Norethindrone Scatters Silver-Stained Nucleolar Organizer Regions of Ishikawa Cells Yasuhiro Yokoyama,1 Kenji Niwa, and Teruhiko Tamaya

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[CANCER RESEARCH 51, 5987-5992, November 1, 1991] Norethindrone Scatters Silver-stained Nucleolar Organizer Regions of Ishikawa Cells Yasuhiro Yokoyama,1 Kenji Niwa, and Teruhiko Tamaya

Department of Obstetrics and (Â¿ynecology,Gifu University School of Medicine, Gifu University, Tsukasa-machi 40, Gifu City, (iifu Prefecture 500, Japan

ABSTRACT dated. Progesterone and estrogen have effects on the nucleolus and Ag-NORs in the endometrial epithelial cell (20-22). If We investigated the effects of sex steroids on silver-stained nucleolar organizer regions (Ag-NORs) and DNA/RNA kinetics in Ishikawa cells. endometrial carcinoma shows the same behavior in response to Norethindrone and its isomer norethynodrel exclusively caused Ag-NORs sex steroids as does its benign counterpart, sex steroids will also have an effect on its Ag-NORs. to scatter in the nuclear matrix, from the nucleolus. No such effect occurred with the other sex steroids tested, including progestational, Therefore, we investigated the effects of progestogens, as well androgenic, and estrogenic compounds. Nuclear argyrophilic substances as the other kinds of sex steroids, on Ag-NORs of endometrial induced by norethindrone, as well as nucleolar ones, were neither DNA carcinoma cells. nor RNA but protein. Electron microscopy showed that norethindrone caused nucleolar segregation, in which the fibrillar components disap peared, and it produced islets, consisting of dense fibrillar materials, in MATERIALS AND METHODS the nucleoplasm. Ag-NORs observed on the fibrillar components in Ishikawa cells are a continuously cultured endometrial carcinoma control nucleoli were translocated onto the dense fibrillar materials in cell line, established by Nishida et al. (23). These cells have undergone the nucleoplasm. Although scattering was preferentially found in the cells 50-70 passages and possess both estrogen and progesterone receptors. synthesizing DNA, the scintillation assay of DNA/RNA kinetics sug The cells used in this study were grown in Eagle's minimum essential gested that scattering was related to the inhibition of RNA synthesis. medium supplemented with glutamine (292 mg/liter) and 10% fetal These results imply that norethindrone preferentially interacts with bovine serum treated with charcoal. Cultures were maintained at 37Â°C, intranucleolar DNA when its duplication is occurring and then interferes in a humidified atmosphere of 5% CO:/95% air. The cells were cultured with rRNA synthesis. Scattering of Ag-NORs might not be caused by in Lab-tek chamber slides or 24-well culture plates, for 2 days, and the hormonal activity of these agents but by a pharmacological effect were then incubated with various sex steroids at IO"4M. derived from their molecular structures. The sources of steroid hormones were as follows. Progesterone, norethindrone, norethynodrel, norethindrone acetate, ethisterone, tes INTRODUCTION tosterone, 17Â«-methyl-testosterone, 19-nor-testosterone, 17Â«-ethinyl- estradiol-3-methyl-ether (mestranol), and ethinyl estradici were pur NORs2 are located on the secondary constrictions of five chased from Sigma Chemical Co. (St. Louis, MO). Norgestrel, danazol, acrocentric chromosomes and can be selectively stained by silver gestrinone, chlormadinone acetate, and medroxyprogesterone acetate staining (1-3). This technique demonstrates nonhistone pro were kindly provided by Wyeth-Ayerst Research (Princeton, NJ), To teins associated with the NORs, rather than the rDNA itself kyo Tanabe Pharmaceutical Inc. (Tokyo, Japan). Lucci Medica Inc. (1, 4). In interphasic cells, Ag-NORs can be observed on the (Tokyo, Japan), Shionogi Pharmaceutical Inc. (Osaka, Japan), and fibrillar centers and peripheral dense fibrillar components of Kyowa Hakkou Inc. (Tokyo, Japan), respectively. Allylestrenol and the nucleoli (4-7). lynestrenol were kindly provided by Organon International B.V. (BH Oss, Netherlands). These sex steroids were dissolved in dimethylsulf- The exact localization of rDNA genes during interphase has oxide, and aliquots were added to the culture medium at the given never been elucidated, but recent investigations have suggested concentrations. The concentration of dimethylsulfoxide in the culture that fibrillar centers might be the sites where they are located medium did not exceed 0.1%. The concentration of dimethylsulfoxide (8-10). Ag-NOR proteins exist in close proximity to rDNA in controls was adjusted to 0.1 %, which had no effect on Ishikawa cells. genes and so may play an essential role in the transcription of Light Microscopy. After incubation for specified time intervals, the rRNA in interphasic nuclei. However, the exact biological role cells were fixed with 6% paraformaldehyde in 0. l M phosphate buffer of these Ag-NOR proteins has not yet been elucidated. (pH 7.2), at 4Â°Cfor 1 h. All sex steroids inhibited cell growth at the concentration of 10~4M. The characteristics of Ag-NORs have been investigated in many types of cells, and differences in the number of Ag-NOR Ag-NOR staining was performed by the method of Howell and Black dots between benign and malignant cells have been demon (2). More than 500 cells in three different fields were examined for strated in some tumors (11-16). Moreover, the staining inten each culture. Digestion tests of argyrophilic substance associated with scattering sity of Ag-NORs has been reported to be related to the activity of the Ag-NORs by norethindrone were conducted using the following of RNA synthesis in fibroblasts (17) and HeLa cell (18) treated treatments: (a) DNase (1 mg/dl) in 0.1 M Tris buffer (pH 7.3) contain with actinomycin D. Thus, the staining of Ag-NORs seems to ing MgCl2 and CaCl2, at 37Â°Cfor 2 h, (b) 0.1% RNase in 0.1 M indicate a transcriptional potential of rDNA. phosphate buffer (pH 6.0), at 37Â°Cfor 1 h, and (c) 0.01% trypsin in Estrogen is associated with a risk of endometrial carcinogen- phosphate-buffered saline, at 37"C for 10 min. The successful extraction esis (19), while some progestogens have been used for the of DNA or RNA was checked by acridine orange fluorescence. treatment of advanced endometrial carcinoma. Sex steroids are Evaluation of DNA and RNA Synthesis. The percentage of inhibition closely associated with the development of endometrial carci of RNA or DNA synthesis by the sex steroids was investigated in the noma, but the mechanism of their effects remains to be eluci- following manner. Approximately 50,000 cells were grown in the culture wells. Forty-eight h later, the cells were reacted with sex steroids at 10~4M and various concentrations of norethindrone. After 12 h of Rcceived 3/12/91; accepted 8/19/91. The costs of publication of this article were defrayed in part by the payment incubation, RNA or DNA synthesis was monitored by pulse-labeling of page charges. This article must therefore be hereby marked advertisement in cells, for 30 min, with 1.0 MCi/ml ['Hjuridine (New England Nuclear, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Boston, MA) or 1.0 ^Ci/ml ['H]thymidine (New England Nuclear), ' To whom requests for reprints should be addressed. ' The abbreviations used are: NOR, nucleolar organizer region: Ag-NOR, respectively. In the former assay, 10 /Â¿molofthymidine were simulta silver-stained nucleolar organizer region. neously added to the medium. 5987

