Human Reproduction Vol.19, No.9 pp.

19571967, 2004 Advance Access publication June 17, 2004

DOI: 10.1093/humrep/deh355

The AG1478 tyrosine kinase inhibitor is an effective suppressor of leiomyoma cell growth
Asher Shushan1,3, Nathan Rojansky1, Neri Laufer1, Benjamin Y.Klein2, Zipora Shlomai2, Rubina Levitzki2, Zipora Hartzstark2 and Hannah Ben-Bassat2
1

Department of Obstetrics and Gynecology and 2Laboratory of Experimental Surgery, Hadassah University Hospital, PO Box 12000, Jerusalem 91120, Israel To whom correspondence should be addressed. E-mail: [email protected]

BACKGROUND: Uterine leiomyomas are the most common benign smooth muscle cell tumours in women. Formation of leiomyomas, still not completely understood, is viewed as a multistep process, with involvement of ovarian steroid hormones, cytokines and growth factors. Our study aimed to identify tyrosine kinase inhibitors as potential signal transduction therapeutics for leiomyomas, underlying the effect of ovarian steroidal hormones. METHODS: The selective epidermal growth factor (EGF) receptor blocker AG1478 was evaluated as a potential target, since EGF has been shown to mediate estrogen action and to play a crucial role in regulating leiomyoma growth. Paired cultures of leiomyoma and normal myometrium samples were established and the suppressive effect of AG1478 on the cells prior and subsequent to steroidal hormone treatment was examined: cell proliferation, recovery after treatment, cell cycle analysis and immunochemical analysis of relevant proteins. RESULTS: Leiomyoma cell growth is effectively blocked by AG1478 and is unaffected by the presence of physiological concentrations of progesterone and estradiol. AG1478 (10 mM) completely suppressed proliferation and the cells did not recover after cessation of treatment. CONCLUSION: The growth-arresting properties of AG1478, unaffected by ovarian steroidal hormones, identify it as a potential lead agent for the non-surgical management of uterine leiomyomas.
Key words: AG1478/growth suppression/leiomyomas/protein tyrosine kinase (PTK) inhibitors/signal transduction therapy

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Introduction Uterine leiomyomas are the most common smooth muscle cell (SMC) monoclonal tumours in women (Buttram and Reiter, 1981; Stewart, 2001a). They are benign tumours, which grow rapidly in response to ovarian steroids (Stewart, 2001b). Fibroids are a major cause of abnormal uterine bleeding and the most cited reason for hysterectomy (Wilcox et al., 1994). Surgical removal through hysterectomy or myomectomy has been the traditional treatment. Currently, however, less invasive surgical techniques such as hysteroscopic removal, uterine artery embolization and focused ultrasound surgery are being examined (Nowak, 2001; Stewart et al., 2003). Surgical treatments are effective in the short term, but the long-term results indicate 51% recurrence in 5 years and 15 26% need secondary surgery (Fedele et al., 1995; Guarnaccia and Rein, 2001). Formation of leiomyomas, still not completely understood, is viewed as a multistep process, with involvement of ovarian steroid hormones, cytokines and growth factors (Sozen and Arici, 2002; Flake et al., 2003; Lefebvre et al., 2003). Previous and present research has opened the way to

new non-surgical treatments, based on the understanding that leiomyomas are SMC steroid-responsive tumours (Liu et al., 1993). A body of research has demonstrated that leiomyomas are affected by estrogen and progesterone and that their response differs compared with that of normal myometrium (Rein et al., 1995; Rein, 2000). Therefore, GnRH agonists that reduce ovarian steroid hormones levels are widely in use (Lethaby et al., 2002). Since leiomyomas are chronic and recurrent, it is advantageous to modify therapies to allow for more prolonged treatment. Indeed, GnRH agonists with addback esteradiol (E2) and progesterone are among the most frequently studied medical therapies (Chegini et al., 2002). Newer therapies are based on the differential expression/ production of growth factors between leiomyomas and normal myometrium. Basic broblastic growth factor (bFGF), suggested as central to the pathogenesis of leiomyomas, may provide a new target therapy especially for women with leiomyoma-related bleeding (Anania et al., 1997). Indeed, interferon-a is a potent inhibitor of bFGF-stimulated cell proliferation in human uterine cells (Lee et al., 1998; Minakuchi et al., 1999). Interferon-a and interferon-b are presently 1957

