Effect of hydrocortisone, adrenalin and actinomycin D on transition of cells to the DNA synthesis phase

EFFECT OF HYDROCORTISONE, ADRENALIN AN 11 ACTINOMYCIN D ON TRANSITION OF CELLS TO THE DNA SYNTHESIS PHASE 0. S. FRANKFURT

Received December 5. 1967

of 11X.1 synthesis is an event in the cell cycle of grcal significancr~ for control of cell division, as in the majority of mammalian tissues transition to the 11X.4 synthesis phase (S phase) almost al\vays r~ults in wII division. Initiation of DNA synthesis in renewing tissues shows that the ccl1 has made a choice betlvcen division and dil’ferentiation in favour of the former. As a result, control of cell division hecomcs that of initiation of DSA synthtasis [l]. On the hasis of the above it hecomcs clear that one of the c*hicf aims in investigating the cell cycle is the analysis of sequential events in the l)rtasy”thetic period that leads to the initiation of DNA synthesis. Important inf’ormntion on this question may he obtained hy stutI;ving changes in transition to the DNA synthesis phase induced hy actions applied to tlif‘f’erent parts of the presynthetic period. The present paper deals \vith Ihr elt’ects of two hormones, hytlrocortisonc anti adrenalin, and of a specific inhibitor of RNA synthesis, actinomyb 1) on transition to the l)NA synthesis phase of squamous epithelial cells of a IIIOUS~ forestomach. In\-estigation of these substances seems to be ncccssary, as hydrocortisone anti adrenalin are hormones playing an importanl part in the control of cell proliferation [B], xvhile actinomycin 1) may give information on the role of RNA synthesis during preparation for the 11X.1 synthesis [13]. MATERIAL AND METHODS

ISITIATION

In the majority of experiments CSHA mice aged approx. 2 months weighing 20 k2 g were used. Experiments with adrenalin (Table 2) used CSHA mice aged 6-8 months weighing 28 +2 g. In all experiments mice received 1 PC/g 3H-thymidine (sp. act 1.4 C/m&‘) injected intraperitoneally and the animals were sacrificed 1 h later. At different times prior to the injection of 3H-thymidine the mice received a suspension of hydrocortisone acetate (Richter, Hungary), saline solutions of adrenalin or actinomycin D (Serva, Heidelberger). All solutions were injected intraperitoneally in 0.2 ml saline per 20 g of weight. Experimental

Cell ISesearch 52

Transition

of cells to Ijll;A

synthesis

223

phme

The methods of preparation for autoradiographs of the forestomach were described previously [Q]. The percentage of labelled basal cells (index labelling, Ii) was determined for 1000 basal cells of squamous epithelium of mouse forestomach. At each time interval 5 mice were used. Values were analysed for significance with the t test. RESULTS Diurnrrl

uariations

of index

labelling

Significant diurnal variations of index labelling in squamous epithelium of mouse forestomach were shown previously [9]. In the present work these diurnal variations \vere studied in detail. Fig. 1 shovvs that between 6 a.m. and 4.30 p.m. the index labelling is low and thereafter increases abruptly. It is evident that many cells enter the S phase during a short time interval: between 4.30 and 6 p.m. Such a high degree of synchrony gives an opportunity to study the effect of different factors on certain parts of the presynthetic period. For example, if a substance is injected at 9 a.m., under its action appear to be the cells for which the DNA synthesis will be initiated in 9 h. Therefore the following experiments were made in accordance with the scheme: substances were injected over the period of 6 a.m. to 3 p.m. and the index labelling was determined at 6 p.m. Efect

