[CANCER RESEARCH 35,1791-1798, July 1975]

The Alcohol and in Rodent and Human Mammary Tumors1

Viviane C. Godefroi, Elizabeth R. Locke,2 Dharm V. Singh,3 and S. C. Brooks

Michigan Cancer Foundation (V. C. G., E. R. L.. D. V. S.. S. C. B.} and Department of , Wayne State University School of Medicine (S C a.], Detroit. Michigan 48201

SUMMARY products as intermediates (21, 23). Several studies also indicate that conjugation may play an essential role Rodent and human mammary tumor systems were in the of , particularly in hepatic tissue investigated to relate the steroid alcohol and estrogen (7, 10, 27). Furthermore, it has been demonstrated that activities to the hormonal dependency of breast carcinoma, unlike normal breast tissue, is active in the tumor as determined by estrogen receptor content. sulfating 3j8-hydroxy-A5- and the 3-phenolic group Unlike the normal mammary gland or the hyperplastic of estrogens (I, 12). If, in fact, sulfate conjugates are in alveolar nodule, rodent mammary neoplasms displayed volved in steroid and metabolism, by significant levels of these two sulfotransferases. In the breast tumor extracts may reflect the 1st stage of a meta hormone-independent mouse tumors produced from out bolic sequence leading to more profound changes in the growth lines D!, D2, and D8, high steroid moiety. Indeed, Adams and Wong (3) have shown sulfotransferase activity was characteristic of the rapidity breast carcinomas to exhibit a "paraendocrine" behavior with which hyperplastic alveolar nodules developed into a that is normally confined to endocrine glands in their neoplasm (Vmax = 52.8 versus 1.8 fmoles/min/mg protein) capacity to produce changes in steroid structure. In addi while sulfotransferase activity was either not detect tion, Dao and Libby (13) have demonstrated correlation able or low (Vmax = 5.5 fmoles). After oophorectomy of between the response of breast cancer patients to adrenal- mice bearing slowly developing tumors, both sulfotransfer ectomy and the relative capacity of their tumors to sulfate ases in the nonregressing neoplasms showed marked in neutral steroids as compared to estrogens. The sulfotrans creases in activity (Vmflxdehydroepiandrosterone = 30.0 ferase pattern of breast tumors, therefore, may be related fmoles; Vmax estrone = 18.5 fmoles). Strain differences to hormonal dependency of the neoplasm, as well as were found to influence the steroid-sulfating capability but eventual prognosis of the disease. not the estrogen receptor content of hormone-dependent rat In this investigation mammary tumors from various mammary tumors. In Wistar-Lewis rats the steroid alcohol rodent model systems and some human breast carcinomas sulfotransferase activity was at least 35 times higher than in were analyzed for levels of the steroid alcohol and estrogen the Sprague-Dawley strain. As was observed in the mouse sulfotransferases and for E2-R4 protein. It was hoped that mammary tumor, Sprague-Dawley rat neoplasms that grew these model systems would provide a valuable tool for in the absence of ovarian hormones contained significantly studying both the development of sulfotransferase activity greater levels of the steroid . Possi during tumor growth and the influence of the hormonal ble correlation between presence of the steroid alcohol milieu upon this sulfating capability. sulfotransferase and the estrogen receptor protein was observed in a limited number of human breast carcinomas. MATERIALS AND METHODS

