ALBERT

Description: The regulation of human microRNA (miRNA) expression is still poorly understood and aberrant epigenetic regulation has recently been implicated in the down-regulation of tumor suppressor miRNAs. In this study, we investigated whether histone modifications would contribute to the dysregulation of miRNAs in lymphoblastic leukemia cells. Using a precursor B-cell acute lymphoblastic leukemia cell line, NALM-6 cells, we demonstrated by miRNA microarray analysis that a specific histone deacetylases inhibitor, trichostatin A (TSA), induced a differential alteration in cellular miRNA expression. A total of 10 miRNAs were down-regulated and 31 up-regulated significantly following TSA treatment. Among TSA-up-regulated miRNAs, miR-22 is an extronic miRNA and resides in the second exon of the non-coding transcript MGC14376. Up-regulation of both miR-22 and MGC14376 was found in NALM-6 cells treated with TSA but not 5-AZA-2’-deoxycytidine, a DNA demethylating agent. Luciferase reporter analysis identified three regions in the promoter of miR-22 and MGC14376 that differentially regulated its transcriptional activation. Although there is a CpG island within the promoter of miR-22 and MGC14376, no obvious methylation was detected at this region in NALM-6 cells. Conversely, H3K27 trimethylation (H3K27triM)-associated histone modification was identified in the first intron of MGC14376 gene and was involved in TSA-induced miR-22 expression. Thus, miR-22 silencing in NALM-6 cells involves H3K27triM-associated histone modification but is independent of DNA methylation, suggesting that methylation-independent H3K27triM histone modification may be an important mechanism for miRNA dysregulation in cancer cells.

Description: Abstract 3621 Increasing evidence suggests that dysregulation of miRNAs plays an important pathological role in various malignant diseases including acute leukemia. To reveal the contributions of aberrant epigenetic modifications to the deregulated miRNA expression in precursor B-cell acute lymphoblastic leukemia, we examined the miRNA expression profile in NALM-6 cells after treatment with the combination of 5-AZA-2'-deoxycytidine (AZA) and trichostatin A (TSA). We found that the treatment significantly increased expression of 34 miRNAs and decreased the expression of 10 miRNAs. One of the most significantly upregulated miRNAs is miR-218, an intronic miRNA that can be transcribed from either pri-miR-218-1 or pri-miR-218-2, residing in the intron of the SLIT2 gene or SLIT3 gene respectively. Interestingly, we detected that pri-miR-218-1 and its host gene SLIT2, but not pri-miR-218-2 and SLIT3, were induced by AZA plus TSA treatment. Consistent with this observation, we showed that the CpG islands in SLIT2 promoter was highly methylated in NALM-6 cells and AZA plus TSA treatment significantly decreased DNA methylaiton in this region. We found that targeting of the 3'untranslated region of CDK6, a bona fide oncogenic factor, by miR-218 resulting in translational repression. Overexpression of miR-218 expression in NALM-6 cells by transfection of miR-218 precursor decreased cellular expression of CDK6 at the protein level, but not at the message level. AZA and TSA treatment decreased CDK6 expression in NALM-6 cells, presumably through upregulating miR-218. Our results indicate that epigenetic regulation plays an important role in controlling miRNA expression in human acute lymphoblastic leukemia cells. Epigenetic silencing of miR-218 may contribute to the overexpression of CDK6 in NALM-6 cells. Disclosures: No relevant conflicts of interest to declare.

Description: All-trans-retinoic acid (ATRA) induces growth inhibition, differentiation, and apoptosis in cancer cells, including acute promyelocytic leukemia (APL). In APL, expression of promyelocytic leukemia protein retinoic acid receptor–α (PML-RARα) fusion protein, owing to the t(15; 17) reciprocal translocation, leads to a block in the promyelocytic stage of differentiation. Here, we studied molecular mechanisms involved in ATRA-induced growth inhibition and myeloid cell differentiation in APL. By employing comprehensive high-throughput proteomic methods of 2-dimensional (2-D) gel electrophoresis and amino acid–coded mass tagging coupled with electrospray ionization (ESI) mass spectrometry, we systematically identified a total of 59 differentially expressed proteins that were consistently modulated in response to ATRA treatment. The data revealed significant down-regulation of eukaryotic initiation and elongation factors, initiation factor 2 (IF2), eukaryotic initiation factor 4AI (eIF4AI), eIF4G, eIF5, eIF6, eukaryotic elongation factor 1A-1 (eEF1A-1), EF-1-δ, eEF1γ, 14-3-3ϵ, and 14-3-3ζ/δ (P 〈 .05). The translational inhibitor DAP5/p97/NAT1 (death-associated protein 5) and PML isoform-1 were found to be up-regulated (P 〈 .05). Additionally, the down-regulation of heterogeneous nuclear ribonucleoproteins (hnRNPs) C1/C2, UP2, K, and F; small nuclear RNPs (snRNPs) D3 and E; nucleoprotein tumor potentiating region (TPR); and protein phosphatase 2A (PP2A) were found (P 〈 .05); these were found to function in pre-mRNA processing, splicing, and export events. Importantly, these proteomic findings were validated by Western blot analysis. Our data in comparison with previous cDNA microarray studies and our reverse transcription–polymerase chain reaction (RT-PCR) experiments demonstrate that broad networks of posttranscriptional suppressive pathways are activated during ATRA-induced growth inhibition processes in APL.

