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RESEARCH REPORT |
1 Departments of Diagnostics and Oral Medicine, Institute of Dentistry, University of Oulu, PO Box 5281, FIN-90014, Oulu, Finland;
2 Department of Clinical Chemistry, Helsinki University Central Hospital, FIN-00029, Helsinki, Finland; and
3 Biomedicum and Oral Pathology Unit, Institute of Dentistry, Laboratory Diagnostics, Helsinki University Central Hospital, University of Helsinki, FIN-00014, Helsinki, Finland;
*corresponding author, Tuula.Salo{at}oulu.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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KEY WORDS: MMP-9 • tumor-associated trypsin-2 • tumor invasion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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Trypsins, secreted in trypsinogen proforms, require activation by enterokinase, a specific trypsinogen activator (Miyata et al., 1998) or autoactivation (Kato et al., 1998). Four trypsinogen isoforms have been cloned (Emi et al., 1986; Tani et al., 1990; Wiegand et al., 1993). The main isoform in malignant tumors is tumor-associated trypsinogen-2 (TAT-2), which is expressed more in metastasizing than in non-metastasizing tumors (Koivunen et al., 1990). Trypsin-2 activates progelatinases in vitro, particularly proMMP-9 at very low concentrations and, less efficiently, proMMP-2 (Sorsa et al., 1997).
Intravasation is the key step of the carcinoma process leading to metastasis (Mignatti and Rifkin, 1993). In a method recently developed for the in vivo study of intravasation and invasion (Kim et al., 1998), tumor cell suspension was inoculated onto the chorioallantoic membrane (CAM) of a chicken egg. Only tumor cells capable of penetrating the blood vessel walls circulated and arrested in vessels of embryonic and extra-embryonic tissues, including the lower CAM. To detect and quantitate the tumor cells in the lower CAM, investigators utilized PCR amplification of human-specific Alu sequences. The intensity of the Alu-PCR band increased with increased content of human cancer cells, dose- and time-dependently.
We created model cell lines overproducing TAT-2 gene and protein to learn whether the overproduction in two human squamous cell carcinoma cell lines of tongue and one non-malignant transformed gingival keratinocyte cell line increased gelatinase (MMP-2 and -9) activation. We also analyzed the effects of TAT-2 overproduction and specific MMP-9 inhibition on the invasive capacity of the cells, using the CAM intravasation and invasion model.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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Reverse Transcriptase-PCR, Cloning, and Sequencing
Total RNA isolated from COLO 205 cells was transcribed to TAT-2 cDNA and amplified with PCR. The 760-bp TAT-2 PCR-product was purified and cloned, by means of a bidirectional TA-cloning kit (Invitrogen, Carlsbad, CA, USA), into a pCR® 3.1 vector. To verify the correct orientation of the TAT-2 gene in the vector and the gene correctness, we performed sequencing using an ABI PRISMTM 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Trypsin-2 expression in HSC-3, SCC-25, or IHGK cell lines was studied with RT-PCR. For details, see the Appendix (www.dentalresearch.org).
Generation of Transfected Cells Secreting TAT-2
The TAT-2 construct was stably transfected into HSC-3, SCC-25, and IHGK cell lines with the use of Lipofectin Reagent (see the Appendix, www.dentalresearch.org). After the G418 selection, the presence of trypsinogen-2 mRNA was estimated by RT-PCR. The amount of TAT-2 protein was measured from serum-free culture medium from TAT-2-transfected and control cells by the immunofluorometric method (Itkonen et al., 1990). Endogenous TATI was measured immunofluorometrically (Osman et al., 1993).
Zymography
Gelatinases were studied by gelatin zymography (see the Appendix, www.dentalresearch.org). The intensities of the separate bands in stained gels from four separate experiments were measured quantitatively by ScionImage software.
Western Immunoblotting
We confirmed the results of gelatin zymography by Western immunoblotting. A polyclonal anti-MMP-9 antibody and a monoclonal antibody against only active MMP-9 were used (see the Appendix, www.dentalresearch.org).
CAM Assays
The CAM (chorioallantoic membrane) assay was done according to Kim et al. (1998), except for a few modifications. Cells (HSC-3, HSC-3+TAT-2, SCC-25, SCC-25+TAT-2, IHGK, and IHGK+TAT-2) with or without enterokinase and TATI were inoculated onto a CAM of 10-day-old chick embryos. After 50 hrs of incubation, the CAMS lining the cavity of the lower eggshell were used for the extraction of genomic DNA. The radioactive PCR produced an Alu-band of 224 bp. The bands from four separate experiments were quantitated by densitometric scanning with ScionImage. Control band intensity was assumed to be 100%; other intensities were compared with that. The effects of MMP-9 inhibitor, CTT-peptide (Koivunen et al., 1999), were studied as previously; the amounts of CTT-peptide and control peptide were 0, 2, 20, and 100 µg/CAM (see the Appendix at www.dentalresearch.org).
