[CANCER RESEARCH 35,1791-1798, July 1975] The Steroid Alcohol and Estrogen Sulfotransferases 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 Biochemistry, Wayne State University School of Medicine (S C a.], Detroit. Michigan 48201 SUMMARY products as intermediates (21, 23). Several studies also indicate that sulfate conjugation may play an essential role Rodent and human mammary tumor systems were in the metabolism of estrogens, particularly in hepatic tissue investigated to relate the steroid alcohol and estrogen (7, 10, 27). Furthermore, it has been demonstrated that sulfotransferase 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-steroids 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 biosynthesis and metabolism, sulfation 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 dehydroepiandrosterone 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 estrone 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 alcohol sulfotransferase. 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 sulfates 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 cholesterol 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. JULY 1975 1791 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 steroid sulfate per min per mg protein and [S] is Tumors were produced from mammary nodule outgrowth the substrate 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 enzymes, 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 enzyme. 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.