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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 7 3452-3459
Copyright © 2002 by The Endocrine Society

Other Original Articles

Survivin Gene Expression in Endometriosis

Masatsugu Ueda, Yoshiki Yamashita, Mikio Takehara, Yoshito Terai, Koji Kumagai, Ken Ueki, Koji Kanda, Hiroyuki Yamaguchi, Daisuke Akise, Yao-Ching Hung and Minoru Ueki

Department of Obstetrics and Gynecology (Ma.U., Y.Y., M.T., Y.T., K.Ku., K.U., K.Ka., H.Y., D.A., Mi.U.), Osaka Medical College, Osaka 569-8686, Japan; and Department of Obstetrics and Gynecology (Y.-C.H.), China Medical College, Taiwan, Republic of China

Address all correspondence and requests for reprints to: Masatsugu Ueda, M.D., Department of Obstetrics and Gynecology, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-8686, Japan. E-mail: . gyn017{at}poh.osaka-med.ac.jp

Abstract

Survivin is a novel inhibitor of apoptosis and is expressed during fetal development and in cancer tissues, but its expression has not been reported in normal adult tissues or benign diseases. We investigated survivin gene and protein expression in a tumor-like benign disease, endometriosis, and correlated them with apoptosis and invasive phenotype of endometriotic tissues. Gene expression levels of survivin, matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1 (MT1)-MMP in 63 pigmented or nonpigmented endometriotic tissues surgically obtained from 35 women with endometriosis were compared with those in normal eutopic endometrium obtained from 12 women without endometriosis. Survivin, MMP-2, MMP-9, and MT1-MMP mRNA expression levels in clinically aggressive pigmented lesions were significantly higher than those in normal eutopic endometrium, and survivin gene expression in pigmented lesions was also higher than that in nonpigmented lesions (P < 0.05). There was a close correlation between survivin and MMP-2, MMP-9, or MT1-MMP gene expression levels in 63 endometriotic tissues examined (P < 0.01). Apoptotic cells detected by the dUTP nick-end labeling were rare in 11 ovarian endometriotic tissues, which showed positive immunohistochemical expression for survivin and MMPs. Our findings suggest that up-regulation of survivin and MMPs may cooperatively contribute to survival and invasion of endometriosis.

ENDOMETRIOSIS, THE PRESENCE of endometrium outside of the uterine cavity, is a common disease, causing abdominal pain, dysmenorrhea, dyspareunia, and infertility in 10–15% of menstruating women (1). Its etiology is unclear but it is thought to be due to the implantation and maintenance of disseminated uterine endometrium, predominantly on the ovary and pelvic peritoneum (2). Clinical observations have led to the assessment that endometriosis is an invasive disease (3, 4, 5). In endometriotic lesions, although derived from normal endometrium, decreased expression of adhesion molecules and increased expression of proteolytic enzymes may contribute to establishment of endometrial glands and stroma at ectopic sites, likely as a behavior of cancer cells (5, 6, 7, 8). Normal epithelial cells undergo apoptosis when they separate from their primary tissue. However, spontaneous apoptosis of ectopic endometrial tissue is impaired in women with endometriosis, and its decreased susceptibility to apoptosis might participate in the growth, survival, and invasion of endometriotic tissue (9, 10). Although there have been some reports on the induction of apoptosis in endometriotic lesions (11, 12), there is no consensus on the mechanism of escape from apoptosis in endometriosis, and little is known on the correlation between survival activity and invasive phenotype in endometriotic cells.

Among the regulators of cell death, inhibitor of apoptosis (IAP) proteins have recently emerged as modulators of an evolutionarily conserved step in apoptosis, which may potentially involve the direct inhibition of terminal effector caspases 3 and 7 (13). Recently, a novel and structurally unique member of the IAP gene family, designated survivin, was identified (14). Unlike other IAP proteins, survivin was found during embryonic and fetal development, was completely down-regulated and undetectable in normal adult tissues, and became prominently reexpressed in all of the most common human cancers (14). However, there has been no report on the biological significance of survivin in endometriosis, an aggressive tumor-like benign disease. In the present study, we investigated survivin gene and protein expression in surgical specimens from patients with endometriosis and correlated them with apoptosis and invasive phenotype of endometriotic tissues.

