Synchronization of mouse L-P59 cells by centrifugal elutriation separation

Printedin Sweden Copyright@ 1977by AcademicPress,Inc. All rightsof reproductionin anyform reserved ISSN 0014-1827

Experimental

Cell Research 105 (1977) 169-177

SYNCHRONIZATION CENTRIFUGAL

OF MOUSE ELUTRIATION

M. L. MEISTRICH,’

L-P59 CELLS

BY

SEPARATION

R. E. MEYN* and B. BARLOGIE3

Section of Experimental Radiotherapy, 1 Department of Physic? and Department of Developmental Therapeutics,3 The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, TX 77030, USA

SUMMARY Cell seoaration bv centrifueal elutriation was emnloved to obtain svnchronized cells from asynchronokpopulations of r&use L-P59 tibroblastceils. The fractions most enriched in specific .ohases of the cell cvcle contained about 90% Gl-ohase cells. 70% S nhase cells and 60% G2+M phase cells, respeciively, as determined by pulse cytophotometry and autoradiography. When replated, cells from separated fractions divided synchronously in culture. The method is rapid and large numbers of cells (3x 108)can be processed in a single run with no loss of viability. This method should be applicable to most cell lines which can be obtained in single-cell suspension.

Bulk synchronization of mammalian cells, i.e. obtaining large quantities of cells at specific phases of the cell cycle, is an important technique in cell biology. However, there are various drawbacks inherent to conventional methods for cell synchronization. Induced synchrony [l] involves blocking cells at specific phases of the cell cycle by the addition of cytostatic, and often cytotoxic drugs, or by the deprivation of essential nutrients from the medium. These treatments usually alter the biochemical balance within the cells and often perturb the subsequent progression of cells through the cycle after release from the block. In addition, such methods yield cells at only one phase of the cycle. Therefore, provision of cells in distinct phases of the cycle either requires the use of a variety of different methods or harvesting cells at sequential times after their release from the

block. Cell synchrony is gradually lost because of the variation in the kinetic parameters of the cell cycle [2]. Natural synchronization [l] involves selection of cells at a specific phase of the cycle. Mitotic selection is the only method that has gained widespread use [3]. It is based on the tendency of cells grown in monolayer cultures to be easily detached from the culture vessel at mitosis. Although this method yields a highly synchronous population at mitosis, it is also subject to synchrony dispersion with time. In addition, there are many cell lines to which this method is not applicable and the yield of mitotic cells is low. Another method for natural synchronization is based on the fact that cell size increases steadily in an exponential manner with age during the cell cycle [4]. The sedimentation rate of a cell is proportional to Exp Cell Res 105 (1977)

170

Meistrich,

Meyn and Barlogie

the two-thirds power of the cell volume, as well as the shape and density. However, shape has very little effect on sedimentation rate and, except for mitosis [5], density is invariant throughout the cell cycle [6]. Therefore, the sedimentation rate primarily reflects the cell volume and, hence, age in the cycle. Sedimentation rate separation performed by centrifugation through a gradient [7, 81is limited in the number of cells separable and in resolution because of wall effects in the centrifuge tube [9]. The Staput method of velocity sedimentation at unit gravity achieves greater resolution; however, it is time-consuming and requires large gradient volumes when large numbers of cells are necessary [l, 10, 111. Zonal centrifugation has been employed to overcome some of the limitations of these methods [ 121. In this paper we shall describe the use of centrifugal elutriation [13, 141to obtain cell separation based on velocity sedimentation. This separation provides fractionation of asynchronously growing cells into a series of naturally synchronous subpopulations. MATERIALS

AND METHODS

Cell characteristics The cell line used in all experiments was the mouse L-P59 originally described by Hsu & Kellogg 1151. These cells have a 22 h cell cycle time with?hhfojlowing cycle parameters: Gl, 9.1 h; S, 9.9 h; G2, 2.3 h;and M, 0.7 h [16]. Cells were grown in McCoy’s 5A medium (GIBCo) supplemented with 20% fetal calf serum (FCS, GIBCo) at 37°C in a humidified 5 % CO, atmosphere.

