J. Cell Sci. 14, 461-473 ('974) 461Printed in Great Britain

SENSITIVITY OF THE CELL DIVISION CYCLE

TO a-AMANITIN IN ALLIUM ROOT TIPS

C. DE LA TORRE, M. E. FERNANDEZ-GOMEZ,G. GIMENEZ-MARTIN AND A. GONZALEZ-FERNANDEZDepartamento de Citologia, Institute de Biologia Celular (C.S.I.C.),Madrid, Spain

SUMMARY

The effect of a-amanitin on the cell cycle in Allium cepa meristematic cells was studied: theGx and G2 periods are prolonged respectively to 1-9 and 1-7 times the normal duration; the5-period is lengthened very little; and the prophase of mitosis is increased to twice the normalduration. It is postulated that real differences in the activity of the non-nucleolar RNA poly-merase might exist in the course of the cell division cycle and that they would account for thehigher sensitivities shown by Glt G2 and prophase.

On the other hand, the interphase nucleolus responds by segregation in the first few hours ofa-amanitin treatment, but recovers its normal structure in continued presence of the drug;and nucleolar reorganization is inhibited in the first few hours in recently formed cells, but theprocess is subsequently speeded up to attain the same value 4 h after the treatment was begunas in untreated cells.

INTRODUCTION

The problem of the kinetics of RNA synthesis in the course of the cell divisioncycle has been the subject of numerous studies, the majority of which suggest thatsynthesis takes place throughout interphase but is absent only from late prophase upto late telophase (see Baserga, 1968; Monesi, 1969).

On the other hand, the problem of the evenness or unevenness of enzyme activitythroughout interphase in the higher organism is a very important one. It is admittedthat some kind of thymidine kinase synthesis takes place at the beginning of the inter-phase 5-period (Hotta & Stern, 1963, in plants; Bollum & Potter, 1959; Mittermayer,Bosselmann & Bremerskov, 1968; Weissmann, Smellie & Paul, i960 in animals) andit has been shown that peaks occur in the activities of certain enzymes in the course ofinterphase, as with glucose-6-phosphate dehydrogenase and lactate dehydrogenase(Klevecz & Ruddle, 1968).

Hence we thought that it would be of interest to study the sensitivity of the cellcycle to a-amanitin, an inhibitor of extranucleolar RNA polymerase (Jacob, Sajdel &Munro, 1971), and so to detect possible changes in the activity of this enzyme.

Parallel studies on nucleolar morphology and rate of nucleologenesis were conductedto elucidate whether nucleolar physiology is or is not impaired by this toxin inmeristems.

462 C. de la Torre and others

MATERIAL AND METHODS

The material used was the root meristem of Allium cepa L. bulbs. They were grown in the darkat 15 ±0-5 °C, in cylindrical receptacles in tap water with constant aeration. The bulbs wereso placed that only their bases remained submerged in the water.

Treatments

Twenty- to thirty-millimetre roots, still attached to the bulbs, were transferred to the differenta-amanitin solutions (i, 10, 50, 100 /tg/ml), under the same conditions as were used for growing.

The a-amanitin was a generous gift from Prof. Th. Wieland, Heidelberg.Labelling with \?H~\thymidine. The bulbs were subjected to [3H]thymidine at a concentration of

5 /tCi/ml and specific activity of 176 Ci/mM in the last 10 min of the treatments. The rootswere immediately fixed and Feulgen-stained. Squashes were prepared on gelatined slides.

For autoradiography, Kodak AR-10 stripping film was used. After 3 weeks' exposure thefilms were developed with Kodak D-igb developer and fixed with ultrarapid acid fixer.

Quantitative procedure. The frequency distribution of cells in Glt S and G2 was obtained byMak's method (Mak, 1965). This method combines autoradiography for detecting cells in theS-period, and Feulgen spectrophotometry of the unlabelled interphase cells to distinguishbetween G1 and G2 cells (2 c and 4 c content of DNA respectively).

