@hem.Piooh.Interactions, 14 (1976) 57+5 0 Elsevier Scientific Pubhshing Company, Amsterdam - Printed in The Netberjan
EFFECTS OF ACTINOMYCIN P) AND PUROMYCIN ON CO INDUCED ENDOREDUPLICATION IN CHINESE IN VlTRO
FABRIZIO PALIlT.1, RUGGER0 RICORDY, PAOLO PERTICONE, SILVANA D’ANDREA and MARCO RIZZONI Centro di Genetica Evoluzionistica de1 C.N.R. and Zstituto di Genetica deNWniue Roma, Rome (Italy) (Received August 2Oth, 1976) (Accepted January 18th, 1976)
SUMMARY
Experiments were darried out to study the events through which the process of endoreduplication occurs after treatment .vith colchicine in Chinese hamster cells cultivated in vitro, and to analyse the differen between the processes leading to normal mitosis and those leading to doreduplication. Chinese hamster cells (line C-125) were treated with colchicine (10”’ for 1 h, in order to induce endoreduplication. Before and after colehicme treatment the cultures were treated at various intervals with actinomy and puromycin. For preparation of mitosis specific RNA synthesis quired, and also synthesis of a specific protein, which is not requi endoreduplication. Moreover this suggests not only that the cells that endoreduplicate do not need new RNA and protein synthesis, but that inhibition of their synthesis apparently favours the processes of endoreduplication. -^.____--_I.__-_-.-.--.--~-
_.. .~_. -
.-- -._. -.---
.“.I____ ___~_..~..__.
INTRODUCTION
Endoreduplication may be utilized to investigate the timin biochemical events in order to define temporal markers in the mammalian cells. A previous study [l] was made of the methods by which the pr endoreduplication OCCW~in Chinese hamster cells cultivated in vi treatment with colchicine. This system gives the possibility of con~~llin Abbreviations: E.I., endoreduplication index; M.I., mitotic index.
and regulating the phenomenon. Treatment with colchicine sets off a series of processes that cause the cells that are in G2 to miss mitosis and to go into a second stage of synthesis (S,) in a highly synchronous manner. The interval between the two syntheses of the endoreduplication (S,, before treatment with colchicine, Sz after treatment) has been called G?. The present study analyses the possible differences between the cells that normally go into mitosis and those which endoreduplicate, and to investigate the nature of G?. A study has been made of the effect of ~hibitors of mRNA synthesis (actinomycin D) and protein synthesis (puromycin) on the endoreduplication induced by colchicine. MATERIALS AND METHODS
A line of Chinese hamster cells, a clonal derivative of line CHEF-125 was used. This line is grown in Puck fibroblast medium containing 15% calf serum. Cells were plated in petri dishes with coverslips. According to a method [I] already developed to induce endoreduplication the day. after plating, the cultures are allowed to grow in the presence of colchicine (10e4 84). Between 20 and 28 h after the beginning of the colchicine treatment endoreduplicated mitoses appear. Actinomycin D, 2.4 yg/ml, or puromycin, 50 Erg/ml,were dissolved in the medium and the solution was sterilized by filtration through a membrane filter (Sartorius pore size 0.95 ~1, At the end of each treatment the cultures were washed twice with fresh medium and allowed to grow in fresh mechum with or without colchicine according to the time of treatment with the inhibitors (Fig. 1). The cultures were fixed after hypotonic treatment in a 1 : 3 mixture of acetic acid/methanol and stained with acetic orcein. Effect of uctinomycin or puromycin on entry into mitosk Cultures were given 90 min pulses of actinomycin D or puromycin at 10, 8.5,7,5.5,4 and 2.5 h before fixing, or a 1 h pulse 1 h before fixing. Effect of u~~i~urny~~nI) or p~r~rny~in on the endored~p~iea~ion induced by the colchicine The plan of the experiment is shown in Fig. 1. The cultures may be divided into two series: (a) treated with actinomycin D or puromycin at 8, 6, 4 or 2 h before colchicine treatment (defined at time 0). The exposures to the drugs were for 2 h. Cultures were fixed 23 or 26 h after colchicine ~eatment; (b) treated with actinomycin D or puromycin at 1, 2, 3 or 4 h after colchicine treatment. Exposure to the drugs was for 1 h. Cultures were fixed 23 or 26 h after colchicine treatment. Control cultures were treated with colchicine alone and fixed 23 or 26 h after treatment ~&olc~~i~e controls). Other control cultures were treated with actinomycin D or puromycin at the same times as the cultures in series 58
TWO -hour
ptilses
One-hour
pulses
of
AD
after
series
PU before
or
colchicine
treatment
treatment
[series
a \
fix
b
A
0
[series
a]
b]
series
A
/
colchicine
fix
\
I
I
I
v
V
v
v
v
v
A
A
I
I
I
I
I
I
I
t
I
1
i
-8
-6
-4
-2
0
1
2
3
4
24
26
A colchicine
I
I
S,
4
G?
