Molecular Human Reproduction, Vol. 7, No. 2, 169-173,
February 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Involvement of annexin V in the antiproliferative effect of GnRH agonist on cultured human uterine leiomyoma cells
Department of Obstetrics and Gynecology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan
Abstract
The objective of this study was to elucidate the role of annexin V, an endogenous inhibitor of protein kinase C (PKC), with regard to the antiproliferative effect of gonadotrophin-releasing hormone (GnRH) agonist (buserelin) on cultured human uterine leiomyoma cells. Uterine leiomyoma tissue was collected from the surgical specimens of patients and cells from 37 specimens (15 cases) were cultured. For up to 96 h after the addition of buserelin to the cultured cells, a time-dependent antiproliferative effect was noted in the group to which 10–5 mol/l buserelin was added. Both the intracellular concentration of annexin V and the expression of annexin V mRNA increased time-dependently with the addition of buserelin. The intracellular concentration of annexin V increased with the addition of PKC activator (12-O-tetradecanoylphorbor-13-acetate; TPA) much as it did with the addition of buserelin, and the rise in the concentration caused by the addition of buserelin was completely attenuated by pretreatment with PKC inhibitor (calphostin C). Our findings suggest that buserelin inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied with an increase in the intracellular concentration of annexin V, mediated, at least in part, by the activation of PKC.
annexin V/GnRH agonist/GnRH receptor/protein kinase C/uterine leiomyoma
Introduction
There have been many clinical reports that gonadotrophin-releasing hormone (GnRH) agonist therapy reduces the size of uterine leiomyomata (Filicori et al., 1983
; Upadhyaya et al., 1983; Matta et al., 1988). It is believed that the mechanism involves a GnRH agonist-driven decrease in pituitary gonadotrophin followed by suppression of ovarian function (Upadhyaya et al., 1983; Matta et al., 1988; Rein et al., 1993). Recent reports, however, suggest that the antiproliferative effect of a GnRH agonist is induced both indirectly through pituitary gonadotrophin and also directly through GnRH receptors in uterine leiomyoma cells (Wiznitzer et al., 1988; Chegini et al., 1996). However, there are only a few reports on the direct action of GnRH agonist on uterine leiomyoma cells (Chegini et al., 1996; Kobayashi et al., 1996, 1997), and no reports dealing with identification of the factor(s) in the intracellular signal transduction system whereby GnRH agonist directly inhibits uterine leiomyoma cells.
Annexin V is a calcium phospholipid-binding protein belonging to the annexin family. This protein was originally studied as a placental anticoagulant protein, but it is now believed that anticoagulation is not the main physiological function of annexin V, either because it is present not only in the placenta but in all the organs of the body or because the concentration of annexin V in blood vessels is extremely low (Shidara et al., 1995). Since annexin V inhibits protein kinase C (PKC) and phospholipase C (PLC), it has been suggested that it is connected with an antiproliferative mechanism (Schlaepfer et al., 1992; Shibata et al., 1992a,b, 1997; Utsumi et al., 1992). Shibata et al. reported that annexin V was involved in an antiproliferative mechanism in endometrial cancer cells (Shibata et al., 1997). Meanwhile, it has been suggested that GnRH agonist activates PKC through GnRH receptors (Imai et al., 1993). In this study we investigated the antiproliferative effect of GnRH agonist on cultured human uterine leiomyoma cells, in association with either the induction of annexin V brought about by GnRH agonist or the relationship between annexin V and PKC.
Materials and methods
Samples and cell culture
Portions of uterine leiomyomata were collected from premenopausal women undergoing either a hysterectomy or enucleation for leiomyomata, and who had not received GnRH agonist therapy until the time of celiotomy. Informed consent was obtained from all patients. The protocol for tissue collection was approved by the Ethical Committee of Akita University Hospital, Japan. The average age of the patients was 43.0 years (range 28–50), and all patients were in the proliferative phase of the menstrual cycle, as confirmed by a basal body temperature chart and histological dating of the endometrium by the method of Noyes et al. (Noyes et al., 1950). Human uterine leiomyoma cells were cultured according to a previously described method (Strawn et al., 1995) with minor modifications. The tissue was minced into fine pieces (1–2 mm), and placed into tubes containing Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories, Grand Island, NY, USA) and a mixture of 0.25% collagenase (Wako, Osaka, Japan) and 400 IU dispase (Godoshusei, Tokyo, Japan), equilibrated at 37°C and mixed continuously. The separated cells were suspended in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco), 50 IU/ml penicillin G (Sigma Chemical Co, St Louis, MO, USA) and 50 µg/ml streptomycin (Sigma). The suspension was incubated at 37°C in 5% CO2 and 95% humidified air. Only primary cultured cells were used in the present study.