Preparation

of cell suspensions

Cells for separation experiments were grown in 32 oz. Brockway prescription bottles and harvested in exponential phase of growth at a density of about 4x IOfi cells/bottle. The cells were removed from the bottles with a solution of 0.025 % trypsin (Worthington) which also contains 20 &ml DNase (deoxyribonuclease I, Sigma) to degrade any free DNA from lysed cells that might cause cell clumping [17]. The trypsin action was stopped by adding an equal volume of medium conExp Cdl Rr.\ IO5 (1977)

taining 5% FCS and DNase. The suspension was examined under a microscope for clumps of cells which were then disruoted bv vieorous oioettine. The cells were pelleted b; low-speed centrifugatioi (150 g for 10 min) and resuspended in 20 ml of medium containing 5% FCS, DNase, and 5 mM 2-naphthol-6,8disulfonic acid (NDA). The NDA further minimizes cell clumping [18].

Cell separation The method of centrifugal elutriation was emoloved . - to separate cells according to their sedimentation rates. The apparatus used, incorporating the Beckman JE-6 elutriator rotor, has been-described previously [18]. When required, the system was sterilized after assembly by pumping 70% ethanol through it the night before the run, keeping the system closed and then pumping ethanol through it again just prior to the run. The entire system was maintained at 4°C. The elutriator medium employed was McCoy’s 5A medium with 5 % FCS and 5 mM NDA. In most experiments the rotor speed was maintained at 1525&10 rpm. The cells were introduced into the chamber at 9:4 ml/min and a total of 70 ml were collected at this flow rate (fraction 1, fl). Then a series of twelve 50 ml fractions (f2-13) were collected by increasing the flow rate of the medium by equal increments up to 25.6 ml/min. Cells remaining in the chamber were washed out after the rotor was stopped (fl4). In some of the earlier experiments the rotor speed was 1750 rpm and flow rates ranging from 12.6 to 34.2 ml/min were used. Equivalent separations were obtained since the sedimentation velocity parameters calculated according to Grabske et al. [19] [(1.93xflow rate)/(rotor speed)2] are identical. The term “sedimentation velocity” as used here is the sedimentation velocity divided -by the gravitational force in the elutriator (in multiples of g, the earth’s gravitational force) and is equal to the-sedimentation velocity at unit gravity. These conditions were selected, based on results of preliminary experiments, to span the range of sedimentation rates of the cells and to subdivide this range into about twelve fractions.

Cell counting and volume analysis Cells were counted on a model ZaI Coulter counter fitted with a 70 pm diameter aperture (84 pm in length). The lower threshold was set to exclude particles with volumes smaller than 340 ym3. The presence of cell clumps and damaged cells was determined by counting in a hemacytometer under a phase contrast microscope. The volume distribution of cells was determined by Coulter volume spectrometry. A multichannel analyzer (Channelyzer II, Coulter Electronics) and plotter were connected to the Coulter counter. The system was calibrated using 18.04 pm diameter latex beads, also supplied by Coulter Electronics. The averaee cell volume for cells in a eiven samole was taken as the modal channel number of the volume distribution. The peak width was estimated using the volume dispersion parameter (4) which is the full

Cell synchronization by centrifugal elutriation r,

IO

6

12 1

14

16

I6

20 I

- 2500

7 2000

- 1500

1000

500

o-

L 2

4

6

6

IO

12

14

0

Fig. I. Abscissa: (bottom) fraction no.; (fop) sedimentation velocity (mm/h/“g”); ordinate: (left) cells/ fraction (X 10e6) (O-O); (right) average cell volume W-P) (- - -1. Separation of exponentially growing L-P59 cells by centrifugal elutriation. The number of cells in each fraction is plotted as a function of fraction number and sedimentation velocity. The average volume of the cells in each fraction and the unseparated cell suspension are calculated from the modal channel number of the Coulter volume distributions. Arrow indicates unseparated cell suspension.

