Acta histochem. 69, 40-49 (1981)

Institute of Histology and General Embryology, University of Fl'ibourg, Switzerland (Dir.: Prof. Dr. G. CO"NTI)

In vitro responses of proliferating and non-proliferating cells to high doses of 17 fJ -estradiol: Cytochemical study

By V ASSILIS GOTZOS, BONA CAPPELLI-GoTzos and RENE BOVET

With 7 figures

(Received September 10, 1980)

Summary The authors studied the effect of 17p-estradiol on the proliferation in vitro of chick embryo

fibroblasts and human macrophages by microdensitometry, cytofluorometry, and autoradiography. For fibroblasts 17p-estradiol shortens the duration of the preparing period to mitosis, particularly of the synthetic phase (S) and has no effect on the duration of mitosis. For macrophages, which have temporarly lost their mitotie capacity, 17p-estradiol cannot induce mitotic division.

Introduction

The effect of 17fJ-estradiol, a "mitogenic" hormone, on the proliferative cell cycle, to our knowledge, is not yet cleared up. Opinions of different authors seem to be con-tradictory. BULLOUGH (1962) estimates that estrogens affect the proliferative cycle by shortening the presynthetic period (GIl and increasing the number of cell of the pro-liferative pool. On the other hand, PERROTTA et al. (1961), PECKHAM et al. (1963), and GALAND et al. (1967) believe that estrogens, especially estradiol, affect DNA synthesis (8). Other authors established an increase of the mitotic index (MI) under the effect of estrogens. It should be emphasized that, according to GALAND et al. (1967), the effects of estrogens on target and non target cells differ only quantitatively.

The aim of the present work was to investigate the effect of high doses of 17 fJ-estradiol on cell cycle kinetics and the possible re-establishment of mitosis in tempo-rarly non proliferating cells.

Material and Methods

'Ve choosed 2 populations fo cells cultivated in vitro differing in their own proliferation: fibro-blasts from a 9-day-old chick embryo and macrophages from human ascitic fluid of patients suffer-ing from a cirrhosis of the liver. Fibroblasts normally show in vitro an important multiplication; macrophages, both in ascitic fluid and t'n vitro, do not multiply by mitosis (their mitotic capacity is temporarly blocked), only in particular conditions they are able to proliferate. Both populations were cultivated in vitro at 38°C in the following medium: 90 % 199 Difeo, 10 % calf serum (Micro-bial. Ass. Inc.), without antibiotics, pH = 7.2( GOTZOS et al. 1974; GOTZOS 1977}_ After 24 h of

In vitro responses of proliferating and non-proliferating cells 41

culture for th9 fibroblasts and after 4 months for macrophages, 17/i-estradiol (Vister, Oalbiochem). previously dissolved in 95 % ethg,nol, was added at the final concentration of 10-4, 10-3, or 10-2 mg! ml, for additional 48 h.

For all experiments, 2 parallel control cultures were prepared and maintained in the same way, one without 17/i-estradiol and the other containing 2 0 / 00 ethg,nol 95 % (same final cOll('pntration as in treated cultures). Since we did not note any difference between thpse 2 controls, we will consider only the one without 17/i-estradiol in our results.

The following analyses were carried out:

a) DXA determination by microdensitometry after the FEULGEX reaetion (fixation by ethanol: acetone [1 : 1 v/v], 24 h at 4°C; hydrolysis with 1 N HCL, 20 min at 56°C) and by cytofluorometry aftpr bis.(4-amino-phenyl).1,3,4.oxadizole (BAO, Oiba) reaction (RUCH 1970).

b) Morphological observations ofliving cells by phase contrast microscopy and mitotic index (MI) determination of ethanol: acetone (1: 1 v/v)-fixed, GIEMsA·stained ePlls.

c) Incorporation of [3H] thymidine ([3H]th) (Radiochemicnl Oentre, Amersham, UK; specific activity 5 Ci/mmol, 0.5/ICi/ml of culture medium, 30 min of incubation at 38 DC) into DNA, studied by autoradiography as previously described (GOTZOZ 1977 a).

