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Liarozole Acts Synergistically with 1,25-Dihydroxyvitamin D₃ to Inhibit Growth of DU 145 Human Prostate Cancer Cells by Blocking 24-Hydroxylase Activity¹

Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305

Address all correspondence and requests for reprints to: David Feldman, M.D., Stanford University School of Medicine, Stanford, California 94305-5103. E-mail: feldman{at}cmgm.stanford.edu

	Abstract

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1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] inhibits the proliferation of many cancer cells in culture, but not the aggressive human prostate cancer cell line DU 145. We postulated that the 1,25-(OH)₂D₃-resistant phenotype in DU 145 cells might result from the high levels of expression of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) induced by treatment with 1,25-(OH)₂D₃. As this P450 enzyme initiates 1,25-(OH)₂D₃ catabolism, we presumed that a high level of enzyme induction could limit the effectiveness of the 1,25-(OH)₂D₃ antiproliferative action. To examine this hypothesis we explored combination therapy with liarozole fumarate (R85,246), an imidazole derivative currently in trials for prostate cancer therapy. As imidizole derivatives are known to inhibit P450 enzymes, we postulated that this drug would inhibit 24-hydroxylase activity, increasing the 1,25-(OH)₂D₃ half-life, thereby enhancing 1,25-(OH)₂D₃ antiproliferative effects on DU 145 cells. Cell growth was assessed by measurement of viable cells using the MTS assay. When used alone, neither 1,25-(OH)₂D₃ (1–10 nM) nor liarozole (1–10 µM) inhibited DU 145 cell growth. However, when added together, 1,25-(OH)₂D₃ (10 nM)/liarozole (1 µM) inhibited growth 65% after 4 days of culture. We used a TLC method to assess 24-hydroxylase activity and demonstrated that liarozole (1–100 µM) inhibited this P450 enzyme in a dose-dependent manner. Moreover, liarozole treatment caused a significant increase in 1,25-(OH)₂D₃ half-life from 11 to 31 h. In addition, 1,25-(OH)₂D₃ can cause homologous up-regulation of the vitamin D receptor (VDR), and in the presence of liarozole, this effect was amplified, thus enhancing 1,25-(OH)₂D₃ activity. Western blot analyses demonstrated that DU 145 cells treated with 1,25-(OH)₂D₃/liarozole showed greater VDR up-regulation than cells treated with either drug alone. In summary, our data demonstrate that liarozole augments the ability of 1,25-(OH)₂D₃ to inhibit DU 145 cell growth. The mechanism appears to be due to inhibition of 24-hydroxylase activity, leading to increased 1,25-(OH)₂D₃ half-life and augmentation of homologous up-regulation of VDR. We raise the possibility that combination therapy using 1,25-(OH)₂D₃ and liarozole or other inhibitors of 24-hydroxylase, both in nontoxic doses, might serve as an effective treatment for prostate cancer.

	Introduction

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THE MAJOR biological action of 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃], the active metabolite of vitamin D, is to maintain calcium homeostasis in the body (1). Recent findings indicate that 1,25-(OH)₂D₃ is also involved in regulating cellular proliferation and differentiation in various target tissues that possess vitamin D receptors (VDR) (1, 2, 3, 4). 1,25-(OH)₂D₃ and less calcemic analogs have been shown to inhibit cell growth in both human prostate carcinoma cell lines (5, 6, 7, 8, 9, 10, 11) and primary cultures of normal and prostate cancers (12). However, 1,25-(OH)₂D₃ showed only minimal inhibition of cell proliferation of DU 145, a human prostate cancer cell line derived from a brain metastasis, despite the presence of substantial amounts of VDR in this cell type (5, 7). The mechanism for the relative unresponsiveness of DU 145 to the antiproliferative action of 1,25-(OH)₂D₃ is not known.