171

One techniaue was that of pulse cytophotometry (identical in pr‘nciple to flow microfluorometry) which yielded the fraction of cells in the Gl, S or G2+M phases of the cell cycle. We used the ICP I I pulse cytophotometer manufactured by Phywe Co., Gottingen, Germany. Cells were stained with mithramycin according to the procedure of Crissman & Tobev f201. The fractions of cells in the Gl. S and G2+M bhases of the cell cycle were calculated according to the evaluation model described by Barlogie etal. [2l]. The fraction of cells in S phase was also determined from the labelling index. About lo6 cells were labelled for 20 min in medium containing 2 &i/ml [“H]TdR (60 Ci/mM, Schwarz-Mann). Autoradiographic techniques have been described previously r??, LLLJ’ The fraction of cells in mitosis (i.e. mitotic index) was scored from the slides prepared for the determination of labelling index. Cell synchrony was also analyzed by following the division of cells as a function of time after plating. Replicate samples of about 3 000 cells were plated into 60 mm culture dishes and incubated at 37°C. Sample dishes were removed at various times, rinsed with saline, fixed for IO min with 95% ethanol and dried. After they were stained with crystal violet (0.5% in 95 % ethanol), they were scored under the microscope bv recording the number of cells/clone in 200 clones. The average number of cells/clone was calculated yielding a multiplicity index which, when plotted as a function of time, provided a measure of cell division.

RESULTS width at half maximum divided by the modal channel number [lo]. The volume dispersion which may be introduced by the Coulter counter was at most 6,,= 0. I4 as determined using latex beads.

Cell viability analysis The integrity of cells could readily be determined by visual examination in a hemacytometer under a phase contrast microscope with results which agreed well with a trypan blue dye exclusion test. Viability (i.e. plating efficiency) was determined by the ability of a single ceil to form a macroscopic colony within IO days after plating in a plastic culture dish. Another test of viability (cell division) was whether a single cell, attached in a culture dish, could undergo at least one division within a time period arbitrarily chosen as the mean cell cycle time multiplied by 1.5 (i.e. 34 h). This cell division was assayed by scoring the number of cells/clone, as described below. The fraction of clones containing two or more cells was calculated. This value yields an upper limit on the viability or proliferative capacity since cells which have lost the capability to attach to the culture dish were not included in the assay.

Determination of cell synchrony Several methods were employed to analyze the degree of synchrony in the various fractions of cells.

Mouse L-P59 cells were used in this study. Single cell suspensions were prepared from exponentially growing monolayer cultures as described in Materials and Methods. These suspensions contained only one clump or pair of cells/200 cells. Greater than 97% of the cells appeared to be intact by phase contrast microscopy; their plating efficiency was about 70%. In each run, 3.5~ 10’ cells were separated by elutriation into 14 fractions and the resulting distribution of cells is shown in fig. 1. On the average, 90% of the cells loaded were recovered in these fractions. Most of the cells sedimented between f3 and f10 corresponding to sedimentation velocities of 9-17 mm/h/g. The average cell volume increased steadily with increasing fraction number and sedimentation rate (fig. 1). This result demonstrated that the separation was E.vp Cd Rrs 105 (1977)

172

Meistrich, Meyn and Barlogie 500

1000

1500

2000

2500

--I

00 Fig. 2. Abscissa: (botrom) channel no.; (zoo) cell vol (pm3); ordinate: cells/channel.

Coulter volume distributions of L-P59 cells in (a) an unseparated cell suspension from an exponerkally growing culture; (b) three fractions separated from that suspension by centrifugal elutriation (-- -, f2; -, f-8; ..‘, f13). The output of the multichannel analyzer is plotted vs channel number and volume.

actually based on cell size. The ratio of the modal volume of cells in f13 to that in f2 was 2.0, which is the expected volume increase for cells traversing the cell cycle. Fractions 1 and 14 were collected, but were not used in any further experiments. Fraction 1 contained subcellular debris and most of the non-intact cells which were present in the original preparation. Fraction 14 contained both very large cells and smaller cells which were washed out of the rotor at the end of the run. The volume distribution of cells in the unseparated cell suspension and in three separated fractions is presented in fig. 2. The unseparated cell suspension showed a broad distribution skewed towards larger volumes. In this case the volume dispersion parameter (full width at half-maximum divided by the modal channel number) was measured to be 6,=0.67. The separated fractions showed nearly symmetrical distriExp CeIIRcsl05 (1977)

