Recovery of HeLa cells from inhibited entry into mitosis induced by p-fluorophenylalanine

Printed in Sweden Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved

Experimental Cell Research92 (1975) 21 l-220

RECOVERY MITOSIS

OF HeLa CELLS INDUCED

FROM

INHIBITED

ENTRY

INTO

BY p-FLUOROPHENYLALANINE

D. N. WHEATLEY

and J. Y. HENDERSON

Department of Pathology, Medical School, University of Aberdeen, Aberdeen AB9 220, Scotland

SUMMARY HeLa cells are inhibited from entering mitosis when maintained in a phenylalanine-deficient medium containing 0.2 mM pFPhe but recover within 2 h of the removal of the analogue or the addition of excess Phe. Recovery is not dependent upon the presence of Phe, however, nor on protein synthesis. Proteins incorporating pFPhe at 32°C do not inhibit the progression of HeLa cells into mitosis. By shifting cells exposed to pFPhe at permissive (32°C) and non-permissive (37°C) temperatures for 2 h to recovery conditions at 32 and 37”C, it has been demonstrated that pFPhe proteins made at 37°C remain inhibitory at both temperatures whereas pFPhe proteins made at 32°C do not become inhibitory at 37°C. These findings imply that the properties of the pFPhe proteins are determined by the temperature at which they are made, and that if two distinct conformations exist, they cannot readily be changed from one to the other by a shift in temperature.

HeLa cells cease to enter mitosis within 15-30 min of addition of 0.2 mM DL-pfluorophenylalanine (pFPhe) to a phenylalanine-deficient culture [ 11. Incorporation of the amino acid analogue into protein is obligatory for inhibition to occur and it has been shown that the analogue proteins are unusually thermosensitive [2], as found in bacteria [3, 41. One important aspect which required investigation was the reversibility of the effect of pFPhe. In this report the kinetics of recovery of HeLa cells are described under a variety of different conditions, including shifting cells from ‘permissive’ to ‘non-permissive’ division temperature [2] and vice versa. The results have significant bearings on the mechanism of action of pFPhe in relation to division control in mammalian cells.

MATERIALS

AND METHODS

The following abbreviations have been used throughout: Phe, phenylalanine; pFPhe, p-fluorophenylalanine; Tyr, tyrosine; TCA, trichloracetic acid; HU, hydroxyurea; C/P, cycloheximide (5 pg/ml) + puromycin (2 pg/ml). HeLa S-3 cells were grown in suspension and monolayer culture as described before [5]. For experiments in suspension culture, cells were grown to 2-3 x lo5 cells/ml and resuspended in phenylalaninefree suspension medium containing 10% dialysed calf serum for 2 h before treatment with the analogue. Monolayer cultures were inoculated at low density and allowed to grow for about 48 h to half-confluency before being transferred to a phenylalanine-deficient basal Eagle’s HeLa medium with 10 % dialysed serum for about 16 h (overnight) prior to analogue treatment. All culture work was carried out in a 37+ 0.5”C hot room. Suspension cultures were incubated at lower temperatures in a Gyrotory water bath shaker G-76 (New Brunswick Scientific Co. Inc., New Brunswick, N.J.) and monolayers in incubators (Laboratory Thermal Equipment Ltd., Greenfield, Oldham, Lanes., UK). In both cases the temperature of incubation was kept within the range 32.0-32.5”C, and for individual experiments the fluctuation was usually less than 0.2”C. Exptl Cell Res 92 (1975)

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RESULTS Basic recovery kinetics

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Fig. I. Abscissa: time (hours); ordinate: % metaphases. (a) Schematic diagram of the protocol adopted in susuension cultures of HeLa cells for nFPhe inhibition and recovery; (b) recovery experiment with HeLa cells. In all figures describing metaphase collection, Colcemid was added at 0 h (unless otherwise stated) at 0.06 .&ml. O-O. Control Phe-treated culture ‘recovered’ in 0.2 mM’m-Phe medium at 4 h; q - q , gFPhe-inhibited culture restored to 0.2 mM DL-Phe medium at 4 h; m-m, pFPhe-inhibited culture given a medium change at 4 h but back into 0.2 mM

