The Biologicalsignificanceof Estradiolreceptorbindingto DNA1

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[CANCER RESEARCH 40, 1050-1053, April 1980] 0008-5472/80/0040-0000$02.00 The BiologicalSignificanceof EstradiolReceptorBindingto DNA1

Sukur Khan, Mark Feldman, and Vincent P. Hollander2

Research Institute of the Hospital for Joint Diseases and Medical center, Mount Sinai School of Medicine, New York, New York 10035

ABSTRACT It is well known that ER complex binds to DNA-cellulose (6, 10, 14, 21, 23, 24). In this study, it will be shown that MTW9- MTW9-D, a rat mammary tumor derived from MTW9 by D, a tumor which regresses after ovex, has a cytosol ER which chronic administration of the dopamine antagonist drug specifically binds to DNA-cellulose. Mt-MtT, a tumor which R33,81 2 shows ovaniectomy-induced regression (OIR). MTW9- does not regress after ovex, has a cytosol receptor which MtT is also a variant of MTW9, grown by coimplantation of a shows much less specific binding to DNA-cellulose. When MtT mammosomatotropic tumor MtTW10, but it does not show OIR. is resected, MTW9 regresses but shows more rapid and com However, when the mammosomatotropic tumor (MtT) is ne plete regression after ovex such that the tumor has been sected, the tumor regresses and shows rapid OIR; implantation converted to one which shows OIR (4). After MtT resection, of MtT into animals bearing MTW9-D prevents OIR following the mammary tumor ER binds to DNA-cellulose, as well as that drug withdrawal. The estradiol receptor (ER) from MTW9-D from MTW9-D. When MtT is implanted into animals bearing cytosol binds to DNA-cellulose significantly more than that from MTW9-D, OIR is prevented and ER binding to DNA-cellulose is MTW9-MtT. After MtT resection, the mammary tumor ER binds significantly reduced. Accordingly, the factor(s) secreted by to DNA-cellulose, as well as ER from MTW9-D, whereas im MtT which prevents OIR may also prevent binding of tumor ER plantation of MtT into animals bearing MTW9-D decreases ER to DNA. binding to DNA-cellulose. The significance of these findings in relation to possible clinical application is discussed. MATERIALS AND METHODS

