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Stromelysin-1 Promoter Mutations Impair Gelatinase B Activation in High Microsatellite Instability Sporadic Colorectal Tumors¹

Departamento de Bioquímica y Biología Molecular II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid [A. M., P. I., C. d. J., R. G-Q., M. B.]; Servicios de Cirugía [A. S-P., A. T., J. L. B.] and Oncología [E. D-R.], Hospital Clínico "San Carlos," 28040 Madrid; and Servicio de Anatomía Patológica, Hospital Puerta de Hierro, 28035 Madrid [S. R. y C.], Spain

	ABSTRACT

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Colorectal cancers from the mutator phenotype pathway display distinctive pathologicalfeatures and confer a lesser aggressiveness than colorectal adenocarcinomas originated by the suppressor pathway. The goal of this work was to test whether tumors developed through the mutator pathway could show a decrease in matrix metalloproteinase (MMP) activity. We evaluated levels and activity of gelatinase A (MMP-2) and gelatinase B (MMP-9), as well as stromelysin-1 (MMP-3) expression in 101 sporadic colorectal tumors in consideration of the microsatellite instability (MSI) status of the groups. Gelatinases were analyzed by ELISA and zymography. The MMP-3 study was performed by real-time quantitative PCR. MMP-9 total levels were significantly higher in MSI-H tumors. However, levels of the active MMP-9 form were significantly much lower in this group of tumors. Data from real-time quantitative PCR indicated that levels of MMP-3 for MSI-L/MSS tumors were much higher as compared with those observed in MSI-H cancers (P = 0.033). Moreover, all MSI-H tumors showed nucleotide insertions and/or deletions in MMP-3 promoter. These mutations were not observed in the group of MSI-L/MSS tumors. Our data indicate that the MMP-3 promoter constitutes a novel target of the defective mismatch repair machinery in sporadic colorectal tumors, resulting in a dramatic decrease in the levels of the active MMP-9 form, which may result in a lessened capacity for invasion.

	INTRODUCTION

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Colorectal cancer poses a major public health problem in Western countries, where there is renewed interest in understanding the basic principles of the molecular biology of colorectal cancer. The colorectal tumorigenesis process is characterized by multiple mutations in common oncogenes and tumor suppressor genes, as well as genomic instability attributable to mismatch repair gene defects (1) .

MSI³ is a characteristic molecular finding in most cases of HNPCC and nearly 13% of all cases of sporadic colorectal cancer (2, 3, 4) . MSI evolves through mutations or epigenetic alterations of the DNA mismatch repair genes and constitutes a very early event in both HNPCC and sporadic colorectal tumorigenesis (5) . Carcinomas with MSI develop an increased intrinsic mutation rate, and it has been suggested that MSI-positive and MSI-negative colorectal tumors might evolve through different mechanisms and different pathways (6 , 7) . Colorectal cancers with MSI-H are believed to display distinctive pathological features and to behave less aggressively than MSS tumors and carcinomas with MSI-L. Thus, similar to HNPCC tumors, sporadic MSI-H tumors seem to have a better prognosis, and the majority are localized to the right colon (8 , 9) . Moreover, according to recent studies, right-sided tumors and colorectal cancers showing high MSI had a better response to adjuvant chemotherapy (10) . At present, we do not understand the molecular basis for the different clinical behaviors of MSI-H and MSS or MSI-L colorectal tumors.

On the other hand, tumor metastasis is a complex multistep process during which tumor cells locally invade the surrounding tissues, penetrate blood or lymphatic vessels, and exit vessels at distant sites to form secondary tumors; proteolytic degradation of the extracellular matrix is an important part of this process (11) . In this context, MMPs, proteins that degrade the components of the extracellular matrix, are abundantly secreted by a variety of tumor and stromal cells and have been implicated in tumor progression (12) . Therefore, overexpression of MMPs may be one part of the multistep process by which the neoplastic cells can proliferate and metastasize. To date, the gelatinases or type IV collagenases (MMP-2 and MMP-9) have been described as playing a significant proteolytic role in colorectal cancer (13) . MMPs are tightly regulated at the levels of transcription, release, and activation (14) . The M_r 72,000 MMP-2 (gelatinase A) is the most widely distributed of the MMPs and is expressed constitutively by most cells including endothelial and epithelial cells (12) . MMP-2 is secreted as an inactive proform; MT1-MMP is the membrane-type MMP that is mainly implicated by its cellular activation (15) . M_r 92,000 MMP-9 (gelatinase B) is produced by inflammatory cells as well as by stimulated connective tissue cells (12) . Pro-MMP-9 conversion to an M_r 82,000 active form progresses via an interacting protease cascade involving stromelysin-1 (MMP-3; Refs. 16 , 17 ).