Human Reproduction vol. 19 no. 9 q European Society of Human Reproduction and Embryology 2004; all rights reserved

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examined as novel treatment modalities, since they were shown to oppose the actions of bFGF in a number of systems (Lee et al., 1998). Recent research has focused on the function of protooncogenes such as Bcl2 and tumour suppressor gene products such as p53 in controlling apoptosis, as well as promoting leiomyoma cell proliferation by reducing the requirements for growth factors (Gao et al., 2001, 2002; Dixon et al., 2002). Matsuo et al. (1997) have reported increased expression of the Bcl2 protein in leiomyoma cells compared with normal myometrium cells, upregulated by progesterone. They suggested Bcl2 as the molecular basis for leiomyoma formation and a vital role for progesterone in the increased expression of this protein (Matsuo et al., 1999; Maruo et al., 2000). Since expression of Bcl2 is downregulated by E2 but upregulated by progesterone, progesterone may also participate in leiomyoma growth through induction of the Bcl2 protein. Shimomura et al. (1998) further demonstrated that E2 and progesterone act in combination to stimulate leiomyoma cell proliferation, through induction of epidermal growth factor (EGF) and EGF receptor (EGFR) expression. Accumulating data suggest that the effects of steroid hormones are mediated by local production of growth factors such as EGF (Hofmann et al., 1984; Sozen and Arici, 2002). Since EGF affects leiomyoma growth, a selective inhibitor of the EGFR such as AG1478 is a potential therapeutic agent for the non-surgical treatment of uterine leiomyomas (Levitzki and Gazit, 1995; Levitzki, 1999, 2001). We present evidence that the AG1478 tyrphostin effectively suppressed proliferation and cell cycle progression in cultures of leiomyoma cells.

approval and informed consent. Primary cell cultures were initiated in HAM/F12:Dulbeccos modied Eagles medium (DMEM) 1:1 with 20% FBS and antibiotics (penicillin 100 U/ml and streptomycin 100 mg/ml). Thereafter, the cell cultures were propagated in phenol red-free DMEM and 10% charcoal-treated FBS, specically for the experiments with ovarian steroid hormones. The experiments were done on secondary and/or tertiary cultures. Cells were maintained at 378C in a humidied incubator containing 5 8% CO2. Exponentially growing cells were used in the experiments. Immunocytochemical staining with a-actin was routinely performed to verify the SMC origin of the cells in culture. Tyrphostins and treatment of cells Stock solutions of 10 mM AG1478 in dimethylsulphoxide (DMSO) were kept at 2708C (Gazit et al., 1996). For the experiments, the tyrphostin was diluted with DMEM containing 10% FBS. The highest concentration of DMSO was 0.1%. Experimental design Cells were seeded at 1 104/well in 96-well microtitre plates and grown for 2 3 days. Thereafter, the medium was replaced with medium containing the tyrphostin. Control cells were grown in medium and in medium with the appropriate concentration of DMSO. Medium was changed after 2 days with new tyrphostin. Two days after the second treatment (4 days treatment), medium was removed, new medium without tyrphostin added, and the cells were grown for another 4 days (rescue/recovery after treatment) followed by determination of growth using the colorimetric methylene blue assay. Calculation of growth inhibition For each tyrphostin concentration used, medium containing only DMSO was used for the control. Thus, for each concentration, the control was taken as 100% growth. Automated microculture methylene blue assay Cell growth was determined by the automated microculture methylene blue assay: the tyrphostin-treated cultures and controls were xed in glutaraldehyde, 0.05% nal concentration, for 10 min at room temperature. After washing, the microplates were stained with 0.01% methylene blue in 0.1 mol/l borate buffer for 60 min at room temperature. Then the microplates were washed extensively and rigorously to remove excess dye, and dried. The dye taken up by cells was eluted in 0.1 mol/l HCl for 60? min at 378C, and the optical density was measured at 620 nm. In preliminary titration experiments, linear readings were obtained for 1 103 4 104 cells/well. Each point of the growth curve experiments is calculated from eight wells. Fluorescence-activated cell sorting (FACS) analysis of DNA content and determination of apoptotic cells Selected samples of cell cultures were FACS analysed for DNA content. Samples of cells treated with AG1478 concentrations for pre-determined periods were dispersed with trypsin 0.25% 1:1 EDTA 0.05% and stained with ethidium iodide in suspension. Cell cycle analysis and determination of the apoptotic cell fraction of the cell populations were carried out with FACS FPAR PLUS (BectonDickinson, Inc., Mountain View, CA). Biochemical activities: western blot (WB) analysis and phosphotyrosine WB analysis for relevant proteins and phosphotyrosine was carried as previously described and according to the manufacturers