INTRODUCTION Generation of Tumors. MTW9 and MtTW10 were obtained MTW93 is a transplantable rat mammary papillary adenocar and transplanted into W/Fu rats as described previously (5). cinoma developed by Kim and Furth (9). We have developed However, in this study, MTW9-D is a tumor grown by chronic sublines from the parent tumor which grow in hosts under administration of R3381 2. Animals received daily s.c. injections different endocrine conditions. These tumor variants differ in of 5 mg of drug in 0.2 ml steroid suspending vehicle (Armour hormonal receptor content and in response to ovex. Pharmaceutical Co., Kankakee, Ill.). Treatment was continued MTW9 can be grown by increasing serum prolactin of the until tumors were 1 to 2 cm in diameter (usually 30 to 50 days host through coimplantation of a mammosomatotropic tumor following implantation). At this state, animals were killed by MtTW10 or by administration of dopamine antagonist drugs. decapitation and mammary tumors were analyzed for ER. The resulting tumors, MTW9-MtT and MTW9-P,4 are similar R3381 2 and other dopamine antagonists (perphenazine, morphologically but differ in ER (3, 5), prolactin receptor (15), spiramide) increased serum prolactin and stimulated the growth and biological response to ovex (4). MTW9-P is our model for of transplantable MTW9 to give tumors which were morpholog a tumor which shows OIR. Kute et al. (12) have recently ically identical. All of these tumors showed OIR, in contrast to suggested that ER-positive tumors which fail to respond to MT-MtT. A description of drug-induced growth of MTW9 is in endocrine therapy have a 45 but no 8S component. Our tumor, preparation. MTW9-MtT ER, has both an 85 and 4S component (3) and is Preparation of [3HJERComplex. Tumors were rapidly re therefore a particularly interesting and useful model for a tumor moved, minced, and washed with 4 volumes of Buffer A (20 which fails to regress after ovex. The mechanism of resistance mM Tnis-1 mM EDTA-30 mM KCI-1 mM mercaptoethanol), pH to regression is not clear but it cannot be attributed to prolactin 7.4 at 0Â°,and then resuspended in 2 volumes of the same or estradiol (4). buffer and homogenized at 0Â°with a Polytron 1OST homoge nizer (Bninkmann Instruments, Inc., Westbury, N. Y.). The ho @ This investigation was supported by Grants P 30 14 194 and CA 10064 mogenate was centrifuged at 190,000 x g for 30 mm. The awarded by the National Cancer Institute, Department of Health, Education, and clear supernatant was incubated at 0Â°for 1 hr with 17@8- Welfare, and American Cancer Society Grant BC-312 awarded by the American [3H]estnadiolat a final concentration of 10 n@in the presence Cancer Society, Inc., New York, N. Y. 2 To whom requests for reprints should be addressed. on absence of 100-fold excess diethylstilbestrol to correct for 3 The abbreviations used are: MTW9, a transplantable mammary carcinoma nonspecific binding. grown in female W/Fu rats; ovex, host ovariectomy; MtTW10, a mammosoma totropic tumor obtained from Dr. U. Kim; MTW9-MtT, MTW9 supported by Activation of [3H]ER Complex. In order to determine the coimplantation of MtTW1 0; ER, estradiol receptor; OlR, ovariectomy-induced optimal activation time and temperature for both tumors, the regression; MTW9-D, a tumor which regresses after host ovariectomy; Mt-MtT, charged cytosols were incubated at various temperatures (4â€” a tumor which does not regress after host ovariectomy; MtT, a mammosomato tropic tumor. 28Â°)for 5 to 90 mm; binding to DNA-cellulose was done as 4 The abbreviation MTW9-P was used in our previous publications to indicate described below. Maximal binding to DNA was found after a variant of MTW9 obtained by perphenazine administration. In this paper, we activation for 30 mm at 20Â°(Chart 1). have used the abbreviation MTW9-D to indicate a variant of MTW9 obtained by administration of the drug 5-chlor-1 -(1 -[3-(2-3-dihydro-2-oxo-1 H-benzimidazol Binding of Activated (3H]ER Complex to DNA. Bindingof 1-yl)propyl]-4-piperidinyl)-1 ,3-dihydro-2H-benzimidazol-2-one. This compound ER complex to DNA-cellulose was determined by a modification (No. R33,812) was generously supplied by Janssen R & D Inc., New Brunswick, of the procedure of Yamamoto and Alberts (22). DNA-cellulose N. J. Received May 25, 1979; accepted December 20, 1979. was prepared by the method of Alberts and Hennick(1). Cellex

