Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu City, Japan
Received 6 November 2001; revised 18 February 2002; accepted 26 March 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angiogenesis is essential for development, growth and advancement of solid tumors. ETS-1 has been recognized as a candidate for tumor angiogenic transcription factor. This prompted us to study the clinical implications of ETS-1-related angiogenesis in uterine cervical cancers.
Patients and methods:
Fifty patients underwent curative resection for uterine cervical cancers. The patients prognoses were analyzed with a 24-month survival rate. In the tissue of 60 uterine cervical cancers, the levels of ets-1 mRNA, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF) and interleukin (IL)-8 were determined by competitive reverse transcriptionpolymerase chain reaction using recombinant RNA and enzyme immunoassay, and the localization and counts of microvessels were determined by immunohistochemistry.
Results:
There was a significant correlation between microvessel counts and ets-1 gene expression levels in uterine cervical cancers. Immunohistochemical staining revealed that the localization of ETS-1 was similar to that of vascular endothelial cells. The level of ets-1 mRNA correlated with the levels of PD-ECGF and IL-8 among angiogenic factors. Furthermore, the prognosis of the 25 patients with high ets-1 mRNA expression in uterine cervical cancers was extremely poor, while the 24-month survival rate of the other 25 patients with low ets-1 mRNA expression was 92%.
Conclusions:
ETS-1 might be a prognostic indicator as an angiogenic mediator in uterine cervical cancers.
Key words: angiogenesis, ets-1, IL-8, PD-ECGF, uterine cervical cancer
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During angiogenesis, ETS-1 is strongly expressed in vascular endothelial cells and the adjacent interstitial cells [8]. Once angiogenesis has finished, ETS-1 expression is distinctly down-regulated [9, 10]. The representative angiogenic factors VEGF and bFGF immediately induce ETS-1 expression in the early stage of angiogenesis, while the inhibition of ETS-1 expression leads to suppression of angiogenesis [11, 12]. The proteases urokinase type-plasminogen activator (u-PA), and matrix metalloprotease (MMP)-1, -3 and -9 conserve an ETS-binding motif, and transcription factor ETS-1 converts vascular endothelial cells to angiogenic phenotypes by inducing u-PA, MMP-1, MMP-3 and MMP-9 and integrin ß3 gene expression [13, 14]. This status prompted us to study whether transcription factor ETS-1 works as an angiogenic mediator, and, if so, which angiogenic factors link to ETS-1 for angiogenesis in uterine cervical cancers. The aim of the study was to formulate an efficient tumor dormancy therapy.
![]() |
Patients and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of internal standard recombinant RNA
Internal standard recombinant RNA (rcRNA) was prepared for competitive reverse transcriptionpolymerase chain reaction (RTPCR) and Southern blot analysis [16] as follows. Deoxynucleic acid construction of the internal standard was originated and synthesized by PCR from a BamHI/EcoRI-ligated fragment of V-erbB (Clontech Laboratories, Palo Alto, CA, USA) with two sets of oligonucleotide primer sequences. The sequences for the first set of primers for ets-1 mRNA (MIMIC ets-1-5' and MIMIC ets-1-3') in the first PCR reaction were as follows: MIMIC ets-1-5', 5'-ATGGAGTCAACCCAGCCTATCGCAAGTGAAATCTCCTCCG-3'; MIMIC ets-1-3', 5'-CCATGCACATGTTGTCTGGGTCTGTCAATGCAGTTTGTAG-3' [17, 18]. The sequences for the second set of primers for ets-1 mRNA (MIMIC ets-1-P and ets-1-3') in the secondary PCR reaction were as follows: MIMIC ets-1-P, 5'-TAATACGACTCACTATAGGATGGAGTCAACCCAGCCTAT-3'; ets-1-3', 5'-CCATGCACATGTTGTCTGGG-3'. The first PCR cycle was conducted in a final volume of 50 µl containing PCR buffer (50 mM KCl, 10 mM TrisHCl pH 8.3, 1.5 mM MgCl2), 0.2 mM deoxyribonucleoside triphosphates (dNTPs), 2 ng of BamHI/EcoRI-ligated DNA fragment of V-erbB, 10 pmol each of the first set of PCR primers and 2.5 U Amplitaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT, USA). The second PCR cycle was conducted in a final volume of 100 µl containing PCR buffer, 0.2 mM dNTPs, 50 pg of the first PCR products, 20 pmol each of the second set of PCR primers and 5 U Amplitaq DNA polymerase. The mixture was amplified for 28 cycles of PCR for 45 s at 94°C for denaturing, 45 s at 55°C for annealing and 90 s at 72°C for extension in a DNA Thermal Cycler (Perkin-Elmer Cetus).
