PMC

Expression of ARK5 mRNA in various cancers (cancer: Ca.) and corresponding normal tissues. A: DNA array analysis of ARK5 expression in 13 different types of cancers (T) and corresponding normal tissues (N). B: Densitometric analysis of ARK5 mRNA expression in colon or rectal cancers (filled bars) and corresponding normal tissues (open bars).

A: Total RNA, protein, and genomic DNA were extracted from each cell line, and Northern blot (N.B.) or Western blot analysis (W.B.) was performed with probe or antibody for ARK5 or β-actin. Southern blot analysis (S.B.) was also performed with probe for ARK5 or PERK. B: Strong association between ARK5 expression and tumor cell invasion activity. Each cell line was seeded on a matrigel-coated invasion chamber (5 × 10⁴ cells/chamber), and cells that had translocated to the lower chamber were counted after 48 hours. Results are shown as means of three experiments, and the bars represent SE values.

A: Quantitative analysis of ARK5 mRNA expression in primary colorectal cancers and liver metastases. Total RNA (100 ng) extracted from 15 samples of non-tumor colon mucosa (normal colon mucosa, N.C.), 41 primary tumors (colorectal cancer, C.Ca.) consisting of Dukes’ B: 19 samples, C: 19 samples, and D: 3 samples, and 15 liver metastases of colorectal cancer (L.M.) were reverse-transcribed, and Q-PCR was performed. Each value was normalized to the amount of GAPDH. B: Expression of Akt-1 and Akt-2 mRNA was also measured by Q-PCR in the same samples of colorectal cancers and their liver metastases.

Tumor cell invasion mediated by the Akt/ARK5 pathway in human colorectal cancer cell lines. A: In vitro invasion activity of DLD-1 (open bars) and D/ARK (filled bars) cell lines exposed to or unexposed (control) to 20 μmol/L LY294002, or 1 μg of the expression vector of dominant negative-Akt-1 (DN-Akt-1), constitutive active-Akt-1 (CA-Akt-1), dominant negative-ARK5 (DN-ARK5), or ARK5-antisense (ARK5-AS) was used. Results are shown as means of three experiments and the bars represent SE values. B: In vitro invasion assay of SW480 cells. Either 20 μmol/L LY294002 or 1 μg of the expression vector of each DN-Akt-1, CA-Akt-1, DN-ARK5, or ARK5/AS was used. Results are also shown as means of three experiments.

Distribution of ARK5 mRNA expression in fresh clinical samples. A: ARK5 and GAPDH expression were examined by RT-PCR in PANC-1 and HepG2 cells. B: In situ hybridization of AR5 mRNA in PANC-1 and HepG2 cells. In situ hybridization was performed by using the antisense and sense probe. C: Tissue sections of non-tumor tissue (normal colon mucosa, N.C.), primary tumor tissue (colon cancer, C.Ca.), and liver metastatic tissue (liver metastases of colorectal cancer, L.M.) were hybridized with the antisense probe for ARK5, and the sections were counterstained with hematoxylin. Nuclear stained cells as brown (diaminobenzidine tetrahydrochloride, DAB) are ARK5 mRNA-positive. As a negative control, in situ hybridization was also performed using the sense probe in the same tissue sections.