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Original Article |
Department of Gynecology and Obstetrics (A.O., M.K., S.F.), Kyoto University Faculty of Medicine, Kawahara-cho, Shogoin, Kyoto 606-507, Japan; Department of Biological Responses (H.M., J.Y.), Institute for Virus Research, Kyoto University, Kawahara-cho, Shogoin, Kyoto 606-8507, Japan; and Department of Obstetrics and Gynecology (T.N., Y.-L.Z., K.K., I.K.), Shinshu University School of Medicine, Matsumoto 390-8621, Japan
Address all correspondence and requests for reprints to: Shingo Fujii, M.D., Department of Gynecology and Obstetrics, Kyoto University Faculty of Medicine, 54, Kawahara-cho, Shogoin, Kyoto 606-8507, Japan. E-mail: . sfu{at}kuhp.kyoto-u.ac.jp
Abstract
Uterine leiomyomas are the most common benign smooth muscle tumors in the myometrium. The expression of redox factor 1 (Ref-1), a DNA repair enzyme and redox-modifying factor, was studied in the myometrium and uterine smooth muscle tumors to investigate the relevance of Ref-1 in the growth regulation of the tumors. Two forms of Ref-1 protein were detected, using three antibodies against different epitopes of Ref-1. The abundance of the large form of Ref-1 was increased in leiomyoma extracts relative to myometrial tissue extracts, and the large form was dominant in cell lines derived from leiomyosarcomas. A single mRNA transcript was detected in the same samples, leading us to hypothesize that the differentially migrating forms are the result of posttranslational modification(s). In vitro incubation of leiomyoma tissue extract lead to a shift from the large form to the small form, and this conversion was inhibited by either protease or phosphatase inhibitors. Finally, the relative abundance of the large form of Ref-1 was found to correlate with proliferating cell nuclear antigen levels, suggesting a correlation with increased proliferation. These results indicate that altered posttranslational modification of Ref-1 is involved in uterine smooth muscle tumorigenesis.
UTERINE LEIOMYOMAS ARE the most commonly occurring benign smooth muscle tumors in the myometrium. About 1 in 800 smooth muscle tumors of the uterus is leiomyosarcoma with very poor prognosis (1). Elucidation of the mechanisms of tumorigenesis is required for better treatment of uterine smooth muscle tumors; however, it has not been clearly understood. Our previous reports showed that leiomyoma and leiomyosarcoma have higher proliferative activity than the myometrium by the analysis of Ki-67 and cell cycle progression factors such as cyclin E and cyclin-dependent kinase 2 (2, 3, 4).
Accumulating evidence has shown that redox activity modulates cellular proliferation and apoptosis (5, 6, 7). Thioredoxin (Trx), a disulfide reductase (5), is one of the key proteins regulating cellular redox conditions. As we reported previously, Trx is widely distributed in the female genital tract, including the ovarian steroidogenic cells, decidua and trophoblast cells, endometrium, and myometrium (8, 9, 10). Trx expression is induced by estrogen (9). Human recombinant thioredoxin stimulates the proliferation of normal fibroblasts and human solid tumor cancer cells (11). Thioredoxin shows thiol-mediated redox activity by regulating the activities of several transcription factors, in concert with redox factor 1 (Ref-1) (6).
Ref-1 was originally identified as a 37-kDa apurinic/apyrimidinic (AP) endonuclease (12, 13). Ref-1 is also a redox-modifying factor that stimulates DNA binding activity of a variety of transcription factors, including activator protein-1 (AP-1) (14, 15), nuclear factor-
B, Myb, and p53 (16, 17). Ref-1 reduces a conserved cysteine residue in the AP-1 DNA-binding domains and facilitates its transcriptional activity (15), and this interaction is potentiated by thioredoxin (6, 18). Thus, Ref-1 seems to be involved in cellular processes such as proliferation, differentiation, and apoptosis.
Recently, a significant increase in Ref-1 expression has been demonstrated in malignant tissues, such as epithelial ovarian cancers, cervical cancer tissues and cell lines, and in germ cell tumors (19, 20, 21). Redox activity, including Trx-Ref-1 system, is suggested to be involved in tumorigenesis of uterine smooth muscle tumors. In the myometrium and leiomyoma, Trx expression was reported not to be different (10). Therefore, to determine whether Ref-1 is involved in the regulation of proliferation of uterine smooth muscle tumors, we analyzed the expression of Ref-1 in the myometrium, leiomyoma, and leiomyosarcoma.
