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Updated: May 12, 2026
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Putative tumour suppressor gene necdin is hypermethylated and mutated in human cancer
L E De Faveri1, C D Hurst, F M Platt
1Section of Experimental Oncology, Cancer Research UK Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Beckett Street, Leeds LS97TF, UK.
Background:
Necdin (NDN) expression is downregulated in telomerase-immortalised normal human urothelial cells. Telomerase-immortalised normal human urothelial cells have no detected genetic alterations. Accordingly, many of the genes whose expression is altered following immortalisation are those for which epigenetic silencing is reported.
Methods:
NDN expression was examined in normal tissues and tumour cell lines by quantitative real-time PCR and immunoblotting. Immunohistochemistry was performed on urothelial carcinoma (UC). Urothelial carcinoma and UC cell lines were subject to HumanMethylation27 BeadChip Array-based methylation analyses. Mutation screening was performed. The functional significance of NDN expression was investigated using retroviral-mediated downregulation or overexpression.
Results:
NDN protein was widely expressed in normal tissues. Loss of expression was observed in 38 out of 44 (86%) of UC cell lines and 19 out of 25 (76%) of non-UC cell lines. Loss of NDN protein was found in the majority of primary UC. Oncomine analysis demonstrated downregulation of expression in multiple tumour types. In UC, tumour-specific hypermethylation of NDN and key CpG sites where hypermethylation correlated with reduced expression were identified. Six novel mutations, including some of predicted functional significance, were identified in colorectal and ovarian cancer cell lines. Functional studies showed that NDN could suppress colony formation at low cell density and affect anchorage-independent growth and anoikis in vitro.
Conclusion:
NDN is a novel tumour suppressor candidate that is downregulated and hypermethylated or mutated in cancer.
Insights
Necdin (NDN) is a novel tumor suppressor candidate. Its expression is lost in many cancers due to hypermethylation or mutation, impacting cell growth and survival.
Area of Science:
- Oncology
- Molecular Biology
- Epigenetics
Background:
- Necdin (NDN) expression is reduced in immortalized human urothelial cells.
- Epigenetic silencing is a common mechanism for gene expression alteration during cell immortalization.
Purpose of the Study:
- To investigate the role of Necdin (NDN) as a tumor suppressor.
- To analyze NDN expression, methylation, and mutation status in various cancers.
Main Methods:
- Quantitative real-time PCR and immunoblotting for NDN expression analysis.
- Immunohistochemistry on urothelial carcinoma (UC) tissues.
- DNA methylation analysis using HumanMethylation27 BeadChip Array.
- Mutation screening and functional studies (downregulation/overexpression).
Main Results:
- NDN protein is widely expressed in normal tissues but lost in 86% of UC cell lines and 76% of non-UC cell lines.
- Tumor-specific hypermethylation of NDN correlated with reduced expression in UC.
- Novel NDN mutations were identified in colorectal and ovarian cancer cell lines.
- NDN suppressed colony formation, anchorage-independent growth, and anoikis in vitro.
Conclusions:
- NDN is a novel tumor suppressor candidate.
- NDN downregulation in cancer is associated with hypermethylation or mutation.
- NDN plays a role in regulating cell proliferation and survival.
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