Statistical Analysis
We performed Scheffé's test to estimate the statistical significance of differences. P values less than 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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). In SCC-25 cells, the increase was highest; control cells produced no TAT-2 protein, but in the transfected clones the mean production was 35 ng/mL (SD + 15.9). In IHGK cells, the TAT-2 production increased from less than 1 ng/mL to 22 ng/mL (SD + 1.7) after transfection. Control HSC-3 cells secreted about 2 ng/mL (+ 1.2) TAT-2, whereas TAT-2-transfected HSC-3 clones produced 7 ng/mL (SD + 1.1) TAT-2. To create model cell lines for gelatinase activation and intravasation studies, we selected clones with the highest increase in TAT-2 production.
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, lanes 1-2 and 5-6). Addition of TATI, an inhibitor of TAT-2, prevented the increased conversion of the 92-kDa proMMP-9 to the 77-kDa active form (Fig. 2A, lanes 3-4). The ratio of active to latent MMP-9 increased up to 4.4-fold (the average increase was 2.2-fold), from 0.79 (+ 0.26) in controls to 1.81 (+ 0.48) in TAT-2-transfected samples (Fig. 2B). In the presence of TATI, the ratio was reduced to 0.73 (+ 0.21) (Fig. 2B). The results were confirmed with Western blotting with anti-MMP-9 antibody (Kjeldsen et al., 1993). TAT-2 significantly increased the amount of active MMP-9 (Fig. 2C, lane 3) compared with control (Fig. 2C, lane 1), control treated with TATI (Fig. 2C, lane 2), or TAT-2-transfected media treated with TATI (Fig. 2C, lane 4). A similar phenomenon was observed with media treated with enterokinase (Fig. 2C, lanes 5-8), except that enterokinase already caused a slight MMP-9 activation in the control sample (Fig. 2C, lane 5). A monoclonal antibody specific for only the active form of MMP-9 (Duncan et al., 1998) showed, in Western immunoblot, that the amount of active MMP-9 in TAT-2-transfected HSC-3 cell media increased significantly compared with the controls (Fig. 2D). The proMMP-9 secreted by IHGK and SCC-25 cells was not affected by TAT-2 overproduction (not shown). The TAT-2 overproduction had no effect on proMMP-2 in HSC-3 (Fig. 2A, lanes 1-6), SCC-25, and IHGK cells before or after TAT-2 transfection (not shown).
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). The inoculation of the TAT-2 activator enterokinase along with the cells increased the intravasation by 24% (+ 11.0%) when compared with untreated controls (Figs. 3A [lanes 1, 2], 3C). However, the increase in the intravasation of TAT-2-transfected HSC-3 cells was higher, 43% (+ 12.4%) (Figs. 3A [lane 3], 3C), and in the presence of both enterokinase and TAT-2, 66% (+ 19.4%) higher compared with control cells (Figs. 3A [lanes 1, 4], 3C). The difference was statistically very significant (p < 0.001) (Fig. 3C). For further investigation of whether TAT-2 was the reason for the increased intravasation, activity of TAT-2 was inhibited by a specific inhibitor. When TATI was inoculated onto the CAMs with the cells, the intravasation of TAT-2-transfected cells was only 15% (+ 2.5%) higher than that of untreated control cells (Figs. 3B [lane 3], 3C). The TAT-2-transfected HCS-3 cells intravasated about 65% less efficiently in the presence of TATI than in its absence (p < 0.05) (Fig. 3C). With enterokinase, TATI reduced the intravasation of transfected cells almost to the level of untreated controls, to only 6% (+ 2.6%) (Figs. 3B [lane 4], 3C). The overproduction of TAT-2 in SCC-25 cells resulted in only a slight increase in intravasation efficiency (Fig. 3D). IHGK cells intravasated very poorly in the CAM model, and TAT-2 had almost no effect on that (Fig. 3E). To confirm that active MMP-9 is one of the key factors in intravasation, we inhibited MMP-9 by specific inhibitor, CTT-peptide (Koivunen et al., 1999), which decreased the intravasation efficiency of HSC-3 cells in a dose-dependent manner (Fig. 3F). The control peptide had no effect (Fig. 3G).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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We have previously shown that TAT-2 activates proMMP-9 in vitro at the lowest molar ratio reported so far, and it can also partially, although less efficiently, activate proMMP-2 (Sorsa et al., 1997). However, the mechanisms of activating gelatinases in vivo, when proteases form complex networks and cascades with their activators, inhibitors, and other regulators, have not been completely clarified. Therefore, we transfected the TAT-2 gene into pre-malignant and malignant oral epithelial cell lines with different metastatic capacities to create model cell lines with elevated TAT-2 protein production in vivo.