of hydrocortisone

on trnnsition

of cells to the DIVA synthesis

phase

\Vhen 5 ,ug/g of hydrocortisone were injected at 6 a.m. or 9 a.m. there was no increase in I, at 6 p.m. If this hormone dose was injected at 12 a.m. or 3 p.m., then at 6 p.m., I, did not differ from the control value (Table 1). This shows that hydrocortisone inhibits the initiation of DNA synthesis and that there is a critical period during the preparation for the DNA synthesis in which the sensitivity of cells to inhibition by hydrocortisone becomes lost. To study the inhibiting effect as a function of the hormone dose, different doses of hydrocortisone were injected at 9 a.m. and I, was determined at 6 p.m. (Table 1). Injection of 2.5 pg/g of hydrocortisone inhibits the transition of cells to the S phase and after injection of 1.25 or 0.5 ,ug/g there is no inhibition. The effect of a large, apparently non-physiological hormone dose (50 pug/g), was also studied. This dose of hydrocortisone inhibits the transition of cells to the S phase when injected both 9 and 6 h before the initiation of DNA synthesis (Table 1). Thus, on injection of a large dose of hydrocortisone, the same as in the case of significantly smaller seemingly physiological doses, a sensitive and a resistant phase are observed. The difference is that the loss of Experimental

Cell Research 52

The duration of hloclting of the transition to the S phase after i n,jcc*iiotl of 5 pg/g of hytirocortisone at 9 a.m. \vas also tirtern~inrtf. III this eslwriment I1 at 9 p.m. \vas 7.5 per cent anti at 12 p.m. 14.X per cent. Thus the wlls ckntcr the S phase approx. 15 h after the injection of h~tir~)~ortisoIir, i.e. ti h Inter than in control mice.

15

0

Fig. l.-Percentage-of cells labelled out of basal cells in squamous epithelium of mouse forestomach vs time of day. Abscissa: Time of injection of 3H-thymidine; ordinate: yO of labelled cells. Fig. 2.-Diagram of mitolic cycle in squamous epithelium of mouse forestomach. (a) Initiation of DNA synthesis sensitive to hydrocortisone and actinomycin; (b) decision on initiation of DNA synthesis (RNA synthesis), stimulated by adrenalin; (c) initiation of DNAsynthesis resistant to hydrocortisone and actinomycin. Fig. 3.--Sequence of events leading to diurnal variations in index labelling. T is the lime between fall in concentration of glucocorticoids and resumption of cells transition to the R phase. t, is the duration of the R phase. is is the duration of the S phase (details in the text). (a) Level of glucocorticoids inhibiting transition to the R phase; (b) transition to R phase: (c) transition to Sphase; (d) index labelling. Experimental

Cell Kesenrch 52

Transition

of cells to DNA4 synthesis

225

phase

One of the most difficult problems in the analysis of the data obtained is to find out what are the doses of hydrocortisone to be considered in the physiological range. Corticosterone in mice is an adrenal hormone of a glucocorticoid activity and we know of no studies involving the comparison of the activity of hydrocortisone and corticosterone injected into the mouse. The l’a~rz 1. Percentage of labelled cells out of the basal cells of squamous epithelium of mouse forestomach at different times after injection of hydrocortisone Time between injections of hydrocortisone and of 3H-thymidinea W 9

6 3

Hydrocortisone,

pg/g weight

50

5

z5

2.3C1.7’ 2.9$-1.4c 11.5 k4.8

6.2i-1.8C 14.6 k 4.1 14 i4.2

7.6 + 3.gC

1.25 lO.S+S.l

0.5 14.7 + 3.3

Control 14.lk5.4”

14.2 k2.4

a All mice received %H-thymidine at 6 p.m. and were killed 1 h later. ’ Control mice in this group received saline 9 h before injection of 3H-thymidine. ’ These values are significantly different from control values (p ~0.05).