The usual protocol in recovery experiments was to pre-incubate cells for 2 h (suspension cultures) or overnight (monolayers) in phenylalanine-free medium which was followed by 2 h of 0.2 mM DL-p-FPhe treatment (fig. 1 a). In asynchronous HeLa cell cultures the metaphase index was usually about 3-3.5 % at the time of pFPhe addition. During the 2 h in pFPhe the index fell to about 1 %, and prophases were rarely seen (fig. 1 b). The presence of 1% metaphases was probably not due to a slow progression of cells into mitosis but a slowing up of the progression out of mitosis of cells which had reached metaphase before pFPhe became fully operative (fig. 2), as described by others [7, 81. On removal of pFPhe from the inhibited cultures and restoration to normal growth medium containing 0.1 mM L-Phe (or 0.2 mM DL-Phe) and colcemid, the metaphase index remained unchanged for about

m-pFPhe.

Cells were counted electronically (Coulter Counter Model Fn). Mitotic (or metaphase) indices were determined bv differential counts on duulicate 1 OOOcell samples at each time point. For this cells were fixed after washing in isotonic saline with a 3 part methanol : 1 part acetic acid mixture, and stained with 0.025 % crystal violet in 1 % acetic acid. Cycloheximide (‘Actidione’, Koch-Light, Colnbrook, Bucks., UK) and puromycin (Sigma, London) were combined in a sterile aqueous solution 100 x concentrated for dilution to approx. 1.8 x 1V M (5 ,ug/ml) and 4.2 x 1O-B M (2 pg/ml) respectively. One hundred times concentrated stock solution were similarly prepared of Colcemid (‘Demecolcine’, CIBA, Horsham, Sussex, UK) for use at 1.5 x 10-’ M, hydroxyurea (British Drug Houses, Poole, Dorset, UK) for 2 x 10-* M, bleomycin (Nippon Kayaku Co., Tokyo) for 3.4 x l@ M,- m-j-fhtorophenylalanine and DL-phenylalanine (Koch-Light) for 2 x 10-* M, and L-phenylalanine (BDH) for 1 x 10-* M. Free amino acid levels in homogenates from 10’ cells cultured in Phe or pFPhe were measured after precipitation of proteins with 10 % trichloracetic acid and aoulication of ether-extracted suoematants to a single-column automatic analyzer Technicon system (Technicon Instruments Co. Ltd., Chertsey, Surrey, UK). Exptl CeNRes 92 (1975)

1

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Fig. 2. Abscissa: time (hours); ordinate: % mitotic cells. Effect of 0.2 mM pFPhe on exit of cells from mitosis. HeLa cells were exposed to Bleomycin at 5 ,ug/ml at 0 h which prevents entry of cells into mitosis after 15 min. Cells in or arriving at mitosis within this time were allowed to progress through mitosis. O-O, Bleomycin in Phe-deficient medium; m-m, Bleomycin and 0.2 mM m-pFPhe in Phedeficient medium.

Inhibition of mitosis by para-fluorophenylalanine

1 h. Between 1 and 2 h there was a small collection of metaphases and from 2 h onwards the rate of collection paralleled the untreated controls (fig. 1 b). This pattern has been consistently found in many experiments indicating that a definite delay period occurs before cells can enter mitosis and that they do not seem to enter mitosis in a synchronous fashion, i.e. they most probably retain their positions relative to one another in the cell cycle. Other interpretations may be equally valid; for example, it is plausible that pFPhe treatment permanently deranged G2 cells which could not subsequently re-enter the cycle. The delay in recovery would represent the loss of the G2 cohort which would be followed by S phase cells entering mitosis after a relatively normal transit time through G2. This possibility was excluded, however, by the use of HU. Cells were exposed to pFPhe (fig. la), except that 2 mM HU was added at the same time as pFPhe to prevent S+ G 2 progression. When cells were reversed after 2 h, HU was continued throughout the

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Fig. 4. Abscissa: time (hours); ordinate: % Colcemidcollected metaphases. Recovery of HeLa cells maintained for different times in 0.2 mM or-pFPhe prior to 0 h.O---0, 15 minpFPhe; O---O, 1 h; O-O, 2 h; 0-O 4 h.