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Materials and methods

Materials The antibodies used to monitor the levels and state of various signalling proteins were: the anti-EGFR external domain (clone F4) monoclonal antibody (catalogue no. 1 428 551; Boehringer Mannheim, Germany); anti-phosphotyrosine mouse monoclonal antibody 4G10 (catalogue no. 05321; Upstate Biotechnology UBI, Lake Placid, NY); mouse monoclonal anti-human antibody Bcl-2 (catalogue no. 05-341; Upstate Biotechnology UBI); anti-human rabbit polyclonal IgG, Stat3 (Upstate Biotechnology UBI); and, for phosphorylated Stat3, mouse anti-phospho Stat3-Tyr705 synthetic peptide (Y704 catalogue no. 06-596; Upstate Biotechnology UBI). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (catalogue no. F2653; Sigma Chemical Company, St Louis, MO) was used for immunouorescence staining. Fetal bovine serum (FBS; USA origin) was from Gibco-BRL (Life Technologies, Inc., Grand Island, NY). Tissue culture media, trypsin-EDTA solution and antibiotics were from Biological Industries (Beit Haemek, Israel). Tissue culture reagents, growth supplements and the ovarian steroid hormones E2 and progesterone were from Sigma Chemical Company. Cell cultures Paired cell cultures of leiomyoma and adjacent normal myometrium tissue samples were established from premenopausal women following hysterectomy conducted for benign disease, after ethical

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recommendation (Ben-Bassat et al., 1997). Briey, cell cultures of leiomyoma and normal myometrium were seeded at 5 105 cells/35 mm plates in DMEM with 10% FCS. After 2 3 days, the cells were washed and tyrphostin at the appropriate concentration was added for pre-determined periods. The reaction was stopped by placing the cultures on ice and washed with ice-cold phosphatebuffered saline (PBS). Whole cells were lysed with buffer, boiled for 5 min and run on a 7 15% SDS polyacrylamide gel for 4 h, then transferred to nitrocellulose paper, at room temperature overnight. Thereafter, the samples were incubated with the appropriate antibody, or with monoclonal anti-phosphotyrosine antibody 4G10, following the manufacturers recommendations. Goat anti-mouse or anti-rabbit uorescent antibody was added (3 ml/30 ml) for 30 min incubation at room temperature. The nitrocellulose membrane was washed with PBS-T, and the ECL (enhanced chemiluminescence) system was applied; then the membrane was exposed to X-rays lms in cassettes. Densitometric analysis was performed with a Fluor-Se Multi-Imager (Bio Rad Laboratories, Hercules, CA) using the Multi-Analyst Program.