Subjects and Methods

Patients and tissue samples

The subjects in this study were women of reproductive age undergoing laparoscopy or laparotomy for suspected endometriosis. At the time of surgery, pelvic organs were examined carefully for the presence and extent of endometriosis. Stages of the disease and macroscopic findings of endometriotic lesions were classified according to the revised American Fertility Society classification (15). Patients who received sex steroid derivatives or GnRH analogs within 1 yr before the operation were excluded in the current study. This study was approved by our institutional review board, and appropriate informed consent was obtained from all women.

Endometriotic tissues for mRNA analysis were removed with biopsy forceps under laparoscopy or laparotomy from 35 women with endometriosis. Of these patients, 2 had stage I, 1 had stage II, 8 had stage III, and 24 had stage IV disease. Control eutopic endometrial tissues were collected by endometrial curettage from 12 women without any findings of endometriosis, which was confirmed with laparoscopy. Cycle phase for each subject was assigned based on histological evaluation of eutopic endometrium or basal body temperature. All tissue specimens were immediately frozen in liquid nitrogen and then stored in -80 C until use.

Tissue samples for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) and immunohistochemistry were obtained from 11 women who underwent ovarian cystectomy or oophorectomy under the diagnosis of chocolate cyst of the ovary. Resected ovarian tissues were fixed in 10% formalin and embedded in paraffin wax. Serial sections of 5 µm each were placed on silanized slides (DAKO Corp., Kyoto, Japan) and maintained at room temperature. Eutopic endometrial tissues collected from seven women without endometriosis as described above were also used for TUNEL and immunohistochemical study.

RNA isolation and cDNA preparation

RNA was extracted from homogenized tissue samples by phenol/chloroform extraction according to the RNA STAT-60 protocol (Tel-Test, Friendswood, TX) according to the supplier’s recommendation. Contaminating residual genomic DNA was removed by digestion with RNase free DNase (Promega Corp., Madison, WI). cDNAs were prepared using at least 2 µg total RNA and SUPERSCRIPT II reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) with random hexamers as primers and were finally dissolved in diethyl pyrocarbonate-treated water and then frozen at -20 C until use.

RT-PCR analysis

Oligonucleotide primers for RT-PCR were designed using a published sequence of survivin (16), matrix metalloproteinase (MMP)-2 (17), MMP-9 (18), membrane type 1 (MT1)-MMP (19), and ß-actin (20) genes and synthesized by the solid-phase triester method. The primers used in this study and the expected sizes from the reported cDNA sequence are shown in Table 1Go.


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  		Table 1. PCR primers and conditions

 
Amplification of cDNA was performed according to the method described previously (20, 21). For PCR, cDNA aliquots were diluted in sterile water, depending on transcript abundance. Three cDNA concentrations for each target gene/ß-actin primer combinations were used. For accurate quantification using this method, measurements must be taken in the linear phase of the reaction, in which cDNA concentration is directly proportional to signal intensity. A range of cDNA concentrations was used to determine the linear phase of the PCR. cDNA template was amplified by PCR in a final volume of 20 µl reaction containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.01% (w/v) gelatin, 200 µM dNTP, 0.5 µM each primer, and 1.25 U Taq polymerase (Perkin-Elmer Corp., Norwalk, CT). After an initial denaturation at 96 C for 3 min, various cycles of denaturation (94 C for 1 min), annealing (57 C for survivin, 58 C for MMP-2, 62 C for MT1-MMP, and 60 C for MMP-9 and ß-actin for 1 min), and extension (72 C for 2 min) for the respective target genes were carried out on a Perkin-Elmer GeneAmp PCR System 9700. The final extension was performed at 72 C for 10 min. The number of cycles in the RT-PCR was determined so as to obtain logarithmic amplification of each gene for semiquantitative analysis of the expression levels of the genes. After visualization of the RT-PCR products by 1.5% agarose gel electrophoresis with ethidium bromide staining, gel images were obtained using the ATTO densitograph UV-image analyzer (ATTO Corp., Tokyo, Japan), and the densities of the products were quantified using the ATTO densitometry software, version 2. The relative expression levels were calculated as the density of the product of each target gene divided by that of ß-actin from the same cDNA. Each analysis was performed in triplicate.

