Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu City, Japan
Received 6 August 2002; revised 3 January 2003; accepted 27 February 2003
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Abstract |
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Gas6, the protein product of the growth arrest-specific gene 6 (gas6), a member of the vitamin K-dependent protein family, was identified as a ligand for the Axl/Sky family of receptor tyrosine kinases. Gas6 acts as a growth-potentiating factor for thrombin-induced proliferation of vascular smooth muscle cells. The aim of the present study was to test for the presence of Gas6 and its receptors Axl and Sky, related to specific growth in uterine endometrial cancers, and to evaluate their plausible growth potential and mechanism.
Materials and methods:
Sixty patients underwent curative resection for uterine endometrial cancers. In uterine endometrial cancers, the mRNA levels and histoscores of Gas6, Axl and Sky were determined by competitive RTPCR using recombinant RNA and immunohistochemical studies, respectively. The rate of proliferation by immunochemistry for Ki67 and the rate of apoptosis by TUNEL were determined.
Results:
The mRNA levels and the histoscores of Gas6 and Axl in well-differentiated endometrial cancers (G1 EC) were significantly higher than in normal uterine endometrium (NE) and in moderately and poorly differentiated endometrial cancers (G2 + G3 EC). The rate of apoptosis in G1 EC was significantly lower than that in NE and in G2 + G3 EC.
Conclusions:
Gas6 and Axl signal transduction is aberrantly stimulated in well-differentiated endometrial cancers, plausibly related to tumor progression due to protection from apoptosis in cancers cells.
Key words: endometrial cancers, growth arrest-specific gene 6, receptor tyrosine kinase Axl
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Introduction |
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Gas6 was identified as a ligand for the Axl/Sky family of receptor tyrosine kinases including Axl (also called Ufo, Ark and Tyro7), Sky (Rse, Brt, Tif, Dtk, Etk-2 and Tyro3) and Mer (c-Eyk, Nyk and Tyro12) [35]. Gas6 binds these receptors with binding affinities in the order of Axl > Sky > Mer [6], and thereafter induces receptor phosphorylation. These receptors are typified by the cell adhesion molecule-related extracellular ligand-binding domain, composed of two immunoglobulin-like motifs and two fibronectin type III motifs.
Gas6 acts as a growth-potentiating factor for thrombin-induced proliferation of vascular smooth muscle cells [7, 8]. Gas6 is also able to induce cell cycle re-entry and protect serum-starved NIH 3T3 cells from cell death by apoptosis [9, 10]. Recently, Gas6 has been shown to induce ß-catenin stabilization and T-cell factor transcriptional activation in mammary cells [11]. Moreover, in the mouse, Axl, Sky and Mer are all normally expressed in Sertoli cells during postnatal development, whereas Gas6 is produced by Leydig cells before sexual maturity, and by both Leydig and Sertoli cells thereafter, suggesting a relationship with the reproductive system [12]. The Axl ligand Gas6 is able to protect E1A transfectants that re-express Axl from serum deprivation-induced apoptosis [13]. In addition, AxlGas6 signaling also activates the anti-apoptotic kinase Akt [14]. The activated Axl kinases in EGF receptorAxl kinase results in signaling events and biological responses dramatically different from those of full-length Axl. While the biological role of Gas6Axl interactions remains to be determined, studies underscore the complexity of ligandreceptor interaction [15]. Acidification prevents endothelial cell apoptosis by Axl activation [16].
This prompted us to investigate the coexpression of gas6 and its receptors Axl and Sky in human uterine endometrial cancer in order to help in the understanding of the mechanism of tumor progression.
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Materials and methods |
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Preparation of internal standard recombinant RNA
Following the procedures described in one of our previous studies [18], the synthesis of internal standard recombinant RNA (rcRNA) was performed. DNA construction of the internal standard was synthesized by PCR from BamHEcoRI fragment of v-erbB (Clontech Laboratories, Palo Alto, CA, USA) with two sets of oligonucleotide primers containing gas6-, Axl- or Sky-specific primer sequences and T7 promoter [1, 1921].
Competitive RTPCR
Total RNA was isolated from the cells by the acid guanidinium thiocyanatephenolchloroform extraction method [22]. Its concentration was determined by UV absorption at 260 and 280 nm. To obtain a standard curve each time, the total RNA (3 µg) and a series of diluted rcRNA (1103 fmol) were reverse-transcribed with 200 IU Moloney murine leukemia virus reverse transcriptase (MMLV-RT) (Gibco BRL, Gaithersburg, MD, USA) in 50 mM TrisHCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 40 IU RNAsin (Toyobo, Osaka, Japan), 10 mM dithiothreitol (DTT), 0.5 mM deoxyribonucleoside triphosphates (dNTPs), and 30 pmol 3'-end specific primers (gas6-3', Axl-3' and Sky-3') at 37°C for 1 h. The reaction was incubated for 5 min at 95°C to inactivate MMLV-RT.
