A human embryonic kidney cell line, HEK293, and human hepatoma cell lines, HepG2 and Alexander, were obtained from the American Type Culture Collection (ATCC). SNU423 and SNU475 cells were obtained from the Korea cell-line bank. All cell lines were grown in monolayers in appropriate media: Dulbecco's modified Eagle's medium for HepG2 and Alexander; Eagle's minimum essential medium for HEK293; and RPMI1640 for SNU423 and SNU475, supplemented with 10% fatal bovine serum and 1% antibiotic/antimycotic solution (Sigma, St. Louis, MO) and maintained at 37°C in air containing 5% CO2 and humidity.
Construction and Infection of Recombinant Adenovirus
Expression of the 20-amino-acid repeat domain of APC
that binds to β
] or that of the entire coding region of AXIN1
was shown to downregulate β
]. Therefore, we constructed adenoviral vectors containing the 20-amino-acid repeat region of wild-type APC
(Ad-APC), the entire coding region of AXIN1
(Ad-Axin) and LacZ
(Ad-LacZ) into pAd-Bg
/ll vector. The recombinant adenoviruses, constructed as described previously [7
], were propagated in HEK293 cells and purified by two rounds of CsCI density centrifugation. HepG2 cells were infected with the viral solutions at 100 MOI and incubated at 37°C for 1 hour, with brief agitation every 15 minutes, followed by addition of culture medium. The infected cells were maintained at 37°C for 72 hours.
Tcf-4 Reporter Assays
The reporter assays were performed as described previously [7
]. Briefly, the reporter plasmid containing four copies of Tcf4-specific DNA-binding sequence (TBE2) in the upstream region of the luciferase gene in pGL3-Basic vector (Promega, Madison, Wl) or mock pGL3- Basic vector (Promega) was cotransfected with a plasmid vector, pRL-TK (Promega), using FuGENE™
6 reagent (Boehringer) according to the supplier's recommendations. The reporter gene activities were measured 72 hours after transfection using dual-luciferase reporter assay system (Promega) according to the manufacturer's protocol.
Preparation of RNA
Total RNAs were isolated from HepG2 cells infected with Ad-APC, Ad-Axin or Ad-LacZ using Trizole reagent (Life Technologies) according to the manufacturer's method.
cDNA Microarray Production
The fabrication of cDNA microarray slides has been described elsewhere [20
]. Two sets of cDNA microarray slides containing a duplicate set of 9216 cDNA spots were used for each analysis of expression profiles to reduce the experimental fluctuation of each signal.
For the preparation of probes, after treatment with 10 units of DNase I, 1 µg of polyA RNA was reverse-transcribed and second-strand synthesis was performed. The double-stranded cDNA was then amplified with Ampliscribe T7 transcription kit (Epicentre Technologies, Madison, Wl) and the amplified RNA was labeled during reverse transcription with Cy-dye (Amersham Pharmacia Biotech, Piscataway, NJ). RNA from HepG2 cells with Ad-LacZ was labeled with Cy3 and that from HepG2 cells with Ad-APC or Ad-Axin was labeled with Cy5.
The Cy3-labeled cDNA derived from HepG2 cells with Ad-LacZ and Cy5-labeled cDNA from cells with Ad-APC or Ad-Axin were mixed with microarray hybridization solution (Amersham Pharmacia Biotech) and formamide at a final concentration of 50%. Hybridization was performed using an automated slide processor (Amersham Pharmacia Biotech) for 16 hours at 42°C. After hybridization, the glass slides were washed in 2xSSC, 0.1% SDS for 10 minutes at 55°C, 0.2xSSC, 0.1% SDS for 10 minutes at 55°C, and 0.1xSSC for 1 minute at room temperature. Subsequently the slides were scanned by an Array Scanner (Molecular Dynamics) and fluorescence intensities of Cy3 and Cy5 for each gene were evaluated by Array Vision software (Amersham Pharmacia Biotech). After subtraction of background signal, the quadruplicated values were averaged for each spot and adjusted so that the mean Cy3 and Cy5 intensities of 52 housekeeping genes for each slide were equal. After the normalization, genes were excluded from further investigation if the intensities of both Cy3 and Cy5 were below 100,000 fluorescence units.