INTRODUCTION Tissues. Normal mammary gland specimens were taken from 2- to 4-month-old W/LEW rats both before and at Steroid have been implicated in biological func various times after (3 hr, 24 hr, 48 hr, 1 week, 2 weeks, 8 tions other than transport, deactivation, or excretion. For weeks, and 12 weeks) intubation of 20 mg DMBA but prior example, the pathway from to dehydroepian to appearance of a tumor. HAN, DMBA-induced mam drosterone can utilize sulfated steroids without hydrolytic mary tumors, and also, mammary neoplasms from trans planted HAN in W/LEW rats were obtained by procedures 1This investigation was supported in part by USPHS Research Grants outlined by Beuving (5). SD mammary tumors were CA-04519 and CA-07177 from the National Cancer Institute and in part induced in 8- to 10-week-old rats with 20 mg DMBA via by an institutional grant to the Michigan Cancer Foundation from the intubation. When desired, oophorectomy was performed United Foundation of Greater Detroit. 2Present address: Department of Biochemistry, Boston University 4The abbreviations used are: E2-R, estrogen receptor; DMBA, 7,12,- School of Medicine, Boston, Mass. 02118. 3Present address: Department of Surgery and Oncology, Wayne State dimethylben/(a)anthracene;[6,7-3H]estrone: [3H]DHEA, HAN, (7-3H]'dehydroepiandrosterone; hyperplastic alveolar nodules; ITLC, [3H)E„ in University School of Medicine, Detroit. Mich. 48201. stant thin layer chromatography; PAPS, 3'-phosphoadenosine 5'-phospho- Received December 18, 1974; accepted March 18, 1975. sulfate; DTT, dithiothreitol; E,, estrone; DHEA, dehydroepiandrosterone.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. V. C. Godefroi et al. either at 3 or 10.5 weeks after appearance of the neoplasm. as fmoles per min per mg protein and [S] is Tumors were produced from mammary nodule outgrowth the concentration or molarity of steroid. lines D,, D2, and D8 in BALB/c mice by a procedure described by Medina (26). Oophorectomy, when needed, was performed on mice 1 month after transplantation of D2 RESULTS or D8 outgrowths. Only female animals were used in the above procedures. Cancer of human breast carcinomas and Sulfating capabilities of neoplasms and other tissues were the hyperplastic epithelium in benign tumors were verified studied by analyzing for levels of 2 , the steroid by pathology. All samples were stored at 70°;no fresh alcohol and estrogen sulfotransferases. Since it had been demonstrated that DHEA, in the presence of [35S]PAPS, tissue was used in these experiments. Preparation of Substrates. [3H]Ei (54 Ci/mmole) and was sulfated at a greater rate than other neutral steroids by [3H]DHEA (21 Ci/mmole) (New England Nuclear, Boston, the steroid alcohol sulfotransferase (2), DHEA was selected Mass.) were purified by ITLC (Gelman Instrument Co., as a substrate of choice for this . E, was chosen as substrate for the estrogen sulfotransferase (31). Ann Arbor, Mich.) (4, 15, 20). Standards were prepared in The optimum pH and concentration of Mg2+ had been absolute ethanol. PAPS was obtained enzymatically by the method of Ro/hin el al. (31). determined earlier for the estrogen sulfotransferase enzyme Preparation of Cytosols. Tissue was minced in 10 mM (31). These same conditions were used also for the steroid Tris-HCl buffer, pH 8.1, containing 20 mM MgCI2 and 0.10 alcohol sulfotransferase with DHEA as substrate since the mM DTT. Homogenization was carried out by a Willems values fell within ranges found by Adams and Edwards (2) Polytron (Brinkmann Instruments, Westbury, N.Y.) twice to be optimum for this enzyme. Before time curves for E! for 15 sec each at a setting of 8 with an interval of 60 sec. and DHEA sulfation were attempted, substrate saturation After ccntrifugation in a Spinco Model L5-50 ultracentri- curves were run on representative tissues to determine fuge at 15,000 x g for 15 min, the resulting supernatant appropriate levels. A typical PAPS saturation curve for fluid was recentrifuged at 100,000 x g for 60 min with a SW DHEA sulfation in a W/LEW rat tumor is shown in Chart 50.1 rotor and buckets equipped with Beckman 305527 1. PAPS was saturating at 35 nmoles for an incubation adapters. All of the above operations were carried out at 4°. time of 30 min. Other tissues with lower DHEA-sulfating The obtained microsome-free cytosol was utilized without activity were incubated for 60 min utilizing 70 to 80 nmoles dilution. An aliquot was taken for Lowry protein analysis PAPS. Chart 2 shows a time curve for DHEA sulfation in (24). a mouse tumor from outgrowth line D, ; linearity extends to Incubation Procedure. Cytosol samples were incubated 60 min incubation time. For E, sulfation also, PAPS was with PAPS and 7-^1 E, or DHEA standards at 37°. saturating at 35 nmoles for the tissues studied; linearity was reached at 45 min. Reaction was stopped by immersing the samples in boiling ITLC proved to be essential in resolving the 3H-labeled water. Blanks for E, and DHEA sulfation were prepared by placing tubes containing cytosol in boiling water before steroid sulfates from contaminating residual tritiated ste addition of appropriate standard. Varying the pmoles of roid or other labeled products present in the ethyl acetate extract. A typical chromatogram is illustrated in Chart 3; added DHEA or E, had no effect on blanks. the blank strip is indicated by shading. Routinely, a 4-cm Extraction and Measurement of Radioactivity. Water (0.15 ml) and 6 /ig carrier steroid sulfate (Schwarz/Mann, spot was cut out for elution with an internal standard Orangeburg, N. Y.) were added to the samples before marker for reference. External standards as markers proved extraction 3 times with diethyl ether. Residual ether in the aqueous layer was removed completely by a stream of 480 p nitrogen before addition of 0.20 g (NH4)2SO4. After 3 extractions with ethyl acetate/ethanol (3/1), the extracts were supplemented with 35 /ig of appropriate steroid sulfate 360 - before evaporating to a small volume for ITLC (11, 29). The resulting spots were eluted with methanol. Aliquots were taken to dryness in counting vials; first, Hyamine (Packard 240 - Instrument Co., Downers Grove, 111.)and then, toluene phosphor were added before determining radioactivity in a 120 - liquid scintillation spectrometer equipped with automatic external standardization to correct for quenching. Controls containing either [3H] or [3H]dehydroepian- drosterone sulfate showed no hydrolysis of steroid sulfate 60 during the outlined procedure. nmol» PAPS Analysis of Kinetic Data. By using PAPS at its saturating Chart 1. PAPS saturation curve for DHEA-sulfating activity in a levels and by varying the steroid concentration, initial W/LEW rat mammary tumor cytosol. Each incubated sample contained velocity measurements were determined. The data were 10" dpm [3H]DHEA and 5.0 /ig DHEA and was analyzed for 1.16 mg submitted to Hofstee (18) plot analysis, i.e., v versus v/[S] protein. Incubation time was 30 min. Saturation was reached at 35 nmoles where v is the velocity of the enzymatic reaction expressed PAPS. DHEA-S, dehydroepiandrosterone sulfate.