Reverse transcription–polymerase chain reaction (RT–PCR)
To examine whether human uterine leiomyoma cells have GnRH receptors, we performed RT–PCR. The total RNA was extracted from the cultured human uterine leiomyoma cells by the acid-guanidinium-phenol-chloroform method (AGPC method; Chomczynski and Sacchi, 1987). First-strand cDNA was obtained from the total cell RNA through the use of oligo (dT) -Latex (Roche, Tokyo, Japan) with Moloney murine leukaemia virus reverse transcriptase (Toyobo, Osaka, Japan). After incubation at 37°C for 60 min, this enzyme was inactivated at 95°C for 10 min. The cDNA was amplified in a 50 µl reaction volume, containing 10 mmol/l Tris–HCI (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl2, 200 µmol/l dNTPs (TaKaRa, Tokyo, Japan), 1.25 IU Taq polymerase (TaKaRa), and 0.2 µmol/l primers. The sequences of the oligonucleotide primers for human GnRH receptor, synthesized according to a previously described method (Imai et al., 1994a) were as follows:
sense: 5'-GACCTTGTCTGGAAAGATCC-3'
antisense: 5'-CAGGCTGATCACCACCATCA-3'
In all, 30 cycles of amplification (denaturation at 90°C for 1 min, followed by annealing at 57°C for 2 min and extension at 72°C for 2 min) were carried out. Human placenta was used as a positive control. Restriction enzyme digestion of PCR products was carried out using HindIII. The DNA product was run on 3.0% agarose gels, and the bands were visualized by ethidium bromide staining on an UV transilluminator.
Proliferation
The cells separated from human uterine leiomyoma tissue (5x105 cells) were plated on 6-well cluster plates (Sumitomo, Tokyo, Japan) in 2 ml medium. After cultured cells became 50% confluent, the medium was changed and 10–5 mol/l and 10–7 mol/l of buserelin (Hoechst Marion Roussel Ltd; Tokyo, Japan) was added according to a previously described method (Shibata et al., 1997). The cells were harvested after 24, 48, 72 and 96 h with the use of 0.25% trypsin and 0.02% EDTA in PBS. Viable cells, determined by Trypan blue exclusion, were counted in a haematocytometer. The buserelin was added and the medium exchanged every 48 h, and each experiment was performed in triplicate.
Determination of intracellular annexin V concentration
The intracellular annexin V concentration was measured by sandwich enzyme-linked immunosorbent assay (ELISA). Viable cells, determined by Trypan blue exclusion, were counted in a haematocytometer and the cells were collected by centrifugation at 800 g for 3 min, washed twice with phosphate-buffered saline (pH 7.4), and sonicated for 20 s in 50 mmol/l Tris–HCI (pH 7.5) containing 5 mmol/l EGTA (pH 7.3), 1 mmol/l phenylmethylsulphonyl fluoride (PMSF), 10 mmol/l 2-mercaptoethanol, and 0.1% Triton X-100 (Sigma). The extract was centrifuged at 18 500 g for 15 min, and annexin V in the supernatant was measured using anti-annexin V monoclonal antibodies (Kowa Co Ltd, Tsukuba, Ibaragi, Japan). After 96-well plates were coated with a 1/100 solution of anti-annexin V monoclonal antibody and washed, the samples were added. Bound annexin V was quantified with a 1/2500 solution of a different anti-annexin V monoclonal antibodies labelled with peroxidase and o-phenylenediamine (Sigma) was used as the colouring reagent. The colour was read at 492 nm with a multiscan spectrophotometer (MPR-A4i, Tosoh, Tokyo, Japan). Recombinant annexin V (Kowa Co Ltd; Nakao et al., 1988) was used for determining standard curves.
