Reversible retention of synchronized HeLa cells in G2 of the mammalian cell cycle

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RETENTION OF SYNCHRONIZED HeLa CELLS IN G2 OF THE MAMMALIAN CELL CYCLE

M. S. Inglis and D. N. Wheatley* Cellular Pathology Laboratory, Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen AB9 2ZD, Scotland ABSTRACT The rate of progression of cycling HeLa cells from G2 into mitosis is greatly reduced by the amino acid analogue, p-fluorophenylalanine, but a small percentage of cells continually escapes into mitosis. Most of a presynchronized population can be held for several h in G2, thereby They are facilitating analyses of cells at this late stage of the cell cycle. reversed from the block by the addition of phenylalanine and resume progression within about 1 h with predictable kinetics but without a significantly enhanced synchrony. The mechanism of action of the analogue is consistent with the hypothesis that synthesis of false proteins interferes with the correct assembly of division-relevant structures. INTRODUCTION The amino acid analogue @luorophenylalanine QFPhe) substitutes for phenylalanine (Phe) in protein when given in a Phe-deficient medium or made available at a 5-10 fold greater concentration than Phe (Richmond, 1962). Cycling cells show a tendency to accumulate in late S phase when the QFPhe is given during the early part of S phase (Mueller and Kajiwara, 1966), or in G2 when given later (Wheatley and Henderson, 1974; 19752). In studies relating specifically to G2, the analogue stops cells entering mitosis within 30 min of addition; this effect is dependent on incorporation of the analogue into protein. It can be explained by cells synthesizing relatively thermolabile analogue proteins which interfere with the utilisation or development of assemblies of ‘division proteins’ (Wheatley and Henderson, 1974; 1975l$. This is emphasised by the fact that analogue proteins synthesized after the transition point i. e. the time when further protein synthesis is no longer required for cells to move from G2 into mitosis - still produce a marked inhibition of progression. +

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The exact nature of G;! arrest with EFPhe has never been adequately described, which is one objective of the present communication. The other is to show how the analogue combined with presynchronizing procedures provides a method of holding cells at a very late stage of the cell cycle. This facilitates a more detailed and leisurely analysis of the final events of the cell cycle, but also allows one to recover normal cell cycle kinetics simply by restoring Phe. It has been successfully applied, at appropriate dose levels and temperatures, to other cellular systems as has already been documented in E. coli by Smith and Pardee (19’70),in Tetrahymena pyriformis by Wheatley, Rasmussen and Zeuthen (1979), and in lymphocytes by Anderson and R$nne (1978). METHODS HeLa S-3 cells growing in suspension culture over an optimal range of from 1 x lo5 to 3 - 4 x lo5 cells/ml were resuspended in a Phedeficient medium containing 5% dialysed serum before analogue or homologue treatment. Cells were counted by means of a FN Coulter counter coupled with a C-1000 channelyzer. Mitotic indices were analysed by acrystal violet staining method previously described (Wheatley and Henderson, 1974). Cells were synchronised b exposing them at 130-150 x lo3 cells/ml to either hydroxyurea (2 x 10 -2 M) or amethopterin (1 x 10e6M, + 5 x 10m5M adenosine) for 16h before reversing them with a change of medium or the addition of 1 x 10w6M thymidine respectively. Effectiveness of synchrony was rmnitored by regular cell sampling in the presence of vinblastine sulpha te (2 x 10m8M) to arrest metaphases, and by cell counting with the Coulter FN machine. RESULTS The ‘arrest’

of G2 cells by pFPhe

Our initial studies suggesting that cells were arrested within about 30 min after addition of 2 x 10V4M QFPhe have been confirmed many times (reviewed by Wheatley, 1978), but the efficacy of the inhibition is highly The use of dependent upon the absence of Phe from the medium. asynchronous cultures where the mitotic index is low makes it more However, difficult to appreciate the degree of inhibition by the analogue. by using a much more detailed and extensive analysis with not less than 10,000 cells being scored from duplicate samples at each time after EFPhe treatment, recent experiments revealed that the prophase index does not fall from 0.8 - 1.0% down to zero but only to about 0.05 - 0.15% in 1 h at The analogue at 5 x 10B4M was marginally more 2 x 10s4MeFPhe. effective in suppressing entry into prophase but considerably better at sustaining this suppression over the following 2 h, whereas in 2 x 10V4M

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EFPhe, the prophase index rose to about half its original and 3 h after treatment (Fig. 1).

level between 2

Time, h

Fig. 1 Effect of PFPhe in Phe-free medium on prophase index in asynchronous HeLa cell culture. Stippled area gives range of controls. Treatments of 2 x 10m4M (o -o), and 5 x 10B4M (o----o) EFPhe. Prophases were scored from first definite appearance of chromosome condensation until prometaphase.

