0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society
Vol. 83, No. 7 Printed in U.S.A.
Separate and Interactive Regulation of Cytochrome P450 3A4 by Triiodothyronine, Dexamethasone, and Growth Hormone in Cultured Hepatocytes* CHRISTOPHER LIDDLE†, BRYAN J. GOODWIN, JACOB GEORGE‡, MICHAEL TAPNER, AND GEOFFREY C. FARRELL§ Storr Liver Unit, Departments of Clinical Pharmacology (C.L., B.J.G.) and Medicine (J.G., M.T., G.C.F.), University of Sydney at Westmead Hospital, Westmead 2145, Australia ABSTRACT CYP3A4, the predominant cytochrome P450 expressed in human liver, is responsible for the metabolism of endogenous steroids and many drugs. On the basis of pharmacokinetic studies in patients with hormonal derangements and the effects of replacement therapy, it has been suggested that iodothyronines decrease CYP3A4-mediated drug metabolism, whereas glucocorticoids and GH enhance CYP3A4 activity. The aim of the present study, using well differentiated human hepatocytes in primary culture, was to examine directly whether hormonal factors regulate CYP3A4 gene expression. Addition of T3 to primary hepatocytes resulted in a marked reduction of CYP3A4catalyzed testosterone 6b-hydroxylase activity and corresponding lev-
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EVERAL clinical studies have suggested that oxidative drug and steroid metabolism are perturbed in disease states associated with altered circulating concentrations of iodothyronines (1–3) and after the therapeutic use of glucocorticoids (4, 5) or GH (6 – 8). The changes in drug and steroid metabolism observed in these studies infer that expression of hepatic cytochrome P450 (P450), CYP3A4, and possibly other P450 enzymes may be subject to extensive and complex hormonal regulation in humans. CYP3A4 is the predominant constitutive P450 of human liver (9). It is responsible for the metabolism of many therapeutic agents, including cyclosporin (10), midazolam (11), erythromycin (4, 12), lidocaine (13), and nifedipine (12, 14). In addition, CYP3A4 is responsible for the 6b-hydroxylation of several endogenous steroids (15) and participates, with a minor contribution from CYP1A2, in the 2- and 4-hydroxylation of estradiol (16). Studies of closely related rat hepatic P450s have demonstrated that hormonal factors are the predominant determinants of constitutive expression of these enzymes. GH and iodothyronines negatively regulate Received September 15, 1997. Revision received February 4, 1998. Accepted March 5, 1998. Address all correspondence and requests for reprints to: Dr. C. Liddle, Department of Clinical Pharmacology, Westmead Hospital, Westmead 2145, Australia. E-mail:
[email protected]. * This work was supported by Project Grants 940026 and 970712 from the Australian National Health and Medical Research Council. † Philip Bushell Postdoctoral Research Fellow of the Gastroenterological Society of Australia. ‡ Recipient of a Australian National Health and Medical Research Council Medical Postgraduate Research Scholarship. § Robert W. Storr Professor of Hepatic Medicine of the University of Sydney.
els of CYP3A4 protein and messenger ribonucleic acid compared to those in untreated cells. Conversely, both dexamethasone and GH treatment substantially increased CYP3A4 gene expression. None of the hormones studied consistently altered the expression of other human cytochrome P450 genes. We conclude that iodothyronines, glucocorticoids, and GH act directly on human hepatocytes to regulate the expression of CYP3A4, and these effects appear to be exerted at a pretranslational level. Altered regulation of hepatic CYP3A4 is, therefore, likely to account for previous observations concerning the effects of endocrine diseases and hormonal treatments on human cytochrome P450-mediated drug and steroid metabolism. (J Clin Endocrinol Metab 83: 2411–2416, 1998)
CYP3A2 in rat liver (17), whereas glucocorticoids positively regulate this enzyme (18). In the present study we sought to examine the separate and interactive effects of T3, GH, and dexamethasone (DEX) on the major constitutive P450s of human liver, namely CYP1A2, CYP2C9, CYP2E1, and CYP3A4, using well differentiated primary cultures of human hepatocytes maintained on Matrigel. We also validated this model by examining the effects of two of these hormones on the expression of other hepatic genes known to be hormonally regulated. Materials and Methods Materials All chemicals were of analytical grade or similar purity and unless otherwise stated were obtained from Sigma-Aldrich (Sydney, Australia) or Merck (Darmstadt, Germany). Williams’ E medium and glutamine were acquired from Life Technologies (Grand Island, NY). Ribonuclease A, ribonuclease T1, deoxyribonuclease I, collagenase (type H), and dispase (type II) were purchased from Boehringer Mannheim, Sydney, Australia, and Promega Biotech (Madison, WI) provided restriction endonucleases, ligase, plasmid vectors, and all reagents for the in vitro transcription of complementary ribonucleic acid (cRNA) probes. [14C]Testosterone, [35S]UTP, and enhanced chemiluminescence Western blot detection reagents were purchased from Amersham International (Aylesbury, UK). Recombinant human GH (Genotropin) was a generous gift from Pharmacia, Sydney, Australia.