It was quite surprising to find that TAT-2 overproduction significantly activated MMP-9 only in transfected HSC-3 cells, in which the increase in TAT-2 production after transfection was lower than in the other two cell lines. Enterokinase, the trypsinogen activator, is known to interfere with the immunofluorometric assay by digesting part of the TAT-2 molecule essential for antibody recognition (Itkonen et al., 1990). Since only the HSC-3 cell line naturally produces TAT-2, it is possible that this cell line has naturally existing enterokinase-like activity, absent from the two less malignant cell lines, interfering with the immunofluorometric assay. Thus, the actual amount of TAT-2 protein in the cell media of TAT-2-transfected HSC-3 cells might be higher than measured. The fact that the addition of enterokinase increased intravasation even in control HSC-3 cells seems to be partly due to enterokinase activating the naturally existing TAT-2 in HSC-3 cells. Enterokinase was also previously shown to be capable of activating proMMP-9 in vitro to some extent (Lukkonen et al., 2000).
Tumor-associated trypsinogen also has a specific inhibitor, TATI (Halila et al., 1988). By measuring the amounts of endogenous TATI levels in all the culture supernatants, we excluded the possibility that the endogenous TATI might inhibit TAT-2. In all of the cell lines studied, the TATI levels were very low—in fact, below the detection limit (not shown). Thus, endogenous TATI levels did not interfere with the experiments. Enterokinase seemed to enhance the inhibitory effect of TATI in the CAM intravasation model. However, the difference between intravasation efficiencies of TATI-treated cells with or without enterokinase was not statistically significant (p = 0.860).
In vivo, significant amounts of progelatinases occur in complex with TIMPs (tissue inhibitor of metalloproteinase) that is likely to prevent accidental or premature activation of proMMPs. Previously, we found that TAT-2 also activated proMMP-9 complexed with TIMP-1, but the activation was clearly slower and less efficient. Trypsin-2 was further shown to degrade TIMP-1 (Sorsa et al., 1997). In addition to gelatinases, TAT-2 can eventually activate other proMMPs and proteinases and directly degrade distinct extracellular matrix and basement membrane components, such as growth factor receptors (Koivunen et al., 1991; Miyata et al., 1998). Therefore, TAT-2 can contribute to matrix degradation and remodeling, both directly and indirectly, via the activation of proteinases. Here, we proved MMP-9 to be the essential enzyme for intravasation: MMP-9 inhibition by a specific inhibitor, CTT-peptide (Koivunen et al., 1999), significantly decreased intravasation in the CAM model. Thus, the MMP-9 activation by TAT-2 seems to be relevant in oral tongue carcinoma invasion.
Different cell lines utilize various, eventually compensatory, mechanisms to activate their secreted and cell-surface-associated proteinases. SCC-25 cells that normally do not produce TAT-2 most likely use different routes to activate progelatinases, and therefore no clear effect on gelatinases was observed after TAT-2 transfection. HSC-3 cells are known to be more invasive than SCC-25 cells (Ramos et al., 1997). In fact, the lack of TAT-2 in normal untransfected SCC-25 cells can be one explanation for that phenomenon.
Recent data on the in vivo relationship between TAT-2 and gelatinases in ovarian tumor cyst fluids provide evidence that TAT-2 levels are significantly associated with proMMP-9, but not proMMP-2 activation (Paju et al., 2001). Furthermore, the reduction of TAT-2 secretion by cultured COLO-205 cells with chemically modified tetracyclines as well as the inhibition of TAT-2 by TATI decreased cell migration and significantly reduced MMP-9, but not MMP-2 activation (Lukkonen et al., 2000). These previous results are in line with our findings, where TAT-2 had no effect on the activity of proMMP-2. The activities of MMP-9 and MMP-2 are obviously regulated by different cascades.
In this study, we provided evidence that the overproduction of TAT-2 in malignant HSC-3 cells is associated with elevated MMP-9 (but not MMP-2) activation, as well as with enhanced intravasation in the in vivo CAM model. These results indicate that TAT-2 and MMP-9 activation play a role in the malignant invasive growth of oral carcinomas.