DU 145 cells have been shown to express high levels of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) after treatment with 1,25-(OH)₂D₃ (5, 7). LNCaP cells can be induced to express low levels of 24-hydroxylase activity [12.6 ± 3.1 x 10^-9 µmol/2 x 10⁶ cells·30 min of 24,25(OH)₂D₃ produced] and are substantially growth inhibited by 1,25-(OH)₂D₃, whereas DU 145 cells can be induced to express very high levels of 24-hydroxylase activity (96.7 ± 39.5 x 10^-9 µmol/2 x 10⁶ cells·30 min) and are minimally growth inhibited (5, 7). As this P450 enzyme initiates the 1,25-(OH)₂D₃ inactivation pathway (1), we (8) and others (7, 13) have considered the possibility that rapid breakdown of 1,25-(OH)₂D₃ by 24-hydroxylase might be the cause of the resistant phenotype in DU 145 cells. In this study, we examine the premise that combination treatment with 1,25-(OH)₂D₃ and an inhibitor of 24-hydroxylase might render DU 145 cells more sensitive to the antiproliferative action of 1,25-(OH)₂D₃.

Combination therapy is often used to enhance the anticancer activity of various agents. Ketoconazole, liarozole, and other inhibitors of P450 enzymes may exhibit anticancer properties via several pathways, including actions on critical enzyme pathways (13, 14). In this study, we examined the possibility that combination treatment with 1,25-(OH)₂D₃ and liarozole, an imidazole derivative with antiprostate cancer properties (15, 16), might result in enhanced growth inhibition of DU 145 cells. Liarozole is known to inhibit several cytochrome P-450 enzymes, including retinoic acid 4-hydroxylase and aromatase (15, 17, 18). It is suspected that the former activity prolongs the half-life of retinoic acid and thereby increases the antiproliferative activity of endogenous retinoic acid when liarozole is administered to patients with prostate cancer (15, 16, 18). Here we show that 1,25-(OH)₂D₃ and liarozole interact synergistically to inhibit DU 145 cell growth. Our data demonstrate, for the first time, the ability of liarozole to directly inhibit 24-hydroxylase activity. The mechanism of liarozole action on DU 145 cells appears to be via inhibition of 24-hydroxylase, which causes a dual effect to prolong 1,25-(OH)₂D₃ half-life and to enhance up-regulation of VDR levels. Additional mechanisms may also play a role.

	Materials and Methods

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Materials
25-Hydroxy-[³H]vitamin D₃ (SA, 12.7 Ci/mmol) was obtained from Amersham Chemical Co. (Arlington Heights, IL). Liarozole fumarate (5-[(3-chlorophenyl)(1H-imidazol-1-yl)methyl]1H-benzimidazole fumarate) was a gift from Dr. C. Bowden (Janssen Research Foundation, Spring House, PA), and 1,25-(OH)₂D₃ was a gift from Dr. M. Uskokovic (Hoffmann-LaRoche, Inc., Nutley, NJ). Aprotinin, pepstatin, and soybean trypsin inhibitor were purchased from Boehringer Mannheim (Indianapolis, IN). Tissue culture media were purchased from Mediatech (Herndon, VA). FBS was obtained from Life Technologies (Gaithersburg, MD). CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS reagent) was purchased from Promega Corp. (Madison, WI). Silica gel TLC plates were purchased from E. M. Science (Darmstadt, Germany). All other reagents, except where indicated, were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture
The DU 145 human prostate carcinoma cell line was obtained from the American Type Culture Collection (Manassas, VA). Cells were routinely cultured in RPMI 1640 medium supplemented with 5% FBS and antibiotics at 37 C in a humidified atmosphere of 5% CO₂.

Assay of cell growth
Cell growth was assessed by measurement of viable cells using the MTS assay. DU 145 cells were trypsinized and seeded at a density of approximately 2,000 cells/well in 96-well tissue culture plates (Falcon, Lincoln Park, NJ) in 200 µl culture medium. The cells were allowed to attach for 24 h, and the medium was replaced with fresh medium containing 5% FBS. Cells were then treated with vehicle (ethanol), 1,25-(OH)₂D₃, and/or liarozole. Triplicate wells were used for each experimental condition. The medium containing vehicle or test compounds was renewed every 2 days during the course of the experiment. After the appropriate incubation period, the cells were processed by replacing them with fresh RPMI 1640 medium containing MTS reagent (100 µl medium plus 20 µl MTS reagent/well). The plates were incubated at 37 C in a humidified atmosphere of 5% CO₂ for approximately 3–4 h. The absorbance at 490 nm was read using an automatic plate reader (Emax Precision Microplate Reader, Molecular Devices, Menlo Park, CA) and was linear up to the highest cell concentration tested (40,000 cells/well).