d) Counting of G2 cells by combined BAO cytofluorometry and [3H]th autoradiography, carried out on the same preparation as follows: [3H]th incorporation (cf. section e), fixation by ethanol: acetone (1: 1 v/v), BAO reaction (d. seetion a), autoradiography by the stripping film method (ApPLETOX 1972). This combined method makes it possible to measure nuelear DNA con-tent by eytofluorometry with incident illumination on the labelled eells (slide up-side down), without the interferell<,e from the silver grains (see also MAK [1965], GAL"TSCHI et al. [1971]).

e) Determination of the lenght of the cell cyele as a whole and of its component phases by the method of the labelled mitoses (QUASTLER and SHERMAX 1959). After 48 h treatment with 17/1-estradiol the eultul'Ps were exposed to [3H]th as in section c) and then changed back to a nonradio-active medium. Samples (4 to 6) were taken at 1 h intervals after the pulse exposure. The percent-age of labelled mitoses was determined by autoradiography of the different samples stained with GIEMSA. The lenght of the cell cycle is given by the interval between the 2 first peaks of the curve; the interval between 0 time and the 50 % point of the first ascedung limb of the curve gives the lenght of 1/2 M + G 2 ; the lenght of the S phg,se (D~A synthetic period) is given by the interval between tk, two 50 % points on the asceding and the descending limb of the first curve; the dura-tion of 1/2 M + G1 is given by subtracting 1/2 M + G2 + S from the lenght of the entire cycle.

The microdensitoll1etric determinations were carried out with a Leitz MPV 2 scanning and integrating mict'Odensitometer linked to a PDP /8 Digital computer for the elaboration of data, and with a Vikers .~ 85/86 scanning and integrating microdensitometer. For cytofluorometry we used a Leitz -,WPT' 1 fluorometer.

Results

In thc fibroblasts after 48 h of treatment with all the doses of 17 ~-estradiol (10-4, 10-3, and 10-2 mg/ml) we noted an increased number of cells containing 4c of DNA (G2) when compared with control cultures (Figs. 1,2; Tab. IA). MI also increased in treated cultures with all the doses of 17 ~-estradiol [(10-4, 10-3, and 10-2 mg/ml); (Table IB)]. The duration of mitosis observed on living cells by phase contrast micro-scopy, did not show any significant variation between control and treated cells (60 to 80 min). The percentage of labelled nuclei after [3H]th incorporation, decreased in treated cells with all the doses [(10-4, 10-3, and 10-2 mg/ml); (Table Ie)]. A labelling of cytoplasm especially at the cytoplasmic expansion extremities was observed with

42 V. GOTZOS, B. CAPPELLI-GOTZOS and H. BovET

%

50

30 coNTROL

10

18 18 36 a.u.

Fig. 1. Nudear DNA content (determined by microdensitometry after FEULGEN reaction) in con-trol and in 17p-estradiol treated chick embryo fibroblasts (10-4, lO-3, and 10-2 mgjml, for 48 h). Ordinate: % of nndei. Abseissa: DNA content per nueleus in arbitrary units (a.n.). 2c: DNA con-tent in telophasie cell nnclei (diploid); 4(': DNA content in prophasic cell nuclei (tetraploid).

12e 12e I

be I

be I

:4e :4e :4e :4c

" 30

20 CONTROL 10-4 10-3 10-2

Fig. 2. Nnc-lear DNA content (determined by eytofluorometry after BAO reac-tion) in control and in 17p-estradiol treated "hick embryo fibroblasts (10-4, lO-3, and 10-2 mgjml, for 48 h), incubated with [3H] th for 30 min. Ordinate: % of nndei. Abscissa: DNA content per nucleus in arbitrary nnits (a.n.). 2 c: DNA content in telophasic "ellundei (diploid); 4 c: DNA content in prophasic cell nne-lei (tetraploid). I',:",;n nnclear DNA eontent of labelled cells (cells in synthetic period or S); for eaeh histogram, Oil the left, nuclear DN A content of G1 cells, and on the right nuc'lear DNA content of G2 cells.