Effects of E2 and progesterone on the biochemical activities of AG1478 Myometrium and leiomyoma paired cells were seeded at 2 105 cells/35 mm plates in DMEM with 10% FCS and grown for 2 days. Thereafter, the cultures were washed, fed with medium containing the steroid: 10 ng/ml E2 or 100 ng/ml progesterone without serum, and starved for 48 h. AG1478 at the appropriate concentration in medium E2 or medium progesterone without serum starvation medium) was added for 4 h. Cells were then stimulated with 30 ng/ml EGF for 10 min. The reaction was stopped by placing the cultures on ice and washed with ice-cold PBS. Immunoblot analysis of relevant proteins was performed on whole cell-lysates. Statistics Data obtained from determination of growth, FACS analysis and densitometry were expressed as meam ^ SD. Statistical analysis was conducted using the Students t-test. Statistical signicance was established at P-values of ,0.05.
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WB analysis for EGFR and phosphotyrosine Cell cultures were seeded at 5 105 cells/35 mm plates in DMEM with 10% FCS and grown for 2 3 days. Thereafter, the cultures were washed, fed with medium without serum and starved for 48 h. Tyrphostin at the appropriate concentration in medium without serum ( starvation medium) was added for 4 h. Cells were then stimulated with 30 ng/ml EGF for 10 min. Placing the cultures on ice and washing with ice-cold PBS stopped the reaction. Whole cells were lysed with hot buffer, scraped, boiled for 5 min and then run on a 7% SDS polyacrylamide gel for 4 h. The cells were then transferred to nitrocellulose paper and incubated overnight at 48C with a monoclonal anti-phosphotyrosine antibody PT-66, following the manufacturers recommendations. Goat anti-mouse uorescent antibody was added for 2 h incubation at room temperature (1 ml/20 ml). After drying, the gels were exposed to X-ray lm in cassettes. Densitometric analysis was performed with a Fluor-Se Multi-Imager (Bio Rad Laboratories) using the Muli-Analyst Program.

Results Growth pattern of leiomyoma and normal myometrium cell cultures The growth of paired leiomyoma and normal myometrium secondary and tertiary cells cultured for up to 21 days was examined. The growth patterns of six myometrium and leiomyoma matched secondary cell cultures are presented in Figure 1. The results indicate a similar growth pattern of actively proliferating cells with no signicant differences between the two cell types. In some cases, the growth rate of primary leiomyoma cells was slower than that of the myometrium cells. Inhibition of growth by AG1478 Since the effects of ovarian steroid hormones on leiomyoma growth are mediated by local production of growth factors such as EGF, we tested the potency of the selective EGFR kinase blocker AG1478 to suppress the proliferation of leiomyoma cell cultures. In parallel, we examined the effect

Effects of E2 and progesterone on cell growth of paired leiomyoma and normal myometrium cell cultures Cell cultures were seeded at 5 105 cells/35 mm plates in DMEM with 10% FCS and cultured for 2 days. Then dose response experiments with the ovarian steroids were performed using 1 20 ng/ml E2 or 10 200 ng/ml progesterone, separately and combined: 5 ng/ml E2 and 10 ng/ml E2 with 10 200 ng/ml progesterone. The cells were grown for another 7 days, followed by determination of growth using the colorimetric methylene blue assay.

Effects of E2 and progesterone on the inhibitory capacity of AG1478 Myometrium and leiomyoma paired cells were seeded at 2 105 cells /35 mm plates in DMEM with 10% FCS and grown for 2 days. Thereafter, the cultures were washed, fed with medium containing the steroid: 10 ng/ml E2 or 100 ng/ml progesterone without serum and AG1478 at 5 and 10 mmo/l in medium without serum E2 or medium without serum progesterone. The cultures were grown further for 7 days, followed by determination of growth using the colorimetric methylene blue assay.

Figure 1. Growth pattern of paired leiomyoma and normal myometrium secondary cell cultures. Cells were seeded at 1 104/well in 96-well microplates and grown in phenol red-free DMEM and 10% charcoal-treated FBS and antibiotics for up to 21 days. Six pairs of leiomyoma and myomertium cell cultures were examined. The results indicate a similar growth pattern of actively proliferating cells with no signicant differences between the two cell types. Growth was determined by the automated microculture methylene blue assay (detailed in Materials and methods).

