Enucleation of human endometrial cells: Nucleo-cytoplasmic distribution of DNA polymerase α and estrogen receptor

J. steroid Biochem. Vol. 24, No. 2. pp. 469-474, Printed in Great Britain. All rights reserved

1986 Copyright

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0022-473 I/86 $3.00 + 0.00 1986 Pergamon Press Ltd

ENUCLEATION OF HUMAN ENDOMETRIAL CELLS: NUCLEO-CYTOPLASMIC DISTRIBUTION OF DNA POLYMERASE a AND ESTROGEN RECEPTOR ACHILLE GRAVANIS and ERLIO GURPIDE Department of Obstetrics, Gynecology and Reproductive Science, Mount Sinai School of Medicine, New York, NY 10029. U.S.A. (Received 4 May 1985) Summary-Nucleo-cytoplasmic distribution of estrogen receptors and DNA polymerase a activity in human endometrial adenocarcinoma cells (HEC-50 line) was evaluated after separation of nuclei following either homogenization or enucleation with cytochalasin B. About 30% of the estrogen receptor was found in the nuclear fraction after homogenization whereas 86% was found in the karyoplasts after enucleation. The total amounts of estrogen receptor per cell after homogenization and enucleation were not significantly different (14,OW17,000 binding sites/cell). Receptor measurements were carried out using the hydroxylapatite method after labeling with [3H]estradiol (5 nM [3H]E, k 500 nM E2) at 30°C for 3 h. About 20% of the DNA polymerase tl activity was found in the nuclear fraction after homogenization, whereas 96% was found in the karyoplasts after enucleation. The average total activity (0.84 Units/lo6 cells) in homogenized cells was about 1 of the activity in karyoplasts. These results indicate that estrogen receptor and DNA polymerase a activity reside in the nucleus in intact HEC-50 cells. DNA polymerase a is translocated to the cytoplasmic fraction and inactivated after homogenization.

INTRODUCTION Homogenization of tissue in buffer solutions can remove proteins from their native intracellular location. For instance, mobilization of mitochondrial enzymes involved in steroid synthesis was found to

occur during homogenization of bovine adrenals, particularly if the tissue had been previously frozen [ 1,2]. Experimental evidence of redistribution of estrogen receptors during homogenization was first presented by Sheridan et al. who found that the estimated values for cytoplasmic/nuclear ratio of estrogen receptor contents increased proportionally to the dilution of the homogenate [3]. In order to avoid solubilization of nuclear-bound proteins, non-aqueous methods have been used for the separation of subcellular fractions after tissue lyophilization. Adopting glycerol as the sedimentation medium, Lynch et al. demonstrated that DNA polymerase c( of rat liver resides in the nucleus, as it would be expected considering its role in DNA synthesis during liver regeneration [4]. Paradoxically, the enzyme is found mostly in the cytoplasm after tissue homogenization in aqueous media [5]. Another approach to preventing artifactual translocation of nuclear proteins to the cytoplasmic fraction is to separate nuclei by treating cells with cytochalasin B, an agent that disrupts microfilaments and allows enucleation under centrifugal forces while preserving the viability of the remaining cytoplasts [6]. Welshons et al., who applied this technique to the study of intracellular distribution of estrogen receptors in GH, rat pituitary cells, found s * 24.2-A

higher concentrations of receptors in a karyoplastrich fraction than in the remaining cytoplasts [7]. Herrick et al. found preferential nuclear localization of DNA polymerase tl in enucleated mouse L929 cells

Val. The purpose of the present study, carried out with human endometrial adenocarcinoma cells (HEC-SO), was to compare estimates of nucleo-cytoplasmic distribution of estrogen specific binders and DNA polymerase c( obtained after enucleation or homogenization in aqueous media. Verification of the concept that estrogen receptor resides in the nucleus in the intact cell was considered to be of enough importance to justify these studies. Receptor distribution has been examined by enucleation in only one cell type [7] and no comparisons of results from parallel experiments on which cells were enucleated or homogenized have been reported. The enucleation studies also offered the opportunity to investigate the intracellular localization of DNA polymerase o! in cells from human endometrium, a tissue that undergoes periodic regeneration and in which the activity of the enzyme is influenced

by ovarian

hormones.

