Workpackage 5
WP5 From Models of DNA damage response and repair mechanism to Humans
(partners involved: 1, 2, 3, 5, 7, 11)
As described above, the identical genetic background in mouse models is very important for the detection of subtle effects of mutations in genome care taking genes. However, the relevance of the results in the mouse model for human disease will have to be validated. Therefore, we will also study the effect of mutations in the human situation. Comparison of the mouse models with human cell lines engineered by siRNA approaches and with cells from human patients will be especially useful in gene profiling and proteomic studies (WP4).
Genome instability syndromes have been known for some time in humans. They have been found to be linked to cancer and ageing-related diseases (e.g. the BRCA genes and breast cancer and MMR genes in inherited and sporadic cancer of the colon and other tissues). Furthermore, genome maintenance genes are now known to be mutated in many tumours. Our efforts will go into three directions: (1) finding novel genes in specific inherited diseases and (2) finding correlations between polymorphisms/mutations MLH1 (in MMR, HR and meiotic recombination) in genome maintenance genes and development of disease and (3) identification of biomarkers to identify dysfunction or activation of various DNA repair and checkpoint pathways.
(1) For several DNA repair pathways, associated human disorders have been identified. NER defects can cause xeroderma pigmentosum (XP), Cockayne's Syndrome (CS), Trichothiodystrophy (TTD) or the UV-sensitive syndrome (UVS), a defect in TLS underlies the Variant form of XP, whereas MMR defects are linked to Hereditary Non-Polyposis Colon Cancer (HNPCC) and BER malfunction has recently been linked to the Multiple Colonic Adenoma syndrome. Mutations in the human UNG gene were recently shown to cause a hyper IgM syndrome (HIGM) and change in somatic hypermutation required for affinity maturation of antibodies. For these disorders, new patients will be recruited in order to identify the affected genes. If a patient falls into a complementation group that is different from the ones identified thus far, we will attempt to clone the gene involved. If the complementation group can be identified, we will determine the mutations of interesting cases.
In other instances, specific human syndromes connected to the repair and genome surveillance pathways have not yet been found. However, it is to be expected that such human conditions exist. For example, until recently, no human condition connected to EJ has been identified. However, several immuno-deficient patients without B or T cells have been found that also show cellular radio sensitivity. These might be expected to harbour a mutation in an EJ gene. Recently, one of these genes was cloned (named Artemis). We are currently investigating a number of these radiosensitive SCID patients for mutations in this or other genes. Similarly, we will try to identify individuals with defects in HR or BER. Subsequently, newly identified genes will be investigated in more detail as described in WP1-4.
(2) The correlation between polymorphisms in genome maintenance genes and disease will be much more difficult to validate. However, it may be of great importance for a rational risk assessment of many environmental and occupational hazards. We will therefore invest some effort into investigating the repair status of cells and pathogenesis. For this purpose, we will establish cell lines from patients and tumours, and investigate various relevant parameters, such as transcription competence, end-joining capacity, formation of radiation induced focal accumulation of HR proteins and MMR parameters. In addition, we will study the repair status of tumours. This may shed light on the risk associated with mutations and polymorphisms in repair genes (e.g. MMR genes in HNPCC samples).
(3) In order to explain, and ultimately predict, how a given cell will respond to a given DNA damaging agent, it is necessary to know which systems of DNA repair and checkpoint signalling are operative in that cell and what the relative contributions of those pathways are. If such knowledge could be obtained for cells derived from a patient’s normal tissues, then it would provide information about that patient’s intrinsic predisposition to cancer and other age-related diseases. Most, if not all cancer cells will have lost one or more of the DNA damage response pathways during tumourigenesis, making them more reliant upon remaining functional pathways. Effective therapy based on DNA repair inhibition will therefore benefit from the knowledge of which pathways are lost and which are still functional in the cancer cells to be treated. For instance, one marker might indicate inactivation of the BRCA1 pathway and a major reliance on the NHEJ pathway. The identification of simple markers for different DNA repair pathways would facilitate investigations by other members of the network and could contribute towards the establishment and analysis of clinical trials that involve DNA damaging agents and/or drugs that interfere with DNA repair or checkpoint pathways. Partner 15 has already identified markers for checkpoint activation in the ATM pathway and will proceed to identify markers for other DNA repair and checkpoint pathways for which there is likely to be clinical relevance. This work will involve proteomic and functional analysis of matched human cell lines and drugs that interfere with DNA repair and checkpoint pathways. Other potential therapeutic benefits from proteomic studies include the possible identification of new candidate drug targets. These could be validated by the various participants involved in work packages WP1 to WP4 and high throughput screens developed by Partner 15 for the use in the identification of novel inhibitors.
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