butions with S, ranging from 0.32 (f2) to 0.37 (f13). The cell volume distributions of f2 and f13 had almost no overlap. This result demonstrated that cells which differ in volume by a factor of two can be almost completely separated by elutriation. Cells of intermediate size (f8) overlapped appreciably in terms of their cell volume distributions with those of fractions containing the smallest and largest cells. This separation on the basis of cell volume actually represented separation on the basis of position of the cell within the cycle. The DNA histograms of an asynchronous population of cells as well as of cells from particular fractions are shown in fig. 3. The percentages of Gl , S and G2+M cells calculated from these histograms are also shown. Fractions 2 and 3 typically contained about 90% Gl phase cells with a small contamination of S phase cells and essentially no G2+M phase cells. Fraction 6 contained Gl phase cells and S phase cells predominantly in the early part of S phase. Fraction 8 was the most enriched in S phase cells (over 70%) with the distribution skewed towards the latter half of the S phase. The fractions most enriched in G2+M cells were f12 and f13 with most of the contamination resulting from late S phase cells. The labelling indices and mitotic indices of the cells from individual fractions were determined (fig. 4). These results indicated that the highest percentage of labelled cells were found in B-fl0. The agreement between autoradiography and pulse cytophotometry indicates that DNA histograms provide a reliable estimate of the percentages of cells in different phases of the cell cycle in the enriched Gl phase and S phase fractions. However, in the later fractions there was a discrepancy between the percentages of S phase cells as determined

b L

L

2

Oii

406060

Fig. 3. Abscissa: channel no. (fluorescence intensity, DNA content); ordinate: counts/ channel (rel. no.). DNA histograms obtained by pulse cytophotometry of (a) an unseparated cell suspension of L-P59 cells (Gl, 36%; S, 54%; G2M, 10%); and (f+j) various fractions of cells separated from that suspension by centrifugal elutriation. (b) f2 (Gl, 90%; S, 10%; G2M, 0%; (c) f6 (Gl, 38%; S, 53%; G2M, 9%); (d) f8 (Gl, 17%; S, 72%;G2M,ll%);(e)flO(Gl,7%;S,34%; G2M, 59%); v) f12 (Gl, 3%; S, 16%; G2M, 81%). The fractions of cells in Gl, S, and G2+M phases, calculated from the histograms, are also presented.

I

e

6 4 P I I

I VA

173

Cell synchronization by centrifugal elutriation

I

60h -%!!!L 20

406080

by labelling index and by pulse cytophotometry. Labelling index showed about 40% of the cells in these fractions to be in S phase, while the DNA histograms indicated only about 20%. We attributed this discrepancy to the presence of very late S phase cells which are difficult to resolve from the G2 peak in the DNA histograms. We considered labelling index to be a more reliable indicator of the percentage of cells in S phase. Therefore, in the G2+M phase enriched fractions, the percentage of S phase cells as determined by autoradiography and the percentage of G 1 phase cells as determined by pulse cytophotometry were subtracted from 100%. The remaining percentage, typically 60 %, was considered to be in the G2+M phase. The ability of separated cells to divide synchronously when placed back into culture was determined by measuring the multiplicity index, or average number of cells per clone, at various times after plating (fig. 5). The unseparated control cell suspension, which was held in the elutriation medium for 2 h prior to replating, showed a “lag” period of 6 h in which no cell division occurred. This was followed by a period of

exponential growth except for an additional “lag” 25 h after replating. This pattern was reproducible and also occurred with cell populations which were merely trypsinized and replated immediately without exposure to low temperatures or to the elutriation medium. We interpreted these results as indicating that after trypsinization and replating, the population of cells may no longer be asynchronous, but that some cells, particularly those in late S and G2, may be preferentially delayed in their progression to division. 100 6 80

/...____< 2

4

6

8

IO

Fig. 4. Abscissa: fraction no.; ordinate: (--); (right) MI (%) (. . .).