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240 gated E,, and for this reason measuring DHEA-sulfating activity was more difficult. Also, the steroid alcohol sulfo showed different ranges for the Km parameter •¿>ISO depending upon the source of the mammary neoplasms: i mouse tumors from outgrowth lines D, and D2, 80 to 520 •¿ ï 120 nM; mouse tumors from line D8, oophorectomized mouse A O tumors from lines D2 and D8, and SD rat neoplasms, 800 to _l i 4,800 nM;and W/LEW rat tumors, 5,600 to 20,400 nM. By I 60 contrast, the estrogen sulfotransferase had values of Km fall within a range of 80 to 520 nM for all the various rodent tumors. Since, optimally, substrate concentrations should

0 IS 30 45 60 lie between 0.1 and 10 Km, preferably with points on each Tim«of Incubation {mini side of Km (18), it was necessary to use 3 sets of standards Chart 2. Time curve for DHEA-sulfating activity in a mouse mam with varying DHEA substrate concentrations to meet these mary tumor cytosol from outgrowth line D,. Each incubated sample requirements. Only 1 set of standards with increasing E, contained 77 nmoles PAPS, 7.5 x 10s dpm [3H]DHEA, and 0.012 ¿ig substrate concentrations was needed for determining estro DHEA and was analyzed for 0.59 mg protein. DHEA-S. dehydroepian- gen sulfotransferase activity. drosterone sulfate. Mouse neoplasms from outgrowth lines D,, D2, and D8 show no regression, but they exhibit continued growth after E, -S

C) 1.2001,000eoos

a.0 Õ 270 600400200•^—^A-1I I 1 I 1.80

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Chart 3. ITLC chromatogram of the ethyl acetate extract of incubated 180 24O 300 MO mouse mammary tumor cytosol from outgrowth line D,. £,-S,estrone sulfate. The incubated sample contained 72 nmoles PAPS, 4.6 x 10sdpm nM Sttroid Subttrott ['H]E„and 3.9 pmoles E,. Shaded portion, blank strip, was derived from Chart 4. Saturation curves for E, (•)and DHEA (O) sulfotransferase an incubation with 4.6 x 10s dpm [3H]E, added to boiled cytosol. Each activities in a human breast tumor (M. Z.) cytosol. Amounts of DHEA cytosol aliquot was analyzed for 0.70 mg protein. used in standards were 7.3, 17, 27, or 36.8 pmoles. Each incubated sample contained 0.38 mg protein. The kinetic parameters, Vm.Mand Km, calcu to be less reliable. Blanks were treated similarly before lated from Hofstee plot analyses, are indicated for each saturation curve. subtracting. Saturation curves (Chart 4) were determined for all tumors and tissues studied in these experiments. However, in order to obtain 2 of the kinetic parameters, Vmaxand Km, the data were analyzed by Hofstee plots (Chart 5). The Hofstee plot was preferred to the Lineweaver-Burk plot for 4.0 2 reasons, (a) In the former graph all points have equal 3.0 weight; in the double reciprocal plot the least accurate points are weighed more heavily, (b) Vmaxand Km values are read directly as an intercept or as the quotient of 2 intercepts in the Hofstee plot, whereas in the Lineweaver- Burk method the kinetic parameters are calculated from the 1.0 reciprocals of the intercepts and a slight error in the reciprocal readings greatly increases the error for Vmaxor O 0.014 0.028 0.042 003« Km- "/ISS The Km values that are indicated in Chart 4 differed in magnitude for the 2 sulfotransferase enzymes. Obviously, Chart 5. A Hofstee plot derived from the data of Chart 4 for E, sulfotransferase activity. The parameter Vm., is equal to the ordinale the steroid alcohol sulfotransferase conjugated DHEA at a intercept, or 4.4 fmoles/min/mg protein; Km is equal to the negative value much slower rate than the estrogen sulfotransferase conju of the slope, or 92 nM.