Primary cell culture Protocols for experiments on human tissue were approved by the human ethics committee of the Western Sydney Area Health Service. Informed consent was obtained from the relatives of organ donors for the use of waste surgical tissue for research. The source of liver for hepatocyte isolation was histologically normal tissue obtained as the surgical waste (usually the discarded right lobe) from reduction hepa-
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tectomies performed before pediatric liver transplantation of sizemismatched donor livers. Donor liver fragments were obtained from the National Liver Transplant Unit, Royal Prince Alfred Hospital (Sydney, Australia) or the Queensland Liver Transplant Unit, Princess Alexandria Hospital (Brisbane, Australia). The procedure for the isolation and culture of human hepatocytes was based on that of Strom et al. (19) and has previously been described in detail (20). In preparation for liver transplantation, liver tissue was een perfused with University of Wisconsin solution (Viaspan, DuPont, Wilmington, DE), and that used for hepatocyte isolation was stored at 4 C before cell dissociation (within 16 h of organ removal). Laminin-rich extracellular matrix (Matrigel) was prepared by extracting propagated Engelbreth-Holm-Swarm tumor as described by Kleinman et al. (21). Matrigel (;400 mL) was applied to 60-mm diameter plastic culture plates (Nunc, Roskilde, Denmark) to provide an attachment surface for hepatocytes. Cells (3.5 3 106 cells/plate) were inoculated into serum-free Williams’ E medium containing 15 mmol/L HEPES, penicillin (100 U/L), ascorbic acid (50 mg/L), and insulin (25 U/L) as the only hormone and applied to the Matrigel-coated culture dishes. Hormones (GH, T3, and DEX) were added to the culture medium as required. Culture medium and hormones were replaced daily for the duration of the experiments. Hepatocytes were harvested on days 2, 4, 6, and 8, as indicated in the figure legends. Medium was aspirated from culture plates, and the cells were overlayed with ice-cold phosphate-buffered saline, pH 7.4, containing 5 mmol/L ethylenediaminetetraacetic acid (EDTA). Hepatocytes, including Matrigel, were scraped from the culture plates with a rubber spatula. Matrigel was allowed to dissolve on ice, and cells were pelleted by centrifugation at 750 3 g for 5 min for use in subsequent assays. For assays of messenger RNA (mRNA), 5 individual plates were pooled for each experimental point. For assays of P450 enzyme activity and protein immunoquantitation, 10 –15 plates were pooled for each experimental point.
Solution hybridization-ribonuclease protection assays Hepatocytes for total nucleic acid (tNA) preparation were lysed in 4 mL lysis buffer [1% (wt/vol) SDS and 10 mmol/L EDTA in 20 mmol/L Tris(hydroxymethyl)aminomethane HCl, pH 7.5] and frozen for later use. tNA was extracted from hepatocytes using proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation as previously described (22). The relative abundance of specific mRNAs for CYP1A2, CYP2C9, CYP2E1, and CYP3A4 as well as insulin-like growth factor I (IGF-I) and tyrosine aminotransferase (TAT) were determined by the solution hybridization assay described by Melton et al. (23), using [35S]UTP (.1000 Ci/mmol)-labeled cRNA probes transcribed in vitro (Riboprobe, Promega Biotech). The complementary DNA templates used for non-P450 cRNA probe synthesis were as follows: IGF-I, bases 136 – 893 of the published sequence (24); and TAT, bases 1417–2051 of the GenBank deposited sequence (accession no. X55675; the TAT complementary DNA was a gift from Dr. V. Luu-The, Centre de Re´cherches en Mole´culaire, Quebec, Canada). The cRNA probes used for P450 mRNA quantitation were previously described (20). The optimal assay temperature was determined for each probe; it was set at 5–10 C below the melting point of the labeled RNA-RNA hybrid. Procedures for hybridization of aliquots of tNA, sample exposure to ribonuclease, and quantitation were previously reported (25). Standard curves were constructed for each assay using tNA extracted from a control subject; these confirmed the linearity of probe hybridization over a wide range of tNA concentrations and allowed the results of individual assays to be compared. Results were expressed as counts per min/mg DNA. All tNA samples were assayed in triplicate.
