Ods reverse phase column

Collected tumors were homogenized with 0.48 N PCA and centrifuged for 5 min at 2,000 rpm. Supernatants were collected and centrifuged for 10 min at 15,000 rpm. Two volumes of 0.5 N tri-n-octylamine in CH₂Cl₂ were added and again centrifuged for 3 min at 15,000 rpm; the supernatant was then analyzed using HPLC system with ODS reverse phase column (250×4.6 mm, 5 μm; Phenomenex), under the analytical method with eluent A (10 mM KH₂PO₄, 10 mM TBA-OH, 0.25% methanol: 50 mM KH₂PO₄, 10 mM TBA-OH, 30% methanol = 6:4, pH 6.7), and eluent B (50 mM KH₂PO₄, 10 mM TBA-OH, 30% methanol, pH 6.7) at a solvent flow rate 0.80 ml/min in a 175-min gradient elution as follows: B 1–23% from 0 to 20 min, B 23–45% from 20 to 130 min, B 45–99% from 130 to 150 min, B 99-99% from 150 to 160 min, B 99-1% from 160 to 160.1 min, and B 1-1% from 160.1 to 175 min.

DNA was extracted from tumors using the DNA Isolation kit for Cells and Tissues (Roche Diagnostics GmbH, Penzberg, Germany). The extracted DNA was diluted to 500 μg/ml with ddH₂O and degraded to nucleosides in a 200-μl reaction mixture consisting of 100 mM Tris-HCl (pH 7.0), 50 mM NaCl, 2.5 mM CaCl₂, 10 mM MgCl₂, 1 U DNaseI (Takara Bio Inc., Tokyo, Japan), 40 μg phosphodiesterase I (Sigma-Aldrich, St. Louis, MO, USA), 2 U alkaline phosphatase (Nippon Gene Co., Ltd., Tokyo, Japan) and 10 μg extracted DNA at 37°C for 2 h. After the reaction, 20 μl of 4.2 N PCA was added to each reaction mixture and centrifuged at 15,000 rpm for 3 min. Sixty microliters of 1 M K₂HPO₄ was added to each sample for neutralization and centrifugation was performed to remove debris. The supernatant was analyzed by HPLC system with ODS reverse phase column (250×4.6 mm, 5 μm; Phenomenex, Shimadzu GLC Ltd., Tokyo, Japan), under the analytical method with eluent A [10 mM sodium phosphate (pH 6.8), 5% CH₃CN], eluent B [10 mM sodium phosphate (pH 6.8), 60% CH₃CN], at a solvent flow rate of 1.0 ml/min in a gradient elution as follows: B 0% from 0 to 5 min, B 0–20% from 5 to 10 min, B 20% from 10 to 15 min, B 20–70% from 15 to 20 min, B 70% from 20 to 25 min, B 70-0% from 25 to 25.1 min, and B 70-0% from 25.1 to 35 min.

Cells were seeded in 175-cm² culture flasks at 1×10⁶ cells and pre-cultured for 24 h, prior to treatment with test compounds. After each treatment, cells were collected and resuspended in 100 μl PBS, and 50 μl of 1.26 N perchloric acid (PCA) was added to the cell suspension. The mixture was left on ice for 10 min and centrifuged for 5 min at 15,000 rpm. Three hundred microliters of 0.5 N tri-n-octylamine dissolved in CH₂Cl₂ was added to the supernatant and centrifuged for 5 min at 15,000 rpm. The supernatant was applied to an HPLC system (Prominence, Shimadzu Corporation, Kyoto, Japan) equipped with an ODS reverse phase column (250×4.6 mm, 5 μm; Phenomenex), under an analytical method utilizing eluent A [10 mM KH₂PO₄, 10 mM tetrabutylammonium hydroxide (TBA-OH), 0.25% methanol, pH 8.0], and eluent B (50 mM KH₂PO₄, 5.6 mM TBA-OH, 50% methanol, pH 6.5) at a solvent flow rate of 0.80 ml/min in a 120-min gradient elution as follows: B 60% from 0 to 10 min, B 60–70% from 10 to 20 min, B 70% from 20 to 45 min, B 70–80% from 45 to 55 min, B 80% from 55 to 80 min, B 80–100% from 80 to 90 min, B 100% from 90 to 100 min, B 100-60% from 100 to 101 min, and B 60% from 101 to 120 min.