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Table I Sulfotransferase activity and E2-R content during various Steroid sulfaiion and receptor content in mouse mammary tumors stages of rodent mammary tumor growth are shown in In the sulfation procedure. 180 mg tissue were homogenized in 3.6 ml Chart 6. The experiments involved SD rats only. Since these buffer. Four aliquots (0.25 ml) of cytosol were incubated with 70 to 80 nmoles PAPS; 4.6 x 10s dpm [3H]E,: and 3.9, 12.5, 21.2, or 30.0 pmoles were DMBA-induced hormone-dependent tumors, all oo- E, for 30 min. Another 4 aliquots were incubated with the same quantity of PAPS; 3.6 x IO5 dpm [3H)DHEA; and 7.8. 40.1, 72.4, or 104.9 pmoles DHEA for 60 min. Two blanks were prepared by adding either 4.6 x 10s Table 2 dpm |3H]E, or 3.6 x 10s dpm ('HjDHEA to boiled cytosol aliquots. Ethyl Steroid sulfation and receptor content in W ¡LEW rat mammary tumors acetate extracts of the 10 samples were chromatographed; then aliquots of Experimental details for the sulfation procedure are given in Table 1 the eluted spots were taken to determine radioactivity. Blanks were with the following changes. Four cytosol aliquots were incubated for 30 min with 50 nmoles PAPS; 3.6 x 10s dpm [3H]DHEA; and 370, 1100, subtracted before analysis of data by Hofstee plots to obtain values for Vm.. . 1800. or 2550 pmoles DHEA. Experimental details for the E2-R procedure In the E2-R procedure. 100 mg tissue were homogeni/ed in 2.0 ml are given in Table 1. buffer. Four aliquots of the diluted supernatant fraction were incubated for 2 hr at 4°with 0.9 x 10", 2.7 x 10", 4.6 x 10", or 9.4 x 10 •¿M17 (VDHEA(fmoles/min/mgprotein)001340±'E,(fmoles/min/mgprotein)00<5E2-R 0-|2,4,6,7-3H]estradiol. Chromatography on four 3.0-g Sephadex G-25 columns followed. The collected void volumes were extracted and aliquots were taken to determine radioactivity. The resulting saturation curve was content(pmoles/liter/mgprotein)0026.7 used to find the level of E2-R present. Specific binding was verified periodically by incubating a 5th supernatant aliquot with 9.4 x 10 * M TissueNormal l70-[2,4,6,7-3H] and a 100-fold excess of unlabeled estradiol. (For details, see Ref. 15). mammarygland"HANTumorSulfation

(VDHEA(fmoles/min/mgprotein)0l.7±)E, E2-Rcontent(pmoles/liter/mgprotein)01.53.202.83.1 770"max (fmoles/min/mgprotein)0.78.9 " Mammary gland supernatants were examined both before and at various limes (see "Materials and Methods") after intubation of DMBA TissueNormal but prior to appearance of a tumor. 6 Mean ±S.E. mammaryglandOutgrowth