recovery phase thereby maintaining the S-G2 inhibition. It can be concluded from fig. 3 that the cells recovering come from the G2 population arrested by the pFPhe treatment, and that there is no obvious synchrony in the recovery. Varying the pulse conditions (a) Length of pulse. The effects of 0.2 mM

OOi

Fig. 3. Abscissa: time (hours); ordinate: % Colcemidcollected metaphases. Demonstration that G2 cells recover from pFPhe inhibition using 2 mM hydroxyurea (HU). O-O, Cells recovered at 0 h in 1 mM L-Phe medium; q - q , cells exposed to HU from time of pFPhe treatment and throughout; W-B, cells in HU throughout but returned to pFPhe medium at 0 h.

pFPhe pulses for different lengths of time on subsequent recovery in HeLa suspension czll cultures is shown in fig. 4. Pulses of 30 min or less fail to develop a sufficient degree of arrest to prevent rapid recovery. Recovery after a 1 h exposure followed a brief delay whereas a 2 h exposure required about 90 min recovery before cells progressed into mitosis; with a 4 h exposure the delay in recovery was only slightly longer than with a 2 h exposure but the absolute number of recovering cells was slightly reduced. Exposure of 6 h gave negligible recovery and was associated with the onset of cellular degeneration. (b) pFPhe dosage. Recovery from low levels of pFPhe (0.05 and 0.1 mM) were Exptl Cell Res 92 (1975)

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Wheatley and Henderson

of experiments. Many of the later experiments-including some of those already discussed-involved addition of excess L-Phe (1 mM) since it ensured an adequate supply of the natural amino acid, and in particular, it allowed the recovery of cells without the perturbation of C 2 + M progression likely to be caused by a medium change. (a) Removal of pFPhe by medium change. -0

i

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Fig. 5. Abscissa: time (hours); ordinate: % Colcemidcollected metaphases. Recovery of HeLa cells by addition of 1 mM L-Phe after a 2 h exposure to -different pFPhe levels. O-O, 0.2 mM DL-pFPhe; O-e, 0.5 mM DLpFPhe; W-U, 0.2 mM Dr.-pFPhe but without addition of Phe at 0 h.

rapid since these levels only partially blccked entry into mitosis in HeLa cell cultures [l]. A 2 h exposure to 0.5 mM pFPhe induced more perturbations in the cells than 0.2 mM and cells recovered from it more slowly and in smaller absolute numbers (fig. 5). Varying the chase(recovery) conditions

There are two ways in which cells can be rescued from pFPhe treatment, the medium can be changed or the natural amino acid can be added since the analogue competes poorly with it [l]. In order to be sure of the exact conditions during recovery, medium changes were carried out in the first series

To ensure that pFPhe was removed, suspension cultures were spun down at 800 g at 37°C for 10 min and resuspended in media with different levels of Phe. Recovery was not dependent on the presence of Phe in the medium, and excess Phe (1 mM or more) did not hasten recovery (fig. 6~). Furthermore it was found that de novo protein synthesis was not necessary since the presence of inhibitors in the rescue medium did not prevent cells entering mitosis (fig. 6~). (b) Reversal by addition of Phe to the medium. Phe and pFPhe compete with each

other in about a ratio of 2.5-5.0: 1, as shown by incorporation studies [l]. Adding an equivalent amount of Phe to pFPheinhibited cultures (0.2 mM) resulted in almost normal recovery. Addition of a IO-fold excess of L-Phe (1 mM) has consistently given excellent recovery even in the presence of C/P (fig. 6b), and has since been adopted as a routine procedure. Phe in a 25-50-fold excess gave no better recovery and at the higher levels produced some adverse effects 6. Abscissa: time (hours); ordinate: % Colcemid-collected metaphases. (a) Recovery of 0.2 mM pFPhe-treated (2 h) cultures by resuspension at 0 h in fresh media containing O-O, 1 mM L-Phe; o--O, Phe deficient; A -A, 1 mM L-Phe + cycloheximide 5 &ml and puromycin 2 pg/ml (C/P); v --- V , C/P only; l - l ,0.2 mM pFPhe. (b) Recovery of cells as in (a) but without a medium change. The following direct additions were made O-O, 1 mM L-Phe; A-A, 1 mM Phe+C/P; V-V,C/Ponly;m--¤, 1 mM L-Tyr; l - 0, saline control. Fig.

