Erythropoietin is involved in growth and angiogenesis in malignant tumours of female reproductive organs
Yoshiko Yasuda5,
Yoshihiko Fujita,
Seiji Masuda1,
Terunaga Musha2,
Koichi Ueda3,
Hayahito Tanaka2,
Hiroyoshi Fujita4,
Takuya Matsuo,
Masaya Nagao1,
Ryuzo Sasaki1 and
Yukio Nakamura2
Department of Anatomy, Kinki University School of Medicine, Osaka-Sayama 589-8511,
1 Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto 606-8507,
2 Department of Obstetrics and Gynaecology, Kyorin University School of Medicine, Mitaka 181-8611,
3 Department of Plastic and Reconstructive Surgery, Osaka Medical College, Takatsuki 590-0451 and
4 Laboratory of Environmental Biology, Hokkaido University, Sapporo 060-8648, Japan
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Abstract
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The accumulating evidence that erythropoietin and erythropoietin receptor are expressed in various non-haematopoietic organs suggests that erythropoietin signalling might be involved in the growth of tumours, but this possibility has never been examined. We found that mRNAs for erythropoietin and erythropoietin receptor are expressed in malignant tumours of female reproductive organs, where erythropoietin levels are higher than in normal tissues. Furthermore, tumour cells and capillary endothelium showed erythropoietin receptor immunoreactivity. To investigate the role of the erythropoietin/erythropoietin receptor pathway in these tumours, we injected mouse monoclonal antibody against erythropoietin or the soluble form of erythropoietin receptor into blocks of tumour specimens and cultured the blocks. After 12 h of injections, these blocks were examined and compared with control blocks injected with mouse monoclonal antibody, heat denatured soluble form of erythropoietin receptor, mouse serum or saline. Tumour cells and capillaries were markedly decreased in a dose-dependent manner after either injection. A marked increase of the cells containing fragmented DNA and the histopathological characteristics of these cells suggest that the decrease in tumour cells and capillary endothelial cells was due to apoptotic cell death. The co-existence of JAK2 and phosphorylated-JAK2, and STAT5 and phosphorylated STAT5, all of which are involved in the mitogenic signalling of erythropoietin, was found frequently in tumour cells and capillary endothelial cells in the untreated blocks. In contrast, most of the phosphorylated-JAK2- or phosphorylated-STAT5-positive cells had disappeared in the experimental blocks. Moreover, reduced tyrosine phosphorylation of STAT5 in the experimental blocks was confirmed by western blotting analysis. The results strongly indicate that erythropoietin signalling contributes to the growth and/or survival of both transformed cells and capillary endothelial cells in these tumours. Thus, deprivation of erythropoietin signalling may be a useful therapy for erythropoietin-producing malignant tumours.
Abbreviations: ADC, adenocarcinoma; ADCC, cervical adenocarcinoma; ADCE, endometrial adenocarcinoma; ADCO, ovarian adenocarcinoma; dsEpoR, denatured soluble form of erythropoietin receptor; E2, estradiol 17ß; Epo, erythropoietin; EpoR, erythropoietin receptor; FCS, fetal calf serum; IP, immunoprecipitate; JAK2, Janus kinase 2; kerat., keratinizing; MAb, monoclonal antibody; non-kerat., non-keratinizing; P-JAK2, phosphorylated-Janus kinase 2; PCNA, proliferating cell nuclear antigen; R2, monoclonal antibody against Epo; SCC, squamous cell carcinoma; sEpoR, soluble form of erythropoietin receptor; P-STAT5, phosphorylated signal transducer and activator of transcription; P-Tyr, phosphotyrosine; STAT5, signal transducer and activator of transcription; TdT, terminal deoxy nucleotidyl transferase; VEGF, vascular endothelial growth factor
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Introduction
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Erythropoietin (Epo) stimulates the formation of red blood cells by preventing apoptotic death of Epo-responsive erythroid precursor cells and by stimulating their proliferation and differentiation (reviewed in refs 13). The foetal liver is the Epo production site essential for foetal erythropoiesis (4,5), but its contribution diminishes after birth (6,7). The kidney is the major Epo production site in the adult, and kidney-derived Epo is responsible for the stimulation of erythropoiesis in adults (1,2). Binding of Epo to Epo receptor (EpoR) in erythroid precursor cells induces receptor homodimerization, allowing autophosphorylation of tyrosine residues in JAK2 associated with the intracellular domain of EpoR. Phosphorylation of JAK2 activates JAK2, which then phosphorylates, the receptor and cellular substrates recruited to the receptor complex. Among these substrates, phosphorylation of a transcriptional factor STAT5 is thought to be responsible for the mitogenic effect of Epo on erythroid precursor cells (reviewed in refs 3 and 8).
