Abstract
BackgroundAneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth and therefore can be exploited therapeutically. Drugs known to increase CIN beyond the therapeutic threshold are currently few in number, and the clinical promise of targeting the CIN phenotype warrants new screening efforts. However, none of the existing methods, including the in vitro micronuclei (MNi) assay, developed to quantify CIN, is entirely satisfactory.MethodsWe have developed a new assay for measuring CIN. This quantitative assay for chromosome mis-segregation is based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows the measurement of HAC loss rate by routine flow cytometry.ResultsUsing the HAC-based chromosome loss assay, we have analyzed several well-known anti-mitotic, spindle-targeting compounds, all of which have been reported to induce micronuclei formation and chromosome loss. For each drug, the rate of HAC loss was accurately measured by flow cytometry as a proportion of non-fluorescent cells in the cell population which was verified by FISH analysis. Based on our estimates, despite their similar cytotoxicity, the analyzed drugs affect the rates of HAC mis-segregation during mitotic divisions differently. The highest rate of HAC mis-segregation was observed for the microtubule-stabilizing drugs, taxol and peloruside A.ConclusionThus, this new and simple assay allows for a quick and efficient screen of hundreds of drugs to identify those affecting chromosome mis-segregation. It also allows ranking of compounds with the same or similar mechanism of action based on their effect on the rate of chromosome loss. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells.
Highlights
Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN)
While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth, and it can be exploited therapeutically
Experimental design for identification of drugs that elevate CIN in cancer cells Figure 2 shows a general scheme of the new assay developed for measuring chromosome instability (CIN) based on the use of a human artificial chromosome (HAC) carrying the encoding the green fluorescent protein (EGFP) transgene
Summary
Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN). Partial reduction of essential mitotic checkpoint components in tumor cell lines caused mild chromosome mis-segregation, but no lethality. These cells were, much more sensitive to low doses of taxol, which enhances the amount and severity of chromosome segregation errors. In another study [3], a set of genes was identified that are repressed in response to taxol treatment and over-expressed in tumors exhibiting CIN The silencing of these genes caused cancer cell death, suggesting that these genes might be involved in the survival of aneuploid cells. Taking into account the fact that aneuploidization per se seems to be a very inefficient path towards cancer and additional hits are necessary for the generation of a cancer cell ([4] and references therein), these and other studies [5,6] indicate that increased destabilization of chromosomes might push genetically unstable cancer cells towards death, whereas more stable normal cells would be able to tolerate such insults
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