Materials and Methods
Materials
Tissue samples were obtained from 14 premenopausal patients (mean age, 45.4 yr; range, 38–51 yr) who were all undergoing hysterectomy for uterine leiomyomas and two patients diagnosed with leiomyosarcoma (33 and 66 yr). All patients gave informed consent for the study, which was approved by the Institutional Review Board. The leiomyomas used in this study were histologically confirmed as ordinary leiomyomas. The leiomyosarcomas used were spindle type. The phase of the menstrual cycle of the premenopausal patients was classified as proliferative (five patients), secretory (six patients), or menstrual (three patients) by endometrial dating (22). All operations were performed under general anesthesia. Leiomyoma, leiomyosarcoma, and myometrial tissue were collected immediately after removal of the uterus.
Cell culture
Tissue samples were collected in DMEM (Nissui Pharmaceutical Co., Tokyo, Japan) with 10% fetal bovine serum (FBS; Biological Industries, Grand Island, NY) and 1% antibiotic-antimycotic solution (Life Technologies, Inc., Grand Island, NY), then transported directly to the laboratory on ice. The fresh tissue was minced into fine pieces and continuously agitated in tubes containing DMEM and 0.4% collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 4 h at 37 C. The cell suspension was centrifuged at 100 x g for 10 min. The cell pellet was suspended in DMEM with 10% FBS and 1% antibiotic-antimycotic solution. A cell solution containing 4 x 104 cells/ml was placed in a 75-ml flask (Becton Dickinson and Co. Laboratories, Lincoln Park, NJ). The cells were incubated at 37 C in DMEM with 10% FBS under 5% CO2 in air. Cultures were determined to be pure smooth muscle cell cultures (>98%) by immunostaining for 
-smooth muscle actin, which is a marker for smooth muscle cells. Cells from passage two of myometrial or leiomyomal cultures were plated at a density of 5 x 105 cells per 6 cm collagen I-coated dish (Corning Laboratory Sciences Co., Corning, NY). After 12 h or 1, 2, 3, 5, 12, 19, or 24 d, the cells were rinsed with cold magnesium-free PBS (Nissui) treated with trypsin and resuspended in magnesium-free PBS. A portion of the cells was stained with 0.2% trypan blue (Nalgene Dainippon Pharmaceutical, Tokyo, Japan), and the number of viable cells was counted in a hemocytometer chamber. More than 95% of collected cells were viable. The remainder was lysed in a cell lysis buffer with protease inhibitors, and 20 µg of total cell extracted protein was analyzed by Western blotting for Ref-1, proliferating cell nuclear antigen (PCNA), and ß-actin. Proteins extracted from cell lines such as SKLMS-1, MES/SA, and SKN, which were derived from leiomyosarcoma, and HeLa cells were also analyzed in this study. SKLMS-1, MES/SA, and HeLa were purchased from American Type Culture Collection (Manassas, VA), and SKN was provided by I. Ishiwata (Ishiwata Obstetrics and Gynecology Hospital, Mito, Ibaraki-ken, Japan).
Western blot analysis
Two samples of leiomyosarcoma, 14 fresh samples of ordinary leiomyoma and associated myometrium from the same patients, were homogenized on ice using a Polytron (Kinematica, Lucerne, Switzerland) until the material turned into liquid. About 100 mg fresh tissue was lysed in 1-ml cell lysis buffer containing 50 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride (Sigma- Aldrich, Tokyo, Japan), 1 µg/ml aprotinin (Roche Molecular Biochemicals, Mannheim, Germany), 1 µg/ml leupeptin (Roche Molecular Biochemicals), and 20 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone (Roche Molecular Biochemicals). Lysates were centrifuged at 13,000 x g for 20 min at 4 C, and the supernatants were stored at -80 C. Western blot analysis was performed as described previously (6). Extracts equivalent to 15–30 µg total protein were separated by SDS-PAGE (12 or 14% acrylamide gel). The relative expression of the 37-kDa Ref-1 band is expressed as a ratio of the corresponding total value. For experiments in which we analyzed the effect of protease inhibitors, total cell extracted protein from SKLMS-1 and tissue lysates from leiomyoma or myometrium were obtained with lysis buffer that did not contain protease inhibitors. Total cell extracted protein from SKLMS-1 (15 µg) was incubated with or without tissue lysates from leiomyoma or myometrium (15 µg) for 45 min at 30 C with or without sodium orthovanadate (10 mM; Wako), or protease inhibitor cocktail (Roche Molecular Biochemicals). Then they were subjected to Western blotting. In addition, various amounts (1–15 µg) of tissue lysates from myometrium or leiomyoma were added to the constant (15 µg) amount of total cell extracted protein from SKLMS-1 and incubated without protease inhibitors or sodium orthovanadate for 45 min at 30 C. They were also subjected to Western blotting.