Liquid scintillation counting was performed using a Beckman Â±SD) or 59 Â±18% of the control level, respectively, whereas LS7500 scintillation counter (Beckman Instruments Inc., Irvine, CA). the thymidine uptake did not decrease. Fig. 5 shows the rela This assay was carried out in quadruplicate. tionship between scattering of Ag-NORs and DNA/RNA ki Relationship between DNA Synthesis and Scattering of Ag-NORs. Cells grown in Lab-tek chamber slides were incubated with norethin- netics at different concentrations of norethindrone. Since the drone at various concentrations. For the last 30 min of the 6-h incu medium was fully saturated at approximately 70 pg/m\ noreth bation period, 5-bromo-2'-deoxyuridine (Sigma) was added to the indrone, the percentage of inhibition and percentage of scatter medium at a final concentration of 10 MM.The cells were then fixed in ing showed limited changes at higher concentrations. RNA 90% ethanol for >30 min. synthesis was inhibited in a concentration-dependent manner, Cells were treated with 0.07 N NaOH to denature DNA, and im- while DNA synthesis was not inhibited up to a concentration munohistochemical staining of bromodeoxyuridine was carried out with of 40 Mg/ml. antibromodeoxyuridine monoclonal antibody (Becton Dickinson Im- Relationship of Ag-NOR Scattering to the S Phase. The munocytometry Systems, Mountain View, CA) and a Vectastain ABC relationship between the phase of the cell cycle and Ag-NOR kit (Vector Laboratories, Burlingame, CA). Following this immunohis- scattering is shown in Fig. 6. After 6 h of exposure to noreth tochemical staining, NOR staining was conducted. The relationship between labeling with bromodeoxyuridine and scattering of Ag-NORs indrone, scattering occurred dose dependently, and the per was investigated in >500 cells from each culture. centage of cells in the S phase decreased. Cells labeled with Electron Microscopy. Cells were fixed with 2.5% glutaraldehyde in bromodeoxyuridine were significantly more likely to show scat 0.1 M phosphate buffer (pH 7.2) for l h at 4Â°Cand with 0.1% osmic tering of Ag-NORs at any concentration of norethindrone (Fig. acid in 0.1 M phosphate buffer (pH 7.2) for an additional l h at room 7). temperature. The cells were dehydrated, detached with propylene oxide, Electron Microscopy. The nucleolus consisted mostly of gran and embedded in Epon. Ultrathin sections were cut and then counter- ular and fibrillar components. Fibrillar centers were not obvious stained with uranyl acetate and lead citrate. in Ishikawa cells. Nucleoplasm consisted mostly of euchromatin For the histochemical study of Ag-NOR localization, the cells were (Fig. 8). After treatment with norethindrone (60 Mg/ml) for 12 fixed with 6% paraformaldehyde in 0. l M phosphate buffer (pH 7.2), at 4Â°Cfor1 h, and were stained for NORs by the same method described h, nucleolar segregation occurred in some nucleoli. Loose fi- above. Cells were then fixed with 1% osmic acid for l h at room temperature, dehydrated, detached with propylene oxide, and embedded in Epon. Ultrathin sections were cut and counterstained with uranyl acetate and lead citrate. The sections were viewed with a Hitachi HU12 electron microscope (75 kV; Hitachi Instruments Inc., Ibaragi, Japan). Statistics. The data obtained were evaluated by the x2 test. Â«85