Northern blot analysis
The expression of annexin V mRNA in cultured human uterine leiomyoma cells was examined by Northern blot analysis. The total RNA was extracted from the cultured human uterine leiomyoma cells by the AGPC method. Samples (16 µg) of total RNA were run on 1.5% agarose gels, transferred to nylon membrane (Zeta-Probe blotting membranes, Bio-Rad, Richmond, USA) and linked by UV linker. The probe was derived from a 1442 bp DNA fragment of annexin V cDNA template (Kowa Co Ltd; Iwasaki et al., 1987) degenerated by EcoRI and HindIII, and labelled with [
-32P]-CTP (Amersham, Buckinghamshire, UK) by the random primer method using the Bca BESTM labelling Kit (Takara). Hybridization of total RNA was performed at 42°C for 30 h in hybridization buffer sodium chloride/sodium citrate [(SCC), 5x Denhardt's solution (50x Denhardt, 1% bovine serum albumin, 1% polyvinylpyrrolidone and 1% Ficoll), 1% sodium dodecyl sulphate (SDS), 50% formamide] with labelled annexin V cDNA after pre-incubation at 42°C for 30 min in the hybridization buffer. After the filters were washed once with 2x SCC and 0.1% SDS at room temperature for 20 min, and twice with 0.4x SCC and 0.1% SDS at 55°C for 5 min each, they were exposed to X-ray film at –70°C. A human G3PDH cDNA probe (Toyobo) was used as the loading control for RNA. Total RNA extracted from human placenta by the AGPC method was used as a positive control. Densitometric analysis of each band was performed with Scan Jet II CX and the numerical analysis of the density of each band was performed with picture analysis soft (NIH Image 1.56). The relative mRNA levels of annexin V were determined as the proportion of the density of each band relative to that of human G3PDH.
Proliferation assay and intracellular concentration of annexin V before and after the treatment with either PKC activator or inhibitor
In the same experiments, after incubation for 48 h, either 10–5 mol/l buserelin or 10–8 mol/l 12-O-tetradecanoylphorbor-13-acetate (TPA; Sigma), a direct PKC activator, was added. In other experiments, 10–8 mol/l calphostin C (Sigma), a specific and potent PKC inhibitor, was added 60 min before exposure to buserelin. The number of viable cells and the intracellular concentration of annexin V were determined after 48 h of drug exposure. The concentrations of TPA and calphostin C were selected based upon the data obtained with a human endometrial cancer cell line (Shibata et al., 1997).
Statistical analysis
The data are presented as the mean ± SD. The antiproliferative effect of GnRH agonist and the intracellular concentration of annexin V after the addition of GnRH agonist were analysed by one-way analysis of variance (ANOVA). The effect of treatment with either PKC activator or inhibitor was analysed by the Fisher's protected least significant difference method and Student's t-test. P < 0.05 was considered to be statistically significant.
Results
Expression of GnRH receptor mRNA in cultured human uterine leiomyoma cells
The expression of GnRH receptor mRNA in cultured human uterine leiomyoma cells was detected using RT–PCR. A single band corresponding to 319 bp was seen (Figure 1). In addition, the PCR product was digested into fragments at 269 and 50 bp by HindIII, confirming that it was GnRH receptor cDNA.
|
Antiproliferative effect of GnRH agonist on cultured human uterine leiomyoma cells
In the control group, the number of viable cells increased time-dependently. The cell number increased to 126% (P < 0.01) relative to that at the initiation of culture (Figure 2A
). In contrast, in the group in which 10–5 mol/l buserelin was added, the viable cell count gradually decreased with the duration of incubation, and the cell number after the addition of buserelin was reduced to 70% of that in the control group at 96 h (P < 0.01). In the cells in which 10–7 mol/l buserelin was added, no inhibiting effect on the cell proliferation was observed (Figure 2B).