Exptl

Cell Res 92 (1975)

Inhibition of mitosis by para-jluorophenylalanine itself. Tyr could not substitute for Phe and allowed no recovery at 1 mM (fig. 6b). Estimations have been made of the movement of the amino acids across the cell membrane into and out of the TCA-soluble pool under the conditions of the recovery experiment outlined above. Table 1 shows that Phe and pFPhe exchange rapidly with one another and that the addition of a lo-fold excess of L-Phe (table 1, d) causes a rapid flushing out of pFPhe to lower levels in 5 min and to undetectable levels in 30 min. Meanwhile Phe pool had reached a 4-fold excess over the pFPhe within that first 5 min. The delay in recovery after addition of 1 mM Phe (fig. 6b) cannot simply be due to a slow return of the availability of the natural amino acid. Table 1. Free amino acids levels in HeLa cells under different conditions of analogue treatment and chase

Treatment

Duration (min)

Percentage of free amino acids (relative to glutamic acid) Phe

pFPhe

(n) Normal medium (b) Phe deficient medium 120 (c) pFPhe 0.2 mM in 5 DL-Phemedium 60

8-10

-

0 0

-

(d) Addition of Phe (1 mM) after 2 h in (c)

41

180

3; 90

0 0

22

8 13 15

9 0 0

10’ HeLa cellswereusedin eachanalysis;they were washedtwice with isotonicsalineand treated with 10% TCA at 4°C from 15min. The supernatantwas extracted3 timeswith etherand the aqueousportion wasevaporatedto dryness.The residuewasdissolved in 0.1 N HCl. PheandpFPhewerewell separated on the Techniconsystem,the latter runningcloserto the referencey-amino-isobutyricacid. The amountsof Phe and pFPhe were calculatedin relation to the gluttic acid pool taken as 100%sincethis amino acidforms the largest pool in HeLa cells and its level remainedconstantunder conditions(u)-(d) above.

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0 8-

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Fig. 7. Abscissa: time(hours);ordinate: % Colcemid-

collectedmetaphases. Dilution and the recovery of suspension-cultured

HeLa cells from 0.2 mM pFPhe treatment given for the 2 h prior to 0 h. At 0 h the following changes were made: dilution with O-O, equal volume conditioned medium containing 2.0 mM Phe; 0 - 0, 3 vol. conditioned Phe-deficient medium; q - q , equal volume of conditioned Phe-deficient medium; W-M, equal volume of 0.2 mM pFPhe-containing Phe-deficient conditioned medium.

The level at which pFPhe blocks entry of HeLa co,lls into mitosis is about 0.2 mM, lower levels being less effective [l]. It was of interest, therefore, to see whether HeLa cells exposed to 0.2 mM pFPhe would recover if the level of analogue was diluted after 2 h to these less effective concentrations. A 4-fold dilution to 0.05 mM allowed moderate recovery but, predictably, less was observed with a 2-fold dilution to 0.1 mM (fig. 7). The maintenance of a critical external concentration of pFPhe is essential for cells to be held in G2; this suggeststhat the physiological mechanism of inhibition is very delicately balanced. Temperature and recovery Thermosensitivity is one of the most interesting sequelae of pFPhe incorporation [l-4]. Careful analysis has been made of the effects of incorporating pFPhe at permissive and non-permissive temperatures in HeLa

cells with respect to their ability to recover in Exptl Cell Res 92 (1975)

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Wheatley and Henderson

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Fig. 8. Abscissa: time (hours); ordinate: % Colcemidcollected metaphases. Temperature shifts and the recovery of HeLa cells following exposure to 0.2 mM DbpFPhe for 2 h at 37°C. O-O, Addition at 0 h of 1 mM L-Phe, kept at 37°C; l - 0, no addition, kept at 37°C; O---O, addition of 1 mM L-Phe, shifted to 32°C; O---O, no addition, shifted to 32°C.