Three slides (one from each triplicate experiment) involving a minimum of 1500 meristematiccells were used to study: the percentage of labelled to unlabelled interphases; and the relativeDNA content of 100 unlabelled interphase nuclei, and of 25-30 telophase nuclei per root squashas well (2 roots for each triplicate experiment).

Unlabelled interphases were grouped according to their content in 2 c or 4 c values (G1 andG2, respectively). A Zeiss photometer microscope was used.

Synchronous binucleate cell population

A caffeine solution (o-i %) was used to 'label' a synchronous cell population as binucleate.A i-h treatment with this drug inhibits cytokinesis in the cells undergoing telophase during thattime. This cell subpopulation is labelled as binucleate in this way, and enters interphase anddevelops synchronously throughout the cell cycle.

Nucleolar morphology. This was studied in roots fixed and impregnated with silver accordingto the technique of Fernandez-Gomez, Stockert, L6pez-Saez & Gimenez-Martin (1969).

OBSERVATIONS

Effect of a-amanitin on the total duration of interphase

Choice of concentration. The duration of interphase was measured in the binucleatecells obtained by labelling with caffeine those cells which were going through telophaseduring the hour when the caffeine treatment was given (Gimenez-Martin, Gonzalez-Fernandez & Lopez-Saez, 1965). As can be seen in Fig. 1, the binucleate cells soformed reached their next biprophase 22-5 h after the administration of caffeine at15 °C when they were kept in water after the caffeine treatment (control roots). Otherbulbs previously treated with caffeine to form binucleate cells were put into the varioussolutions of a-amanitin and the moment when the first binucleate cells reached theirbiprophase was recorded. The duration of interphase of these cells was protracted bya factor of 1-5 with 10 fig/ml a-amanitin solution (Table 1), while 1 /tg/ml seemed notto have any effect. Concentrations of 50 /tg/ml and more prolonged the duration ofinterphase by a factor of more than 2-4. The dose of 10/ig/ml was then chosen forsubsequent experiments to study the effect of the drug on each of the phases of the

464 C. de la Torre and others

interphase, since it did not produce total inhibition or blockage at any point in theinterphase, but only significant prolongation of it.

Effect of a-amanitin on the kinetics of the meristematic cells population

An analysis of the mitotic and phase indices during the period of treatment witha-amanitin at io/«g/ml is recorded in Table 2.

Table 1. Minimum duration of the interphase of binucleate cellsin control and a-amanitin-treated meristems

Treatment

Controla-amanitin, fig/m\

1

1 0

5O1 0 0

Duration, h

22-s

22-533-o

> 54-0> 54-o

Factor of protraction

i - o

i - o

i -5> 2-4> 2-4

Table 2. Percentage of meristematic cells in the various phasesof mitosis during continuous treatment with a-amanitin*

Hours oftreatment

oi

36

1 2

2 4

* Each valuebulbs from each

Mitotic index±S.D.

I I - 6 + 2 1I I - 3 ± I - 8

15-4 + 2-78-8±i-39"4± !'S8-9±i-7

represents the meanseparate experiment)

Prophase

5-5578-85-85 85-9

percentage

Metaphase

i - 51-53- ii - o1-2

i - o

of over 3000

Anaphase

1 1I - 32-O

o-8i - o0 9

cells from 6

Telophase

3 52-8

i -S1-2

i - 4I - I

bulbs (2 parallel

We observe that the mitotic index (M.I.) at 3 h is higher than in the control (o h),mainly owing to the increase in percentage of prophases. From the 6th hour onwardsthe percentage of prophases remains slightly higher than the value found in the control,while the mitotic index is lower, which seems to point to a differential prolongation ofthe duration of the prophase. Approximate steady-state kinetics have been attainedfrom the 6th hour of treatment, for the frequency distributions of mitotic phasesat 6, 12 and 24 h are quite similar.