Induction of 24-hydroxylase activity
24-Hydroxylase enzyme activity was assayed in a cell suspension system slightly modified from the method previously described (19). A 100-mm² confluent DU 145 culture, growing under standard conditions, was treated for various times (0.5, 3, 6, 16, and 20 h) with either vehicle (ethanol) or 10 nM 1,25-(OH)₂D₃. Cells were then rinsed with 10 ml PBS and incubated with 10 ml culture medium at 37 C in a humidified atmosphere of 5% CO₂ for approximately 30 min to remove 1,25-(OH)₂D₃. Cells were then trypsinized and resuspended at 10⁶ cells/200 µl RPMI 1640 containing 10 mM HEPES with 1% FBS. The cells were incubated for 30 min at 37 C with 1.0 nM [³H]25-OHD₃ and 1.0 µM 25-(OH)D₃. The reaction was terminated by the addition of 750 µl methanol-chloroform (2:1) and 20 µl 24,25-(OH)₂D₃. The metabolites were extracted three times with 200 µl chloroform. The organic extracts were combined, dried with a Speed-Vac (Savant Instruments, Farmingdale, NY) and dissolved in a 90:10 mixture of hexane-isopropanol. The production of [³H]24,25-(OH)₂D₃ was quantitated by TLC on silica gel/aluminum foil plates developed in methylene chloride-ethyl acetate (1:1) run with authentic standards. The TLC strips were cut into 14 fractions and placed individually in minicounting vials. This TLC system produced good separation of [³H]25-(OH)₂D₃ from [³H]24,25-(OH)₂D₃.

Inhibition of 24-hydroxylase activity
Time-course studies indicate that induction of 24-hydroxylase activity could be detected at 3 h by 10 nM 1,25-(OH)₂D₃ treatment with a plateau at approximately 20 h. Therefore, the conditions selected for studying the inhibition of 24-hydroxylase activity by liarozole were 20-h induction, 10⁶ cells, 1 nM [³H]25-(OH)D₃, and 30-min incubation with various concentrations of liarozole (1, 10, 50, and 100 µM).

Determination of 1,25-(OH)₂D₃ half-life
The half-life of 1,25-(OH)₂D₃ in DU 145 cells was determined by measuring the residual unmetabolized [³H]1,25-(OH)₂D₃ in the conditioned medium at various time points after addition. Confluent DU 145 cells were treated for various times with [³H]1,25-(OH)₂D₃ in the presence or absence of liarozole. Two hundred microliters of conditioned medium were mixed with 750 µl methanol-chloroform (2:1), and the metabolites were extracted with 200 µl chloroform. Chloroform extraction was repeated three times. The organic extracts were dried with a Speed-Vac and dissolved in a 90:10 mixture of hexane-isopropanol. The disappearance of [³H]1,25-(OH)₂D₃ and the production of [³H]1,24,25-(OH)₃D₃ were quantitated by a TLC system using methylene chloride-ethyl acetate (1:3). After 145 min of development, the TLC strips were dried and fractionated by cutting regions identified as 1,25-(OH)₂D₃ and 1,24,25-(OH)₃D₃ by comigration of authentic standards. This TLC system gave good separation between [³H]1,25-(OH)₂D₃ and [³H]1,24,25-(OH)₃D₃. The Rf value for 1,25-(OH)₂D₃ was 0.667, and that for 1,24,25-(OH)₃D₃ was 0.333.

Western blot analysis of vitamin D receptor (VDR)
Cell monolayers grown in RPMI 1640 supplemented with 5% charcoal-stripped serum in 100-mm dishes were incubated with ethanol vehicle, 1,25-(OH)₂D₃ (0.1, 1, and 10 nM), and/or liarozole (10 µM) for 4 days. After 4 days of incubation, cells were harvested, and Western blot analysis was performed as described previously using anti-VDR monoclonal antibody (9A7) (20). The experiment was repeated twice with similar results.

