Association of aberrant ASNS imprinting with asparaginase sensitivity and chromosomal abnormality in childhood BCP-ALL

Abstract Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remain unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, but ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. The ASNS CpG island is largely unmethylated in normal hematopoietic cells, but it is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knockin mice. In 3 childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL patients with favorable karyotypes but is mostly unmethylated in BCP-ALL patients with poor prognostic karyotypes. Higher ASNS methylation is associated with higher L-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene as a result of aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.

Download Full-text

Association of allele-specific methylation of the ASNS gene with asparaginase sensitivity and prognosis in T-ALL

Blood Advances ◽

10.1182/bloodadvances.2021004271 ◽

2021 ◽

Author(s):

Koshi Akahane ◽

Shunsuke Kimura ◽

Kunio Miyake ◽

Atsushi Watanabe ◽

Keiko Kagami ◽

...

Keyword(s):

Gene Cluster ◽

Cell Lines ◽

Cpg Island ◽

Lymphoblastic Leukemia ◽

Methylation Status ◽

Aberrant Methylation ◽

Imprinted Gene ◽

Allele Specific ◽

Allele Specific Methylation

Asparaginase therapy is a key component of chemotherapy for T-cell acute lymphoblastic leukemia (T-ALL) patients. Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, while leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Since the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite PCR products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from childhood T-ALL patients in Japanese cohorts (n = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In childhood T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively showed ASNS hypomethylation status. These observations demonstrate that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

Download Full-text

Hypermethylation-Associated Silencing of miR-125a and miR-125b: A Potential Marker in Colorectal Cancer

Disease Markers ◽

10.1155/2015/345080 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 23

Author(s):

Hui Chen ◽

Zhiying Xu

Keyword(s):

Colorectal Cancer ◽

Cpg Island ◽

Methylation Status ◽

Pcr Analysis ◽

Aberrant Methylation ◽

Dna Hypermethylation ◽

Expression Levels ◽

Qrt Pcr ◽

Potential Marker ◽

Potential Biomarker

Background. MicroRNAs (miRNAs) have been found to be downregulated in human colorectal cancer (CRC), and some of them may function as tumor suppressor genes (TSGs). Aberrant methylation triggers the inactivation of TSGs during tumorigenesis.Patients and Methods. We investigated the methylation status of miR-125 family in CRC tissues and adjacent nontumor tissues by using bisulfite sequencing PCR (BSP). The expression levels of the two miRNAs were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR).Results. The methylation frequency of miR-125a and miR-125b was higher in CRC tissues. QRT-PCR analysis showed that miR-125a and miR-125b were significantly downregulated in CRC tissues. Moreover, the expression levels of miR-125a and miR-125b were inversely correlated to CpG island methylation in CRC.Conclusions. Our results suggest that DNA hypermethylation may be involved in the inactivation of miR-125a and miR-125b in CRC, and hypermethylation of miR-125 is a potential biomarker for clinical outcome.

Download Full-text

Epigenetic Status Of CEBPA Promoter and Expression Of CEBPα Correlates With Molecular Genetic Subtype and CD2 Aberrant Expression In B Cell Precursor ALL

Blood ◽

10.1182/blood.v122.21.2573.2573 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2573-2573

Author(s):

Lucie Slamova ◽

Julia Starkova ◽

Karel Fiser ◽

Eva Fronkova ◽

Leona Rezkova Reznickova ◽

...

Keyword(s):

B Cell ◽

Early Phase ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Methylation Status ◽

