Glycosaminoglycan Synthesis and Elevated during Early G1 Is Depressed during Mitosis

35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from twoto three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis. In their pioneering work on glycosaminoglycan (GAG) ~ synthesis by cultured cells, Kraemer and Tobey (14) showed that a significant fraction of trypsin-releasable (i.e., surface) GAGs was shed during mitosis. Although the Chinese hamster ovary (CHO) cells analyzed were grown in suspension, viewed from the perspective of recent studies of cell adhesion (e.g., reference 32), it seemed likely that the shedding of GAGs was related to the familiar ease with which mitotic cells are detached from monolayers. We hypothesized that any effect of shedding would be reinforced by a coordinate reduction in GAG synthesis that would delay replacement of surface GAGs, and that a rapid resumption of GAG synthesis in early G i would be required for cell reattachment. A parallel motivation for our studies arose from the accumulating evidence that membrane vesicle-mediated transport is depressed during mitosis. This is shown clearly in work from our laboratory demonstrating depression of endocytosis ( 1, 2) and transferrin recycling (35) and from that of Warren and colleagues showing a decrease in surface transferrin receptors (41), and in depression of histamine secretion (9). These studies focus primarily on fusion and budding events at the plasma membrane. In addition, the insertion of the G t Abbreviations used in this paper. CHO, Chinese hamster ovary; GAG, glycosaminoglycan. protein of vesicular stomatitis virus is also depressed during mitosis (42), which suggests a defect in its processing through the Golgi apparatus. Since carbohydrate chains of GAGs are largely assembled within the Golgi apparatus by multiple enzymes (31), a general defect in processing should affect GAG synthesis. Moreover, the apparent localization of sulfation to the trans-Gol~ apparatus (7, 44) would serve to focus on later steps of the processing sequence. We describe here the rates of labeled sulfate incorporation into CHO cells during mitosis, in their subsequent movement into G1, and in late G2. We followed incorporation using both biochemical and autoradiographic techniques. Our results indicate that the rate of GAG biosynthesis parallels the normal cycle of ceU-substrate detachment associated with the rounding up of mitotic cells and their subsequent reattachment after cell division. In addition, the depressed incorporation of label during mitosis indicates an associated defect in Golgi apparatus processing that is consistent with a generalized defect in membrane-vesicle transport. MATERIALS AND METHODS Cell Culture and Collection of Mitotic Cells: CHO cells were grown in a 5% CO2 atmosphere in RPM1 1640 supplemented with 10% fetal bovine serum (Gibco Laboratories, Inc., Grand Island, NY). Cells were plated 1086 THE JOURNAL Of CELL BIOLOGY • VOLUME 101 SEPTEMBER 1985 1086 1093 © The Rockefeller University Press • 0021-9525/85/09/1086/08 $1.00 on N ovem er 6, 2017 jcb.rress.org D ow nladed fom into 100-um-diam plastic tissue culture dishes (Falcon Labware, Oxnard, CA). For some experiments, 1 mM B-umbelliferyl-n-xyloside (Sigma Chemical Co., St. Louis, MO) was added to the cultures 6-7 h before sulfate labeling. Mitotic cells were collected by selective detachment (6). Cultures were partially synchronized by the addition of 5 mM thymidine overnight. 5 h after removal of the thymidine, mitotic cells were detached by rinsing the dish gently with the culture medium; the collected medium was diluted into ice-cold phosphate-buffered saline containing 1% bovine serum albumin (BSA). Fresh medium was added to the plates and rinsing was repeated every 30 rain. The mitotic index for each collected sample was determined before pooling from an aliquot stained with Hoechst dye 33242 (Sigma Chemical Co.); samples with <70% mitotics were discarded. A precise mitotic index was determined after the pulse-labeling. Since the mitotic interval of CHO is -25 min, and agents to arrest mitosis were purposely not employed, the percent mitoties decreased during the 15 min labeling period (described below); mitotic indices of 50-65 % at the conclusion of the pulse interval were typical. This implies that the activity of extracts is dominated by mitotic cells but mixed with appreciable contributions from interphase cells. Radiolabeling Procedures: For short-term radiolabeling, cells were rinsed in warm pulse medium (70 mM sodium chloride, 30 mM Tris, pH 7.2, 5 mM magnesium acetate, 0.5 mM EDTA, 50 mM potassium phosphate, 0. I% wt/vol BSA, and 75 mM glucose; 270-280 mOsmol (16); and incubated in pulse medium with 10 uCi/ml H23sSO, (specific activity 4.2 mCi mmol) at 37"C. Cells were routinely incubated for 15 min, or, to examine the kinetics of sulfate incorporation, up to l h. In this defined pulse medium mitotic cells incorporated enough sulfate to quantify before their progression into Gl. Subsequent plating efficiency and growth of cells incubated with pulse medium for 1 h were equivalent to those