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of this compound on the growth of normal myometrium cell cultures. For each tyrphostin concentration used, medium containing only DMSO was used for the control. The results demonstrate that AG1478 inhibited the growth of leiomyoma and myometrium cell cultures (IC50 5.6 mmol/l for leiomyoma and 5.7 mmol/l for myometrium cell cultures, respectively). AG1478 is already effective at $ 10 mmol/l and the cells remained arrested after withdrawal of the compound on day 4, as monitored on day 8. A similar pattern of growth inhibition was obtained for normal myometrium cell cultures (Figure 2). Recovery after treatment The cells were treated with medium containing the tyrphostin for 4 days, then the medium containing the tyrphostin was removed and new medium without the compound was added.

The cells were grown further for another 4 days. The period between day 4 and day 8 indicates recovery of the proliferation activity, i.e. rescue of the cells after cessation of treatment (Figure 2). AG1478 at $ 10 mmol/l exerted $ 90% inhibition and no recovery of the cells after cessation of treatment. After treatment with 1 mmol/l AG1478, the cells resumed growth and complete survival was obtained. The self-renewal capacity of leiomyoma and normal myometium cell cultures after treatment with AG1478 is almost the same. Points of growth arrest in the cell cycle and apoptosis To explore more directly the mechanism of growth suppression by AG1478, cell cycle analysis was performed on the leiomyoma and myometium cell cultures after treatment with this compound. Cells exposed to AG1478 were assessed by FACS analysis on day 2 after initiating treatment. The results indicate that AG1478 alters the cell cycle distribution
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Figure 2. The inhibitory effect of AG1478 on the proliferation and rescue after treatment of leiomyoma and myometrium cell cultures. The cells were treated with AG1478, and medium was changed after 2 days with new tyrphostin. Two days after the second treatment (4 days treatment), medium was removed, new medium without tyrphostin added, and the cells were grown for another 4 days to determine recovery after treatment, followed by determination of growth using the automated microculture methylene blue assay. AG1478 at $ 10 mmol/l exerted $ 90% inhibition and no recovery of the cells after cessation of treatment.

Figure 3. The effect of AG1478 on the cell cycle phase distribution and apoptosis of leiomyoma and myometrium cell cultures. The cells were treated with various concentrations of AG1478 and harvested on day 2. Cell cycle distribution and apoptosis were determined by FACS analysis. AG1478 alters the cell cycle distribution in a dose-dependent manner. The proportion of cells in G1 was increased and the proportion of cells in S and G2/M decreased, with no effect on the apoptotic cell fraction.

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Figure 4. Biochemical activities of AG1478 (western immunoblot analysis). Leiomyoma and myometrium paired cell cultures were grown for 2 days. Thereafter, the cultures were washed, fed with medium without serum and starved for 48 h. AG1478 at the appropriate concentration in starvation medium was added for 4 h. The cells were then stimulated with 30 ng/ml EGF for 10 min and analysed. AG1478 inhibited EGFR autophosphorylation in a dose-dependent manner that is enhanced greatly in the presence of the ligand. AG1478 did not alter the level of EGFR or the expression of the apoptotic proteins Bcl2 and Bax as indicated by densitometric analysis (data not shown). Densitometric analysis after normalization is presented for inhibition of EGFR phosphorylation with mean ^ SD.

(Figure 3). After one treatment with $ 10 mmol/l AG1478 for 2 days, the proportion of cells in G1 is increased and the proportion of cells in S and G2/M is decreased, with no effect on the apoptotic cell fraction (Figure 3). Biochemical activities of AG1478 Since treatment of the leiomyoma and normal myometium cell cultures with AG1478 induced growth arrest and changes in the cell cycle, we examined relevant proteins that could be linked to these processes. AG1478 induced a dose-dependent inhibition of EGFR autophosphorylation that is enhanced greatly in the presence of the ligand. AG1478 is already effective at 0.1 mmol/l ($ 61% inhibition in the presence of the ligand and $ 25% in its absence compared with control).

