SW620, a human colon carcinoma cell line, was provided by Dr A.T. Fojo (National Cancer Institute, Bethesda, MD, USA). RPMI-1640 medium was purchased from Nissui Seiyaku Co. (Tokyo, Japan), and fetal calf serum (FCS) was from JRH Biosciences (Lenexa, KS, USA). A rabbit anti-ERK polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A rabbit anti-MVP polyclonal antibody was prepared using a glutathione S-transferase (GST)-MVP (aa 694–794) fusion protein as an antigen (4). Multiple tissue cDNA panels were from Clontech Laboratories Inc. (Mountain View, CA, USA). Anti-α-tubulin antibody was from Calbiochem (Darmstadt, Germany).

SW620 and ACHN cells, a human renal adenocarcinoma cell line, were grown in RPMI-1640 containing 10% FCS, 2 mM glutamine and 100 U/ml of penicillin at 37ºC in a 5% CO₂ humidified atmosphere. Cell cultures were maintained in exponential growth by media replacement every 2–3 days. Cells were provided with fresh media the day before use for experimentation.

Imaging was conducted using a confocal laser scanning microscope (FV500; Olympus Corp., Tokyo, Japan). SW620 cells (1×10⁶) were incubated for 24 h at 37ºC. After being carefully washed twice with PBS, SW620 cells were fixed with 3% formaldehyde for 30 min at room temperature. They were then washed twice with PBS and permeabilized with 100% methanol for 20 min at room temperature. After blocking for 30 min with 3% skimmed milk in PBS, cells were incubated for 1 h with a polyclonal antibody against MVP diluted in PBS containing 3% skimmed milk. After washing three times in PBS, cells were incubated for 20 min with FITC-conjugated goat anti-rabbit IgG diluted 1:100 in PBS containing 3% skimmed milk. Cells were then washed three times in PBS and samples were examined by confocal microscopy. Fluorescence was imaged at an excitation wavelength (λ_ex) = 488 and with an emission bandpass filter set for FITC.

Samples were subjected to 6 or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli. Gel proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (Immobilon-P transfer membrane; Millipore, Bedford, MA, USA) using the Bio-Rad Trans-Blot SD apparatus. The membrane was treated with a buffer [350 mM NaCl, 10 mM Tris-HCl (pH 8.0), 0.05% Tween-20] containing 3% skimmed milk for 1 h and incubated with the indicated antibody (1:1,000) in the buffer containing 3% skimmed milk for 1 h. Following four washes with the buffer (10 min each), the membrane was incubated with a peroxidase-conjugated horse anti-rabbit IgG diluted 1:1,000 in the buffer containing 3% skimmed milk for 1 h. The membrane was washed with buffer, and developed using the enhanced chemiluminescence western blotting detection system (Amersham Pharmacia, Buckinghamshire, UK).

The human MVP promoter ranging from −407 and −78 to +66 was amplified from the genomic DNA of SW620 cells as previously described (22). The PCR product was cloned into the pT7 Blue-2 vector (Merck Biosciences, San Diego, CA, USA) and sequenced. Following digestion with KpnI and SacI, the MVP promoter fragment was gel-purified and ligated into the KpnI and SacI-linearized pGL3-Basic vector (Promega, Madison, WI, USA). The resulting constructs were designated pMVP407 and pMVP78 (22). SW620 cells were grown in RPMI-1640 medium containing 10% FCS, 2 mM glutamine, and 100 U/ml of penicillin at 37ºC in a 5% CO₂ humidified atmosphere. Cells (3.0×10⁵/well in 6-well plates) were transfected with 1 μg of luciferase reporter plasmid (pGL3, Promega) or 100 ng of control plasmid (pRL-TK; Promega). Following incubation for 24 h with the transfected cells, the luciferase assay was performed using the Dual-Luciferase Reporter Assay System following the manufacturer’s protocol (Promega). Luminescence assays were performed using a luminometer (TD-20/20 Luminometer; Turner Designs, Sunnyvale, CA, USA). All experiments were performed in triplicate and the results were normalized to pRL-TK activity.

Small interfering RNA (siRNA) duplexes were synthesized using the Silencer^® siRNA construction kit (Ambion Inc.). A target site within the gene was chosen from the mRNA sequence of USF1. Following selection, the target site was searched with the National Center for Biotechnology Information Blast to confirm its specificity. The siRNA used in the present study consisted of a 21-nucleotide sense strand and a 21-nucleotide antisense strand with a two-nucleotide overhang at the end. Sequences were as follows: USF1-siRNA sense, 5′-GGAAGGUGCAGUGGCUACUGG-3′, starting at nucleotide 54 from the AUG start codon of the human USF1 coding sequence; USF1-siRNA antisense, 5′-GGCCAGUAG CCACTGCACCUU-3′; siRNA was transfected to cells using Lipofectamine 2000™ according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).