Taking into account that examination of MMPs in relationship to microsatellite status of sporadic colorectal tumors has not been investigated, the goal of this work consists of determining the total levels and activity of MMP-2 and MMP-9 in sporadic colorectal tumors classified according to their microsatellite status. Thus, data reported here could provide a molecular base to understand the lesser aggressiveness that has been associated with high MSI tumors that develop through the mutator phenotype pathway.

	MATERIALS AND METHODS

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Tissue Samples.
One hundred and one colorectal adenocarcinomas and their corresponding control tissue samples were obtained from patients who underwent surgery at "San Carlos" and "Puerta de Hierro" Hospitals in Madrid. Informed consent was obtained from patients before investigation. All tissue samples were snap-frozen in liquid nitrogen immediately after surgery and stored at -80°C. Cryostat-sectioned, H&E-stained samples from each tumor block were examined microscopically by two independent pathologists to confirm the presence of >80% tumor cells. Paired normal tissues from the same patient, used as controls, were also microscopically confirmed. Clinicopathological parameters of tumors as well as the follow-up of patients were provided in 88 cases.

Microsatellite Analysis.
Microsatellite analysis was performed to classify tumors in groups of high and low instability. Thus, genomic DNA from tumor and control samples was isolated as described by Blin and Stafford (18) . DNA samples were amplified using PCR and investigated for MSI using five polymorphic human repeat DNA markers that map on different chromosome loci: BAT25 in 4q12, BAT26 in 2p16.3–p21, D5S346 in 5q21-22, D17S250 in 17q11.2–q12, and D2S123 in 2p16. This microsatellite panel is recommended as a "reference" panel by the American Joint Commission on Cancer (19 , 20) . PCR products were obtained after a multiplex PCR analysis using the HNPCC Microsatellite Instability test (Roche Molecular Biochemicals, Mannheim, Germany). The generated, fluorescently labeled PCR products, corresponding to tumor and control tissues, were then investigated on an automated ABI PRISM 310 Genetic Analyzer (Applied Biosystems) to evaluate MSI status of colorectal tumors. Thus, MSI was detected by comparing the length of microsatellite alleles amplified from tumor DNA and DNA from normal tissue in the same individual. To confirm the reproducibility of the experiments, all cases showing MSI were examined at least twice by an independently performed PCR and electrophoresis. Adenocarcinomas showing MSI on two or more of the markers considered were classified as MSI-H, whereas cases with only one altered marker were classified as MSI-L, and cases without MSI were considered as stable (MSS). In all analyses for clinicopathological associations with MSI, MSI-L and MSS tumors were grouped together and indicated as MSI-L/MSS.

Detection of TGFßRII and BAX Mutations.
All samples showing MSI-H were tested to detect mutations affecting repeat sequences located in coding regions of the TGFßRII and BAX genes. Two regions of TGFßRII, the first one containing a 10-deoxyadenine repeat and the latter containing a 3-GT repeat, were amplified. Thus, we obtained a 90-bp region (nucleotides 677–766) by using the forward primer 5'-AGATGCTGCTTCTCCAAAGTGC-3' and the reverse primer 5'-TTGCACTCATCAGAGCTACAGG-3' and a 123-bp region (nucleotides 1887–2009) by using the forward primer 5'-ACTGAGTGCTGGGACCACG-3' and the reverse primer 5'-AGGAATCTTCTCCTCCGAGC-3'. Moreover, a 94-bp region encompassing the (G)8 tract in BAX was amplified with primers reported by Rampino et al. (21) .

