Strong Association of ARK5 with Tumor Invasion and Metastasis

G. Kusakai et al.
Materials and Methods
Homology Search for Subunit Structures of ARK5
and AMPK Catalytic Subunits. Based on their reported
amino acid sequences, a BLAST search analysis was
used to detect a putative consensus catalytic peptide
sequence of the AMPK family members AMPK-α1,
AMPK-2, MELK, SNARK, and ARK5. A homology
search was performed by computer analysis using
GENETYX 10.1 software.
Cell Lines and Culture. All human carcinoma cells
were cultured in Dulbecco's modified Eagle Medium
(DMEM: NISSUI, Japan) supplemented with 10%
fetal bovine serum, 2 mM L-glutamine and 25 mM
HEPES-NaOH (pH 7.3) at 37°C under an atmosphere
of 5% CO 2 in air.
Reverse Transcription-PCR (RT-PCR) and Quantitative-RT-PCR
(Q-RT-PCR). Total RNA was isolated
from cancer cells by the AGPC method using ISOGEN
(NIPPONGENE, Japan), and 1 µg of total RNA was
reverse transcripted into cDNA with oligo-dT primers
and AMV-reverse transcriptase (TaKaRa, Japan)
according to the manufacturer's instructions. The
cDNA was subjected to 25 cycles of PCR amplification
on a PCR Thermal Cycler 480 (TaKaRa, Japan), and the
PCR primers used for ARK5 detection were: forward,
5'-ATGCTAAGTACCCTCTGAATG-3', and reverse,
5'-GCAACAAGCAGTCAGTCGATC-3'. PCR products
were fractionated by agarose electrophoresis,
stained with ethidium bromide, and visualized under
UV light. GAPDH served as a control for PCR.
A Light-Cycler (Roche, Germany) was used to
detect the number of ARK5 and GAPDH gene copies.
The amplification mixture contained cDNA derived
from 100 ng of total RNA. The PCR primers used for
GAPDH detection were: forward, 5'-AGGGCTG-
GTTTTAACTCTGGT-3', and reverse, 5'-CCC-
CACTTGATTTTGGAGGGA-3'. Q-PCR was performed
under cycling conditions of 95°C for 10 min,
followed by 40 cycles of denaturation at 95°C for 10
sec, annealing at 60°C for 10 sec, extension at 72°C for
20 sec.
Transfection and Selection. For transfection, 1˘10 5
cells were seeded into each well of a 6-well plate, and
ARK5-full length expression vector was transfected
into DLD-1 and PANC-1 cells with TransFast transfection
reagent (Promega Corp., USA) according to the
manufacturer's instructions with some modifications.
Stable transfectants were selected with G418.
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Immunoblotting. Cells were washed with PBS containing
1mM sodium ortho-vanadate, 10mM sodium
fluoride and 25 mM sodium glycerophosphate. Cells
were collected, and lyzed with a buffer containing 1%
sodium dodecyl sulfate (SDS), 1 mM SOV4 and 10
mM Tris-HC1 (pH 7.4). Proteins were separated on a
10% SDS polyacrylamide gel, and transferred onto a
polyvinylidene difluoride membrane. A polyclonal
antibody to human SNARK was made by immunizing
rabbits with a peptide based on the sequence of
SNARK (KKPRQRESGYYSSPEPS) and it was found
to crossreact with ARK5 (1). Membranes were incubated
with the anti-SNARK antibody at dilution of
1:1000 and anti-rabbit antibody conjugated with horseradish
peroxidase (HRP) was used as a second antibody.
For visualisation, Western blotting detection
reagent (ECL: Amersham Pharmacia Biotech, UK) and
Hyperfilm ECL (Amersham Pharmacia Biotech, UK)
were used.
Migration Assay. In vitro migration assay was performed
with a matrigel-coated invasion chamber (Becton-DickinsoN,
USA). DLD-1 cells (5˘10 4 /chamber)
were seeded into each chamber, and the media in the
inner and outer chamber were changed after 6 hrs. The
cells that had invaded were counted under a phase-contrast
microscope after 48 hrs.
In vivo Liver Metastasis Assay in SCID Mice. SCID
mice (CB-17/Icr Crj-scid, 5-week-old males) were
anesthetized with pentobarbital-Na (Nembutal:
ABBOTT, USA). Ventrotomy was performed, the
spleen was exposed, and 200 µl of serum free DMEM
containing 1˘10 6 PANC-1 or P/ARK cells was injected
under the splenic capsule of ten mice each. The mice
were sacrificed 5 weeks later, and liver metastasis was
assessed by counting the number microscopically. The
liver metastases were stained with hematoxylin and
eosin (H.E.) and confirmed pathologically.
Results
Subunit Structures of ARK5 and AMPK Catalytic
Subunits. The AMPK family consists of five members,
AMPK-α1, AMPK-α2, MELK, SNARK, and ARK5
recently identified (Fig.1). As shown in Fig. 1, all
AMPK members have a putative consensus catalytic
domain structure. ARK5 contained the putative consensus
catalytic peptide sequence at amino acids 55 -
305, and an Akt-phosphorylation site (RXRXXS) was
found near the C-terminal (Fig.1). The C-terminal sub-