With $ 1.0 or $ 10 mmol/l AG1478, the autophosphorylation inhibition increased (65 and 74% in the presence of the ligand and 50 and 46% in its absence) (Figure 4). The results also indicate that AG1478 did not alter the level of EGFR in the cells. These ndings are in accordance with conclusive evidence that AG1478 specically and potently inhibits ligand-induced autophosphorylation of the EGFR. Thereafter, the effect of AG1478 on protein expression of two major regulatory proteins of apoptosis, Bcl2 (antiapoptotic) and Bax (pro-apoptotic), was examined. AG1478 had no effect on the expression of the Bcl2 or Bax proteins, in accordance with the FACS analysis showing no detectable changes in the apoptotic cell fraction (Figure 3). The leiomyoma cell cultures of this biopsy sample show a higher 1961

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level of the anti-apoptotic Bcl2 protein than the normal myometrium cell cultures, as reported by Matsuo et al. (1997). However, WB analysis of the Bcl2 and Bax as well as of the EGFR proteins in additional matched leiomyoma and myometrium cell cultures did not demonstrate a consistent and/or clearly dened differential level of protein expression in these cultures (see further Figure 8). Ovarian steroids do not affect AG1478 inhibitory activities The ovarian steroid-dependent growth potential of leiomyomas is well established (Stewart, 2001a,b). It has also been suggested that E2 and progesterone may act in combination to stimulate the proliferation potential of leiomymas (Matsuo et al., 1997, 1999; Maruo et al., 2000). Therefore, we examined the potency of AG1478 to suppress the proliferation of leiomyomas cultured with E2 and progesterone, separately and combined. The results of the dose response experiments with E2 or progesterone indicated that 1 20 ng/ml E2 or 10 200 ng/ml progesterone had no effect on the proliferation of leiomyoma and myometrium cell cultures. Further experiments to examine the combined effect of 5 or 10 ng/ml E2 with 10 200 ng/ml progesterone on the proliferation of

leiomyomas and myometrium cells also indicated no signicant effects (Figure 5 summarizes a representative experiment with normal myometrium cell cultures, and Figure 6 summarizes a representative experiment with leiomyoma cell cultures). The effect of AG1478 on cell proliferation of leiomyoma and myometrium cells cultured with 10 ng/ml E2 or 100 ng/ml progesterone (these concentrations are within the physiological tissue concentrations found in leiomyomas and myometrium; Matsuo et al., 1997) showed no signicant inuence of E2 or progesterone on the inhibition of cell growth by AG1478 (Figure 7). Similarly, cell cycle analysis of leiomyoma and myometrium cells cultured with the ovarian steroids and treated with AG1478 showed no signicant effect of E2 or progesterone on the cell populations or on the fraction of apoptotic cells (data not shown). Finally, WB analysis was performed to examine the inhibitory capacity of AG1478 on EGFR autophosphorylation and on the apoptosis-related proteins Bcl2 and Bax of leiomyoma cells cultured with E2 (10 ng/ml) or progesterone (100 ng/ml). Densitometric analysis of the results indicated that 5 and 10 mmol/l AG1478 effectively inhibited EGFR

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Figure 5. Effect of E2 and progesterone separately and in combination on proliferation of myometrium cell cultures. Myometrium cells were grown for 2 days (paired with the leiomyoma cultures presented in Figure 6). Thereafter, the cultures were washed and fed with medium containing the steroids: 1 20 ng/ml E2 (upper left) or 10 200 ng/ml progesterone (upper right), 5 ng/ml E2 and 10 200 ng/ml progesterone (lower left) or 10 ng/ml E2 and 10 200 ng/ml progesterone (lower right). At the indicated times, growth was determined by the automated microculture methylene blue assay (detailed in Materials and methods). E2 or progesterone separately and in combination do not affect proliferation of myometrium cell cultures.