Fisher Exact Test ◽

Cell Precursor ◽

Aberrant Expression ◽

Mll Gene

Abstract CCAAT/enhancer binding protein alpha (CEBPα) is one of the crucial transcription factors involved in hematopoietic differentiation and leukemogenesis. CEBPα promotes myeloid differentiation by up-regulation of lineage specific genes and by cell proliferation arrest. Epigenetic regulation of CEBPα expression through DNA methylation has been demonstrated in acute myeloid leukemia (AML) (Figueroa et al, Cancer Cell, 2010). However, only limited data are available regarding CEBPA promoter methylation and its expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Methylation status of CEBPA promoter (-295 to -593bp upstream of the transcription start site (TSS), 24 CpG dinucleotides) was analyzed by bisulfite sequencing. Five subgroups of BCP-ALLs were analyzed: MLL gene rearranged (n=5), hyperdiploid (n=6), mBCR-ABLpos(n=5), ETV6-RUNX1pos(n=6) and other BCP-ALLs (no hyperdiploidy, MLL gene rearrangement, BCR-ABL or ETV6-RUNX1 fusion gene (“BCP-others”, n=29)). CEBPA promoter was hypermethylated in MLL-rearranged, hyperdiploid and ETV6-RUNX1pos BCP-ALL (5/5, 6/6 and 4/6 respectively). Surprisingly CEBPA promoter was hypomethylated in all mBCR-ABLpos cases (5/5). In subgroup of other BCP-ALLs both hypermethylation (10/29) and hypomethylation of CEBPA promoter (19/29) were detected (Figure 1A). In previous study we found association of CD2 (LFA-2) aberrant expression and switch to the monocytic lineage during the early phase of treatment in BCP-ALLs (Slamova et al, ASH 2012). We were interested if a possible link between hypomethylation of CEBPA promoter correlates with aberrant expression of CD2. There was a significant association between aberrant expression of CD2 antigen and hypomethylation in CEBPA promoter in BCP-others (Fisher exact test, p<0.0001). Interestingly, in the only hypomethylated ETV6-RUNX1pos case we found aberrant CD2 expression on blasts, which is exceptional in ETV6-RUNX1pos ALL. We next asked whether methylation of CEBPA promoter correlates with CEBPα expression. It is generally accepted that promoter hypomethylation is often associated with increased expression of the relevant gene. Our data prove that in general, this holds true also for BCP-ALL. However, in two genetically defined subsets we observed either high expression despite hypermethylation (MLL-rearranged ALL) or low expression despite hypomethylation (mBCR-ABLpos ALL) (Figure 1B). In BCP-others hypomethylation of CEBPA promoter was significantly associated with upregulation of myeloid antigens (CD14 and/or CD33) and downregulation of B cell marker CD19 on blasts during the first weeks of the treatment (Fisher test, p=0.0009). In summary Methylation status of CEBPA promoter correlates with genetic subtypes of BCP-ALL. The notion that hypomethylation leads to overexpression was confirmed in majority of BCP-ALLs, while in mBCR-ABLpos and MLL gene rearranged BCP-ALL it did not follow this pattern. Hypomethylation of CEBPA promoter in BCP- others correlates with CD2 expression on blasts and increased CEBPα gene expression. During the early phase of the treatment in other BCP-ALLs with hypomethylated CEBPA promoter increase of myeloid and decrease of B lymphoid markers on blasts was observed. Supported by: GACR P301/10/1877, GACR P304/12/2214, GAUK 914613, UNCE 204012, NT13462, NT12397- 4, project for conceptual development of research organization (Ministry of Health, CZ) 00064203, the FACS Aria instrument was supported by EU-Prague project CZ.2.16/3.1.00/24022 Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Aberrant DNA methylation and epigenetic inactivation of Eph receptor tyrosine kinases and ephrin ligands in acute lymphoblastic leukemia

Blood ◽

10.1182/blood-2009-05-222208 ◽

2010 ◽

Vol 115 (12) ◽

pp. 2412-2419 ◽

Cited By ~ 58

Author(s):

Shao-Qing Kuang ◽

Hao Bai ◽

Zhi-Hong Fang ◽

Gonzalo Lopez ◽

Hui Yang ◽

...