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APPENDIX |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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Reverse Transcriptase-PCR
The RT-reaction was done according to the instructions of the manufacturer, with 3 µg of total RNA and 2.8 pmol/L of TAT-2 gene-specific antisense primer. The antisense primer was 5'-ATGGGATCCTTAGCTGTTGGCAGCTATGGT-3', and the sense primer was 5'-CTGGCTAGCACCATGAATCTACTTCTGATC-3'. The TAT-2 PCR-reaction was performed with 2 U DynazymeTMEXT DNA polymerase (Finnzymes, Espoo, Finland), 150 ng of each primer, 200 µmol/L dNTP (Promega, Madison, WI, USA), and 0.5 mmol/L MgCl2 in a final volume of 50 µL of 1 x EXT buffer (Finnzymes). After initial denaturation (5 min at 95°C), 25 cycles were performed (1 min at 95°C, 1 min 15 sec at 54°C, and 3 min at 72°C), followed by the final extension of 10 min at 72°C.
Generation of Stably Transfected Cells Secreting Human TAT-2
A 1-µg quantity of control or TAT-2 plasmid and 2.5 µL of Lipofectin reagent were incubated with 30% confluent cells in 24-well dishes (Nunclon, Roskilde, Denmark) for 5 hrs in 250 µL of serum- and antibiotic-free medium. Cells were grown in normal medium for three days and then placed under G418 (LifeTechnologies, Paisley, Scotland) selection (300 µg/mL for HSC-3 and SCC-25 cells and 600 µg/mL for IHGK cells), either directly (HSC-3 and SCC-25) or after being replated 1:5 (IHGK).
Gelatin Zymography
Gelatin zymography was performed in 10% SDS-PAGE that had been cast in the presence of 1 mg/mL fluorescently (2-methoxy-2,4-diphenyl-3-[2H]furanone; Fluka, Ronkonkoma, NY, USA) labeled gelatin (O'Grady et al., 1984). Samples were prepared in non-reducing loading buffer. After electrophoresis, SDS was removed by 2.5% Triton X-100 to renature the gelatinases. Gels were then incubated in 50 mmol/L Tris-HCl buffer (pH 7.8, 150 mmol/L NaCl, 5 mmol/L CaCl2, 1 µmol/L ZnCl2) overnight at 37°C. The degradation of gelatin was visualized under long-wave UV light. Gels were also stained with 0.5% Coomassie blue R-250.
Western Immunoblot Analysis
Samples of serum-free conditioned medium with or without enterokinase treatment were concentrated at least three-fold, separated on 12% SDS-polyacrylamide gels, stained with Coomassie blue R-250 to ensure that the amount of protein was the same in all samples, de-stained (Dionisi et al., 1995), and transferred to a nylon membrane (Immobilon-P, Millipore, Bedford, MA, USA). Non-specific binding was blocked with 5% non-fat dry milk in TBS for 1 hr at room temperature. The blots were incubated with a polyclonal anti-MMP-9 antibody (dilution 1:1000; antibody described in Kjeldsen et al., 1993) or a monoclonal antibody against only active MMP-9 (dilution 1:10; kindly provided by Dr. John Fothergill; antibody described and characterized in Duncan et al., 1998) for 20 hrs at room temperature, followed by a biotinylated secondary antibody (DAKO, Glostrup, Denmark) diluted 1:500 for 1 hr at room temperature. After being washed in TBS, the blots were incubated at room temperature for 50 min in avidin biotin complex-horseradish peroxidase (DAKO) diluted 1:500. The proteins were detected by means of the enhanced ECL kit (Amersham LifeScience, Buckinghamshire, UK) and Hyperfilm ECL high-performance chemiluminescence film (Amersham). The intensities of the bands from triplicate experiments were analyzed by ScionImage software.
CAM Assay
The frozen CAMs were crushed to fine powder, suspended in digestion buffer (100 mmol/L NaCl, 10 mmol/L Tris-Cl, pH 8.0, 25 mmol/L EDTA, pH 8.0, 0.5% SDS, 0.1 mg/mL proteinase K), and incubated at 50°C for 18 hrs. The samples were extracted with phenol/chloroform/isoamylalcohol (25:24:1) and centrifuged for 10 min at 1700 x g. The DNA in the aqueous phase was precipitated with 0.5 vol of 7.5 mol/L ammonium acetate and 2 vol of ethanol, centrifuged for 2 min at 1700 x g, washed, dried, and re-suspended in sterile water.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received January 28, 2002; Last revision August 29, 2002; Accepted October 2, 2002
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONAPPENDIXREFERENCES |
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