Drug-induced genome instability: translation from yeast to human
Summary of the proposed research
Genomic instability is implicated in various disorders, like cancer, neurological diseases, cellular senescence and aging, and is dependent on genetic factors and xenobiotics. For instance, drug-induced DNA damage can lead to chromosomal instability and aneuploidy. We have identified in yeast various DNA damage response (DDR) genes, some of which are conserved in humans, that prevent cells from drug-induced genome instability. We will study these genes in detail and examine their human orthologs as potential biomarkers for diagnostic or therapeutic purposes.
Paracetamol is a common over-the-counter drug, which can cause severe hepatotoxicity when overdosed. At high concentrations, the drug is also toxic in the yeast Saccharomyces cerevisiae. Paracetamol-tolerant yeast cells were identified in a genome-wide loss-of-function screen in collaboration with Dr. Fred van Leeuwen (AvL-NKI, Amsterdam). Deletion of any of these genes results in paracetamol resistance. Four of these genes are linked to DDR, including IXR1, a gene without a clear human counterpart, previously identified in a cisplatin resistance screen in yeast (Huang et al., 2005). Interestingly, these deletion strains induce genomic instability upon paracetamol exposure, suggesting loss of a cell-cycle checkpoint control. These findings implicate these genes in protecting cells from illegitimate, drug-induced genome alterations. We focus initially on two of the identified genes, which encode E2 ubiquitin-conjugating enzymes with clear orthologs in humans.
In this project, we want to characterize the drug-induced genome instability further. Furthermore, we will investigate the (poly-)ubiquitination of potential target proteins and use GFP-tagged versions of the proteins to perform intracellular (co-)localization studies.
Because oncogenesis is often accompanied by mis-segregation of chromosomes, we plan to investigate the role of the human orthologs in cell lines in combination with anti-tumor drugs that affect mitosis. We will silence genes by RNAi in cell lines (e.g. primary and Human Papilloma Virus-immortalized keratinocytes (Dr. Steenbergen, CCA,VUmc)) to translate the yeast hits to genome instability in response to anti-tumor drugs in human cells. Also, we intend to screen human tumor databases for mRNA expression levels of relevant genes to test their potential as diagnostic biomarker.
The collaboration provides complementary expertise on the identification of yeast genes important for drug-tolerance (FEW, NKI) and basic genetic and epigenetic mechanisms (FALW), as well as on (tumor) cell lines and tumor microarray databases within the Cancer Center Amsterdam (VUmc).