	Results

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Combination effect of 1,25-(OH)₂D₃ and liarozole on DU 145 cell growth
DU 145 cells are only minimally responsive to the antiproliferative effect of 1,25-(OH)₂D₃ (5, 7, 8). In our current studies, DU 145 cells were treated with increasing concentrations of 1,25-(OH)₂D₃ (1, 10, and 100 nM) over a time course of 2, 4, and 6 days (Fig. 1, A and B). The growth of DU 145 cells was not significantly inhibited by the lower concentrations of 1 and 10 nM; however, at the highest concentration (100 nM), there was a slight growth inhibition of approximately 20% on day 6. Similarly, as shown in Fig. 1, C and D, liarozole failed to inhibit the proliferation of DU 145 cells at 1 and 10 µM, but 100 µM resulted in 50% growth inhibition at 4 days and 60% growth inhibition at 6 days. However, 100 µM liarozole is a toxic dose and when administered to patients at these concentrations it causes hypervitaminosis A. Neither 10 nM 1,25-(OH)₂D₃ nor 1 µM liarozole had any antiproliferative effect when used alone. However as shown in Fig. 1, E and F, the combination treatment caused 60% growth inhibition. These data indicate that 1,25-(OH)₂D₃ and liarozole interact synergistically to inhibit DU 145 cell growth.

View larger version (12K): [in this window] [in a new window]   Figure 1. Dose-response effect of 1,25-(OH)2D3, liarozole, and the combination on DU 145 cell growth over a time course of 6 days. Cells were plated at approximately 2000 cells/well in 96-well tissue culture plates in 200 µl medium with the indicated concentrations of hormone. Media were changed every 2 days. Cell proliferation was estimated using the MTS assay. Data are expressed as the mean ± SD (n = 3) in the left panels. The right panels show a single representative experiment comparing treatment to vehicle and expressed in absorbance units. *, Significant changes (P < 0.05) compared with the ethanol control. A and B, Treatment with 1,25-(OH)2D3. C and D, Treatment with liarozole. E and F, Treatment with a combination of 1,25-(OH)2D3 (10 nM) and liarozole (1 µM).

View larger version (7K): [in this window] [in a new window]   Figure 2. Time course of the effect of 1,25-(OH)2D3 or liarozole on 24-hydroxylase activity in DU 145 cells. Cells were treated with 10 nM 1,25-(OH)2D3, 10 µM liarozole, or ethanol vehicle, and enzyme activity was measured at 0.5, 3, 6, 16, and 20 h. At 20 h, 10 nM 1,25-(OH)2D3 induced a 27-fold rise in 24-hydroxylase activity compared with the effect of vehicle. This is a representative experiment that was performed twice with similar results.

View larger version (7K): [in this window] [in a new window]   Figure 3. Dose-dependent effect of liarozole on 24-hydroxylase activity in DU 145 cells. Cells were treated with 10 nM 1,25-(OH)2D3 for 20 h. Treated cells were subsequently incubated with liarozole at various concentrations (0, 1, 10, 50, and 100 µM) for 30 min before enzyme activity was measured. This is a representative experiment that was performed three times with similar results.

View larger version (6K): [in this window] [in a new window]   Figure 4. Effect of liarozole on 1,25-(OH)2D3 half-life. DU 145 cells were incubated with [3H]1,25-(OH)2D3 (0.5 nM) plus unlabeled 1,25-(OH)2D3 (10 nM) in the absence or presence of 10 µM liarozole. Conditioned media were collected at various time points (0, 4, 24, 32, and 48 h), and the residual amount of unmetabolized [3H]1,25-(OH)2D3 was determined by TLC. This is a representative experiment performed twice with similar results.

View larger version (29K): [in this window] [in a new window]   Figure 5. A, Western blot analysis of VDR levels in DU 145 cells in response to various treatments. Cells were treated with ethanol or increasing concentrations of 1,25-(OH)2D3, liarozole, or 1,25-(OH)2D3 and liarozole for 4 days. High salt extracts were prepared, and 100 µg protein were loaded onto an 8% SDS-PAGE. After gel transfer, the blot was probed with anti-VDR monoclonal antibody 9A7, and the signal was detected using the enhanced chemiluminescence method. B, Densitometric analysis of Western blot. The pixel intensities of VDR bands were quantitated using a laser densitometer.