the presence or absence of the analogue after a shift between these different temperatures. Recovery of pFPhe-treated cells following a period of chilling to 4°C is also discussed here. (a) pFPhe exposure at non-permissive temperatures. After a 2 h pre-incubation in

Phe-free medium, HeLa cell cultures were treated for 2 h with 0.2 mM pFPhe at 37°C. Subsequently half the cultures were treated with 1 mM L-Phe whereas the other half were given saline. Two sets of duplicates were prepared from each group, one set remaining at 37°C (non-permissive) and the other set being incubated at 32°C in the gyrotory shaker water bath. The temperature fell to 32°C in approx. 4 min. Colcemid was added to all cultures and metaphase collection assessed. The results (fig. 8) show that no recovery occurred at 37°C in the continued presence of pFPhe but excellent collection for 2 h onwards was seen in those cultures given 1 mM Phe. The cultures shifted to 32°C in the continued presence of Exptl Cell Res 92 (197.5)

pFPhe showed a very slow recovery; cells looked in poor condition after 6 h although some prophases were definitely present. This recovery pattern suggests a reasonable degree of recovery compared with cultures kept at 37°C in the continuing presence of pFPhe, though far less than was found in cultures given Phe during the 32°C chase. The reason for this only partial recovery at the permissive temperature is still obscure and further experiments are in progress to analyse this particular temperature shift more thoroughly. This experiment has been repeated more than 5 times. The amount of recovery at 32°C in the presence of pFPhe has been very small for 5-6 h, but thereafter cells begin to enter mitosis at the same rate as the 32°C Phe control. Since exposure of HeLa cells from the outset to pFPhe at 32°C does not inhibit entry into mitosis ([2], see also fig. 9), the above experiment establishes that the analogue proteins made during a prior

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Fig. 9. Abscissa: time (hours); ordinate: % Colcemidcollected metaphases. Temperature-shift and the recovery of HeLa cells from 0.2 mM DL-pFF%e for 2 h at 32°C. o-0. Addition at 0 h of 1 mM L-Phe, shifted to 37°C; .6-O. no addition, shifted to 37°C; O---O, addttton of 1 mM L-We, kept at 32°C; O---O, no addition, kept at 32°C; A -A, 1 mM L-PIE added+ C/P, shifted to 37°C; A-A, C/P only added, shifted to 37°C.

Inhibition of mitosis by para-fluorophenylalanine

exposure to 37°C do not immediately lose their inhibitory powers following a shift to the lower temperature, otherwise these cultures would have given a similar metaphase collection curve to the 32°C Phe-supplemented control.

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(b) pFPhe exposure at permissive temperatures. These experiments were the reciprocal

of those described above. Cells exposed to pFPhe at 32°C (permissive) and shifted after 2 h to 37°C showed excellent ‘recovery’ in the presence of 1 mM Phe with no detectable setback (fig. 9). Cultures shifted to 37°C in the continued presence of pFPhe became inhibited within the first hour, but not immediately, at this non-permissive temperature. Cultures remaining at 32°C with or without pFPhe continued their steady progression into mitosis. The absence of a delay in collection when cells were transferred from pFPhe at 32°C to Phe-containing medium at 37”C, and the small collection at 37°C in the continued presence of pFPhe, demonstrate quite conclusively that pFPhe proteins made at 32°C do not become inhibitory on elevation of the temperature to 37°C. It was also shown (fig. 9) that the progression of cells into mitosis occurred for several hours in the presence of C/P treatment at 37°C in pFPhe medium, i.e. shifting from 32 to 37°C does not inhibit progression in the continued presence of pFPhe if analogue incorporation at 37°C is suppressed. The relevance of these experiments to the important question of whether temperature shifts can alter the functional status of existing protein molecules is discussed later. (c) Chilling of pFPhe-exposed

cells to 4°C.

