422

Preliminary

notes

sence of serum, is due to the increase in area of cell/substrate apposition in manganese-containing medium. Thus manganese appears to have an indirect rather than specific effect on cell adhesion. The increase in number of cells remaining on the substrate in MSBK plus serum, over SBK plus serum, indicates that here the area of cell substrate apposition affects the strength and speed of cell attachment. In conclusion, this study of early adhesion of Iibroblasts to glass demonstrates a striking effect of serum on the fine structure of cell/substrate relationships and reaffirms Rabinovitch & De Stefano’s and Yasuda’s [3, 4, 51 findings on the effects of manganese on cell spreading. In addition, further work using the system described here might well elucidate the stages of development of cell/substrate plaques and associated filamentous structures [lo], together with the effects of divalent cations and various synthetic or protein coated surfaces on this development. We would like to thank Dr J. E. M. Heaysman for her help in the preparation of this manuscript.

References 1. Rqjaraman, R, Rounds, D E, yen, S B S & Rembaum, A, Exp cell res 88 (1974) 327. 2. Witkowski, J A & Brighton, W D, Exp cell res 70 (1972) 41. 3. Rabinovitch, M & De Stefano, M J, J cell biol 59 (1973) 165. 4. Yasuda, K, J cell sci 15 (1974) 269. 5. Rabinovitch, M & De Stefano, M J, Exp cell res 79 (1973) 423. 6. Maroudas, N G, Nature 254 (1975) 695. 7. Lagunoff, D & Curran, D E, Exp cell res 75 (1972) 117 __,. 8. Takeichi, M, Exp cell res 68 (1971) 88. 9. Heaysman, J E M & Pegrum, S M, Exp cell res 78 (1973) 71. 10. kbercrombie, M, Heaysman, J E M & Pegrum, S M, Exo cell res 67 (1971) 359. 11. Dutton’, R C, Webb&, A J, Johnson, S A & Baier, R E, J biomed mater res 3 (1969) 13. 12. Revel, J P & Wolken, K, Exp cell res 78 (1973) 1. 13. Taylor, A C, Exp cell res 8, suppl. (1%1) 154. Exptl

Cell

Rcs 96 (1975)

14. Easty, G C, Easty, D M & Ambrose, E J, Exp cell res 19 (1960) 539. 15. Branton, D, Proc nad acad sci US 55 (1966) 1048. Received May 12, 1975 Revised version received July 16, 1975

Hydroxyurea synchronization fetal spleen cells

of bovine

DEBORAH S. PARRIS, R. C. BATES and E. R. STOUT, Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 9 USA Summary. Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80-85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2-4 h following removal of HU. The rapid induction of DNA synthesis--in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 4050% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.

The use of hydroxyurea (HU) in synchronizing transformed or stable diploid mammalian cells has been well established [l-7]. In these cell lines, HU is a superior agent for synchronizing cells at the Gl/S border because it effectively but reversibly inhibits DNA synthesis [5, 61 without significantly affecting the synthesis of RNA and protein [3, 8, 91. Although HU was previously shown to inhibit DNA synthesis in primary rabbit kidney cells [lo], little information is available concerning the use of HU as a synchronizing agent of fetal cells or the effect of HU on RNA and protein synthesis in these cells. Synchronized fetal

Preliminary

-20

-16

60-

- n

40-

-*

lo-

-4

Fig. I. Abscissa: time after HU removal (hours); ordinafe: (l&r) q - - -0, TCA-insoluble [3H]uridine incorporation (% of control); n - - -m, TCA-insoluble [“Cl amino acids incorporation (% of control); O-O, percentage nuclei incorporation rH]TdR into HClO,-insoluble material; (righr) O-O, TCAinsoluble [3H]TdR incorporation (cpmx lO-3/lO5 cells).

or primary cells may be required (i) to study the events which occur in the cell cycle of normal cells compared with those which occur in transformed cells; (ii) to determine if changes in cycle-specific events occur during differentiation of normal mammalian tissues, or (iii) to study cell cycle-dependent replication of viruses with a limited host cell range. The data presented in this report demonstrate that HU is effective in producing highly synchronous populations of bovine fetal spleen (BFS) cells.