Antibodies
Three different anti-Ref-1 antibodies were used for Western blot analysis. A rabbit polyclonal antibody that recognizes the epitope corresponding to amino acids (aa) 299–318 (1:1000 dilution; clone 20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), a mouse monoclonal antibody that recognizes the epitope corresponding to aa 3–142 (1:250 dilution; clone 15, Transduction Laboratories, Inc., Lexington, KY), and anti-apurinic/apyrimidinic endonuclease (APE/Ref-1) mouse monoclonal antibody IgG2b, (1:2000 dilution; clone 13B8E5C2, Novus Biologicals Inc., Littleton, CO) were used in this study. Others were anti-PCNA antibody (mouse monoclonal antibody, 1:1000 dilution; DAKO Corp., Glostrup, Denmark), anti--smooth muscle actin antibody (1:100 dilution; DAKO Corp.), and anti-ß-actin antibody (mouse monoclonal antibody, 1:12,000 dilution; AC-15; Biomakor, Rehovot, Israel). Anti-ß-actin antibody was used as the internal standard.
Northern blot analysis
The coding region of Ref-1 was amplified from total RNA of a leiomyoma case by RT-PCR using Ref-1-specific primers that were chosen according to the published sequence (13, 23) (primer 1, 5'-ACACGCATGCTTAGGAAGATGGAAGGC-3'; and primer 2, 5'-ACACGTCGACCCACTCACATCTAATCC-3'). Total RNA was extracted as described previously (24), using the acid guanidinium-phenol-chloroform method. Northern blotting was performed as previously described (25).
Statistical analysis
The data are presented as box plots. The significance of difference was assessed using the Mann-Whitney U test or the Kruskal-Wallis rank test, and Scheffé’s F test was used to examine the significance of differences in expression. Differences were considered significant when P values were less than 0.05.
Results
Two forms of Ref-1 protein from the same Ref-1 mRNA
Western blot analysis revealed the expression of Ref-1 in myometrial tissue, leiomyoma, and leiomyosarcoma. Although only a 37-kDa band was detected in previous reports, two bands that correspond to molecular sizes 35 and 37 kDa were detected in all samples from the myometrial tissue, leiomyoma, and leiomyosarcoma (Fig. 1A
), irrespective of the antibodies against Ref-1 used in this study. Western blot analysis of all cell lines investigated here, showed a dominant 37-kDa band (Fig. 1, A and B). To examine whether these two forms of Ref-1 are derived from splice variants of Ref-1, the size and expression of Ref-1 mRNA were analyzed by Northern blotting. A single 1.6-kb band of Ref-1 mRNA was observed in all samples from the myometrium, leiomyoma, and the cell lines, SKN and SKLMS-1 (Fig. 1C). These data collectively suggest that the two forms of Ref-1 result from posttranslational protein modification.
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We then examined the nature of these two forms of Ref-1 by Western blotting using tissue lysates from the myometrium or leiomyoma. Because proteolysis and phosphorylation/dephosphorylation are known as important mechanisms of posttranslational modification, we first analyzed the mechanisms using protease inhibitors or phosphatase inhibitors. When tissue lysates were incubated without protease inhibitors or sodium orthovanadate for 45 min at 30 C, only a trace amount of the large form was detected and the small form appeared clearly. Addition of protease inhibitors or sodium orthovanadate in the lysates prevented this shift (Fig. 2A), suggesting that protein processing mechanisms such as phosphorylation/dephosphorylation or proteolysis are involved in the regulation of the two forms. In contrast, extracted protein from SKLMS-1 showed the predominance of the large form of Ref-1 even after incubation without protease inhibitors and sodium orthovanadate. These results led us to hypothesize that some indispensable component to process the Ref-1 protein exists in leiomyoma or myometrial extracts, but does not exist or decrease in SKLMS-1 extracts. To test the hypothesis, an extract from leiomyoma or myometrium was added to an extract from SKLMS-1 cells, and the pattern of the two forms of Ref-1 was examined by Western blotting. The small form of Ref-1 was indeed detected in SKLMS-1 extract after the addition of leiomyoma or myometrial extract (Fig. 2B, lane 6), indicating the existence of necessary components to process Ref-1 protein in leiomyoma or myometrial extracts. To analyze those components, we tested the effects of a phosphatase inhibitor and protease inhibitors on combined SKLMS-1 and leiomyoma or myometrial extracts. In the presence of protease inhibitors or sodium orthovanadate, the shift from the large form into the small form was prevented (Fig. 2B, lanes 7 and 8). By incubating a constant amount of SKLMS-1 protein with various amounts of myometrial or leiomyoma lysate, the shift from 37 kDa to 35 kDa occurred dose dependently (Fig. 2C).