12

14

(kff)

Ll (%)

Labelling indices (LI) and mitotic indices (Ml) of L-P59 cells in each fraction from an elutriator separation and in the corresponding unseparated cell suspension. Arrows indicate unseparated cell suspension. Exp Cd Res 105 (1977)

174

4 I

Meistrich, Meyn and Barlogie

b

3

2

0

IO

..:. ..A 20

30

40

50

Fig. 5. Abscissa: time after plating (hours); ordinate: average no. of cells/clone. Average number of cells/clone at various times after plating the cells from (a) an unseparated cell suspension (0-O) held at 4°C in elutriator medium for 2 h; (b) fractions of cells separated by centrifugal elutriation (m-B, f12 (G2M); 0 ... 0, B (S); A---A, f2 631).

The three separated fractions of cells (fig. 5 b) showed growth characteristics distinctly different from the unseparated control suspension. The Gl phase population (f2) showed no cell division for 18 h; 66% of the cells divided between 18 and 27 h; 87 % of the cells had divided by 34 h. Between 34 and 44 h only 6% of the cells passed through their second division. The cells entered a second wave of division at 44 h and 43 % of the cells had divided again by 52 h. Cells from f8, which was enriched in S phase cells, showed negligible division for 8 h but then divided between 8 and 21 h. No cell division occurred between 21 and 28 h and then division resumed as cells passed through their second mitosis. The synchrony of cell division of this population, as measured by the maximum slope of the curve, was not as great as f2. Exp Cell RPS 105 (1977)

Cells from f12, which was enriched in late S and G2+M phase cells, showed little cell division for 6 h after replating, but 66% of the cells divided by 14 h as opposed to 34 % in the unseparated control suspension. A second wave of division began at about 26 h. We have determined the viability of the cells separated by elutriation in two ways. In the first method, which may also be used with cells which do not form colonies, viability was indicated by the fraction of cells which went through their first cell division within 1.5 cell cycle times (table 1). Most cells from all fractions passed through at least one cell division within 34 h with no delay induced by the elutriation process. The second method involved the use of plating efficiency. Table 1 shows that maintaining cells in elutriation medium in the cold has no effect on plating efficiency. Also, no reduction in plating efficiency was seen with the fractions of cells enriched in Gl and S phase cells. The G2+M cells showed a slight reduction in plating efficiency. This difference in plating efficiency between Gl and S vs G2+M cells was confirmed in two subsequent experiments. We could not distinguish whether the lower Table 1. Viability of cells after separation by centrifugal elutriation Fraction of cells undergoing Plating efficiency division (9%) m) Control (trypsinized and replated) Control (held in elutriation medium at 4°C for 2 h) Separated fractions Fraction 2 (G 1) Fraction 8 (S) Fraction 12 (G2+M)

93

70

88

77

;: 90

:i 61

Cell synchronization by centrifugal elutriation plating efficiency was a result of trypsinization and replating of the G2+M cells or whether the elutriation process had an effect on these cells. To analyse the influence of cell load on the separation, we varied the cell load by two orders of magnitude. When 3.6~ lo6 cells were run, the distribution of cells as a function of fraction number was indistinguishable from that shown in fig. 1. When 3.0X 10s cells, with a total cell volume of 0.5 ml, were separated, the distribution of cells was shifted towards slower sedimentation rates as expected from previous studies with large numbers of testicular cells [18]. Even though a broader range of flow rates (at 1520 r-pm) was used (i.e., 6.8 to 25.6 ml/mm) in order to include slower sedimentation rates, cells were found predominantly in the lower numbered fractions. Nevertheless, fractions were obtained which contained 86 % Gl phase cells, 65% S phase cells and 77% late S+G2+M phase cells, respectively, as determined by pulse cytophotometric analysis. DISCUSSION We have used the method of centrifugal elutriation, which takes advantage of the fact that cells increase in size through the cell cycle [4], to separate cells into fractions enriched at specific phases. Several factors may limit the purity of the separated fractions. The volume dispersion of cells at a particular age in the cell cycle is sufficiently great that even if one were able to select cells of a precisely defined volume, they would not be at precisely the same age. Anderson et al. [4] demonstrated that Chinese hamster ovary cells, even when selected at mitosis, have a volume dispersion with a S, of 0.42. Another factor is the inability of the elutriator to achieve a per12-771814

