Changes in patterns of protein synthesis during the mammalian cell cycle

Experimental

Cell Research 56 (1969) 117-121

CHANGES IN PATTERNS OF PROTEIN SYNTHESIS DURING CELL CYCLE G. M. KOLODNY

THE MAMMALIAN

and P. R. GROSS

Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139, USA

SUMMARY The pattern of protein synthesis during the G2 period of interphase in HeLa cells has been compared with the pattern of synthesis during an entire cell cycle in exponential growth. The patterns, as revealed by co-electrophoresis of the two populations of radioactive proteins on acrylamide gels, are very different. The differences are not due to selective or discontinuous degradation of proteins made radioactive during long or short exposures to tracer amino acids, nor do they arise from errors in the method of separation and counting. Similar differences are found between other phases of the cell cycle and the cycle as a whole, and between phases studied in pairs. Thus the reproductive cycle of eukaryotic cells not only involves translational shifts, an involvement already known to exist in consequence of studies in individual proteins, but it involves translational shifts of a magnitude sufficient for detection in bulk separation of the soluble proteins.

Synchronized cultures of microorganisms show periodic increases in the activity of various enzymes. The synthetic bursts represented by such increases have been related to sequences of transcription [4, 6, 91. Eukaryotic cells of higher organisms also show changes in enzyme activities during the generative cycle. Lactate dehydrogenase and glucose-6-phosphate dehydrogenase increase intermittently through the cell cycle in cultured Chinese hamster cells, for example [7], suggesting that synthesis is itself intermittent, or that complex cycles of synthesis and degradation are the source of variation in activity. Histones of HeLa cells are synthesized in the cytoplasm, and the synthesis is coincident with the S phase, i.e., with DNA replication [lo]. In this case, the evidence favors control of protein synthesis via precisely timed transcription of the appropriate messenger RNA [l]. Progress through the cycle of cellular reproduction is generally assumed to be associated with altered patterns of translation. In order to understand the mechanisms by

which these alterations are regulated, we shall have to know how widespread, among the very large populations of proteins found in higher cells, are the shifts in synthesis pattern. Our experiments were done in order to determine whether translational shifts during a mammalian cell cycle are of sufficient magnitude to be detected in bulk separations of the soluble proteins, rather than by assay of individual species. We discuss here. data that do reveal large-scale differences in synthesis pattern between phase G2 (from the end of DNA synthesis to the beginning of karyokinesis) and the whole cell cycle. The same techniques have been applied to other phases of the cycle, and yield similar results, hence only the G2 studies will be described in detail. The essential experimental design was as follows: Proteins were labeled with 3H-leucine or 14C-leucine during G2 and during the whole cycle : cells containing these molecules were lysed and their soluble proteins separated by acrylamide gel electrophoresis. Distributions of radioactivity referable to each isotope were Exptl Cell Res 56

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G. M. Kolodny & P. R. Gross

determined over the length of the gel, and these distributions were analyzed in such a way as to reveal significant differences between them.

obtained. The mean value of all such ratios was then determined for each gel. A normalized ratio, e, was then

obtained for each fraction by:

(dpm H3/dpm C14h ” = (dpm H3/dpm C14)8vepBge

METHODS HeLa cells (S-3) with a 22-h intermitotic time were maintained in spinner cultures at 37°C in Joklik’s modification of SMEM, with 10 % horse serum added. Monolayer cultures were prepared by transferring 40 ml of spinner culture (5 xlOi cells/ml) to 32-oz.-flint glass bottles (Brockway Glass Co.). All exueriments were carried out upon populations of cells-in exponential growth. For extraction of proteins, cells were collected as described below and resuspendedin a hypotonic buffer containing 0.01 M NaCI. 0.01 M tris (hvdroxvmethvl) aminomechane at pH 7.4, and 0.0015 &I- MgCI,. Lysis was accelerated by several passages of a Dounce homogenizer pestle. Centrifugation of mitotic cell lysates at 10,000 g for 10 min yielded no discernible precipitate. The “supernatant” solution was carefully collected and recentrifuged, this time at 105,000 g. The 105,000 g supernatants were dialysed for 16 h against electrophoresis buffer prepared in 8 M urea, and the contents of the dialysis sacs were used as samples for electrophoresis. All manipulations beginning with harvesting of the cells were performed at C#C. Procedures for electrophoresis were essentially as described by Davis [2], except that solutions were made up in 8 M urea instead of water. This insured that all proteins in the very complex mixtures would remain in subunit form, and it also prevented aggregation during dialysis and elctrophoretic separation. The buffer system was tris-glycine at pH 8.3. Running gels 85 mm long, containing 5 % acrylamide, were cast with 2.5 % spacer gels on top. Diameters were 6 mm. Samples were layered onto the spacer gels with the aid of a string dauber and pipette. Electrophoretic migration was allowed to proceed at 4°C for 4 h at 2 mA/sample. The gels were removed by the conventional rimming procedure, injecting water from a 5 cc syringe through a clean needle. Some were stained with amido black in 7 % acetic acid and then destained in the acid alone. These gels all showed identical band patterns, which were extremely complex and contained much hyperfine band structure. Duplicate unstained gels were fixed overnight in 7 % acetic acid and then sliced transversely, after freezing, at 0.5 mm thickness. The slices were cut with a precision tissue slicer, modified for such use by Loening [8]. Following a method devised in this laboratory by Terman [14], slices were placed (in adjacent pairs for this work) into scintillation vials, and the proteins were brought into solution with the aid of 0.3 ml NCS (a product of Nuclear-Chicago Corp.) by shaking at 37°C for 12-20 h. Fifteen ml of toluene-based scintillation fluid were then added to each vial. The scintillant mixture contained 4 g/l PPO and 0.5 g/l POPOP. Liquid scintillation spectrometry was done on a Beckman LS-250 Scintillation Counter, with automatic quench correction to compensate for possible slight inhomogeneities in gel slice thickness and in gel solubilization. All samples were counted to 1 % error or less, and the data pFesented are corrected for background and for quenching in each fraction. The number of disintegrations/min (dpm) was computed for each isotope in each gel fraction, and for each fraction a ratio of the two isotopic radioactivities was Exptl

Cell

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Values of these normalized isotope ratios are plotted as a function of fraction number (which is proportional to electrophoretic mobility). For an ideal mixture of two protein populations, labeled with SH-leucine and 14Cleucine, respectively, but otherwise identical, the value of Q should everywhere be unity. Significant deviations from this value indicate differences in the two groups of labeled proteins. The basic design for experiments on stage-dependent changes in protein synthesis pattern was to obtain distributions of 0 for proteins labeled briefly during a particular cycle stage with 3H-leucine relative to proteins labeled during the entire cycle with 14C-leucine, all in the same cell population. This procedure circumvents possible differences in protein extractability between cells collected at different stages of the cycle. Since the cultures were growing exponentially, proteins labeled during an entire cycle could be assumed to represent all possible proteins soluble under the extraction conditions employed. Where necessary, the variances in Q can be computed and the significance of differences between variances for different gels can be. tested by ordinary means (e.g., Snedecor’s F-test). ‘*C-leucine (250 me/m mole) and 3H-leucine (5 c/m mole) were from the New England Nuclear Corp.

RESULTS Control experiments

Three kinds of control are needed to establish the validity of this method. (a) Variances in Q must be small when two identical protein populations, differing only as to isotopic label, are mixed and processed as described. (b) Breakdown of radioactive proteins, accumulated during a 24-h period, must be small during a subsequent 24-h period of exponential growth, and there must not be discontinuities in the decay curve. (c) Breakdown of proteins labeled during a short exposure to tracer, e.g., 2 h, must be small and not show a discontinuous pattern of degradation (or “turnover”) during the succeeding few hours. The results of experiments performed to establish these control conditions are shown in figs 1, 2, and 3. Media were gently discarded from 2 12-h monolayer cultures. Forty ml of Ca2+-leucinedeficient medium with 10% horse serum were then added to each. (Horse serum contains 2 mg/l leucine, which is is the concentration in

Protein synthesis during the cell cycle

SMEM [15).] One culture received in addition 2 PC/ml 14C-leucine while the other received 10 PC/ml “H-leucine. Both cultures were incubated for 2 h thereafter. All cells were collected by scraping and washing from the bottles. The cell mixture was processed as described, yielding a mixture of two populations of proteins identical except for 3H in half of it and 14C in the other half. In fig. 1 we plot the normalized dpm ratios for a gel upon which these proteins were separated. This is a typical result, and it is evident from it that the absolute variance introduced by all experimental errors is quite small. Spinner cultures (3 X lo5 cells/ml) were given 0.03 PC/ml 14C-leucine under conditions that allowed continuous incorporation of the tracer amino acid for 24 h. The cultures were then centrifuged and the cells were washed twice with normal medium. They were then resuspended and allowed to continue growth for another day. Cell counts showed that growth was normal. At intervals, 10 ml samples were taken. Proteins were precipitated and processed [ 121for scintillation counting of their radioactivity. All samples had more than 10,000 cpm and were counted to 0.5 % error or less. The activities per sample, I .4 1.3 1.2