The sequences of primers used to amplify the gas6, Axl and Sky genes were as follows: 5'-TGCTGTCATGAAAATCGCGG-3' (gas6-5'; 13281347), 5'-CATGTAGTCCAGGCTGTAGA-3' (gas6-3'; 15941613), 5'-GGTGGCTGTGAAGACGATGA-3' (Axl-5'; 18201839), 5'-CTCAGATACTCCATGCCACT-3' (Axl-3'; 21032122), 5'-CACTGAGCTGGCTGACTAAGCCCC-3' (Sky-5'; 27192742) and 5'-AATGCATGCACTTAAGCAGCAGGG-3' (Sky-3'; 30393062) synthesized by Rikaken Co. Ltd (Nagoya, Japan). PCR with reverse-transcribed RNAs as templates (1 µl) and 5 pmol of each specific primer was carried out using a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT, USA) with 0.5 IU Amplitaq DNA polymerase (Perkin-Elmer Cetus) in a buffer containing 50 mM KCl, 10 mM TrisHCl, pH 8.3, 1.5 mM MgCl2 and 0.2 mM dNTPs. Amplification was performed for 38 cycles at 94°C for 45 s for denaturing, 55°C for 45 s for annealing, and 72°C for 90 s for extension. To assess the quality of the RNA samples, all samples were confirmed by RTPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression (GAPDH-5'; 7196, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and GAPDH-3'; 10301053, 5'-CATGTGGGCCATGAGGTCCACCAC-3').
Southern blot analysis
Amplified PCR products were applied to 1.2% agarose gel for electrophoresis performed at 100 V, and capillary transferred to a nylon membrane (Immobilon-S; Millipore, Burlington, MA, USA) for 20 h using 10 x standard sodium citrate solution (SSC; 1.5 M NaCl, 0.15 M sodium citrate, pH 7.0). After blotting, the membrane was dried at 75°C and then cross-linked by ultraviolet irradiation (33 000 µJ/cm2 at 254 nm). The membrane was prehybridized in hybridization buffer [1 M NaCl, 50 mM TrisHCl, pH 7.6, 1% sodium dodecyl sulfate (SDS)] at 42°C for 2 h, and then in the same solution with the biotinylated gas6, Axl or Sky gene-specific oligonucleotide probe (gas6 probe: 5'-TCGTCTGGATGGCTGCATG-3', Axl probe: 5'-TGTCTGTTTCCAGGGTTCTG-3', Sky probe: 5'-TTGATGGAAGTGGGCCAGTC-3') and biotinylated internal standard gene-specific oligonucleotide probe (5'-TGTTATACAGGGAGTGAAA-3') simultaneously to detect specific genes, or hybridized with biotinylated gas6-, Axl- or Sky-5' (10 pmol/µl; Rikaken Co. Ltd) to detect their precise intensities at 42°C for 24 h. The membrane was washed with 2 x SSC for 15 min at room temperature, then with 2 x SSC for 15 min at 42°C, and finally with 0.5 x SSC for 15 min at 42°C. The detection reaction for hybridized biotin was performed using a Plex Chemiluminescent Kit (New England BioLabs, Beverly, MA, USA). Kodak XAR-5 film (Eastman Kodak, Rochester, NY, USA) was exposed to the membrane for 15 min. The strength of the signal recorded on the film was analyzed densitometrically, using Bio Image (Millipore).
