Combination of suberoylanilide hydroxamic acid with heavy ion therapy shows promising effects in infantile sarcoma cell lines

Human sarcoma cell lines (KHOS24-OS and A-204), as well as the human osteoblast hFOB 1.19 were obtained from the American Type Culture Collection (ATCC; Rockville, MD).

SAHA was obtained from Alexis Biochemicals (Lörrach, Germany). Primary monoclonal mouse antibodies against Rad51, Ku70 and Ku80, p21 and p53 were obtained from Abcam (Cambridge, UK). Primary monoclonal mouse antibodies against ß-actin as well as a secondary antibody for immunoblot experiments were purchased from CellSignaling Technology (Danvers, MA, USA). For the flow cytometry experiments as well as immunoblots, γH2AX antibody Alexa Fluor^® 488 anti-H2A.X-phosphorylated (Ser139) was obtained from BioLegend (San Diego, USA).

Clonogenic assays were performed as described previously [7]. In brief, exponentially growing tumor cells were plated in T25 culture bottles at appropriate numbers to give an estimated 50-250 colonies/flask and were incubated with medium containing 0 to 5 μM SAHA. Incubation of SAHA with the respective LD₂₀ and LD₅₀ for each cell line started 24 h before XRT/HIT. Incubation was stopped after 5 days. Monolayers were stained with 0.5% crystal violet for 10 minutes. Plates were stained with 0.1 M sodium citrate (pH 4) in ethanol 100% (3:1) for another 10 minutes. Afterwards, plates were dried for 48 to 72 h and colonies were counted manually. Survival was defined as the ability of cells to form colonies (≥ 50 cells).

Surviving fractions were obtained by normalizing the plating efficiencies (cell number/plated cell) to the respective control values. Each experiment was done in triplicate and at least three independent repetitions were performed. In combination experiments, the survival rates after different doses of radiation were normalized to the treatment with SAHA given alone.

Following a theoretical concept of combination effects by Steel and Peckham [15, 16] the range of additivity was calculated from the response to the LD 20 of the single agent. This range is encompassed by the prediction of independent cell killing (accounted for by normalizing the radiation survival rates to SAHA toxicity, see above) and a theoretical survival curve that can be obtained if the fraction of cells surviving drug treatment is formally treated as being irradiated with an isoeffective dose D¢. Assuming that the radiation sensitivity coefficients were ax and bx, one readily finds that the theoretical survival curve (normalized to drug toxicity) can be written as SF = exp(- a p D- bxD 2) with a p = ax + 2bxD¢. For graphical representation of the combination effect in excess of independent cell killing, both the experimental survival fraction (cell number/plated cells) and the fitted survival curves were multiplied with the averaged surviving fraction after SAHA exposure alone.

Cells were seeded in T25 culture plates at a density of 1 × 10⁶ cells per plate 24 h before HIT. In the SAHA experiments, 0.5-1 μM SAHA was added 24 h before HIT. At certain time points after HIT, cells were harvested and centrifuged (800 g). Cells were washed with PBS several times and then fixed with 3% paraformaldehyde (PFA, Sigma) for 10 min at room temperature (RT). Ice-cold methanol (90%) was added and samples were kept on ice for another 30 min. Afterwards, samples were washed three times in 0.5% BSA/PBS re-suspended in 100 μl 0.5% BSA/PBS and incubated for 10 min at room temperature. Cells were stained for γH2AX by 1 h incubation at RT in 10 μl antibody plus 90 μl 0.5% BSA/PBS per sample. Finally, cells were washed three times with 0.5% BSA/PBS. Cells were further stained with DAPI for cell cycle analysis for 30 min at RT and analyzed simultaneously with the γH2AX staining. The samples were analyzed directly on a "Galaxy Pro"- Flowcytometer from Partec (Münster, Germany). The relative fluorescence intensity in the gated areas was calculated using the multiparameter "Flow max" software from Partec as described in a further report. For the detection of apoptotic cells we used Nicoletti stain measured in a FACS Calibur Flow cytometer (Becton Dickinson Cytometry Systems, San Jose, CA) as described in our earlier report [7].

To assess the mean extent of DNA damage at a particular phase of the cell cycle, the mean values of γH2AX immunfluorescence were calculated separately for G_0/1, S and G₂/M cells by the computer-interactive "gating" analysis. Cells in S and G₂/M have 1.5 and 2.0 times higher γH2AX mean immunofluorescence respectively, compared to cells in G_0/1 because of the increase of DNA and histone content during the cell cycle. Therefore, the data has to be normalized for DNA (histone) content by dividing the mean γH2AX immunofluorescence of S- and G₂/M-phase cells by 1.5 and 2.0, respectively. Finally, a low level of γH2AX immunofluorescence is seen in the untreated cells which represent an "intrinsic" γH2AX phosphorylation. Therefore, the γH2AX immunofluorescence level of the untreated controls has to be subtracted from the immunofluorescence level of the treated cells in order to get the γH2AX immunofluorescence level which is treatment-related. Then multiparametric analysis was done on a Galaxy pro flow cytometer (PARTEC, Münster, Germany) by stimulating the fluorochromes DAPI with mercury 100 W vapour lamp, H2AX-FITC with a 488 nm air cooled argon laser and measuring the fluorescence intensities at 530/30 nm and apoptotic cells stained with Nicoletti stain/DAPI/propidium iodide (PI) at 610/20 nm. The green fluorescent FITC, and red fluorescent PI, was measured in the logarithmic mode, DAPI stained DNA measured in linear mode. Analysis and calculation the Flow Max software (Partec, Münster, Germany was used. Each analysis represents 5000 cells.

Immunoblot analysis was performed as reported previously [7]. In brief, following treatment, cells were lysed with lysis buffer (0.5 M tris/Cl, pH 6,8, SDS, 87% Glycerin, DTT 1 M ad Aqua 100 ml). Following this, 1 ml lysate was incubated with 1 μl benzonase for 15 min at 37°C. 40 μg of protein extracts underwent electrophoresis onto a 12% polyacrylamide gel (Pierce Biotechnology Inc., Roxford, IL, USA) under reducing conditions. The separated proteins were transferred onto nitrocellulose membranes (Amersham Pharmacia Bioscience, Piscataway, NJ). The membranes were then incubated for 45 minutes in blocking buffer (tris-buffered saline with 0.1% Tween (TBS-T) and 5% nonfat dry milk), followed by incubation with specific primary antibodies at 1:1000 dilution at -4°C for 24 h or at room temperature for 1 h. After being washed with TBS-T buffer three times, the membrane was incubated with anti-mouse IgG secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:1000 dilution for 1 h at room temperature. The signals were visualized with the ECL+ detection system and autoradiography.

The nuclear organization of chromatin and γHSAX in KHOS-24 OS cells upon treatment with SAHA, HIT and both in combination was observed by laser scanning microscopy using a Zeiss LSM 700, equipped with a 63 × oil objective. Images were acquired with pinhole-size 1 Airy at pixel-size 100 nm. Cells were fixed with buffered 2% formaldehyde, chromatin stained with DAPI and γH2AX revealed by IHC using Alexa 488-charged secondary antibody as reporter.

All experiments were performed at least twice. Furthermore, each experiment was done in duplicate. Clonogenic assays were performed in triplicate. Combination studies were evaluated using student's t test with the resulting p value representing a two-sided test of statistical significance.