Total cellular RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). RT-PCR was performed with the SuperScript One-Step RT-PCR system and gene-specific primers according to the manufacturer’s instructions (Invitrogen). Reaction mixtures containing total RNA (500 ng of each), 0.2 μM of each primer, and an enzyme mixture composed of SuperScript II RT, Platinum Taq DNA polymerase, were maintained at 50ºC for 20 min, then at 94ºC for 2 min, and PCR was performed as follows: 30 cycles at 94ºC for 15 sec, 55ºC for 30 sec, and 70ºC for 30 sec. Primers for RT-PCRs were designed based on human sequences in GenBank. These sequences used the following primers: MVP (GenBank accession no. NM_017458), 5′-CAGGATGTGTATGTGCTGT CGG-3′ and 5′-GCTGGAGGCTCTTAGCTGTGTC-3′; USF1 (GenBank accession no. NM_ 007122), 5′-ATGAAGGGG CAGCAGAAAACA-3′ and 5′-TTAGTTGCTGTCATTCT TGA-3′ and GAPDH (GenBank accession no. NM_002046), 5′-AGAACATCATCCCTGCCTCTACTGG-3′ and 5′-AAAG GTGGAGGAGTGGGTGTCGCTG-3′.

Total cellular RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). One microgram of RNA was reverse transcribed using a first-strand cDNA synthesis kit (ReverTra Ace^®; Toyobo, Osaka, Japan). Human MVP and GAPDH gene expression levels were assayed by real-time reverse PCR (PRISM 7900HT; Applied Biosystems, Foster City, CA, USA) according to the technical specifications. Human GAPDH was used for normalization. The quantification of target gene expression was obtained with the comparative cycle threshold method according to the instructions of the manufacturer.

Cells were fixed with 1% formaldehyde for 10 min at 37ºC to cross-link protein to DNA. A chromatin immunoprecipitation (ChIP) assay was carried out using a ChIP assay kit (Upstate Biotechnology) according to the manufacturer’s instructions. The soluble DNA fraction was mixed with an anti-USF-1 antibody (Santa Cruz Biotechnology) or non-immunized mouse IgG (Santa Cruz Biotechnology) and the precipitated DNA was amplified with primers for the MVP promoter, 5′-GCCAGCTGGCTCCAAG GTAG-3′ (sense) and 5′-GGCAGGGCAAGGCAGGCCAA-3′ (antisense).

At the start of the present study, we examined the tissue expression profile of MVP mRNA by performing PCR. MVP was found to be expressed in the heart, placenta, lung, liver, kidney and pancreas in normal tissues (Fig. 1).

To examine if the MVP protein was expressed in cancer cell lines, we cultured human colon adenocarcinoma SW620 cells. As shown in Fig. 2A, the MVP protein was expressed in SW620 cells. It is known that MVP can shuttle between the nucleus and the cytoplasm. We examined the localization of MVP in SW620 cells by immunofluorescence. MVP was mainly localized in the cytoplasm of SW620 cells (Fig. 2B).

To confirm basal MVP gene transcription, we cloned human MVP promoter fragments and inserted them into a luciferase reporter vector. One construct, pMVP407, contained all the known conserved promoter elements, Y-box, Myo D, GATA, E-box, Sp1, p53 and STAT of the MVP promoter (22). Following transfection of the individual plasmids into SW620 cells, the cells and MVP promoter activity was monitored by a luciferase assay (Fig. 3). Luciferase activity in pMVP407 and pMVP263-transfected cells was 200-fold higher than that of cells transfected with the control luciferase vector. The level of luciferase activity was gradually reduced as the fragment became shorter. Basal luciferase activity in pMVP78-transfected cells was significantly lower than that in pMVP102-transfected cells. The construct pMVP78 lacks the E-box binding site of the construct pMVP102. These findings suggest that the E-box-binding motif may play a crucial role in maintaining basal MVP promoter activity.

USF1 and USF2 are members of the eukaryotic evolutionary conserved Basic-Helix-Loop-Helix Leucine Zipper transcription factor. They interact with high affinity to cognate E-box regulatory elements and are ubiquitously expressed. To examine whether the expression of USF1 regulates the expression of MVP, SW620 cells transfected with siRNA duplexes targeting USF1 or green fluorescent protein (GFP) as a control were cultured. As shown in Fig. 4A, the introduction of siRNA against USF1 resulted in a downregulation in MVP expression in SW620 cells. To examine the effects in ACHN cells, ACHN cells were transfected with siRNA duplexes targeting USF1 or green fluorescent protein (GFP). The introduction of siRNA against USF1 also resulted in a downregulation in MVP expression in ACHN cells. These results indicate that the expression of MVP is affected by the expression of USF1 in SW620 cells.

To determine if MVP mRNA expression was altered by the introduction of siRNA against USF1, we examined the siRNA against USF1 on the expression levels of MVP in SW620 cells by RT-PCR and real-time RT-PCR. RT-PCR and real-time RT-PCR analyses confirmed that the introduction of siRNA against USF1 in SW620 cells downregulated the expression of USF1 and consequently suppressed MVP mRNA levels in SW620 cells (Fig. 5A and B). These results suggest that USF1 modulates the expression of MVP.

To test for in vivo USF1 binding to the MVP promoter, we performed a ChIP assay using the antibody against USF1. The chromatin immunoprecipitated by the antibody was then amplified by PCR with primers specific for MVP. As shown in Fig. 6, USF1 bound the MVP promoter in SW620 cells.