l.I I.0L#dbeeJe 1 I-

0.90 0.80

I 0.70 0.60

i I IO

I 20

I 30

I 40

I 50

I 60

, 70

I 60

Abscissa: Fraction number; ordinate: e. Fig. 1. Values of the normalized isotope ratio, e (see text), as a function of fraction number for an acrylamide gel on which a mixture of proteins labeled with 3H-leucine and W-leucine was senarated. Electroohoretic miaration anodal (to the right). Cells growing exponentiilly in culture were labeled with 2 &ml r4C-leucine for 2 h. These were mixed with an equal number of cells from another culture labeled similarly, but with 3H-leucine at 10 &ml. Soluble proteins fom the mixture were separated by electrophoresis at 2 mA for 4 h. Deviations of e from unity represent errors in the method.

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60t

Abscissa: Time, h; ordinate: % of initial activity. Fig. 2. Degradation during a 24-h period of proteins labeled and accumulated during a prior 24-h period in culture. Spinner cultures (two separate runs, indicated by circles and squares, respectively) were exposed to 0.03 PC/ml r4C-leucine for 24 h. After replacement of the medium with fresh medium lacking tracer leucine, 10 ml aliquots were taken at intervals for assay of protein radioactivity. Growth continued logarithmic during this “chase” period. Least squares fit of the data points (not plotted) gives slope corresponding to loss of proteins at 0.75 %/h.

referred to the initial value, are plotted in fig. 2. If there were a large-scale and differential breakdown of proteins in any phase of the cell cycle, this would be revealed by a sharp early decline of counts in a plot like that of fig. 2 (which combines data from two separate experiments). There is no such decline. Instead, the decay is steady (within limits of precision of the method) and, from a least-squares analysis of the data, at a rate of less than 0.75 %/h. Because some cells certainly die of trauma induced by the stirring and other mechanical features of culture conditions, the true value may be smaller. The value obtained is lower than that observed by different methods in the laboratory of Eagle [3], but similar to that found elsewhere for mouse fibroblasts [16]. The breakdown or turnover revealed by “chase” experiments like these is too small to produce Q variances in stage comparisons that are significantly higher than those seen in controls like that represented in fig. 1. The final control concerns the possibility of large-scale turnover or degradation of proteins synthesized during a short exposure to tracer amino acid. An experiment to test this possibility is represented by fig. 3. The procedure was like that of the 24-h labeling control, except that Exptl Cell Res 56

120 G. M. Kolodny & P. R. Gross

40 r 20 0 il

20

! 40

I 60

I 80

I 100

I 120 +dm

Abscissa: Time, m; ordinate: % of initial activity. Fig. 3. Results of an experiment like that of fig. 2, except labeling of proteins was for 2 h with 0.16 ,uc/ml Wleucine. Plotted are percentages of initial radioactivity per 10 ml culture in acid-precipitable proteins. Result shows that decay of radioactive proteins accumulated during a short exposure to tracer is not larger than expetted from the decay in uniformly labeled cultures (fig. 2).

exposure to 14C-leucine was for 2 h only, followed by a long exposure to normal unlabeled medium. There is no significant decline of counts during the first 2 h following the pulse beyond that expected from the behavior of labeled proteins accumulated during an entire cell cycle. Again, such levels of protein breakdown or turnover would not be expected to produce large variances in e in the course of experiments designed to test for differences in labeling pattern between a particular short phase of the cycle and the cycle as a whole. Stage comparison

A typical comparison experiment is represented by the data in fig. 4. Monolayer cultures were prepared as described. Fourteen hours later, the media (including dead cells and debris) were gently discarded and each bottle received 40 ml fresh medium with +,th the normal concentration of leucine and 3 ,uc of 14C-leucine/ml. Incorporation of label into proteins was allowed to continue for 24 h. The medium was then discarded and there was substituted for it fresh leucinefree medium containing 60 PC/ml 3H-leucine. Two hours later, cells in mitosis were harvested from all bottles by gently rocking each one about 20 times [I 11. Of the cells so collected, 75 % or more showed mitotic figures under the microExptI Cell Res 56

!:1 \’ l.ni’ / I

.60 -

I I I I I I IO 20 30 40 50 60 70 80 Abscissa: Fraction number; ordinate: Q. Fig. 4. Plot of Q vs. fraction number for a gel upon which were separated proteins in a mixture containing G2labeled proteins (2 h, 60 &ml *H-leucine) and uniformly labeled proteins (24 h, 3 ,uc/ml ‘“C-leucine) from the same cells. See text for description of cuiture and selection techniques. Electrophoresis as in fig. 1. The deviations of @from unity are large and distributed over the entire range of electrophoretic mobilities. Large variance in values of @ for the set indicates that the pattern of translation in G2 is different from that carried out through the reproductive cycle as a whole.