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The expression pattern of the two forms of Ref-1 was different in leiomyoma and myometrial tissue (Fig. 3A). The small form was strongly expressed in the myometrial tissue, whereas the large form was strongly expressed in leiomyoma, as well as leiomyosarcoma (Fig. 1A
). The ratio of the large form band intensity to the total band intensity was significantly higher in leiomyoma than in myometrial tissue (P < 0.0001; Fig. 3B). Because the ovarian sex steroid hormones fluctuate during the menstrual cycle and cause a variety of effects on myometrial tissue, we analyzed the effect of sex steroid hormones on Ref-1 expression in myometrial and leiomyomal tissue during the menstrual cycle. The high ratio of the large form intensity to total band intensity in leiomyoma was seen regardless of the phases of the menstrual cycle (Fig. 3C; P < 0.05 and P < 0.01). Quantitative information of Ref-1 expression was normalized against ß-actin expression, which showed no changes during the menstrual cycle in either myometrial or leiomyomal tissue. In the two cases of myometrial lysate, a band migrating faster than 35-kDa form was observed (Fig. 3A).
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Next, we examined the relationship between the pattern of expression of the two forms of Ref-1 and the proliferative activity marker PCNA. PCNA expression was usually higher in leiomyoma than myometrium during the menstrual cycle and was associated with the expression of the large form of Ref-1 (Fig. 4A). To confirm the relationship between the expression of the large form of Ref-1 and the expression of PCNA, we examined the expression in a primary culture of myometrial smooth muscle cells between 12 h and 24 d after the start of the culture period (Fig. 4B). Representative pictures of the smooth muscle cells cultured for 1, 3, and 19 d are shown in Fig. 4Bc. By the second day of culture, both forms of Ref-1 were detected. However, from approximately the third day of culture, the small form of Ref-1 began to disappear; this change was associated with increased intensity of the PCNA band (Fig. 4B, a and b) and was observed by 12th day at the latest. By the 19th day of culture, when the cells were confluent, the small form of Ref-1 reappeared, and the intensity of the expression of PCNA expression decreased (Fig. 4B, b and c). Similar results were obtained in the primary culture of smooth muscle cells from leiomyoma.
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In this study, Western blot analysis for Ref-1 in protein extracted from the myometrium, leiomyoma, and leiomyosarcoma revealed two different sized forms of Ref-1, at molecular weight 37,000 and 35,000 based on electrophoretic mobility. These two forms of Ref-1 were also confirmed by immunodetection using three different antihuman Ref-1 antibodies. These results suggested that the two forms of Ref-1 are structurally very similar. Only a single band was detected with Northern blot analysis, suggesting that the two forms are not products of different splice variants. An alternative explanation is that the two forms detected by Ref-1 antibodies are posttranslational modifications of the Ref-1 protein. The Ref-1 protein has been reported to be changed by reduction/oxidation of intramolecular disulfide bonds. In in vitro studies, the additional Ref-1 band has been reported as the oxidized form of Ref-1 resulting from the oxidizing conditions (18). In our experiment, however, the patterns of expression of these two forms were not sensitive to reduction by 2-mercaptoethanol (data not shown). Moreover, treatment of tissue extracts with diamide, a sulfhydril-oxidizing agent (10–1000 µM) did not result in any change in the pattern of the two forms (data not shown). It therefore seems unlikely that these two forms result from reduction/oxidation of intramolecular disulfide bonds in Ref-1. Interestingly, the two forms are seen in the myometrium, leiomyoma, and leiomyosarcoma, whereas the large form was predominant and the small form was hardly detectable in cell lines, such as SKLMS-1, SKN, MES/SA, and HeLa. Addition of small amounts of tissue lysates from leiomyoma or myometrium to SKLMS-1 extract resulted in the appearance of the small form of Ref-1, indicating that components necessary for Ref-1 processing exist in tissue lysates from leiomyoma or myometrium. These effects were suppressed by sodium orthovanadate, which is a potent tyrosine phosphatase inhibitor, suggesting that a dephosphorylation step is required for Ref-1 processing. It also indicates that tissue lysates from leiomyoma and myometrium contain endogenous phosphatase and that the large form of Ref-1 is a phosphorylated form of Ref-1. Ref-1 has several putative sites for tyrosine phosphorylation based on amino acid sequence. The Ref-1 protein is reported to be phosphorylated by the serine/threonine kinases such as casein kinases I and II and protein kinase C (26, 27). In our study, treatment with these kinases did not show clear shifting of Ref-1 from the small form to the large form, raising the possibility that the other kinases are involved in Ref-1 modification. The Ref-1 protein may also be modified by other mechanisms such as proteolysis. Indeed, treatment with protease inhibitors also suppressed the shift from the large to the small form. In addition, in the two cases of myometrial lysate, a band migrating faster than 35-kDa form was observed, although specificity and the significance of this band are currently unclear. It might be a much processed form of 35-kDa form. We thus consider that Ref-1 protein is modified at multiple steps. The mechanisms of Ref-1 protein modification are under investigation.