|
= 10–5 mol/l buserelin group (n = 20); 
= 10–7 mol/l buserelin group (n = 12); • = control group. *P < 0.05 compared with control, **P < 0.01 compared with control.Change in intracellular concentration of annexin V and expression of annexin V mRNA after the addition of GnRH agonist
The mean intracellular concentration of annexin V in human uterine leiomyoma cells before the addition of GnRH agonist was 1.31 ± 0.17 pg/cell in the control group, and it gradually decreased over the duration of incubation (Figure 3
). In contrast, the concentration of annexin V increased in a time-dependent manner after buserelin was added. The concentration rose to 126% (P < 0.01) and 161% (P < 0.01) of that in the control group, at 48 and 72 h incubation respectively, after it was added. The expression of annexin V mRNA in human uterine leiomyoma cells was examined by Northern blotting. At 24 and 72 h incubation annexin V mRNA levels rose to134 and 176% respectively relative to that at the initiation of culture, after the addition of buserelin (Figure 4). Thus, annexin V mRNA expression increased in a time-dependent manner.
|
|
Effects of PKC activator or inhibitor on cell proliferation and intracellular concentration of annexin V
The number of viable cells 48 h after the addition of either buserelin or TPA was significantly reduced to 83% (P < 0.01) and 85% (P < 0.01) respectively, compared with the control group. In contrast, the number of viable cells was similar to the control level when cells were treated with calphostin C before the addition of buserelin (Figure 5A
). The intracellular concentration of annexin V 48 h after the addition of either buserelin or TPA rose significantly to 136% (P < 0.01) and 142% (P < 0.01) respectively, compared with the mean level (1.17 pg/cell) in the control group. In contrast, the concentration of annexin V remained at the control level when cells were treated with calphostin C before the addition of buserelin (Figure 5B). There was no significant difference between the concentration of annexin V with calphostin C alone and that of the control group (data not shown).
|
Discussion
In recent years, it has been demonstrated that GnRH receptors are present not only in the pituitary gland but also in ovarian and endometrial cancer cells (Imai et al., 1993, 1994b), suggesting the presence of an autocrine or paracrine mechanism in the cell. These reports propose that GnRH agonist inhibits the growth of tumour cells either through a decrease in ovarian oestrogen production as the result of the down-regulation of pituitary GnRH receptors or through direct inhibitory action. It is suggested that GnRH agonist may act directly on uterine leiomyoma cells because GnRH receptor mRNA was detected in cultured human uterine leiomyoma cells (Wiznitzer et al., 1988; Chegini et al., 1996). Kobayashi et al. then reported that GnRH agonist exerted a direct inhibitory action on cultured human uterine leiomyoma cells (Kobayashi et al., 1997). In this study, we also demonstrated the presence of GnRH receptor mRNA and the antiproliferative effect of 10–5 mol/l buserelin on human cultured leiomyoma cells.
PKC is one of the most important factors in the intracellular signal transduction system, and activated PKC is believed to regulate cell proliferation by phosphorylating various substrates. Several studies have reported that PKC is activated by GnRH in pituitary gonadotrophs (Hirota et al., 1985; Naor et al., 1985; Hori et al., 1992). The initial phase of GnRH action involves G protein-mediated stimulation of phospholipase C, leading to the formation of inositol trisphosphate (IP3) and diacylglycerol. Subsequently, IP3 induces the release of intracellular calcium, and diacylglycerol activates PKC, resulting in multiple cellular responses to GnRH (Kimura et al., 1999). GnRH agonist also activates PKC in ovarian cancer cells at the initial step of cell response (Imai et al., 1993). It might be possible that a similar mechanism occurs in human uterine leiomyoma cells. Thus, GnRH agonist would exhibit cell responses, including growth inhibition through the activation of PKC.
Our data showed that the PKC activator, TPA and GnRH agonist had the same effects on the growth inhibition and the annexin V expression of human uterine leiomyoma cells. These effects of GnRH agonists were attenuated by the inhibition of PKC activity with calphostin C. These results suggest that the activation of PKC, presumably by GnRH agonist, initiates annexin V synthesis in this system.
There have been several reports on the relationship between the annexin family and cell proliferation. We previously reported that annexin V plays an important role in antiproliferative effects on endometrial cancers (Shibata et al., 1997), and that the expression level of annexin V in uterine cancer tissues is lower than that in normal tissues (Karube et al., 1995). It has also been reported that annexin VI inhibits cell proliferation (Theobald et al., 1994, 1995) and that annexin II inhibits DNA synthesis (Kumble et al., 1992). These facts suggest that the annexin family, including annexin V, may inhibit cell proliferation.