175

feet volume separation. However, the volume dispersion factors obtained with the elutriator are slightly better than those obtained with the Staput [lo]. Density variations of cells are too small to account for the dispersion in the volumes of individual fractions [lo]. Cell separation by elutriation can be used to obtain synchronized populations of cells in two ways. Firstly, the various fractions of cells can be used directly. Fraction 2 is most enriched in Gl cells and f13 in G2 cells. The intermediate fractions are enriched in cells in “compartments”of the cell cycle 1yingbetweenearlyG ltolateG2 (fig. 3). These cells can be directly analysed for cellular parameters which vary through the cycle. Elutriation has the advantage of providing cells at all phases of the cell cycle simultaneously which greatly facilitates the processing of samples for further analysis. Sufficient quantities of cells can be obtained for biochemical experiments. Analysis of drug and radiation sensitivity of the cells in the different fractions has been performed [23]. These experiments show a ten-fold difference in radiosensitivity of the cells from the different fractions confirming their relative homogeneity. The second way in which this method can be used for synchronization is to replate the most highly purified fraction of cells, the Gl fraction. Then, samples removed at various times after replating would be synchronized in subsequent stages of the cell cycle. The selection of Gl cells by this technique is comparable to the method of mitotic shake-off. Yen et al. [24] have recently shown that cells which are small at the time of mitosis had a longer cell cycle than cells which are large at mitosis. Therefore, it is possible that our method of selecting cells on the basis of volume might yield a more synchronous population of cells than miE.rpCdlR6,J 10.5 (1977)

176

Meistrich, Meyn and Barlogie

totic selection. The fact that the 13,values shown that fewer cell clumps are present in for Chinese hamster ovary cells obtained by trypsinized monolayer cultures than in elutriation [25] are smaller than those ob- suspension cultures. However, the prestained by mitotic selection, supports this ence of clumped cells is not a problem when possibility. The sharpness of the wave of only selecting Gl or S phase cells, because cell division of separated Gl cells (fig. 56) the clumps sediment more rapidly [25]. Synchronization by elutriation can be provides evidence of the synchrony obapplied to cells from established lines in cultained. The procedure of centrifugal elutriation ture, primary cultures, normal tissues and has several major advantages over conven- tumors and, therefore, has a wider range of tional methods of cell synchrony. Any utility than conventional synchronization method of natural synchronization is pre- methods. The present study has utilized ferable to induced synchrony because cells mouse L-P59 cells, a cell line which had are not deliberately perturbed by the syn- been difficult to synchronize by convenchronization procedure. Since no gradient tional methods [26]. In addition, we have is required, the cells may be kept in their applied this synchronization method to usual culture medium. Centrifugal elutria- Chinese hamster ovary cells and human tion is a rapid method. Separations are com- lymphoma Tl cells grown in monolayer pleted within 50 min. This time may be culture. Grabske et al. [27] has reported further reduced, if desired, by increasing separation of Gl phase Chinese hamster rotor speed and flow rates or by collecting ovary cells grown in suspension culture by fewer fractions. If only G I cells are desired, elutriation. We have recently achieved the separation may be completed within separation on the basis of cell cycle position of mouse L-P59 cells and fibrosarcoma cells 20 min. Synchronization by centrifugal elutriation grown as solid tumors in vivo [28]. In the should be dependent simply upon the cells tumor studies, elutriation also achieves a having a homogenous size at a specitic partial separation of the smaller, more point in the cell cycle and uniformly in- slowly sedimenting normal cells within the creasing their size during the cell cycle. tumor mass from the tumor cells. One limiThis property should be valid for many cell tation of velocity sedimentation separation types. Hence, synchronization by centri- of tumor cells is that such a heterogeneous fugal elutriation should be applicable to any population contains cells of different densuch cell type which can be obtained in sities [29]. This limitation may be overcome by selecting cells banding at homogenous single-cell suspension. The latter limitation may require new methods for preparing densities and then subjecting them to secells from tissues as well as cells which tend paration by centrifugal elutriation. The fractionation of L-P59 cells by centrito form tight junctions in culture. Comparison of our experience with different fugal elutriation provides subpopulations in cells grown in monolayer culture has Gl and G2+M, the purity of which comshown that the frequency of clumped cells pares favorably with other methods of nain the cell suspension is 0.5% with L-P59 tural and induced synchronization. In parcells, but varies between 1% and 20% in ticular, the synchrony of the Gl-enriched different experiments with Chinese hamster sample is as good as that obtained by plating ovary cells [2.5]. Other workers [4] have cells selected at mitosis. The synchrony of E.I/I Cdl Rc,.\ 105 (IY77)