1050 CANCERRESEARCHVOL. 40

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.@ ______0 5 30 45 60 90 E Time in mtnutes a â€˜0 C 0 @ 20 .0 @. B 0 ! 100 E 5 30 45 60 :1 80 Time inminutes Chart 2. Time course of [â€˜H]ERbindingto DNA-cellulose at 20Â°.â€¢,ER @60 prepared from MT-D;0, ER prepared from MT-MtT tumor; Each point represents 0 the average of duplicate samples from a representative experiment. Duplicate values agreed within 10%. @40 .@ + + 4 2 20 of diethylstilbestrol. Binding to DNA by a 3H-labeled receptor containing diethylstilbestrol was similar to the binding of a labeled receptor to cellulose which contained no DNA. Binding @@@ â€”@I-_@-5 30 45 60 90 was expressed as fmol [3H@estradiolbound per mol charged Time in minutes activated receptor complex contained in the incubation tube Chart 1. Influence of activation temperature for [3H]ERfrom MTW9-D (A) and (input). Care was taken when comparing experimental groups MTW9-MtT (B@on the binding to DNA-cellulose. 0, 20Â°activation; 0, 28Â° activation; + , 2â€”4Â°actIvation; â€¢,15Â° activation; Each point represents the that comparisons were done at approximately the same inputs average of duplicate samples from a representative experiment. Duplicate values of [3H]ER. This was done by using the appropriate dilutions of agreed wIthin 10%. changed cytosols. Input was determined prior to the incubation of changed activated receptor complex with DNA-cellulose by grade cellulose (Bio-Rad Laboratories, Richmond, Calif.) was a conventional dextran-coated charcoal technique with minor treated in a similar manner except that no DNA was used. The variations (11). cellulose was used as a control for the measurement of binding to DNA-cellulose. The DNA-cellulose suspension was re RESULTS washed on the day of use, resuspended in Buffer B (Buffer A Binding of MTW9-D and MTW9-MtT to DNA-Cellulose. plus 0.2% bovine serum albumin and 10% glycerol), and ad Chart 3 shows the specific binding of estradiol from MTW9-D justed to contain 10 @sgofDNA pen0.1 ml of suspension. The or MTW9-MtT to DNA-cellulose. When compared in terms of DNA-cellulose suspension was then incubated with known either protein concentration of cytosol or radioactive input, concentrations of charged activated [3H]ER complex in a total MTW9-D cytosol receptor binds significantly more to DNA than volume of 0.5 ml at 200 for 30 mm. Optimal time for maximum does MTW9-MtT cytosol receptor under the experimental con binding at 200 for both tumor cytosols was found to be 30 mm ditions used. (Chart 2). The incubation was carried out in a water bath with Effect of MtT Resection or Implantation on DNA Binding gyrotatory shaking to ensure uniform suspension of ONA-cel of MTW9 Receptor. When MtTWI 0 is nesected from animals lulose. At the end of the incubation period, the mixture was beaning MTW9-MtT, the mammary tumor regresses. If such chilled with 2.0 ml of Buffer C at 0@(Buffer B containing 0.5 resected animals are also ovaniectomized, the regression of @LMnonradioactive estradiol) and centrifuged at 2500 x g at 2Â° mammary tumor is faster and more complete than with MtT for 5 mm. The supernatant was removed by aspiration, and the resection alone (5). These observations are consistent with the DNA-cellulose pellet was washed once more with 2.0 ml of hypothesis that MtTW10 secretes a factor which prevents OIR. Buffer C. The 2500 x g pellet was then extracted with 1 ml of Our observations that transplantation of MtTW10 into animals ethanol at 37Â°for 15 mm with constant shaking. A suitable bearing MTW9 converts the tumor to one which no longer aliquot of the 2500 x g supernatant was assayed for radioac regresses after ovaniectomy strengthens this hypothesis (4). tivity. The second wash with Buffer C completely removed all The hypothesis suggests that MtT resection not only would traces of binding by free [3H]estradiol to DNA. The specific remove the source of the factor which inhibited OIR but also binding of(3H]ER to DNA-cellulose was determined by subtract would enable the mammary tumor cytosol ER to bind to DNA ing the radioactivity bound to DNA by a cytosol receptor to the same degree as MTW9-D, a tumor which invariably prepared as above, but to which was added a 100-fold excess shows OIR.

APRIL 1980 1051

a)us A 0

11) U z a Chart 3. ER bound to DNA-cellulose as a function of total radio 0 0 activity input (A) or as a function of the protein concentration in the @0 receptor preparations (B). A, cytosol prepared from MT-D tumor;@, cytosol prepared from MT-MtT tumor; Each point represents the average of duplicate samples from a representative experiment. Du plicate values agreed within 10%.

.1 0.2 03 04 0.5 0 2.0 30 40 5.0 [3H] ER applied/tube (pmol) Protein concentration (mq)/tube