Keyword(s):

Dna Methylation ◽

Acute Lymphoblastic Leukemia ◽

Cpg Island ◽

Lymphoblastic Leukemia ◽

Methylation Status ◽

Eph Receptors ◽

Raji Cells ◽

Eph Receptor ◽

Phosphorylated Akt ◽

Ephrin Ligands

Eph receptors and their ephrin ligands are involved in normal hematopoietic development and tumorigenesis. Using methylated CpG island amplification/DNA promoter microarray, we identified several EPH receptor and EPHRIN genes as potential hypermethylation targets in acute lymphoblastic leukemia (ALL). We subsequently studied the DNA methylation status of the Eph/ephrin family by bisulfite pyrosequencing. Hypermethylation of EPHA2, -A4, -A5, -A6, -A7, -A10, EPHB1, -B2, -B3, -B4, EFNA1, -A3, -A5, and EFNB1 and -B2 genes was detected in leukemia cell lines and primary ALL bone marrow samples. Expression analysis of EPHB4, EFNB2, and EFNA5 genes demonstrated that DNA methylation was associated with gene silencing. We cloned the promoter region of EPHB4 and demonstrated that promoter hypermethylation can result in EPHB4 transcriptional silencing. Restoration of EPHB4 expression by lentiviral transduction resulted in reduced proliferation and apoptotic cell death in Raji cells in which EPHB4 is methylated and silenced. Finally, we demonstrated that phosphorylated Akt is down-regulated in Raji cells transduced with EPHB4. These results suggest that epigenetic silencing by hypermethylation of EPH/EPHRIN family genes contributes to ALL pathogenesis and that EPHB4 can function as a tumor suppressor in ALL.

Download Full-text

Methylation profiling in acute myeloid leukemia

Blood ◽

10.1182/blood.v97.9.2823 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2823-2829 ◽

Cited By ~ 233

Author(s):

Minoru Toyota ◽

Kenneth J. Kopecky ◽

Mutsumi-Ohe Toyota ◽

Kam-Wing Jair ◽

Cheryl L. Willman ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Correlation Coefficients ◽

Aberrant Methylation ◽

Independent Events ◽

Methylation Defects ◽

Acute Myeloid

Abstract Aberrant methylation of multiple CpG islands has been described in acute myeloid leukemia (AML), but it is not known whether these are independent events or whether they reflect specific methylation defects in a subset of cases. To study this issue, the methylation status of 14 promoter-associated CpG islands was analyzed in 36 cases of AML previously characterized for estrogen-receptor methylation (ERM). Cases with methylation density of 10% or greater were considered positive. Seventeen cases (47%) were ERM+ while 19 cases were ERM−. Hypermethylation of any of the following,p15, p16, CACNA1G,MINT1, MINT2, MDR1,THBS1, and PTC1 (2 promoters), was relatively infrequent (6% to 31% of patients). For each of these CpG islands, the methylation density was positively correlated with ERM density (rank order correlation coefficients, 0.32-0.59; 2-tailedP ≤ .058 for each gene). Hypermethylation ofMYOD1, PITX2, GPR37, andSDC4 was frequently found in AML (47% to 64% of patients). For each of these genes as well, methylation density was positively correlated with ERM density (correlation coefficients 0.43 to 0.69, P ≤ .0087 for each gene). MLH1 was unmethylated in all cases. Hypermethylation of p15,MDR1, and SDC4 correlated with reduced levels of expression. There was an inverse correlation between age and the number of genes methylated (P = .0030). It was concluded that CpG-island methylation in AML results from methylation defects in subsets of cases. These results have potential implications for the classification and prognosis of AML and for the identification of patients who may benefit from treatment with methylation inhibitors.

Download Full-text

B-Cell Precursor Acute Lymphoblastic Leukemia Cells Create a Self-Reinforcing Niche Independent of the CXCR4/CXCL12 Axis

Blood ◽

10.1182/blood.v126.23.1297.1297 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1297-1297

Author(s):