Recently two sites (brain and uterus) have been shown to produce Epo with new physiological functions. In the brain, there is a paracrine Epo/EpoR system that is independent of the endocrine system in adult erythropoiesis (915); neurons express EpoR (9,10) and astrocytes produce Epo (10,11,15). Epo production in the kidney, liver and brain is hypoxia-inducible (1,2,5,10,15,16). We have shown that there is another paracrine Epo/EpoR system in the mouse uterus and that Epo plays an important role in uterine angiogenesis via EpoR expressed in vascular endothelial cells of the uterine endometrium (17). Furthermore, Epo production in uterine tissue is stimulated by estradiol-17ß (E2). Because oxygen concentration was believed to be the major regulator of Epo production, E2 stimulation of uterine Epo production was surprising, but it provided evidence of Epo function in E2-dependent cyclical angiogenesis in the uterus (17). Recently we have demonstrated that Epo mRNA is expressed in normal human cervix, endometrium and ovary, and that JAK2, EpoRphosphotyrosine, and STAT5 are expressed at the Epo-responsive sites in these organs (18). Taken together with the mitogenic action of Epo and the properties of Epo production (hypoxia- and hormone-inducibility), which are deeply related to tumorigenesis, the paracrine or autocrine Epo/EpoR operation may contribute to the development and progression of tumours of female reproductive organs.
In this paper we report that Epo and EpoR mRNA are expressed in malignant tumours of the female reproductive organs and that the interception of Epo signalling by the injection of a monoclonal antibody against Epo or the soluble form of EpoR (sEpoR) into tumour blocks dramatically reduces capillaries and causes tumour cell destruction. sEpoR is an extracellular domain of EpoR, which is capable of binding with Epo as the antibody does (19,20).
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Materials and methods
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Sampling materials
Immediately after the resection of malignant tumours, some blocks of resected masses were put into liquid nitrogen or fixed in Zamboni solution, 10% buffered neutral formalin and Karnovsky solution. Others were put into MEM (Gibco BRL, Gaithersburg, MD) containing streptomycin (80 µg/ml, Meiji, Tokyo, Japan) and penicillin (100 IU/ml, Meiji). The frozen materials were stored at 80°C until the beginning of the biochemical analyses. The fixed materials in Zamboni solution were processed for immunohistochemistry as described previously (21). The other fixed materials in formalin and Karnovsky solution were processed for TdT assay and electron microscopy, respectively. The materials in MEM with antibiotics were stored at 4°C until the beginning of the treatment within 24 h after the resection. The patients gave informed consent for these specimens to be used for the experiments.
RTPCR amplification
Total RNA was prepared with the total RNA isolation kit (Trizol, Gibco BRL). When samples did not show the band for Epo or EpoR with the use of total RNA, mRNA of each sample was used. Each total RNA was dissolved in elution buffer, 10 mM TrisHCl buffer containing 1 mM EDTA (pH 7.4), and extracted with aqueous solution containing guanidinium thiocyanate and N-auroyl sarcosin. The solution was absorbed onto oligo (dT)-cellulose columns (Pharmacia). After the columns were washed with high- and low-salt solutions, mRNA was eluted with elution buffer at 65°C. Total amounts and concentrations of mRNA were demonstrated spectrophotometrically.
