BMC Cancer 2007, 7:187 http://www.biomedcentral.com/1471-2407/7/187 Background Matrix metalloproteinase (MMP)-1 (Collagenase-1) is a major proteinase of the MMP family that specifically degrades type I collagen, which is a major component of the extracellular matrix (ECM), as well as other fibrillar collagens of types II, III, V and IX [1,2]. MMP-3 (Strome- lysine-1) is responsible for degradation of type IV colla- gen, which forms the basement membrane, and collagen V, IX, X [3]. MMP-3 also has a role in activation of proMMP-1 in tumor tissue into the active form of MMP-1 [4]. Gene expression data in our previous report demon- strated enhanced expression of MMP family genes in OSCC tissues, and suggested correlation of high expres- sion levels of MMP-1 and MMP-3 with aggressive behav- ior, such as metastasis, and clinical prognosis [5]. Similar correlations have been reported in pharyngeal, colon and various other tumor types [6-8]. Colocalization of MMP-1 and MMP-3 with destruction of ECM in the invasive front of cancer tissue suggests a direct role in cancer invasion [9]. It has also been shown that a function of MMPs affects susceptibility to different kinds of carcinoma. To date, polymorphisms of the promoter domain have been described in MMP-1, -3, -9, -12 to influence the expres- sion level of the genes [10]. A 2G type of single nucleotide polymorphism (SNP) at -1607 bp site in the promoter domain of MMP-1 creates a sequence, 5'-GGA-3,' that is the core recognition sequence of the binding site for Ets family transcription factors. The 2G type promoter results in higher transcription activity of the MMP-1 gene than does a 1G type promoter [11]. A 5A type promoter at - 1171 bp site of MMP-3 is also known to have a twofold higher transcriptional activity in vitro than that of a 6A type [12]. It has been documented that the 2G type SNP of MMP-1 confers increased susceptibility to colorectal [13], ovarian [14], lung [15], endometrial [16], renal cell [17] and head and neck [18] cancers; and the 5A type SNP of MMP-3 is associated with an increased susceptibility to breast cancer [19]. An in situ hybridization study revealed that MMP-1 expression in normal oral mucosa is controlled at a low level, whereas a remarkably elevated expression level is observed in cases of oral epithelial dysplasia, which becomes even higher in cases of OSCC [20]. It has also been documented that cases of oral epithelial dysplasia showing high expression level of MMP-1 developed into OSCC at higher frequency than cases with low expression of MMP-1 [21]. Taking all the observations noted above into consideration, it seems likely that MMPs may serve as key factors in all stages of the OSCC progression from car- cinogenesis in the early precancerous condition to the advanced invasive and metastatic phases. The purpose of this study is to document the effect of genomic polymorphisms of MMP genes in the develop- ment of OSCC. We compared genotype distribution in the promoter domains of the functional SNPs that influence the transcriptional activity of MMP-1 and MMP-3 between OSCC patients and healthy control groups. Multivariate analysis was effectively used to assess correlations among parameters such as OSCC, the genotypes, age and sex. In this report, we describe the role of functional SNP of the MMP-1 gene in susceptibility to OSCC and, as a remarka- ble finding; we discuss the crucial impact of the MMP-1 2G allele in the development of OSCC in younger individ- uals. Methods Study subjects The tested cases were all unrelated native Japanese com- prising 170 cases of OSCC (107 males, 63 females; aver- age age 56.5 ± 13.9 years) who were histopathologically diagnosed as differentiated squamous cell carcinoma. The controls comprised 164 (104 males, 60 females) healthy subjects who did not have a history of malignant tumors and were frequency-matched to the cases by age (± 5 years; average age 51.5 ± 14.7 years). All OSCC subjects were patients who had been treated in the Dental Department of Niigata University Medical and Dental Hospital, Spe- cial Dental Care and Oral Surgery, Shinshu University Hospital, and Division of Oral Surgery, Nagaoka Red Cross Hospital. Blood samples were taken after obtaining the patients' informed consent to participate in the study and proc- essed anonymously. All cases were diagnosed histopatho- logically as OSCC. The study protocol was approved by the ethics committees of each institution. Genotyping of MMP-1 and MMP-3 5 ml of blood was obtained from the subjects and used as the source of peripheral blood lymphocytes. 0.2% NaCl was added for destruction of red blood cells, followed by addition of TNE buffer, 10% SDS and Proteinase K (MERCK Co., Darmstadt, Germany). After incubation at 58°C for more than six hours, genomic DNA was extracted by phenol-chloroform treatment and ethanol precipitation. The reaction was performed in a 25 µl vol- ume made up of 70 ng genomic DNA, 2.25 µl of the spe- cific forward/reverse primer (10 µM), 1 µl of TaqMan® MGB probes (5 µM), 12.5 µl of TaqMan® Universal Master Mix (Applied Biosystems, Foster City, CA, USA). PCR cycling conditions were 10 min of initial denaturation at 95°C followed by 35 cycles of 15 sec denaturation at 92°C and 1 min of one step annealing/extension at 60°C for MMP-1 or 62°C for MMP-3 (ABI Prism® 7900 HT Sequence Detection System, Applied Biosystems). Combi- nation of the probe/primer used for the MMP-1 SNP were: Page 2 of 9 (page number not for citation purposes)