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 fraction 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 permeability. 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. apparent rates of both DNA and RNA synthesis. Incorpora- These results are summarized in Fig. 3. A semi-logarithmic

'0 6: (I) I00 w2 4 Q 4 180 ()_flW' 2 I 60 6 z 0 0 40- 0 0 20 40 60 80 HOURS 20 1 FIG. 1. Growth of CHO on glass coverslips. Cells were plated 0 10 20 30 40 50 at a density of 14,000 per coverslip in F-12 medium. After attach- HOURS ment and growth for 44 hr, the medium was withdrawn and re- FIG. 3. Effect of 1 mM Bu2cAMP on incorporation of nucleo- placed with fresh medium (zero time) with (V V) or without sides into acid-insoluble material. By use of the data of Fig. 2, (a-*) 1 mM Bu2cAMP. The arrow indicates the time at which incorporation by cells in the presence of Bu2cAMP is expressed as the morphological transformation caused by Bu2cAMP was first a percentage of the control values measured at the same time. observable. v *-4, thymidine; A/ A, uridine; O-O, deoxycytidine. Downloaded by guest on September 24, 2021 3544 Cell Biology: Hauschka et al. Proc. Nat. Acad. Sci. USA 69 (1972)

kinase activity, for example, shows an increase during exponential growth, then a decline in stationary phase in L-cells (16). Kit et al. (17) found uridine kinase to behave in a similar fashion in mouse L-M fibroblasts. In chick-embryo fibroblasts and mouse 3T3 cells, the 3- to 5-fold reduction in uridine incorporation in dense cultures is not related to the rate of RNA synthesis (still 70% of normal) but to reduced uptake of uridine (15), a process in which uridine kinase has been strongly implicated (18). Of interest here is the observation that Bu2cAMP can prematurely cause the incorporation rate of some precursors to mimic that normally found only in stationary-phase cultures. 20 30 MINUTES THYMIDINE UPTAKE AND INTRACELLULAR POOLS FIG. 4. Rate of thymidine uptake at 1°. Coverslip cultures in Our initial results suggested that depression of the apparent early log phase were given fresh medium, with (0 0) or with- rates of DNA and RNA synthesis by Bu2cAMP was an out (O -O) 1 mM Bu2cAMP. 9 hr later, the dishes were chilled artifact caused by perturbation of one or more of the pro- to 10 and pulsed for various lengths of time with [3H]dT (2 jCi/ cesses involved in incorporation of an exogenous nucleoside ml, 0.125 ,M). Coverslips were washed extensively in Earle's precursor into nucleic acid. Incorporation of [3H ]dT, for solution at 00, dried, and counted. About 98% of the cell-as- example, requires entry, phosphorylation by thymidine sociated radioactivity was soluble in acid under these conditions. kinase, and enzymatic polymerization into DNA from a pool of thymine nucleotides that is also supplied by the thymidy- plot of Fig. 3 (not shown) reveals a rather abrupt decrease of late synthetase reaction. 1 mM Bu2cAMP caused no change in U and dT incorporation to 50% of the control rate by 40 min, the following parameters: generation time (16 hr); % of cells followed by a slower decline (half-time about 4 hr) to only 7% in the S-phase, as judged by autoradiography (50-55%); of the control. and rate of entry into the S-phase (data not shown) after Large variations in the rate of precursor incorporation over release from G1 arrest by isoleucine deprivation (11). By the culture cycle (sparse-exponential-stationary) is shown several criteria the cells were growing vigorously in Bu2cAMP, by the control curves in Fig. 2. Increasing incorporation albeit with altered morphology. Therefore, transport, phos- during early stages of the culture cycle is attributed to the phorylation, and the pool of dT and its derivatives were in- recovery of the cells from the late-log metabolic state that vestigated. existed at the time of planting. [3H]dT and [3H]U both reach Preliminary studies showed that the rate of uptake of a maximum rate of incorporation in mid-log