Reverse-transcription (RT) reaction was performed with an avian myeloblastosis virus (AMV) reverse transcriptase, random nanomer primer and 3 µg of each RNA in a volume of 20 µl, or oligo (dT) adaptor primer and 2 µl of each mRNA in a volume of 21 µl with the use of a Takara RNA LAPCRTM Kit (AMV) Ver. 1.1. PCR primer for Epo, EpoR (21) and ß-actin (10) were described previously. PCR cycles and conditions for denaturation, annealing, and elongation were 38 cycles, 30 sec at 94°C, 30 sec at 65°C and 30 sec at 72°C of Epo; 38 cycles, 30 sec at 94°C, 30 sec at 60°C and 30 sec at 72°C of EpoR and ß-actin. The amplified DNA was fractioned by electrophoresis and stained with ethidium bromide.
Determination of Epo
Normal and malignant specimens were homogenized in 4 (w/v) vol of 0.1 M phosphate buffer, pH 7.4, with an ultrasonic homogenizer (Tomy, Japan). The homogenate was freezethawed three times. The supernatant was used for assay after centrifugation at 1 200 g for 10 min and at 10 000 g for 20 min. Epo protein was measured in triplicate with an enzyme-linked immunoassay (22) with recombinant human Epo (gift from Snow Brand Milk Product Co., Japan) as the standard. The protein content of each sample was determined with a protein assay kit (BioRad) with bovine serum albumin (Sigma) as the standard. At least two independent determinations were performed on each sample. The results are summarized in Table I
.
Immunohistochemistry and antibodies
After 5 h of fixation in Zamboni solution, samples were processed for light microscopic immunohistochemistry. The methods have been described previously (23). The following antibodies were used as the primary antibody: anti-EpoR (1:250; 23), anti-Factor VIII (1:250; DAKO, Glostrup, Denmark), anti-proliferating cell nuclear antigen (PCNA) (1:1; DAKO), PCNA negative control (1:1; DAKO). The cryosections mounted on glass slides were treated with 3% H2O2 in 30% methanol for 15 min to remove endogenous peroxide and processed for detection with ABC complex (Vector, Burlingame, CA) and diaminobenzidine (Dojin, Wako, Osaka, Japan). The staining specificity of the antibody preparation of EpoR was assessed with supernatants of the incubation mixture of antibody and antigen (sEpoR). No antigen for anti-Factor VIII was available, so rabbit IgG (DAKO) (1:250) was used. The staining specificity of EpoR antibody was confirmed with the use of cell lines expressing EpoR (23).
Double staining with each pair of antibodies, anti-JAK2 (Santa Cruz, CA) and anti-phospho-JAK2 (P-JAK2) antibodies (Upstate Biotechnology, Lake Placid, NY), and anti-STAT5 (Transduction, KY) and anti-phosphorylated STAT5 (P-STAT5; Santa Cruz, CA) antibodies was performed on the same sections, respectively. Cryosections were incubated with a mixture of goat antiserum against peptide of JAK2 (1:2000) and rabbit IgG against mouse peptide of P-JAK2 (1 µg/ml) in PBS containing 0.5% goat serum, 0.25%
-carrageenan and 0.3% Triton X-100 for 812 h, washed twice with PBS, covered with biotinylated anti-rabbit IgG (10 µg/ml; Chemicon, CA) for 1h at room temperature, washed twice and then incubated with Texas Red Avidin D (5 µg/ml; Vector), FITC-labelled donkey antibody against goat IgG (10 µg/ml; Santa Cruz) and 10% normal rabbit serum for 1h. For double staining with anti-STAT5 and anti-P-STAT5, a mixture of mouse IgG against peptide of STAT5 and goat IgG against peptide of P-STAT5 was followed by processes similar to those in JAK2P-JAK2 except for the use of Texas Red conjugated donkey anti-goat IgG (5 µg/ml; Proto) and FITC-labelled donkey anti-mouse IgG (10 µg/ml; Jackson, PA).
