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Estradiol-17β Sulfotransferase Activity in Canine Osteosarcoma D17 Cells
Biochemical and Biophysical Research Communications 273, 505–508 (2000) doi:10.1006/bbrc.2000.2984, available online at http://www.idealibrary.com on
Estradiol-17 Sulfotransferase Activity in Canine Osteosarcoma D17 Cells J. I. Raeside, H. L. Christie, L. Forster, and R. L. Renaud Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Received May 22, 2000
Estrogen sulfatase and sulfotransferase (EST) activities are present in breast cancer tissues but there are no reports on EST in cancerous bone cells. We incubated [ 3H]estradiol-17 with cells from a canine osteosarcoma D17 line for periods up to 24 h. Radioactive steroids were recovered from the media and separated into unconjugated and conjugated fractions using Sep-Pak C18 cartridges. The conjugate fraction was solvolyzed and the resulting free steroids were obtained from a second C18 cartridge. Little metabolism was apparent in 4 h of incubation, but by 24 h as much as one half of the radioactivity was seen in the conjugate fraction. Most of the conjugates were recovered as sulfates in all three experiments. HPLC profiles showed a limited metabolism of estradiol to other compounds except for estrone, which was clearly present in both free and sulfate fractions. These results suggest that EST may have a role in the local metabolism of estrogens in bone. © 2000 Academic Press Key Words: estrogen metabolism; sulfotransferase; sulfatase; bone; osteosarcoma; canine.
Estradiol-17 (E2) is known to play a role in normal, as well as in cancerous, bone cells. Its presence in bone results from estrogen synthesis and metabolism (1). Several studies have reported on steroid-convertingenzyme activities in various osteoblast and osteoblastlike cells in culture (1– 4). Further evidence in support of the importance of steroid metabolism by bone cells themselves is given by the demonstration of expression of messenger ribonucleic acid (mRNA) for several key enzymes, such as aromatase (P-450arom), 17hydroxysteroid dehydrogenase (17-HSD), and steroid sulfatase (4 – 8). Thus, bone has the capacity to form the biologically potent estrogen from steroids in the peripheral blood circulation (Scheme 1). Estrone sulfate is the major form of estrogen in blood and so provides an important source for estradiol biosynthesis in bone.
A parallel situation is seen in breast cancer tissue where estrone sulfate is quantitatively the most important source for estradiol formation (9). High levels of estrogen sulfatase in the tissues provide the first step leading to the bioavailability of estradiol (9 –11). Although the presence of estrogen sulfotransferase has been reported in human mammary cancer cell lines (9, 11) and primary mammary carcinoma (10), it seems that the balance between the activities of the two enzymes favors hydrolysis of estrogen sulfates. No comparable data are available for bone cells. In the course of studies on cultures of bone cells we noted metabolism of estradiol which suggested the presence of estrogen sulfotransferase activity. MATERIALS AND METHODS Cell culture. Canine osteosarcoma D17 cells (ATCC No. CRL6248) were used as part of a study to observe the direct effects of estradiol-17 on bone. The cell line was maintained in our department (Dr. J. LaMarre) in modified Eagle’s MEM with non-selective amino acids, 2% penicillin/streptomycin and 10% FBS in 10 ml of medium in T75 cell culture flasks. For incubation with estradiol-17, cells were plated out and grown to confluency in 6-well plates (Costar) in 1.5 ml of culture medium. [ 3H]Estradiol-17 (0.5 ⫻ 10 6 cpm) was added to the confluent cells in 1.5 ml of culture medium. Radiolabeled [2,4,6,7- 3H]estradiol (83 Ci/mmol) was from Amersham Canada, Ltd. (Oakville, ON). Incubations were done in duplicate in 3 experiments and media were removed after fixed times (0, 1, 4, 12 and 24 h). Cells were washed with 2 ⫻ 0.5 ml saline (PBS) which was added to the appropriate sample and frozen until later processing. The washed cells were used for other aspects of the study and were not available for extraction of steroids. Analytical procedures. Unconjugated and conjugated steroids in the media were recovered separately by solid phase extraction (SPE, using Waters Sep-Pak C 18 cartridges) with diethyl ether and methanol, respectively, as described previously (12). The ether and methanol extracts were evaporated to dryness separately under nitrogen at ⬍45°C. The conjugated material was hydrolyzed by solvolysis (overnight at 45°C in trifluoroacetic acid/ethyl acetate; 1/100; v/v) to obtain a “sulfate” fraction as free steroids from a second Sep-Pak cartridge, used as described above. The amount of radioactive material recovered from each incubation and at each fractionation step was determined by taking an aliquot for liquid scintillation counting (LSC) in 5 ml cocktail (Ecolite; ICN, Costa Mesa, CA). HPLC profiles of the unconjugated and hydrolyzed steroids were generated with a binary solvent gradient of acetonitrile/water on a
505
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 273, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SCHEME 1. Metabolism of steroids in bone to illustrate the potential significance of estrogen sulfotransferase.