EXPERIMENTAL CelI cultures

Cells from the human endometrial line HEC-50, established by Kuramoto et al. from a specimen of poorly differentiated adenocarcinoma [8], were cultured in Eagle’s minimal essential medium (MEM, Grand Island Biochemical Co., Grand Island, N.Y., 469

470

ACHILLE GRAVANIS and ERLIOGWRPIDE

GIBCO) supplemented with 15% fetal bovine serum (FBS, Flow Laboratories, Rockville, Md) and 1% of an antibiotic-antimycotic mixture (GIBCO), using T 3013 plastic flasks (Falcon Plastics, Los Angeles, Calif.). Cells were maintained at 37’C in a humidified atmosphere of 95% air-S% CO,. Enucleation

When cultures approached confluency the flasks were filled with 50ml of Eagle’s MEM containing 20pg/ml cytochalasin B (Sigma Chemical Co., St Louis, MO.) and placed in Teflon-lined cups of a prewarmed HB-4 rotor. After centrifugation in a Sorvall RC-SB centrifuge (3000 g, 90 min, 35”(Z), the karyoplasts were gently suspended in the enucleation medium, transferred to a plastic tube, centrifuged and washed twice by resuspension in Hanks’ balanced salt solution (HBSS, Flow Laboratories). Whole cells and cytoplasts present in these preparations were removed by incubating the mixture with MEM-FBS for 30 min in culture flasks; under these conditions only karyoplasts remain in suspension. The anuclear cells (cytoplasts) and the small proportion of nucleated cells remaining attached to the plastic surface were removed by treatment with 1% trypsin in Ca *+-free HBSS for 1Omin at 37°C collected by centrifugation for 5 min at 800g and washed once with MEM-FBS and then twice with HBSS. Measurement of estrogen receptor levels

Whole cells or cytoplasts were homogenized in 2.4ml of Tri-sucrose buffer (20mM Tris, 0.5 M sucrose, 1 mM MgCl*, 1 mM dithiothreitol, pH 7.4) and aliquots were taken to measure DNA [9] and protein [lo] contents. The homogenate from whole cells was centrifuged at 1500 g for 10 min to separate nuclei; cytosol was obtained by centrifugation of the homogenates at 105,OOOg for 60min. Karyoplasts obtained after enucleation and nuclei obtained after homogenization of whole cells were suspended in 2.4 ml of a Tris-sucrose-0.5 M KC1 buffer (Tris-sucrose buffer with 0.5 M KCI) for 30 min and then centrifuged at 15,000g to prepare nuclear extracts Aliquots (0.2 ml) of cytosol from whole cells or cytoplasts and aliquots (0.2ml) of nuclear extracts from karyoplasts or nuclei isolated from homogenates were incubated with 5 nM [3H]estradiol (sp. act. 100 Ci/mmol, Amersham Corp., Arlington Heights, Ill.) for 3 h at 30°C in the presence or absence of a loo-fold molar excess of unlabeled estradiol. 0.25 ml of hydroxylapatite (HAP, Bio-Rad, Richmond, Calif.) was added to each tube and after approx 30 min at 0°C the pellet containing nuclei and HAP was separated by centrifugation. The levels of specifically bound [3H]estradiol were estimated from differences in the radioactivity content of pellets from tubes with or without unlabeled estradiol.

