Quantitation of Inhibition of DNA Methylation of the Retinoic Acid Receptor β Gene by 5-Aza-2′-deoxycytidine in Tumor Cells Using a Single-Nucleotide Primer Extension Assay

doi:10.1006/abio.2000.4562

Analytical Biochemistry

Volume 281, Issue 1, 15 May 2000, Pages 55-61

https://doi.org/10.1006/abio.2000.4562 Get rights and content

Abstract

The expression of several cancer-related genes has been reported to be silenced by DNA methylation of their promoter region. 5-Aza-2′-deoxycytidine (5-AZA-CdR), a potent and specific inhibitor of DNA methylation, can reactivate the in vitro expression of these genes. In future clinical trials in tumor therapy with 5-AZA-CdR a method to quantitate its inhibition of methylation of specific tumor suppressor genes would provide important data for the analysis of the therapeutic efficacy of this analogue. We have modified the methylation-sensitive single-nucleotide primer extension assay reported by Gonzalgo and Jones (Nucleic Acids Res. 25, 2529–2531, 1997). Genomic DNA was treated with bisulfite and a fragment of the promoter region of the human retinoic acid receptor β (RARβ) gene, a tumor suppressor gene, was amplified using seminested PCR. Using two different primers we quantitated the inhibition of methylation produced by 5-AZA-CdR at two specific CpG sites in the RARβ promoter in a human colon and a breast carcinoma cell line. The results obtained with the modified assay show a precise and reproducible quantitation of inhibition of DNA methylation produced by 5-AZA-CdR in tumor cells.

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Cited by (7)

The effects of cladribine and fludarabine on DNA methylation in K562 cells
2003, Biochemical Pharmacology
The effects of the antileukemic adenosine analogues, 2-chloro-2′-deoxyadenosine (cladribine) and 9-β-d-arabinosyl-2-fluoroadenine (fludarabine), on DNA methylation were studied in a cell line K562. It was previously found that both drugs inactivated SAH hydrolase, an enzyme which participates in the “active methyl” cycle. The study examined the effects of these drugs on three aspects of DNA methylation: (i) activity of endogenous C-5 DNA methyltransferase; (ii) capacity of genomic DNA (gDNA) to accept methyl groups, transferred from S-adenosylmethionine by the bacterial methyltransferase, SssI; (iii) estimation of changes of methylated cytosine levels in gDNA, using methylation-dependent restriction analysis. Cladribine and fludarabine inhibited C-5 DNA methyltransferase, with ed₅₀ values of 3.5 and 47.0 μM, respectively, after 24 hr cell growth in the presence of the drugs. After 48 hr growth of cells with cladribine (0.1 μM) or fludarabine (3 μM), the capacity of DNA to accept methyl groups, in the presence of exogenous bacterial SssI methylase, increased by approximately 1.8 and 1.6 times, respectively, compared to control DNA. Digestion of gDNA with endonucleases HpaII and BssHII followed by SssI DNA methylation, indicated that cladribine (0.1 μM) reduced the level of methylated cytosines in both CpG islands and CCGG sequences, sensitive to HpaII restriction enzyme. Inhibition of DNA methylation by fludarabine was observed mainly in CpG dinucleotide located within sequences sensitive to HpaII. The perturbation of DNA methylation was considered as a complex process. Our findings for cladribine and fludarabine should be regarded as an extra element of their antileukemic efficacy.
Potential of 5-aza-2′-deoxycytidine (Decitabine) a potent inhibitor of DNA methylation for therapy of advanced non-small cell lung cancer
2001, Lung Cancer
Although new agents and drug combinations have increased the response rate in advanced non-small cell lung cancer (NSCLC), long-term survivors are rare. There is an urgent need to develop new chemotherapeutic approaches for disease. In a previous pilot phase I–II study on 5-aza-2′-deoxycytidine (5-AZA-CdR) in patients with stage IV NSCLC, we observed several interesting responses, including one patient that was still alive (68 months) at the time of publication of our results. In the present report, we want to point out the long-term follow up of this patient, who survived 81 months, and discuss the interesting mechanism of action of 5-AZA-CdR that may have been responsible for this interesting response. 5-AZA-CdR is a potent inhibitor of DNA methylation. Recent progress in this field has shown that aberrant methylation of the promoter region of tumor suppressor genes inhibits their expression. This epigenetic event can contribute to tumorigenesis. Since 5-AZA-CdR can reactivate these genes by blocking DNA methylation, it has the potential to reverse tumorigenesis. This novel mode of action makes it an interesting agent to investigate for the chemotherapy of malignant disease, including lung cancer.
A modified protocol for bisulfite genomic sequencing of difficult samples
2009, Biological Procedures Online
Allelic methylation bias of the RARB2 tumor suppressor gene promoter in cancer
2008, Genes Chromosomes and Cancer
Study on the relationship between the RAR-β gene expressive defection and its methylation
2007, Academic Journal of Xi'an Jiaotong University
Methylation matters
2001, Journal of Medical Genetics

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¹: To whom correspondence should be addressed at Centre de Recherche Pédiatrique, Hôpital Ste-Justine, 3175 Côte Ste-Catherine, Montréal, Québec H3T 1C5, Canada. Fax: (514)-345-4801.

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Regular ArticleQuantitation of Inhibition of DNA Methylation of the Retinoic Acid Receptor β Gene by 5-Aza-2′-deoxycytidine in Tumor Cells Using a Single-Nucleotide Primer Extension Assay

Abstract

Leukemia Res.

Pharmacol. Ther.

Anal. Biochem.

Biochem. Pharmacol.

Cancer epigenetics comes of age

Nature Genet.

5′ CpG islands methylation is associated with transcriptional silencing of the tumor suppressor gene p16/CDKN2/MTS1 in human cancers

Nat. Med.

Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma

Proc. Natl. Acad. Sci. USA

Frequency and parental origin of hypermethylated Rb1 alleles in retinoblastoma

Hum. Genet.

Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B1

Cancer Res.

Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cells lines

Cancer Res.

Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas

Proc. Natl. Acad. Sci. USA

Distinct patterns of E-cadherin CpG island methylation in papillary, follicular, Hurle's cell, and poorly differentiated human thyroid carcinoma

Cancer Res.

Association between CpG islands methylation and microsatellite instability in colorectal cancer

Cancer Res.

Pilot study of 5-aza-2′-deoxycytidine (Decitabine) in the treatment of poor prognosis acute myelogenous leukemia patients: Preliminary results

Leukemia

Regular Article
Quantitation of Inhibition of DNA Methylation of the Retinoic Acid Receptor β Gene by 5-Aza-2′-deoxycytidine in Tumor Cells Using a Single-Nucleotide Primer Extension Assay

Hypermethylation-associated inactivation indicates a tumor suppressor role for p15^INK4B1