42

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V. GOTZOS, B. CAPPELLI-GOTZOS and H. BovET

12e I

:4e

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18

be

10-3

I :4e be I

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36 a.u.

Fig. 1. Nudear DNA content (determined by microdensitometry after FEULGEN reaction) in con-trol and in 17p-estradiol treated chick embryo fibroblasts (10-4, lO-3, and 10-2 mgjml, for 48 h). Ordinate: % of nndei. Abseissa: DNA content per nueleus in arbitrary units (a.n.). 2c: DNA con-tent in telophasie cell nnclei (diploid); 4(': DNA content in prophasic cell nuclei (tetraploid).

12e I 12e I be I be I

:4e :4e :4e :4c

" 30

2J II CONTROL II 10-4 II 10-3 II 10-2

Fig. 2. Nnc-lear DNA content (determined by eytofluorometry after BAO reac-tion) in control and in 17p-estradiol treated "hick embryo fibroblasts (10-4, lO-3, and 10-2 mgjml, for 48 h), incubated with [3H] th for 30 min. Ordinate: % of nndei. Abscissa: DNA content per nucleus in arbitrary nnits (a.n.). 2 c: DNA content in telophasic "ellundei (diploid); 4 c: DNA content in prophasic cell nne-lei (tetraploid). I',:",;n nnclear DNA eontent of labelled cells (cells in synthetic period or S); for eaeh histogram, Oil the left, nuclear DN A content of G1 cells, and on the right nuc'lear DNA content of G2 cells.

In vitro respon.ses of prolifprating and non-proliferating cells 43

Table 1. A: percE'ntages df G2 cells in control and in 17(1-pstradiol treated chick embryo fibro-blasts. Each perwmtagc was calculated fmm histogmms of preparations after the combination of BAO cytofluorollletry and [3H]th autoradiography, carried out in at least 500 cells. B: mitotic index [%0] in c-ontrol and in 17(1-estradiol tr·pated chick embryo fidroblasts. Each value WdS cal-culated from data from nlOrp than ij 0:10 celk C: perc-entages of [3H]th labelled nuclei in control and ill 17(1 estradiol treated chiek embryo fibrobhlsts. Eaeh perepntage was calculated from data from more than 1 000 cells

control 17{:i-estradiol for 48 It [mg/ml]

10-! 10-3 10-2

A G2 cells [OAl] 20.15 25.00 30.15 27.80 19.00 26.30 29.70 24.90 17.{iO 28.15 24.80 26.30 18.10 29.4() 26.20 28.15 18.71 ± 1.13* 27.21 ± 1.95* 27.7l ± 2_56* 26.78 ± 1.49*

B mitotic index [%0] 16.90 23.10 24.70 32.40 15.GO 25.()O 26.45 26.35 19.30 29.10 27.30 28.90 20.10 31.30 30.25 29.70 17.90 30.10 27.80 30.10 17.96 ± 1.81 * 27.84 ± 3.40* 27.30 ± 2.01 * 29.49 ± 2.15*

C [3H]th labelled nudei 1%] 16.20 8.00 7.30 6.90 14.00 9.15 8.45 9.15 15.15 7.80 7.40 8.10 14.16 9.09 8.10 7.70 15.30 7.90 7.60 14.9(j ± 0_90* 8.38 ± 0.69* 7.81 ± 0.56* 7.89 ± 0.82*

* avel'ctge t B.D.

the highest dose [(10-2 mg/ml); (Fig. 3a and b)J. DNAase digestion confirmed the presence of neosynthesized DNA.

By the method of the labelled mitoses (Fig. 4) we could establish the lenght of the proliferative cycle of fibroblasts treated with 10-2 mg/ml of 17p-estradiol. After 48 h of action (Fig. 5B) the total length was about 13 h; 5 h shorter than in control fibro-blasts (Fig. 5A). This shortening is to be imputed to the preparing period to mitosis (GI + S -t- G2), particularly to the presynthetic (GI ) and synthetic (8) phases.