Since prophase - as scored here (see legend to Fig. 1) - is the shortest phase of the cell cycle occupying about 6 - 10 min, a detectable index throughout treatment suggests that cells must be moving very slowly into mitosis in the presence of the analogue. The apparent absence of a corresponding accumulation of c-metaphases over incubations of 4 - 6h in the presence of the analogue is due to their slow disintegration at 3’7’C in the continued presence of the alkaloid. Further evidence to confirm this apparently low rate of progression came from experiments in which metaphases were removed from monolayer cultures of HeLa cells treated with EFPhe by a repeated shake-off procedure (Terasima and Tolmach, 1963) at 30 min intervals, and new cohorts of c-metaphases were detected in each decantation of medium, although the nature of this experiment precluded accurate quantification of the rate of progression through prophase. pFPhe treatment of synchronised cells The ratj.onale for earlier studies (Wheatley

and Henderson,

1974;

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19752 and b)being carried out on asynchronous HeLa cells was (i) the difficulty of obtaining well-synchronized cultures towards the later stages of the cell cycle and (ii) the avoidance of unbalanced growth (Rueckert and Mueller, 1960) which might of itself alter the response of cells to subsequent treatment. However, cells synchronized at the GI/S boundary as previously described, have a standard G2 time, and unbalanced growth proved to have no significant affect on the responses of G2 cells to the analogue. After alignment at the GI/S boundary with hydroxyurea, the medium was changed and the cells moved on through S and G2 relatively synchronously. Treatment with PFPhe at the end of S phase blocked progression into mitosis, but the synchrony procedure magnified the slow filtering through of cells beyond this block. This had not been apparent in asychronous cultures until careful analyses were made on thousands of cells as in Fig. 1. Two findings of note were, first, that the eFPhe arrest was not complete at 37’C: and second, only the rate at which cells reached division was altered, i. e. it took much longer for EFPhe-treated cells to pass the transition from G2 into M. In this respect, the behaviour of mammalian cells in response to QFPhe is similar to that in Tetrahymena (Wlneatley, Rasmussen and Zeuthen, 1979). Since the majority of cells in a synchronized culture will be in G2 from approximately 7 to 9 h after reversal of the G1/S block, staggered 2 h pulse-treatments with analogue were started at 6, 7, 8 and 9 h after Analogue and Controls received Phe instead of analogue. reversal. homologue treated cultures were subsequently ‘rescued’ by the addition of Phe to a final concentration of 1 x 10B3M except for one analogue treated culture in each set which was given Dulbecco’s saline without Phe to The results are monitor the effects of continued exposure to analogue. shown in Fig. 2 2 - A. With pulses from 6 - 8 h (Fig. 2 a), the analogue treated cultures The non-reversed showed a delay of about 1 h compared with controls. culture showed a slow but steady rise in metaphases from 11 h onwards. The effect of a pulse from 7 - 9 hr after reversal were very similar (cf. Fig. 2 b with Fig. 2 a). Delaying pulse treatment by a further hour (from 8 to 10 h after reversal, Fig. 2 c), produced only a small delay of about 30 min in the The percentage of cells taking part in the synchronous mitotic wave. synchrony was now very close to that of the control whereas in the earlier Cells given prolonged analogue pulses, a lower asymptote was reached. This ability exposure entered mitosis at about 50% the rate of controls. of a 2 h pulse of EFPhe to delay entry into mitosis was completely abolished when treatment began at 9 h (Fig. 2 d). It is important to note, however, that very few control cells began to enter mitosis by 9 h after

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reversal (approximately 2% above the figure at 6 h), and another elapsed before half the dividing population were seen as arrested metaphases.