Waters C18 column and system, with absorbance monitored at 280 nm (12). Fractions (1 ml) were collected automatically (LKB RediFrac; Pharmacia, Kalamazoo, MI), and aliquots taken for (LSC) in the first 2 experiments. For the third experiment a radiodetector (Packard 505TR) was used for a direct scan of radioactivity (cpm), along with UV (280 nm) absorbance in the chromatography.
RESULTS The time course of conjugation in the metabolism of [ 3H]estradiol-17 by bone cells is shown in Table 1. It is clear that the rate of conjugate formation was not rapid TABLE 1
Conjugation of [ H]Estradiol-17 by Bone Cells 3
Experiment 1 Time (h)
Total
0 1 4 12 24
1.3 4.3 — — 30.7
Experiment 2
Sulfate
Total
25
4.2 — 3.2 7.6 14.1
Experiment 3
Sulfate
Total
Sulfate
6.7 9.5
2.1 — — 18.3 57.8
14.1 47.1
Note. Values are given as means of duplicate incubations, based on percentage distribution of cpm at SPE (SEP-PAK), before and after solvolysis.
FIG. 1. (a) HPLC profiles of unconjugated metabolites of [ 3H]estradiol-17 formed by bone cells after 1 h (solid, dark) and 24 h incubation (interrupted line) are shown. Authentic standards of estradiol (E2) and estrone (E1) were included for reference in the chromatography and are seen along with background absorption at 280 nm (thin line). (b) HPLC profile of metabolites of [ 3H]estradiol liberated from the steroid sulfate fraction (24 h) by solvolysis (Experiment 1; about 25% appeared as “sulfates”). See legend to Fig. 1a for more details.
but by 12 h of incubation there had been an appreciable amount of radiolabeled substrate metabolized in this manner. It is also evident that the major component in the conjugate fraction was the sulfate part. In fact, almost half of the substrate was present in a sulfoconjugated form after 24 h of incubation in the third experiment. No further work has been done, as yet, to identify the lesser amounts of other possible form(s) of conjugation. The HPLC profiles for the unconjugated steroids in the media after 1 h and 24 h of incubation (Fig. 1a) show that no definite metabolism could be detected within the first hour, but by 24 h there was evidence of formation of estrone from the substrate. From the same medium sample, the sulfate fraction after solvolysis revealed a noticeable amount of metabolism, mainly to compounds less polar than estradiol (Fig.
506
Vol. 273, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. HPLC profiles of (a) unconjugated and (b) solvolyzed metabolites of [ 3H]estradiol (Experiment 3; about 50% appeared as “sulfates”). Radiodetection was made online, resulting in lower counting efficiency (cpm). See legend to Fig. 1a for more details.
