Antagonistic effects of sequential administration of BL1521, a histone deacetylase inhibitor, and gemcitabine to neuroblastoma cells.

Additive to synergistic induction of apoptosis has been reported as a result of sequential incubation of cancer cells with a histone deacetylase inhibitor (HDACi) and gemcitabine (dFdC), a deoxycytidine analogue with proven anti-tumour activity. This study shows that sequential treatment of two neuroblastoma cell lines with BL1521, an HDACi, and dFdC resulted in strong antagonism despite a minor increase of dFdCTP incorporation into the DNA of one cell line. Furthermore, no difference in the deoxycytidine kinase activity was observed in response to BL1521. In conclusion, cancer cells that respond to HDACi with a cell cycle arrest and differentiation may no longer be sensitive to dFdC.

Gene expression profiling in response to the histone deacetylase inhibitor BL1521 in neuroblastoma.

Neuroblastoma is a childhood tumor with a poor survival in advanced stage disease despite intensive chemotherapeutic regimes. The new histone deacetylase (HDAC) inhibitor BL1521 has shown promising results in neuroblastoma. Inhibition of HDAC resulted in a decrease in proliferation and metabolic activity, induction of apoptosis and differentiation of neuroblastoma cells. In order to elucidate the mechanism mediating the effects of BL1521 on neuroblastoma cells, we investigated the gene expression profile of an MYCN single copy (SKNAS) and an MYCN amplified (IMR32) neuroblastoma cell line after treatment with BL1521 using the Affymetrix oligonucleotide array U133A. An altered expression of 255 genes was observed in both neuroblastoma cell lines. The majority of these genes were involved in gene expression, cellular metabolism, and cell signaling. We observed changes in the expression of vital genes belonging to the cell cycle (cyclin D1 and CDK4) and apoptosis (BNIP3, BID, and BCL2) pathway in response to BL1521. The expression of 37 genes was altered by both BL1521 and Trichostatin A, which could indicate a common gene set regulated by different HDAC inhibitors. BL1521 treatment changed the expression of a number of MYCN-associated genes. Several genes in the Wnt and the Delta/Notch pathways were changed in response to BL1521 treatment, suggesting that BL1521 is able to induce the differentiation of neuroblastoma cells into a more mature phenotype.

Histone deacetylase inhibitor BL1521 induces a G1-phase arrest in neuroblastoma cells through altered expression of cell cycle proteins.

Histone deacetylase inhibitors (HDACi) have been discovered as potential drugs for cancer treatment. The effect of BL1521, a novel HDACi, on the cell cycle distribution and the induction of apoptosis was investigated in a panel of MYCN single copy and MYCN amplified neuroblastoma cell lines. BL1521 arrested neuroblastoma cells in the G1 phase and induced up to 30% apoptosis. Downregulation of CDK4, upregulation of p21(WAF1/CIP1) and an increase of hypophosphorylated retinoblastoma protein were observed, indicating a possible mechanism for the cell-cycle arrest. BL1521 also induced downregulation of p27, which may underlie the observed induction of apoptosis.

The novel histone deacetylase inhibitor BL1521 inhibits proliferation and induces apoptosis in neuroblastoma cells.

Neuroblastoma is a childhood cancer arising from the sympathetic nervous system. Disseminated neuroblastoma has a poor prognosis despite intensive multimodality treatment. Histone deacetylases (HDACs) were recently discovered as a potential target for pharmacological gene therapy in cancer. HDACs have an important function in regulating DNA packaging in chromatin, thereby affecting the transcription of genes. In this paper, we tested the efficacy of a newly developed histone deacetylase inhibitor, BL1521, on neuroblastoma in vitro by investigating the changes in: acetylation of histone H3, in situ HDAC activity, p21(WAF1/CIP1) and MYCN expression, metabolic activity, proliferation, morphology and the amount of apoptosis present. BL1521 inhibited the in situ HDAC activity of a panel of neuroblastoma cell lines by at least 85%. Western analysis showed an increase of histone H3 acetylation in neuroblastoma cells after incubation with BL1521. Northern analysis showed an increase in the expression of p21(WAF1/CIP1) and a decrease in the expression of MYCN in neuroblastoma cells after incubation with BL1521. Proliferation as well as the metabolic activity of neuroblastoma cells decreased significantly in response to treatment with BL1521, regardless of the MYCN status of the cells. BL1521 induced poly-(ADP-ribose) polymerase cleavage in a time- and dose-dependent manner, indicating the induction of apoptosis. Furthermore, when compared to the HDAC inhibitors Trichostatin A and 4-phenylbutyrate, BL1521 has an intermediate efficacy. Our results show that BL1521 is a potent inhibitor of HDAC and that HDACs are an attractive target for selective chemotherapy in neuroblastoma.

Histone deacetylases (HDACs): characterization of the classical HDAC family.

Transcriptional regulation in eukaryotes occurs within a chromatin setting, and is strongly influenced by the post-translational modification of histones, the building blocks of chromatin, such as methylation, phosphorylation and acetylation. Acetylation is probably the best understood of these modifications: hyperacetylation leads to an increase in the expression of particular genes, and hypoacetylation has the opposite effect. Many studies have identified several large, multisubunit enzyme complexes that are responsible for the targeted deacetylation of histones. The aim of this review is to give a comprehensive overview of the structure, function and tissue distribution of members of the classical histone deacetylase (HDAC) family, in order to gain insight into the regulation of gene expression through HDAC activity. SAGE (serial analysis of gene expression) data show that HDACs are generally expressed in almost all tissues investigated. Surprisingly, no major differences were observed between the expression pattern in normal and malignant tissues. However, significant variation in HDAC expression was observed within tissue types. HDAC inhibitors have been shown to induce specific changes in gene expression and to influence a variety of other processes, including growth arrest, differentiation, cytotoxicity and induction of apoptosis. This challenging field has generated many fascinating results which will ultimately lead to a better understanding of the mechanism of gene transcription as a whole.

A physiological threshold for protection against menadione toxicity by human NAD(P)H:quinone oxidoreductase (NQO1) in Chinese hamster ovary (CHO) cells.

NAD(P)H:quinone oxidoreductase 1 (NQO1) has often been suggested to be involved in cancer prevention by means of detoxification of electrophilic quinones. In the present study, a series of Chinese hamster ovary (CHO) cell lines expressing various elevated levels of human NQO1 were generated by stable transfection. The level of NQO1 over-expression ranged from 14 to 29 times the NQO1 activity in the wild-type CHO cells. This panel of cell lines, allowed investigation of the protective role of NQO1 in quinone cytotoxicity. It could be demonstrated that menadione toxicity was significantly reduced in all NQO1-transfected CHO clones compared to the wild-type cells, but the clones did not show differences in their level of protection against menadione. This observation pointed at a critical threshold concentration of NQO1 above which a further increase does not provide further protection against quinone cytotoxicity. Additional studies in which the NQO1 activity was inhibited by dicoumarol showed that only dicoumarol concentrations of about five times the EC(50) for NQO1 inhibition were able to reduce NQO1 levels below the apparent threshold, making the cells more sensitive. The level of this threshold was estimated to be in the range of base line NQO1 activities observed in several tissues and species. Thus, the results of the present study indicate that beneficial effects of NQO1 induction by, for example, cruciferous vegetables might be absent or present depending on the NQO1 activity threshold for optimal protection and the basal level of NQO1 expression in the tissue and species of interest.