DNA nick-end labeling

DNA breaks were detected in situ by TUNEL according to the method of Gavrieli et al. (22), with some modifications as described previously (23, 24). Paraffin sections were de-waxed, rehydrated through a graded alcohol series, and washed with PBS. Subsequently, the tissues were digested with 20 µg/ml proteinase K (Sigma, St. Louis, MO) for 15 min at room temperature and then washed with distilled water and subsequently with PBS. The tissues were incubated with a solution containing 2% H2O2 in PBS to inhibit endogenous peroxidase activity and then washed with PBS. TdT buffer solution [100 mM potassium cacodylate, 2 mM cobalt chloride, and 0.2 mM dithiothreitol (pH 7.2)] containing 0.3 U/µl TdT (Oncor, Gaithersburg, MD) and 0.04 nmol/µl digoxigenin-dUTP (Oncor) were added to cover the tissues, which were then incubated in a humidified atmosphere for 60 min at 37 C. The tissues were washed with buffer solution containing 300 mM sodium chloride and 30 mM sodium citrate for 30 min at 37 C to terminate the reaction and then washed with PBS. They were subsequently incubated with anti-digoxigenin-peroxidase complex for 30 min at room temperature and stained with a solution of 0.05% 3,3'-diaminobenzidine and 0.01% H2O2 in Tris-HCl buffer (DAB solution) at pH 7.6 for 3–6 min at room temperature. The sections were counterstained with hematoxylin. Negative controls were obtained by omitting TdT from the buffer solution.

Immunohistochemistry

For immunohistochemical staining, rabbit antihuman survivin polyclonal antibody (SURV11-A; Alfa Diagnostic International, San Antonio, TX) and mouse antihuman MMP-2, MMP-9, and MT1-MMP monoclonal antibodies (Fuji Photo Film Co., Ltd., Toyama, Japan) were used for avidin-biotin-peroxidase complex procedure. After deparaffinization, sections were treated with a solution containing 2% H2O2 in PBS to inhibit endogeneous peroxidase activity. For survivin staining, the slides were then immersed in a 10-mM citrate buffer (pH 6.0) and heated in an autoclave for 5 min at 120 C to retrieve masked antigens. The slides were incubated overnight at 4 C with primary antibodies at a 1:50 dilution and washed with PBS. Biotinylated goat antirabbit or horse antimouse immunogloblin (DAKO Corp.) was then added to the sections for 30 min at room temperature. Peroxidase-conjugated avidin (DAKO Corp.) was applied after the sections were washed with PBS. Peroxidase activity was detected by exposure of the sections to the DAB solution as described above. The sections were counterstained with hematoxylin. Normal rabbit or mouse IgG was used as a substitute for the primary antibody for the negative controls.

Evaluation of histochemical staining

Semiquantitative evaluation of TUNEL and immunohistochemical staining was performed according to the method reported by Harada et al. (25), with some modifications. The slides were evaluated independently by two investigators in a blind fashion. Three different fields containing 100 glandular cells per field were examined at x400 magnification. The percentage of positive epithelial cells was scored as: 0, no positive cells; 1, 0–25% positive; 2, 25–50% positive; 3, more than 50% positive. To indicate intra- and interobserver variations, the staining result was expressed as mean ±SE for each case. For semiquantitative evaluation of the expression of survivin and MMPs in stromal components, three different fields containing at least 200 stromal cells around the glands were examined and the percentage of positive stromal cells was scored as described above.