Injection materials
sEpoR was prepared by the method of Nagao et al. (20) and diluted with saline (Ohtsuka, Tokyo, Japan). Monoclonal mouse antibody to Epo (R2), a gift from the Snow Brand Milk Product Co. (Tokyo, Japan), was dissolved in saline. sEpoR denatured by incubation at 56°C for 1 h, mouse monoclonal antibody against S-100 (MAb) (Cosmo, Tokyo) dissolved with saline, saline and mouse serum were used as controls. To these injection materials, Evans blue dye (Merck, Darmstadt, Germany) was added to yield a final concentration of 0.25% in each solution for colouring.
Organ culture
The time between resection and beginning of culture was <24 h. Specimens were cut into approximately 5 x 5 x 7 mm blocks with a razor blade. After removal of the damaged part of the tissue, 1520 solid pieces of a fresh sample were selected under a dissecting microscope. Some blocks from each sample were fixed at the time of this procedure. A block was put into a plastic petri dish (Falcon 3002) and weighed. Into several parts of each block, one of the following solutions (0.51.0 µl/mg of tissue) was injected with a 32 gauge needle and a microsyringe (Hamilton, Reno): 0.4, 0.2 or 0.08 mg/ml of sEpoR, 0.4 mg/ml of denatured sEpoR, 16 or 8 mg/ml of R2, 8 mg/ml of MAb, serum or saline. The petri dish was covered and incubated at 37°C with 5% CO2 in air for the first 90 min. The second injection was done without removal of the former solution released from the block, and incubation was continued for 60 min under the same conditions. The process was repeated 24 times, and the block was removed from the petri dish, put on a metal grid (Falcon, 3014) in a well filled with MEM and 10% FCS (Gibco BRL) in a multiwell dish (Falcon, 3047) and incubated for 8.5 h or longer. Almost all procedures were finished within 12 h after the beginning of the treatment except for SCC-51. Then the blocks were examined under a dissecting microscope and fixed in Zamboni solution, 10% neutralized buffered formalin or Karnovsky solution.
Immunoprecipitation and western blotting
Two specimens, ADCE-73 and ADCO-64, were treated four times with 0.4 mg/ml of sEpoR, 0.4 mg/ml of R2 or saline, and 0.4 mg/ml of sEpoR or saline, respectively, and processed as above mentioned. The blocks were frozen in liquid nitrogen. These samples were lysed in 500 µl lysis buffer (60 mM TrisHCl, pH 6.8, 1 mM sodium vanadate, 10% glycerol, 1% Triton X-100) containing a Protease Inhibitor Cocktail (Roche Diagnostics). The insoluble material was discarded by centrifugation, and 100 µg protein lysates were incubated with polyclonal anti-STAT5 antibody (Santa Cruz, CA) for 2 h. Immunocomplexes were then collected with protein G-Sepharose (Amersham Biosciences) for an additional hour, washed in lysis buffer and subjected to 7.5% SDSPAGE. After transfer onto Clear Blot membrane-p (Atto, Japan), proteins were detected with a monoclonal anti P-Tyr antibody (Upstate, NY). Blots were developed with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Amersham). For reprobing, the membranes were incubated in a ST buffer (62.5 mM TrisCl, pH 6.7, 2% SDS, and 2 mM ß-mercaptoethanol) for 30 min at 60°C.
Capillary density
In all specimens, sections stained with Factor VIII antibody were used. In each specimen positively stained cross- and longitudinally-sectioned capillaries were counted in 100 areas of 13.1 x 10-2 mm2 under 200-fold magnification in every third section.
TdT assay and electron microscopy for apoptosis
Samples were fixed in 10% neutral formalin for 6 h and the cryosections were processed by the TdT-mediated dUTPbiotin nick end-labelling (TUNEL) method (24) with an in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Control slides for each sample were incubated in the same reaction mixture without TdT. Following treatment with peroxidase-conjugated anti-digoxigenin antibody, the sections were coloured with peroxidasebenzidine reaction, and then stained with haematoxylin (Delafield, Merck). For positive controls, cryosections of adult rat testis were processed in the same way. Samples fixed in Karnovsky solutions were postfixed with 1% OsO4, and then processed for electron microscopy.