Materials and Methods Stationary BFS cells, prepared from 5-7 month fetuses by the method of Youngner [I I], were synchronized by seeding 1 to I .6x IO5 cells/ml of synchronization medium into Leighton tubes (1 ml/tube) or into 60x 15 mm Petri dishes (5 ml/dish). Synchronization medium consisted of minimum essential medium containing 10% lamb serum and varying concentrations of HU (Calbiochem). Radioactive compounds were purchased from AmershamlSearle. The rates of DNA and RNA synthesis were determined by adding [3H]thymidine (C3H]TdR, 18.4 Cilmmole) or [3H]uridine (28 Ci/ mmole) directly to the medium to a final concentration of 0.5 &i/ml for 15 and 60 min, respectively. To determine the rate of protein synthesis, the medium

notes

423

was removed and similar medium without amino acids but containing 3% lamb serum was added. Then r4C protein hqdrolysate (55 mCi/mAtom) was added for 1 or 2 h to a final concentration of 0.1 uCilm1. The addition of medium without amino acids did not affect the availability of amino acids to the cells since uptake was linear for at least 2 h. Petri dish cultures of cells were assayed for the amount of [3H]uridine and 14C amino acids incoroorated into TCA-insoluble material bv the method of kegan & Chu [12]. TCA-soluble pools were assayed at 4°C bv nrecinitating sonicated cellular material prepared as above in 3 % TCA for 10 min and centrifuging at 800 g. Aliquots from the supematant were added to vials containing Dimilume (Packard) and counted by liquid scintillation spectrometry. Cpm for both TCA-soluble and insoluble material were standardized on the basis of nrotein content bv the method of Lowry et al. [13]. Coverslips containing cells which had been pulse-labeled with [3H]TdR were fixed in 1% HClO,, rinsed in water, and dried. The rate of DNA svnthesis was determined bv breakine the coverslipsV into liquid scintillation -vials and solubilizing overnight at 25°C in 0.5 ml Soluene 350 (Packard). Toluene-based scintillation fluid was then added, and the samples counted as above. Following acid fixation, some coverslips were prepared for autoradiography using NTB-2 photographic emulsion (Kodak) and developed 2 to 3 days later. Cells containing more than 10 grains over the nucleus were defined as specifically labeled. In all cases, 300 to 500 cells/slide were scored.

Results and Discussion

DNA synthesis, as determined by autoradiography, was not completely inhibited when BFS cells were exposed to concentrations of HU below 0.8 mM for up to 48 h. However, at concentrations from 0.8 to 4 mM, incorporation of [3H]TdR into acid-insoluble material was completely inhibited 24 h after stationary cells were seeded in synchronization medium. At intervals from 24 to 48 h after seeding, cells were released by washing from the HU block and labeled with [3H]TdR. Examination of autoradiograms revealed that the maximum percentage of cells synthesizing DNA was obtained following release of cells exposed to 2 mM HU for 32 h. The kinetics of acid-insoluble [3H]TdR incorporation for cells synchronized by this procedure was determined by pulselabeling cells with [3H]TdR before and Exprl

Cell

Res 96 (1975)

424

Preliminary

notes

after release from HU (fig. 1). Before HU at 32 h (immediately before release, release from the HU block, synthesis of fig. 1). The lower rates of incorporation at DNA did not occur above background this time are not due to a decrease in transport of uridine or amino acids since the levels. However, immediately after release, cells were synthesizing DNA and within TCA-soluble pools of these compounds in 2 h, 80 to 85 % of the cells contained specifiHU-treated cells are similar to those in uncally labeled nuclei. The maximum rate of treated cells. Upon removal of HU, BFS DNA synthesis was observed 2 to 4 h after cells begin to rapidly synthesize DNA, release of cells from HU. At 9 to 10 h after synthesize RNA and protein at increased release, the rate of DNA synthesis had levels (not shown), and subsequently decreased to 50% of the level observed at divide. Therefore, the reduced levels of 2 h, and by 16 h, only background levels of RNA and protein synthesis seen in BFS DNA synthesis were occurring. Between 18 cells in the absence of DNA synthesis may and 22 h after release, cells began to underprevent the cells from entering a state of go mitosis synchronously as demonstrated irreversible unbalanced growth. by the presence of high percentages of These results demonstrate that HU is a mitotic figures in stained preparations (re- superior synchronizing agent for BFS cells sults not shown). because (i) HU treatment induces large The relative toxicity of HU for cells syn- numbers of BFS cells to enter and exit S chronized with this agent appears to vary. phase synchronously; (ii) HU has relaAlthough HU differentially kills Chinese tively low toxicity for BFS cells; and (iii) hamster cells in S phase [2], the drug extended exposure of BFS cells to HU does produces no differential killing of HeLa not result in irreversible unbalanced cells [3]. Furthermore, HeLa and mouse L growth. In general, optimum conditions cells suffer no loss in viability through 18 h for obtaining synchronized BFS cells did not vary for the populations of cells exof exposure to HU and thus are more resistant to the cytotoxic effect of HU than amined in this laboratory. However, due to are Chinese hamster cells [2, 3, 51. BFS the heterogeneity of fetal cells, it may be cells are even more resistant to the cyto- necessary to determine optimum concentratoxic action of this drug, remaining viable tions and times of exposure to this drug before fetal cells of other types can be effecfor at least 72 h in 2 mM HU as determined tively synchronized. by trypan blue exclusion tests (unpublished results). The authors thank Theresa Tunstall for excellent Although HU has been shown to have technical assistance. little effect on RNA and protein synthesis in established cell lines [3, 8, 91, its effect References I. Sinclair. W K. Science 150 (1965) 1729. in fetal cells has not been studied. RNA and 2. - Can&r res’27 (1967) 297.‘ protein synthesis in BFS cells seeded in 3. Pfeiffer. S E & Tolmach. L J. Cancer res 27 (1967) 124. 2 mM HU occur at normal rates until DNA 4. Kim, J H, Gelbard, A S & Perez, A G, Cancer res synthesis is completely inhibited (8 h before 27 (1967) 1301. 5. Adams, R L P & Lindsay, J G, J biol them 242 release, fig. 1). The synthesis of these (1967) 1314. macromolecules continues in the absence of 6. Tobey, R A & Crissman, H A, Exp cell res 75 (1972) 460. DNA synthesis but at 40 to 50 % of the rates 7. Plageman, P G W, Richey, D P, Zylka, J M & observed in cells seeded in medium without Erbe, J, Exp cell res 83 (1974) 303. Erprl Cdl Rrs 96 (1975)