Statistical analysis

All statistical calculations were carried out using StatView statistical software (Tokyo, Japan). Gene expression levels of survivin and MMPs in endometriotic tissues were compared with those in normal eutopic endometrium, and the significance of differences was determined by the Mann-Whitney U test. The Spearman rank correlation coefficient was also used to analyze the relation between survivin and MMP gene expression levels in endometriotic tissues. A level of P less than 0.05 was accepted as statistically significant.

Results

A total of 63 endometriotic tissues for mRNA analysis was obtained from 35 women with endometriosis. They included 43 pigmented (red or black; blueberry spot, blood breb, and chocolate cyst) and 20 nonpigmented (white or yellow-brown; serous breb and surface elevation) lesions. Gene expression levels of survivin, MMP-2, MMP-9, and MT1-MMP in these endometriotic lesions were compared with those in normal eutopic endometrium obtained from 12 women without endometriosis.

Figure 1Go shows an example for mRNA expression of survivin, MMP-2, MMP-9, and MT1-MMP in normal endometrium and in endometriotic tissues. Their relative gene expression levels in comparison with ß-actin expression ranged widely among the lesions examined; however, there was no significant difference in the expression level of survivin and MMPs between stages of the disease and cyclic phases of the patients (data not shown). As can be seen in Fig. 2Go, gene expression levels of survivin, MMP-2, MMP-9, and MT1-MMP in pigmented lesions were significantly higher than those in normal eutopic endometrium (P = 0.0056, 0.0324, 0.0021, and 0.0349), respectively. Moreover, survivin gene expression in pigmented lesions was statistically higher than that in nonpigmented lesions (P = 0.0446), whereas there was no significant difference in MMP-2, MMP-9, and MT1-MMP gene expression levels between two groups. As shown in Fig. 3Go, a–c, there was a close correlation between survivin and MMP-2, MMP-9, or MT1-MMP gene expression levels in 63 endometriotic tissues examined, with coefficient correlations of 0.356, 0.663, and 0.411 (P = 0.0042, <0.0001, and 0.0008), respectively. Interestingly, relative gene expression levels of MMP-2 were also well correlated with those of MT1-MMP, with a coefficient correlation of 0.636 (P < 0.0001) (Fig. 3dGo).



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  		Figure 1. RT-PCR analysis of survivin, MMP-2, MMP-9, and MT1-MMP in normal endometrium and endometriotic tissues. Lanes 1 and 2 correspond to samples from normal eutopic endometrium, lanes 3 and 4 from nonpigmented lesions, and lanes 5 and 6 from pigmented lesions, respectively. The primers and the expected sizes from the reported cDNA sequence are shown in Table 1Go.

 


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  		Figure 2. Gene expression levels of survivin (a), MMP-2 (b), MMP-9 (c), and MT1-MMP (d) in pigmented and nonpigmented endometriotic tissues and normal eutopic endometrium. Values are expressed as relative expression levels of each target gene in comparison with ß-actin expression. Box, 25–75% distribution; bars, 10–90% distribution. N.S., Not significant.

 


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  		Figure 3. Correlation between survivin and MMP-2 (a), MMP-9 (b), or MT1-MMP (c), and between MMP-2 and MT1-MMP (d) gene expression levels in 63 endometriotic tissues examined. Each point represents the mean of triplicates.

 
Seven specimens from normal endometrium and 11 from ovarian endometriosis were examined for apoptosis and expression of survivin and MMPs. Table 2Go shows the overall results of histochemical staining outcomes. TUNEL and immunohistochemical staining for cases 5 and 9 are shown in Figs. 4Go and 5Go, respectively. The negative control slides for TUNEL, survivin, and MMPs exhibited no specific staining. The apoptotic epithelial cells positive for TUNEL were detected in 2 of 7 and only 1 of 11 cases in eutopic and ectopic endometrium, respectively. Glandular epithelial cells positive for survivin and MMPs were observed in all 11 specimens from ovarian endometriosis regardless of the menstrual cycle phase. However, survivin, MMP-2, MMP-9, and MT1-MMP expression in normal endometrial gland were relatively weak and detected in one, two, two, and three of seven specimens, respectively. On the other hand, stromal cells positive for survivin, MMP-2, MMP-9, and MT1-MMP were detected in 4, 11, 7, and 8 of 11 specimens from ovarian endometriosis, respectively, whereas MMP-2 expression in normal endometrial stroma was weak and detected in 2 of 7 specimens. Survivin, MMP-9, and MT1-MMP expression were not observed in stromal cells from normal eutopic endometrium.