	Discussion

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As discussed earlier, at concentrations that are nontoxic, neither liarozole nor 1,25-(OH)₂D₃ alone substantially inhibited cell growth. Yet the combination of 1 µM liarozole and 10 nM 1,25-(OH)₂D₃ resulted in significant synergistic antiproliferative effects. Furthermore, this synergy is observed at a pharmacologically relevant concentration for both compounds. Based on this observation, we explored the possible mechanisms behind this synergy. We discovered that liarozole directly inhibited 24-hydroxylase activity in addition to inhibiting the already known P-450 enzymes, such as 4-hydroxylase and aromatase (15, 17, 18). As 24-hydroxylase is the initial enzyme for inactivating 1,25-(OH)₂D₃, we measured 1,25-(OH)₂D₃ half-life, and indeed, it was prolonged from 11 to 31 h. Therefore, by preventing rapid inactivation of 1,25-(OH)₂D₃ and prolonging the exposure time of cells to active hormone, DU 145 cells were able to respond to its antiproliferative effect. This provided the first possible mechanism for the synergistic activity of the 1,25-(OH)₂D₃/liarozole combination.

It is known that receptor regulation is an important mechanism for modulating target cell responsiveness to hormone (20, 23, 24). We explored the possibility that homologous up-regulation of the VDR would be enhanced in the presence of liarozole. Western blot analysis demonstrated a 5-fold increase in VDR protein level when cells were treated with 10 nM 1,25-(OH)₂D₃ and 10 µM liarozole compared with that after treatment with 1,25-(OH)₂D₃ alone. DU 145 cells treated with 1,25-(OH)₂D₃ alone only demonstrated a slight up-regulation of the VDR protein level. The possible explanation for this observation is that 1,25-(OH)₂D₃ is a potent stimulus of 24-hydroxylase activity; therefore, this would induce rapid degradation of 1,25-(OH)₂D₃, causing only a transient homologous up-regulation of VDR in DU 145 cells. Augmentation of VDR up-regulation has previously been reported using ketoconazole to inhibit 24-hydroxylase in a similar manner (25, 26). Enhanced VDR up-regulation is the second contributing mechanism explaining the liarozole synergistic interaction with 1,25-(OH)₂D₃. The increase in both ligand and receptor is a plausible mechanism for the enhanced antiproliferative activity of the 1,25-(OH)₂D₃/liarozole combination therapy in DU 145 cells. It is of interest that analogs of 1,25-(OH)₂D₃ designed to prevent 24-hydroxylation, such as 19-nor-25,26-hexafluoro-1,25-(OH)₂D₃, inhibit DU145 cell proliferation (9).

Our data suggest that DU 145 cells are more responsive to the antiproliferative effect of 1,25-(OH)₂D₃ when both its hormone and receptor are increased. Although the presence of VDR is essential for 1,25-(OH)₂D₃ activity (27), the level of VDR abundance in different prostate cancer cell lines by itself is not necessarily predictive of the amplitude of hormonal response (5, 28). However, in a given cell, increased abundance of receptor does appear to predict the extent of hormonal responsiveness, and increased or decreased receptor levels are usually correlated with increased and decreased responsiveness, respectively (20, 23, 24).

It should be noted that Zhao et al. have shown that combination therapy with either ketoconazole or liarozole and 1,25-(OH)₂D₃ or its analogs is cell type specific (13). That finding supports the concept that differences in cellular metabolism can at least partially explain the different potencies of various vitamin D analogs and differences in antiproliferative activity between different cancer cells. The fact that some cells are substantially growth inhibited by 1,25-(OH)₂D₃ alone (LNCaP and primary cultures) and other cells are not (DU 145) depends on a combination of factors, including, but not limited to, VDR abundance and inducible 24-hydroxylase activity (5, 7, 28). Liarozole in combination with 1,25-(OH)₂D₃ improves both parameters; by increasing VDR abundance and inhibiting 24-hydroxylase activity, it allows the otherwise resistant DU 145 cell to be growth arrested by 1,25-(OH)₂D₃. In preliminary experiments, liarozole also augmented the growth inhibitory activity of 1,25-(OH)₂D₃ in PC-3 and LNCaP cells, but to a much lesser extent (data not shown) than shown here for DU145 cells. The smaller augmentation was probably due to the greater antiproliferative activity of 1,25-(OH)₂D₃ alone in these cells (5) as well as the lesser induction of 24-hydroxylase in these cell lines (7), making the liarozole action to inhibit 24-hydroxylase less essential for 1,25-(OH)₂D₃-mediated growth inhibition.

