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Analysis of methylation-sensitive transcriptome identifies GADD45a as a frequently methylated gene in breast cancer

Oncogene volume 24, pages 2705–2714 (2005)Cite this article

Abstract

Treatment of the breast cancer cell line, MDAMB468 with the DNA methylation inhibitor, 5-azacytidine (5-AzaC) results in growth arrest, whereas the growth of the normal breast epithelial line DU99 (telomerase immortalized) is relatively unaffected. Comparing gene expression profiles of these two lines after 5-AzaC treatment, we identified 36 genes that had relatively low basal levels in MDAMB468 cells compared to the DU99 line and were induced in the cancer cell line but not in the normal breast epithelial line. Of these genes, 33 have associated CpG islands greater than 300 bp in length but only three have been previously described as targets for aberrant methylation in human cancer. Northern blotting for five of these genes (α-Catenin, DTR, FYN, GADD45a, and Zyxin) verified the array results. Further analysis of one of these genes, GADD45a, showed that 5-AzaC induced expression in five additional breast cancer cell lines with little or no induction in three additional lines derived from normal breast epithelial cells. The CpG island associated with GADD45a was analysed by bisulfite sequencing, sampling over 100 CpG dinucleotides. We found that four CpG's, located approximately 700 bp upstream of the transcriptional start site are methylated in the majority of breast cancer cell lines and primary tumors but not in DNA from normal breast epithelia or matched lymphocytes from cancer patients. Therefore, this simple method of dynamic transcriptional profiling yielded a series of novel methylation-sensitive genes in breast cancer including the BRCA1 and p53 responsive gene, GADD45a.

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Acknowledgements

We are indebted to Nancy Glover for her ongoing technical assistance. This work was supported by the NCI funded Early Detection Research Network Grant, CA84955 awarded to JRM.

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  1. Wei Wang

    Present address: Department of Medicine, University of Washington, Seattle, WA, 98195, USA

Authors and Affiliations

  1. Department of Surgery, Duke University Medical Center, Durham, NC, 27710, USA

    Wei Wang, Gudrun Huper, Yueqi Guo, John A Olson Jr & Jeffrey R Marks

  2. Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC, 27710, USA

    Susan K Murphy

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Correspondence to Jeffrey R Marks.

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Wang, W., Huper, G., Guo, Y. et al. Analysis of methylation-sensitive transcriptome identifies GADD45a as a frequently methylated gene in breast cancer. Oncogene 24, 2705–2714 (2005). https://doi.org/10.1038/sj.onc.1208464

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