phase (30 hr). [3H]dT from the medium at 370 was reduced 3- to 4-fold by Incorporation of [3H]dT then drops sharply during entry into treatment for 13 hr with 1 mM Bu2cAMP, and that the stationary phase, while [3H ]U shows a more gentle decline, in steady-state rate of incorporation into acid-insoluble material keeping with the relatively high levels of RNA synthesis was reduced about 7-fold. Equilibration of label with the maintained during this period (14, 15). Aside from the acid-soluble pool was complete by 15 min in control cells, initial stimulation by fresh medium, [3H]dC incorporation but was not achieved in Bu2cAMP-treated cells within proceeds at a slowly decreasing rate over the culture cycle. 40 min. Since the initial rate of uptake of [3H]dT into the was in Such fluctuations in precursor incorporation are a combined acid-soluble pool at least 3-fold smaller the presence result of changes in the absolute rates of nucleic acid synthesis of Bu2cAMP, it appears that transport is the primary process and changes in nucleoside kinase activities. Thymidine controlled by Bu2cAMP, and that reduced labeling of acid- insoluble material is a consequence of inhibited transport of [3H ]dT. 40F TABLE 1. Acid-soluble pool size -130- coLi -- Total acid- 01 a ----U Conditions Pulse soluble cpm -'-.20 a_ Control 15 min at 370 147,000 u- 10 +Bu2cAMP 15 min at 370 24, 100 Control 40 min at 10 77,800 +aminophylline 40 min at 10 12,200 0 10-8 10-7 10-6 10-5 10-4 10-3 10-2 +Bu2cAMP 40 min at 10 2,180 Bu2cAMP, M +aminophylline 40 min at 10 1,340 FIG. 5. Effect of Bu2cAMP on thymidine uptake at 10. and Bu2cAMP Coverslip cultures in early-log phase were given fresh medium containing various concentrations of Bu2cAMP. 13 hr later, some Fresh medium with or without additives (aminophylline, dishes were chilled to 10, pulsed for 40 min with [3H] dT (2 ,Ci/ml, 0.5 mM; Bu2cAMP, 1 mM) was added to cells 20 hr before the 0.125 jM), then washed and counted (0) as in Fig. 4. Parallel pulse. Duplicate cultures in mid-log phase (2.2 X 106 cells per cultures were washed after 13 hr with fresh, warm medium to bottle) were pulsed with [3H]dT at 2 ,.Ci/ml. After six washes remove the Bu2cAMP, and were then incubated for an additional with cold F-12 medium (salts and glucose only), three sequential 2 hr at 370 before measurement of [3H]dT uptake at 10 (U). extractions with cold 0.5 M HCl04 were pooled and counted. Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Nucleoside Transport of Chinese Hamster Cells 3545

The study of uptake at low temperature is often advan- TABLE 2. Thymidine kinase activity tageous, in that depletion of the intracellular pool by metabolic events is severely inhibited, and transport processes may Experiment Control +Bu2cAMP Ratio be more clearly discerned. We have studied the rateof uptake of A Crude extract 34.6 15.6 2.2 [3H]dT at 10 (Fig. 4), under conditions where DNA synthesis Dialyzed 18.9 10.6 1.8 is more than 98% inhibited. The uptake is linear, and the B Crude extract 22.2 10.2 2.2 rate of uptake by the control cells is 15-fold less than it is at Dialyzed 11.1 5.9 1.9 370 (corresponding to a Qjo of 2.1). The large (21-fold) inhibition of uptake by 1 mM Bu2cAMP suggests a significant Cells grown in the presence or absence of 1 mM Bu2cAMP for permeability barrier to thymidine in the presence of this com- 20 hr were harvested in mid-log phase with 0.5 mM EDTA- pound. Fig. 5 demonstrates the sigmoidal dependence of 0.15 M NaCl, collected by centrifugation, washed twice with inhibition on Bu2cAMP concentration, with half-maximal cold F-12 medium (salts and glucose only), resuspended in 2.0 ml inhibition occurring at about 6 MuM Bu2cAMP. Significant of buffer A (0.1 M Tris HCl-l mM EDTA-5 mM 2-mercapto- recovery of the ability to transport [3H]dT is observed within ethanol, pH 7.5), and sonicated for 15 sec. After centrifugation for 2 hr after the Bu2cAMP is washed away with fresh medium 30 min at 40,000 X g, portions of the supernatants