e--------
SP
Fig. 1. General scheme of the experiment for studying the effects of actinomycin D (AD) and puromycin (PU) on the progression of the cells through the endoreduplication cycle induced by the colchicine.
Q ar.d b, but they were not treated with colchicine at time 0 and were fixed together with the corresponding cultures treated with colchicine (actinomytin D-puromycin controls) after a 4 h pretreatment with colchicine to accumulate mitoses. Untreated control cultures were fixed after a 4 h treatment with colchicine. RESULTS
The effect of actinomycin and puromycin on entiy into mitosis Observations of the M.I. were carried out on 2000 cells. The results are shown in Fig. 2. A reduction in the M.I. was observed in the cultures treated at the times immediately preceding fixation, the effect occurring earlier with puromycin; on the other hand, the cultures treated 10-7 h before fixation displayed a low increase in the M.I. The results are in good agreement with those obtained by other authors 1231. The effect of actinomycin D or puromycin on the endoreduplication induced by colchicine In the untreated controls 0.5% of endoreduplicated cells was observed in even 200 mitoses; for the controls treated with actinomycin D or puromytin alone (actinomycin D-puromycin controls) the same value was found on average. For the cultures treated with colchicine alone, a percentage of 15% en-
59
10 9 8
7 6 5 4 3 2 1 0 -10
-6.36
-7
-5.30
-4
-2.30
-1
Fig. 2.Abscissa: time (II) of administration of 90-min pulses of AD (o- - - - - -0) and BU (0~). Ordinate: MI. (%): the horizontal line represents the control value; vertical bars represent the standard error. Effects of AD and PU on the MI. of normal cells.
doreduplication was observed, which agrees with the data already reported PI. To assess the effect of the actinomycin D or puromycin treatments on the colchicine-induced endoreduplication, a more sensitive parameter was used: namely the E.I. which corresponds to the relative frequency of endoreduplicated mitoses in the overall population (mitoses + interphases) calculated on 2000 cells. The results are shown in Fig. 3. There were no differences between the response to the actinomycin D treatment and the response to the puromycin treatment whether these were carried out before or after the administration of colchicine. For the series of cultures subjected ,to the treatments before the colchicine treatment (series a), it was observed that: (1) the treatments with the two substances have no effect on the E.I. when they are carried out between time -2 and time 0 (treatment with colchkine); (21 they result in a reduction of about 0.1-0.2% in the E.I. however if they occur at times prior to t = -2. For the series of cultures subjected to the treatments after the colchicine treatment (series b), a gradual and continuous increase is observed in the E.I., values of about 4-8s being reached, so that microscopic examination revealed numerous endoreduplicated metaphases iz-~the same field (Fig. 4). No differences were observed as regards the two times of fixation. The M.I. follows a course parallel to that of the E.I. (Fig. 3); the difference between the M.I. and the E.I. is more or less constant and is similar to that found in controls treated with colchicine (colchicine controls).
60
AD FIX 23
PkJ FIX 23 75 13 - 12 11 - 10 9-0 7-4 5-4 32 1 0
AD FIX 26
PU FIX 26
14 13 12
i-4, \ \
,&’ IX
i !f”
11 10 9 % 7
“Er/~~t -* +___--_____________ t 4
5
4
3
2 1
~
0
8
-6
-4
-2
01234
Fig. 3. Abscissa: time (h) of administration of pulses of AD and PU in relation to the time of administration of the colchicine (t = 0 h). Before colchicine treatment the pulses are of 2-h duration; afterwards, 1 h. Ordinate: M.I. ((I- - - - - -0) and E.I. (C-----Q): the horizontal lines represent the control values; vertical bars represent the standard error. The effect of AD and PU on ~olchicine-induced endoredupli~ation.