Preparation of microsomes Microsomes were prepared from cultured hepatocytes as follows. After harvesting, cells were suspended in ice-cold buffer (0.1 mol/L potassium phosphate, 0.25 mol/L sucrose, and 1 mmol/L EDTA, pH 7.4) and homogenized using a Potter-Elvehjem homogenizer. The 10,000 3 g supernatant (25 min) was centrifuged for 60 min at 105,000 3 g, and the microsomal pellet was resuspended in 50 mmol/L potassium phosphate buffer containing 20% glycerol and 1 mmol/L EDTA, pH 7.4, and stored at 270 C for later use.
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Immunoquantitation of P450 proteins Microsomal proteins were quantitated by immunoblotting as previously described (26). The primary antibodies used were as follows. Rabbit anti-CYP3A4 (a gift from Dr. F. P. Guengerich, Vanderbilt University, Nashville, TN) detects several CYP3A subfamily proteins; therefore, recombinant human CYP3A4 protein (Gentest Corp., Woburn, MA) was used as a standard. Antirabbit CYP1A2, raised in sheep, recognized a single band in human liver tissue and was a gift from Dr. P Maurel (INSERM U-128, Montpellier, France). Again, a recombinant CYP1A2 protein standard (Gentest Corp.) was included.
Testosterone hydroxylase assay Testosterone 6b-hydroxylase activity was used to determine the catalytic activity of CYP3As as previously described (27). Thin layer chromatographic separation of products was performed according to the method of Waxman et al. (28), and testosterone metabolite quantification was accomplished using a PhosphorImager (model SI, Molecular Dynamics, Sunnyvale, CA).
Other assays Hepatic microsomal protein was estimated by the method of Lowry et al. (29), with BSA as standard. The DNA content of the tNA samples was measured using the fluorescent DNA-binding dye Hoecst-33258 according to the procedure of Lebarca and Paigen (30).
Results Viability of human primary hepatocytes and validation of hormone responsiveness
As determined by phase contrast microscopy, cultured hepatocytes maintained a rounded, differentiated appearance throughout the 8-day duration of the experiments. Cellular integrity was confirmed by the low level of leakage of intracellular enzymes; alanine aminotransferase and lactate dehydrogenase were virtually undetectable in the culture medium after day 2. To establish the validity of the primary human hepatocyte culture system for physiological studies, responsiveness to DEX and GH was determined by estimating TAT and IGF-I mRNA, respectively. As previously described for rat primary hepatocytes in culture (22), cells were found to require an adjustment period of approximately 4 days before full responsiveness to added hormonal stimuli was achieved. Thus, on day 2 in culture, DEX and GH treatment resulted in 25% and 68% increases in TAT and IGF-I mRNA, respectively, compared to untreated cells. By day 4 in culture, the respective increases were 66% and 300%. Effects of hormone treatments on microsomal CYP3A enzyme activity and protein expression
Testosterone 6b-hydroxylation, predominantly mediated by CYP3A4 in human liver (15), declined rapidly with time in culture, so that by day 8, testosterone 6b-hydroxylase activity was 3.7% of that in freshly isolated hepatocytes (Fig. 1). Exposure of cells to T3 (1029 mol/L) exacerbated this loss of enzyme activity (Fig. 1). In contrast, treatment of hepatocytes with GH (100 ng/mL) or DEX (1028 mol/L) markedly increased CYP3A catalytic activity, such that by day 6 in culture, testosterone 6b-hydroxylase activity approached that observed in freshly isolated hepatocytes (Fig. 1). Expression of CYP3A proteins in microsomes prepared from control and hormone-treated human hepatocytes was
HORMONAL REGULATION OF CYP3A4
FIG. 1. Cytochrome P450 CYP3A catalytic activity in cultured human hepatocytes. Microsomes were prepared using differential ultracentrifugation from cultured human primary hepatocytes treated continuously with hormones from day 1 in culture and harvested on the days indicated (10 –15 culture dishes/experimental point). CYP3A activity was determined by the 6b-hydroxylation of 14C-labeled testosterone. Testosterone metabolites were separated by thin layer chromatography and quantitated using a PhosphorImager. The dashed line represents testosterone 6b-hydroxylase activity in freshly isolated human hepatocytes. DEX, 1028 mol/L; T3, 1029 mol/L; GH, 100 ng/mL.