Bob de Rooij ◽

Roel Polak ◽

Rob Pieters ◽

Monique L. Den Boer

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Leukemic Cells ◽

Cell Precursor ◽

Cxcr4 Antagonist

Abstract Background Acute lymphoblastic leukemia (ALL) cells create a leukemic niche that protects malignant cells from the effects of cytostatic agents and immune cells by altering their bone marrow microenvironment. This malignant process can be counteracted by impairing the homing of leukemic cells towards the bone marrow. Hematopoietic cells express the chemokine receptor CXCR4 and migrate towards its ligand CXCL12, which is actively produced by MSCs in the bone marrow. Therefore clinical trials have been initiated using the CXCR4 antagonist AMD3100 (Plerixafor) during leukemia treatment. However, these trials, as well as priming of AML in more than 4000 patients using a CXCR4 dependent mechanism, have not resulted in improved overall survival rates. This suggests that CXCR4 inhibition is not sufficient to disrupt leukemic niches. Objectives In this study we investigated how leukemic cells regulate the chemoattractive properties of their microenvironment. Results Here we show, using an ex vivo niche model with primary MSCs, that B-cell precursor ALL (BCP-ALL) cells affect their healthy microenvironment without altering CXCL12 secretion. Using a transwell migration assay we studied the chemoattractive properties and chemokine secretion patterns of several cell types and co-cultures. We confirmed that BCP-ALL cells migrate towards a CXCL12 gradient produced by primary MSCs (11-fold more migrated cells compared to background, p < 0.001). Inhibition of CXCR4 by AMD3100 reduced migration towards MSCs by 80% (p < 0.01). BCP-ALL cells migrated even more towards co-cultures of BCP-ALL cells and primary MSCs (24-fold more migrated cells compared to background, p < 0.001). Strikingly, this ex vivo leukemic niche did not produce higher levels of CXCL12 compared to MSC mono-cultures. Moreover, the induced migration towards MSC-ALL co-cultures could not be inhibited by AMD3100 treatment, indicating that BCP-ALL cells enhance the chemoattractive properties of their microenvironment in a CXCL12-independent manner. In contrast to BCP-ALL cells, the migration of CD34+ progenitor cells towards co-cultures of BCP-ALL cells and MSCs was significantly reduced (0.8-fold compared to migration towards MSCs, p < 0.05). Similar results were observed when we studied the migratory behavior of MSCs. MSCs actively migrated towards BCP-ALL cells (1.7 fold compared to background, p < 0.001), while migration of MSCs was significantly reduced towards MSC-ALL co-cultures (0.4-fold compared to migration towards BCP-ALL, p < 0.001). To find candidate factors influencing this process, we quantified the secreted levels of 64 cytokines in co-cultures of patient-derived BCP-ALL cells and MSCs. We observed leukemia-driven cytokine secretion patterns that were not influenced by the source of primary MSCs. In contrast to unaltered levels of CXCL12, we observed significant inductions of MCP-1/CCL2 and MDC/CCL22 (CCR4-ligands), IL8 and GRO-1 (CXCR1/2-ligands) and IP10/CXCL10 (CXCR3-ligands). Conclusion Our data indicate that leukemic cells alter the chemoattractive properties of their microenvironment, resulting in the secretion of multiple chemokines into the leukemic niche. This leukemic niche is highly potent in attracting BCP-ALL cells and repels the influx of healthy hematopoietic cells and MSCs using a CXCL12-independent mechanism. Furthermore, our results identify candidate factors that might be valuable future therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Epigenetic Modification of Death Receptor Genes for TRAIL and TRAIL Resistance in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

Genes ◽

10.3390/genes12060864 ◽

2021 ◽

Vol 12 (6) ◽

pp. 864

Author(s):

Atsushi Watanabe ◽

Kunio Miyake ◽

Koshi Akahane ◽

Kumiko Goi ◽

Keiko Kagami ◽

...