DISCUSSION
It is considered cific target setting the regulation of duced by different
that colchicine having entered the cell reacts with a speoff the process of endoreduplication. The alteration in the cell cycle that causes endoreduplication may be inagents in different stages of the cell cycle and in different
Fig. 4. (a,b) The appearance of a culture treated with AD 4 h after treatment with colchicine: the endoreduplicated ceils arc indicated by arrows. On the right hand side there is an endoredupl~~ted cell at 1000 X magnification.
experimental material; for .example, in human leucocytes, in C, with (Ymercaptoethanol [4 f and with &mercaptoethanol [5,6] ; in stage S with X-rays [7$3] ; in S with HAS in root tips of Zeta mays [Q] ; in GZ with 8azaguanine in root tips of P&m sutivum [lo J and also in G2 in human leucocytes with colcemid [ll] ; and in all stages in the L strain cells with podophyllic acid [ 12 ] . In the ca,se of colchicine-induced endoreduplication, the cells that endoreduplicate at the moment of treatment are in late S and especially in Gz [1].It is assumed that the target for the induction of the endoreduplication is present in this stage. This may occur either through direct damage to specific targets of colchicine, for example the protein subunits of the microtubules [13] or through indirect damage. As a consequence of the alteration in the cyto-skeleton the cell cycle is altered and the cell undergoes endoreduplication rather than mitosis. In this respect an interesting model is proposed by Lin and Walden [Qf suggesting the presence of “‘transition factors” that regulate the passage from one stage of the cycle to the following by inhibiting through a feed-back mechanism the activity of an initiator of the stage just ended. According to these authors in encioreduplication there occurs a blocking of the tr~sition factor for the passage from stage S to stage G2 or from Gz to mitosis; there would then be no inhibition of the 62
initiator of the synthesis, thus enabling a second synthesis (S2) to occur without the cell having completed its normal cycle. The first type of experiment was intended to confirm, for the present cell line, the results already observed by other authors [2,3]. Treatment with actinomycin D or puromycin in G2 produces an inhibition of mitosis (reduction in the M.I.), by preventing the synthesis of a specific mRNA or a specific protein necessary for mitosis to proceed. We also wished to investigate the consequences of treatments carried out at times further removed from the entry of the cell into mitosis so as to compare the effects on entry into mitosis with those on the formation of the target fcpr endoreduplication. As regards the M.I., it is observed that the earlier treatment,s (probably during the S stage) do not produce any considerable alterations in the way the cell cycle proceeds; there is only a mitotic delay. The second type of experiment was intended to show, on the one hand, the effects of the inhibitors on the formation of the colchicine “target” (series a), and on the other hand the effects of the inhibitors on the possible formation of RNA and proteins necessary for the Sz stage, synthesised just before this stage as a result of the stimulation with colchicine (series b). In the cultures in series (L, it is observed that there is a uniform reduction in the percentage of endoreduplicated cells in the cultures from time -8 to time -2, the latter time corresponding in the cycle to an advanced G?. From this time onward, there is a recovery up to the level of control values (Fig. 3) which would indicate that actinomycin D and puromycin have no effect if administered immediately prior to colchicine stimulation. The results show that, whereas the inhibiting activity of actinomycin D and puromycin on the entry into mitosis affects structures that are formed in the G2 stage just before mitosis, the same inhibiting activity of these substances on the endoreduplication affects different structures (the “target” of the colchicine for endoreduplication) which form in a period of the cycle prior to the late S stage. We may therefore consider that from time -2 (G? stage) everything necessary is ready for colchicine to trigger the process of endoreduplication. That the