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Figure 6. Effect of E2 and progesterone separately and in combination on the proliferation of leiomyoma cell cultures. Leiomyoma cells were grown for 2 days. Thereafter, the cultures were washed and fed with medium containing the steroids: 1 20 ng/ml E2 (upper left) or 10 200 ng/ml progesterone (upper right), 5 ng/ml E2 and 10 200 ng/ml progesterone (lower left) or 10 ng/ml E2 and 10 200 ng/ml progesterone (lower right). At the indicated times, growth was determined by the automated microculture methylene blue assay (detailed in Materials and methods). E2 or progesterone separately and in combination do not affect proliferation of leiomyoma cell cultures.

autophosphorylation and did not alter the level of EGFR, or the expression of the Bcl2 and Bax proteins in the cells cultured with E2 and/or progesterone. Similar results were obtained with the myometrium control cultures (Figure 8). Discussion We have shown that the AG1478 tyrphostin has a marked anti-proliferative activity on leiomyoma cell cultures and causes changes in their cell cycle distribution. AG1478 is very effective in inhibiting leiomyoma cell growth and inducing irreversible growth arrest without cytotoxicity at micromolar concentrations. Rescue experiments in which we measured the self-renewal capacity of the cells after removal of the tyrphostin on day 5 conrm the growth inhibition experiments. AG1478 blocked the growth of leiomyoma cell cultures mainly by increasing the fraction of cells in G0/G1 (with a concomitant decrease in the fraction of cells in S and G2 M) without inducing apoptosis. Leiomyomas are apoptosis resistant. However, recently it was shown that AG1478 increased apoptosis induced by the death ligand CD95L in some human glioma cell lines through inhibition of EGFR autophosphorylation (Steinbach et al., 2002). Thus, inhibition of EGFR might improve

the efcacy of cancer therapies based on ligand-mediated apoptosis. Previous and present biochemical analyses of AG1478 show that this tyrphostin effectively inhibits EGFR autophosphorylation in a dose-dependent manner, without an effect on the level of EGFR expression (Levitzki, 1992; Gazit et al., 1996; Ben-Bassat et al., 1997; Ben-Bassat and Klein, 2000). The EGF EGFR system plays a crucial role in regulating uterine leiomyoma growth, possibly by mediating estrogen action (Huet-Hudson et al., 1990; Shimomura et al., 1998). Growth of these tumours is considered to depend on ovarian steroid hormones, evident by their remarkable reduction in the menopause and by GnRH analogue treatment that creates a pseudo-menopausal state (Friedman et al., 1993, 1994; Stewart, 2001b). Maruo et al. (2000) suggested that the ovarian steroid hormones E2 and progesterone act in combination to stimulate the proliferative potential of leiomyomas through induction of EGF and EGFR. The present results demonstrate that E2 and progesterone do not affect the inhibitory activity of AG1478. AG1478 suppressed the proliferation of leiomyoma cells treated with E2 or progesterone separately and in combination. Nevertheless, the observation that no signicant effect of E2 and progesterone on the proliferation of cultured myometrial cells could 1963

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Figure 7. Effect of E2 and progesterone on the growth-suppressive activity of AG1478. Leiomyoma and myometrium paired cell cultures were grown for 2 days. Thereafter, the cultures were washed, fed with medium containing the steroids: 10 ng/ml E2 or 100 ng/ml progesterone and AG1478 at 5 and 10 mmol/l concentration. The cells were grown further for 7 days, followed by determination of growth using the colorimetric methylene blue assay. E2 or progesterone did not affect the growth-suppressive activity of AG1478.

be demonstrated might be considered as a major limitation of this model. A possible explanation could have been the loss of the steroid receptors in the in vitro model (Severino et al., 1996), yet we found that our tissue culture samples were positive for these receptors (by WB analysis and immunohistochemistry; data not shown). Another possible explanation might be associated with the cell proliferation assay. We measured cell proliferation by the automated microculture methylene blue assay and it might be that another assay, such as thymidine incorporation, could have been more signicant for this purpose. Finally, however, we believe that this resistance to steroids of our model demonstrates that in vivo, E2.and progesterone act as promoters of leimyoma cell proliferation, but there are other paracrine and autocrine growth factors that locally mediate this proliferation. In the in vitro model, these growth factors might not always be activated by the same signals and, therefore, the proliferative effect of E2 and progesterone could not be demonstrated. The EGFR gene is often amplied or overexpressed or both in diverse types of tumours. Usually, the overexpression of the EGFR is also accompanied by the autocrine or paracrine expression of its ligands, producing persistent enhanced stimulation of EGF-dependent pathways. Therefore, a suc1964