examined by immunoblotting on nitrocellulose membranes, using a polyclonal antihuman CYP3A4 antiserum (Fig. 2). At least four protein bands could be resolved. CYP3A4 was identified by the use of a recombinant CYP3A4 protein standard. The identities of the other bands are uncertain, although it is likely that some correspond to the other CYP3A proteins recognized in human liver, namely CYP3A5 and CYP3A7. In close approximation to the results of testosterone 6bhydroxylase activity, CYP3A4 protein expression decreased throughout the culture period, and by day 4 it was 16% of that in freshly isolated hepatocytes. As expected from the testosterone 6b-hydroxylase activities, treatment of human hepatocytes with DEX (1028 mol/L) markedly increased CYP3A4 protein expression (to 180% of that in fresh hepatocytes on day 6). GH (100 ng/mL) was also found to be a potent positive regulator of CYP3A4 protein expression; values were 360% of those in freshly isolated hepatocytes on day 6. In contrast, exposure of cells to T3 (1029 mol/L) completely abrogated CYP3A4 protein expression. Interestingly, the application of hormone treatments did not influence the intensity of the other protein bands detected by the anti-CYP3A antibody (Fig. 2). Effect of GH on CYP3A4 mRNA expression
Addition of hormones to primary hepatocyte cultures produced changes in the expression of CYP3A4 mRNA that closely mirrored the findings for protein expression (Fig. 3). Thus, DEX and GH addition increased CYP3A4 mRNA by 740% and 910%, respectively (day 6 data pooled from experiments carried out on four individual livers; Table 1). T3 treatment suppressed CYP3A4 mRNA expression throughout the 8-day culture period.
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FIG. 2. Cytochrome P450 CYP3A4 protein in cultured human hepatocytes. A, Microsomes were prepared from hepatocytes harvested on days 4 and 6 in culture, as indicated below each lane (samples from freshly isolated hepatocytes and day 2 and 8 controls were also included). Proteins were separated by reduced SDS-PAGE (7.5% gel), transferred to a nitrocellulose membrane, probed with a polyclonal antihuman CYP3A antibody, and detected by an enhanced chemiluminescence technique. CYP3A4 was positively identified through the use of a recombinant protein standard, as indicated by the arrow. B, Results of densitometric scanning of the CYP3A4 protein band. F, Freshly isolated hepatocytes; Stds, CYP3A4 recombinant protein standards. DEX, 1028 mol/L; T3, 1029 mol/L; GH, 100 ng/mL.