1b). A radioactive peak coincident with the nonradioactive standard of estrone was noted, as was seen previously in the case of the unconjugated fraction (Fig. 1a). These findings were confirmed in another experiment (Experiment 3) where estrone was the principal metabolite formed from estradiol in both the unconjugated and sulfo-conjugated fractions (Fig. 2). Metabolic activity was greater in this experiment (about 50% conjugation), and small quantities of some polar products were detected in the unconjugated fraction.
solvolysis step to liberate estradiol from the steroid conjugate fraction of media extracts. Nearly half of the radioactivity in one experiment was found in the sulfate fraction after a 24 h incubation. Unfortunately, no estrogen sulfates were included as substrates in our studies so it is not possible to compare estrogen sulfatase and sulfotransferase activities in the bone cells, as has been done for human primary breast tumors (10). Estrone sulfate is extensively hydrolyzed in mammary tissues; however, in one breast cancer cell line (MDAMB-468) the presence of very strong estrogen sulfotransferase has also been shown (9). Normal breast tissues contain estrogen sulfatase and sulfotransferase but the activities are lower than those in tumors (10). Whether normal bone cells show a lower level of EST than we have found with an osteosarcoma cell line remains for further study. Postmenopausal bone loss is primarily due to estrogen deficiency. Impaired local production as well as degradation of estradiol in bone cells might contribute to the development of osteoporosis (7). Estradiol metabolism in our study revealed two ways in which bioavailability of the potent estrogen could be reduced. In addition to the production of estradiol sulfate by EST, there was some formation of estrone (17-HSD), as seen on chromatography of both the unconjugated and sulfate fractions. Evidence of other metabolic products was slight, including other forms of conjugation. However, minor differences between the HPLC profiles for the sulfate and unconjugated steroid fractions give support to the view that EST is present in the canine bone cancer cells. The question of specificity has not been addressed in our studies, but clearly a more general phenol sulfotransferase could be involved (15). Lastly, if the balance of the two opposing enzymes, EST and sulfatase has importance for determining the level of estradiol in mammary tissues, it may also have relevance for bone cells. High levels of EST might be seen as counterproductive in bone in cases of osteoporosis but beneficial in bone tumor cells. In this regard, agents known to stimulate EST in breast cancer cell lines are now available (17) and could be tested in bone cells. ACKNOWLEDGMENT We thank Dr. Jonathan LaMarre for his interest and for making the bone cells available for this study.
REFERENCES
DISCUSSION In the course of some studies on cultures of bone cells we have encountered metabolism of estradiol which suggests the presence of sulfotransferase activity. Since we have found no reports of such enzyme activity in bone (13, 14), we now provide evidence in support of this view. It is based principally on the ability of a
507
1. Purohit, A., Flanigan, A. M., and Reed, M. J. (1992) Estrogen synthesis by osteoblast cell lines. Endocrinology 131, 2027–2029. 2. Nawata, H., Tanaka, S., Tanaka, S., Takayanagi, R., Sakai, Y., Yanase, T., Ikuyama, S., and Haji, M. (1995) Aromatase in bone cell: Association with osteoporosis in postmenopausal women. J. Steroid Biochem. Mol. Biol. 53, 165–174. 3. Fujikawa, H., Okura, F., Kuwano, Y., Sekizawa, A., Chiba, H.,
Vol. 273, No. 2, 2000
4.
5.
6.
7.
8. 9.
10.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Shimodaira, K., Saito, H., and Yanaihara, T. (1997) Steroid sulfatase activity in osteoblast cells. Biochem. Biophys. Res. Commun. 231, 42– 47. Dong, Y., Qiu, Q. Q., Debear, J., Lathrop, W. F., Bertolini, D. R., and Tamburini, P. P. (1998) 17-Hydroxysteroid dehydrogenase in human bone cells J. Bone Miner. Res. 13, 1539 –1546. Eyre, L. J., Bland, R., Bujalska, I. J., Sheppard, M. C., Stewart, P. M., and Hewison, M. (1998) Characterization of aromatase and 17-hydroxysteroid dehydrogenase expression in rat osteoblast cells. J. Bone Miner. Res. 13, 996 –1004. Sasano, H., Uzuki, M., Sawai, T., Nagura, H., Matsunaga, G., Kashimoto, O., and Harada, N. (1997) Aromatase in human bone cells. J. Bone Miner. Res. 12, 1416 –1423. Jakob, F., Siggelkow, H., Homann, D., Kohrle, J., Adamski, J., and Schutze, M. (1997) Local estradiol metabolism in osteoblastand osteoclast-like cells. J. Steroid Biochem. Mol. Biol. 61, 167– 174. Saito, H., and Yanaihara, T. (1998) Steroid formation in osteoblast-like cells. J. Int. Med. Res. 26, 1–12. Pasqualini, J. R., Schatz, B., Varin, C., and Nguyen, B. L. (1992) Recent data on estrogen sulfatases and sulfotransferases activities in human breast cancer. J. Steroid Biochem. Mol. Biol. 41, 323–329. Tseng, L., Mazella, J., Lee, L. Y., and Stone, M. L. (1983) Estrogen sulfatase and estrogen sulfotransferase in human mammary carcinoma. J. Steroid Biochem. 19, 1413–1417.