RESULTS Light Microscopy. Ag-NORs were observed in the nucleoli of Ishikawa cells during interphase. Among the sex steroids tested, only norethindrone and its isomer norethynodrel caused mor phological changes in the Ag-NORs. Treatment with these agents caused the emergence of Ag-NORs into the nuclei. The nuclear Ag-NORs showed stronger NOR staining than did nucleolar ones. We designated this morphological feature "scat tering" (Fig. 1). The nucleolar Ag-NORs in cells with the scattering of Ag-NORs induced by treatment with 20 ng/ml Fig. 1. Morphological changes of Ag-NORs in Ishikawa cells caused by norethindrone at 20 (ig/ml for 12 h. X 1350. Some cells show scattering of Ag- norethindrone for 12 h became obscure. At 60 tig/m\ noreth NORs in nuclei, while others show the intranucleolar localization of Ag-NORs. indrone, the nucleoli of the cells with the scattering dwindled in size (Fig. 2). None of the other sex steroids caused any changes in the distribution of the Ag-NORs or nucleolar morphology. Fig. 3 shows the percentage of cells showing scattered Ag-NORs after 12 h of incubation with 10~4 M sex steroids. After 12 h of treatment with norethindrone and norethynodrel, 74% and 25% of the cells, respectively, showed scattering of the Ag-NORs, while the cells incubated with the other sex steroids did not show scattering. The other sex steroids could not cause the scattering at any concentrations. In order to identify the argyrophilic substance related to the scattering, digestions with various enzymes were performed. The scattered Ag-NORs observed with norethindrone treat ment, as well as the nucleolar Ag-NORs, were completely eliminated by trypsin treatment, but not by either RNase or DNase treatment. DNA and RNA Kinetics. DNA/RNA synthesis after 12 h of exposure to the various sex steroids is shown in Fig. 4. The Fig. 2. Scattering caused by norethindrone at 60 pg/ml for 12 h. x 1350. Note the shrinkage of nucleoli and marked decrease of nucleolar Ag-NORs in the cells percentage of radioactive undine uptake in the presence of showing scattering. In contrast, cells without scattering have large nucleoli in norethindrone or norethynodrel decreased to 44 Â±10% (mean which Ag-NORs are localized. 5988

