A new assay for measuring chromosome instability (CIN) and identification of drugs that elevate CIN in cancer cells

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.

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

“…S2B). The fraction of cells without the HAC-GFP in the control line agreed well with previous studies (53). We further confirmed that loss of fluorescence detected by flow cytometry corresponds with HAC loss events using fluorescent in situ hybridization (Fig.…”

“…Based on this list of dCIN genes, we then chose 20 candidate genes for functional testing in human cells (Table S4). The dCIN phenotype in human cells was examined using a quantitative assay based on a nonessential human artificial chromosome (HAC) (53) (Fig. 3A).…”

“…The HAC-based assay can detect CIN induced by chemical agents that destabilize chromosomes (53) but had yet to be tested with genetic perturbations. As a proof-of-principle experiment, we overexpressed CCNE2 and CCND1, two known human dCIN genes, in HAC-GFP-containing cells.…”

“…Human fibrosarcoma HT1080 cells with alphoid tetO -HAC-GFP were cultured in DMEM (Life Technologies) with 10% (vol/vol) FBS. To maintain the HAC, HT1080 cells were grown in 5 μg/mL of blasticidin (Sigma) as previously described (53). RH30 (alveolar RMS, PAX3-FOXO1) cells were cultured in DMEM (Gibco BRL Life Technologies) supplemented with 10% heat-inactivated FBS (HyClone) and 1% antibiotic-antimycotic (Gibco BRL Life Technologies).…”

“…A minimum of 4 × 10 4 events were acquired for each sample. The presence of HAC was confirmed by FISH analysis as previously described (53).…”