Measurement of DNA polymerase u activity

Whole cells were homogenized at 4°C in 0.5 ml of Tris-sucrose buffer and the homogenate was centrifuged at 15,000g for 15 min to obtain a nuclear pellet. This nuclear preparation and karyoplasts obtained by enucleation were suspended in 1 ml of Tris-sucrose-O.5 M KCI at 4C. sonicated for 20 s (Ultrasonic Inc., N.Y.) and kept in the same buffer at 4°C for I h. At the end of this period, the suspensions were centrifuged at 15,000g for 15 min and the extracts were used to measure DNA polymerase activity [l 11. The activity was also measured in cytoplasts lysed in Tris-sucrose-0.5 M KC1 buffer and in supernatants from whole cell homogenates, after mixing with an equal volume of Tris-sucrose-l M KC1 buffer to bring the concentration of KCI down to 0.5 M. Enzymatic activity was measured by the method of Bertazzoni et af.1121, based on the incorporation of [3H]thymidine (sp. act. 50 Ci mmol) into DNA activated bv treatment with DNAse I from bovine pancreas (Sigma). Twenty ~1 of the samples were mixed with 30 ~1 H,O and 200 ~1 of a potassium phosphate (25 mM)-dithiothreitol (0.125 mM) buffer, pH 7.2, containing dATP, dCTP, dGTP, [3H]TTP (62.5 PM each), MgClz a-mercaptoethanol (10 mM), (1.25 mM) and activated DNA (225 pgg/ml). After 5, 10 and 15 min of incubation at 37°C 0.05 ml aliquots were transferred onto fiber glass filters (Whatman GF/C). The filters were washed at 0-4”C with 5% trichloroacetic acid (TCA)-1% sodium pyrophosphate and then twice with TCA to remove soluble radioactivity. In order to test for effects of cytochalasin B on DNA polymerase c(, cultures of HEC-50 cells (5 x 106cells/dish) in MEM-FBS were incubated for 1.5 h at 37°C with cytochalasin B (20pg/ml). The final concentration in the medium of the solvent in which cytochalasin B was dissolved (dimethylsulfoxide, DMSO) was 0.5%. Cells were then collected by treatment with 0.5% trypsin in 0.02% EDTA for 5 min at 37°C washed once with MEMFBS and twice with HBSS. The cell pellet was homogenized in 0.5 ml of Tri-sucrose-l M KC1 buffer and sonicated for the measurement of DNA polymerase SI activity as described above. Control experiments without cytochalasin B but with DMSO at the same final concentration were carried in parallel. RESULTS Enucleation

The appearance of HEC-50 cells in monolayer culture before and after enucleation is shown in Figs 1A and 1B, respectively. The efficiency of the enucleation procedure was 89 f 2.1 as determined by fixing, staining with eosin-hematoxylin, and counting the nucleated and anuclear cells that remained attached to the flask after treatment with cytochalasin B and centrifugation. Forty different low power ( x 200)

Enucleation of human endometrial cells

(A)

(W Fig. I, Cultures of HEC-50 cells before (A) and after (B) enucleation. Eosin-hematoxylin ethanol fixation ( x 400).

fields were examined for a total count of about 1000 cells plus cytoplasts per flask. The enucleated cells remained attached and excluded trypan blue for at least 3 days. Cytoplasts could be removed from the flask by 0.05% trypsin4.02% EDTA treatment and replated after washing with MEM-FBS. Estrogen receptor The distribution between nucleus and cytoplasm of specific binding sites for estradiol was studied using two different fractionation methods: (a) homoge-

staining after

nization in Tris-sucrose buffer followed by separation of nuclei and cytoplasm by centrifugation, and (b) enucleation by treatment of attached cells in monolayer cultures with cytochalasin B followed by collection of free karyoplasts and attached cytoplasts. As shown in Table 1, about 32% of the total cellular estrogen receptor was found in the nuclear fraction after homogenization. In contrast, about 86% of the total intracellular estrogen receptor was found in the karyoplasts. This difference in distribution was statistically significant (P < 0.001, paired Student’s t-test).

ACH~LLEGRAVANIS and

472

Table 1, Nucleo-cytoplasmic

distribution of estrogen receptors in HEC-50 cells

Fractionation Exp. no.

1

Enucleation

Homogenization

2

Enudeation

Homogenization

3

Enucleation

Aver. *SE

..--.

procedure Homogenization

Homogenization Enucleation

ERLIO GURPIDE

Fraction

Specific [‘H]E, binding sites cell

% Nuclear

Cytosof Nuclear Total Cytoplast Karyoplast Total

8600 5200 I3800 2100 8500 10600

38

Cytosol Nuclear Total Cytoplast Karyoplast Total

12000 4800 16800 1600 13000 14600

29

Cytosol Nuclear Total Cytoplast Karypolast Total

15000 6000 21000 1700 16000 17700

29

Totai Total

DNA polymerase ct Results on distribution of DNA poiymerase a activity between nuclei and cytosol after homogenization, and between karyoplasts and cytoplasts after enucleation, are shown in Table 2. Only about 20% of the activity was found in the nuclear fraction of homogenized cells whereas 96% of the activity was found associated with karyoplasts. The results presented in Table 2 also indicate that the total enzymatic activity homogenates is about f of the total activity measured after enucleation, probably due to loss of activity measured after homogenization. Table 3 shows results from measurements of DNA Table 2. Nucleo-cytoplasmic

80

89

90

17OOOi2100 14000 f 2100

32 f 3.0 86 f 5.5

NS

P < 0.001

polymerase tl activity in homogenates of HEC-50 cells in the presence or absence of cyt~halasin B. The enzymatic activity in homogenates is similar to the total activity estimated by adding nuclear and cytosolic activities after homogenization (Table 2). These results also demonstrate that cytochalasin B has no effect on DNA polymerase c( activity. DISCUSSION

The results reported here provide further evidence indicating that estrogen receptors and DNA poly merase a activity are removed from nuclei during homogeni~tion. In addition, it demonstrates that

distribution of DNA polymerase a activity in HEC-50 cells DNA poly. activity Units

Exp. no.

