Abstract
Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene (“loss of signal” assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this “loss of signal” assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this “gain of signal” assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The “gain of signal” assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level.
Highlights
Abnormal chromosome number is a known feature of most solid tumors and is often accompanied by an elevated rate of chromosome missegregation termed chromosome instability (CIN) [1]
The human artificial chromosome (HAC) developed in hamster host Chinese hamster ovary (CHO) cells carries the mCherryFP transgene and a constitutively expressed short hairpin RNA against EGFP
After transfer of the mCherry-shRNAHAC into HT1080-EGFP-expressing cells, the HAC is maintained as a non-essential 47th chromosome (II) due to the presence of its functional kinetochore and the cells maintaining the HAC become red (II) (Figure 1A and 1B)
Summary
Abnormal chromosome number (aneuploidy) is a known feature of most solid tumors and is often accompanied by an elevated rate of chromosome missegregation termed chromosome instability (CIN) [1]. The gain or loss of entire chromosomes leads to large-scale changes in gene copy number and expression levels. Mutations in CIN genes are thought to be an early event in tumor development, predisposing cells to the accumulation of genetic changes leading to progression to a cancerous state [2,3,4]. A significant fraction of human CIN genes remains unidentified and in part this is due to the lack of a simple assay to detect CIN in vertebrate cells, similar to that developed for yeast cells [5]. There is an important need to develop improved assays for measuring chromosome transmission fidelity in human cells
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