were dialyzed at over most of against buffer A. Activity is expressed as pmol of [14C]thymidyl- 37°. The concentration range which the ate produced perMAg of cell protein in 60 min at 37°. uptake inhibition takes place is satisfying close to measured values of intracellular cyclic AMP concentration. For instance, Otten et al. (9) report endogenous cyclic AMP con- tion of uptake in Fig. 3 after Bu2cAMP addition, it is not centrations of 0.6 ,uM to 11.0,uM in mouse fibroblasts. Activa- expected that direct inhibition of thymidine kinase by this tion of cyclic AMP-dependent protein kinases, a commonly cyclic AMP analog should occur. presumed mechanism for the diverse effects of cyclic AMP, is Reviewing the evidence that nucleoside uptake involves observed at concentrations ranging from 10 nM to 10MuM (19). nucleoside kinases, we find that in all cases where uptake is Further properties of the acid-soluble thymine nucleotide reduced, kinase activity is also low; stimulation of uptake is pool are shown in Table 1. The total acid-soluble label at accompanied by increased kinase activity. Cells in crowded or 370 is reduced sharply by Bu2cAMP, with a larger effect confluent cultures have decreased permeability to exogenous occurring at low temperatures (10). Aminophylline also metabolites, as compared to sparse, rapidly growing cultures causes reduction of the pool size, both alone and synergistically (15, 16, 23). Transformation by viruses (21), addition of with Bu2cAMP. Because this drug is known to inhibit the fresh serum (23), dilution to lower cell densities (16, 20), and phosphodiesterase that degrades endogenous cyclic AMP, hormonal stimulation of growth-as in liver regeneration such a result is expected if cyclic AMP is involved in the after partial hepatectomy (24)-rapidly stimulate precursor control of dT transport. uptake, with concomitant increase in nucleoside kinase Acid-soluble pools extracted from [14C]dT-labeled cells activities. While enzymes that show similar growth-related were analyzed for nucleotide content. Most of the pool variation of activity are numerous [thymidine kinase (16, (about 83%) from cells labeled at 370 in the presence or 21, 25), uridine kinase (17, 20), choline kinase (26), and absence of 0.3 mM Bu2cAMP wasphosphorylated. At 10, the ribonucleotide reductase (25)1, there are other activities that control cells had a more extensively phosphorylated pool remain relatively constant [DNA polymerase (25), uridine (81%) than did the Bu2cAMP treated cells (60%). The phosphorylase (17), and lactate dehydrogenase (27)]. salient conclusion from these data is that the size of the radio- The mechanism for nucleoside uptake by mammalian cells active acid-soluble pool can be greatly reduced by growth in is generally believed to be "facilitated diffusion" (28, 29), the presence of Bu2cAMP (Table 1), but there is no substan- meaning that freely diffusing nucleosides are phosphorylated tial change in the composition (fraction phosphorylated) of intracellularly by specific kinases and rendered incapable of the thymidine derivative pool under these conditions. easy exit by virtue of the negatively charged phosphate group. Hence, almost all intracellular label in an uptake THYMIDINE KINASE, TRANSPORT, experiment involving low extracellular concentrations of AND CYCLIC AMP labeled nucleosides represents phosphorylated derivatives Numerous correlations between nucleoside kinase activities (30, 31). Exceptional cases, where there is little or no phos- and the transport of nucleosides (18, 20, 21) led us to measure phorylation, occur when the transport system is saturated thymidine kinase in extracts of control and Bu2cAMP- (30), or when the pertinent nucleoside kinase is absent (32). treated cells. Comparative assays were done under con- Our studies of thymidine uptake were performed at very low ditions where enzyme concentration was shown to be the thymidine concentrations (0.125 uM), where kinase-facili- limiting