Immunohistochemical staining
Immunohistochemical staining was conducted using formalin-fixed paraffin-embedded sections of tissues by the avidinbiotinperoxidase complex (ABC) method [23]. Four-micrometer sections of formalin-fixed paraffin-embedded tissues were cut with a microtome and dried overnight at 37°C on a silanized slide (Dako, Glostrup, Denmark). Samples were deparaffinized in xylene at room temperature for 80 min and washed with a graded ethanol/water mixture and then with distilled water. The samples were soaked in citrate buffer and then microwaved at 100°C for 10 min [24]. The following steps were used. Before addition of the primary antibodies, endogenous peroxidase activity was blocked by incubation in methanol containing 1% H2O2 for 20 min, followed by 60 min incubation with normal donkey serum to reduce background staining. The primary antibodies, goat anti-human Gas6, Axl and Sky antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), were incubated at 4°C for 8 h, followed by incubation with the biotinylated secondary antibody (donkey anti-goat, IgG; Santa Cruz Biotechnology) for 30 min and ABC complex for 30 min. The primary and secondary antibodies were used at 1:80 and 1:100 dilutions, respectively. The peroxidase-binding sites were demonstrated by the 3,3'-diaminobenzidine tetrahydrochloride (DAB) method. A phosphate-buffered solution instead of the primary antibody was used in the protocols for negative controls. The results of immunohistochemical staining for Gas6, Axl and Sky were semiquantitatively evaluated as described by McCarty et al. [25]. Each stained section was given a histochemical score (histoscore) calculated by the formula: (i + 1) x Pi, in which i = nuclear staining intensity (range 14; 0 indicates no staining) and Pi = percentage of stained nuclei of cells [25].
Western blot analysis
All steps were carried out at 4°C. Frozen tissues of uterine endometrial cancers (wet weight, 1020 mg) were homogenized in a homogenizing buffer [5 mM TrisHCl (pH 7.4), 5 mM NaCl, 1 mM CaCl2, 2 mM O,O'-bis(2-aminoethyl)ethyleneglycol-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, Lucerne, Switzerland). This suspension was incubated for 30 min on ice, and centrifuged in a microfuge at 10 000 g for 30 min to obtain the supernatant. The protein concentration of samples was measured by the method of Bradford [26]. Sample concentrations were adjusted to 10 µg/30 µl in the lysis buffer. Thirty microliters of each sample (containing 10 µg protein) were mixed with 20 µl sample buffer containing 10% glycerol, 60 mM Tris pH 6.8, 2% SDS and 0.02% bromophenyl blue, boiled for 5 min, and subjected to SDSpolyacrylamide gel electrophoresis on 4% polyacrylamide gel. The proteins were then transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore). The blots were incubated with the primary antibodies against Gas6, Axl and Sky each at concentrations of 1:500 overnight at 4°C after blocking for non-specific staining with 5% bovine serum albumin (BSA) in Tris-buffered salineTween 20. The reaction was visualized with an enhanced chemiluminescence detection system using an ECL kit (Amersham, Amersham, Bucks, UK).
To block the specific bands (antigens Gas6, Axl and Sky), the primary antibodies (goat anti-human Gas6, Axl and Sky antibodies, respectively) were incubated with five-fold blocking peptide [sc-1936 P, sc-1096 P and sc-1095 P, respectively (Santa Cruz Biotechnology)] in phosphate-buffered saline (PBS) at 4°C overnight. The mixture of the primary antibody and the blocking peptide instead of the primary antibodies was used in the protocols for blocking.
Determination of apoptotic index and rate of proliferation
Apoptosis studies were performed using Apoptag, an apoptosis detection kit (S7100-kit; Oncor, Gaithersburg, MD, USA) that detects by a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) procedure. Briefly, 4 µm thick paraffin-embedded tissue sections were mounted on silanized-slides (Dako). Tissue sections were deparaffinized with xylene and rehydrated in a series of graded ethanol similar to tissues stained for immunohistochemistry. Protein digestion was done with 20 mg/ml proteinase K (Sigma Chemical Co., St Louis, MO, USA) for 15 min at room temperature. Tissues sections were rinsed in distilled water for 2 min followed by quenching of endogenous peroxidase with 3% H2O2 in PBS for 30 min at room temperature. Samples were rinsed in PBS, excess liquid was removed by blotting, and a 1 x equilibrium buffer (kit part no. S7100-1) was applied for 1015 s at room temperature. Residues of digoxigeninnucleotide (digoxigenin-11-dUTP and dATP) were added to DNA using TdT enzyme (kit part no. S7100-3) in a reaction buffer (kit part no. S7100-2) and by incubating the samples in a humidified chamber at 37°C for 1 h. The tissue sections were then placed in a pre-warmed stop/wash buffer (kit part no. S7100-4) for 30 min at 37°C. Next, samples were incubated with anti-digoxigenin (kit part no. S7100-5) conjugated to peroxidase and visualized using DAB, followed by counterstaining with Mayers hematoxylin and dehydration with a series of graded ethanol.