tumorlineD,D,IVI).D."Sulfation

0.7"l.8± ±2.45.5 0.430.0 ±2.618.5 ±7.852.8 ±7.400.9 ±20.419.7 ± 9.6max ±0.2-

" Mean ±S.E. "Tumors were taken after oophorectomy. oophorectomy is performed and thus are classified as hormone independent (26). As expected, E2-R levels in tumors from these 3 lines were found to be very low (Table 1). The mouse tumor from outgrowth line D8 showed high DHEA sulfokinase activity (52.8 fmoles/min/mg protein) 2 4 6 B IO 12 and no E, sulfation, whereas the neoplasms from lines D, Wtths of Mammary Tumor Growth (Sproguc-DOMlty Rat«) and D2 showed low DHEA sulfation and substantial E, sulfation. After oophorectomy, E! sulfation was detectable Chart 6. Sulfotransferase activities and E2-R content during various in tumors from outgrowth line D8 and both DHEA- and stages of SD rat mammary tumor growth (for experimental details, see E,-sulfating activities were increased markedly in tumors Table 1). , tumor growth rate in intact rats. Lower, range of 4 tumor sizes at various times of measurement. Tumor activities (mean ±S.E.) from line D2 (30.0 and 18.5 fmoles/min/mg protein, analy/.ed at 12 weeks showed: Vm„DHEA, 8.9 ±2.9 fmoles/min/mg respectively). No E2-R and no sulfotransferase activity protein; Vma. E,, 7.9 ±2.8 fmoles/min/mg protein; E2-R. 30.6 ±3.1 could be discerned in the normal mammary gland. pmoles/liter/mg protein. Analysis at 3 weeks of I tumor from an intact rat W/LEW rats have been shown to produce mammary gave: Vm„DHEA, 2.9 fmoles/min/mg protein: Vmax E,, 125.0 fmoles/ nodules that on transplantation eventually produce mam min/mg protein; E2-R, 25.3 pmoles/liter/mg protein. Upper, measure mary tumors (5). No differences in E2-R levels or in sul- ments of 2 mammary tumors on separate rats that were oophorectomized fating capabilities could be determined between neoplasms (OvX). at 3 weeks. - -, appearance of ovarian hormone-independent that arose from transplanted HAN and tumors that were neoplasms. The 2 additional tumors analyzed in this group fused with induced by DMBA intubation. As seen in Table 2, these adjacent mammary tumors and could not be measured. Analysis at 11 hormone-dependent tumors exhibited high amounts of E2-R weeks of these 4 tumors gave: V,,,,, DHEA, 36.5 ±15.5 fmoles/min/mg protein; Vma, E,, 7.0 ±4.2 fmoles/min/mg protein; E2-R, 25.4 ±3.6 and remarkably high DHEA sulfotransferase levels (1340 pmoles/liter/mg protein. Lower, - -, regression of a large neoplasm after fmoles/min/mg protein). Neither sulfation nor E2-R could oophorectomy at 10.5 weeks. Analyzed at 12.5 weeks, this tumor gave: be detected in HAN or in the normal mammary gland that Vma, DHEA. 2.3 fmoles/min/mg protein; Vm„.E„0; E2-R, 4.5 pmoles/ was obtained either before or after DMBA treatment. liter/mg protein.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. Sulfotransferases in Mammary Tumors phorectomi/.ed tumors regressed in size (Chart 6). Neo Table 3 plasms from animals that were oophorectomized at 3 weeks Steroid sulfation and receptor conteníin human breast tumors reappeared with time as ovarian hormone-independent Experimental details for sulfation procedure are given in Table 1 with tumors and by 11 weeks had become large. These ovarian the following changes. The amounts of DHEA used for all tissues except hormone-independent tumors contained the same high MCF-7 were 7.3, 17.0, 27.0, or 36.8 pmoles; the amounts of DHEA used levels of E2-R as were found in neoplasms from nonoo- for MCF-7 were 7.8, 40.1, 72.4, or 104.9 pmoles. The MCF-7 cytosol was incubated for 30 min regardless of substrates used. phorectomized SD rats. Stimulation of such tumors by Experimental details for the E2-R procedure are given in Table 1 with nonovarian estrogens remains a possibility. Oophorectomy the following change. Six aliquots of the diluted MCF-7 supernatant frac markedly increased the steroid alcohol, but not the estro tion were incubated with 0.9 x 10 »,2.7x 10 '. 4.6 x 10 ", 9.4 x 10 ', gen, sulfotransfease activity. On the other hand, large 14.3 x 10-', or 23.7 x 10-•M170[2,4,6,7-3H]estradiol. tumors that were analyzed 2 weeks after oophorectomy, i.e., during regression, were low in sulfating ability and, as also Sulfation(Vm.J shown by Vignon and Rochefort (32), contained less E2-R. content In a nonoophorectomized rat, a younger mammary tumor (pmoles/ min/mg min/mg liter/mg taken after a growth period of 3 weeks displayed a very high response"YesYesYesYesYesNoNoNoNo level of estrogen sulfotransferase. PatientV.S."MM."E. protein)0.4002.805.11.46.025.00.4400.003.200004.5001.50E,(fmoles/protein)2.30.41.632.75.813.78.233.5151.00.812.61.29.91.50.23.61.40.51.16.14.41.7E,-Rprotein)337.6117.879.076.859.835.234.721.820.520.4174.03.60000000000Clinical Analyses of recurrent human breast tumors are shown in Table 3. The sulfating capabilities of 10carcinomas that are positive with respect to E2-R can be compared to 10 F.A. carcinomas with negative or borderline levels of E2-R. All K.H.Z."N.M.L. tumors from these oophorectomized or postmenopausal women showed E, sulfation to be present. However, DHEA M."J.M.D. sulfation proved to be less prevalent since it was evident in 7 of 10 neoplasms that were positive with respect to E2-R and F."B.C."MCF-7CM.Gi.Z.G.H.K.N.L."E. in 3 of 10 E2-R-negative tumors. The cell culture that was derived from a breast tumor (MCF-7) was high in DHEA- sulfating activity as well as in E2-R content. Hyperplastic epithelium tissue did not contain measurable levels of the steroid alcohol sulfotransferase or E2-R. P.»M. V.M.G."A.F."M.S."M.Z."HyperplasticepitheliumDHEA(fmoles/ DISCUSSION

Two enzymes are required in order to synthesize PAPS from inorganic sulfate in cells: the sulfate adenyltransferase and the adenylsulfate kinase. A 3rd enzyme, a sulfotransfer ase, utilizes PAPS to sulfate substrates such as steroids. Thus, when [35S]SO42~ and a steroid substrate are incu 0Clinical response to hormonal manipulation consisting of either adrenalectomy or administration of 5 to 10 mg estrogen per day. bated with a supernatant preparation from tumor tissue, 3 6 Blanks for these patients indicate no recurrence of disease at this enzymes must be present in order to synthesize the sulfated time. steroid . Use of [35S]PAPS, or nonradioactive cCell culture of human breast tumor (8). PAPS plus a 3H-labeled steroid, requires the presence of only 1 enzyme, a sulfotransferase, in the incubation to form endogenous competitive inhibitors. Since are a sulfated product. Optimum conditions for converting known to be associated with microsomes, the routine use of inorganic [35S]SO42~to a labeled, sulfated steroid product microsome-free cytosols obtained from 100,000 x g centrif- would be difficult, if not impossible, to achieve since the ugation would minimize the possibility of sulfatases being most favorable pH and concentrations of Mg2+ and DTT present during sulfation incubations. Thus only 100,000 x g would have to be determined for each enzyme and, if cytosols were utilized in these experiments. The levels of different, could not be used in the same incubation. On the endogenous steroids found in rodent and human mammary other hand, the requirements of 1enzyme can be satisfied as tumors are not known and, unless determined by radioim- was done in these experiments, which involved only the munoassay or isotope dilution methods, must be considered steroid alcohol sulfotransferase or the estrogen sulfotrans a variable in calculations involving analysis of sulfating ferase. enzyme activity. However, if enzyme levels are expressed in Published procedures to data (6, 13, 22) determine pmoles terms of the kinetic parameter, Vmax,it is not necessary to steroid sulfate per mg protein produced by tumor prepara determine endogenous steroid levels since the quantity Vmax tions at 1 level of substrate. Data of this kind represent a is independent of substrate concentration and remains singular point on the saturation curve. The position of this unchanged in the presence of endogenous competitive point on the curve may vary greatly from tumor to tumor inhibitors. For this reason all sulfotransferase activities in depending on, for example, the presence and concentration the tissues studied were expressed as values of Vmai. The of sulfatases, endogenous steroids used as substrate, or wide range of Km values obtained for DHEA sulfation

