Mutants of Chinese hamster ovary cells have been found that no longer produce heparan sulfate. Characterization of one of the mutants, pgsD-677, showed that it lacks both N-acetylglucosaminyl-and glucuronosyltransferase, enzymes required for the polymerization of heparan sulfate chains. pgsD-677 also accumulates 3-to 4-fold more chondroitin sulfate than the wild type. Cell hybrids derived from pgsD-677 and wild type regained both transferase activities and the capacity to synthesize heparan sulfate. Two segregants from one of the hybrids reexpressed the dual enzyme deficiency, the lack of heparan sulfate synthesis, and the enhanced accumulation of chondroitin sulfate, suggesting that all of the traits were genetically linked. These fin gs indicate that the pgsD locus may represent a gene involved in the coordinate control of glycosaminoglycan formation.Proteoglycans consist of a core protein and one or more covalently attached glycosaminoglycan chains. Typical animal cells produce proteoglycans bearing chondroitin (dermatan) sulfate or heparan sulfate chains, but the composition varies considerably among different cells (1, 2). To study the regulation of proteoglycan composition, we have isolated Chinese hamster ovary (CHO) cell mutants defective in glycosaminoglycan biosynthesis (3-6). Many of these mutants bear mutations in genes involved in the formation of both heparan sulfate and chondroitin sulfate chains (3, 5). Here we describe a CHO cell mutant, pgsD-677, that specifically lacks heparan sulfate. The mutation in pgsD-677 affects both N-acetylglucosaminyl (GlcNAc)-and glucuronosyl (GlcA)-transferase activities required for heparan sulfate polymerization, suggesting that some form of coordinate regulation of these enzymes exists.EXPERIMENTAL PROCEDURES Cell Cultures. CHO cells (CHO-Ki) were obtained from the American Type Culture Collection (CCL-61). All mutants were identified by colony autoradiography (7), and the purity of each strain was ensured by its isolation from cultures containing only mutant colonies. Cells were maintained in Ham's F12 (8) medium (Mediatech, Washington) supplemented with 10% (vol/vol) fetal bovine serum (HyClone) or in sulfate-deficient medium (4).Cell fusion studies required the isolation of a CHO-K1 subline resistant to thioguanine and ouabain (OT-1). Wildtype cells were treated with 10 ,uM 6-thioguanine in hypoxanthine-free F12 medium supplemented with dialyzed fetal bovine serum. A resistant mutant was isolated and then treated with mutagen (7), and a ouabain-resistant clone was selected in growth medium containing 1 mM ouabain. The introduction of these markers did not alter the proteoglycan composition of the cells.Cell hybrids were generated by co-plating 2 x 105 cells of pgsD-677 and OT-1 in individual wells of a 24-well plate. After overnight incubation, the mixed monolayers were treated for 1 min with 50% (wt/wt) poly(ethylene glycol) (PEG 3320) prepared in F12 medium without serum (9). After 1 day the cells were harvested with trypsin, and multiple 100-mm-diam...

The role proteoglycans play in tumor formation was examined by measuring the tumorigenicity of proteoglycan-deficient Chinese hamster ovary cell mutants in nude mice. When 10(7) cells were injected subcutaneously, mutants with less than about 15% of the wild-type level of proteoglycan synthesis did not produce tumors. Mutants defective in the synthesis of heparan sulfate proteoglycans also did not form tumors, whereas mutants with altered chondroitin sulfate proteoglycans were tumorigenic. Tumors arose from mixtures of wild-type and nontumorigenic mutant cells and contained both cell types, suggesting that wild-type cell proteoglycans enabled mutant cells to survive. The failure of heparan sulfate-deficient mutants to form tumors depended on the ability of the host to mount a B cell-mediated immune reaction.

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