extrapolation, sometimes applied, of the amount of hormone secreted by human adrenal to the dose injected into the mouse is very doubtful for many reasons. Physiological doses of corticosterone for rodents are known. For example, 7-15 ,uglg of corticosterone is the dose necessary for normalisation of growth in adrenalectomized rats [17]. It may be supposed that 2.5-5 pug/g of hydrocortisone is a dose displaying a glucocorticoid activity that corresponds to the maximum value of the physiological range. It is evident that 50 ,ug/g of hydrocortisone is far above the physiological value. The fact that it was possible to explain the diurnal variations in the index labelling on the basis of the results obtained with 5 ,ug/g of hydrocortisone (see discussion) is an argument in favour of the physiological value of this dose. Eflect

of adrenalin

on transition

of cells to the DNA

synthesis

phase

Adrenalin inhibits the transition of cells from the G2 phase to mitosis and was shown to play an important part in the control of cell proliferation [3]. In this connection it was important to study the effect of adrenalin on the transition of cells to the S phase. The results obtained were quite unexpected. 15-lx1811

Experimenfal Cell Research 52

Time between injections of adrenalin and of 3H-thymidinca (h) 9 12 15

i\drenalin, 2.5 27 (1X-34) 15.8 (16.X-69.5) 35.1 (1X-64)

@g/g weight 1

23 (15-37)

0.25 11.9 (10&13.(i) -. -

13.:s i(i-23) 23 I1 X-27) lS.;r ilCi-26.1)

a All mice received adrenalin at 9 a.m., 3H-thymidine 9-15 h laler and were killed injection of 3H-thymidine. b Control mice received 3H-thymidine al the same time as corresponding experimcnlat

1 h after groups.

p.m. the increase \vas far more significant and reached in individual mice such estremely high values as 65-69 per cent. Approx. 9 h were neccssar! for the appearance of the stimulating effect of adrenalin on transition 01’ cells to the S phase, i.e. when adrenalin was injected at 12 a.m. or 3 p.m. there \+-as no increase in I, at 6 p.m. above control values. As can be seen from Table 2 injection of 1 ,ug/g of adrenalin stimulated the transition of cells to the S phase and no stimulation was observed after injection of 0.25 pg/g. Table 2 lists the results of one of the three performed Stimulation of the transition of cells to the S experiments using adrenalin. phase \vas attained in all three experiments. From the 34 mice \\-hich rcceived 2.5 pg/g of adrenalin in these three experiments in 16 mice I1 \vas above 30 per cent, that is a higher value than that met in control mice. As can be seen from the paper by Bullough and Laurence 1.5 , another effect of adrenalin on the cell cycle, blocking of transition of cells to mitosis, appeared at lower doses than the stimulation of transition of cells to the S The stimulation by adrenalin of cell phase, observed in our experiments. transition to the S phase seems to be a non-physiological efyect, as the I, values attained after stimulation were never met in normal mice. Experimentul

Cell Research 52

Trnnsition Erect

of

actinomycin

of cells

D on transition

227

to DNA synthesis phase of cells

to

the DLVA synthesis phase

Actinomycin D is a specific inhibitor of RNA synthesis and its effect on the initiation of DNA synthesis was studied by several workers [ 10, 13, 181. It was shown that actinomycin D blocks the initiation of DNA synthesis. When the action of actinomycin D begins only several hours prior to initiation of DNA synthesis there is a decrease in the inhibition of the transition of cells to the DNA synthesis phase. This research shobvs the important role of RNA synthesis in preparation for DNA synthesis. The aim of our experiments was to elucidate whether the disappearance of sensitivity to the inhibiting effect of actinomycin D several hours prior to initiation of DNA synthesis was observed in the case of the epithelium of mouse forestomach. If such an effect exists, it was important to find out whether the loss of sensitivity to hydrocortisone and actinomycin D coincide in time. The action of two doses of actinomycin D (0.1 pug/g and 1 ,ug/g) was studied (Table 3). Injection of 0.1 pg/g of actinomycin D gave the same effect as that of ,5 pg/g of hydrocortisone: the transition to the S phase was blocked if the substance was injected 9 h and not blocked if injection was made :I-6 h prior to initiation of DNA synthesis. A large dose of actinomycin D inhibits the transition to the S phase whether injected 9, 6 or 3 h prior to initiation of DNA synthesis. DISCUSSION