phenomenon of inhibition is specific for endoreduplication is also shown by the fact that no regular alteration is shown in the values of the M.I., although they vary in a wide range, as far as series a is concerned (Fig. 3). As regards the cultures in series b - those treated with actinomycin D or puromycin after stimulation with colchicine - a considerable increase in the E.I. may be observed, compared with the controls. The times at which the treatments are carried out correspond to the part of the G? stage extending from the colchicine treatment to the beginning of the S2 stage. This should correspond to a G1 stage, in which there would be an increase in the RNA and protein synthesis for starting the second phase of DNA synthesis. What is observed suggests not only that the cells that endoreduplicate have 63
no need of a new BNA and protein synthesis but that in fact the inhibition of their synthesis even favours the process of endor~upli~tion. Almost identical results - albeit in a different system - have been obtained by Nag1 [14) in root tips of A&urn curinatum in which the treatments with actinomycin D or f1 histone more strongly inhibit the mitosis of the endoreduplication. That the increase in the E.I. is real is also shown by analysis of the M.1, The increase in MI. in the cultures treated with the subs~nces after colchicine is merely due &o the increase in the endoreduplicated cells that go into mitosis (Fig. 3). It is suggested therefore that in normal conditions colchicine triggers the process of endoreduplication in a greater number of cells than appear endoreduplicated in mitosis and this would occur because in the cells induced by colchicine to endoreduplica~ there may occur regulation phenomena which by blocking the regular course of the endoreduplication cycle lower the percentage of endoreduplicated mitoses. The action of actinomycin D and puromycin would inhibit these regulation processes with a consequent increase in the percentage of endoreduplicated cells.
We are grateful to Mr.
L
Frediani for his Pxcellent technical assistance.
1 M, Rizzoni and F. Palitti, Regulatory mechanism of cell division, Exp. Cell Res. 77 (1973) 456. 2 0.1. Epifanova, I.M. Smolenskaia, M.V. Sevastianova and A.G. Kurdyomova, Effects of actinomycin D and puromycin on the mitotic cycle in synchronized cell culture, Exptl. Cell Res., 58 (lQ69) 401. 3 R.A. Tobey, D.F. Petersen, E.G. Anderson and T.T. Puck, Life cycle analysis of m,ammalian cells, III. Tbe inhibition of division in Chinese hamster cells by puromytin and actinomycin, Biophys. J., 6 (1966) 567. 4 H.G. Schwarzacher and W. Schnedl, Position of labelled chrom&ids in diplochromosomes of endoreduplicated cells after uptake of tritiated thymidine, Nature, 269 (1966) 197. 5 J-F. Jackson, Polyploidy and endoreduplication in human leucocyte cultures treated with ~-mer~aptoethanol, Exptl. Cell Res., 31 (1963) 194. 6 J.F. Jackson and D. Killander, DNA-synthesis in phytohemagglutinin=stimuIated human leucocyte culdures treated with flmmercaptoethanol, Exptl. Cell Res., 33 (1963) 459. 7 A.G. Bell and D.G. Baker, X-Irradiation-induced polyploidy in humrm leucocyte cultures, Exptl. Cell Res., 38 (1965) 144. 8 C.E. Nasjleti, J.H. Walden and M.H. Spencer, Polyploidization and aberration of human chromosomes induced in vitro and in vivo with ionizing radiations, J. Nucl. Med., I(lQ66) 159. 9 M.S. Lin and D.B. Walden, Endoreduplication induced by hydroxylamine sulfate in Zeu mays root tip nuclei, Exptl. Cell Res., 86 (1974) 47. 16 V. Nuti Ronchi, S. Avanzi and F. D’Amato, Chromosome endo~duplication (endopolyploidy) in pea root meristems induced by 8*azaguanine, Caryologia, 18 (1965) 599.
64
11 B. Herreros, A. Guerro and E. Romo, Colcemid-induced polypioidy, Lancet, II (1966) 500. 12 B. Neskovic, A. Milosavijevic, M. Pantelic, M. Bugarski, 2. Ajdaric, R. Tomin and S. Nikolic, Signs of different cellular activities in intermitotic correlated with mitosis, Neoplasma, 14 (1967) 585. 13 SE. Maiawista and K.G. Bensch, Human polymorphonuciear leukocytes: demonstration of microtubules and effect of colchisine, Science, 156 (1967) 521. 14 W. Nag], Differential inhibition by actinomycin D and histone ft of mitosis and endomitosis in Allium carinatum. 2. Pflenzenphysiol., 63 (1970) 316.