Distribution of meristematic cells in the interphase periods after 6 h of treatment

In order to study any possible differential effect of a-amanitin upon the duration ofthe interphase periods Glt S and G2, a 6-h treatment was given with the drug, becauseit seemed that a steady state had been reached after this interval of time (Table 2).

The distribution of the cells in interphase was studied by combining autoradio-graphy and cytophotometry (Mak, 1965; Gonzalez-Fernandez, Gimenez-Martin &

a-amanitin and the division cycle 465

de la Torre, 1971). After a pulse with tritiated thymidine, the percentage of cells inS-period was calculated and subsequently the number of unlabelled interphase cellswith a 2c DNA content was cytophotometrically determined (G^), as well as that ofcells with a 4c DNA content (G2).

Variations are observed in the percentages of cells in each stage of interphase witha decrease in the percentage of cells engaged in DNA synthesis and an increase in thosein Gx and G2 (Table 3).

Table 3. Percentages of interphase cells in the Glt S and G2 periods

Labelled interphases* Unlabelled interphasesf ± S.D.±S.D. , A

s(5-period) 2c(Gj) 4c(G2)

Control 50-1 ±6-8 27-413-4 22-4 + 3-4a-amanitin, 37-9±5-i 3S'4 + 2-6 26-712-5

io/tg/ml, 6 h

* Percentage of labelled interphase cells after a pulse of tritiated thymidine.t Percentages of unlabelled interphase cells with 2 c and with 4 c DNA content (G1 and G2,

respectively).

Table 4

Controla-amanitin,

10 /tg/ml, 6Factor of

protraction

. Calculation of the

Minimumduration of

interphase inbinucleate

cells

22-0 (a, b)33-o (6)

hi -5

absolute duration

Mean durationof interphase

in mono-nucleate cells

26-2 (a)38-4 (c)

i -5

ofGlf

S

1 3 1

14-5

I - I

S and G2 in

Duration'1"A

G,

7-2136

19

hours

of

G2

S-9IO-2

1-7

(a), from Gonzalez-Fernandez et al. (1966).(6), direct measurement of minimum interphase in the binucleate cells.(c), calculated from the ratio minimum interphase/mean interphase.{d), calculated by multiplying values in Table 3 by values (a) and (c) of the second column.

Calculation of the absolute duration of the Glt S and G2 periods

Since the duration of interphase did, in fact, vary, we calculated the absoluteduration of each of the phases (Table 4). For this purpose we used the control valuesdetermined for the same material under identical experimental conditions by Gonzalez-Fernandez, Lopez-Saez & Gimenez-Martin (1966) and Gonzalez-Fernandez et al.

In absolute values, we observe a slight increase in duration of the >S-period whencells are treated with a-amanitin while the Gx period is prolonged up to 1-9 times andthe G2 period 1-7 times the corresponding value observed in the control cells.

Bip

rop

has

e B

imet

aph

ase

Bit

elo

ph

ase

2n

d-o

rde

r '2

I b

inu

clea

te

2 *

Fig

. 2.

Sch

emat

ic r

epre

sent

atio

n of

th

e m

etho

d us

ed t

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st t

he

effe

ct o

f 10 pg

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a-am

anit

in o

n pr

ogre

ss o

f m

itos

is.

Th

e t

imes

w

hen

bim

etap

hase

s, b

itel

opha

ses

and

2nd-

orde

r bi

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eate

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ls a

ppea

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are

show

n in

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d t

reat

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ells

.

a-amanitin and the division cycle 467

Analysis of the effect of a-amanitin on mitosis

In order to study possible effects of a-amanitin on the progress of mitosis, binucleatecells were used in the way set out in Fig. 2. We studied the time when the first binu-cleate cells reached metaphase, anaphase, telophase and interphase when left in waterafter the caffeine treatment or when they were put into the a-amanitin solution fromthe 22nd hour after caffeine labelling when the first biprophases were about to appear.The duration of prophase (marked by the appearance of the first bimetaphase) is twiceas great as in the controls. Metaphase and anaphase were protracted 1-5 times whiletelophase lasted about as long as in the control.