The macrophages after 4 months in vitro do not enter mitosis, but are still able to synthesize nllelear DNA (GoTzos et al. 1977). We te8ted the same doses of 17p-estra-

Fig. 3a and b. 17(1-estradiol treated fibroblasts (10-2 mg/ml, for 48 h) post-stained with GIEMSA,

incubated with [3H]th, for 30 min. Note the presence of silver grains at the cytoplasmic expansion. extremities, revealing DNA synthesis. X 1,100. c: living macrophages after 4, months in vitro. X 1,000. d: fixed maerophages after 4 months in vitro (ethanol: ac-etone, 1: 1 v/v, phase contrast). X 1,000.

44

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Fig. 4. Labelled mitoti c fibroblas ts t,·eated with 17p-estradiol (10 - 2 mgfml, for 48 h). a: prophase; b: metaphase; c: anaphase ; d: telophase), 8 h a fter the incubat.ion with [3H]th (30 min); post-stained GIE~ISA . X 1,1>00.

46 V. GOTZOS, B. CAPl'ELLI-GOTZOS awl R. BOVET

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46 V. GOTZOS, B. CAPl'ELLI-GOTZOS awl R. BOVET

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In vitro responses of proliferating and non-proliferating cells

I I 18c

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Fig. 6. Nudear DNA content (determined by eytofluorometry after BAO reaction) in control and in 17p-estradiol treated (10-2 mg/ml, for 48 h) human maerophages, from non tumoral ascitic fluid, after 4 months in vitro. Ordinate: % of nuelei. Abscissa : DNA content per nueleus in arbitrary units (a.u.). 2<:, 4o, 8c: DNA contents calculated from mesures inleukocyes from the same ascitic fluid. Dotted parts of the histogram represent nuclei containing more than 45 a.u. (2 c content plus 50 %). This arbitrary separation was done for a better comparison between the behaviour of the control and of the treated cells.

% %

A B T . .1

50 50 1 T ..

T 1 1 . .1

! T l ! .. ..

10 0

CONTROL CONTROL

Fig. 7. Percentages of human macrophages containing more than 45 a.u. of DNA, in control and in 17p-estradiol treated cultures (10-4, 10-3 , and 10-2 mg/ml, for 48 h). A and E: cell strains from 2 patients suffering from cirrhosis. Each percentage was calculated from date obtained from 300 nuclei on different samples.

Fig. 5. Determination of the length of the cell cycle in control (A) and in 17 p-estradiol treated fibroblasts [(E); (10-2 mg/ml, for 48 h) by the method of the labelled mitoses. Ordinate: % of 1,1belled mitoses. Abscissa: time [h] after the exposure to [3H]th. Each point represents the per-centage calculated from 4 to 6 samples. T: and Tp linear representation of the length (h) of the entire "yell' and of its component

phases. T length of the entire cycle.

= length of the half mitosis. = length of the postsynthetic period.

1/2 M + 02 = length of the half mitosis and the postsynthetic period. = length of the presynthetic period. = length of the synthetic period.

be %

30

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In vitro responses of proliferating and non-proliferating cells

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Fig. 6. Nudear DNA content (determined by eytofluol'ometry after BAO reaction) in control and in 17p-estradiol treated (10-2 mg/ml, for 48 h) human maerophages, from non tumoral ascitic fluid, after 4 months in vitro. Ordinate: % of nuelei. Abscissa : DNA content pel' nueleus in arbitrary units (a.u.). 2<:, 4o, 8c: DNA contents calculated from mesures inleukocyes from the same ascitic fluid. Dotted parts of the histogram represent nuclei containing more than 45 a.u. (2 c content plus 50 %). This arbitrary separation was done for a better comparison between the behaviour of the control and of the treated cells_

%

j A

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%

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Fig. 7. Percentages of human macrophages containing more than 45 a.u. of DNA, in control and in 17{I-estradiol troated cultures (10-4, 10-3 , and 10-2 mg/ml, for 48 h). A and E: cell strains from 2 patients suffering from cirrhosis. Each percentage was calculated from date obtained from 300 nuclei on different samples.