Since the recovery of cells from pFPhe exposure at 37°C is not dependent on the presence of the natural amino acid or on renewed protein synthesis, the inhibitory principle is presumably removed quite rapidly by some enzymatic or physical process. To distinguish

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Fig. 10. Abscissa: time (hours); ordinate: % Colcemidcollected metaphases. Chilling of HeLa cells at recovery. Cells were exposed for 2 h to 0.2 mM pFPhe. At 0 h some were stored at 4°C by swirling in iced water; others were left to recover immediately at 37°C by addition of 1 mM L-Phe. O-O, Normal HeLa cells, unchilled; O---O, normal culture chilled from 0 h to 2 h; B---W, pFPhe-treated cells chilled from 0 h to 2 h but allowed to recover at 37°C though in the continued presence of 0.2 mM pFPhe; q - q , pFPhetreated culture recovered in 1 mM-Phe at 0 h without chilling; A - A, pFPhe-treated culture chilled from 0 h to 2 h and recovered at 37°C in presence of 1 mM L-Phe.

between these two, pFPhe-exposed cells were allowed to recover immediately or after a 2 h period at 4°C. If an enzymatic process is required to remove the inhibitory pFPhe principle, cells would be expected to show a longer delay (i.e. excluding the 2 h at 4°C) than the controls. The results (fig. 10) support the opposite interpretation-cells recover faster after the 2 h chilling than cells recovered in Phe-containing medium immediately, i.e. the delay at 37°C is about 90 min in unchilled controls while it is negligible in the chilled controls when measured from the time they were returned with Phe to 37°C. The inhibitory pFPhe principle must have been removed at 4°C almost as effectively as at 37”C, which suggests a physical sequestration or inactivation rather than enzymatic degradation. Exptl Cell Res 92 (1975)

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Wheatley and Henderson

DISCUSSION HeLa cells can be prevented from entering mitosis by pFPhe provided the analogue is available at ‘physiological’ levels of about 0.2 mM of the racemic mixture. There is no doubt that the analogue interferes with many other cellular processes mainly by becoming incorporated in proteins and gradually suppressing protein synthesis over a number of hours [l, 21. The ability of cells to survive for some time, however, shows that the effect on entry of cells into mitosis is relatively specific, especially as this inhibition can be abolished with inhibitors of protein synthesis (ref. [I] and figs 6 and 9). The onset of severe pFPhe-induced inhibition [2] of protein synthesis results in a generalized toxicity from which cells fail to recover. In most of our experiments, pFPhe treatment has been limited to 2 h but they will survive 4 h without significant loss in numbers or viability. Under these conditions cells can recover from pFPhe within a short space of time and enter mitosis. Since the recovery process has almost invariably been monitored by Colcemid collection, no information is reported here concerning the ability of cells to progress through mitosis successfully although it has been shown that the analogue does make it more difficult for cells to do so (fig. 2 and ref. [7]). It is of particular interest that recovery from pFPhe inhibition is dependent upon neither a supply of the natural amino acid (Phe) nor protein synthesis. This suggests that an inhibitory principle is formed from pFPhe which is sequestered, inactivated or destroyed quite rapidly after the analogue amino acid has been removed. Indeed, the process must be highly dynamic because reduction of pFPhe to ‘sub-physiological’ levels (0.1 mM or less) is sufficient to allow some recovery (fig. 7). Exptl

Cell Res 92 (1975)

The simplest recovery procedure is to rescue the cells with L-Phe. At 1 mM, Phe becomes 10 times more abundant than the L-pFPhe (0.1 mM of the 0.2 mM racemic mixture) and in addition is preferentially incorporated at up to 5 times the level of pFPhe. Both Phe and pFPhe enter HeLa cells easily and pFPhe forms a larger pool than Phe (table 1, and unpublished results). It is noteworthy that pFPhe was removed very quickly from the cell pool in the presence of 1 mM Phe (table 1) suggesting that the competition between Phe and pFPhe for transport into cells definitely favours the natural analogue. In these experiments, Tyr allows no recovery, as might be expected, since Tyr cannot be used as a precursor for Phe. This agrees with the findings of others [9, 101. The recovery of cells from an arrested G2 condition has been established by the HU experiments in fig. 3. In other experiments it has been demonstrated that with 2 mM HU HeLa cells in G2 can be easily delineated by this technique [l l] and are not ‘contaminated’ by S phase cells until about 30 h after treatment when a small percentage begin to leak through into mitosis (Brown & Wheatley, unpublished results). Since the pFPhe experiments involve only 6-8 h of exposure to HU, cells in S phase are held there and cells in G2 must have been arrested by the pFPhe. The recovery pattern establishes that it is these G2 cells that are released into mitosis. A problem which is not so readily answered is whether the cells come into mitosis in strictly the same order as they were arrested in G2, i.e. the late G2 cells should enter before early G2 cells, and it is conceivable that the opposite might occur if greater setbacks are experienced by the cells nearest to mitosis [3, 121. Although recovery results rarely show clear evidence of synchrony, there have been occasions when some sugges-