Keyword(s):

T Cells ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Methylation Status ◽

Leukemia Cells ◽

Cell Surface Expression ◽

Clinical Samples ◽

Cell Precursor ◽

Trail Resistance

Immunotherapies specific for B-cell precursor acute lymphoblastic leukemia (BCP-ALL), such as anti-CD19 chimeric antigen receptor (CAR) T-cells and blinatumomab, have dramatically improved the therapeutic outcome in refractory cases. In the anti-leukemic activity of those immunotherapies, TNF-related apoptosis-inducing ligand (TRAIL) on cytotoxic T-cells plays an essential role by inducing apoptosis of the target leukemia cells through its death receptors (DR4 and DR5). Since there are CpG islands in the promoter regions, hypermethylation of the DR4 and DR5 genes may be involved in resistance of leukemia cells to immunotherapies due to TRAIL-resistance. We analyzed the DR4 and DR5 methylation status in 32 BCP-ALL cell lines by sequencing their bisulfite PCR products with a next-generation sequencer. The DR4 and DR5 methylation status was significantly associated with the gene and cell-surface expression levels and the TRAIL-sensitivities. In the clinical samples at diagnosis (459 cases in the NOPHO study), both DR4 and DR5 genes were unmethylated in the majority of cases, whereas methylated in several cases with dic(9;20), MLL-rearrangement, and hypodiploidy, suggesting that evaluation of methylation status of the DR4 and DR5 genes might be clinically informative to predict efficacy of immunotherapy in certain cases with such unfavorable karyotypes. These observations provide an epigenetic rational for clinical efficacy of immunotherapy in the vast majority of BCP-ALL cases.

Download Full-text

Hypermethylation of the p15INK4B Gene in Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v90.4.1403.1403_1403_1409 ◽

1997 ◽

Vol 90 (4) ◽

pp. 1403-1409 ◽

Cited By ~ 6

Author(s):

Toshiki Uchida ◽

Tomohiro Kinoshita ◽

Hirokazu Nagai ◽

Yohsuke Nakahara ◽

Hidehiko Saito ◽

...

Keyword(s):

Kinase Inhibitor ◽

Malignant Tumors ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Hematologic Malignancies ◽

Genetic Alterations ◽

Refractory Anemia ◽

Aberrant Methylation ◽

Cyclin Dependent Kinase Inhibitor

Previous studies have shown that the cyclin-dependent kinase inhibitor (CDKI) genes p15INK4B and p16INK4A are frequently inactivated by genetic alterations in many malignant tumors and that they are candidate tumor-suppressor genes. Although genetic alterations in these genes may be limited to lymphoid malignancies, it has been reported that their inactivation by aberrant methylation of 5′ CpG islands may be involved in various hematologic malignancies. In this study, we investigated the p15INK4B and p16INK4A genes to clarify their roles in the pathogenesis of myelodysplastic syndrome (MDS). Southern blotting analysis showed no gross genetic alterations in either of these genes. However, hypermethylation of the 5′ CpG island of the p15INK4B gene occurred frequently in patients with MDS (16/32 [50%]). Interestingly, the p15INK4B gene was frequently methylated in patients with high-risk MDS (refractory anemia with excess blasts [RAEB], RAEB in transformation [RAEB-t], and overt leukemia evolved from MDS; 14/18 [78%]) compared with patients with low-risk MDS (refractory anemia [RA] and refractory anemia with ring sideroblast [RARS]; 1/12 [8%]). Furthermore, methylation status of the p15INK4B gene was progressed with the development of MDS in most patients examined. In contrast, none of the MDS patients showed apparent hypermethylation of the p16INK4A gene. These results suggest that hypermethylation of the p15INK4B gene is involved in the pathogenesis of MDS and is one of the important late events during the development of MDS.

Download Full-text

Adipose Tissue LPL Methylation is Associated with Triglyceride Concentrations in the Metabolic Syndrome

Clinical Chemistry ◽

10.1373/clinchem.2017.277921 ◽

2018 ◽

Vol 64 (1) ◽

pp. 210-218 ◽

Cited By ~ 15

Author(s):

Daniel Castellano-Castillo ◽

Isabel Moreno-Indias ◽

José Carlos Fernández-García ◽

Juan Alcaide-Torres ◽

Inmaculada Moreno-Santos ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Syndrome ◽