Preliminary 8. Young, C W & Hodas, S, Science 146 (1964) 1172. 9. Pollak, R D & Rosenkranz, H S, Cancer res 27 (1967) 1214. 10. Adams, R L, Abrams, R & Lieberman, I, J biol them 241 (1966) 903. 11. Youngner, J S, Proc sot exp biol med 85 (1954) 202. 12. Regan, J D & Chu, E H Y, J cell bio128 (1966) 139. 13. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. Received June 23. 1975

Transport of thymidine during the cell cycle in mitotically synchronized CHO cells L. E. HOPWOOD, Department Colorado USA

of State

W. C. DEWEY and W. HEJNY,

Radiology University,

and Fort

Radiation Collins,

CO

Biology, 80523,

The kinetics of total uptake of thymidine into the cell were determined for cells which had been mitotically synchronized, plated into scintillation vials and pulsed with five concentrations of [3H]thymidine at various times during the cell cycle. From Lineweaver-Burk plots of these rates, V,, and K, values were determined for the transport of thymidine. The V,,, values ranged from a low of 2.0.pmoles/ min/106 cells in mid-G1 to a high of 99.7 in mid-S before a decline in late S and G2.K, values displayed only a 5-fold range in values.

Summary.

Thymidine at a concentration less than 2 PM is taken up by mammalian cells by means of a passive mediated transport process, also known as facilitated diffusion. The initial thymidine uptake into the total cell compartment displays typical saturation kinetics: from first order at low concentrations to zero order at high concentrations. Using the Lineweaver-Burk plot of the Michaelis-Menten equation (and the initial uptake rates for each concentration), one can obtain values for V,,, and K,. Similar to the interpretation for enzyme kinetics, V,,, is proportional to the number of transport sites and K, is inversely proportional to their binding strength [l]. The variation of thymidine uptake during the cell cycle could be related to any of several factors that fluctuate during the cell

notes

425

cycle: (1) DNA synthesis; (2) thymidine kinase activity; and (3) cell surface area. These three possibilities would give rise to different patterns of thymidine uptake in synchronous cultures. (1) If thymidine transport were related to DNA synthesis, it should be practically zero during G 1 (only those asynchronous cells in S would take up thymidine) but should increase rapidly during early S to peak at mid S, and then decline [2]. During S phase, the thymidine taken up by the cell initially goes into the acid-soluble pool where it is rapidly phosphorylated and then incorporated into acidinsoluble DNA. Then, total uptake parallels acid-insoluble incorporation while uptake into the acid-soluble fraction reaches a plateau [l]. (2) If thymidine transport depended on thymidine kinase activity, its rate should be proportional to thymidine kinase activity, i.e., uptake would increase linearly throughout S and plateau in G2 at ten-fold the Gl value [3]. Although phosphorylation of thymidine by thymidine kinase is separate from the transport of thymidine across the membrane, there is evidence that the formation of dTTP may regulate the transport of thymidine [l]. (3) Finally, if transport were merely a function of increased surface area, it should increase almost linearly throughout the cycle, and by a factor of only 1.6 between early Gl and late G2. Materials and Methods CHO cells were removed from a liquid N, freezer every 3-4 months and maintained as stock monolayer cultures in McCoy’s 5a medium with 10% calf and 5 % fetal calf sera in an atmosphere of 6 % CO, at 37°C. Three davs before svnchronization the cells were transferred-to modified McCoy’s, omitting bactopeptone and using dialysed calf and fetal calf sera at the above concentrations (Betten’s special medium). In both media, the sera were heat-inactivated at 55°C for 30 min. Synchronous cultures were obtained by selectively detaching mitotic cells from Blake bottle cultures by shaking every 10 min for 10 set at 37.S”C [2, 41. The Exprl

Cell

Res 96 (1973)