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Alteration of Nucleoside Transport of Chinese Hamster Cells By Proc. Nat. Acad. Sci. USA Vol. 69, No. 12, pp. 3542-3546, December 1972 Alteration of Nucleoside Transport of Chinese Hamster Cells by Dibutyryl Adenosine 3':5'-Cyclic Monophosphate (thymidine and uridine uptake/thymidine kinase/DNA and RNA synthesis) PETER V. HAUSCHKA, LEIGHTON P. EVERHART, AND ROBERT W. RUBIN Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo. 80302 Communicated by Keith R. Porter, September 21, 1972 ABSTRACT Cultured Chinese hamster ovary cells served alterations that they induce in plasma membrane showed no significant change in generation time or frac- tion in the S-phase in the presence of 1 mM N6,02'-di- properties. Bu2cAMP causes a large decrease in the agglutin- butyryl adenosine 3': 5'-cyclic monophosphate. Growth ability of mouse fibroblasts by wheat-germ agglutinin (8), continued for at least two generations after expression of and increased adhesion to plastic surfaces (10). We studied the the morphological transformation induced by this cyclic effect of Bu2cAMP on DNA synthesis in CHO cells; our AMP analog. Despite identical growth rates, apparent attention soon focussed on metabolite transport, because it rates of DNA and RNA synthesis (incorporation of [3Hl- thymidine or [IHluridine) were reduced up to 15-fold in appeared that this process was most severely affected by log phase by 1 mM cyclic nucleotide. PIHiDeoxycytidine Bu2cAMP. incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [,;H]thymidine demonstrated MATERIALS AND METHODS an inhibition of transport rate dependent on the concen- Chinese hamster ovary cells (line CHO) were originally ob- tration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 10 was decreased 21-fold tained from Dr. Donald F. Petersen. Monolayer cultures by 1 mM cyclic nucleotide; half-maximal inhibition were grown at 370 in a moist atmosphere of 5% C02-95% air occurred at 6 MM. At 370, the pool size of acid-soluble in Ham's F-12 nutrient medium supplemented with 10% thymidylate was strongly reduced by 1 mM cyclic nucleo- fetal-calf serum (Flow Laboratories, Bethesda, Md.), penicillin, tide, and synergistic reduction of the pool size was found was with 0.5 mM aminophylline. Phosphorylation of the acid- and streptomycin; thymidine omitted. Serum was soluble intracellular label was unaffected by dibutyryl dialyzed at 40 against Earle's balanced salt solution. Washed cyclic AMP. Inhibition of thymidine uptake is attributed glass coverslips (25 mm, round) served as the growth surface to an observed decrease in thymidine kinase activity and were placed in 35-mm plastic petri dishes (Falcon caused by growth in 1 mM dibutyryl cyclic AMP, and possi- Plastics) containing 2.0 ml of medium. Large numbers of bly to a simultaneous alteration in membrane permeabil- ity. Kinase-facilitated uptake of other metabolites may be culture dishes were identically prepared by aseptically dis- regulated in a similar fashion by cyclic AMP. pensing the stirred cell suspension with a Labindustries repipet. Cells were counted on duplicate coverslips after Adenosine 3':5'-cyclic monophosphate has proven to be a trypsinization, vigorous suspension with a pipet, and dilution very common regulatory substance in biological systems. Its with 0.15 M NaCl-0.01% trypsin; a Coulter Counter (model diverse functions include regulation of transcription in F) was used. N,02'-dibutyryl adenosine 3':5'-cyclic mono- bacteria (1), intercellular communication in slime molds (2), phosphate (Bu2cAMP) was obtained from Sigma Chemicals. and mediation of hormone action in mammalian tissues (3). Apparent rates of DNA and" RNA synthesis at 370 were Recently, cyclic AMP has been implicated in the control of measured under the following labeling conditions: [3H]dT cell growth and differentiation. Hsie and Puck (4) were (Schwarz-Mann, 16 Ci/mmol, 2 ACi/ml, 15 min); [3H]dC among the first to suggest that cyclic AMP might control (New England Nuclear, 8.75 Ci/mmol, 2 MCi/ml, 60 min); cellular differentiation, and the concept has been expanded ['H]U (New England Nuclear, 27.4 Ci/mmol, 1 juCi/ml, by studies on macrophage and granulocyte cells (5) and 15 min). Incorporation was terminated by washing coverslips mouse-adrenal tumor cells (6). The dibutyryl cyclic AMP twice in cold Earle's solution (50 ml). After 10 min in ethanol- (Bu2cAMP)-induced morphological transformation of Chinese acetic acid 3:1, dehydration with 95% and absolute ethanol, hamster ovary (CHO) cells (4, 7) is coincident with greatly hydrolysis for 10 min in 1 N HCl at 250, and rinsing with H20 increased collagen production (7). Sheppard (8) clearly and absolute ethanol, the dry coverslips were counted in a showed that spontaneously- and virally-transformed mouse gas-flow planchet counter (11). cell lines could be restored to contact-inhibited growth by Total uptake of [3H]dT by log-phase cells was