The second PCR products were purified with a Gene Clean kit (Bio 101 Inc, La Jolla, CA, USA) and transcribed with 100 U T7 RNA polymerase (Gibco-BRL, Gaithersberg, MD, USA) in a final volume of 100 µl containing T3/T7 buffer (40 mM TrisHCl, pH 8.0, 8 mM MgCl2, 2 mM spermidine-(HCl)3, 25 mM NaCl), 0.1 M dithiothreitol (DTT), 10 mM ribonucleotide triphosphate, 40 U RNase inhibitor (Promega, Madison, WI, USA), 20 mM template DNA and 10 µCi of DNase (Takara Shuzo, Kyoto, Japan) at 37°C for 5 min to remove the DNA template. The products were subsequently extracted with water-saturated [-32P]UTP (New England Nuclear, Boston, MA, USA) as a tracer. The reaction was incubated at 37°C for 1 h, and then treated with 70 U of RNase-free phenol/chloroform and passed through a Sephadex G50 II column (Boehringer Mannheim, Mannheim, Germany). The amount of transcribed internal marker was calculated from the total radioactivity of the transcribed RNA.
Competitive RTPCR Southern blot analysis
Total RNA was isolated from tissues using the acid guanidium thiocyanatephenolchloroform extraction method [19].
To obtain a standard curve each time, the total RNA (3 µg) and a series of diluted recombinant RNA for ets-1 mRNA (1100 fmol) were reverse transcribed for 1 h at 37°C in a 20 µl volume with a mixture of 200 U Moloney murine leukemia virus reverse transcriptase (MMLV-RTase; Gibco-BRL) and the following reagents: 50 mM TrisHCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 40 U RNAsin (Toyobo, Osaka, Japan), 10 mM DTT, 0.5 mM dNTPs and 30 pmol 3'-end specific primer as detailed below. The reaction was incubated for 5 min at 94°C to inactivate the MMLV-RTase.
The sequences of primers to amplify the genes of ets-1 (ets-1-5' and ets-1-3') were: ets-1-5', 5'-ATGGAGTCAACCCAGCCTAT-3' (exon 5); ets-1-3', 5'-CCATGCACATGTTGTCTGGG-3' (exon 6). The sizes of PCR products for ets-1 mRNA and its internal standard rcRNA were 288 bp and 440 bp, respectively. PCR with reverse-transcribed RNAs as templates (1 µl) and 5 pmol of each specific primer was carried out using a DNA Thermal Cycler with 0.5 U Amplitaq DNA polymerase in a buffer containing 50 mM KCl, 10 mM TrisHCl, pH 8.3, 1.5 mM MgCl2 and 0.2 mM dNTPs in a 20 µl volume. Thirty cycles of amplification for ets-1 mRNA were performed at 94°C for 45 s for denaturing, 55°C for 45 s for annealing and 72°C for 90 s for extension.
Amplified PCR products (8 µl) were electrophoresed with 1.2% agarose gel in a 100 V constant voltage field. PCR products were capillary-transferred to an Immobilon transfer membrane (Millipore Corp., Bedford, MA, USA) for 16 h. The membrane was dried at 80°C for 30 min, then ultra violet (UV)-irradiated to fix PCR products tightly. These PCR products were prehybridized in buffer (1 M NaCl, 50 mM TrisHCl, pH 7.6, 1% sodium dodecyl sulfate) at 42°C for 1 h, and hybridized in the same solution with the biotinylated oligodeoxynucleotide probe (ets-1-DT, 5'-TGGTATTGAGCATGCCCAGT-3' for the ets-1 gene), synthesized from the sequences of ets-1 cDNA between the specific primers, and the corresponding biotinylated internal standard gene-specific oligonucleotide probe (MIMIC-DT, 5'-GCAGATGAGTATCTTGTCCC-3') simultaneously to check gene specificity at 65°C overnight. They were also hybridized with the biotinylated ets-1-5' probe (10 pmol/ml) to determine the signal intensity under the same conditions. Specific bands hybridized with the biotinylated probes were detected with Plex Luminescent Kits (Millipore Corp.), and X-ray film was exposed on the membrane at room temperature for 10 min. The quantification of Southern blot was carried out with Bio Image (Millipore, Ann Arbor, MI, USA).