cessful potent inhibitor of the EGFR kinase has the chance of becoming a broad-spectrum inhibitor. To this point, two quinazoline derivatives of AG1478, AG1517/SU5271, are effective suppressors of growth of psoriatic keratinocytes (Powell et al., 1999; Ben-Bassat and Klein, 2000). More recently, AG1478 has been selected for clinical development for glioma multiformis in combination with cisplatin (Nagane et al., 2001). AG1478 did not alter the expression of the apoptosisrelated proteins Bcl2 (anti-apoptotic) and Bax (pro-apoptotic) in the leiomyoma and myometrium cultures, in accordance with the FACS analysis showing no detectable increase in the apoptotic cell fraction. Further, AG1478 had no effect on the expression of the Bcl2 and Bax proteins in leiomyoma and myometrium cells cultured with the ovarian steroids E2 or progesterone. WB analysis of the Bcl2 and Bax proteins in matched leiomyoma and myometrium cell cultures did not demonstrate consistent and/or a clearly dened differential expression level of these proteins between the two types of cells. Previous reports concerning Bcl2 expression in leiomyomas demonstrated overexpression of this protein in leiomyomas compared with normal myometrium (Matsuo et al.,

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Figure 8. Effect of E2 and progesterone on the biochemical activities of AG1478. Leiomyoma and myometrium paired cell cultures were grown for 2 days. Thereafter, the cultures were washed, fed with medium containing the steroid: 10 ng/ml E2 or 100 ng/ml progesterone without serum and starved for 48 h. AG1478 at the appropriate concentration in starvation medium E2 or starvation medium progesterone was added for 4 h. The cells were then stimulated with 30 ng/ml EGF for 10 min and analysed. E2 or progesterone do not affect the biochemical activities of AG1478 as indicated by densitometric analysis (western immunoblot analysis).

1997). A possible reason for this discrepancy could be the monoclonal origin of individual broids leading to differences between broids even from the same individual uterus. Matsuo et al. (1999) suggested that since Bcl2 protein expression is upregulated by progesterone, but downregulated by E2, progesterone might participate in leiomyoma development through induction of Bcl2. The molecular mechanism that initiates transformation of myometrium smooth muscle cells into leiomyomas during the reproductive years is still not completely understood (Myers et al., 2002; Lefebvre et al., 2003). Ovarian steroids are important for leiomyoma growth, and GnRH analogue

therapy is often used for their medical management. However, several in vitro studies have provided support for the direct action of GnRH by demonstrating changes in cell growth, cell cycle progression, apoptosis and expression of several growth factors, proteases and their inhibitors in the myometrium and leiomyoma cells, as well as in other steroid-sensitive cell types (Flake et al., 2003; Lefebvre et al., 2003). Recent in vitro studies provided additional evidence that a GnRH agonist (antide) and a progesterone antagonist (RU468) induce leiomyoma regression through an interactive mechanism that alters cell growth and suppresses transforming growth factor-b (a DNA synthesis stimulator) production. 1965

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These changes are mediated in part through GnRH receptoractivated signal transduction (Chegini et al., 2002; and references therein). In summary, we have identied the AG1478 tyrphostin as a potent inhibitor of leiomyoma and myometrium cell cultures. It selectively inhibits autophosphorylation of EGFR and downstream signal tranduction events, including suppression of cell proliferation and cell cycle progression at micromolar concentrations. A number of tyrosine kinase inhibitors, including getinib-Iressa-ZD1839 (inhibitors of autophosphorylation of EGFR), are progressing through clinical development and are beginning to provide new treament options for a range of malignancies (Levitzki, 2001). Our results suggest that the specic EGFR inhibitor combined with selective GnRH agonists may be useful as a future therapeutic approach for medical treatment of uterine broids. Tyrphostins are the rst signal transduction agents to be used in clinical practice. Acknowledgements
Supported in part by The Hadassah Medical Organization-Women Health Grant (S.A.) and the MJF Foundation.

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