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Results
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Expression of EpoR and Epo mRNA
Sixteen tumour specimens, nine ADC of ovary, endometrium or cervix, five SCC, one endometrial carcinosarcoma and one cervical lymphoma, and one normal ovary and one normal endometrium were examined with RTPCR for the expression of EpoR and Epo mRNA. Figure 1
shows the results of RTPCR. Ten out of 16 tumour specimens showed high expression of both EpoR and Epo mRNA, and two specimens (ADCO-65 and ADCC-36) showed a weak expression of EpoR mRNA, and two specimens (ADCO-65 and SCC-30) showed a weak expression of Epo mRNA. As a control for EpoR mRNA, we used UT-7, which is a human megakaryoblastic leukaemia cell line established and reported by Komatsu et al. (25). Also as a control for Epo mRNA, one of the human hepatoma cell lines, HepG2 was used (26). One specimen (ADCE-51) was also used for injection of sEpoR (Figure 4
).
Content of Epo protein
Although the protein content of malignant and normal specimens was comparable, the average Epo protein in the malignant tumours was significantly higher than that in normal ovarian and endometrial specimens (P < 0.05; Table I
). Three malignant specimens (ADCO-K-65; ADCE-T-51; ADCC-S-36) and one normal specimen of ovary (H-47) were also processed for RTPCR analysis (Figure 1
).
Haematological examination
Haematological data (mean ± SEM, n = 35) of these patients just before operation were 12.03 ± 0.36 g/dl haemoglobin, 36.12 ± 1.03% haematocrit and 396.08 ± 10.42x104/ml red blood cell count. These values were within the normal range and ruled out anaemia and polycythemia.
Epo-responsive sites in tumours
Sections stained with anti-EpoR antibody of all tumours, three ADCO, six SCC and three ADCE, showed the EpoR immunoreactivity as shown in Figure 2
, which shows the loci of EpoR in SCC-38 (Figure 2A
) and in ADCO-56 (Figure 2C
). In both tumours, the malignant cells and capillary endothelium in the intervening tissues showed positive immunoreactivity to EpoR antibody (Figure 2A and C
). The staining specificity of the EpoR antibody was confirmed in each section adjacent to those shown in Figure 2A and B
; no immunoreactivity was detectable in malignant cells or capillary endothelium (Figure 2C and D
).
Alteration of histopathological features in tumour blocks
When the experimental blocks showed severe degenerative and necrotic changes, we determined whether the changes were due to the storage conditions or had already been seen before culture by comparing the histological preparations at the time of resections, and omitted the data from the experimental results. Moreover, as shown in Table II
, dead cells of the tissue blocks were significantly less before culture than those after treatment with controls (P < 0.01) except for SCC-44. Consequently, 17 experimental and 14 control blocks immunostained were examined for evaluation of the treatments. Injection of R2 or sEpoR into the blocks decreased the EpoR-expressing (Figure 3A
) malignant cells (Figure 3B
) and capillary endothelial cells due to their destruction (Figure 3C
), resulting in many vacant foci of the tumour tissues (Figure 3A and B
). Moreover, a few PCNA-positive cells were detectable in the damaged regions (Figure 3B
). In contrast, a great many cells with chromatin condensation and fragmented nuclei were discernible (Figure 3B
). Sections of all tumours were also adopted for TdT assay. Almost all nuclei of the cells in these destroyed foci reacted to TdT assay (Figure 3D
), indicating that the death of these cells was caused by apoptosis. Furthermore, some sections of blocks were processed for electron microscopy. The nuclei of the experimental malignant cells showed chromatin condensation that is typical for apoptosis (Figure 3E
).
The control blocks treated with saline, denatured sEpoR, serum or MAb did not show the vacant damaged foci seen in the experimental tissues (Figure 3F and G
), but they had rigid capillary networks with intact endothelial cells (Figure 3H
), many PCNA-positive cells (Figure 3G
), and a few cells undergoing apoptotic death (Figure 3I
) and no chromatin condensation (Figure 3J
).