PCR amplifications were performed in a thermocycler (Gene Amp PCR System 2400; Perkin-Elmer, Norwalk) and were carried out in a 20-µl volume containing 200 ng of genomic DNA, 0.6 µM of each primer, 0.2 mM of each deoxynucleoside triphosphate, 10 mM Tris Cl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, and 2 units of Taq Gold DNA polymerase (Perkin-Elmer). PCR conditions were as follows: enzyme activation and initial denaturation at 95°C for 12 min; 40 cycles at 95°C for 20 s, at 58°C for 20 s, and at 72°C for 30 s. The final extension was at 72°C for 7 min. PCR products were sequenced on an automated DNA Sequencer (PRISM 310; Applied Biosystems), using the sense primers described above. All samples were examined at least twice by an independently performed PCR and electrophoresis.

MMP-2 and MMP-9 Concentrations by ELISA.
MMP-2 and MMP-9 protein total levels were determined from normal and tumor sample homogenates by ELISA (Oncogene, Cambridge, MA). The total protein concentration of each tissue sample was determined by the method of Bradford (22) . Duplicate tissue sample homogenates were diluted when necessary (MMP-2, 1:20 for nontumor and tumor samples; MMP-9, 1:20 in the case of nontumor tissues and 1:60 for tumor tissues). Before performing the assays, possible protein degradation was evaluated by determining alkaline phosphatase activity (23) .

ELISAs were performed following the assay protocols. Briefly, the MMP-2 and MMP-9 enzyme immunoassays used two antibodies. An antibody, specific for the human MMP-2 or MMP-9 protein, was immobilized onto the surface of microtiter wells. The samples to be assayed and biotinylated detector monoclonal antibodies were pipetted into the wells and allowed to incubate for 2 h, during which time any MMP-2 or MMP-9 that was present bound to the capture and detecting antibodies. Unbound material was washed away, and horseradish peroxidase-conjugated streptavidin was added, which binds to the detector antibodies. The horseradish peroxidase catalyzed the conversion of the chromogenic substrate tetramethylbenzidine from a colorless solution to a color solution, the intensity of which is proportional to the amount of MMP-2 or MMP-9 protein in the sample. The colored reaction products were quantified using a microplate reader set at 450 nm (Benchmark; Bio-Rad). Quantitations were achieved by the construction of a standard curve using known concentrations of MMP-2 or MMP-9. Results were expressed as nanograms of MMP-2 or MMP-9 per milligram of protein.

MMP-2 and MMP-9 Activity by Gelatin Zymography.
Gelatinases (MMP-2 and MMP-9) were detected by gelatin zymography under nondenaturing conditions. Tissue homogenates, containing 50 µg of total proteins, were electrophoresed down 8% SDS-polyacrylamide gels copolymerized with 1 mg/ml gelatin at 100 V for 6 h (Vertical slab gel; Hoefer, Pharmacia Biotech). After electrophoresis, gels were washed twice in 2.0% Triton X-100 for 30 min at room temperature with shaking to remove SDS. Zymograms were subsequently developed by incubation overnight at 37°C in collagenase buffer [0.2 M NaCl, 5 mM CaCl₂, 1% (v/v) Triton X-100, and 0.02% NaN₃ in 50 mM Tris-HCl, pH 7.4]. Zymograms were stained with 1 w/v Coomassie blue G-250 dissolved in 30% methanol containing 10% v/v glacial acetic acid at room temperature for 60 min. Gels were destained in the same solution but without the Coomassie blue stain.

In all cases, appropriate controls were included in the gel. Gelatinolytic activity was visualized as a clear band against a dark background of stained gelatin and quantitated by densitometry using a Bioimage Analyzer (GS-710; Bio-Rad) linked to Quantity-One software (Bio-Rad). Thus, the area and absorbance of each latent and active gelatinase band were determined for each sample. The enzyme activity levels were expressed as the fold-increase in expression of the M_r 92,000, 82,000, 72,000, and 62,000 bands in tumor relative to that measured in the corresponding adjacent normal mucosa and referred to total levels of the corresponding metalloproteinase.