scope. These cells had therefore received their exposure to 3H-leucine during the G2 phase (immediately preceding mitosis), while their 14C-containing proteins ’ had been accumulated during an entire cell cycle, and could be considered to represent the entire extractable population (vide supra). There are, as shown in fig. 4, very large fluctuations in e over the entire mobility spectrum. No such Q fluctuations are ever seen in controls (e.g., fig. l), and the variance of gels like the one in fig. 4 is significantly higher than that of control gels. The probability for the null hypothesis in their variance ratios (i.e., that the variance of the Q population in fig. 4 is not significantly greater than the Q variance for the gel of fig. 1) is far below 0.001. CONCLUSIONS The pattern of protein synthesis in G2 is different from that for the whole cell cycle, and no significant part of the difference is attributable

Protein synthesis during the cell cycle

to differential degradation. Experimental design eliminates differential protein extraction as the source of the observed variation in e. The pattern of protein synthesis in this phase must therefore differ from the pattern in other phases, and the differences are sufficiently large to be detectable in bulk protein populations. Experiments like this one have been done for proteins labeled in other phases, compared with 24-h labeled proteins, and for phases compared with each other. They have also been done with proteins extracted in sodium dodecyl sulfate (SDS) and separated on detergent-containing gels [13]. The latter method has the advantage of bringing all proteins into solution and hence *obviating, in certain types of experiments (not the ones discussed here), selective extraction errors. Gel-electrophoretic separations in SDS are, however, inferior to those obtained by the method used, since separation of detergenttreated proteins is determined not by charge and molecular volume, but by molecular volume alone. All of these experiments have shown however, like the one whose result is given in fig. 4, that the pattern of protein synthesis changes from one stage of the cell cycle to another. In fact, there are significant differences in labeling pattern between proteins synthesized in early and late Gl, and between proteins synthesized just before and just after release of contact inhibition in neonatal rat heart cells (Kolodny & Gross, results in preparation for the press). The differences in radioactivity pattern could arise because different proteins are being synthesized at different stages. Data mentioned in the introduction to this paper show that such differences certainly play a part. Variations could equally well arise, however, from altered rates of synthesis among a large and constant set of proteins, and in fact this too must play a not insignificant role, since synthesis of particular

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proteins is not likely to be turned on and off discontinuously, i.e., in square pulses. Electrophoretic patterns like the ones that generate the distributions shown are produced by (at least) hundreds of proteins. It is unlikely that any peak of radioactivity (or of stain density in stained gels) contains a single, or even a few, polypeptide species. Thus we can offer a conclusion only about the whole pattern. But that conclusion can be firm: the pattern changes from one stage of the cellular reproductive cycle to another. If it is true, as has been argued elsewhere [5], that cells become “differentiated” with respect to one another as a result of changing patterns of protein synthesis, then orderly progress through the cell cycle, even during exponential growth, requires the same fundamental processes. G. M. K. is an Advanced Academic Fellow of the James Picker Foundation. These studies were supported by grants from the National Institutes of Health (GM13560-03, l-SO%PRO7047-01), the National Science Foundation (GB-5760), and the American Cancer Society (E-285).

REFERENCES 1. Borun, T W, Scharff, M D & Robbins, E, Proc natl acad sci 58 (1967) 1977. 2. Davis, B 3, Ann NY acad sci 121 (1964) 404. 3. Eagle, H, Piez, K A, Fleischman, R & Oyama, V I, biol them 234 (1959) 592. 4. Gorman, J, Taruo, P, Laberge, M & Halvorson, H, Biochem biophys res comm 15 (1964) 43. Gross, P R, Ann rev biochem 37 (1968) 631. 2: Kates, J R & Jones, R F, Biochim biophys acta 145 (1967) 153. 7. Klevecz, R R & Ruddle, F H, Science 159 (1968) 634. a. Loening, U E, Biochem j 102 (1967) 251. 9. Masters, M, Kuempel, P L & Pardee, A B, Biochim biophys res comm 15 (1964) 38. 10. Robbins, E & Borun, T W, Proc natl acad sci 57 (1967) 409. 11. Robbins, E & Marcus, P I, Science 144 (1964) 1152. 12. Siekevitz, P, J biol them 195 (1952) 549. 13. Summers, D F, Maizell, J V & Darnell, J E, Biochemistry 54 (1965) 505. 14. Terman, S A, Ph.D. thesis. In preparation. 15. Westfall, B B, Peppers, E V, Sanford, K K & Earle, W R, J natl cancer inst 15 (1954) 27. 16. Zetterberg, A, Exptl cell res 43 (1966) 526. Received November 14, 1965

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