Oral carcinoma cell lines derived from different sites were used: oral floor, HSC2, KOSC2 and Ho1u1; tongue, HSC3 and OSC19; and gingiva, TSU and Ca9.22. They were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan) or RIKEN BRC Cell Bank (Tsukuba, Japan). Cells were cultured in RPMI-1640 or DMEM supplemented with 10% fetal bovine serum and 100 U/ml of penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO). HSC2 cells, which marginally express MALT1, stably expressing full-length wild-type MALT1 (wtMALT1, _wtMALT1HSC2 cells), the NH₂ terminal death and Ig-like domains-deleted dominant-negative MALT1 (ΔMALT1, _ΔMALT1HSC2 cells) or vector alone (_mockHSC2 cells) were previously established (12). The wtMALT1 and ΔMALT1 cDNA cloned into pEBMulti-Hyg with a FLAG-tag (Wako Pure Chemical Industries Ltd., Osaka, Japan) or vector alone were transiently transfected into oral carcinoma cells by electroporation. Cells were harvested 24 h after the transfection and used for analyses. The short interfering RNA (siRNA) targeting MALT1 (#18601 siRNA) and a negative control siRNA (Silencer Negative Control #1 siRNA; Ambion, Austin, TX) were used.

The _wtMALT1HSC2 and _mockHSC2 cells scraped from the culture dish were centrifuged at 1,500 rpm for 7 min at 4°C. The supernatant was discarded and the cell pellets were washed with ice-cold PBS three times. The pellets were solubilized in lysis buffer containing 0.1% NP-40 on ice and sonicated for 30 sec three times. The lysate was centrifuged at 15,000 rpm at 4°C for 15 min to remove insoluble materials. The resultant supernatant was collected and the protein concentration was determined by BCA protein assay (Pierce, Rockford, IL). Protein extracts (30 μg) were applied to a one dimensional (1-D) SDS-PAGE gel (10% total acrylamide) and stained with Coomassie Brilliant Blue R-250. All experiments were repeated three times from independent experiments. The gels were put on a flatbed scanner, and protein bands were compared using the Image J 1.46r (15). Protein bands of interest that were electrophoresed at different positions or with different intensities were subjected to MALDI-TOF mass spectrometry (MS) analysis.

Protein bands differentially detected between the cells were excised from the gel. The gel bands were digested by proteomics grade trypsin (Roche Diagnostics GmbH, Mannheim, Germany), and tryptic peptides were extracted from gels with 1% trifluoroacetic acid in 50% acetonitrile and dried using a vacuum pump. The digest was analyzed by MALDI-TOF MS on a Voyager DE-PRO MALDI-TOF (Applied Biosystems, Foster city, CA) with a nitrogen laser (337 nm). The analyte mixture (1 μl) was mixed with 1 μl of saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid, and spotted on a MALDI target plate and dried at room temperature. Ion acceleration was set at 20 kV. Mass spectra were obtained by averaging 200 laser shots. Calibration of the spectra was externally calibrated with peptide mass standards (Applied Biosystems). Row data were analyzed using the computer software provided by the manufacturer as monoisotopic masses. Average of proteins from MALDI-TOF MS spectra was achieved using the MASCOT database (MASCOT ver. 2.0, Matrix Science Inc., Boston, MA) (16). Monoisotopic peptide mass spectra were matched against the SWISS-PROT or NCBI non-redundant databases set at ±1.2 kDa peptide tolerance, limited to the Homo Sapiens proteins.

Total RNA extracted from _wtMALT1HSC2 and _mockHSC2 cells was reverse transcribed to cDNA by MultiScribe Reverse Transcriptase (Applied Biosystems) and subjected to real-time PCR using the StepOne Real-time PCR system (Applied Biosystems). PCR conditions were 95°C for 20 sec followed by 40 cycles of 95°C for 1 sec and 60°C for 20 sec. The TaqMan probes (Applied Biosystems) specific for KRT8 (Hs01595539_g1), KRT18 (Hs02827483_g1), KRT5 (Hs00361185_m1) and KRT14 (Hs00265033_m1) were used. Expression levels (n=4) were normalized against GAPDH (Hs02758991_m1). Levels of gene expression (2^−ΔΔCt) were determined by the standard curve method (17).

Total cell lysates were immunoblotted with a standard protocol. The lysates in the SDS sample buffer containing 1 mM phenylmethanesulfonyl fluoride and protease inhibitor cocktail (Roche Diagnostics GmbH) were size-fractionated by SDS-PAGE gels under reducing conditions and electrotransferred to PVDF membranes. The membrane was probed with antibodies specific to K5 (clone PRB-160P, Covance, Princeton, NJ), K8 (clone Ks 8.7, Progen Biotechnik GmbH, Heidelberg, Germany), K14 (clone LL002, Cell Marque, Rocklin, CA), K18 (clone DC 10, Dako, Glostrup, Denmark), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), FLAG-M2 or β-actin (Sigma-Aldrich).

Normal oral epithelium covering the oral floor and the side edge of tongue taken from patients who underwent surgery for non-tumorous diseases or positioned at the far distal site from carcinomas that were histologically considered normal by a pathologist (HY) were used for controls. All tissues were obtained with a written consent of the patient and with approval by institutional review boards of the Nippon Dental University. Unstained formalin-fixed and paraffin-embedded sections were treated with microwave (500 W) in 0.01 M sodium citrate buffer, pH 6.0, and incubated with antibodies against K5, K8, K14 or K18 followed by biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA). After treatment with avidin-biotin complexes (Vector Laboratories), the color was developed with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich).