Using light microscopy, Apoptag-labeled sections of uterine endometria and endometrioid adenocarcinomas of the uterine endometrium were evaluated for the presence of DAB-stained (brown) fragmented nuclear material. Their hematoxylineosin-stained sections were also evaluated for the presence of apoptotic nuclei morphological criteria such as intensely basophilic nuclei (compaction of chromatin), cell membrane blebbing, karyorrhexis, cell shrinkage with formation of apoptotic bodies, absence of a significant inflammatory response, and phagocytosis of dying cells by adjacent normal cells and macrophages, used to diagnose apoptosis [27]. The cells were examined by light microscopy and divided into four to 10 regions. A minimum of 100 cells were counted in each region and the apoptotic index was determined as the mean percentage of positively stained cells in a section [28].
The rate of cell proliferation was evaluated using the mouse anti-human Ki67 monoclonal antibody (Dako) directed against a nuclear antigen present in cells that are in G1, S, G2 or M phases of the cell cycle. Quiescent G0 cells are not recognized [29]. The Ki67 monoclonal primary antibody was applied to tissue at a 1:50 dilution in 1% BSAPBS and incubated at 4°C overnight, followed by incubation with the biotinylated secondary antibody (1:100) (donkey anti-mouse IgG; Santa Cruz Biotechnology) for 30 min and ABC complex for 30 min. The sections were examined by light microscopy at x400. Each section was divided into four to 10 regions; a minimum of 100 cells were counted in every region excluding degenerated and necrotic areas. The results were expressed as the mean percentage of positively stained cells in a section [30].
Statistics
We performed total RNA isolation and competitive RTPCRSouthern blot analysis in triplicate in three different parts of each individual sample. Statistical analysis was performed with Students t-test for two comparisons. Correlation coefficients were determined by Pearsons correlation coefficient. Differences were considered statistically significant at P <0.05. Data are expressed as means ± SD.
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Results |
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Discussion |
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The dissociation constants of the binding of Gas6 to Axl, Sky and Mer are 0.4, 2.7 and 29 nM, respectively [6]. Mer might have an original ligand other than Gas6. In the present study, the percentage of Sky mRNA levels to Axl mRNA levels were 22% in NE, 7% in G1 EC and 17% in G2 + G3 EC. Taking into account the dissociation constants, the effect of Gas6 on NE and EC appears to be exerted mainly via Axl.
Since Axl/Sky family receptors have cell-transforming activity through overexpression, they are thought to be involved in tumor progression and in normal cell proliferation, but little is known of the physiological functions of these receptors. Axl and Sky are not expressed in primary colon cancer, but are expressed in normal colon mucosa and hepatic metastasis [39]. Axl is not expressed in normal thyroid tissues, but is weakly expressed in thyroid tumors, and its expression is detected in 90% of thyroid carcinomas [42]. Axl expression occurred in
60% of non-small-cell lung cancer cell lines, which grow adherently, and in normal bronchial epithelial cells, but not in cell lines of small-cell lung cancer origin. Axl expression appears to be a consequence of cellular adhesion and possibly influences differentiation in lung cancers [44]. Although initially isolated as candidate growth factor receptors, members of the Axl family are not mitogenic unless they are overexpressed at high levels in tumor cell lines [45]. In addition, Axl was initially derived in studies searching for novel growth factor receptors, and Axl has been found to be overexpressed in some leukemia cell lines [46]. Initial studies focused on the role of the kinase as a mitogenic stimulus, but recent studies have suggested Axl is only a weak mitogen unless overexpressed [47]. Axl gene expression is observed in cutaneous malignant melanoma. The biological significance of the coexpression of Gas6 and receptor tyrosine kinases in normal uterine and uterine endometrial cancers still remains to be clarified, but it might be related to the proliferation of uterine endometrium and the development and progression of endometrial cancers. The previous studies comparing eutopic and ectopic endometria showed different mechanisms of proliferation control [4851]. In the current study, the levels of Gas6 and Axl mRNAs in G1 EC were significantly higher than those in G2 + G3 EC and in NE. The proliferative activity in G2 + G3 EC was significantly higher than that in G1 EC, which might be related to the higher potential for advancement of poorly differentiated endometrial cancers. On the other hand, G1 EC was more protected from apoptosis than G2 + G3 EC, plausibly due to overexpression of Gas6 and Axl in G1 EC. This evidence is plausibly related to aberrantly stimulated growth of G1 EC as a unique differentiation pathway in Gas/Axl function.
In conclusion, the present study is the first to demonstrate the coexpression of Gas6 and receptor tyrosine kinases Axl and Sky, and the high expression of Gas6 and Axl, in G1 EC. The signal transduction of Gas6 and Axl, as a unique differentiation pathway, might be involved in the development and growth of G1 EC due to protection from apoptosis in cancers cells.
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Acknowledgements |
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Footnotes |
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