Fig. 5. Determination of the length of the cell cycle in control (A) and in 17 p-estradiol treated fibroblasts [(E); (10-2 mg/ml, for 48 h) by the method of the labelled mitoses. Ordinate: % of 1,1belled mitoses. Abscissa: time [h] after the exposure to [3H]th. Each point represents the per-centage calculated from 4 to 6 samples. T: and Tp lineal' representation of the length (h) of the entire "yell' and of its component

phases. T 1/2 :M G2

length of the entire cycle. = length of the half mitosis. = length of the postsynthetic period.

1/2 M + G2 = length of the half mitosis and the postsynthetic period. G1

8 = length of the presynthetic period. = length of the synthetic period.

48 V. GOTZOS, B. CAPPELLI·GOTZOS and n. BOVET

diol (10-4, 10-3, and 10-2 mg/ml, for 48 h) on different strains of human macrophages, obtained from ascitic fluids, after 4 months in vitro [(Fig. 3 c, d); (BOVET et al. 1976)J. With tho lower doses of 17j3-ostradiol (10-4 and 10-3 mg/ml) nuclear DNA content was not different hetween treated and control macrophages, meanwhile with the highest dose (10-2 mg/mi) the number of cells containing more than 2c of DNA increased in treated culturos (Figs. 6, 7). This increased number was not accompanied by the entering into mitosis of the colIs. Tho MI remained at 0.00%0 both for control and treated macrophages.

Discussion

In this paper we did not want to compare chick embryo fibroblasts with human macrophages, which are cells gE'netically quite different, but only to consider how their proliferation capacities respond to 17 tJ-estradiol. Our results showed that 17 tJ-estradiol has an effed on proliferative ('ell cycle of fibroblasts (proliferating cells) and of macrophages (non proliferating cells).

In chick embryo fibroblasts it inereased the number of the mitotic (MI) and of the Gz ('ells, meanwhile it de (Teased the percentage of the synthetic cells (8). These results have to be inter-preted on the basis of the alterations of the length of the different phases of the cell cycle observed in treated ('ells. In particular, the shortening of DNA synthesis duration may explain the deerease of 8 (·pll percentage ([3H] th labelled cells). It should bp emphasized that this effect on 8 phase is inconsistent with the results obtained by other authors (PERROTTA 1962, BVLLOUGH and LAD"-REXCE 1966, EPIFA"OYA 1971), possibly because they investigated the "target cells" from uterine and vaginal epithelia.

The fad th'lt in fibroblasts the lenght of mitosis did not vary and thp lenght of Gz phase w;:ts almost invarying, may accunt for the inereased number of treated cells in these phases. This is supported also be the shortening of the G1 and S phases that should bring a greater number of eells into Gz and mitosis. These results suggest that 17tJ-estradiol act at the level of the first half of the cell cyele (G1 and S) without having any effect on Gz cells going into mitosis and on the mitosis itself.

This assumption could be eonfil'lned by treating cells which do not enter mitosis, with this hormone. In the experiment with macl'ophages, indeed, 17tJ-estradiol alone could not make cells to enter mitosis, even if its action was evident: the percentage of the C'ells C'ontaining more than 2 c of DNA increased in treated maC'rophages.

It should also be stated that, on the 2 cellular types tested, 17 tJ-estradiol differed in its effect: on macrophages it acted only at the highest dose. For this reason, in this experiment, the effects of 17tJ-estradiol cannot be ('onsidercd as hormonal effects, but rather as an in vitro collateral action.

vVe conclude saying that the high doses of 17-tJ'estradiol tested on chick embryo fibroblasts and on human macrophages accelerate the metablic pathways ruling the first half of the cell cyele (G1 and S). The appellation "mitogenic" for 17p-estradiol at high doses and for the cell populations tested in this study, has not t[) be conceived in the meaning of a trigger for mitosis, but as a factor being able to increase th£' rate of "dl pl'Oliferation by its action on basal metabolism.

