A Novel Multiplexed, Image-Based Approach to Detect Phenotypes That Underlie Chromosome Instability in Human Cells

Abstract

Chromosome instability (CIN) is characterized by a progressive change in chromosome numbers. It is a characteristic common to virtually all tumor types, and is commonly observed in highly aggressive and drug resistant tumors. Despite this information, the majority of human CIN genes have yet to be elucidated. In this study, we developed and validated a multiplexed, image-based screen capable of detecting three different phenotypes associated with CIN. Large-scale chromosome content changes were detected by quantifying changes in nuclear volumes following RNAi-based gene silencing. Using a DsRED-LacI reporter system to fluorescently label chromosome 11 within a human fibrosarcoma cell line, we were able to detect deviations from the expected number of two foci per nucleus (one focus/labelled chromosome) that occurred following CIN gene silencing. Finally, micronucleus enumeration was performed, as an increase in micronucleus formation is a classic hallmark of CIN. To validate the ability of each assay to detect phenotypes that underlie CIN, we silenced the established CIN gene, SMC1A. Following SMC1A silencing we detected an increase in nuclear volumes, a decrease in the number of nuclei harboring two DsRED-LacI foci, and an increase in micronucleus formation relative to controls (untreated and siGAPDH). Similar results were obtained in an unrelated human fibroblast cell line. The results of this study indicate that each assay is capable of detecting CIN-associated phenotypes, and can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer.

Reduced SKP1 Expression Induces Chromosome Instability through Aberrant Cyclin E1 Protein Turnover

“…To gain novel insight into the genes and pathways regulating chromosome stability in humans, these 164 candidate genes, along with positive (SMC1A) and negative controls (siControl) [6,7], were subjected to a single-cell quantitative imaging microscopy (scQuantIM) screen in two distinct and karyotypically stable human cell lines (hTERT and HT1080). These genes, along with positive (SMC1A) and negative (siControl) controls, were silenced using a custom-arrayed siGENOME (Dharmacon) library.…”

“…A siGENOME library (Dharmacon, Lafayette, CO, USA) containing a pool of 4 distinct siRNA duplexes (6.25 pmol) in each well that target a specific candidate gene or in separate wells, positive and negative controls [6,7], was employed in the scQuantIM screen. Standard reverse transfection with DharmaFECT 2 transfection reagent (Dharmacon, Lafayette, CO, USA) was performed according to manufacturer's instructions.…”

“…Standard reverse transfection with DharmaFECT 2 transfection reagent (Dharmacon, Lafayette, CO, USA) was performed according to manufacturer's instructions. Cells were seeded, allowed to grow for 4 (HCT116) to 6 days (hTERT) at 37 • C, fixed (4% paraformaldehyde) and counter-stained (Hoechst 33342) to visualize nuclei [7]. Subsequent direct tests employed ON-TARGETplus siRNA duplexes (Dharmacon, Lafayette, CO, USA) in a pooled format as detailed elsewhere [6].…”

Chromosome Instability; Implications in Cancer Development, Progression, and Clinical Outcomes

“…A large number of single cell approaches including microscopy, flow cytometry, next generation DNA sequencing, and copy number analysis have been devised that are capable of assessing CIN phenotypes, such as changes in chromosome numbers by standard cytogenetic staining [23] or the use of chromosome enumeration probes and whole chromosome paints [24,25], along with more recent technological advancements including single cell DNA sequencing or copy number variation [26,27]. Single cell approaches have also been developed to quantify and compare surrogate markers of CIN (e.g., CIN-associated phenotypes), including micronucleus formation [28,29] and changes in nuclear areas [29] or human artificial chromosomes [30]. Conceptually, micronuclei are extra nuclear bodies that are found outside the primary nucleus and are hallmarks of CIN that typically arise due to chromosome missegregation events [31][32][33], while changes in nuclear areas and human artificial chromosomes are associated with small and large (i.e., ploidy) scale changes in DNA content, respectively [30,[34][35][36][37][38].…”

“…Conceptually, micronuclei are extra nuclear bodies that are found outside the primary nucleus and are hallmarks of CIN that typically arise due to chromosome missegregation events [31][32][33], while changes in nuclear areas and human artificial chromosomes are associated with small and large (i.e., ploidy) scale changes in DNA content, respectively [30,[34][35][36][37][38]. These approaches typically involve quantitative imaging microscopy or flow cytometry that are each capable of rapidly assessing CIN-associated phenotypes in hundreds-to-thousands of cells [28,29,39]. In any case, numerous complementary single cell approaches have been developed that can provide critical insight into the prevalence of cell-to-cell heterogeneity and CIN within experimental and clinical contexts.…”