Related Experiment Videos
De novo methylation of selective CpG dinucleotide clusters in transformed cells mediated by an activated N-ras
N P Kisseljova1, E S Zueva, V S Pevzner
1Institute of Carcinogenesis, Cancer Research Center, Kashirskoye shosse 24, Moscow 115478, Russia.
Abstract:
We have explored a possible role of an activated N-ras oncogene in aberrant methylation of CpG clusters in DNAs of transformed cells. Using three lines of hamster cells transformed by Rous sarcoma virus (RSV) and the method of detection of CpG islands as clustered sites for methylation-sensitive restriction enzymes we have demonstrated that in each cell line the transcribed RSV proviruses are integrated in the vicinity of sequence containing the cluster of unmethylated CpG dinucleotides. Two out of three examined CpG clusters had hypermethylation patterns in N-ras-neo- but not in neo-transfected variants of the cell lines. De novo methylation of CpG dinucleotides correlated with transcriptional inactivation of adjacent RSV proviruses that was related neither to the lack of transcriptional factors binding RSV long terminal repeat (LTR) nor to the transcriptional incompetence of the LTR, as measured by reporter gene assays with the LTR cloned from DNA of these cells. These data suggest that activation of N-ras signal transduction pathway in transformed cells may be relevant to long-term inactivation of selective genes by hypermethylation of their CpG islands.
Insights
Activated N-ras oncogenes may trigger aberrant DNA methylation in transformed cells. This CpG island hypermethylation correlates with gene silencing, suggesting a role in long-term gene inactivation.
Area of Science:
- Molecular Biology
- Cancer Genetics
- Epigenetics
Background:
- Aberrant DNA methylation is a hallmark of cancer.
- The role of specific oncogenes, like N-ras, in driving these epigenetic changes is not fully understood.
Purpose of the Study:
- To investigate the potential role of activated N-ras oncogene in aberrant CpG island methylation in Rous sarcoma virus (RSV)-transformed hamster cells.
- To determine if N-ras activation influences the methylation status of CpG clusters and subsequent gene expression.
Main Methods:
- Utilized three lines of RSV-transformed hamster cells.
- Employed methylation-sensitive restriction enzyme analysis to detect CpG islands.
- Compared methylation patterns in N-ras-transfected versus control (neo-transfected) cell variants.
- Assessed transcriptional activity using reporter gene assays with the RSV long terminal repeat (LTR).
Main Results:
- Transcribed RSV proviruses were found near unmethylated CpG clusters in all cell lines.
- Two of three examined CpG clusters exhibited hypermethylation in N-ras-transfected cells, but not in neo-transfected controls.
- De novo CpG methylation correlated with transcriptional inactivation of adjacent RSV proviruses.
- Inactivation was independent of transcriptional factor binding or LTR competence.
Conclusions:
- Activation of the N-ras signal transduction pathway in transformed cells is implicated in the long-term inactivation of genes via CpG island hypermethylation.
- N-ras-mediated epigenetic modifications may contribute to cellular transformation and gene regulation in cancer.