% of cells with scattered Ag NORs brillar materials were observed to migrate from the nucleolus 10 20 30 40 50 60 70 80 90 100 toward the nucleus, and patches of dense fibrillar materials Norethindrone emerged in the nucleoplasm (Fig. 9). An additional 6 h later, the fibrillar components disappeared out of the nucleoli, and Norethynodrel only condensed granular components remained. Many islets Norethindrone acetate consisting of dense fibrillar materials emerged in the nucleo Norgestrel plasm (Fig. 10). Ag-NORs were localized on the dark staining area of the Lynestrenol nucleolar body (Fig. 11). Most granular components and nu- Allylestrenol cleolar interstices were negative for staining. After treatment with norethindrone (60 /Â¿g/ml)for 12 h, Ag-NORs were stained Testosterone irregularly in such nucleoli and spottily in the nucleoplasm (Fig. Ethisterone 12). The Ag-NORs in the nucleoplasm corresponded to dense Ud-Methyltestosterone fibrillar materials. The loose fibrillar materials migrating from Mestranol the nucleolus were free from staining. The staining intensity of nucleolar Ag-NORs was heterogeneous. Ethinyl Estradici Progesterone DISCUSSION MPA With transmission electron microscopy, three morphologi cally distinct components are usually observed in interphasic Chlormadinoneacetate Gestrinone Danazol 19-nortestosterone

Fig. 3. Percentage of cells showing scattering of Ag-NORs after 12 h of exposure to 10~4M sex steroids. Ag-NORs were evaluated in >500 cells in three different fields for each culture. MPA, medroxyprogesterone acetate.

% Synthesis 20 40 60 80 100 120 140 160 180 200

Norethindrone ! 20 40 60 80 Norethynodrel (Concentration Fig. 5. Relationship of the percentage of cells with scattered Ag-NORs to RNA Norethindrone acetate and DNA kinetics at different concentrations of norethindrone. All cells were Norgestrel treated for 12 h. The percentage of cells showing scattering of Ag-NORs increased dose dependently. The medium was fully saturated with norethindrone at approx Lynestrenol imately 70 Mg/ml. O, percentage of synthesis of DNA; â€¢¿,percentageof synthesis of RNA; G, percentage of cells showing scattering of Ag-NORs. Allylestrenol Testosterone 50 100 Ethisterone 170-Methyltestosterone

Mestranol Ethinyl Estradiol Progesterone MPA Chlormadinoneacetate Gestrinone *P<0.001 Danazol â€¢¿Intact Ag-NORs in S phase 19-nortestosterone SJ8 Scattering Ag-NORs in S phase ^^ Scattering Ag-NORs in the other phase Ewl Intact Ag-NORs in the other phase Fig. 6. Relationship between the scattering of Ag-NORs and DNA synthesis. Fig. 4. Percentage of inhibition of DNA and RNA synthesis after 12 h of The cells were double-stained with Ag and avidin-biotin complex immunoperox- exposure to 10"* M sex steroids. Synthetic activity was evaluated by 30 min of idase methods for bromodeoxyuridine, 10 fimo) of which were pulse-labeled in pulse labeling with 1 nCi/mi ['Hjthymidine (DNA) or 1 /jCi/ml [3H]uridine the last 30 min of a 6-h incubation with various concentrations of norethindrone. (RNA). The experiments were performed in quadruplicate. MPA, medroxy At every concentration of norethindrone, S phase cells were significantly more progesterone acetate. likely to show scattering. 5989

both may be relevant substances. Therefore, the scattering of â€¢¿f* Ag-NORs may be associated with the translocation of rDNA. During the past several years, numerous investigations have . validated the usefulness of the enumeration of Ag-NORs for cancer diagnosis in certain tumors (11-15). In addition, rela tions of the number of Ag-NOR dots in tumor cells to their proliferative activity (16, 26-28) and prognosis (29, 30) have been reported. However, it is not entirely certain whether Ag- NORs are an index for the transcriptional activity of rDNA. t As shown in this study, norethindrone appeared to increase Ag- f 1 NOR dots in Ishikawa cells, but the total RNA synthesis and the proliferative activity clearly decreased. Under this experi f mental condition, Ag-NOR dots merely indicated possible lo cations of rDNA in Ishikawa cells, and the number was related neither to RNA synthetic activity nor to proliferative activity. Cells labeled by bromodeoxyuridine were liable to show scat- Fig. 7. Cells double-stained with Ag and immunohislochemislry for bromo deoxyuridine. Dark cells (arrows) were positive for bromodeoxyuridine. x 1330.