The staining specificity of Factor VIII and PCNA antibody was confirmed in sections of SCC and ADCE; no immunoreactivity was discernible (data not shown). The positive control for TdT assay was confirmed in sections of rat testis according to the manufacturers recommendation (Oncogene). The results were similar to the previous experiments (27) and the data are not shown.
Inhibition of proliferation and death of tumour blocks
The viability of cells in the experimental and the control blocks was compared. Cells with positive PCNA-reactivity and dying cells were counted in a definite area of serial sections separated by 14 µm in 24 blocks of the representative tumours. The data are summarized in Table II
. In the tumours examined, the percentage of living cells with PCNA (proliferation rate) was significantly lower, but of dead cells (death rate) was significantly higher in the experimental blocks than those in the blocks before culture or in the blocks after saline, denatured sEpoR or MAb injection (P < 0.001, P < 0.01, P < 0.05, Table II
).
Changes in vascularization
The vascularization in the blocks was compared by an analysis of the capillary density in all experimental and control blocks (Figure 4
). All experimental blocks had significantly fewer capillaries than did the controls (P < 0.001 and P < 0.01). Moreover, the blocks treated for 12 h with four injections of a low dose of sEpoR (0.08 or 0.2 mg/ml; SCC-44, ADCO-68) had significantly fewer capillaries than did those injected twice with a high dose (0.4 mg/ml; SCC-51, P < 0.001) (Figure 4
). The effectiveness of four injections of sEpoR in destroying capillaries was similar to that of five specimens treated with three or four injections of R2 (Figure 4
).
Abolishment of JAK2 phosphorylation
The expression of Epo and EpoR mRNA, the presence of EpoR and the effect of sEpoR on the tumours strongly point to the process of Epo signalling. To confirm this, we explored the effect of the injection of sEpoR or R2 on the phosphorylation of JAK2 in the tumour blocks, since Epo signal transduction proceeds through phosphorylation of JAK2, EpoR and other substrate proteins, including STAT5, by activated JAK2.
The location of JAK2, P-JAK2, STAT5 and P-STAT5 was examined immunohistochemically in eight ADC and eight SCC. In all tumours, JAK2, P-JAK2, STAT5 and P-STAT5 were present in tumour cells and capillary endothelial cells in the tumours: all substances were detectable in the cytoplasm of the responsive cells. STAT5 and P-STAT5 were additionally seen in the nucleus (data not shown).
Next, the co-existence of each pair, P-JAK2 and JAK2, and P-STAT5 and STAT5, was demonstrated with a double staining technique (Figure 5AH
). There were many cells positive for P-JAK2 and JAK2 in the sections prepared from the tumour blocks cultured after the injection of saline (Figure 5A and B
). In the sections derived from the blocks injected with R2, JAK2 was found in many cells but P-JAK2 had largely disappeared (compare Figure 5C with D
). Similarly, cells positive for P-STAT5 and STAT5 in the nucleus were often discernible in the sections from control blocks (Figure 5E and F
), but cells positive for P-STAT5 in the nucleus were rarely seen although many cells positive for nuclear STAT5 were present in the sections from experimental blocks (compare Figure 5G
with H).
Moreover, western blotting analysis of the STAT5 immunoprecipitates revealed the weak tyrosine phosphorylation of STAT5 in the lysates from the experimental blocks compared with saline controls, but the expression of STAT5 was comparable (Figure 6
).

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Fig. 6. Tyrosine phosphorylation of STAT5 in tumour blocks treated in vitro. Cell lysates were immunoprecipitated with anti-STAT5. Western blotting with anti-P-Tyr shows weak or no band in lysates of blocks treated with sEpoR and R2, but strong in the controls.
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Discussion
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The present studies revealed that malignant tumours of female reproductive organs produce Epo and EpoR, and that the tumour cells themselves and capillary endothelial cells are sites responsive to the Epo signal. Furthermore, the injection of sEpoR or Epo-monoclonal antibody into the tumour blocks suppressed proliferative activity and caused apoptotic death of the cells. These results strongly indicate that Epo signal transduction occurs in a paracrine or autocrine manner and contributes to the survival and/or growth of tumour cells.