Description: Abstract 3464 Poster Board III-352 MicroRNA-22 is one of the miRNAs frequently downregulated in human ALL cells and may play an important anti-tumor role in normal hematopoiesis. Histone modification and DNA methylation can have different roles in gene silencing in cancer. To investigate whether histone modifications would contribute to the dysregulation of miRNA-22 in acute lymphoblastic leukemia (ALL), the effect of a histone deacetylase inhibitor, trichostatin A (TSA), on miRNA-22 expression of primary ALL cells was analyzed by real-time PCR. The total number of patients included to this study is 33, including 26 samples of leukemia (18 of ALL and 8 of acute myeloid leukemia) and 7 normal controls. All patient blood samples were collected at the time of diagnosis. We detected a lower expression of pri-miR-22 in PMBCs from ALL patients compared with that from the health volunteers. Treatment with TSA significantly increased pri-miR-22 expression in PMBCs from ALL patients, but not in cells from the health volunteers. Whereas PMBCs from ALL patients and AML patients showed comparable levels of pri-miR-22. TSA treatment had no effect on pri-miR-22 expression in PMBCs from AML patients, suggesting TSA-mediated upregulation of miR-22 transcription in ALL but not AML malignant cells. Moreover, we used MPS assay to analyze the methylation status at the promoter element of miR-22 gene in primary human specimens. No DNA hypermethylation was detected in PMBCs from the health volunteers and patients with either ALL or AML. These data provide further evidence that miR-22 silencing in ALL cells may be DNA methylation-independent. In contrast, accumulation of the repressive histone marker H3K27 trimethylation (H3K27triM) was indentified around the transcriptional start point of the gene, which reduced by TSA treatment. In conclusion, we showed that histone modification is involved in miRNA dysregulation in human ALL cells. Specifically, the silencing of miR-22 in ALL cells is associated with the accumulation of histone modification in its promoter element of miR-22 gene but independent of DNA methylation. The accumulation of H3K27triM may be a novel epigenetic mechanism for miR-22 silencing in ALL. Disclosures No relevant conflicts of interest to declare.

Description: Arsenic trioxide (ATO) induces differentiation and apoptosis in acute promyelocytic leukemia (APL). Compared with other leukemia cells, the APL-derived NB4 cells are particularly sensitive to ATO-induced apoptosis. Several reports indicate that in NB4 cells, apoptosis occurs in part by a mechanism that involves the inhibition of glutathione peroxidase, one of the enzymes that regulate the levels of H2O2 in the mitochondria, and that is present only at low levels in NB4 cells. Peroxirredoxin III (Prx III) is a mitochondria-specific H2O2-scavenger member of the thioredoxin-dependent peroxidases. NB4 cells express high levels of Prx III; however the role of Prx III in ATO-induced apoptosis in NB4 cells has not been investigated. We studied here whether Prx III is regulated during ATO-induced apoptosis in NB4 cells and whether the depletion of Prx III further sensitized these cells to ATO-induced apoptosis. The protein and mRNA levels of Prx III were decreased during ATO-induced apoptosis of NB4 cells. The down-regulation of Prx III occurred prior to the accumulation of reactive oxygen species, reduction in the mitochondrial membrane potential and apoptosis. Depletion of Prx III enhanced the ATO-induced mitochondrial damage, and also promoted cytochrome-c release, and caspase-3 and caspase-9 activation. Prx III is downregulated early during ATO-induced apoptosis, facilitating in this way the mitochondria-depending apoptotic events triggered by the accumulation of H2O2.