Transcriptional signature of histone deacetylase inhibition in multiple myeloma: Biological and clinical implications -- Data Supplement - HTML Page - 6759SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Materials and Methods
Materials.
The proteasome inhibitor PS-341 (bortezomib) was obtained from Millennium Pharmaceuticals (Cambridge, MA); the immunomodulatory thalidomide analog IMID-1 was obtained from Celgene (Warren, NJ); doxorubicin, dexamethasone, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. Rabbit polyclonal antibodies against XBP-1, Aurora A, Aurora B, hsp90, hsp70, myb, and tubulin were purchased from Upstate Biotechnologies (Lake Placid, NY), Calbiochem, Cell Signaling, Santa Cruz Biotechnology, Pharmingen, or R & D Systems; and the Enhanced Chemiluminescence (ECL) kit, which includes the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies, was from Amersham Pharmacia. Global Gene Expression Profiling of Suberoylanilide Hydroxamic Acid (SAHA)-Treated Multiple Myeloma (MM) Cells.
Total RNA was extracted and purified with the Qiagen RNeasy kit (Qiagen, Valencia, CA). Five micrograms of total RNA was used in the first-strand cDNA synthesis with T7-d(T)₂₄ primer [GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)₂₄] and Superscript II (GIBCO/BRL, Rockville, MD). The second-strand cDNA synthesis was carried out at 16°C by adding Escherichia coli DNA ligase, E. coli DNA polymerase I, and RNase H to the reaction, followed by T4 DNA polymerase to blunt the ends of newly synthesized cDNA. The cDNA was purified through phenol/chloroform and ethanol precipitation. The purified cDNA was incubated at 37°C for 5 h in an in vitro transcription reaction, using the BioArray High Yield RNA Transcript Labeling kit (Enzo Diagnostics, Farmingdale, NY), to produce cRNA labeled with biotin. Affymetrix Chip Hybridization.
cRNA (20 m g) was fragmented by incubating in buffer containing 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, and 150 mM MgOAc at 94°C for 35 min. The hybridization mixture containing 15 m g of adjusted fragmented cRNA mixed with Eukaryotic Hybridization controls (contains control cRNA and oligonucleotide B2) was hybridized with a preequilibrated human U133A Affymetrix chip at 45°C for 16 h. After hybridization, cocktails were removed, chips were washed in a fluidic station with low-stringency buffer (6× standard saline phosphate with EDTA, 0.01% Tween 20, and 0.005% antifoam) for 10 cycles (two mixes per cycle) and high-stringency buffer (100 mM N-morpholinoethanesulfonic acid, 0.1 M NaCl, and 0.01% Tween 20) for four cycles (15 mixes per cycle); stained with streptavidin phycoerythrin; incubated with normal goat IgG and biotinylated mouse anti-streptavidin antibody; and restained with streptavidin phycoerythrin. Chips were scanned in an HP ChipScanner (Affymetrix, Santa Clara, CA) to detect hybridization signals. Gene Expression Profiling Data Analysis.
Scanned image output files were visually examined for major chip defects and hybridization artifacts and then analyzed with Affymetrix GENECHIP MICROARRAY ANALYSIS SUITE 5.0 software (Affymetrix). The image from each GeneChip was scaled such that the average intensity value for all arrays was adjusted to a target intensity of 150. Expression analysis files created by GENECHIP MICROARRAY ANALYSIS SUITE 5.0 software were exported as flat text files to Microsoft EXCEL for further analysis. Data analysis identified signals with at least two-fold difference between SAHA-treated samples and respective controls. These results were screened for P < 0.0025 by Student’s t test, to identify induced or repressed transcripts. Data were visualized by using the RAINBOW program (developed by Charles Bailey and Towia Libermann, Beth Israel Deaconess Medical Center, Boston) that enables representation of data in color format according to their values on a logarithmic scale. Annotations and informations for all genes were retrieved using the NetAffx web site (www.affymetrix.com/analysis/index.affx) and UnChip (unchip.org:8080/bio/unchip) and added to the data file. Annotated data were sorted according to functional relationships. MTT Colorimetric Survival Assay
. Cell survival was examined using the MTT colorimetric assay as described (1). Cells were plated in 48-well plates at 70–80% confluence and then treated as indicated. At the end of each treatment, cells were incubated with 1 mg/ml MTT for 4 h at 37°C; a mixture of isopropanol and 1 M HCl (23:2, vol/vol) was then added under vigorous pipetting to dissolve the formazan crystals. Dye absorbance (A) in viable cells was measured at 570 nm, with 630 nm as a reference wavelength. Cell viability was estimated as a percentage of the value of untreated controls. All experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average value ± SD of representative experiments. Effect of SAHA in MM-1S Cells Overexpressing Constitutively Active Akt
. To evaluate the role of the antiapoptotic kinase Akt in SAHA-induced apoptosis, MM-1S cells were stably transfected with vectors encoding a constitutively active (myristoylated) form of Akt (Upstate Biotechnologies) or empty (neo) vector by using Lipofectamine 2000 according to the instructions of the manufacturer. Forty-eight hours later, the cells were incubated in growth medium containing G418 (500 m g/ml, Life Technologies) to select pools of stable clones that were subsequently treated with SAHA (2.5-10 m M for 36 h). The overexpression of Akt in transfected cells was confirmed by immunoblotting against the myc-tag of the myristoylated Akt. Immunoblotting Analysis.
Immunoblotting analysis was performed as described (1). Evaluation of NF-k B and p53 Activity.
The DNA-binding activities of NF-k B, p53, and HIF-1a were quantified in nuclear extracts of SAHA-treated MM-1S cells by ELISA format with the respective TransAM Transcription Factor Assay kits for NF-k B p65, p53 and HIF-1a (Active Motif North America, Carlsbad, CA) according to the instructions of the manufacturer, as described (2-5). Nuclear extracts were prepared as described (6). Telomerase Activity Assay
. Telomerase activity was assayed with the TRAPEZE Telomerase Detection kit (Oncor, Gaithersburg, MD) as described (7). Each extract of cells was diluted 1:40 so that an aliquot of 2.0 m l corresponded to 250 cells for cell lines. After incubation for 20 min at 30°C, PCR amplification was performed with 30 cycles at 94°C for 30 s, at 58°C for 30 s, and at 72°C for 60 s, and the PCR products were analyzed by electrophoresis on 12% polyacrylamide nondenaturating gels and stained with Sybr Green I (Molecular Probes). The gels were photographed by using a digital camera and an UV transilluminator (Alpha Innotech Corporation, San Leandro, CA). Telomerase activity was assessed by determining the ratio of the entire telomerase ladder to that of the internal control by using NIH IMAGE analysis software. Determination of 20S Proteasome Activity.
20S Proteasome activity was measured at 37°C as described (8, 9) by monitoring the release of 7-amido, 4-methyl coumaric acid (MCA) during degradation of the fluorogenic substrate suc-Leu-Leu-Val-Tyr-MCA by extracts of SAHA-treated and control MM-1S cells. Statistical Analysis.
Statistical significance was examined by a two-way ANOVA, followed by Duncan’s post hoc test. In all analyses, P < 0.05 was considered statistically significant.

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