EFFECTS OF ACTINOMYCIN P) AND PUROMYCIN ON CO INDUCED ENDOREDUPLICATION IN CHINESE IN VlTRO
FABRIZIO PALIlT.1, RUGGER0 RICORDY, PAOLO PERTICONE, SILVANA D’ANDREA and MARCO RIZZONI Centro di Genetica Evoluzionistica de1 C.N.R. and Zstituto di Genetica deNWniue Roma, Rome (Italy) (Received August 2Oth, 1976) (Accepted January 18th, 1976)
SUMMARY
Experiments were darried out to study the events through which the process of endoreduplication occurs after treatment .vith colchicine in Chinese hamster cells cultivated in vitro, and to analyse the differen between the processes leading to normal mitosis and those leading to doreduplication. Chinese hamster cells (line C-125) were treated with colchicine (10”’ for 1 h, in order to induce endoreduplication. Before and after colehicme treatment the cultures were treated at various intervals with actinomy and puromycin. For preparation of mitosis specific RNA synthesis quired, and also synthesis of a specific protein, which is not requi endoreduplication. Moreover this suggests not only that the cells that endoreduplicate do not need new RNA and protein synthesis, but that inhibition of their synthesis apparently favours the processes of endoreduplication. -^.____--_I.__-_-.-.--.--~-
_.. .~_. -
.-- -._. -.---
.“.I____ ___~_..~..__.
INTRODUCTION
Endoreduplication may be utilized to investigate the timin biochemical events in order to define temporal markers in the mammalian cells. A previous study [l] was made of the methods by which the pr endoreduplication OCCW~in Chinese hamster cells cultivated in vi treatment with colchicine. This system gives the possibility of con~~llin Abbreviations: E.I., endoreduplication index; M.I., mitotic index.
and regulating the phenomenon. Treatment with colchicine sets off a series of processes that cause the cells that are in G2 to miss mitosis and to go into a second stage of synthesis (S,) in a highly synchronous manner. The interval between the two syntheses of the endoreduplication (S,, before treatment with colchicine, Sz after treatment) has been called G?. The present study analyses the possible differences between the cells that normally go into mitosis and those which endoreduplicate, and to investigate the nature of G?. A study has been made of the effect of ~hibitors of mRNA synthesis (actinomycin D) and protein synthesis (puromycin) on the endoreduplication induced by colchicine. MATERIALS AND METHODS
A line of Chinese hamster cells, a clonal derivative of line CHEF-125 was used. This line is grown in Puck fibroblast medium containing 15% calf serum. Cells were plated in petri dishes with coverslips. According to a method [I] already developed to induce endoreduplication the day. after plating, the cultures are allowed to grow in the presence of colchicine (10e4 84). Between 20 and 28 h after the beginning of the colchicine treatment endoreduplicated mitoses appear. Actinomycin D, 2.4 yg/ml, or puromycin, 50 Erg/ml,were dissolved in the medium and the solution was sterilized by filtration through a membrane filter (Sartorius pore size 0.95 ~1, At the end of each treatment the cultures were washed twice with fresh medium and allowed to grow in fresh mechum with or without colchicine according to the time of treatment with the inhibitors (Fig. 1). The cultures were fixed after hypotonic treatment in a 1 : 3 mixture of acetic acid/methanol and stained with acetic orcein. Effect of uctinomycin or puromycin on entry into mitosk Cultures were given 90 min pulses of actinomycin D or puromycin at 10, 8.5,7,5.5,4 and 2.5 h before fixing, or a 1 h pulse 1 h before fixing. Effect of u~~i~urny~~nI) or p~r~rny~in on the endored~p~iea~ion induced by the colchicine The plan of the experiment is shown in Fig. 1. The cultures may be divided into two series: (a) treated with actinomycin D or puromycin at 8, 6, 4 or 2 h before colchicine treatment (defined at time 0). The exposures to the drugs were for 2 h. Cultures were fixed 23 or 26 h after colchicine ~eatment; (b) treated with actinomycin D or puromycin at 1, 2, 3 or 4 h after colchicine treatment. Exposure to the drugs was for 1 h. Cultures were fixed 23 or 26 h after colchicine treatment. Control cultures were treated with colchicine alone and fixed 23 or 26 h after treatment ~&olc~~i~e controls). Other control cultures were treated with actinomycin D or puromycin at the same times as the cultures in series 58
TWO -hour
ptilses
One-hour
pulses
of
AD
after
series
PU before
or
colchicine
treatment
treatment
[series
a \
fix
b
A
0
[series
a]
b]
series
A
/
colchicine
fix
\
I
I
I
v
V
v
v
v
v
A
A
I
I
I
I
I
I
I
t
I
1
i
-8
-6
-4
-2
0
1
2
3
4
24
26
A colchicine
I
I
S,
4
G?