Decision on DNA synthesis period of cell cycle

rind

existence of the R phase in the presynthetic

The effect of three substances on the transition of cells to the S phase was studied. The results obtained show that to the end of the presynthetic period there is a phase which differs from the preceding part of the presynthetic, period in the reaction to the factors affecting the transition of cells to the S phase. Sensitivity of cells to the inhibiting action of 5 ,ug/g of hydrocortisone on transition to the S phase is lost at some time ranging from 6 to 9 h prior to initiation of DNA synthesis. It will be of interest to note that the same 6-9 h are necessary for the appearance of the stimulating effect of adrenalin on transition to the S phase. Evidently within the presynthetic period 6-9 h prior to initiation of DNA synthesis there is a critical transition which corresponds to the start of processes inevitably leading to the initiation of DNA synthesis. After this transition hydrocortisone in physiological doses does not Experimental

Cell Research 52

ac*t on transition IO the S phase. ;\clrenalin also acts 011this c~rilical lra114ilion, this inducing it in cells in \\-hic~h, in the absence of slim~llation b\- :r(lr~~rralin, transition \VOLIICI ow~r later or nol al all. The cslremely high \.alt~tb 01’ inctcb\ lahelling is in fayonr of the latter possibility. ‘I’a1%1.13. Percenttrge of lnbelled cells out o/’ the lmsol cells ~~‘.s~~~~~III~o~Is epitllelirru~ of‘ mouse forestonwch nt diferent times after injection o/’ nctinom~gc~i~l I) Time

between injections of actinomycin 11 and of 3H-thymidinea (h) 9 li 3

.\ctinomycin D, ,ug/g weight 1

0.1

4.4 5 26 6.7f4.2” 8.5 -t 1 .ib

5.3 +2.tb 10.9 i -1.2 12 i2.x

(hntrol

I”.:! “4.5

a All mice received 3H-thymidine b These values are significantly

at 6 p.m. and were killed 1 II later. different from control values (p --O.(C).

Experiments using actinomycin D sho\v that this critical transition is c’onnected with the synthesis of RN,4 necessary for synthesis of DSA. It is of interest to note that the dose of actinomyrin D which in our experiments displayed an inhibiting action only prior to the critical transition described above is close to that inhibiting stimulation of RNA synthesis in the regenerating liver and showing no effect on the RNA synthesis in normal liver 110’. Thus, this dose of actinomycin I> presumably inhibits the RNA synthesis connected with the preparation for DNA synthesis. RNA synthesis in the last part of the presynthetic period in the squamous epithelium of the forestomach is evidently connected with the preparation for DNA synthesis same as for some other tissues [ 13, 18]. It may be supposed that the decision on initiation of DK’A synthesis consists in the synthesis of specific RNA. For the cells studied in our experiments this synthesis comes to its close (i-9 h prior to initiation of DNA synthesis. The part of the presynthetic period after decision on DNA synthesis which is characterized by the resistance to hydrocortisone and actinomycin I> \\-ill be denoted as R phase (resistant phase) (Fig. 2). Diurnal uuriutions in the glucocorticoid variations in index lubelling

leoel us a mechanism lending to diurnal

Diurnal variations in the percentage of cells synthesizing DNA \vere found in many tissues of adult mammals [7, l-2, 15, 16, 191. These tiinrnal varia-

