Journal of the National Cancer Institute Advance Access published online on July 10, 2007
JNCI Journal of the National Cancer Institute, doi:10.1093/jnci/djm044
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Histone Deacetylase 1 Gene Expression and Sensitization of Multidrug-Resistant Neuroblastoma Cell Lines to Cytotoxic Agents by Depsipeptide
Affiliations of authors: Developmental Therapeutics Program, Institute for Pediatric Clinical Research (NK, ZW, RS, CPR), Division of Hematology– Oncology (NK, ZW, LJ, RS, CPR), Childrens Hospital Los Angeles, University of South California, Los Angeles, CA; Departments of Pediatrics (NK, RS, CPR), Pathology (ED, TJT, CPR), and Preventive Medicine (RS), Keck School of Medicine, University of Southern California, Los Angeles, CA
Correspondence to: Nino Keshelava, MD, Division of Hematology–Oncology, MS# 57, Childrens Hospital Los Angeles, University of Southern California, 4650 Sunset Blvd, Los Angeles, CA 90027 (e-mail: nkeshelava{at}chla.usc.edu).
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 difference, 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 associated 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.
| CONTEXT AND CAVEATS Prior knowledge More than 50% of patients treated for high-risk neuroblastoma die, possibly from drug resistance acquired during therapy. Although such drug resistance is often caused by loss of p53 function, other mechanisms are also likely to induce drug resistance. Study design Molecular study in human neuroblastoma cell lines to identify genes that confer drug resistance in the presence of functional p53. Contribution The histone deacetylase 1 (HDAC1) gene was found to be expressed at higher levels in multidrug-resistant neuroblastoma cells than in drug-sensitive neuroblastoma cells. Knockdown of HDAC1 expression made neuroblastoma cells more sensitive to chemotherapeutic agents. The histone deacetylase inhibitor depsipeptide enhanced cytotoxicity in multidrug-resistant neuroblastoma cell lines of four agents commonly used to treat neuroblastoma independent of the p53 status of the cell. Implications HDAC1 is a potential therapeutic target in multidrug-resistant neuroblastomas. Limitations The genome-wide screen used to identify genes whose overexpression is associated with drug resistance may not detect all potentially relevant genes.
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Manuscript received November 13, 2006; revised April 6, 2007; accepted June 7, 2007.
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J Natl Cancer Inst 2007 99: 1057.
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