetic novelty may subsequently be determined by complementation analysis with selected members of 10 recessive, glycosylation-defective complementation groups defined by this laboratory. In an attempt to identify new complementation groups, 11 Chinese hamster ovary membrane mutants independently isolated in different laboratories have been investigated for their lectin resistance and complementation properties. Only one new complementation group was defined by these studies. The remaining 10 mutants fell into complementation group 1, 2, 3, or 8. Although no evidence for intragenic complementation was observed, indirect evidence for different mutations within some genes was obtained. Seven of the independent isolates fell into complementation group 1, reflecting the high probability of isolating the Lec1 phenotype from Chinese hamster ovary populations. The results emphasize the importance of performing a genetic analysis before biochemical characterization of putative new membrane mutants.

“…(i) A large population of mutagenized wild-type (51-llB) cells was permitted to incorporate [3H]fucose for 24 h under stand-VOL. 1,1981 on May 9, 2018 by guest http://mcb.asm.org/ ard growth conditions.…”

Section: Resultsmentioning

confidence: 99%

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Hirschberg¹,

Baker²,

Perez³

et al. 1981

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wildtype cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5-to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.Glycoprotein components of mammalian cell surfaces are thought to play vital roles in such processes as regulation of cell growth, intercellular recognition, and cellular adhesion (8,31,32). A powerful approach for investigating relationships between glycoprotein structure and functions and for elucidating the biosynthesis of the oligosaccharide moieties of these molecules is to examine the phenotypes of somatic cell mutants with altered glycoprotein synthesis (5,10,14,16,18,20,22). One notably successful tactic for the isolation of such glycoprotein mutants has been the selection of cells resistant to cytotoxic lectins (4, 6, 9, 11, 23-25, 30, 33). Such resistance presumably reflects alterations in cell surface glycoproteins, particularly those which are part of or adjacent to the lectin receptors. Recent studies have suggested that more than one sugar may be involved in the surface recognition system for some lectins (2).We have initiated studies based on an alternative method for selection of glycoprotein mutants with altered oligosaccharide moieties. Our