Transition

of’ cells to DNA synthesis phnse

229

tions may be explained on the basis of our experiments on the action of hydrocortisone on transition to the S phase and reported information on diurnal variations in glucocorticoid level [ll 1. The sequence of events leading to diurnal variations in the index labelling may be described as follows: At some time of the day the level of glucocorticoids in blood increases and the transition of cells to the R phase is blocked but cells which already come to the R phase pass through it and enter the S phase. Consequently index labelling falls to a minimum in a time equal to the duration of R and S phases after the amount of glucocorticoids increases to a level inhibiting the transition to the R phase. Some time (z) after the decrease in the glucocorticoids level transition of cells to the R phase is renewed. After a time z plus duration of the R phase the cells which were blocked before transition to the R phase pass ito the S phase and consequently index labelling reaches its maximum value. In order that diurnal variations in I, (shown in Fig. 1) take place, transition of cells to the S phase must stop at approx. 11 p.m. and that to the R phase at approx. 3 p.m. This corresponds to the achievement of maximum concentration of corticosterone in blood of mice at 4 p.m. [ll] The transition of cells to the S phase resumes approx. 15 h after injection of hydrocortisone. Taking into account the rapid metabolism of injected hydrocortisone [S] the duration of the blocking action after a decrease in concentration of glucocorticoids in blood must be 6 h as nearly 9 h are necessary for passing the R phase. Thus in order for the increase in I1 to take place at 6 p.m., the secretion of glucocorticoids must fall approx. 15 h before it. The minimum level of glucocorticoids at 4-6 a.m. [ll] is consistent with this. The whole sequence of events is illustrated in Fig. 3 where the duration of the R phase is taken as 8 h and z as 6 h with a view to obtaining the real fluctuations in I,. It may be concluded that diurnal variations in the level of glucocorticoids are the chief factors responsible for diurnal variations in the index labelling of the mouse forestomach epithelium. The same mechanism may be valid for other tissues as for some tissues the maximum and minimum values of I, coincide in time [ 161. Such a coincidence is possible if t,, tR and t are equal. If these values differ, then the diurnal curve for index labelling must also be different. This model was constructed assuming complete inhibition of transition to the R phase or absence of inhibition. It is evident that in a real situation inhibition at the maximum level is only partial and when the concentration of glucocorticoids is low there also may be some inhibition. The model gives a good explanation for the maximum and minimum values of I 1 lvithout describing the range between these values. Experimental

Cell Research 52

I<\-itlently,

glri~ocwrti~oicls

is c~ncb01’ Ilic factors regulating

Ihcb ralcx 01’ c*cII

proliferation in squamous cpithrlium. This regulation is c*arricd 0111 t)v th(~ inhibilion of Iransition to the I< 1)h:tse. ,-\clrenalcclomy significantly inc*rcases the mitotk activity in squamo~is epithelium :20 Absence 01’ fil~ic~oc~c~rlic~c~i(l~ appears to be the main cause for this increase.

to the R phase, is :I region of Transition to the S phase or, more precisely, It was sho\vn (Frankfurl, to be the cell cycle affected by glu~ocorticoids. published) that in the hyperplastic epithelium of the mouse forestonlach, in the case when hpperplasia appeared some months after single administration of carcinogen, hydrocortisone inhibited the transition of cells to the S phase. In the hgperplastic as \\-cl1 as in the normal epithelium the sensilivity to hydrocortisone is lost G-9 h prior to initiation of DSA synthesis. Ho\vever, the transition to the R phase is less sensitive to hytlrocortisone in the hypes’This dill’crcnces is plastic epithelium than that of the normal epithelium. evident from a lesser decrease of I, in the hyperplastic comparecl IO the normal epithelium after injection of the same dose of h~dro~ortisoIi(,. Hyclrocortisone in doses of 5 or 50 ,ug/g has no influence on the transition of~clls to the S phase in papillomas of the mouse forestomach. It is of intewst lo correlate the above with the rate of cell proliferation in these types of cpithrlium. Cell proliferation \\-as accelerated in hypcrplastic epithclium anti avc~clcratctl This \vas clearly seen from lhc I, values considerably more in papillomas. in the morning hours, that is at the time \vhen the transition to the S phase \vas inhibited I)\- endogenous ~lucocorticoic1s. At 9 a.m. I, for the normal ant1 hyperplastic epithelium and papillomas \\-as 3.6, 19.4 and 47.3 1x.r cent, respectively. It is of interest to note also that diurnal variations in I, IVCIY observed in the hyprrplastic epithelium but not in papillomas. It seems that the change in the sensitivity of cells to hytlrocortisonc during carcinogcncsis is one of the factors leading to acceleration of cell proliferation. Another important indication of the role of glucocortiwicls in the carcinogenesis of squamous epithelium may be found in the paper by ‘l’rainin -21 . He has shown that the level of glucocorticoids is responsible for the frec1uenc! of tumors: feeding of hytlrocortisone decreases anti atlrenale~tomy increases the frequency of tumors. In ‘I’rainin’s experiments [21 j mice received GO-80 pg of hydrocortisone per day \vhich is near to the dose rwvl in our c’xperiIt may be sul~pos~d that the merits that inhibits transition to the R-phase. action of h~drocol.tisonf in Trainin’s mechanism of the anticarcinogenic