11. Pasquilini, J. R., and Chetrite, G. S. (1999) Estrone sulfatase versus estrone sulfotransferase in human breast cancer: Potential clinical applications. J. Steroid Biochem. Mol. Biol. 69, 287– 292. 12. Raeside, J. I., Renaud, R. L., and Christie, H. L. (1997) Postnatal decline in gonadal secretion of dehydroepiandrosterone and 3hydroxyandrosta-5,7-dien-17-one in the newborn foal. J. Endocrinol. 155, 277–282. 13. Hobkirk, R. (1993) Steroid sulfation: Current concepts. Trends Endocrinol. Metab. 4, 69 –74. 14. Strott, C. A. (1996) Steroid sulfotransferases. Endocrine Rev. 17, 670 – 697. 15. Bernier, F., Solache, I. L., Labrie, F., and Luu-The, V. (1994) Cloning and expression of cDNA encoding human placental estrogen sulfotransferase. Mol. Cell. Endocrinol. 99, R11–R15. 16. Petrotchenko, E. V., Doerflein, M. E., Yoshimitsu, K., Pedersen, L. C., and Negishi, M. (1999) Substrate gating confers steroid specificity to estrogen sulfotransferase. J. Biol. Chem. 274, 30019 –30022. 17. Chetrite, G. S., Kloosterboer, H. J., Philippe, J. C., and Pasquilini, J. R. (1999) Effect of Org OD14 (LIVIAL) and its metabolites on human estrogen sulfotransferase activity in the hormone-dependent MCR-7 and T-47D, and the hormoneindependent MDA-MB-231, breast cancer cell lines. Anticancer Res. 19, 269 –275.
508
Estradiol-17 Sulfotransferase Activity in Canine Osteosarcoma D17 Cells J. I. Raeside, H. L. Christie, L. Forster, and R. L. Renaud Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Received May 22, 2000
Estrogen sulfatase and sulfotransferase (EST) activities are present in breast cancer tissues but there are no reports on EST in cancerous bone cells. We incubated [ 3H]estradiol-17 with cells from a canine osteosarcoma D17 line for periods up to 24 h. Radioactive steroids were recovered from the media and separated into unconjugated and conjugated fractions using Sep-Pak C18 cartridges. The conjugate fraction was solvolyzed and the resulting free steroids were obtained from a second C18 cartridge. Little metabolism was apparent in 4 h of incubation, but by 24 h as much as one half of the radioactivity was seen in the conjugate fraction. Most of the conjugates were recovered as sulfates in all three experiments. HPLC profiles showed a limited metabolism of estradiol to other compounds except for estrone, which was clearly present in both free and sulfate fractions. These results suggest that EST may have a role in the local metabolism of estrogens in bone. © 2000 Academic Press Key Words: estrogen metabolism; sulfotransferase; sulfatase; bone; osteosarcoma; canine.
Estradiol-17 (E2) is known to play a role in normal, as well as in cancerous, bone cells. Its presence in bone results from estrogen synthesis and metabolism (1). Several studies have reported on steroid-convertingenzyme activities in various osteoblast and osteoblastlike cells in culture (1– 4). Further evidence in support of the importance of steroid metabolism by bone cells themselves is given by the demonstration of expression of messenger ribonucleic acid (mRNA) for several key enzymes, such as aromatase (P-450arom), 17hydroxysteroid dehydrogenase (17-HSD), and steroid sulfatase (4 – 8). Thus, bone has the capacity to form the biologically potent estrogen from steroids in the peripheral blood circulation (Scheme 1). Estrone sulfate is the major form of estrogen in blood and so provides an important source for estradiol biosynthesis in bone.