3h

The results in Fig. 2 show, first, that the ‘escape’ of cells into Second, the mitosis in the continued presence of analogue is confirmed. recovery of cells from 2h exposure to analogue was delayed by only about 1 h when given at the most critical times of 6 - 8 or 7 - 9 h (Figs. 2 2 and 11). And third, the released cells showed a slightly slower rate of entry into division but the degree of synchrony itself was unaffected, i. e. no further synchronisation had been achieved, or alternatively little or none of it had been lost. In the next experiment, cells were exposed to 5 x 10V4M PFPhe in the same way as in Fig. 2 except that exposures were of 2, 4 or 6 h duration either before the 9th h (Fig. 3 2) or after the 7th h of reversal In this way the 2 h pulses of Fig. 3 2 and from hydroxyurea (Fig. 3 b). The results are complicated in the case of exposures before b overlap. 7 h by the action of EFPhe on S phase (2). Thus a 6 h exposure to EFPhe from 3 h after reversal of hydroxyurea caused a delay of 4 h before cells slowly began entering mitosis (curve 3). The 4 h pulse treatment gave an intermediate response and the 2 h produced the least delay. This was also confirmed by a comparison between the 2 h pulse curves in Fig. 3 -a and Fig. 3 b, which corresponded to the 2 h pulse in Fig. 2 b. Fig. 3 b shows the delaying effects of longer pulses from 7 h onwards. yt illustrates particularly clearly the fact that the cell populations had already begun to enter division by 11 - 12 h, i.e. at or before rescue with Phe in curves 2 and 3, and continued to accumulate in metaphase thereafter at much the same rate. Prophase activity was prominent from about 10 h onwards from the mitotic analyses made on these preparations. DISCUSSION PFPhe slows cell progression through G2 and the entry of cells into mitosis. Cells which are already at the last stages of G2 are not sensitive (Fig. 2 c and d) but they probably would have experienced metaphase delay (Sisken and Wilkes, 1967), although this has not been followed here. We have already shown that the inhibiting effects of EFPhe are only evident after analogue has been incorporated into protein (Wheatley and Henderson, 1974). Therefore sufficient time must elapse for a critical amount of analogue protein to be formed before an effect is observed. This may still be ineffective if critical contamination of the pool of division proteins occurs after sufficient normal molecules have already been co-opted for division purposes (Wheatley, Rasmussen and Zeuthen, 1979).

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h after S reversal

Fig. 2 (above) Effect of 2 h pulses of 2 x 10V4M EFPhe on synchronous HeLa cell cultures starting from 6,7,8 and 9 h after G1/3 synchrony. Controls received 2 x 10D4M Phe, and pulses were terminated by addition of 1 x 10m3M Phe, except which was maintained in analogue.

Fig. 3 (right) Effect of 5 x 10D4M pulses of EFPhe of 2,4 or 6 h duration on synchronous HeLa cell cultures given in (a) for hours preceding the 9th h after reversal of Gl/S synchrony, and in (b) for hours following the 7th h after reversal. Only one control is included in each case. A 11 cultures in this experiment were rescued with 1 x 10B3M Phe .

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The pronounced slowing of entry of cells into mitosis with EFPhe expresses the changed probability per unit time that a cell in the synchronized population will move from G2 into mitosis (Talavera and It does not move cells out of phase Basilica, 1978 ; Wheatley, 1977). with one another and is therefore devoid of the ability either to enhance or dissipate their synchrony in the later stages of the cell cycle (Fig. 2 per unit and 3), as would be expected of an agent which alters probability The analogue has its value in delaying cells in G2, and in allowtime. ing one to reverse the inhibition by a simple manoeuvre - the addition of Phe - while retaining an almost unperturbed synchronous wave of division (Fig. 2). It is possible, however, by longer exposure to EFPhe to produce a reasonable degree of parasynchrony in both HeLa cells (Sunkara, Rao and Wheatley, unpublished) and Tetrahymena (Zeuthen, 1964). The most valuable asset of analogue pulse treatment is to separate two distinct processes, i. e. the synthesis and the utilization of specific It can be combined with other groups of proteins involved in cell division. treatments, such as temperature (Smith and Pardee, 1970; Wheatley, Zeuthen and Rasmussen, 1979) or pressure (Walker and Wheatley, 1979) and thereby allow a much improved dissection of cell cycle events. ACKNOWLEDGEMENTS The financial assistance of the Cancer Research Campaign, the Carnegie Fund and Medical Endowments Research Trust are acknowledged. Mrs D Selbie provided technical assistance and Miss A H Mackay prepared the manuscript, to whom we are most grateful. REFERENCES Andersen,

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Received:

26th

June

1979

1979

Accepted:

9th

July

1979