of control cultures. Furthermore, mitotic cells in pulse medium progressed quantitatively into G l. For analysis of glycopeptide and protein synthesis, cells were incubated at 37"C for from 10 min to l h with either 20 #Ci/ml o-[6-3H]glucosamine (specific activity, 31 Ci/mmol) in pulse medium in which glucose had been exchanged for sucrose in order not to inhibit glucosamine transport and supplemented with 0. l mM inorganic sulfate, or with 20/~Ci/ml L-[3H]tyrosine (specific activity, 52.1 Ci/mmol) in Ham's F-12 nutritive mixture (Gibeo Laboratories, Inc.) supplemented with 10% fetal bovine serum. After radiolabeling in monolayers or in suspension, cells were washed three times with ice-cold PBS. For biochemical characterization of labeled products, cells were sonicated into buffer A (50 mM sodium phosphate, pH 8.0, l M sodium chloride) and further subjected to SDS PAGE, enzyme studies, and molecular-sieve and ion-exchange chromatography. For light autoradiography, samples were fixed in 2% glutaraldehyde in 0.1 M cacodylate, pH 7.4, for 30 rain at room temperature. Cells were washed three times with 0. l M cacodylate buffer. To produce more adherent pellets, all cell samples were incubated with BSA (10% wt/vol) after fixation and rinsed again. Autoradiography of Thick Sections: The protocol of Oliver (28) was generally followed for light microscopy autoradiography. 1-/~m-thick epoxyembedded sections were mounted on subbed slides and dipped in NTB-2 (Eastman Kodak Co., Rochester, NY) nuclear track emulsion (l:l vol/vol, 42"C, 60% relative humidity). After drying, the coated slides were stored refrigerated for 2 wk in bakelite boxes containing dessicant, then developed in full strength D19 (Eastman Kodak Co.) at 13"C for 2.5 min and counterstained with methylene blue. The cells were photographed using a Zeiss 40x oil Planapo objective. Prints (8.5 x l I in) of the center portion of each negative were used for analysis. For mitotic populations, all cells identifiable as mitotics on each print were scored. Interphase cells, with a clear nuclear envelope, were also scored on the same prints; these were selected in order from one corner of the print to reduce bias in selection. In nonmitotic samples all cells with a defined nuclear envelope were scored. The area of each selected cell was determined using a sonic digitizer (Graf Pen, Science Accessories Corp., Southport, CT), and grains over cells were counted by eye. Data are expressed as grains per area. These ratios were determined by dividing the sum of grains by the sum of areas for each population sample. Standard deviations were derived from the estimate of variance for ratios as published by Weibel (43). For distribution analyses, ratios of grains per area for individual cell profiles were determined and plotted. Characterization of Radiolabeled Products: The extent of incorporated radiolabel was routinely assayed by gel filtration on Bio-Gel P-6DG (Bio-Rad Laboratories, Richmond, CA) for [35S]sulfate and Bio-Gel P-2 for [3H]glucosamine. Aliquots were applied to 0.6 x 8.0 cm columns; excluded fractions were collected directly into scintillation vials. Counts were normalized to the protein content of the original cell suspension, determined by the method of Lowry et al. (22) using BSA as standard. Enzymatic and chemical treatments for specific analyses were preceded by pronase digestion in 2 mg/ml S. griseus protease (Type VI, Sigma Chemical Co.) 1.5 mM CaCI2 at 37"C for 18-24 h. Free label was then removed by column chromatography on Bio-Gel P-6DG or P-2. Excluded fractions were pooled for subsequent chondroitinase and nitrous acid digestions or chloroform/methanol extraction. Chondroitinase AC or ABC (Sigma Chemical Co.) was added to a final concentration of 2.5 U/mg and allowed to incubate for 18-24 h at room temperature. Nitrous acid deamination, initiated by adjustment to 1.8 M CH3COOH and 0.24 M NaNO3, was allowed to proceed for 90 min (20). The extent of degradation was estimated after fractionation on BioGel P-6DG (for chondroitinases) or P-10 (for nitrous acid). Lipids were extracted according to the chloroform/methanol treatment of Heifetz and Snyder (8), and phases were counted directly. Stepwise ion-exchange chromatography was adapted from the basic procedure of Kraemer (15). Free label was first removed by gel filtration on Bio-Gel P-6DG concomitant with buffer exchange into 10 mM ammonium acetate, pH 7.0. Excluded fractions were pooled and subjected to pronase digestion as outlined above. The samples were then applied to 1-2 ml DEAE-cellulose (Sigma Chemical Co.) columns equilibrated in the same buffer; elution proceeded with 1.5-2 times the column volume for each subsequent step of 0.5, 1.1, and 2.0 M ammonium acetate, pH 7.0. SDS PAGE was performed in 5-15% gradient slab gels according to the method of Laemmli (17). Proteins were fixed and stained with Coomassie Brilliant Blue R in acidic isopropanol. Radiolabeled materials were detected by fluorography of Enlightning (New England Nuclear, Boston, MA) impregnated gels by exposing Kodak SB-5 X-ray film to the dried gels at -70"C.