In the competitive RTPCR Southern blot analysis for ets-1 mRNA, only two predicted sizes of DNA fragment were detected with ets-1-DT and ets-1-5' simultaneously to check specificity and determine quantity, respectively. As a negative control, no ets-1 mRNA was detected without reverse transcription in 30 cycles of PCR. The levels of ets-1 mRNA were determined using a standard curve and a serial dilution of rcRNA in competitive RTPCR Southern blot analyses, as shown in Figure 1.
|
Vessels were counted in the five highest density areas at magnification x200 (0.785 mm2 per field). Microvessel counts were expressed as the mean numbers of vessels in these areas [20]. Microvessel density was evaluated by counting microvessels.
Enzyme immunoassay for determination of bFGF, VEGF, PD-ECGF and IL-8 antigens
All steps were carried out at 4°C. Tissues of uterine cervical cancers (wet weight 1020 mg) were homogenized in HG buffer (5 mM TrisHCl, pH 7.4, 5 mM NaCl, 1 mM CaCl2, 2 mM ethyleneglycol-bis-[ß-aminoethyl ether]-N,N,N',N'- tetraacetic acid, 1 mM MgCl2, 2 mM DTT, 25 µg/ml aprotinin, and 25 µg/ml leupeptin) with a Polytron homogenizer (Kinematics, Luzern, Switzerland). This suspension was centrifuged in a microfuge at 10 000 g for 3 min to obtain the supernatant. The protein concentration of samples was measured by the method of Bradford [21] to standardize VEGF, bFGF, PD-ECGF and IL-8 antigen levels.
Basic FGF, VEGF and IL-8 antigen levels in the samples were determined by a sandwich enzyme immunoassay using a Human bFGF Quantikine kit, a Human VEGF Quantikine kit and a Human IL-8 Quantikine kit (all from R&D Systems, Minneapolis, MN, USA), respectively. PD-ECGF antigen levels were determined by the method described by Nishida et al. [22]. The levels of bFGF, VEGF, PD-ECGF and IL-8 were standardized with the corresponding cellular protein concentrations.
Statistics
Survival curves were calculated using the KaplanMeier method and analyzed by the log-rank test. The levels of ets-1 mRNA, VEGF, bFGF, PD-ECGF and IL-8 were measured from three samples taken from each tissue, and the assay for each sample was carried out in triplicate. Differences were considered significant at P <0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the current study, transcription factor ETS-1 was dominantly expressed in vascular endothelial cells and their adjacent interstitium, but not in uterine cervical cancer cells, while ets-1 mRNA levels correlated with microvessel density observed in the immunohistochemical staining for factor VIII-related antigen. Generally, distinct ETS-1 expression in vascular endothelial cells has been recognized as evidence of accelerated angiogenesis [810]. The present data reveal that ets-1 mRNA levels increased with increasing disease stage, and correlated with patient prognosis in uterine cervical cancers. Therefore, ETS-1 might be activated as a direct angiogenic mediator for the initiation and maintenance stages of angiogenesis, and may possibly be an excellent indicator of patient prognosis in uterine cervical cancers.
Furthermore, from the results of this study, it appears that ETS-1 expression might be positively regulated by the major angiogenic factors PD-ECGF and IL-8, but not by bFGF or VEGF, notwithstanding the fact that bFGF and VEGF are known to induce ETS-1 expression in vascular endothelial cell lines [11, 12]. Therefore, even if PD-ECGF and IL-8 can be suppressed by some agents, angiogenesis might be suppressed only transiently, which could lead to temporary suppression of tumor growth and secondary spreading. In such a case, other angiogenic factors would be induced and would link to ETS-1 in the search for alternate angiogenic activation, as a kind of tolerance to angiogenic inhibitors. Therefore, suppression of the major angiogenic factors along with suppression of ETS-1 recruitment might be more effective as a tumor dormancy therapy than as mere suppression of major angiogenic factors. A specific inhibitor for ETS-1, transdominant mutant ETS-1, has already been shown to act as a dominant negative molecule, and can be used as an efficient tool for angiogenic inhibition [38].