The two forms of Ref-1 showed different characteristic patterns in the myometrium, leiomyoma, and leiomyosarcoma; a high ratio of the small form of Ref-1 band intensity to total band intensity for Ref-1 was consistently observed in the myometrium, whereas a high ratio of the large form to total band intensity was characteristic in leiomyoma and leiomyosarcoma. Given that the ratio of the large form of Ref-1 to total band intensity did not change during the menstrual cycle, modification of the Ref-1 protein does not seem to be affected by hormonal regulation. Protein phosphorylation/dephosphorylation has been recognized as a critical regulatory element in signal transduction, in processes such as cell proliferation, motility, and differentiation (28, 29). Differential phosphorylation of Ref-1 might be caused by disregulation of signal transduction in uterine smooth muscle tumors. In leiomyoma, activity of kinases, such as tyrosine kinase, might be augmented. Alternatively, activity of phosphatase or some specific protease might be suppressed. These possibilities should be further analyzed.
Ref-1 acts on mutagenic apurinic/apyrimidinic (baseless) sites in DNA as a critical member of the DNA base excision repair pathway. Ref-1 also stimulates the DNA binding activity of transcription factors, such as Fos/Jun (AP-1) and p53. Phosphorylation of Ref-1 by casein kinase II was reported to stimulate the DNA binding ability of AP-1 in COS cells (26). Studies are needed to examine whether the two different forms of Ref-1 in the myometrium, leiomyoma, and leiomyosarcoma function in the same way.
In the present study, we showed that the expression patterns of the two forms of Ref-1 were associated with the cellular proliferation status. During the growing phase of smooth muscle cells obtained from both leiomyoma and myometrial tissue, the intensity of the small form (thought to be the dephosphorylated, processed Ref-1) diminished, and it increased when the cells reached confluence. The predominance of the large form in leiomyoma may be related to the higher proliferative activity of leiomyoma cells than that of myometrial cells in vivo. Leiomyosarcomas, which possess higher proliferative activities than leiomyomas (3, 4), showed much clearer predominance of the large band of Ref-1 compared with those of leiomyomas. Studies using cell lines, such as SKLMS-1, SKN, MES/SA, and HeLa, which possess much higher proliferative activities, showed much clearer predominance of the large form, further suggesting the relationship between the predominance of the large form and higher proliferative activities. It can be speculated that Ref-1 inactivation via posttranslational modification, hence the change into the small form, is repressed in proliferating cells. The higher expression of the large form of Ref-1, as observed in almost all leiomyoma and leiomyosarcoma samples, may contribute to the selective growth advantage of these tissues. In addition, in cell lines in which quite strong predominance of the large form of Ref-1 exists, the dephosphorylation or processing system for Ref-1 might be suppressed, resulting in continuous activation of Ref-1. Collectively, we here propose that altered posttranslational modification of Ref-1 protein is involved in the pathogenesis of leiomyoma and leiomyosarcoma.
Acknowledgments
We gratefully acknowledge Dr. Toshihiko Toki, Dr. Akiko Horiuchi, Dr. Akira Nishiyama, Dr. Masaya Ueno, Dr. Yong-Won Kwon, and Dr. Ken Fukuhara for advice and support and Yoshimi Yamaguchi for technical assistance.
Footnotes
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by a grant-in-aid for research for the future from the Japan Society for the Promotion of Science.
Abbreviations: aa, Amino acid(s); AP-1, activator protein 1; FBS, fetal bovine serum; PCNA, proliferating cell nuclear antigen; Ref-1, redox factor 1; Trx, thioredoxin.
Received December 13, 2001.
Accepted April 18, 2002.
References
B in vitro. Gene 145:197–203[CrossRef][Medline]
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