A parallel situation is seen in breast cancer tissue where estrone sulfate is quantitatively the most important source for estradiol formation (9). High levels of estrogen sulfatase in the tissues provide the first step leading to the bioavailability of estradiol (9 –11). Although the presence of estrogen sulfotransferase has been reported in human mammary cancer cell lines (9, 11) and primary mammary carcinoma (10), it seems that the balance between the activities of the two enzymes favors hydrolysis of estrogen sulfates. No comparable data are available for bone cells. In the course of studies on cultures of bone cells we noted metabolism of estradiol which suggested the presence of estrogen sulfotransferase activity. MATERIALS AND METHODS Cell culture. Canine osteosarcoma D17 cells (ATCC No. CRL6248) were used as part of a study to observe the direct effects of estradiol-17 on bone. The cell line was maintained in our department (Dr. J. LaMarre) in modified Eagle’s MEM with non-selective amino acids, 2% penicillin/streptomycin and 10% FBS in 10 ml of medium in T75 cell culture flasks. For incubation with estradiol-17, cells were plated out and grown to confluency in 6-well plates (Costar) in 1.5 ml of culture medium. [ 3H]Estradiol-17 (0.5 ⫻ 10 6 cpm) was added to the confluent cells in 1.5 ml of culture medium. Radiolabeled [2,4,6,7- 3H]estradiol (83 Ci/mmol) was from Amersham Canada, Ltd. (Oakville, ON). Incubations were done in duplicate in 3 experiments and media were removed after fixed times (0, 1, 4, 12 and 24 h). Cells were washed with 2 ⫻ 0.5 ml saline (PBS) which was added to the appropriate sample and frozen until later processing. The washed cells were used for other aspects of the study and were not available for extraction of steroids. Analytical procedures. Unconjugated and conjugated steroids in the media were recovered separately by solid phase extraction (SPE, using Waters Sep-Pak C 18 cartridges) with diethyl ether and methanol, respectively, as described previously (12). The ether and methanol extracts were evaporated to dryness separately under nitrogen at ⬍45°C. The conjugated material was hydrolyzed by solvolysis (overnight at 45°C in trifluoroacetic acid/ethyl acetate; 1/100; v/v) to obtain a “sulfate” fraction as free steroids from a second Sep-Pak cartridge, used as described above. The amount of radioactive material recovered from each incubation and at each fractionation step was determined by taking an aliquot for liquid scintillation counting (LSC) in 5 ml cocktail (Ecolite; ICN, Costa Mesa, CA). HPLC profiles of the unconjugated and hydrolyzed steroids were generated with a binary solvent gradient of acetonitrile/water on a
505
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 273, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SCHEME 1. Metabolism of steroids in bone to illustrate the potential significance of estrogen sulfotransferase.
Waters C18 column and system, with absorbance monitored at 280 nm (12). Fractions (1 ml) were collected automatically (LKB RediFrac; Pharmacia, Kalamazoo, MI), and aliquots taken for (LSC) in the first 2 experiments. For the third experiment a radiodetector (Packard 505TR) was used for a direct scan of radioactivity (cpm), along with UV (280 nm) absorbance in the chromatography.
RESULTS The time course of conjugation in the metabolism of [ 3H]estradiol-17 by bone cells is shown in Table 1. It is clear that the rate of conjugate formation was not rapid TABLE 1
Conjugation of [ H]Estradiol-17 by Bone Cells 3
Experiment 1 Time (h)
Total
0 1 4 12 24
1.3 4.3 — — 30.7
Experiment 2
Sulfate
Total
25
4.2 — 3.2 7.6 14.1
Experiment 3
Sulfate
Total
Sulfate
6.7 9.5
2.1 — — 18.3 57.8
14.1 47.1
Note. Values are given as means of duplicate incubations, based on percentage distribution of cpm at SPE (SEP-PAK), before and after solvolysis.