Table 1 cellulose. Such binding is specific in that the binding requires Effect of MtT resection or implantation on DNA-cellulose binding of MTW9-MtT steroid and activation of receptor (14, 22). Studies in this p for MTW9-MtT versus MtT resection, <0.001 ; p for MTW9-MtT versus laboratory (6â€”8)haveshown that ER shows preference for AT MTW9-D, <0.001 ; p for MIT resection versus MTW9-D, <0.1 ; p for MTW9-D versus MTW9-D + MtT, <0.001. regions of double-stranded DNA and that substitution of the 5- [â€˜H]ERMTW9-D+MTW9-MITMtT-resectionMTW9-DMIT70.1102.5149.620.592.0112.1154.024.39.093.4139.020.233.080.698.033.117.5119.188.569.4fmol bound to DNA-cellulose/pmol methyl group in thymidine with halogens strengths binding to DNA, according to the electronegativity ofthe substituted atom. DNA-cellulose does not appear to bind ER by acting as a simple polyanion: tRNA does not compete (8). For these rea sons, we looked for differences in DNA binding of ER from 2 sublines of MTW9, one which shows OIR and one of which does not. We felt that this system was useful for relating OIR 224.8 and DNA binding of ER. By appropriate changes in host hor 455a 106.6 64.9a 91.6 monal environment, OIR can either be induced or prevented .26.9Mean 36.8a and DNA binding should change accordingly if the concept has 37.483.3 utility. The realization of this prediction relates OIR and ER binding to DNA, at least in the MTW9 system. Failure of ER 3.0aÂ±SE 5.7 Â±7.798.6 Â±6.3131.5 Â±16.224.5 Â± binding to DNA may represent an intrinsic lack of nuclear sites Mock operated. in some receptors (19) similar to defects found in corticoid translocation in lymphoma cells (17), or it may represent loss Table 1 shows that, 3 days after MtT resection and after a of DNA-binding sites in ER by proteolytic enzymes present in 20 to 30% reduction in MTW9 diameter, ER from MTW9-MtT certain mammary tumors (18). Alternatively, the differences in showed an increase of binding to DNA equivalent to that shown DNA binding observed in the 2 tumor sublines might not reflect by MTW9-D. Mock operation on animals bearing Mt-MtT had differences in the receptor per se but might rather be due to no effect on the binding of ER to DNA. Although there is different levels of â€˜â€˜inhibitor'â€˜orâ€˜â€˜activator'â€˜substancesin the considerable variation among tumors of the same type, the cytosol, which would bind to ER and directly influence the difference between groups is large enough to be clearly signif DNA-binding site. Such substances have been postulated to icant. Table 1 also shows that implantation of MtT into animals influence rec@ptonbinding in a number of different systems (2, with MTW9 tumors significantly reduced ER binding to DNA; 20). A different level of these molecules in the 2 tumors could MTW9-D tumors were grown as described in â€˜â€˜Materialsand indicate genetic differences brought on by clonal selection of Methods' â€˜(1to 2 cm in diameter), at which time the drug was some subpopulation of cells by the MtT. These possibilities are withdrawn, and MtT was implanted s.c. When MtT became under study and will be reported elsewhere. palpable and MTW9 growth was evident(4 to 6 weeks following We will not discuss in this paper the relationship between implantation), animals were killed and tumors were analyzed binding of ER to DNA with in vivo nuclear binding or the for ER binding to DNA cellulose. ER content per mg cytosol possible heterogeneity of tumor receptor for DNA-binding sites. protein, which was 2 to 3 times higher in MTW9-D than in These questions are under active study. Here, we discuss a MTW9-MtT, did not change following MtT implantation or re possible test which correlates OIR with ER binding to DNA and section (data not shown). These data support the concept that forms the basis of our plan to study hormone-sensitive and DNA binding differences between the 2 receptors may be hormone-resistant human tumors. Only about one-half of the related to functional differences. patients with mammary cancers which contain ER achieve remission through endocrine therapy, although the correlation DISCUSSION may be considerably improved in patients with tumors binding both estradiol and progesterone (13). If patients could be High-affinity ER proteins from the uterus (1 6, 21 , 22), mam selected by a simple extension of present tests for tumor mary gland (14), and mammary tumors (12) bind to DNA cytosol steroid receptors, therapy would be improved and the