Literature

ApPLETON, T. C., Stripping film autoradiography, In: Autoradiography for Biologists (ed. P. B. GAHAX), pp. 33--49, Academic Press, London-New York 1972.

BOVET, R., GOTZOS, V., CAPPELLT-GOTZOS, B., et CONTI, G., Etude cytofluorometl'ique des teneurs de l' ADN SUI' des cplIules mesotheliales per·jtoneales humaines cultivees in vitro en presence de 17tJ-()(,stradiol, Aeta Anat. 95, 1()2 (1970).

In vitro responses of proliferating and non-proliferating cells 49

BULLOUGH, vV. S., The control of mitotic ac·tivity in adult mammalian tissues, Biol. Rev. 37, 307 - 342 (1962).

- and LAUREKCE, E. B., Carcinogenesis, In: Advances in Biology of Skin (eds. MONTAGNA, W., and DOBSON, R. L.), pp. 1-36, Vol. 7, Pergamon, Oxford 1966.

EPIFANOVA, O. 1., Effeets of Hormones on the Cell Cycle, In: Thc Cell Cycle and cancer (ed. BA-SERGA, R.), pp. 143-182, Marcel Dekker, New York 1971.

GALAND, P., RODESCH, F., LEROY, F., and CHRETIEN, ,J., Hadioautographic evaluation of the estrogen-dependent proliferativE' pool in the stem cell eompartment of the mouse uterine and vaginal epithelia, Exp. Cell. Res. 48, 595 - 604 (1967).

GAUTSCHI, J. R., SCHINDLER, R., and HURNI, C., Studies on the division cycle of mammalian cells. V. Modifications of time paranwters by different steady-state culture conditions, J. Cell. BioI. 51,653-663 (1971).

GOTZOS, V., Culture in dtro de cellules de liquide peritoneal humain, Acta Anat. 98,281- 294 (1977). Histochemical study of nucleic acids and proteins by cytophotometry and histoautoradio-graphy in fibroblasts eultivated in hyperoxia, Acta Histoehem. 58, 141-155 (1977 a). CAPPELLI-GOTZoS, B., and BOVET, R., Cytofluorometric and histoautoradiographic study on DNA and protein synthesis of human mesothelial cells, Experientia 33, 266-267 (1977). SPRECA, A., et CONTI, G., Effets de O-(tl-hydroxyaethyl)-Rutosidea (HR) sur les fibroblastes d'embryon de ponlet cultivos in vitro en diff6rentes concentrations d'oxygime. 1. Etude mol'-phobiochimique, Acta Anat. 90, Hll-178 (1974).

MAK, S., Mammalian cell cycle analysis using microspectrophotometry combined with autoradio-graphy, Exp. Cell. Res. 39, 286-289 (1965).

PECKHAM, B., LADIKSKY, J., and KIEKHOFER, vv., Autoradiographic investigation of estrogen response meehanisfIls in rat vaginal epithelium, Am. J. Obst. Gyn. 87, 1, 710 (1963).

PERROTTA, C. A., Initiation of cell proliferation in the vaginal and uterine epithelia of the mouse, Am. J. Anat. HI, 195-·-201 (1962).

- QUASTLER, H., and STALEY, N., Proliferation in the vaginal epithelium of the mouse as shown by autoradiography with tritiated thymidine, An. Rec. 139, 2(i3 (1961).

QUASTLER, H., and SHERMAN, F. G., Cell population kinetics in the intestinal epithelium of the mouse, Exp. Cell. Res. 17,420-438 (1959).

RUCH, F., Principles and some applications of cytofluorometry, In: Introduction to quantitative pytochemistry-II (eds. VVIED, G. L., and BAHR, G. F.), pp. 431-450, Academic Press, New York and London 1970.

Address: Dr. VASSILIS GOTZOS, Institute of Histology and General Embryology, University of Fribourg, CH - 1700 Fribourg.

4 Acta histochem. Bu. 09