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. y. C. Godefroi et al. would indeed suggest the presence, in these neoplasms, of indicate a possible dependence of these enzymes on the varying amounts of sulfatases and/or endogenous neutral hormonal milieu of the host. In the porcine uterus it has steroids that are acting as competitive inhibitors. been noted previously that the estrogen sulfotransferase In order to facilitate the detection of low enzyme activity, appears only after the initial estrous cycle and that this utilization of a labeled substrate of high specific activity is enzyme cannot be detected in the 1st follicular phase (9). essential. The specific activity of available inorganic While inconclusive, these observations suggest involvement [35S]SO42 varies from 0.65 to 1.0 Ci/mmole whereas of the gonadotrophins, possibly the luteinizing hormone, in [35S]PAPS can be obtained at 0.2 to 3.0 Ci/mmole, and the activation of certain sulfotransferases. [3H]DHEA and [3H]E, can be obtained at 21 and 40 In the rat, development of HAN in response to DMBA is Ci/mmole, respectively. Thus with [3H]E„rather than more efficient in the W/LEW strain (5). Both the normal [35S]SO42 , as the labeled substrate the sensitivity of the W/LEW mammary gland and mammary glands removed analysis would increase by a factor of at least 40. To at various times after DMBA intubation did not contain compensate for the lower specific activity of [3H]DHEA as measurable sulfotransferase activity (Table 2). Supernatant compared to [3H]E,, the sensitivity of the steroid alcohol fractions from pooled HAN also did not display sulfokinase sulfotransferase assay may be improved by doubling the activity. Not until the actual development of the mammary incubation time. tumor did the capacity to sulfate DHEA and E, appear. One additional factor was instrumental when a procedure Similarly, only neoplasms possessed measurable E2-R. for analyzing these tissues was selected. In experiments with Strain differences were found to influence steroid-sulfat- [35S]PAPS, difficulty was encountered in isolating the ing capabilities, but not the E2-R content, of hormone- 35S-labeled steroid sulfate from the many radioactive sul- dependent rat mammary tumors. In W/LEW rats the ste fated products present. By contrast, when a 3H-labeled roid alcohol sulfotransferase levels were at least 35 times steroid was used as a substrate, the tritiated steroid sulfate higher than in the SD strain (Table 2; Chart 6). was readily separated by ITLC from traces of labeled As was observed in mouse tumors from the outgrowth impurities and residual tritiated substrate that remained line D2, rat neoplasms that grew in the absence of ovarian after ether extraction. The identity of products obtained hormones contained significantly greater amounts of the from all incubations, whether with 35S-labeled or 3H-labeled steroid alcohol sulfotransferase. On the other hand, estro substrates, was verified routinely in this laboratory. gen sulfotransferase and E2-R levels were the same in Therefore, the described tritiated steroid substrate proce tumors from intact rats as in tumors that grew after dure was selected as the method of choice in order to: (a) oophorectomy (Chart 6). Again, steroid alcohol sulfotrans have complete control over optimum enzyme requirements; ferase activity within the mammary neoplasm showed a (b) evaluate 2 kinetic parameters, Vmai[and Km: and (c) dependence on the hormonal milieu of the rodent since this attain the highest possible sensitivity so as to detect low activity greatly increased when plasma gonadotrophic con amounts of enzyme. centrations were increased. Because of the low enzyme levels encountered in many These results point to the possible paraendocrine activity tissues studied, a reducing agent, DTT, was used in all of certain mammary tumors as reported earlier by Adams cytosol preparations. The MCF-7 cell culture, when frozen and Wong (3) and by Dao et al. (14) and Griffiths et al. (16). and stored for 7 days at -70°, lost 30% of its DHEA-sulfat- Recent experiments in this laboratory have shown that ing activity. Increasing the concentration of DTT in the estrogen and steroid alcohol sulfotransferase activities exist buffer to 10 mM eliminated this loss (J. Rozhin. A. Huo. in the normal porcine uterus and that estrogen sulfation and S. C. Brooks, unpublished data from this laboratory). varies directly in relation to the estrous cycle (28). This For mammary tumors a loss of activity on freezing and would indicate that sulfokinase activity not only is charac storage is to be expected. However, all tissues in these teristic of endocrine or hepatic tissues but also appears in studies were treated similarly with immediate freezing and normal "target" tissues of steroid hormones. Mammary then storage at -70°. tumors may display high levels of these 2 sulfotransferases In Table 1, sulfotransferase activity in mouse tumors simply as a magnification of very low activities in the from outgrowth lines D,, D2, and D8 seems to be related to normal gland. the rapidity with which HAN develop into neoplasms; lines The value of an E2-R assay in predicting clinical response D, and D2 are slow growing with little sulfating ability, to endocrine therapy has been established (25). However, whereas line D8 is fast growing with high DHEA sulfation. attempts to correlate sulfating activity with E2-R content or After oophorectomy in the slower-growing line (D2), with clinical response to endocrine ablation have been marked increases for both E, and DHEA sulfation in the inconclusive. Dao and Libby (13) have suggested that high tumor were observed. In the outgrowth line D8, oophorec estradiol-sulfating ability relative to DHEA sulfation, or no tomy resulted in detectable estrogen sulfotransferase activ sulfating enzyme activity at all, results in poor clinical ity in the tumor, whereas levels of the extremely active response to adrenalectomy. However, in their attempts to steroid alcohol sulfotransferase did not change significantly. substantiate this work, Leung et al. (22) and Braunsberg et The presence of an endogenous steroid before oophorec al. (6) independently found no correlation between estrogen tomy is not a factor since the parameter Vmax is not binding (as an indication of a hormone-dependent tumor) dependent on substrate concentration. These major in and sulfating capacity. creases in activity of the steroid alcohol and estrogen The data in Table 3 suggest possible correlation between sulfotransferases in mammary tumors after oophorectomy the presence of detectable quantities of the steroid alcohol