MMP-3 Expression by Real-Time Quantitative PCR.
MMP-3 expression was evaluated in a pool of samples showing MSI-H or MSI-L/MSS by real-time quantitative PCR. Two-step reverse transcription-PCR was performed following the TaqMan Gold reverse transcription-PCR and SYBR Green PCR Master Mix kit protocols (PE Applied Biosystems). Specific primers to amplify a region of MMP-3 mRNA (GenBank GI: 13027803) were established using the Primer Express Software (PE Applied Biosystems). Thus, we obtained a 115-bp region (nucleotides 834–949) by using the forward primer 5'-TGGCATTCAGTCCCTCTATGG-3' and the reverse primer 5'-AGGACAAAGCAGGATCACAGTT-3'. PCRs were performed in a 7700 Sequence Detector ABI PRIMS (PE Applied Biosystems). PCR conditions were as follows: AmpliTaq Gold activation and initial denaturing at 95°C for 10 min; 40 cycles of denaturing at 95°C for 15 s and annealing/extension at 60°C for 1 min. Real-time quantitative PCR for glyceraldehyde-3-phosphate dehydrogenase was also performed on the same samples to correct for any residual differences in the initial level of RNA in the specimens by expressing the amount of MMP-3 as a ratio to the amount of glyceraldehyde-3-phosphate dehydrogenase. The products of real-time quantitative PCR were verified on agarose gels.

Analysis of Mutations in MMP-3 Promoter.
Next, we performed a study to detect the presence of possible mutations affecting the MMP-3 promoter in all samples showing MSI-H status. Moreover, we investigated these abnormalities in a pool of 20 MSI-L/MSS tumors that were considered as controls. In both MSI-H and MSS samples, determinations were established in genomic DNA from tumor and nontumor tissues. This analysis was performed by sequencing PCR products containing a region located between nucleotides 607 and 809 of the MMP-3 promoter (GenBank GI: 11093513). Thus, we obtained a 202-bp region by using the forward primer 5'-AATTCACATCACTGCCACCA-3' and the reverse primer 5'-GCCTCAACCTCTCAAAGTGC-3'. PCR and sequencing conditions were the same as we have described in this report for detection of TGFßRII and BAX mutations.

Statistical Analysis.
When possible, microsatellite status was assessed for potential associations with a number of clinicopathological parameters, including Dukes’ stage, tumor location, differentiation grade of the primary tumor, and recurrence. Associations of categorical variables were assessed using the ² test. P < 0.05 was judged to be significant. Moreover, levels and activity for MMP-2 and MMP-9 and MMP-3 expression were correlated with microsatellite status of tumors by the Student’s t test. The differences among multiple groups were analyzed by ANOVA.

	RESULTS

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DNA samples from patients affected by sporadic colorectal cancer (n = 101) were classified in relation to MSI. For this purpose, DNA samples from tumor and nontumor tissues were subjected to a study using the five microsatellite markers recommended by the American Joint Commission on Cancer. Thus, our data indicated that 20 cancers showed instability at two or more of the markers considered, and these samples were classified as MSI-H tumors. A total of 81 samples, showing instability in only one locus or without instability, were considered MSI-L/MSS tumors.

Microsatellite status was correlated with clinicopathological features of tumors in the 88 cases in which these variables were provided (Table 1) . There were no significant differences in Dukes’ stage and tumor differentiation distribution between patients with MSI-H and MSI-L/MSS tumors. However, MSI-H tumors, as expected, showed a marked predilection for the proximal colon. In fact, 8 of the 13 MSI-H tumors (61.5%) were localized in the right colon. In contrast, 59 of 75 MSI-L/MSS tumors (78.6%) were localized in the distal colon or in the rectum (P = 0.011). Moreover, distant metastases during the follow-up time (median, 28.6 months; range, 8–52 months) were detected more frequently among patients with MSI-L/MSS tumors (19% versus 0%; P = 0.126).