Preparation of A6555 cDNA and Isolation of the Entire Coding Sequence of MARKL1
The A6555 cDNA spotted on the microarray was prepared by RT-PCR using a primer set, A6555F (5′-CCTAAAGA-CTGGAGAATCTGG-3′), and A6555R (5′-AAGAAGTGGC-TTAGAGTCCAGTG-3′) designed from the sequence of Hs.8312 in UniGene database (http://www.ncbi.nlm.nih.gov/UniGene/
). By a computer search in the human EST database in NCBI with the BLAST program, we identified 58 EST sequences. To obtain the sequence of the 5′ region of A6555, we further searched for genomic sequences in genomic databases (http://www.ncbi.nlm.nih.gov/blast/blast.cgi
), and found three sequences. By the use of exon prediction programs, GENSCAN (http://ccr-081.mit.edu/GENSCAN.html
) and Gene Recognition and Assembly Internet Link (http://grail.genome.ad.jp/Grail-1.3/
), we could identify 10 exon-like sequences. RT-PCR experiments using a primer set, A6555F2 (5′-ATGT-CTTCGCGGACGGTG-3′) and A6555R2 (5′-CCAGATT-CTCCAGTCTTTAGG-3′), which were designed in the exon-like sequence and the sequence of Hs. 8312, respectively, produced a 2733-base product and subsequently its sequence was determined.
5′ Rapid Amplification of cDNA Ends (5′RACE)
5′RACE using Marathon cDNA amplification kit (Clontech, Palo Alto, CA) was performed according to the manufacturer's instructions. For the amplification of the 5′ part of MARKL1 cDNA, a gene-specific reverse primer (5′-GCTGGGATTCAGCTGGGTTTTGTCG-3′) and the AP1 primer supplied in the kit were used. The cDNA template was synthesized from human testis mRNA. The nucleotide sequences of the PCR products were determined directly by the use of the gene-specific reverse primer with an ABI PRISM 377 DNA sequencer (Applied Biosystems) according to the manufacturer's instructions.
Northern Blot Analysis
Human multiple-tissue blots (Clontech) were hybridized with a 32P-labeled A6555 cDNA. Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at -80°C for 24 hours.
The entire coding region of MARKL1 was amplified by RT-PCR with a primer set, MARKL1EGFP-F (5′-CCCCGTCGACCCGGAGAAGATGTCTTCGCGG-3′) and MARKL1EGFP-R (5′-TCAGAATTCGAGGTCGTTGGA-GATGCGGGTG-3′). The product was digested with SalI and EcoRI, and cloned into the appropriate cloning site of a plasmid vector, pEGFP-N2 (Clontech). Another product amplified with a different primer set, MARKL1FLAG-F (5′-CCCGGAATTCGGAGAAGATGTCTTCGCG-GACGG-3′) and MARKL1FLAG-R (5′-CTCAGTCGAC-GAGGTCGTTGGAGATGCGGGTG-3′) was digested with EcoRI and SalI, and cloned into pFlag-CMV-5a (Kodak). HEK293 cells were transfected with either pEGFP-MARKL1 or pFlag-MARKL1 and fixed with PBS containing 4% paraformaldehyde. Cells tranfected with pFlag-MARKL 1 was stained by anti-Flag monoclonal antibody (Sigma) and by rhodamine-conjugated anti-mouse secondary antibody. Nuclei were stained with 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI; Boehringer Mannheim). Subcellular localization of the protein was visualized by fluorescent image using Eclipse E800 (Nikon).
Clinical Materials and RT-PCR
Twenty sets of primary HCCs and their corresponding noncancerous liver tissues were obtained with informed consent from surgical specimens of patients who underwent hepatectomy in Kyoto University Hospital. Among them, nine cases of HCCs revealed nuclear accumulation of β-catenin by immunohistochemical staining. Total RNAs were extracted from these nine HCCs and their corresponding noncancerous tissues. Ten micrograms of total RNA was reversely transcribed for single-stranded cDNAs using d(T)12–18 primer (Amersham Pharmacia Biotech) with Superscript II reverse transcriptase (Life Technologies). Each single-stranded cDNA was diluted for subsequent PCR amplification by monitoring glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a quantitative control. The primers used for amplification were GAPDHF (5′-ACAACAGCCTCAAGATCATCAG-3′), GAPDHR (5′-GGT-CCACCACTGACACGTTG-3′), MARKL1F (5′-CCTAAA-GACTGGAGAATCTGG-3′) and MARKL1R (5′-AAGA-AGTGGCTTAGAGTCCAGTG-3′). Each PCR was carried out in a 20-µl volume of PCR buffer (TAKARA), and amplified for 4 minutes at 94°C for initial denaturing, followed by 25 (for GAPDH) or 30 (for MARKL1) cycles of 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 30 seconds, in the Gene Amp PCR system 9600 (Perkin-Elmer, Foster City, CA).
Alexander, SNU423, and SNU475 cells were transfected with sense S-oligonucleotide (5′-TCGGACACGGTGAG-TG-3′) or antisense S-oligonucleotide (5′-CACTCACCGT-GTCCGA-3′) encompassing the first exon-intron boundary designed to suppress the expression of MARKL1 using Lipofectin Reagent (Gibco BRL, Grand Slam, NY). Cells were maintained for 1 week, and then fixed with 100% methanol and stained by Giemsa solution.