factor in the rate of thymidylate production. The 'tated transport is dominant over the nonspecific diffusional substrate concentration was well above the measured Km process. value of about 4 uM (data not shown). Table 2 shows that The apparent.discrepancy between the extent of inhibition the specific activity of thymidine kinase is halved by of transport (3- to 21-fold) and the extent of thymidine growth in the presence of Bu2cAMP. Dialyzed cell extracts kinase reduction (2-fold) by Bu2cAMP may be rationalized were found to have a similar 2-fold ratio of activities, thus by several arguments: (i) in vivo levels of the enzyme may eliminating inhibitory metabolites as a possible cause of differ more than our in vitro measurements suggest; (ii) the the decreased thymidine kinase activity. Fluctuations in degree of saturation of the kinase with substrate may be dTTP and dCTP affect thymidine kinase in other systems different in the control and Bu2cAMP-treated cells because of (12, 22). No inhibition by cyclic AMP or Bu2cAMP (0.5 mM) variations in the intracellular levels of thymidylate, dTTP, was observed in vitro. From the relatively slow rate of inhibi- dCTP, and other regulatory metabolites; (iii) BU2cAMP Downloaded by guest on September 24, 2021 3546 Cell Biology: Hauschka et al. Proc. Nat. Acad. Sci. USA 69 (1972) may cause a true change in the permeability of the plasma Barkley, D. S. (1967) Proc. Nat. Acad. Sci. USA 58, 1152- membrane to thymidine, thus amplifying the difference in 1154. by limiting free of the 3. Butcher, R. W., Robison, G. A. & Sutherland, E. W. thymidine kinase activities diffusion (1970) Control Processes, in Multicellular Organisms- substrate. Conceivably, we have observed two simultaneous Ciba Foundation Symposium, eds., Wolstenholme, G. E. W., phenomena. The rapid inhibition of thymidine and uridine & Knight, J., (J. and A. Churchill, London), pp. 64-85. incorporation by 50% (Fig. 3) in the first 40 min may be 4. Hsie, A. W. & Puck, T. T. (1971) Proc. Nat. Acad. Sci. USA related to changes in the plasma membrane or in specific 68, 358-361. 5. Landau, T. & Sachs, L. (1971) Proc. Nat. Acad. Sci. USA membrane-bound carriers. Change in the adhesiveness of 68, 2540-2544. Bu2-cAMP-treated fibroblasts (10), and inhibition of uridine 6. Masui, H. & Garren, L. D. (1971) Proc. Nat. Acad. Sci. and choline permeation (29) are also observed over a short USA 68, 3206-3210. time interval (<1 hr). The more slowly expressed inhibition 7. Hsie, A. W., Jones, C. & Puck, T. T. (1971) Proc. Nat. of incorporation shown in Fig. 3 over the period from 1 to 20 Acad. Sci. USA 68, 1648-1652. 8. Sheppard, J. R. (1971) Proc. Nat. Acad. Sci. USA 68, hr (half-time about 4 hr) might then be a consequence of the 1316-1320. decreasing thymidine kinase activity. 9. Otten, J., Johnson, G. S. & Pastan, I. (1971) Biochem. Involvement of thymidine kinase in transport would Biophys. Res. Commun. 44, 1192-1198. suggest a membrane location, but the activity is clearly 10. Johnson, G. S. & Pastan, I. (1972) Nature New Biol. 236, 247-249. "soluble" in some studies (refs. 12, 33, and present paper), and 11. Everhart, L. P. (1972) Exp. Cell Res., in press. in other cases it is membrane-associated (34) or nuclear (22). 12. Bresnick, E. & Karjala, R. J. (1964) Cancer Res. 24, 841- The existence of two classes of thymidine kinase (33) may 846. partially resolve this discrepancy. A heavy form of the 13. Tobey, R. A. & Ley, K. D. (1970) J. Cell Biol. 46, 151-157. 14. Enger, M. D. & Tobey, R. A. (1972) Biochemistry 11, 269- enzyme is elevated in tumor extracts (33), and growth- 277. related changes in the intracellular distribution and membrane 15. Weber, M. J. & Rubin, H. (1971) J. Cell. Physiol. 77, 157- binding of thymidine kinase are now known (34). Control 168. by cyclic AMP may be exerted through dissociation of the 16. Weissman, S. M., Smellie, R. M. S. & Paul, J. (1960) Rio- heavy form after phosphorylation by a protein kinase. chim. Biophys. Acta 45, 101-110. 