1

Fractionation procedure Homogenization

Enucleation

2

Homogenization

Enucleation

3

Homogenization

Enucleation

Aver. & SE

Homogenization Enucleation

% Nuclear

106 cells

Fraction Cytosol Nuclear Total Cytoplast Karyoplast Total

0.59 0.15 0.74 0.21 7.9 8.1

20

Cytosol Nuclear Total Cytoplast Karyoplast Total

0.61 0.16 0.77 0.22 5.5 1.0

21

Cytosol Nuclear Total Cytoplast Karyoplast Total

0.82 0.19 1.0 0.82 5.7 6.0

19

Total Total

I pm01 TTP incorp. ‘DNA polymerase OLunit = -min

0.84 k 0.08 6.6 f 0.75

91

96

95

P < 0.001

20 rt 0.58 96 + 0.58

P < 0.001

Enucleation of human endometrial cells

473

Table 3. DNA polymerase a activity in homogenates of HEC-50 cells: effects of cytochalasin B DNA polymerase 01activity

Homogenate (0.5% DMSO) Homogenate + Cvtochalasin B*

(Units/IO6cells)

Exp. I

Exp. II

Exp. III

I.!

0.91

1.0

1.0 kO.10

I.1

0.89

0.96

0.98 2 0. I I

X+SD

*20 gg/ml, I .3 h incubation at 37 ‘C. Dimethylsu~foxide (DMSO) present at 0.5% concentration.

DNA polymerase a is partially inactivated during the preparation of homogenates. Enucleation was shown not only to be a useful method to avoid artifactual mobilization of nuclear proteins but also to prevent inhibitory or degradative processes that affect DNA polymerase a during homogenization. This and other laboratories have shown the presence of unoccupied estrogen binders (“available receptors”) in nuclear preparations 1131,a finding that does not conform with the view that estrogen receptors reside in the cytoplasm and are translocated to the nucleus only after being “activated” by binding to estrogens, as generally accepted [ 141. Other observations challenging the classical view include the finding of “inactive” 8-9s form of the estrogen receptor in nuclei isolated from rat uterus [ 151and the immunocytochemical detection with monoclonal antibodies of estrogen receptor in nuclei but not in cytosol of breast tumors from postmenopausal women [ 161. The results of enucleation experiments reported by Welshons et a1.171 and those presented here demonstrate the preferred nuclear location of the estrogen receptor. It should be emphasized, however, that these findings do not affect the basic postulate regarding early events in estrogen action, viz., that in viuo binding of the hormone with the receptor produces changes in the affinity of the binding of the receptor to chromatin components which are considered to be necessary for hormonal action, Whether the “activation” occurs in the nucleus or the cytoplasm is not a fundamental tenet in the original proposal. It is of interest that HEC-50 endometrial adenocarcinoma cells, in spite of the presence of specific 13H]E2 binders in the nucleus, have not been found to respond to estrogens. In contrast, cultures of another human endometrial adenocarcinoma cell line (Ishikawa), established by Nishida et aZ.[l7], have been shown to respond to estrogens by increasing cell numbers and progesterone receptor levels [ 181,as well as DNA polymerase c( activity (Gravanis et al., unpublished). Comparison of the affinity of estrogen binders to karyoplasts in responsive and unresponsive endometrial cells in the presence of excess E, may serve to identify differences in the quality of receptors and acceptors in these two cell types. Measurements of DNA polymerase GIactivity in karyoplasts will allow a more realistic evaluation of the regulation of this activity by estrogens and its changes under physiological conditions. Further-

more, recombination of karyoplasts with cytoplast contents may reveal cytoplasmic factors responsible for inhibition of the nuclear activity. Acknowledgements-We are grateful to Dr H. Kuramoto, Kitasato University, Japan, for making Ishikawa cells avail-

able to us. This work was supported by grants HD 07197, awarded by the National Institute of Child Health and Human Development, and CA 15648, awarded by National Cancer Institute.

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