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. Sulfotransferases in Mammary Tumors sulfotransferase enzyme and the E2-R protein. To date, for Ro/hin for the unlabeled PAPS. The technical assistance of Louisa Halmi those 8 patients in whom the disease has recurred and who is gratefully acknowledged. were treated by hormonal manipulation, a positive clinical réponsewasmatched in all 4 cases with the presence of E2-R REFERENCES in the tumor. Three of 4 of these carcinomas also contained the steroid alcohol sulfotransferase. Tumors from the 4 1. Adams, J. B. Enzymatic Synthesis of Steroid Sulfates. II. Presence of patients who failed to respond to treatment did not contain Steroid Sulfokinase in Human Mammary Extracts. J. Clin. Endo- significant E2-R levels and only 1 contained sulfated crinol. Metab., 24: 988 996, 1964. DHEA. Since the estrogen sulfotransferase was found in all 2. Adams, J. B.. and Edwards, A. M. Enzymic Synthesis of Steroid carcinomas, it is possible to view the data as supporting Sulphates. VII. Association-Dissociation Equilibria in the Steroid those of Dao and Libby (13), who found DHEA sulfation to Alcohol Sulphotransferase of Human Adrenal Gland Extracts. Bio- be greater than estradiol sulfation in patients who responded chim. Biophys. Acta, 167: 122 140, 1968. to adrenalectomy. Obviously, many more tumors must be 3. Adams, J. B., and Wong, M. Paraendocrine Behavior of Human analyzed before credence can be placed on such findings. Breast Cancer. In: T. L. Dao (ed.). Estrogen Target Tissues and Interpretation of these results is difficult at this time. Neoplasia, pp. 125 150.Chicago: University of Chicago Press, 1972. However, some information available from experiments in 4. Barton, G. M., Evans, R. S., and Gardner, J. A. F. Paper Chromatog- this laboratory and from the literature may be mentioned. raphy of Phenolic Substances. Nature, 170: 249-250, 1952. 5. Beuving, L. J. Mammary Tumor Formation within Outgrowths of The estrogen sulfotransferase, which is ubiquitous to both Transplanted Hyperplastic Nodules from Carcinogen-Treated Rats. J. rodent and human mammary tumors, may be involved in Nati. Cancer Inst., 40: 1287-1289, 1968. limiting nuclear activity of the hormone. For example, it has 6. Braunsberg, H., Killen, E., and Melville, E. Studies on Steroid been observed in the porcine uterus that the time of least Sulphation and Binding by Human Malignant Tumours. European J. uterine estrogen sulfotransferase activity, i.e., ovulation, Cancer. 10: 13 20, 1974. coincides with the greatest migration of E2-R complex to 7. Brooks, S. C., and Horn, L. Hepatic Sulfation of Estrogen Metabo the nucleus; conversely, when estrogen sulfation is highest, lites. Biochim. Biophys. Acta, 231: 231 241, 1971. the least amount of E2-R complex enters the nucleus (28). 8. Brooks, S. C., Locke, E. R., and Soule, H. D. Estrogen Receptor in a Thus hormonal differentiation of mammary tumor tissue in Human Cell Line (MCF-7) from Breast Carcinoma. J. Biol. Chem. response to estrogen may be limited by the increased 248: 625\ 6253, 1973. estrogen sulfotransferase in the neoplasm relative to normal 9. Brooks. S. C., Pack, B. A., and Horn. L. The Influence of Sulfation and Estrogen Metabolism and Activities. In: T. L. Dao (ed.). Estrogen tissue. This is also evidenced by the abnormally high levels of unbound cytoplasmic E2-R in hormone-dependent mam Target Tissues and Neoplasia, pp. 221 236. Chicago: University of Chicago Press. 1972. mary neoplasms of rodents with functioning ovaries. 