In this study, we reported the up-regulation of annexin V in the antiproliferative effect of GnRH agonist on human uterine leiomyoma cells. GnRH agonist induced annexin V expression at both the mRNA and the protein level in a time-dependent manner. Annexin V is considered to possess an antiproliferative effect because it works as an endogenous inhibitor of PKC in the cell (Shibata et al., 1992a,b; Sato et al., 2000) and an inhibitor of PLC in vitro (Utsumi, 1992; Shibata et al., 1997). Therefore, the GnRH agonist-induced annexin V may inhibit the activated PKC, resulting in the inhibition of uterine leiomyoma cell proliferation.
Taken together, these results suggest that the antiproliferative effect of GnRH agonist occurs through PKC and that once GnRH agonist activates PKC, annexin V synthesis is initiated and this may finally inhibit cell proliferation, at least in part, through the inhibition of activated PKC.
In this study we have investigated the mechanism(s) by which GnRH agonist directly inhibits human uterine leiomyoma cell proliferation after the addition of GnRH agonist. Further research is required on the association of annexin V and PKC in intracellular signal transduction.
Notes
1 To whom correspondence should be addressed. E-mail: obgyn{at}med.akita-u.ac.jp 
References
Chegini, N., Rong, H., Dou, Q. et al. (1996) Gonadotropin-releasing hormone (GnRH) and GnRH receptor gene expression in human myometrium and leiomyomata and the direct action of GnRH analogs on myometrial smooth muscle cells and interaction with ovarian steroids in vitro. J. Clin. Endocrinol. Metab., 81, 3215–3221.[Abstract]
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Anal. Biochem., 162, 156–159.[ISI][Medline]
Filicori, M., Hall, D.A., Loughlin, S.J. et al. (1983) A conservative approach to the management of uterine leiomyoma: Pituitary desensitization by a luteinizing hormone-releasing hormone analogue. Am. J. Obstet. Gynecol., 147, 726–727.[ISI][Medline]
Hirota, K., Hirota, T., Aguilera, G. et al. (1985) Hormone-induced redistribution of calcium activated phospholipid dependent protein kinase in pituitary gonadotrophs. J. Biol. Chem., 260, 3243–3246.
Hori, H., Uemura, T. and Minaguchi, H. (1992) Effects of GnRH on protein kinase C activity of rat granulosa cells. In Lunenfeld, B. (ed.), Basic Science of GnRH Analogues, Plus a Review of the Management of Precocious Puberty, Advances in the Study of GnRH Analogues. Vol. 2. Parthenon Publishing Group, Carnforth, Lancs, UK, pp. 85–97.
Imai, A., Ohno, T., Fujii, T. et al. (1993) Gonadotropin-releasing hormone stimulates phospholipase C but not protein phosphorylation/dephosphorylation in plasma membrane from human epithelial ovarian cancer. Int. J. Gynecol. Cancer, 3, 311–317.[ISI][Medline]
Imai, A., Ohno, T., Iida, K. et al. (1994a) Gonadotropin-releasing hormone receptor in gynecologic tumors. Frequent expression in adenocarcinoma histologic types. Cancer, 74, 2555–2561.[ISI][Medline]
Imai, A., Ohno, T., Iida, K. et al. (1994b) Presence of gonadotropin-releasing hormone receptor and its messenger ribonucleic acid in endometrial carcinoma and endometrium. Gynecol. Oncol., 55, 144–148.[ISI][Medline]
Iwasaki, A., Suda, M., Nakao, H. et al. (1987) Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta. J. Biochem., 102, 1261–1273.
Karube, A., Shidara, Y., Hayasaka, K. et al. (1995) Suppression of calphobindin I (CPB I) production in carcinoma of uterine cervix and endometrium. Gynecol. Oncol., 58, 295–300.[ISI][Medline]
Kimura, A., Ohmichi, M., Kurachi, H. et al. (1999) Role of mitogen-activated protein kinase/extracellular signal-related kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line. Cancer Res., 59, 5133–5142.
Kobayashi, Y., Nikaido, T., Zhai, Y.L. et al. (1996) In-vitro model of uterine leiomyomas: formation of ball-like aggregates. Hum. Reprod., 11, 1724–1730.