17. Kit, S., Valladares, Y. & Dubbs, D. R. (1964) Exp. Cell Because the concentration of cyclic AMP decreases under Res. 34, 257-265. the same conditions discussed above [sparse cultures and 18. Scholtissek, C. (1968) Biochim. Biophys. Acta 158, 435- rapid growth (9), addition of fresh serum (35), trypsinization 447. (35), and viral transformation (35) ], where both kinase 19. Langan, T. A. (1968) Science 162, 579-580; Miyamoto, E., Kuo, J. F. & Greengard, P. (1969) J. Biol. Chem. 244, activities (36) and general metabolite transport are increased, 6395-6402; Goodman, D. B. P., Rasmussen, H., DiBella, the control of these latter properties may be mediated by F. & Guthrow, C. E., Jr. (1970) Proc. Nat. Acad. Sci. USA cyclic AMP. Previous studies of the regulation of transport by 67, 652-659. cyclic AMP have focussed on secretory processes (37, 38). 20. Plagemann, P. G. W., Ward, G. A., Mahy, B. W. J. & In these cases, transport is stimulated by increased cyclic Korbecki, M. (1969) J. Cell. Physiol. 73, 233-250. 21. Hare, J. D. (1970) Cancer Res. 30, 684-691. AMP, presumably because microtubules are involved in the 22. Adams, R. L. P. (1969) Exp. Cell Res. 56, 49-54. translocation of secretory vesicles. Assembly of microtubule 23. Cunningham, D. 1). & Pardee, A. B. (1969) Proc. Nat. Acad. structures is enhanced by Bu2cAMP (4,39). Sci. USA 64, 1049-1056. We have found strong inhibition of thymidine transport by 24. Bollum, F. J. & Potter, V. R. (1959) Cancer Res. 19, 561- Bu2cAMP; the uptake of uridine, but not deoxycytidine, is 565. 25. Cory, J. G. & Whitford, T. W. (1972) Cancer Res. 32, 1301- probably similarly controlled by kinase regulation. Although 1306. thymidine is not an essential nutrient for CHO cells, its trans- 26. Plagemann, P. G. W. (1969) J. Cell Biol. 42, 766-782. port behavior may serve as an index for the uptake of other 27. Ward, G. A. & Plagemann, P. G. W. (1969) J. Cell. Physiol., compounds that are essential for growth. Choline, for instance, 73, 213-231. 28. Jacquez, J. A. (1962) Biochim. Biophys. Acta 61, 265-277. is a required precursor for membrane synthesis by mammalian 29. Plagemann, P. G. W. & Roth, M. F. (1969) Biochemistry cells in culture. Choline kinase is strongly implicated in the 8, 4782-4789. uptake of choline by rat hepatoma cells, and the enzyme 30. Lindberg, U., Nordenskjold, B. A., Reichard, P. & Skoog, activity fluctuates during the culture cycle in the same L. (1969) Cancer Res. 29, 1498-1506. is also 31. Plagemann, P. G. W. (1971) J. Cell. Physiol. 77, 241- manner as the nucleoside kinases (26). Sugar transport 258. facilitated by kinases or phosphotransferases (40), and may 32. Schuster, G. S. & Hare, J. D. (1971) In Vitro 6, 427-436. be equally sensitive to control by cyclic AMP. It stands as an 33. Okuda, H., Arima, T., Hashimoto, T. & Fujii, S. (1972) obvious possibility that cell proliferation is controlled by Cancer Res. 32, 791-794. cyclic AMP-induced limitation of metabolite uptake. 34. Baril, E., Baril, B. & Elford, H. (1972) Proc. Amer. Assc. Cancer Res. 13, 84, Abstr. 334. 35. Sheppard, J. R. (1972) Nature New Biol. 236, 14-16. We thank Drs. David M. Prescott and Keith R. Porter for 36. Nordenskjold, B. A., Skoog, L., Brown, N. C., & Reichard, helpful discussions. Supported by The Jane Coffin Childs P. (1970) J. Biol. Chem. 245, 5360-5368. Memorial Fund, NIH Grants 5-FO2 GM 50354 and 1-FO2 37. Prince, W. T., Berridge, M. J. & Rasmussen, H. (1972) NS51, 111-01, and NCI Grant 1 RO1 CA 12302-01 CBY. Proc. Nat. Acad. Sci. USA 69, 553-557. 38. Peach, M. J. (1972) Proc. Nat. Acad. Sci. USA, 69, 834- 1. de Crombrugghe, B., Chen, B., Gottesman, M., Pastan, 836. I., Varmus, H. E., Emmer, M. & Perlman, R. L. (1971) 39. Porter, K. R., Puck, T. T., Hsie, A. W. & Kelley, D. (1972) Nature New Biol. 230, 37-40. J. Cell Biol. in press. 2. Konijn, T. M., van de Meene, J. G. C., Bonner, J. T. & 40. Roseman, S. (1969) J. Gen. Physiol. 54, 138s. Downloaded by guest on September 24, 2021