Dna Methylation ◽

Adipose Tissue ◽

Cpg Island ◽

Methylation Status ◽

The Metabolic Syndrome ◽

Postprandial Triglycerides ◽

Gene And Protein Expression ◽

Lpl Gene

Abstract BACKGROUND DNA methylation is one of the epigenetic mechanisms that regulate gene expression. DNA methylation may be modified by environmental and nutritional factors. Thus, epigenetics could potentially provide a mechanism to explain the etiology of metabolic disorders, such as metabolic syndrome (MetS). The aim of this study was to analyze the level of DNA methylation of several lipoprotein lipase (LPL)-promoter-CpG dinucleotides in a CpG island region and relate this to the gene and protein expression levels in human visceral adipose tissue (VAT) from individuals with and without MetS. METHODS VAT samples were collected from laparoscopic surgical patients without and with MetS, and levels of LPL mRNA, LPL protein, and LPL DNA methylation were measured by qPCR, western blot, and pyrosequencing. Biochemical and anthropometric variables were analyzed. Individuals included in a subset underwent a dietary fat challenge test, and levels of postprandial triglycerides were determined. RESULTS We found higher levels of DNA methylation in MetS patients but lower gene expression and protein levels. There was a negative association between LPL methylation and LPL gene expression. We found a positive association between LPL methylation status and abnormalities of the metabolic profile and basal and postprandial triglycerides, whereas LPL gene expression was negatively associated with these abnormalities. CONCLUSIONS We demonstrate that LPL methylation may be influenced by the degree of metabolic disturbances and could be involved in triglyceride metabolism, promoting hypertriglyceridemia and subsequent associated disorders, such as MetS.

Download Full-text

Aberrant Promoter Methylation of the ABCG2 Gene in Renal Carcinoma

Molecular and Cellular Biology ◽

10.1128/mcb.00650-06 ◽

2006 ◽

Vol 26 (22) ◽

pp. 8572-8585 ◽

Cited By ~ 87

Author(s):

Kenneth K. W. To ◽

Z. Zhan ◽

Susan E. Bates

Keyword(s):

Dna Methylation ◽

Promoter Methylation ◽

Renal Carcinoma ◽

Cpg Island ◽

Transmembrane Protein ◽

Methylation Status ◽

Luciferase Reporter ◽

Restriction Enzyme Digestion ◽

Aberrant Methylation ◽

Histone Deacetylase 1

ABSTRACT ABCG2 is a ubiquitous ATP-binding cassette transmembrane protein that is important in clinical drug resistance. Little is known about the mechanism(s) regulating the expression of ABCG2. We hypothesized that DNA methylation could play a role in the epigenetic regulation of ABCG2 gene expression. The promoter methylation status of three renal carcinoma cell lines was assessed with restriction enzyme digestion-coupled PCR and bisulfite genomic sequencing. Both UOK121 and UOK143, with known methylation of the VHL promoter, showed induction of ABCG2 expression after 5-aza-2′-deoxycytidine (5-aza-dC) treatment, suggesting that aberrant methylation of the ABCG2 gene was associated with gene silencing. In vitro methylation of the ABCG2 promoter-driven luciferase reporter vector resulted in a significant inhibition of transcription. Our data suggested that the ABCG2 gene is regulated coordinately at both histone and DNA levels. A chromatin immunoprecipitation assay demonstrated that the methylated promoter in UOK121 and UOK143, but not the unmethylated one in UOK181, is associated with the methyl CpG binding domain proteins (MBDs), MBD2 and MeCP2. Histone deacetylase 1 and a corepressor, mSin3A, were identified binding to the promoter region containing the CpG island, thereby suppressing ABCG2 transcription. Reactivation of ABCG2 was achieved by treatment with 5-aza-dC, a demethylating agent, concomitant with the release of MBDs from the promoter. Furthermore, the association of methylated lysine 9 on histone H3, a hallmark of promoter methylation, with the promoter was reduced following 5-aza-dC treatment. These data suggest that DNA methylation-dependent formation of a repressor complex in the CpG island contributes to inactivation of ABCG2.

Download Full-text