Inhibition of mitosis by para-fluorophenylalanine

tions of parasynchrony occurred, particularly after temperature shifts. Because of the time scale (G2 is about 2.5 h or approximately one tenth of the cycle time of HeLa cells) and the small population of cells involved, it is unlikely that a high degree of synchrony of the G2 cohort entering mitosis would be readily observed during recovery. In other systems in which pFPhe has produced a synchronisation, e.g. E. coli [3] and Tetrahymena [12], pFPhe treatment has been given for relatively longer periods of the generation time to achieve this effect. Perhaps the most significant of the recovery experiments have been those concerned with temperature shifts. We have already reported that 0.2 mM pFPhe is effective in HeLa cells at 37°C but does not inhibit the progression of cells at 32°C. Two questions were posed: (i) when pFPhe is incorporated into protein at 32°C (permissive), do the proteins become inhibitory at 37°C and (ii) when pFPhe is incorporated at 37°C (nonpermissive), does the inhibitory action persist or disappear at 32”C? The results of experiments described in figs 8 and 9 are unambiguous and confirmatory in their complementarity. Inhibitory pFPhe proteins made at 37°C do not become functional again at 32°C so as to allow normal progression of cells into mitosis. Similarly, pFPhe proteins made at 32°C do not become inhibitory at 37°C. Since pFPhe incorporation is of much the same order at 32°C as at 37°C [2], an almost immediate suppression would be expected in the presence of C/P and Phe at 37°C in the latter situation if the proteins had changed their conformation with the temperature (fig. 9). The most reasonable conclusion which can be drawn is that proteins incorporating pFPhe at 32°C and at 37°C have different conformational structures and that shifts in temperature do not result in corresponding changes of

219

existing molecules from the one conformational state to the other. Thus the properties of pFPhe proteins arl: determined by the temperature at which they are initially made. A more simple explanation could be that the inhibitory principle formed at 37°C is not made at all at 32°C. The behaviour of pFPhe-treated cells at different temperatures and the consequences of shifting cells from one temperature to another may have particular relevance to the phenomenon of growth in temperaturesensitive mutants [13, 141. The existence of thermosensitive proteins with several possible conformational structures responsible for the altered behaviours at different temperatures has still to be demonstrated, although it is implicit in findings such as those of Kang & Markovitz [4]. Investigations are presently being undertaken into the critical levels of pFPhe proteins required for inhibition and their rate of disappearance from the cytoplasm and nucleus during chase with 1 mM Phe. Overall rates of turnover of Phe and pFPhe proteins are similar but some of the soluble proteins from cell lysates are removed faster than others (to be published) and it is here that pFPhe proteins responsible for inhibition may be found. We wish to acknowledge the assistanceof Dr Linda Fothergill of the Department of Biochemistry, Marischal College, in this University. We also thank Mrs Marget Inglis for technical assistance. This work was supported by a grant from the Cancer ResearchCampaign.

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8. Sisken, J & Iwasaki, T, Exptl cell res 55 (1969) 161. 9. Kruh, J & Rosa, J, Biochim biophys acta 34 (1959) 561. 10. Richmond, M H, Biochem j 77 (1960) 121. 11. Wheatley, D N, Int symp on adriamycin, p. 47. Springer-Verlag, Berlin, Heidelberg, New York (1972).

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12. Rasmussen, L & Zeuthen, E, Compt rend trav lab Carlsberg 32 (1962) 333. 13. Naha, P M, Nature 233 (1969) 1384. 14. Wang, R J, Nature 248 (1974) 76. Received July 8, 1974