Both MMP-2 and MMP-9 levels were greater in colorectal tumors than in normal tissues. Thus, mean ± SE values were: 9.66 ± 1.17 versus 6.37 ± 1.63, P = 0.107, in the case of MMP-2; and 53.89 ± 5.70 versus 6.57 ± 0.87, P = 0.001, for MMP-9. MMP-2 levels in tumor tissues correlated significantly with right-side colon location (P = 0.009; data not shown), whereas insignificant associations were detected between MMP-9 levels and clinicopathological features of tumors. When we compared MMP-2 and MMP-9 levels in sporadic colorectal tumor tissues with microsatellite status, a significant association was detected for MMP-9; MSI-H was the group of cases showing significantly greater levels of this protein (P = 0.001; Table 3A ).

We evaluated MMP-3 expression in the two groups of sporadic colorectal cancers, MSI-H and MSI-L/MSS. This analysis was performed taking into account the role of MMP-3 in the activation of MMP-9. This study was possible only in the 39 cases in which RNA was available (12 MSI-H tumors and 27 MSI-L/MSS tumor samples). Thus, data from real-time quantitative PCR indicated higher levels of MMP-3 expression for MSI-L/MSS tumors when we compared MMP-3 expression in MSI-H cancers (P = 0.033; Table 4 ).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Examples of MMP-3 promoter mutations in MSI-H sporadic colorectal tumors. The MMP-3 promoter region between nucleotides 681 and 751 is shown. For each one of the samples, nucleotide sequence corresponding to normal (NT) and tumor (T) tissue from the same patient are indicated. Bold letters indicate nucleotide insertions or single changes in tumor tissue compared with nontumor samples. - -, nucleotide deletions in tumors.

	DISCUSSION

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Colorectal adenocarcinomas originated by the suppressor and the mutator pathways differ in several pathological features. Tumors with widespread MSI are located predominantly in the proximal colon and demonstrate poor differentiation, mucinous or medullary architecture, and prominent peritumoral lymphocytic infiltration more often than MSS and MSI-L tumors (8) . Furthermore, patients with MSI-H tumors have a more favorable survival than patients with MSI-L/MSS colorectal carcinomas. At present, no reports exist to explain a molecular basis that could help us to understand the different behaviors shown by both groups of tumors. Thus, to contribute to the knowledge of molecular changes involved in the apparent lesser invasive capacity shown by MSI-H sporadic colorectal cancers, we used a panel of microsatellite markers that allowed us to clearly identify the subgroup of tumors with widespread MSI. These tumors were classified as MSI-H tumors and, according to our data, demonstrated clinicopathological features inherited in cancers that develop through the mutator phenotype pathway, such as a marked preference to locate in the proximal colon or a reduced metastatic potential. To perform this study, we used a panel of five microsatellite markers that had been validated previously as a reference panel. Our data indicated that one of these markers (BAT25) was unstable in a very high percentage of MSI-H colorectal tumors, and it was sufficient alone to recognize virtually the 90% of tumors with an MSI-H phenotype. Moreover, we confirmed the tumor characterization by screening mono- and dinucleotide repeated sequences on coding regions of TGFßRII and BAX genes. Thus, these analyses allowed us to classify our tumor population in two subgroups, those showing a high MSI rate (MSI-H) and those exhibiting a low or null rate of MSI (MSI-L/MSS).

As it has been mentioned above, the biological basis of the more favorable clinical course of patients with MSI-H adenocarcinomas is still undetermined. It has been suggested that the high mutational load of MSI-H tumors may be detrimental to their growth and metastatic potential (27) . An alternative explanation could be that these tumors elicit a strong host immune response arising as a result of a high mutation rate of tumor-associated antigens (28) . Our hypothesis was that tumors of the mutator phenotype pathway could show a lesser invasive capacity, and therefore it could exhibit a decrease in MMP activity. To confirm this hypothesis, we evaluated the levels and activity of MMP-2 and MMP-9 in the two groups of sporadic colorectal cancers that emerged from the previous analysis, MSI-H and MSI-L/MSS. To select MMPs to be investigated in this work, we considered the significant role of MMP-2 and MMP-9 in the transformation of a tumor from the benign to the malignant state by enabling the tumor cells to infiltrate blood vessels and lymphatics allowing metastasis to a distant site, according to previous reports (29) .

