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ABT-737 is a pan-Bcl-2 inhibitor that has a wide range of single-agent activity against acute lymphoblastic leukemia (ALL) cell lines and xenografts. A relationship between expression of myeloid cell leukemia 1 (Mci-i), an antiapoptotic member of the Bci-2 family of proteins, and resistance to ABT-737 has been reported for various cancers. The synthetic cytotoxic retinoid N-(4-hydroxyphenyi)retinamide (4-HPR) is known to generate reactive oxygen species (ROS), and ROS have been shown to activate c-Jun kinase (JNK), which in turn phosphorylates and inhibits Mci-i. Thus, we investigated whether 4-HPR-mediated inactivation of Mci-i could act synergistically with ABT-737 to promote leukemia cell death. Cytotoxicity was determined using the fluorescence-based DIMSCAN assay. Synergy was defined as a combination index (CIN) less than 1. The expression of Bcl-2 family messenger RNAs was measured by real-time reverse transcription-polymerase chain reaction, and caspase activity was measured enzymati- cally. Changes in Bcl-2 family proteins and release of mitochondrial cytochrome cwere detected by immu- noblotting. ROS, apoptosis, mitochondrial membrane depolarization, and phospho-JNK were measured by flow cytometry. Gene silencing was by small interfering RNA (siRNA). All statistical tests were two-sided. ABT-737 decreased Mci-i protein expression in ABT-737--sensitive ALL cell lines but not in ABT-737-resistant lines. Using the antioxidant ascorbic acid and siRNA-mediated knockdown of JNK, we showed that 4-HPR decreased Mci-i via ROS generation (that phosphorylates JNK) in ABT-737-resistant cell lines. Combining ABT-737 with 4-HPR enhanced the mitochondrial apoptotic cascade (percentage of cells with depolarized mitochondrial membrane at 6 hours, ABT-737 vs ABT-737 plus 4-HPR: 24.5% vs 45.5%, difference = 20.1%, 95% Cl = 18.9% to i3.9%; P< .001) arid caused caspase-dependent, synergistic multilog cytotoxicity in all seven ALL cell lines examined (mean CIN = 0.57, 95% Cl = 0.37 to 0.87), with minimal cytotoxicity for nor- mal lymphocytes. An increase of Mci-i protein in response to ABT-737 is one mechanism of ABT-737 resistance that can be overcome by 4-HPR, resulting in synergistic cytotoxicity of ABT-737 combined with 4-HPR in ALL cell lines. [ABSTRACT FROM AUTHOR]

Background Genes that are overexpressed in multidrug-resistant neuroblastomas relative to drug-sensitive neuroblastomas may provide targets for modulating drug resistance. Methods We used microarrays to compare the gene expression profile of two drug-sensitive neuroblastoma cell lines with that of three multidrug-resistant neuroblastoma cell lines. RNA expression of selected overexpressed genes was quantified in 17 neuroblastoma cell lines by reverse transcription—polymerase chain reaction (RT-PCR). Small-interfering RNAs (siRNAs) were used for silencing gene expression. Cytotoxicity of melphalan, carboplatin, etoposide, and vincristine and cytotoxic synergy (expressed as combination index calculated by CalcuSyn software, where combination index < 1 indicates synergy and >1 indicates antagonism) were measured in cell lines with a fluorescence-based assay of cell viability. All statistical tests were two-sided. Results A total of 94 genes were overexpressed in the multidrug-resistant cell lines relative to the drug-sensitive cell lines. Nine genes were selected for RT-PCR analysis, of which four displayed higher mRNA expression in the multidrug-resistant lines than in the drug-sensitive lines: histone deacetylase 1 (HDAC1; 2.3-fold dif- ference, 95% confidence interval [CI] = 1.0-fold to 3.5-fold, P = .025), nuclear transport factor 2-like export factor (4.2-fold difference, 95% CI = 1.7-fold to 7.6-fold, P = .0018), heat shock 27-kDa protein 1(2.5-fold difference, 95% CI = 1.0-fold to 87.7-fold, P = .028), and TAF12 RNA polymerase II, TATA box—binding protein—associated factor, 20 kDa (2.2-fold, 95% CI = 0.9-fold to 6.0-fold, P = .051). siRNA knockdown of HDAC1 gene expression sensitized CHLA-136 neuroblastoma cells to etoposide up to fivefold relative to the parental cell line or scrambled siRNA—transfected cells (P<.001). Cytotoxicity of the histone deacetylase inhibitor depsipeptide was tested in combination with melphalan, carboplatin, etoposide, or vincristine in five multidrug-resistant neuroblastoma cell lines, and synergistic cytotoxicity was demonstrated at a 90% cell kill of treated cells (combination index < 0.8) in all cell lines. Conclusion High HDAC1 mRNA expression was correlated with multidrug resistance in neuroblastoma cell lines, and inhibition of HDAC1 expression or activity enhanced the cytotoxicity of chemotherapeutic drugs in multidrug—resistant neuroblastoma cell lines. Thus, HDAC1 is a potential therapeutic target in multidrug—resistant neuroblastoma. [ABSTRACT FROM AUTHOR]