e--------
SP
Fig. 1. General scheme of the experiment for studying the effects of actinomycin D (AD) and puromycin (PU) on the progression of the cells through the endoreduplication cycle induced by the colchicine.
Q ar.d b, but they were not treated with colchicine at time 0 and were fixed together with the corresponding cultures treated with colchicine (actinomytin D-puromycin controls) after a 4 h pretreatment with colchicine to accumulate mitoses. Untreated control cultures were fixed after a 4 h treatment with colchicine. RESULTS
The effect of actinomycin and puromycin on entiy into mitosis Observations of the M.I. were carried out on 2000 cells. The results are shown in Fig. 2. A reduction in the M.I. was observed in the cultures treated at the times immediately preceding fixation, the effect occurring earlier with puromycin; on the other hand, the cultures treated 10-7 h before fixation displayed a low increase in the M.I. The results are in good agreement with those obtained by other authors 1231. The effect of actinomycin D or puromycin on the endoreduplication induced by colchicine In the untreated controls 0.5% of endoreduplicated cells was observed in even 200 mitoses; for the controls treated with actinomycin D or puromytin alone (actinomycin D-puromycin controls) the same value was found on average. For the cultures treated with colchicine alone, a percentage of 15% en-
59
10 9 8
7 6 5 4 3 2 1 0 -10
-6.36
-7
-5.30
-4
-2.30
-1
Fig. 2.Abscissa: time (II) of administration of 90-min pulses of AD (o- - - - - -0) and BU (0~). Ordinate: MI. (%): the horizontal line represents the control value; vertical bars represent the standard error. Effects of AD and PU on the MI. of normal cells.
doreduplication was observed, which agrees with the data already reported PI. To assess the effect of the actinomycin D or puromycin treatments on the colchicine-induced endoreduplication, a more sensitive parameter was used: namely the E.I. which corresponds to the relative frequency of endoreduplicated mitoses in the overall population (mitoses + interphases) calculated on 2000 cells. The results are shown in Fig. 3. There were no differences between the response to the actinomycin D treatment and the response to the puromycin treatment whether these were carried out before or after the administration of colchicine. For the series of cultures subjected ,to the treatments before the colchicine treatment (series a), it was observed that: (1) the treatments with the two substances have no effect on the E.I. when they are carried out between time -2 and time 0 (treatment with colchkine); (21 they result in a reduction of about 0.1-0.2% in the E.I. however if they occur at times prior to t = -2. For the series of cultures subjected to the treatments after the colchicine treatment (series b), a gradual and continuous increase is observed in the E.I., values of about 4-8s being reached, so that microscopic examination revealed numerous endoreduplicated metaphases iz-~the same field (Fig. 4). No differences were observed as regards the two times of fixation. The M.I. follows a course parallel to that of the E.I. (Fig. 3); the difference between the M.I. and the E.I. is more or less constant and is similar to that found in controls treated with colchicine (colchicine controls).
60
AD FIX 23
PkJ FIX 23 75 13 - 12 11 - 10 9-0 7-4 5-4 32 1 0
AD FIX 26
PU FIX 26
14 13 12
i-4, \ \
,&’ IX
i !f”
11 10 9 % 7
“Er/~~t -* +___--_____________ t 4
5
4
3
2 1
~
0
8
-6
-4
-2
01234
Fig. 3. Abscissa: time (h) of administration of pulses of AD and PU in relation to the time of administration of the colchicine (t = 0 h). Before colchicine treatment the pulses are of 2-h duration; afterwards, 1 h. Ordinate: M.I. ((I- - - - - -0) and E.I. (C-----Q): the horizontal lines represent the control values; vertical bars represent the standard error. The effect of AD and PU on ~olchicine-induced endoredupli~ation.
