Differential TERT promoter methylation and response to 5-aza-2'-deoxycytidine in acute myeloid leukemia cell lines:TERT expression, telomerase activity, telomere length, and cell death

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

BACKGROUND: Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

METHODS: HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity Pathway Analysis.

RESULTS: Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

CONCLUSION: This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.

Hepatocyte growth factor (HGF) protects c-met-expressing Burkitt''s lymphoma cell lines from apoptotic death induced by DNA damaging agents

The relative sensitivity of neoplastic cells to DNA damaging agents is a key factor in cancer therapy. In this paper, we show that pretreatment of Burkitt's lymphoma cell lines expressing the c-met protooncogene with hepatocyte growth factor (HGF) protects them from death induced by DNA damaging agents commonly used in tumour therapy. This protection was observed in assays based on morphological assessment of apoptotic cells and DNA fragmentation assays. The protection was dose- and time-dependent — maximal protection requiring pre-incubation with 100 ng/ml HGF for 48 h. Western blotting analysis and flow cytometric studies revealed that HGF inhibited doxorubicin- and etoposide-induced decreases in the levels of the anti-apoptotic proteins Bcl-XL, and to a lesser extent Bcl-2, without inducing changes in the pro-apoptotic Bax protein. Overall, these studies suggest that the accumulation of HGF within the microenvironment of neoplastic cells may contribute to the development of a chemoresistant phenotype.

Effects of the aurora kinase inhibitors AZD1152-HQPA and ZM447439 on growth arrest and polyploidy in acute myeloid leukemia cell lines and primary blasts.

BACKGROUND:
Aurora kinases play an essential role in the orchestration of chromosome separation and cytokinesis during mitosis. Small-molecule inhibition of the aurora kinases has been shown to result in inhibition of cell division, phosphorylation of histone H3 and the induction of apoptosis in a number of cell systems. These characteristics have led aurora kinase inhibitors to be considered as potential therapeutic agents.
DESIGN AND METHODS:
Aurora kinase gene expression profiles were assessed in 101 samples from patients with acute myeloid leukemia. Subsequently, aurora kinase inhibitors were investigated for their in vitro effects on cell viability, histone H3 phosphorylation, cell cycle and morphology in acute myeloid leukemia cell lines and primary acute myeloid leukemia samples.
RESULTS:
The aurora kinase inhibitors AZD1152-HQPA and ZM447439 induced growth arrest and the accumulation of hyperploid cells in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. Furthermore, both agents inhibited histone H3 phosphorylation and this preceded perturbations in cell cycle and the induction of apoptosis. Single cell cloning assays were performed on diploid and polyploid cells to investigate their colony-forming capacities. Although the polyploid cells showed a reduced capacity for colony formation when compared with their diploid counterparts, they were consistently able to form colonies.
CONCLUSIONS:
AZD1152-HQPA- and ZM447439 are effective apoptosis-inducing agents in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. However, their propensity to induce polyploidy does not inevitably result in apoptosis.

Fluorescence (FISH) and chromogenic (CISH) in situ hybridisation in prostate carcinoma cell lines: comparison and use of virtual microscopy

Chromogenic in situ hybridisation (CISH) has become an attractive alternative to fluorescence in situ hybridisation (FISH) due to its permanent stain which is more familiar to pathologists and because it can be viewed using light microscopy, The aim of the present study is to examine reproducibility in the assessment of abnormal chromosome number by CISH in comparison to FISH. Using three prostate cell lines - PNTIA (derived from normal epithelium), LNCAP and DU145 (derived from prostatic carcinoma), chromosomes 7 and 8 were counted in 40 nuclei in FISH preparations (x100 oil immersion) and 100 nuclei in CISH preparations (x40) by two independent observers. The CISH slides were examined using standard fight microscopy and virtual microscopy. Reproducibitity was examined using paired Student's t-test (P