To investigate the relative importance of interactions among these hormonal effects, experiments were carried out with combinations of GH and T3 as well as DEX and T3. T3 treatment of hepatocytes partially abrogated the inductive effect of GH on CYP3A4 mRNA, but did not have a consistent effect on DEX-mediated induction (data not shown). Effects of hormone additions on CYP1A2, CYP2C9, and CYP2E1 expression
In contrast to the striking effects of hormone treatments on CYP3A4, no consistent change in mRNA expression was observed for some other constitutively expressed hepatic P450s; CYP1A2, CYP2C9, and CYP2E1 (Fig. 4). As previous clinical studies of drug metabolism had implied that CYP1A2 regulation may be influenced by hormonal status, CYP1A2 protein was also examined using immunoblotting and an antihuman CYP1A2 antiserum. In keeping with the mRNA findings, no significant effect of hormone treatment on CYP1A2 protein expression was observed (Fig. 5). Discussion
A number of factors may contribute physiologically and pathophysiologically to the highly variable constitutive expression of hepatic P450 enzymes in man. These factors include age (31, 32), gender (5, 32, 33), nutritional status (34), and cytokine production (33). The present data indicate that one way in which such constitutional variables may affect
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FIG. 3. Cytochrome P450 CYP3A4 mRNA expression in cultured human hepatocytes. tNA were prepared using phenol-chloroform extraction from cultured human primary hepatocytes treated continuously with hormones from day 1 in culture and harvested on the days indicated. CYP3A4 mRNA was determined by a ribonuclease protection assay. Each experimental point represents the mean 6 SD of triplicate determinations of total nucleic acid samples from five pooled culture dishes. DEX, 1028 mol/L; T3, 1029 mol/L; GH, 100 ng/mL. TABLE 1. Effects of hormone treatments on CYP3A4 mRNA expression in primary cultures of human hepatocytes maintained in culture on Matrigel for 4 or 6 days CYP3A4 mRNA (% of control)
Day 4 Day 6 Pa
DEX
T3
GH
418 (150 –1420) 738 (165–1846)
46 (36 –53) 37 (13– 89)
342 (213–974) 908 (175–1670)
0.01
0.03
0.03
All treatments commenced on day 1 in culture. Data were pooled from four experiments using different donor livers and expressed as the median with the range in parentheses. DEX, Dexamethasone (1028 mol/L); T3, 1029 mol/L; GH, recombinant human GH (100 ng/ mL). a By Willcoxon’s signed rank test (day 6 data).
hepatic expression of P450 enzymes is via modulation of hormone concentrations. The results of the present study demonstrate that CYP3A4, the major hepatic P450 protein in human liver, can be regulated by several hormones at physiologically relevant concentrations. Experiments in rodents have demonstrated that most constitutive hepatic P450 enzymes are subject to hormonal regulation. In male rats, the activity of microsomal ethylmorphine N-demethylase is about 5-fold greater than that in female liver (35). This difference is due to the relative levels of constitutive P450 proteins, as detected by their immunochemical quantitation, and in parallel by hepatic levels of the P450-specific mRNAs. Using these approaches, it has also been demonstrated conclusively that the pattern of pituitary GH release is the major determinant of the sex difference in hepatic P450 expression in rats (36). Sex hormones act indirectly by influencing the pituitary release of GH and appear to have little or no direct effect on hepatic P450 expression. Other hormones, in particular iodothyronines (37– 40) and glucocorticoids (17, 18, 41, 42), also contribute to the regulation of constitutive P450s in rats as well as modulate xe-
FIG. 4. Cytochromes P450 CYP1A2 (A), CYP2C9 (B), and CYP2E1 (C) mRNA expression in cultured human hepatocytes. Cell treatments and mRNA quantitation were performed as described in Fig. 3. Each experimental point represents the mean 6 SD of triplicate determinations of total nucleic acid samples from five pooled culture dishes. DEX, 1028 mol/L; T3, 1029 mol/L; GH, 100 ng/mL.
nobiotic-induced expression of P450s. These experiments have provided a paradigm for hormonal regulation of at least some P450s, but, to date, it has been uncertain whether such hormonal factors are physiologically or pharmacologically
HORMONAL REGULATION OF CYP3A4
FIG. 5. Cytochrome P450 CYP1A2 protein in cultured human hepatocytes. Microsomes were prepared from hepatocytes harvested on days 4 and 6 in culture, as indicated below each lane (samples from freshly isolated hepatocytes and day 2 and 8 controls were also included). Proteins were separated by reduced SDS-PAGE (7.5% gel), transferred to a nitrocellulose membrane, probed with a polyclonal antihuman CYP1A2 antibody, and detected by an enhanced chemiluminescence technique. CYP1A2 was positively identified through the use of a recombinant protein standard, as indicated by the arrow. F, Freshly isolated hepatocytes; Std, CYP1A2 recombinant protein standard. DEX, 1028 mol/L; T3, 1029 mol/L; GH, 100 ng/mL.