DISCUSSION
It is considered cific target setting the regulation of duced by different
that colchicine having entered the cell reacts with a speoff the process of endoreduplication. The alteration in the cell cycle that causes endoreduplication may be inagents in different stages of the cell cycle and in different
Fig. 4. (a,b) The appearance of a culture treated with AD 4 h after treatment with colchicine: the endoreduplicated ceils arc indicated by arrows. On the right hand side there is an endoredupl~~ted cell at 1000 X magnification.
experimental material; for .example, in human leucocytes, in C, with (Ymercaptoethanol [4 f and with &mercaptoethanol [5,6] ; in stage S with X-rays [7$3] ; in S with HAS in root tips of Zeta mays [Q] ; in GZ with 8azaguanine in root tips of P&m sutivum [lo J and also in G2 in human leucocytes with colcemid [ll] ; and in all stages in the L strain cells with podophyllic acid [ 12 ] . In the ca,se of colchicine-induced endoreduplication, the cells that endoreduplicate at the moment of treatment are in late S and especially in Gz [1].It is assumed that the target for the induction of the endoreduplication is present in this stage. This may occur either through direct damage to specific targets of colchicine, for example the protein subunits of the microtubules [13] or through indirect damage. As a consequence of the alteration in the cyto-skeleton the cell cycle is altered and the cell undergoes endoreduplication rather than mitosis. In this respect an interesting model is proposed by Lin and Walden [Qf suggesting the presence of “‘transition factors” that regulate the passage from one stage of the cycle to the following by inhibiting through a feed-back mechanism the activity of an initiator of the stage just ended. According to these authors in encioreduplication there occurs a blocking of the tr~sition factor for the passage from stage S to stage G2 or from Gz to mitosis; there would then be no inhibition of the 62
initiator of the synthesis, thus enabling a second synthesis (S2) to occur without the cell having completed its normal cycle. The first type of experiment was intended to confirm, for the present cell line, the results already observed by other authors [2,3]. Treatment with actinomycin D or puromycin in G2 produces an inhibition of mitosis (reduction in the M.I.), by preventing the synthesis of a specific mRNA or a specific protein necessary for mitosis to proceed. We also wished to investigate the consequences of treatments carried out at times further removed from the entry of the cell into mitosis so as to compare the effects on entry into mitosis with those on the formation of the target fcpr endoreduplication. As regards the M.I., it is observed that the earlier treatment,s (probably during the S stage) do not produce any considerable alterations in the way the cell cycle proceeds; there is only a mitotic delay. The second type of experiment was intended to show, on the one hand, the effects of the inhibitors on the formation of the colchicine “target” (series a), and on the other hand the effects of the inhibitors on the possible formation of RNA and proteins necessary for the Sz stage, synthesised just before this stage as a result of the stimulation with colchicine (series b). In the cultures in series (L, it is observed that there is a uniform reduction in the percentage of endoreduplicated cells in the cultures from time -8 to time -2, the latter time corresponding in the cycle to an advanced G?. From this time onward, there is a recovery up to the level of control values (Fig. 3) which would indicate that actinomycin D and puromycin have no effect if administered immediately prior to colchicine stimulation. The results show that, whereas the inhibiting activity of actinomycin D and puromycin on the entry into mitosis affects structures that are formed in the G2 stage just before mitosis, the same inhibiting activity of these substances on the endoreduplication affects different structures (the “target” of the colchicine for endoreduplication) which form in a period of the cycle prior to the late S stage. We may therefore consider that from time -2 (G? stage) everything necessary is ready for colchicine to trigger the process of endoreduplication. That the phenomenon of inhibition is