DFM'

Fig. 8. Control cell fixed with 2.5% glutaraldehyde and I % osmic acid and Fig. 9. Cell treated with norethindrone at 60 ng/ml for 12 h, fixed with 2.5% contrasted with maini acetate and lead citrate, x 17,000. FC, fibrillar compo glutaraldehyde and 1% osmic acid, and contrasted with uranyl acetate and lead nents; (if. granular components. citrate. Note the nucleolar segregation, x 17,000. GC, granular component; LFM, loose fibrillar materials; DIM, dense fibrillar materials.

nucleoli (24, 25). The fibrillar center is a possible area where rDNA exists, the fibrillar component usually exists at the periphery of the fibrillar centers and is the site of pre-rRNA synthesis, and the granular component is the main body of the nucleolus, which is composed of ribunucleoprotein particles, the terminal products of rRNA synthesis in the nucleolus. The fibrillar center is a conspicuous structure in many kinds of cells but in some cells is not obvious and may be absent (25). In Ishikawa cells, the fibrillar center was not obvious and the Ag- NORs were observed mostly on the fibrillar components. Thus, in Ishikawa cells, the fibrillar components will be the sites where rDNA exists and pre-rRNA is synthesized. Norethin- drone exclusively affected this specific site, causing inhibition of RNA synthesis. -*-* - Norethindrone segregated the nucleolar structure of Ishikawa cells and caused the following changes: (a) disappearance of fibrillar components from the nucleolus, (b) condensation of the granular components, and (c) appearance of dense fibrillar â€¢¿Â»v materials in the nucleoplasm. The scattering of Ag-NORs ob served in Ishikawa cells appears to be due to the argyrophilic nature of dense fibrillar materials emerging in the nucleoplasm. Fig. 10. Cell treated with norethindrone at 60 g/ml for 18 h, fixed with 2.5% glutaraldehyde and 1% osmic acid, and contrasted with uranyl acetate and lead Because the dense fibrillar materials structurally resemble the citrate, x 9,000. NO, nucleolus. Arrows, dense fibrillar materials. Nucleolus fibrillar components of the nucleolus and are also argyrophilic, consists only of the granular components. 5990

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1991 American Association for Cancer Research. NORETHINDRONE AFFECTS NUCLEOLAR ORGANIZER REGIONS ther the androgenic nor the estrogenic agents used in this study cause such changes, suggesting that scattering was not related to the pure hormonal effects of those agents. The other 19-nortestosterone progestogens used in this study, including noreth indrone acetate, norgestrel, lynestrenol, and allylestrenol, have the same multipotentiality as norethindrone but did not cause scattering. These results indicate that scattering of Ag-NORs is a pharmacological effect derived from the molecular structure of these two compounds. The facts that only a few kinds of progestational agents, which contain an acyl group at the 170-position, are used clinically against endometrial carcinoma, that the progesterone receptor content of the tumor is not always a key factor for the efficacy of progestogen therapy, and that a high loading dose of progestational agents is necessary for their efficacy (35) suggest that the mechanism of action of progestogens against endometrial carcinoma is not due to their hormonal effects but Fig. 11. Localization of Ag-NORs in control cells. After fixation with 6% to a pharmacological effect derived from their molecular paraformaldehydc, the cell was stained for NORs and then fixed with 1% osmic acid. After counterstaining with uranyl acetate and lead citrate, silver deposits structures. corresponded to fibrillar components in the nucleolus. x 8,000. NO, nuclcolus. There are no available reports on the efficacy of norethin drone against endometrial carcinoma. As shown in this study, the site of action of norethindrone is quite different from that of the other progestogens. This specificity suggests that noreth indrone might be effective for tumors that are resistant to current progestational agents.

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5992

Yasuhiro Yokoyama, Kenji Niwa and Teruhiko Tamaya

Cancer Res 1991;51:5987-5992.

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