Transition

231

of cells to DNA synthesis phase

experiments [al] consists in inhibition of cell transition to the R phase. Long ago, Bullough [2] has shown that inhibition of cell proliferation is a mechanism of anticarcinogenic action of caloric restriction. As to the stimulation of carcinogenesis by adrenalectomy [21] it may be supposed that its mechanism consists in the removal of inhibition by glucocorticoids of the squamous epithelial cell transition to the R phase. It is consistent with the statement made by Bullough [-I] that a weakening of homeostatic regulation of cell proliferation, occurring \vith age as a result of a weakening of the adrenal cortex function, is favourable for carcinogenesis. In general such a depentience of carcinogenesis on the level of glucocorticoids points to the very important part played by acceleration of cell proliferation in carcinogenesis.

SUMMARY

Injection of 5 ,ug/g of hydrocortisone or 0.1 pg/g of actinomycin D 9 h before the transition of cells to the S phase inhibited the initiation of DNA synthesis in squamous epithelial cells of the mouse forestomach. \Yhen these substances were injected 3 or 6 h before the transition of cells to the S phase there \\-as no efI’ect on initiation of DNA synthesis. Injection of 1 or 2.5 ,ug/g of adrenalin stimulated the transition of cells to the S phase, the stimulating effect appearing 9 h after injection of adrenalin. ‘This shows that at some time ranging from 6 to 9 h before the transition to the S phase, the cell made a decision on initiation of DNA synthesis and that this decision \vas connected with the synthesis of RNA necessary for DNA synthesis. A part of the presynthetic, period between decision on, and initiation of DNA synthesis was named as the R phase. Diurnal variations in index labelling \vere explained as a result of inhibition by glucocorticoids of the transition of cells to the R phase. REFERENCES 1. BASERGA, R., Cancer Res. 25, 581 (1965). 2. BULLOUGH, W. S., Bril. J. Cancer 4, 329 (1950). 3. BULLCIUGH, W. S., in EYELOT and M~~HLBOCK (eds). Cellular Control Mechanisms Cancer, p. li4. Elsevier, Amsterdam, 1964. ~ ” 4. BUI,LOUGH, W. S., Cancer Res. 25, 1683 (1965). 5. BULLOUGH, W. S. and LAURENCE, E. B., Proc. Roy. Sot., ser B, 154, 540 (1961). 6. BULLOUGH, LX’. S. and RYTGMAA, T., Nature 205, 573 (1965).

7. C~UJXAK, M. G., Dokl. Akad. Nauk 8. DOUGHERTY,

T. F., BERLINER,

and

USSR 149, 960 (1463).

M. J,., SCHNEEBELI,

G. L. and BERMSER,

D. L.,

Ann.

S.Y.

Acad. Sci. 113, 825 (1964). 9. FR.ASKFURT, 0. S., Expil Cell Res. 46, 603 (1967). 10. FUJIOKA, RI., KOGA, 31. and LIEBERMAN, I., J. Biol. Chem. 238, 3101 (1963). Experimental

Cell Research 52

Exfxrimenfal

Cell Kesecrrch 52