The duration of the whole bimitosis was 6 and 10 h in the control and in thea-amanitin-treated cells, respectively.

80

70

60

SO

40

30

20

10

5 6Time, h

10

Fig. 3. Percentage of nucleolar segregation in interphasic cells during continuoustreatment with 10 /tg/ml a-amanitin.

Effect of a-amanitin on the nucleolus

Effect on fully organized nucleoli. The effect of 10/ig/ml a-amanitin on the fullyorganized nucleolus was studied in silver-impregnated meristematic cells. This tech-nique has the advantage of displaying features that can otherwise be seen only byelectron microscopy for it has been shown that fibrous moieities have significantlyhigher silver affinities than granular ones. We observed the transient appearance ofnucleolar segregation, i.e. a redistribution of the fibrous and granular portions ofnucleoli (Fig. 6). This phenomenon reaches a maximum after 3 h of treatment, andrecovery is complete between the 6th and the 10th hours (Fig. 3).

Effect on nucleoli in process of formation. The problem of nucleolar reorganization31 CEL 14

C. de la Torre and others

a-amanitin and the division cycle 469

was studied in freshly formed binucleate cells in the way set out in Fig. 4. Thesebinucleate cells are very convenient for this sort of study, for they are labelled at amoment in the cycle — cytokinesis - which coincides with nucleolar formation. Thedynamics of these cells appearing with completely organized nucleoli in the controlsand in a-amanitin-treated meristems were studied. The values found are seen inFig. 5. There is an initial slowing down of this process and a subsequent speeding up toattain roughly the same value as in untreated cells 4 h after caffeine labelling.

100 -

90 -

80 -

70 -

60 -

50 -

40 -

30 -

20 -

10 -

FFE

JNE

U

- 1 2Time, h

Fig. 5. Percentage of binucleate cells with fully organized nucleoli in control (0— iand a-amanitin-treated (0 0 ) cells.

DISCUSSION

We know that DNA synthesis in eukaryotic organisms occurs in the course ofa well defined period of interphase, namely the 5-period. On the other hand, RNAsynthesis seems to take place throughout interphase including the 5-period (Baserga,1968; Monesi, 1969). We know that a-amanitin inhibits the extranucleolar RNApolymerase (Stirpe & Fiume, 1967; Sekeris & Schmid, 1972; Lindell, Weinberg,Morris, Roeder & Rutter, 1970; Kedinger, Gniazdowski, Mandel, Gissinger &Chambon, 1970), and accordingly, a dramatic drop in the number of nuclear fibrilswhich are considered to be a morphological expression of freshly synthesized extra-nucleolar RNA, has been reported (Petrov & Sekeris, 1971).

The use of this drug at a dosage which slows down, but does not block, progress31-2

470 C. de la Torre and others

of the cells through interphase enabled us to obtain different responses from cellsgoing through different parts of the interphase. The relative distribution of cells inGv S and G2 is found to be radically altered (Table 3). We supposed, from study of themitotic indices (Table 2), that a steady state has been reached in the dynamics of thecells after 6 h in presence of the drug such that the percentage of cells in a given phaseis directly proportional to the duration of the phase in question. The total duration ofinterphase was determined by the use of binucleate cells (33 h, compared with 22-5in the control, see Table 1). The prolongation is found to take place particularly atGx and G2, while the 5-period was hardly lengthened at all (Table 4). These differen-tial sensitivities might be interpreted as due to differences in the activity of the enzymeinhibited, namely the extranucleolar RNA polymerase. Our data seem to be in agree-ment with those of Kim & Perez (1965) who detected 2 peaks in RNA synthesis,situated precisely in the G1 and G2 periods.