We found that total MMP-2 and MMP-9 levels were higher in colorectal tumors than in the control tissues. Our results are in line with reports by Zeng et al. (29) and Heslin et al. (13) . Within the tumor tissues, when we considered the levels and activity of MMP-2 and MMP-9, we observed some interesting differences between MSI-H and MSI-L/MSS cancers. Surprisingly, whereas no differences were detected in the case of MMP-2, MMP-9 total levels were significantly higher in the group of tumors showing high rates of MSI (MSI-H); levels of the activated form of MMP-9 were the lowest in this group of tumors. To investigate these differences in synthesis and activity of MMP-9, we considered that overproduction of proMMP-2 and proMMP-9 is necessary but not sufficient for the development of the invasive phenotype, whereas activation of both MMP-2 and MMP-9 is required for full acquisition of the invasive colorectal phenotype (29) . Thus, activation is a critical step in the regulation of MMP-dependent proteolytic activity. In fact, MMP-2 and MMP-9 are increased in cancers from different origins, including colorectal cancer, relative to corresponding normal tissue. However, other MMPs such as stromelysin-1 (MMP-3) may also contribute to colorectal cancer progression (29) . In addition, previous studies demonstrated that purified proMMP-9 was activated by MMP-3 directly (30 , 31) , and coexpression of MMP-9 and MMP-3 has been reported in tumor tissues (32) . These data suggest that proMMP-9 activation by MMP-3 results in colorectal cancer progression and metastasis (32) . Our data from real-time quantitative PCR analysis indicated significantly lower levels of MMP-3 expression in MSI-H tumors when we made comparisons with levels found in the group of MSI-L/MSS cancers. These results are in agreement with the decreased levels of the active MMP-9 form in the first group of mentioned cancers. Stromelysin-1 (MMP-3) exhibits several activities that would make it a particularly good tumor marker. In addition to activating gelatinase B, MMP-3 can degrade numerous extracellular matrix components and release several cell surface molecules, including E-cadherin, a known contributor to cancer development (33) .

In another context, Ye et al. (24) reported the presence of a common polymorphism in the MMP-3 promoter that was associated with progression of atherosclerosis and demonstrated that expression of the MMP-3 construct with 6A at the polymorphic site was lower than a construct containing 5A. Taking into account that this region of MMP-3 promoter, located between nucleotides 699 and 704 (GenBank GI: 11093513), constitutes a repeated mononucleotide locus that could be a target for the mutator phenotype cancer pathway, we next investigated possible alterations at this level in the subgroup of sporadic colorectal MSI-H tumors as well as in a pool of MSI-L/MSS tumors that were used as controls. According to our results, MMP-3 promoter deletions and/or insertions in repeat sequences can be observed in all tumors grouped as MSI-H, and the presence of these mutations correlated with a decreased MMP-3 expression. However, none of the MSI-L/MSS tumors analyzed showed these alterations. Thus, in this work, we demonstrate that the MMP-3 promoter emerges as a novel target of the defective mismatch repair machinery inherited in cancers from the replication error phenotype pathway (1) . Recently, it has been suggested that the presence of the 5A polymorphism at the MMP-3 promoter may be one of the risk factors for the development and/or progression of cancer, especially in mammary tumors (34) .

In conclusion, data reported here indicate that the MMP-3 promoter constitutes a novel target of the defective mismatch repair machinery inherited in colorectal cancers from the replication error phenotype pathway. This fact results in a decreased expression of MMP-3 and a lesser activation of proMMP-9. Thus, tumors that develop through the mutator phenotype pathway could confer a minor invasive capacity. Therefore, our data may contribute to an understanding of the molecular basis of the favorable prognosis of patients with sporadic colorectal cancer affected by tumors showing high rates of MSI (MSI-H).

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Grants from Aventis-Pharma and Sanofi-Synthelabo, Spain.

² To whom requests for reprints should be addressed, at Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain. Phone: 34-91-394-17-77; Fax: 34-91-394-17-79; E-mail: benito{at}farm.ucm.es

³ The abbreviations used are: MSI, microsatellite instability; MSI-H, high-frequency MSI; MSI-L, low-frequency MSI; HNPCC, hereditary nonpolyposis colorectal cancer; MMP, matrix metalloproteinase; MSS, microsatellite stable.

Received 1/23/02. Accepted 5/ 1/02.

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