10. Creange, J. E., and Szego, C. M. Sulphation as a Metabolic Pathway It has been suggested that the steroid alcohol sulfotrans for Oestradiol in the Sea Urchin Strongylocentrotus franciscanus. ferase may be involved in the biosynthesis of dehydro- Biochem. J., ¡02:898-904, 1967. sulfate from in the 11. Crépy,O., Judas, O., and Lachese, B. Méthodesde Detection des adrenals, thereby preserving substrate from the activity of Steroi'des ConjugèsSéparésparChromatographie en Couche Mince. the 3/3-hydroxysteroid dehydrogenase and from subse J. Chromatog., 16: 340 344, 1964. quent conversion to (19, 23). However, 12. Dao, T. L., and Libby, P. R. Conjugation of Steroid Hormones by Normal and Neoplastic Tissues. J. Clin. Endocrinol. Metab., 28: ovaries that actively synthesize gonadal steroids (and not 1431-1439, 1968. corticosteroids) from DHEA have very low or negligible steroid alcohol sulfotransferase activity (17). Steroid 3- 13. Dao, T. L., and Libby, P. R. Conjugation of Steroid Hormones by Breast Cancer Tissue and Selection of Patients for Adrenalectomy. sulfates have not been shown to be required for the for Surgery, 66: 162-166, 1969. mation of or estrogens. Therefore, if certain 14. Dao, T. L., Várela,R., and Morreal, C. 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Dao (ed.), Estrogen Target Tissues and Neoplasia, SD rat mammary neoplasm present after oophorectomy pp. 151 162. Chicago: University of Chicago Press, 1972. also regressed either after adrenalectomy or on admin 17. Hochberg, R. B., Ladany, S., and Lieberman, S. Cholesterol Sulfate: istration of estradiol benzoate (30). Like the hormone-de Some Aspects of Its Biosynthesis and Uptake by Tissues from Blood. pendent human breast tumor, these rat mammary neo Endocrinology, 94: 207 213, 1974. plasms contained high levels of the steroid alcohol 18. Hofstee, B. H. J. Non-Inverted Versus Inverted Plots in Enzyme sulfotransferase and E2-R (Chart 6). Kinetics. Nature, 184: 1296-1298, 1959. 19. Jaffe, R. B., and Payne, A. H. Gonadal Steroid Sulfates and . ACKNOWLEDGMENTS IV. Comparative Studies on Steroid Sulfokinase in the Human Fetal Testis and Adrenal. J. Clin. Endocrinol. Metab., 33: 592 596, 1971. The authors are indebted to Dr. A. Singhakowinta and Dr. R. 20. 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21 Lebeau, M. C., Alberga, A., and Baulieu, E. E. Adrenal Biosynthesis 3-53. New York: Academic Press, Inc., 1973. of Dehydroisoandrosterone Sulfate. Biochem. Biophys. Res. Com 27. Miya/aki, M., Yoshi/awa, I., and Fishman. J. Directive O Methyla- mun., 17: 570-572, 1964. tion of Estrogen Catechol Sulfates. Biochemistry.*. 1669 1672. 1969. 22 Leung, B. S., Fletcher, W. S., Lindell, T. D., Wood, D. C., and 28. Pack, B. A., and Brooks, S. C. Cyclic Activity of Estrogen Sulfotrans- Krippaehne, W. W. Predictability of Response to Endocrine Ablation ferase in the Gilt Uterus. Endocrinology, 95: 1680 1690, 1974. in Advanced Breast Carcinoma. A Correlation to Estrogen Receptor 29. Payne, A., and Mason, M. Application of Glass Fiber Chromatogra- and Steroid Sulfurylation. Arch. Surg., 106: 515 519, 1973. phy for Separation of Some Neutral and Phenolic Steroid Sulfates. 23 Lieberman, S. Steroid Sulfates as Biosynthetic Intermediates. In: L. Anal. Biochem., 26: 463 464, 1968. Martini, F. Fraschini, and M. Motta (eds.). Proceedings of the Second 30. Pearson, O. H., Molina, A., Butler, T. P., Llerena. L.. and Nasr, H. International Congress on Hormonal Steroids, pp. 22 36. New York: Estrogens and Prolactin in Mammary Cancer. In: T. L. Dao (ed.). Excerpta Medica Foundation, 1967. Estrogen Target Tissues and Neoplasia, pp. 287-305. Chicago: 24. Lowry, O. H., Rosebrough, N. J., Farr. A. L., and Randall, R. J. University of Chicago Press, 1972. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 31. Rozhin, J., Soderstrom, R. L., and Brooks, S. C. Specificity Studies 193: 265 275, 1951. on Bovine Adrenal Estrogen Sulfotransferase. J. Biol. Chem., 249: 25. McGuire, W. L., Carbone, P. P., and Vollmer, E. P. (eds.), Estrogen 2079 2087, 1974. Receptor in Human Breast Cancer. New York: Raven Press, 1975. 32. Vignon, F., and Rochefort, H. Endocrinologie et Cancerologie. 26. Medina, D. Preneoplastic Lesions in Mouse Mammary Tumorigene- Compi. Rend., 275: 103 106, 1974. sis. In: H. Busch (ed.). Methods in Cancer Research, Vol. 7, pp.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. The Steroid Alcohol and Estrogen Sulfotransferases in Rodent and Human Mammary Tumors

Viviane C. Godefroi, Elizabeth R. Locke, Dharm V. Singh, et al.

Cancer Res 1975;35:1791-1798.

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