The real-time cell electronic sensing assay based on electrical impedance readings in cell monolayers plated in wells containing built-in gold electrodes was performed. We have used the analyzer (xCELLigence RTCA-DP), 16-well E-plates and the integrated software (Roche Diagnostics GmbH). The RTCA-DP system works by measuring the electronic impedance at the cell-sensor electrode interface integrated on the bottom of E-plates. The _wtMALT1HSC2 cells, _ΔMALT1HSC2 cells and _mockHSC2 cells were plated at a density of 1×10⁴ cells/well installed on the analyzer. The analyzer and the installed plates were placed in a standard cell culture incubator, at 37°C in a humidified atmosphere of 5% carbon dioxide and air. Cells were allowed to adhere to plates overnight and subjected to the analysis.

Doubling time of _wtMALT1HSC2 cells, _ΔMALT1HSC2 cells and _mockHSC2 cells were statistically analyzed by Kruskal-Wallis test using JMP 7.0.1 (SAS Institute Inc., Cary, NC).

Protein bands of _mockHSC2 and _wtMALT1HSC2 cell lysates were compared by 1-D SDS-PAGE. Sixteen bands were differentially detected between them (9 in _mockHSC2 cells and 7 in _wtMALT1HSC2 cells, Fig. 1), and subjected to the MALDI-TOF MS analysis. Six bands were not specified because of the non-satisfactory spectrum or the insufficient confidence of database screening to yield unambiguous results. Finally, 6 and 4 proteins were specified in _mockHSC2 and _wtMALT1HSC2 cells, respectively (Table I). It includes K5 and K14 in _mockHSC2 cells and K8 and K18 in _wtMALT1HSC2 cells. Since alterations of keratin expression associate with phenotypic changes of carcinoma cells (5,6), we focused on the keratin regulation by MALT1 in this study.

The differential keratin expression was validated for its protein and gene expressions. K5 and K14 proteins were predominantly expressed in _mockHSC2 cells and negligibly in _wtMALT1HSC2 cells (Fig. 2A). Suppression of K5 and K14 expression by MALT1 was supported by the increased expression in _ΔMALT1HSC2 cells. K8 was not detected in _mockHSC2 and _ΔMALT1HSC2 cells but abundant in _wtMALT1HSC2 cells. K18 was also strongly detected in _wtMALT1HSC2 cells. The differential keratin expression was confirmed at the mRNA level (Fig. 2B). When compared to _mockHSC2 cells, genes encoded by K5 (KRT5) and K14 (KRT14) were downregulated and upregulated in _wtMALT1HSC2 cells and _ΔMALT1HSC2 cells, respectively. The _wtMALT1HSC2 cells upregulated K8 (KRT8) and K18 (KRT18) expression and _ΔMALT1HSC2 cells downregulated them.

To exclude the possibility of long-term effect of MALT1 expression on the keratin expression, MALT1 cDNA was transiently transfected into the parental HSC2 cells (Fig. 3). It dose-dependently downregulated K5 and K14 and upregulated K8 and K18. Since there is a possibility that the keratin alterations are specific to HSC2 cells, we transiently transfected wtMALT1 and ΔMALT1 cDNA in a set of oral carcinoma cells (Fig. 4). In contrast to the predominant reduction of K5 and K14 expression by wtMALT1 in most of carcinoma cells, wtMALT1 upregulated K8/18, especially in oral floor carcinoma cells.

HSC2 cells and KOSC2 cells were established from carcinomas of the non-keratinized oral floor epithelial origin (18,19). Since the oral cavity is covered by non-keratinized and keratinized epithelium, keratin expression in non-keratinized oral floor and keratinized tongue epithelium were verified by the immunostaining. K8 and K18 were stained at the basal cells of oral floor epithelium but not the tongue epithelium, and K5 and K14 vice versa (Fig. 5). The sublingual gland staining confirmed a previous study (20); serous cells, intercalated and striated ducts were strongly positive for K8 and K18 and faintly for K5, and excretory ducts for K8, K5 and K14. Mucous cells were negative for these keratins.

Since keratin substitution closely associates with proliferation status of squamous epithelial cells (21), effects of MALT1 on cell proliferation were examined using the real-time sensing RTCA-DP system. Fig. 6A illustrates the remarkable enhancement of _ΔMALT1HSC2 cell and suppression of _wtMALT1HSC2 cell proliferation compared to the _mockHSC2 cells. Doubling time of _mockHSC2 cells was 11.1±0.3 h, and _ΔMALT1HSC2 cells and _wtMALT1HSC2 were 8.4±0.5 and 21.3±2.1 h, respectively (P<0.01). Decreased proliferation of _wtMALT1HSC2 cells was not due to the cell death because they did not increase the TUNEL reactivity (Fig. 6B) and the trypan blue staining (data not shown). The _wtMALT1HSC2 cells ceased cyclin D1 expression but restored it by the siRNA against MALT1 in a dose-dependent manner (Fig. 6C and D). The _ΔMALT1HSC2 cells and _mockHSC2 cells did not respond to the siRNA because of the lack of siRNA-binding site in a ΔMALT1 gene construct and the marginal expression of endogenous MALT1, respectively.