Kobayashi, Y., Zhai, Y.L., Iimura, M. et al. (1997) Effects of a GnRH analogue on human smooth muscle cells cultured from normal myometrial and from uterine leiomyomal tissues. Mol. Hum. Reprod., 3, 91–99.
Kumble, K.D., Iverson, P.L. and Vishwanatha, J.K. (1992) The role of primer recognition proteins in DNA replication: inhibition of cellular proliferation by antisense oligodeoxyribonucleotides. J. Cell Sci., 101, 35–41.
Matta, W.H., Stabile, I, Shaw, R.W. et al. (1988) Doppler assessment of uterine blood flow changes in patients with fibroids receiving the gonadotropin-releasing hormone agonist Buserelin. Fertil. Steril., 49, 1083–1085.[ISI][Medline]
Nakao, H., Nagoya, T., Zaino, Y. et al. (1988) ELISA of calphobindin using monoclonal antibodies. Blood Vessel, 19, 205–207.
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.
Naor, Z., Zer, J., Zakut, H. et al. (1985) Characterization of pituitary calcium activated phospholipid dependent protein kinase; Redistribution by gonadotropin-releasing hormone. Proc. Natl Acad. Sci. USA, 82, 8203–8207.
Rein, M.S., Barbieri, R.L., Welch, W. et al. (1993) The concentration of collagen-associated amino acids are higher in GnRH agonists-treated uterine myomas. Obstet. Gynecol., 82, 901–905.
Sato, H., Ogata, H. and De Luca, L.M. (2000) Annexin V inhibits the 12-O- tetradecanoylphrbol-13-acetate-induced activation of Ras/extracellular signal-regulated kinase (ERK) signaling pathway upstream of Shc in MCF-7 cells. Oncogene, 19, 2904–2912.[ISI][Medline]
Schlaepfer, D.D., Jones, J. and Haigler, H.T. (1992) Inhibition of protein kinase C by annexin V. Biochemistry, 31, 1886–1891.[Medline]
Shibata, S., Sato, H. and Maki, M. (1992a) Calphobindins (placental annexins) inhibit protein kinase C. J. Biochem. Tokyo, 112, 552–556.
Shibata, S., Sato, H. and Maki, M. (1992b) Calphobindin I (annexin V) inhibits protein kinase C. Tohoku J. Exp. Med., 166, 479–481.[ISI][Medline]
Shibata, S., Sato, H., Ota, H. et al. (1997) Involvement of annexin V in antiproliferative effects of gonadotropin-releasing hormone agonists on human endometrial cancer cell line. Gynecol. Oncol., 66, 217–221.[ISI][Medline]
Shidara, Y., Kimura, N. and Tanaka, T. (1995) Investigation of placental anticoagulant protein Calphobindin. Clin. Gynecol. Obstet., 49, 1307–1317.
Strawn, E.J., Novy, M.J., Burry, K.A. et al. (1995) Insulin-like growth factor I promotes leiomyoma cell growth in vitro. Am. J. Obstet. Gynecol., 172, 1837–1843.[ISI][Medline]
Upadhyaya, M.D., Doody, M.C. and Googe, P.G. (1983) Histopathological changes in leiomyomata treated with leuprorelin acetate. Fertil. Steril., 54, 811–814.
Utsumi, T. (1992) Inhibitory effects of calphobindins on phospholipase A2 and phospholipase C. Acta. Obstet. Gynecol. Jpn., 44, 793–799.
Theobald, J., Smith, P.D., Jacob, S.M. et al. (1994) Expression of Annexin VI in A431 carcinoma cells suppresses proliferation: a possible role for Annexin VI in cell growth regulation. Biochem. Biophys. Acta, 1223, 383–390.[Medline]
Theobald, J., Handy, A., Patel, K. et al. (1995) Annexin VI has tumour-suppressor activity in human A431 squamous epithelial carcinoma cells. Br. J. Cancer, 71, 786–788.[ISI][Medline]
Wiznitzer, A., Marbach, M., Hazum, E. et al. (1988) Gonadotropin-releasing hormone specific binding sites in uterine leiomyomata. Biochem. Biophys. Res. Commun., 152, 1326–1331.[ISI][Medline]
Submitted on May 30, 2000; accepted on November 14, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||