determined addition of Bu2cAMP and theophylline to the medium. by washing pulse-labeled coverslips for 45 sec in six 100-ml Otten et al. (9) found an inverse relationship between growth volumes of Earle's solution at 00, drying, and counting. Acid- rate and endogenous levels of cyclic AMP in 12 mouse-fibro- insoluble counts from duplicate coverslips were subtracted blast cell lines. The involvement of cyclic AMP and from the total incorporation for estimation of the acid-soluble Bu2cAMP in growth control is interesting in light of the ob- pool. Total uptake at 10 was measured after cooling culture dishes for 15 min on an iced metal plate. Abbreviations: CHO, Chinese hamster ovary (cells); Bu2cAMP, Acid-soluble pools were extracted from saline-washed, N6,0'-dibutyryl cyclic AMP. pulse-labeled cells with 0.5 M HC104 or 0.5 N HCl. Analysis 3542 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Nucleoside Transport of Chinese Hamster Cells 3543 for total thymine nucleotides (% phosphorylation) involved binding to DEAE-cellulose filter discs (Whatman DE81) and washing with 1 mM ammonium formate and ethanol to remove dT quantitatively (12). Quenching was minimized by using [14C]dT (New England Nuclear, 54.5 Ci/mol, 2 ACi/ml) for pool analysis. Thymidine kinase was assayed in 0.1-ml reaction mix- tures consisting of 5 mM MgCl2, 5 mM ATP, 2.5 mM 2- mercaptoethanol, 0.5 mM EDTA, 150 mM Tris HCl (pH 7.5), 92 AM ['4C]dT (0.5 ,4Ci), and 50-80 Asg of extract protein (12). Phosphorylation of dT was linear for more than 60 min at 37°. Protein of extracts that had been dialyzed against phosphate buffer was determined by a modified microbiuret method. PRELIMINARY OBSERVATIONS Fig. 1 indicates the rate of growth of CHO cells in the presence or absence of 1 mM Bu2cAMP. The generation time of about 16 hr is essentially unaffected by Bu2cAMP. Within HOURS 5 hr after addition of Bu2cAMP, the morphological change FIG. 2. Incorporation of nucleosides into acid-insoluble cell At various times the culture curve characteristically produced by this compound (4, 6, 7) is material. during cycle (growth shown in Fig. 1), tritiated nucleosides were added to culture clearly observable. Treated cells become extended and dishes at 370, and acid-insoluble radioactivity was determined. flattened, and are oriented in swirling patterns with neigh- Top: [3H]uridine; middle: ['H]deoxycytidine; bottom: ['H]thymi- cells. at boring These cells undergo least two complete dine; * -, control; O-O, 1 mM Bu2cAMP. rounds of division in the presence of Bu2cAMP; their altered morphology is maintained through iyost of each cell cycle. tion of ['H)dT into DNA and ['H]U into RNA is depressed Saturation density of the Bu2cAMP cultures is slightly by as much as 15-fold during the culture cycle. Superficially, lower than that of the controls, and it is possible that the these data suggest that the nucleic-acid complements of the property of contact-inhibition of growth has been restored. cells should be drastically reduced by Bu'cAMP. This con- Hsie and Puck (4) showed that clonal growth of CHO cells, clusion is unlikely for two reasons. First, the Bu2cAMP- achieved by plating at low cell 4ensity, provides conditions treated cells. continue to multiply normally for several where multilayered growth can .occur, apparently without generations. Second, sonicated suspensions of control and significant depletion of the nutrient medium. These authors treated cells (108 cells per ml) have identical absorbance found inhibition of multilayering in the presence of Bu2cAMP (A2,0 = 0.58 i 0.02) after dialysis and centrifugation, and testosterone, and concluded that contact-inhibited suggesting similar amounts of total nucleic acid per cell. growth had been restored by this treatment. Our untreated The pattern of ['H]dC incorporation in Fig. 2 is obviously CHO cultures reach stationary phase at about the same time different from the ['H]dT and ['HJU data, thus strengthening as a crowded monolayer is achieved. This coincidence stems the interpretation that Bu2cAMP causes a relatively specific from nutrient depletion (11, 13). Accurate measurement of inhibition of incorporation of the latter precursors and that it multilayered growth by these transformed cells is difficult be- does not act generally to depress the rates of DNA and RNA cause weak adhesion of CHO to substrates causes unavoidable synthesis. Stimulation of ['H]dC incorporation is caused by losses of cells during changes of medium. the use of fresh medium at 0 hr; this effect was not observed Incorporation of radioactive nucleosides into acid-insoluble with the other nucleosides. Bu2cAMP reduces the stimulation cell material is generally used as an index of the rate of nucleic by about 50%, but after 10 hr only a small difference remains acid 2 a on synthesis. Fig. shows clear effect of Bu2cAMP the between the rates- of incorporation in the two cultures.
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