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. Estradiol Receptor Binding to DNA detailed study of the mechanisms of binding ER to DNA would 10. King, R. B., and Gordon, J. Involvement of DNA in acceptor mechanism for uterine estradiol receptor. Nature (Lond.), 240: 185â€”187,1972. become of special importance. 11. Korenman, S. G., and Dukes, B. A. Specific estrogen binding by the cytoplasm of humanbreast carcinoma. J. Clin. Endocrinol. Metab., 30: 639â€” 645, 1970. ACKNOWLEDGMENTS 12. Kute, T. E., Heidemann, P., and Wittliff, J. L. Molecular heterogeneity of cytosolic forms of estrogen receptors from human breast tumors. Cancer We wish to thank Annie Miller for her valuable technical assistance and Drs. Res., 38: 4390-4393, 1978. John Stevens and Edward Diamond for their continuous interest in our work and 13. McGuire, W. L. Steroid receptors in human breast cancer. Cancer Res. , 38: constructive criticism of this manuscript. 4289-4291, 1978. 14. Park, D. C., and Wittliff, J. L. Assessment of â€˜â€˜activation'â€˜of estrogen receptors in lactating mammary gland using DNA-cellulose binding. Bio chem. Biophys. Res. Commun., 78: 251-258, 1977. REFERENCES 15. Powell, B. L., Diamond, E. J. , Koprak, S., and Hollander, V. P. Prolactin binding in ovariectomy-responsive and ovariectomy-nonresponsive rat mam 1. Alberts, B., and Herrick, G. DNA-cellulose chromatography. Methods En mary carcinoma. Cancer Res., 37: 1328â€”1332,1977. zymol., 210: 198â€”217,1971. 16. Ratajczak, T., and Hahnel, R. Chromatographic and other properties of the 2. Chamness, G. C., Jennings, A. W., and McGuire, W. L. Estrogen receptor estrogen receptors from human myometrium. J. Steroid Biochem., 7: 185â€” binding to isolated nuclei. A nonsaturable process. Biochemistry, 13: 327â€” 197, 1976. 331, 1974. 17. Sibley, C. H. . and Tomkins, G. Mechanism of steroid resistance. Cell, 2: 3. DIamond, E. J., Giladi, M., Khan, S., and Hollander, V. P. Estradiol binding 221â€”227,1974. In ovarlectomy-responsive and non-responsive rat mammary carcinoma. 18. Sherman, M. R., Pickering, L. L., Rollwagen, F. M., and Miller, L. K. Mero Cancer Res., 37: 1852-1856, 1977. receptors: proteolytic fragments of receptors containing the steroid-binding 4. Diamond, E. J., Khan, S., Koprak, S., and Hollander, V. P. Inhibition of site. Fed. Proc., 37: 167â€”173, 1978. ovarlectomy-induced rat mammary tumor regression by a mammosomato 19. Shymala, G. Estradiol receptors in mouse mammary tumors: absence of the tropictumor.CancerRes.,38: 2239-2245, 1978. transfer of bound estradiol from the cytoplasm to the nucleus. Biochem. 5. DIamond,E. J., Koprak, S., Shen, S. K., and Hollander, V. P. The conversion Biophys. Res. Commun., 46: 1623â€”1630,1972. of an ovarlectomy-nonresponsive to an ovariectomy-responsive mammary 20. Simons,5. S., Jr., Martinez, H. M., Garcea, R. L., Baxter, J. D., and Tomkins, tumor strain. Cancer Res., 36: 77â€”80,1976. G. M. Interactions of glucocorticoid receptor steroid complexes with accep 6. Kallos, J., Fasy, T. M., Hollander, V. P., and Bick, M. D. Estrogen receptor tor sites. J. Biol. Chem., 251: 334â€”343,1976. has enhanced affinity for bromodeoxyuridine-substituted DNA. Proc. NatI. 21 . Toft, D. Interaction of uterine estrogen receptors with DNA. J. Steroid Acad. Scm.U.S. A., 75: 4896-4900, 1978. Biochem., 3: 515â€”522,1972. 7. KalIos, J., Fasy, T. M., Hollander, V. P., and Bick, M. D. Estrogen receptor 22. Yamamoto, K. R., and Alberts, B. In vitro conversion of estradiol-receptor can distinguish among various halodeoxy-uridine substituted DNAs. FEBS protein to its nuclear form: dependence on hormone and DNA. Proc. NatI. Leff., 98: 347-349, 1979. Acad. Sci. u. S. A., 69: 2105-2109, 1972. 8. Kallos, J., and Hollander, V. P. Assessment of specificity of estrogen 23. Yamamoto,K. A., and Alberts, B. On the specificity of the binding of estradiol receptor-DNA interaction by a competitive assay. Nature (Lond.), 2 72: 177â€” receptor protein to deoxyribonucleic acid. J. Biol. Chem., 249: 7076â€”7086, 179, 1978. 1974. 9. Kim, U., and Furth, J. Relation of mammotropes to mammary tumors. IV. 24. Yamamoto,K. R., and Alberts, B. The interaction of steroid-receptor protein Development of highly hormone dependent mammary tumors. Proc. Soc. with genome: an argument for the existence of undetected specific sites. Exp. Biol. Med., 105: 490-492, 1960. Cell, 4: 301-310, 1975.

APRIL 1980 1053

Sukur Khan, Mark Feldman and Vincent P. Hollander

Cancer Res 1980;40:1050-1053.

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