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  		Table 2. Histochemical staining outcomes

 


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  		Figure 4. Histochemical staining of normal eutopic endometrial tissue (case 5 in Table 2Go). Intense TUNEL signals are observed in the small nuclei of glandular epithelial cells (a; arrows), but the immunoreactivity of survivin (b), MMP-2 (c), and MT1-MMP (d) is negative both in epithelial and stromal cells. Original magnification, x400.

 


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  		Figure 5. Histochemical staining of ectopic endometrial tissue from ovarian endometriosis (case 9 in Table 2Go). Glandular epithelial cells show negative staining for TUNEL (a), but the immunoreactivity of survivin (b), MMP-2 (c), and MT1-MMP (d) is positive both in epithelial and stromal cells. The immunoreactivity of MMP-9 is similar to that of MMP-2 (data not shown). Original magnification, x400.

 
Discussion

Survivin is a novel inhibitor of apoptosis and is expressed during fetal development and in cancer tissues, but its expression has not been reported in normal adult tissues or benign diseases (13, 14). Very recently, Konno et al. (16) demonstrated that survivin gene and protein expression was detected in normal human endometrium and that survivin could play an important role in physiological homeostasis during the normal menstrual cycle. It could be expected that survivin is also expressed in ectopic endometriotic tissues, however, there has been no investigation on the biological role of survivin in endometriosis.

MMPs, enzymes that are important for extracellular matrix turnover, have recently been implicated in invasion and development of endometriosis (8). MMPs appear to be overexpressed in endometriotic lesions and contribute to establishment of endometrial glands and stroma at ectopic sites (26, 27, 28). Although sex steroids and GnRH agonists have been shown to regulate MMP expression in a model of experimental endometriosis (27, 28, 29), limited investigation has focused on gene expression of MMPs in human peritoneal endometriotic lesions (8, 30). Moreover, it is still unclear how endometriotic cells expressing MMPs escape from apoptosis during the development of endometriosis.

In the present study, we investigated gene expression of survivin and MMPs in eutopic and ectopic endometrial tissues and found that survivin, MMP-2, MMP-9, and MT1-MMP mRNA expression levels in pigmented endometriotic lesions were significantly higher than those in normal eutopic endometrium. Moreover, survivin gene expression in pigmented lesions was higher than that in nonpigmented lesions. In patients with endometriosis, visual findings of peritoneal endometriotic foci are often recognized as typical red or black pigmented lesions and nonpigmented lesions seem to occur earlier during the development of the disease (31, 32). We previously reported that pigmented lesions, such as blueberry spot, blood breb and chocolate cyst, were clinically more active and aggressive (33). It is likely that up-regulation of survivin and MMP expression may cooperatively contribute to survival and invasion of endometriotic tissues. Interestingly, there was a close correlation between survivin and MMP-2, MMP-9, or MT1-MMP gene expression levels in 63 endometriotic tissues examined. Invasive endometriotic cells may escape from apoptosis by expressing a higher level of survival gene survivin.

To confirm this hypothesis, we then performed histochemical study for eutopic and ectopic endometrial tissues. Endometriotic lesions rarely contained apoptotic epithelial cells, and only 1 of 11 cases showed positive staining for TUNEL. Jones et al. (34) also demonstrated that apoptotic cells were rare in ovarian endometriosis, and there was no significant difference in the number of apoptotic cells between eutopic and ectopic endometrium. Interestingly, immunohistochemical expression of survivin and MMPs in glandular epithelial cells was positive for all 11 specimens from ovarian endometriosis, and survivin expression in ectopic endometrium was extremely stronger than that in eutopic endometrium. Konno et al. (16) reported that survivin protein expression in normal endometrium was strongest in the late secretary phase and was not detected in the proliferative phase. In this study, survivin was identified in one normal endometrial sample in the late secretary phase, however, it could not be detected in four samples in the early secretary phase. The cyclic change of survivin protein during the normal menstrual cycle should be further elucidated.