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Fujimoto J, Sakaguchi H, Hirose R et al. Expression of vascular endothelial growth factor (VEGF) and its mRNA in uterine cervical cancers. Br J Cancer 1999; 80: 827833.[ISI][Medline]
3. Fujimoto J, Ichigo S, Hori M et al. Expression of basic fibroblast growth factor and its mRNA in advanced uterine cervical cancers. Cancer Lett 1997; 111: 2126.[ISI][Medline]
4. Fujimoto J, Ichigo S, Sakaguchi H et al. The expression of platelet-derived endothelial cell growth factor in uterine cervical cancers. Br J Cancer 1999; 79: 12491254.[ISI][Medline]
5. Fujimoto J, Sakaguchi H, Hirose R et al. Clinical implication of expression of platelet-derived endothelial cell growth factor (PD-ECGF) in metastatic lesions of uterine cervical cancers. Cancer Res 1999; 59: 30413044.
6. Fujimoto J, Sakaguchi H, Aoki I, Tamaya T. The value of platelet-derived endothelial cell growth factor as a novel predictor of advancement of uterine cervical cancers. Cancer Res 2000; 60: 36623665.
7. Fujimoto J, Sakaguchi H, Aoki I, Tamaya T. Clinical implications of expression of interleukin 8 related to angiogenesis in uterine cervical cancers. Cancer Res 2000; 60: 26322635.
8. Wernert N, Raes MB, Lassalle P et al. c-ets proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularization and other forms of angiogenesis in humans. Am J Pathol 1992; 140: 119127.[Abstract]
9. Kola I, Brppkes S, Green AR et al. The Ets-1 transcription factor is widely expressed during murine embryo development and is associated with mesodermal cells involved in morphogenic process such as organ formation. Proc Natl Acad Sci USA 1993; 90: 75887592.
10. Maroulakou IG, Papas TS, Green JE. Differential expression of ets-1 and ets-2 proto-oncogenes during murine embryogenesis. Oncogene 1994; 9: 15111565.
11. Iwasaka C, Tanaka K, Abe M et al. Ets-1 regulates angiogenesis by inducing the expression of urokinase-type plasminogen activator and matrix metalloproteinase-1 and the migration of vascular endothelial cells. J Cell Physiol 1996; 169: 522531.[ISI][Medline]
12. Tanaka K, Abe M, Sato Y. Roles of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in the signal transduction of basic fibroblast growth factor in endothelial cells during angiogenesis. Jpn J Cancer Res 1999; 90: 647654.[ISI][Medline]
13. Oda N, Abe M, Sato Y. ETS-1 converts endothelial cells to the angiogenic phenotype by inducing the expression of matrix metalloproteinases and integrin ß3. J Cell Physiol 1999; 178: 121132.[ISI][Medline]
14. Sato Y, Abe M, Tanaka K et al. Signal transduction and transcriptional regulation of angiogenesis. Adv Exp Med Biol 2000; 476: 109115.[ISI][Medline]
15. FIGO News. Int J Gynecol Obstet 1989; 28: 189193.
16. Fujimoto J, Hirose R, Sakaguchi H, Tamaya T. Expression of oestrogen receptor- and -ß in ovarian endometriomata. Mol Hum Repro 1999; 5: 742747.