As regards mitosis, the inhibition of non-nucleolar RNA synthesis particularlyaffects prophase, which is appreciably lengthened, and this is in accord with the obser-vations of Gonzalez-Fernandez, Fernandez-Gomez, Stockert & Lopez-Saez (1970)in the same material, using different inhibitors of RNA synthesis.

Despite the in vitro selective nature of the action of a-amanitin on a non-nucleolarenzyme, the nucleolus initially responds in our experiment by the phenomenon ofnucleolar segregation, pointing to the transient inhibition of rRNA synthesis that thisdrug showed in vivo (Jacob et al. 1971; Sekeris & Schmid, 1972; Tata, Hamilton &Shields, 1972). This could be interpreted in three ways.

(1) If nucleolar activity is regulated by control from outside.In this respect, it is relevant that irradiation of extranucleolar nucleoplasm produces

lesions in the nucleolus itself (Montgomery, Reynolds & Cook, 1966). Brachet, Hubert& Lievens (1972) postulate that some kind of messenger would be necessary for thefunction of the nucleolar organizer genes to interpret the blockage of embryo develop-ment at the blastula stage by a-amanitin, when nucleoli must appear.

(2) If the condensation of chromosomes with a nucleolar organizer (NOR) phy-sically interacts with NOR activity impairing the transcriptional activity of theribosomal cistrons. The fragmentation of hepatic nucleoli (Marinozzi & Fiume, 1971)is interpreted by Bucci, Nardi, Mancino & Fiume (1971) on the basis of their obser-vations on oocytes of Triturus, as a secondary effect due to chromosomal condensationproduced by the drug.

(3) If the toxin has a direct action on post-transcriptional rRNA maturation.Serfling, Wobus & Panitz (1972) showed that a-amanitin does delay processing of therRNA precursor.

In any case, the recovery of normal morphology and physiology during treatmentmakes this effect of a-amanitin more intriguing and difficult to explain.

In short, the use of a-amanitin, an inhibitor of extranucleolar RNA polymerase,points to: a greater sensitivity to this inhibition in the Gx and G2 periods, which becometwice as long; a lengthening of prophase in preference to other phases of mitosis;and transient effects on the interphase nucleolus, in which it produces segregation, andan initial brake on nucleolar reorganization.

a-amanitin and the division cycle 471

Detection of different responses of the cell cycle to enzyme inhibition seems to bea promising way of bridging the great gap in our knowledge of control of cell develop-ment along its division cycle.

This work has been partially supported by the III Plan de Desarrollo (Spain). We wish tothank Miss M. L. Martinez and M. C. Partearroyo for their skilful technical assistance.

For the generous gift of a-amanitin we are greatly indebted to Professor Wieland, Universityof Heidelberg.

REFERENCES

BASERCA, R. (1968). Biochemistry of the cell cycle: a review. Cell Tissue Kinetics 1, 167-191.BOLLUM, F. J. & POTTER, U. R. (1959). Nucleic acid metabolism in regenerating rat liver. IV.

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BRACHET, J., HUBERT, E. & LIEVENS, A. (1972). The effects of a-amanitin and rifampicins onamphibian egg development. Rev. suisse Zool. 79, 47—63.

Bucci, S., NARDI, I., MANCINO, G. & FIUME, L. (1971). Incorporation of tritiated uridine innuclei of Triturus oocytes treated with a-amanitin. Expl Cell Res. 69, 462-465.

FERNANDEZ-GOMEZ, M. E., STOCKERT, J. C , LOPEZ-SAEZ, J. F. & GIMENEZ-MARTI'N, G. (1969).Staining plant cell nucleoli with AgNO3 after formalin-hydroquinone fixation. Stain Technol.44, 48-49.

GIMENEZ-MARTIN, G., GONZALEZ-FERNANDEZ, A. & L6PEZ-SAEZ, J. F. (1965). A new method oflabelling cells. J. Cell Biol. 26, 305-309.

GONZALEZ-FERNANDEZ, A., LOPEZ-SAEZ, J. F. & GIMENEZ-MARTIN, G. (1966). Duration of thedivision cycle in binucleate cells. Expl Cell Res. 43, 255-267.