Cell synchronization by centrifugal elutriation the late S+G2+M enriched sample is also as good as can be obtained by other methods, with the exception of the use of agents which generally are highly cytotoxic to block cells at the S-G2 boundary. In conclusion, the speed, simplicity and general applicability of elutriation separation makes it particularly attractive for preparative scale cell synchronization. We thank Patricia Trostle and Susan Reed for expert technical assistance. This work was supported by grants CA-04484, CA-17364 and CA-06294 from the NC1 of NIH.

REFERENCES 1. Whitmore, G F, In vitro 6 (1971) 276. 2. Anderson, E C & Petersen, D F, Exp cell res 36 (1964) 423. 3. Terasima, T & Tolmach, L J, Exp cell res 43 (1963) 424. 4. Anderson, E C, Bell, G I, Petersen, D F & Tobey, R A, Biophys j 9 (1969) 246. 5. Wolff, D A & Pertoft, H, J cell biol55 (1972) 579. 6. Anderson, E C, Petersen, D F & Tobey, R A, Bionhvs i 10 (1970) 630. 7. Sinclair,“R & Bishop, D H L, Nature 205 (1965) 1212. 8. Morris, N R, Cramer, J W & Reno, D, Exp cell res 48 (1967) 216. 9. Boone, C W, Harrell, G S & Bond, H E, J cell biol 36 (1968) 369. 10. MacDonald, H R & Miller, R G, Biophys j 10 (1970) 834. 11. Shall, S, Methods in cell biology (ed D M Prescott) vol. 7, p. 269. Academic Press, New York (1973).

177

12. Probst. H & Maisenbacher. J. Exu cell res 78 (1973) 335. 13. Lindahl. P E. Nature 161 (1948) 648. 14. Glick, D, von Redlich, D,‘Juhos, E T & McEwen, C R, Exp cell res 65 (1971) 23. 15. Hsu, T C & Kellogg, D S, J natl cancer inst 24 (1960) 1067. 16. Dewey, W C & Humphrey, R M, Radiat res 16 (1960) 503. 17. Meistrich, M L, J cell physiol 80 (1972) 299. 18. - Methods in cell biology (ed D M Prescott) vol. 15. Academic Press, New York (1976). In press. 19. Grabske, R J, Lake, S, Gledhill. B L & Meistrich, M L, J cell physiol 86 (1975) 177. 20. Crissman, H A B Tobey, R A, Science 184 (1974) 1297. 21. Barlogie, B, Drewinko, B, Johnston, D A, Biichner, T, Hauss, W H & Freireich, E .I, Cancer res 36 (1976) 1176. 22. Barranco, SC & Humphrey, R M, Mutation res 11 (1971) 421. 23. Meyn, R E, Trostle, P K, Reed, S J & Meistrich, M L, Radiat res (1976). In press. 24. Yen, A, Fried, J, Kitahara, T, Strife, A & Clarkson, B D, Exp cell res 95 (1975) 295. 25. Meistrich, M-L, Grdina, D J & Meyn, R E, Growth kinetics and biochemical regulation of normal and malignant cells. Williams & Wilkins, Baltimore, Md (1976). In press. 26. Thompson, L H & Humphrey, R M, Int j radiat biol 15 (1969) 181. 27. Grabske, R J, Lindl, PA, Thompson, L H &Gray, J, J cell biol 67 (1975) 142a. 28. Grdina, D J, Lee, L Y, Hittleman. W & Meistrich, M L. In preparation. 29. Grdina, D J, Basic, I, Mason. K A &Withers, H R, Radiat res 63 (1975) 483. Received June 2 1, 1976 Accepted August 19. 1976

Erp Cdl Rrs 105 (1977)