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Methylation and Cervical Tumors

#3312


Methylation as Epigenetic Factor in Progression of Cervical Tumors Associated with Human Papilloma Virus Infection

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • MED-OTH/Other/Medicine
  • MED-DRG/Drug Discovery/Medicine

Status
8 Project completed

Registration date
28.06.2005

Completion date
25.02.2010

Senior Project Manager
Melnikov V G

Leading Institute
State Research Center of Virology and Biotechnology VECTOR, Russia, Novosibirsk reg., Koltsovo

Supporting institutes

  • Cancer Research Center, Russia, Moscow\nSiberian Branch of RAS / Institute of Chemical Biology and Fundamental Medicine, Russia, Novosibirsk reg., Novosibirsk

Collaborators

  • Northwestern University / Feinberg School of Medicine, USA, IL, Chicago\nUniversity of Massachusetts / Medical School, USA, MA, Worcester

Project summary

The progression of tumors is characterized by genetic and epigenetic changes of cellular genome. At epigenetic changes it is observed as a rule aberrations in levels of gene expressions without changes of their structure. One of the most studied changes of similar type is DNA hypermethylation which is described for the majority of known human tumors. Thus occurs cytosine methylation which can be registered by means of methyl-sensitive or methyl-specific polymerase chain reaction and thus it is accessible to the analysis. Methylation is most precisely shown for so-called CpG islands, localized or in promoter areas of genes, or in their first exons. Methylation of these areas causes sharp reduction or full suppression of gene expression.

Cervical tumors are the second most frequent women's cancer. Cervical tumors and cervical tumor cell lines cultivated in vitro represent a suitable model for the analysis of the role of methylation in tumor progression. This disease has several steps in its development that can be clearly distinguished one from another by clinic-morphological criteria; human papilloma viruses (HPV) from the high-risk group, which contain transforming genes E6 and E7, capable to inactivate tumor-suppressor genes (p53 and Rb), are identified as etiological agents of this disease. Modification in host-cell gene functioning is considered as important events in HPV-mediated carcinogenesis. Many of these host-cell genes are not detected yet.

Project goal: the detection of methylation pattern alterations in tumor cells harboring transforming genes of human papilloma viruses.

The following set of experiments will be done for the achievement of this aim:

  1. Detection of genes inactivated in tumors by hypermethylation of CpG islands.
  2. Analysis of the interaction of Dnmts with proteins of DNA replication complexes in tumor cells and searching for effective direct inhibitors of Dnmts.

For realization of this Project the bank of frozen cervical tumor samples, characterized by clinic-morphological criteria and by HPV expression, will be used. The methylation-sensitive arbitrary primed PCR will be used for the detection of CpG islands which are aberrantly methylated in tumors. The status of methylated CpG islands will be verified by sequencing of DNA treated with sodium bisulphite. All these methods are well developed in Laboratory of Viral Molecular Biology, Cancer Center.

For the analysis of the proteins that can interact with Dnmts, new method of selective photoaffinity modification of DNA-binding proteins, which has been elaborated in Laboratory of Bioorganic Chemistry of Enzymes during last few years, will be utilized. This method is based on ability of DNA containing dNMP moieties with photoreactive group to react with DNA binding proteins. In specific conditions under UV light exposure, this interaction results in cross-linking of DNA to DNA binding proteins. This technique was successfully applied for the analysis of protein interactions with the specific DNA structures in DNA replication and DNA repair complexes (see References). This approach allows also indirect evaluation of protein-protein interaction in complexes, components of which covalently links with the photoreactive analogs of specific DNA structures. This can be done by analysis of the radioactively labeled DNA-protein adducts formed under UV-irradiation of the radioactive photoreactive DNA structures preincubated with the protein systems of the whole cell extract or reconstituted from the purified proteins. The following enzymes and protein factors will be purified for this search: DNA methyltransferase I, DNA polymerase α, replication protein A (RPA), processivity factor (PCNA), flap endonuclease I etc.

The interaction of Dnmt 1 with oligonucleotides (ONT), designed in accordance with the existing ideas about the structure of a potential inhibitors of Dnmts, will be investigated, and corresponding association constants will be evaluated by techniques and methods developed in Laboratory of Protein Biochemistry (see References). Oligonucleotides with high specificity and maximal affinity to Dnmt 1 will be used as a material for the synthesis of photoreactive derivatives of ONT, which will be utilized for photoaffinity modification of DNA-interacting proteins in reconstituted systems and in cellular extracts of neoplastic and normal cells. Identification of inpidual proteins cross-linked to DNA can be performed by different techniques including immunoprecipitation by specific antibodies (see References). The goal of this analysis consists in identification of Dnmt-interacting proteins pattern in normal and neoplastic cell extracts. These ONT may be also useful as the potential inhibitors of Dnmt1. The combination of the high affinity of the photoreactive ONT to Dnmt1 and their ability to a covalent linkage with proteins allows to expect the selective irreversible inhibition of the enzyme. We propose to check the level of stability of Dnmt1 inactivation in living cells, toxicity and influence of Dnmt1 inhibitors on level of methylation of CpG islands of the genes, described in point 1 of this project (conjointly with lab. of Prof. F. Kisseljov).


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