We also evaluated survivin and MMP expression in stromal components of eutopic and ectopic endometrium. Konno et al. (16) demonstrated that stromal cells of eutopic endometrium were completely negative for survivin. Our present results revealed that survivin was not detected in stromal components of normal eutopic endometrium, however, 4 of 11 cases with ectopic endometrium showed positive immunostaining for survivin in stromal cells around the glands. Jones et al. (34) reported that bcl-2 up-regulation in stromal cells may prevent apoptosis in ectopic endometrium. We speculate that endometrial stromal cells expressing survivin may antagonize caspase-3-mediated apoptosis and promote the development of endometriosis, but its molecular mechanism should be further elucidated. Rodgers et al. (35) reported a consistent expression of MMP-2 throughout the menstrual cycle in the stromal components of cycling endometrium. In contrast, Wenzl and Heinzl (36) were not able to detect MMP-2 expression in the stromal components of eutopic endometrium, but they observed strong MMP-2 immunoreactivity both in glandular epithelial and stromal cells of ectopic endometrium and stated that ectopic endometriotic tissue has a greater capacity to invade due to higher proteinase expression. In this study, 2 of 7 cases with normal eutopic endometrium showed positive immunostaining for MMP-2 in glandular epithelial and stromal cells, whereas all 11 cases with ectopic endometrium showed stronger MMP-2 expression not only in gland but also in stroma. Moreover, MMP-9 and MT1-MMP immunoreactivity in stromal cells was detected only in ectopic endometriotic tissue. MMP expression in stromal components of ectopic endometrium may be also related to up-regulation of MMP gene expression and invasive phenotype of endometriosis.

Endometriotic epithelial cells that expressed MMP-2 were also positive for MT1-MMP. MMP-2 has been shown to be activated by MT1-MMP, which possesses a functional transmembrane domain and acts as cell-surface receptor and activator for proMMP-2 (19, 37, 38). Relative gene expression levels of MMP-2 were well correlated with those of MT1-MMP in endometriotic lesions as described above. The up-regulated invasiveness of endometriotic cells by MMP-2 may be closely linked to its activation by MT1-MMP. These results suggest that overexpression of both survivin and MMPs in endometriotic tissues may contribute to escape from apoptosis and development of the disease.

The molecular interaction between survivin and MMPs in endometriotic tissues is still unclear. However, recent studies have demonstrated that highly metastatic cancers exhibit a higher resistance to apoptotic cell death compared with low metastatic forms (39, 40) and that survivin expression is closely associated with invasive phenotype of oral (41), esophageal (42), and ovarian (43) cancers. Yoshida et al. (43) reported that the expression of survivin enhanced invasive activity of ovarian cancer cells by up-regulation of MMP-2. Survivin expression may provide a strong advantage factor for tumor progression, both affording protection from broad apoptosis-inducing environmental stimuli and maintaining proper mitotic progression of the proliferating and invasive population (41). Additional studies are needed to clarify the molecular events that co-regulate the expression of survivin and MMP genes in the development of endometriosis.

To the best of our knowledge, this is the first report to demonstrate survivin gene expression in endometriosis and to highlight the relation between survival and invasion of endometriotic cells. These observations are potentially important in understanding the pathogenesis of an aggressive tumor-like benign disease, endometriosis.

Acknowledgments

We are grateful to Dr. N. Tanigawa (Department of Surgery, Osaka Medical College, Osaka, Japan) for his interest and advice. We also thank several colleagues for collecting clinical materials and K. Sato for technical assistance.

Footnotes

This work was supported in part by High-Tech Research Program of Osaka Medical College.

Abbreviations: IAP, Inhibitor of apoptosis; MMP, matrix metalloproteinase; MT1, membrane type 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling.

Received August 8, 2001.

Accepted April 4, 2002.

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