17. Vanden Hevvel JP, Tyson FL, Bell DA. Construction of recombinant RNA templates for use as internal standards in quantitative RTPCR. Biotech 1993; 14: 395398.[ISI]
18. Watson DK, McWilliams MJ, Lapis P et al. Mammalian ets-1 and ets-2 genes encode highly conserved proteins. Proc Natl Acad Sci USA 1988; 85: 78627866.[Abstract]
19. Chomcznski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanatephenolchloroform extraction. Anal Biochem 1987; 162: 156159.[ISI][Medline]
20. Maeda K, Chung Y, Ogawa Y et al. Thymidine phosphorylase/platelet-derived endothelial cell growth factor expression associated with hepatic metastasis in gastric carcinoma. Br J Cancer 1996; 73: 884888.[ISI][Medline]
21. Bradford MA. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 315323.[ISI][Medline]
22. Nishida M, Hino A, Mori K et al. Preparation of anti-human thymidine phosphorylase monoclonal antibodies useful for detecting the enzyme levels in tumor tissues. Biol Pharm Bull 1996; 19: 14071411.[ISI][Medline]
23. Nakayama T, Ito M, Ohtsuru A et al. Expression of the ets-1 proto-oncogene in human gastric carcinoma: correlation with tumor invasion. Am J Pathol 1996; 149: 19311939.[Abstract]
24. Ito T, Nakayama T, Ito M et al. Expression of the ets-1 proto-oncogene in human pancreatic carcinoma. Mod Pathol 1998; 11: 209215.[ISI][Medline]
25. Nakayama T, Ito M, Ohtsuru A et al. Expression of the ets-1 proto-oncogene in human thyroid tumor. Mod Pathol 1999; 12: 6168.[ISI][Medline]
26. Kitange G, Kishikawa M, Nakayama T et al. Expression of the Ets-1 proto-oncogene correlates with malignant potential in human astrocytic tumors. Mod Pathol 1999; 12: 618626.[ISI][Medline]
27. Saeki H, Kuwano H, Kawaguchi H et al. Expression of ets-1 transcription factor is correlated with penetrating tumor progression in patients with squamous cell carcinoma of the esophagus. Cancer 2000; 89: 16701676.[ISI][Medline]
28. Ito Y, Miyoshi E, Takeda T et al. Expression and possible role of ets-1 in hepatocellular carcinoma. Am J Clin Pathol 2000; 114: 719725.[ISI][Medline]
29. Ito Y, Miyoshi E, Takeda T et al. Ets-1 expression in extrahepatic bile duct carcinoma and cholangiocellular carcinoma. Oncology 2000; 58: 248252.[ISI][Medline]
30. Ozaki I, Mizuta T, Zhao G et al. Involvement of the ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma. Cancer Res 2000; 60: 65196525.
31. Watabe T, Yoshida K, Shindoh M et al. The Ets-1 and Ets-2 transcription factors activate the promoters for invasion-associated urokinase and collagenase genes in response to epidermal growth factor. Int J Cancer 1998; 77: 128137.[ISI][Medline]
32. Kitange G, Shibata S, Tokunaga Y et al. Ets-1 transcription factor-mediated urokinase-type plasminogen activator expression and invasion in glioma cells stimulated by serum and basic fibroblast growth factors. Lab Invest 1999; 79: 407416.[ISI][Medline]
33. Nakada M, Yamashita J, Okada Y et al. Ets-1 positively regulates expression of urokinase-type plasminogen activator (uPA) and invasiveness of astrocytic tumors. J Neuropathol Exp Neurol 1999; 58: 329334.[ISI][Medline]
34. Kitange G, Tsunoda K, Anda T et al. Immunohistochemical expression of Ets-1 transcription factor and the urokinase-type plasminogen activator is correlated with the malignant and invasive potential in meningiomas. Cancer 2000; 89: 22922300.[ISI][Medline]
35. Valter MM, Hugel A, Huang HJ et al. Expression of the ets-1 transcription factor in human astrocytomas is associated with fms-like tyrosine kinase-1 (Flt-1)/vascular endothelial growth factor receptor-1 synthesis and neoangiogenesis. Cancer Res 1999; 59: 56085614.
36. Pande P, Mathur M, Shukla NK et al. Ets-1: a plausible marker of invasive potential and lymph node metastasis in human oral squamous cell carcinomas. J Pathol 1999; 189: 4045. [ISI][Medline]
37. Tsutsumi S, Kuwano H, Asao T et al. Expression of ets-1 angiogenesis-related protein in gastric cancer. Cancer Lett 2000; 60: 4550.
38. Nakano T, Abe M, Tanaka K et al. Angiogenesis inhibition by transdominant mutant Ets-1. J Cell Physiol 2000; 184: 255262.[ISI][Medline]