FIG. 1. (a) HPLC profiles of unconjugated metabolites of [ 3H]estradiol-17 formed by bone cells after 1 h (solid, dark) and 24 h incubation (interrupted line) are shown. Authentic standards of estradiol (E2) and estrone (E1) were included for reference in the chromatography and are seen along with background absorption at 280 nm (thin line). (b) HPLC profile of metabolites of [ 3H]estradiol liberated from the steroid sulfate fraction (24 h) by solvolysis (Experiment 1; about 25% appeared as “sulfates”). See legend to Fig. 1a for more details.
but by 12 h of incubation there had been an appreciable amount of radiolabeled substrate metabolized in this manner. It is also evident that the major component in the conjugate fraction was the sulfate part. In fact, almost half of the substrate was present in a sulfoconjugated form after 24 h of incubation in the third experiment. No further work has been done, as yet, to identify the lesser amounts of other possible form(s) of conjugation. The HPLC profiles for the unconjugated steroids in the media after 1 h and 24 h of incubation (Fig. 1a) show that no definite metabolism could be detected within the first hour, but by 24 h there was evidence of formation of estrone from the substrate. From the same medium sample, the sulfate fraction after solvolysis revealed a noticeable amount of metabolism, mainly to compounds less polar than estradiol (Fig.
506
Vol. 273, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. HPLC profiles of (a) unconjugated and (b) solvolyzed metabolites of [ 3H]estradiol (Experiment 3; about 50% appeared as “sulfates”). Radiodetection was made online, resulting in lower counting efficiency (cpm). See legend to Fig. 1a for more details.
1b). A radioactive peak coincident with the nonradioactive standard of estrone was noted, as was seen previously in the case of the unconjugated fraction (Fig. 1a). These findings were confirmed in another experiment (Experiment 3) where estrone was the principal metabolite formed from estradiol in both the unconjugated and sulfo-conjugated fractions (Fig. 2). Metabolic activity was greater in this experiment (about 50% conjugation), and small quantities of some polar products were detected in the unconjugated fraction.
solvolysis step to liberate estradiol from the steroid conjugate fraction of media extracts. Nearly half of the radioactivity in one experiment was found in the sulfate fraction after a 24 h incubation. Unfortunately, no estrogen sulfates were included as substrates in our studies so it is not possible to compare estrogen sulfatase and sulfotransferase activities in the bone cells, as has been done for human primary breast tumors (10). Estrone sulfate is extensively hydrolyzed in mammary tissues; however, in one breast cancer cell line (MDAMB-468) the presence of very strong estrogen sulfotransferase has also been shown (9). Normal breast tissues contain estrogen sulfatase and sulfotransferase but the activities are lower than those in tumors (10). Whether normal bone cells show a lower level of EST than we have found with an osteosarcoma cell line remains for further study. Postmenopausal bone loss is primarily due to estrogen deficiency. Impaired local production as well as degradation of estradiol in bone cells might contribute to the development of osteoporosis (7). Estradiol metabolism in our study revealed two ways in which bioavailability of the potent estrogen could be reduced. In addition to the production of estradiol sulfate by EST, there was some formation of estrone (17-HSD), as seen on chromatography of both the unconjugated and sulfate fractions. Evidence of other metabolic products was slight, including other forms of conjugation. However, minor differences between the HPLC profiles for the sulfate and unconjugated steroid fractions give support to the view that EST is present in the canine bone cancer cells. The question of specificity has not been addressed in our studies, but clearly a more general phenol sulfotransferase could be involved (15). Lastly, if the balance of the two opposing enzymes, EST and sulfatase has importance for determining the level of estradiol in mammary tissues, it may also have relevance for bone cells. High levels of EST might be seen as counterproductive in bone in cases of osteoporosis but beneficial in bone tumor cells. In this regard, agents known to stimulate EST in breast cancer cell lines are now available (17) and could be tested in bone cells. ACKNOWLEDGMENT We thank Dr. Jonathan LaMarre for his interest and for making the bone cells available for this study.