Another possible mechanism for the enhanced antiproliferative effect in the presence of liarozole is its ability to inhibit retinoid metabolism, leading to increased retinoid levels (15, 18, 29). Retinoids have been shown to inhibit various cancer cell lines, including prostate cancer (30), and to be synergistic with 1,25-(OH)₂D₃ in inhibiting prostate cancer cell growth (10, 31). In fact, the beneficial effect of liarozole in patients with prostate cancer is attributed to this activity (15, 18, 29). We investigated the possibility that this activity might be contributing to the growth inhibition in our experiments. We treated DU 145 cells with a combination of 1,25-(OH)₂D₃ and increasing concentrations of retinoic acid to mimic the liarozole effect. We observed only a slight enhancement of growth inhibition (data not shown). As liarozole alone had no antiproliferative activity, and retinoids were not added in our standard combination experiments, we believe that the inhibition of retinoid metabolism does not substantially contribute to the effects that we have seen in cultured cells. However, in patients, the ability of liarozole to inhibit retinoid metabolism would be expected to further enhance the synergistic activity that we have demonstrated.

The mechanism(s) by which 1,25-(OH)₂D₃ inhibits the growth of prostate cancer cells is complex, multifactorial, and different in different cell lines. Several investigators have reported that treatment with 1,25-(OH)₂D₃ causes LNCaP cells to accumulate in the G₁ phase of the cell cycle (10, 11). 1,25-(OH)₂D₃ also elicits a reduction of cyclin-dependent kinase 2 activity and an increase in the level of hypophosphorylated retinoblastoma (Rb) protein, which is a critical regulator of the G₁/S checkpoint (11). Interestingly, DU 145 cells lack functional Rb protein. However, ectopic expression of functional Rb in DU 145 cells was not sufficient to restore the growth response to 1,25-(OH)₂D₃ (32). Others have found that growth inhibition of prostate cancer cells by a potent vitamin D analog involves the induction of p21^waf1, p27^kip1, and E-cadherin (9). In addition, we have demonstrated that 1,25-(OH)₂D₃ significantly regulates androgen receptor gene expression, which contributes to the regulation of LNCaP cell growth (33). Therefore, the mechanism by which 1,25-(OH)₂D₃ inhibits cell proliferation involves multiple signaling pathways and differs in various prostate cancer cell lines.

In summary, our data suggest that liarozole directly inhibits 24-hydroxylase activity, thereby effectively prolonging the 1,25-(OH)₂D₃ half-life. The increase in the 1,25-(OH)₂D₃ half-life resulted in enhanced up-regulation of VDR protein levels. We believe that this combination of increased 1,25-(OH)₂D₃ hormone levels as well as augmented VDR abundance represents the principal mechanism for the synergistic effect of 1,25-(OH)₂D₃ and liarozole in our experiments. However, additional mechanisms may play a role in the synergistic effect of this combination in DU 145 cells. In conclusion, the novel combination of liarozole and 1,25-(OH)₂D₃ therapy may serve as an effective treatment regimen for prostate cancer patients.

	Acknowledgments

 
We thank Dr. J. W. Pike for the anti-VDR monoclonal antibody (9A7); Dr. M. Uskokovic, Hoffmann-LaRoche, Inc. (Nutley, NJ), for providing 1,25-(OH)₂D₃; and Dr. C. Bowden, Janssen Research Foundation (Spring House, PA) for providing liarozole.

	Footnotes

 
¹ This work was supported by Stanford Medical Scholar Program (Resident & Pfeiffer), NIH Grant DK-42482, and a grant from the American Institute for Cancer Research. Portions of this work Were presented at the American Federation for Medical Research, Carmel, California, February 1998.

Received June 17, 1998.

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