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.

GATA2 regulates the erythropoietin receptor in t(12;21) ALL.

The t(12;21) (p13;q22) chromosomal translocation resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality in children with acute lymphoblastic leukemia (ALL). The erythropoietin receptor (EPOR), usually associated with erythroid progenitor cells, is highly expressed in ETV6/RUNX1 positive cases compared to other B-lineage ALL subtypes. Gene expression analysis of a microarray database and direct quantitative analysis of patient samples revealed strong correlation between EPOR and GATA2 expression in ALL, and higher expression of GATA2 in t(12;21) patients. The mechanism of EPOR regulation was mainly investigated using two B-ALL cell lines: REH, which harbor and express the ETV6/RUNX1 fusion gene; and NALM-6, which do not. Expression of EPOR was increased in REH cells compared to NALM-6 cells. Moreover, of the six GATA family members only GATA2 was differentially expressed with substantially higher levels present in REH cells. GATA2 was shown to bind to the EPOR 5'-UTR in REH, but did not bind in NALM-6 cells. Overexpression of GATA2 led to an increase in EPOR expression in REH cells only, indicating that GATA2 regulates EPOR but is dependent on the cellular context. Both EPOR and GATA2 are hypomethylated and associated with increased mRNA expression in REH compared to NALM-6 cells. Decitabine treatment effectively reduced methylation of CpG sites in the GATA2 promoter leading to increased GATA2 expression in both cell lines. Although Decitabine also reduced an already low level of methylation of the EPOR in NALM-6 cells there was no increase in EPOR expression. Furthermore, EPOR and GATA2 are regulated post-transcriptionally by miR-362 and miR-650, respectively. Overall our data show that EPOR expression in t(12;21) B-ALL cells, is regulated by GATA2 and is mediated through epigenetic, transcriptional and post-transcriptional mechanisms, contingent upon the genetic subtype of the disease.

Screening for miRNA expression changes using quantitative PCR (Q-PCR).

In this section, we describe the use of Applied Biosystems TaqMan Array microRNA Card Set 3.0 to identify miRNA expression in a given RNA sample. This array set includes an array "A" and an array "B" which each have 384 wells that contain specific forward, reverse, and probe oligoinucleotides for measuring the expression of individual miRNAs during a Real-Time PCR (Q-PCR) reaction. Array "A" includes assays for profiling the comparatively higher expressed and better characterized miRNAs. Presently there are 1,048 mature miRNAs annotated in miRBase (release 16). The relatively small amount of miRNAs in comparison to protein-coding genes makes this format a viable option for measuring genome-wide miRNA expression changes. The Applied Biosystems TaqMan miRNA array set 3.0, which includes two separate arrays, can profile 754 miRNAs.

Microarray for epigenetic changes: gene expression arrays.

Microarrays can be used to examine changes involving all aspects of the epigenetic interactions. As the relationship between DNA methylation, histone modifications, and gene expression is elucidated, an important aspect to investigate is how the epigenetic status regulates the cell through differential expression of genes. This can provide data from work including experimental drug investigations from cell line models, or more directly patient comparison data.

Detecting DNA methylation using the methylated CpG island amplification and microarray technique.

Methylated CpG island amplification and microarray (MCAM) is a two-color array technique that quantifies DNA methylation by hybridizing equimolar amounts of treated vs. control cell-line DNA to an array platform. Sample preparation, hybridization, and scanning are performed over 1 week, but multiple samples can be prepared simultaneously, allowing for high-throughput processing and data acquisition. One microarray slide is required for each sample assayed, with a control sample hybridized with each test sample.

Detection and analysis of DNA methylation by pyrosequencing.

Pyrosequencing is a "sequencing by synthesis" technique which can be used to quantify DNA methylation at specific CpG sites within the target region of interest. Biotin labelled polymerase chain reaction (PCR) products form the template for base-pair nucleotide incorporation causing a light emitting cascade reaction resulting in the formation of a pyrogram and the calculation of the percentage methylation for each site. Prior to pyrosequencing, it is essential to bisulphite-convert the DNA sample and then perform locus-specific PCR for the region of interest. One of the PCR primers needs to be biotinylated and a separate sequencing primer is required for the pyrosequencing itself.