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(1970). Effect produced by inhibitors of RNA synthesis on mitosis. Expl Cell Res. 60,320-326.

GONZALEZ-FERNANDEZ, A., GIMENEZ-MARTIN, G. & DE LA TORRE, C. (1971). The duration ofthe interphase periods at different temperatures in root tip cells. Cytobiologie 3, 367-371.

HOTTA, Y. & STERN, H. (1963). Molecular facets of mitotic regulation I. Synthesis of thymidinekinase. Proc. natn. Acad. Sci. U.S.A. 49, 648-654.

JACOB, S. T., SAJDEL, E. M. & MUNRO, H. N. (1971). Mammalian RNA polymerases and theirselective inhibition by amanitin. In Advances in Enzyme Regulation, vol. 9 (ed. G. Weber),pp. 169-181. Oxford: Pergamon.

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K I M , J. H. & PEREZ, A. G. (1965). Ribonucleic acid synthesis in synchronously dividing popu-lations of HeLa cells. Nature, Lond. 207, 974-975.

KLEVECZ, R. R. & RUDDLE, F. H. (1968). Cyclic changes in enzyme activity in synchronizedmammalian cell cultures. Science, N. Y. 159, 634-636.

LINDELL, T. J., WEINBERG, F., MORRIS, P. W., ROEDER, R. G. & RUTTER, W. J. (1970). Specificinhibition of nuclear RNA polymerase II by a-amanitin. Science, N. Y. 170, 447-449.

MAK, S. (1965). Mammalian cell cycle analysis using microspectrophotometry combined withautoradiography. Expl Cell Res. 39, 286-289.

MARINOZZI, V. & FIUME, L. (1971). Effects of a-amanitin on mouse and rat liver cell nuclei.Expl Cell Res. 67, 311-322.

MITTERMAYER, C , BOSSELMANN, R. & BREMERSKOV, V. (1968). Initiation of DNA synthesis ina system of synchronized L-cells: Rhythmicity of thymidine kinase. Europ. J. Biochem. 4,487-489.

MONESI, V. (1969). DNA, RNA, and protein synthesis during the mitotic cell cycle. In Handbookof Molecular Cytology (ed. A. Lima-de-Faria), pp. 472-499. Amsterdam and London: NorthHolland Publishing.

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PETROV, P. & SEKERIS, C. E. (1971). Early action of a-amanitin on extranucleolar ribonucleo-proteins, as revealed by electron microscopic observation. Expl Cell Res. 69, 393-401.

SEKERIS, C. E. & SCHMID, W. (1972). Action of a-amanitin in vivo and in vitro. FEBS Letters,Amsterdam 27, 41-45.

SERFLING, E., WOBUS, U. & PANITZ, R. (1972). Effect of a-amanitin on chromosomal and nucleo-lar RNA synthesis in Chironomus thummi polytene chromosomes. FEBS Letters, Amsterdam20, 148-152.

STIRPE, F. & FIUME, L. (1967). Studies on the pathogenesis of liver necrosis by a-amanitin.Biochem. J. 105, 779-782.

TATA, J. R., HAMILTON, M. J. & SHIELDS, D. (1972). Effects of a-amanitin in vivo on RNApolymerase and nuclear RNA synthesis. Nature, New Biol. 238, 161-164.

WEISSMANN, S. M., SMELLIE, R. M. S. & PAUL, J. (i960). Studies on the biosynthesis of deoxy-ribonucleic acid by extracts of mammalian cells. IV. The phosphorylation of thymidine.Biochim. biophys. Ada 45, 101-110.

{Received 1 August 1973)

ct-amanitin and the division cycle 473

10

mm

6AFig. 6. Silver impregnation of nucleolar material in interphase cells: A, untreated cells;

B, nucleolar segregation in cells treated with 10/tg/ml a-amanitin. x 1200.