relevant regulators of hepatic P450-mediated metabolism in man. Studies in patients with thyrotoxicosis have demonstrated that an excess of circulating iodothyronines is associated with increased 2-hydroxylation of estrogens to form catechol estrogens (1, 2), whereas in hypothyroidism this reaction is barely detectable (1). Conversely, cortisol 6b-hydroxylation, a major catabolic pathway for glucocorticoids in humans, is enhanced in hypothyroidism and impaired in states of iodothyronine excess (3). Estradiol 2-hydroxylation is catalyzed by CYP1A2 and one or more CYP3A proteins (16), most likely CYP3A4, whereas steroid 6b-hydroxylase activity is mediated principally by CYP3A subfamily proteins (15). It follows that exposure to increased iodothyronine concentrations would be expected to down-regulate P450s of the 3A subfamily and possibly also up-regulate CYP1A2. In the present study, T3 was a potent negative regulator of CYP3A4 expression in cultured human hepatocytes, an effect that was predominantly pretranslational, as indicated by the parallel changes in mRNA and protein levels. There is also evidence that GH can affect human hepatic P450-mediated oxidative metabolism. Replacement GH therapy in deficient children increased clearance of theophylline by 50%, but decreased that of amobarbital (6). Conversely, in another study of GH-deficient children, replacement with recombinant GH reduced caffeine clearance by 20% (7). Because these drugs are metabolized by several P450s, it has been difficult to ascribe the changes to individual enzymes, although it is likely that CYP1A2 and CYP3A4 are involved (43– 45). We recently observed that antipyrine metabolism is also impaired in GH-deficient adults, and this can be reinstituted after replacement therapy with recombinant GH (8). The present study clarifies these clinical findings by showing that GH treatment of cultured human hepatocytes markedly up-regulates CYP3A4 expression at a pretranslational level. Moreover, the inductive effect of GH appeared to be quantitatively similar to that produced by dexamethasone, the classical inducer of CYP3A subfamily enzymes. The positive regulatory effect of GH could be only partially suppressed by T3 treatment. Glucocorticoids are inducers of CYP3A subfamily genes in man (4, 5) as well as many other species. For example, measurement of CYP3A-catalyzed erythromycin-N-demethylase activity in patients before and after the institution of glu-
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cocorticoid therapy revealed a 55% increase after treatment (5). Recent studies have shown that CYP3A mRNA and protein are up-regulated by exposure to dexamethasone in both primary human hepatocytes and liver cell tumor-derived cell lines (42, 46). The present study confirms the responsiveness of CYP3A4 to glucocorticoid induction in primary cultures of human hepatocytes. In addition, we found that glucocorticoid induction is only slightly abrogated by T3 treatment In summary, the present study provides detailed information concerning the regulatory effects of iodothyronines, glucocorticoids, and GH on CYP3A4 expression. These hormonal effects are exerted directly on hepatocytes and operate principally at the pretranslational level, as indicated by the high concordance among CYP3A4 mRNA expression, protein concentration, and enzyme activity. Moreover, these effects are gene specific, as hepatic CYP1A2, CYP2C9, and CYP2E1 showed no consistent effects of hormonal manipulation. These findings have implications for understanding how constitutional and disease-related factors alter hepatic steroid and drug metabolism and may be of clinical utility in forecasting the types of drug for which changes in disposition are likely to occur in endocrinological disorders. Acknowledgments The authors acknowledge the contribution of Glenda Baulderstone and Associate Professor Stephen Lynch, Queensland Liver Transplant Unit, Princess Alexandria Hospital (Brisbane, Australia), and the National Liver Transplant Unit, Royal Prince Alfred Hospital (Sydney, Australia), for assistance in obtaining the liver tissue used in this project.
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Progesterone Progestins, and Antiprogestins in the Next Millennium Jerusalem, Israel September 1–3, 1999 A Satellite Symposium of the Sixth International Congress on Hormones and Cancer—Jerusalem, Israel (September 5–9, 1999). For further information, please contact: Irving M. Spitz, M.D., D.Sc., Institute of Hormone Research, Shaare Zedek Medical Center, P.O. Box 3235, Jerusalem, 91031 Israel. Telephone: 1972-2-655-5188; Fax: 1972-2-652-2018; E-mail:
[email protected]; Internet: http://www.weizmann.ac.il/Biological– Regulation/antiprog/