Epo production has been reported in a variety of human tumours, particularly renal cell carcinoma and haemangioblastoma (2830). Cerebellar haemangioblastomas are highly vascularized tumours in which blood-filled capillaries are separated by intervascular stroma cells. Interestingly, mRNAs of Epo and VEGF are expressed in all haemangioblastoma specimens analysed, and cells expressing both mRNAs are neoplastic stromal cells (30). The precise role of Epo expression in haemangioblastoma remains speculative. The finding that brain capillary endothelial cells express EpoR (31), however, suggests that Epo and VEGF might co-operate in the capillary formation of haemangioblastoma as they do in cyclical angiogenesis in the uterine endometrium (17).
Expression of EpoR was found in the capillary endothelial cells of tumours from female reproductive organs, and these endothelial cells died when the Epo signal was blocked. These findings suggest that Epo supports the survival of endothelial cells. Cultured endothelial cells have shown that Epo is involved in angiogenesis through its mitogenic action similar to that on erythroid precursor cells (3135). A variety of angiogenic factors, including VEGF, which is also hypoxia-inducible (36), and their possible involvement in tumour formation have been reviewed (37,38). Here we emphasize that in some tumours Epo stimulates the proliferation of tumour cells not only by delivering its mitogenic signal to these cells but also by stimulating capillary formation. In fact, Epo plays a critical role in E2-dependent cyclical angiogenesis in the uterus via EpoR expressed in vascular endothelial cells (17).
Tumour-induced upregulation of Epo mRNA sometimes results in secondary erythrocytosis (2830). In the present studies the Epo levels in the ovarian and endometrial ADC extracts ranged from 8.86 to 124.85 mU/g of tissue, while in the normal specimens, they ranged from 6.40 to 39.09 mU/g of tissue. The wide range of Epo levels in the malignant tumours may be due to estrogen differences among the patients and the pathological characteristics of the tumours. We did not measure the blood Epo levels in the patients, but none of them showed polycythemia. The serum Epo level in adults is 525 mU/ml (1). The Epo levels in the extracts of renal carcinomas of patients with and without erythrocytosis were 342.3 mU/ml (144.4 mU/g tumour) and 4.8 mU/ml (1.2 mU/g tumour), respectively, and in the normal kidney tissue of these patients, it was 7.8 mU/ml (1.0 mU/g tumour) (39). Thus Epo production by the tumours examined in the present studies appears to be sufficient to affect the microenvironment within the tumour, but not large enough to influence the Epo level in the systemic circulation.
In conclusion, Epo signal transduction appears to have a dual effect on the growth of tumours in the female reproductive organs. Epo acts directly to stimulate proliferation and/or survival of transformed cells and capillary endothelial cells. As sEpoR or Epo antibody can block both effects, the administration of these substances may be a promising way to treat female reproductive organ-derived cancers. In a separate paper, we have reported that the blockade of Epo signalling on xenografts of uterine and ovarian tumours leads to the destruction of tumours in nude mice (27). Thus, our findings provide evidence that local injections of sEpoR or R2 into malignant tumours may be effective therapy for Epo-producing tumours.
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Notes
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5 To whom correspondence should be addressed Email: y1126yas{at}med.kindai.ac.jp 
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Acknowledgments
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We thank Dr. Alice S.Cary for reading and correcting the manuscript, Hidetoshi Higashiguchi for photographic help, Mariko Chikuma and Kazuhiko Inoue for producing sEpoR, Mie Onozaki, Yoshitaka Horiuchi and Katsumi Okumoto for technical assistance, and Keiko Yamashita for preparing the manuscript. This work was partly supported by funds from Life Insurance Association of Japan to Yoshiko Yasuda and from a grant-in-aid from the Research for the Future Program in The Japan Society for the Promotion of Science to Masaya Nagao.
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Received October 8, 2001;
revised October 8, 2002;
accepted July 10, 2002.