REFERENCES
DISCUSSION In the course of some studies on cultures of bone cells we have encountered metabolism of estradiol which suggests the presence of sulfotransferase activity. Since we have found no reports of such enzyme activity in bone (13, 14), we now provide evidence in support of this view. It is based principally on the ability of a
507
1. Purohit, A., Flanigan, A. M., and Reed, M. J. (1992) Estrogen synthesis by osteoblast cell lines. Endocrinology 131, 2027–2029. 2. Nawata, H., Tanaka, S., Tanaka, S., Takayanagi, R., Sakai, Y., Yanase, T., Ikuyama, S., and Haji, M. (1995) Aromatase in bone cell: Association with osteoporosis in postmenopausal women. J. Steroid Biochem. Mol. Biol. 53, 165–174. 3. Fujikawa, H., Okura, F., Kuwano, Y., Sekizawa, A., Chiba, H.,
Vol. 273, No. 2, 2000
4.
5.
6.
7.
8. 9.
10.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Shimodaira, K., Saito, H., and Yanaihara, T. (1997) Steroid sulfatase activity in osteoblast cells. Biochem. Biophys. Res. Commun. 231, 42– 47. Dong, Y., Qiu, Q. Q., Debear, J., Lathrop, W. F., Bertolini, D. R., and Tamburini, P. P. (1998) 17-Hydroxysteroid dehydrogenase in human bone cells J. Bone Miner. Res. 13, 1539 –1546. Eyre, L. J., Bland, R., Bujalska, I. J., Sheppard, M. C., Stewart, P. M., and Hewison, M. (1998) Characterization of aromatase and 17-hydroxysteroid dehydrogenase expression in rat osteoblast cells. J. Bone Miner. Res. 13, 996 –1004. Sasano, H., Uzuki, M., Sawai, T., Nagura, H., Matsunaga, G., Kashimoto, O., and Harada, N. (1997) Aromatase in human bone cells. J. Bone Miner. Res. 12, 1416 –1423. Jakob, F., Siggelkow, H., Homann, D., Kohrle, J., Adamski, J., and Schutze, M. (1997) Local estradiol metabolism in osteoblastand osteoclast-like cells. J. Steroid Biochem. Mol. Biol. 61, 167– 174. Saito, H., and Yanaihara, T. (1998) Steroid formation in osteoblast-like cells. J. Int. Med. Res. 26, 1–12. Pasqualini, J. R., Schatz, B., Varin, C., and Nguyen, B. L. (1992) Recent data on estrogen sulfatases and sulfotransferases activities in human breast cancer. J. Steroid Biochem. Mol. Biol. 41, 323–329. Tseng, L., Mazella, J., Lee, L. Y., and Stone, M. L. (1983) Estrogen sulfatase and estrogen sulfotransferase in human mammary carcinoma. J. Steroid Biochem. 19, 1413–1417.
11. Pasquilini, J. R., and Chetrite, G. S. (1999) Estrone sulfatase versus estrone sulfotransferase in human breast cancer: Potential clinical applications. J. Steroid Biochem. Mol. Biol. 69, 287– 292. 12. Raeside, J. I., Renaud, R. L., and Christie, H. L. (1997) Postnatal decline in gonadal secretion of dehydroepiandrosterone and 3hydroxyandrosta-5,7-dien-17-one in the newborn foal. J. Endocrinol. 155, 277–282. 13. Hobkirk, R. (1993) Steroid sulfation: Current concepts. Trends Endocrinol. Metab. 4, 69 –74. 14. Strott, C. A. (1996) Steroid sulfotransferases. Endocrine Rev. 17, 670 – 697. 15. Bernier, F., Solache, I. L., Labrie, F., and Luu-The, V. (1994) Cloning and expression of cDNA encoding human placental estrogen sulfotransferase. Mol. Cell. Endocrinol. 99, R11–R15. 16. Petrotchenko, E. V., Doerflein, M. E., Yoshimitsu, K., Pedersen, L. C., and Negishi, M. (1999) Substrate gating confers steroid specificity to estrogen sulfotransferase. J. Biol. Chem. 274, 30019 –30022. 17. Chetrite, G. S., Kloosterboer, H. J., Philippe, J. C., and Pasquilini, J. R. (1999) Effect of Org OD14 (LIVIAL) and its metabolites on human estrogen sulfotransferase activity in the hormone-dependent MCR-7 and T-47D, and the hormoneindependent MDA-MB-231, breast cancer cell lines. Anticancer Res. 19, 269 –275.
508