specific for endoreduplication is also shown by the fact that no regular alteration is shown in the values of the M.I., although they vary in a wide range, as far as series a is concerned (Fig. 3). As regards the cultures in series b - those treated with actinomycin D or puromycin after stimulation with colchicine - a considerable increase in the E.I. may be observed, compared with the controls. The times at which the treatments are carried out correspond to the part of the G? stage extending from the colchicine treatment to the beginning of the S2 stage. This should correspond to a G1 stage, in which there would be an increase in the RNA and protein synthesis for starting the second phase of DNA synthesis. What is observed suggests not only that the cells that endoreduplicate have 63
no need of a new BNA and protein synthesis but that in fact the inhibition of their synthesis even favours the process of endor~upli~tion. Almost identical results - albeit in a different system - have been obtained by Nag1 [14) in root tips of A&urn curinatum in which the treatments with actinomycin D or f1 histone more strongly inhibit the mitosis of the endoreduplication. That the increase in the E.I. is real is also shown by analysis of the M.1, The increase in MI. in the cultures treated with the subs~nces after colchicine is merely due &o the increase in the endoreduplicated cells that go into mitosis (Fig. 3). It is suggested therefore that in normal conditions colchicine triggers the process of endoreduplication in a greater number of cells than appear endoreduplicated in mitosis and this would occur because in the cells induced by colchicine to endoreduplica~ there may occur regulation phenomena which by blocking the regular course of the endoreduplication cycle lower the percentage of endoreduplicated mitoses. The action of actinomycin D and puromycin would inhibit these regulation processes with a consequent increase in the percentage of endoreduplicated cells.
We are grateful to Mr.
L
Frediani for his Pxcellent technical assistance.
1 M, Rizzoni and F. Palitti, Regulatory mechanism of cell division, Exp. Cell Res. 77 (1973) 456. 2 0.1. Epifanova, I.M. Smolenskaia, M.V. Sevastianova and A.G. Kurdyomova, Effects of actinomycin D and puromycin on the mitotic cycle in synchronized cell culture, Exptl. Cell Res., 58 (lQ69) 401. 3 R.A. Tobey, D.F. Petersen, E.G. Anderson and T.T. Puck, Life cycle analysis of m,ammalian cells, III. Tbe inhibition of division in Chinese hamster cells by puromytin and actinomycin, Biophys. J., 6 (1966) 567. 4 H.G. Schwarzacher and W. Schnedl, Position of labelled chrom&ids in diplochromosomes of endoreduplicated cells after uptake of tritiated thymidine, Nature, 269 (1966) 197. 5 J-F. Jackson, Polyploidy and endoreduplication in human leucocyte cultures treated with ~-mer~aptoethanol, Exptl. Cell Res., 31 (1963) 194. 6 J.F. Jackson and D. Killander, DNA-synthesis in phytohemagglutinin=stimuIated human leucocyte culdures treated with flmmercaptoethanol, Exptl. Cell Res., 33 (1963) 459. 7 A.G. Bell and D.G. Baker, X-Irradiation-induced polyploidy in humrm leucocyte cultures, Exptl. Cell Res., 38 (1965) 144. 8 C.E. Nasjleti, J.H. Walden and M.H. Spencer, Polyploidization and aberration of human chromosomes induced in vitro and in vivo with ionizing radiations, J. Nucl. Med., I(lQ66) 159. 9 M.S. Lin and D.B. Walden, Endoreduplication induced by hydroxylamine sulfate in Zeu mays root tip nuclei, Exptl. Cell Res., 86 (1974) 47. 16 V. Nuti Ronchi, S. Avanzi and F. D’Amato, Chromosome endo~duplication (endopolyploidy) in pea root meristems induced by 8*azaguanine, Caryologia, 18 (1965) 599.
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11 B. Herreros, A. Guerro and E. Romo, Colcemid-induced polypioidy, Lancet, II (1966) 500. 12 B. Neskovic, A. Milosavijevic, M. Pantelic, M. Bugarski, 2. Ajdaric, R. Tomin and S. Nikolic, Signs of different cellular activities in intermitotic correlated with mitosis, Neoplasma, 14 (1967) 585. 13 SE. Maiawista and K.G. Bensch, Human polymorphonuciear leukocytes: demonstration of microtubules and effect of colchisine, Science, 156 (1967) 521. 14 W. Nag], Differential inhibition by actinomycin D and histone ft of mitosis and endomitosis in Allium carinatum. 2. Pflenzenphysiol., 63 (1970) 316.
