Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis

Abstract

Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a "dicentric" chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

Fig. 1

Induction of an ectopic kinetochore causes nuclear abnormalities and DNA damage in 3T3-LacO cells. a Stable cell lines expressing GFP-CENP-T ΔC-LacI or GFP-LacI were generated in 3T3 cells containing a lacO array on chromosome 3. Growth in the presence of 10 mM IPTG prevented LacI–lacO interactions. Representative immunofluorescence images show 3T3-LacO cells after removal of IPTG for 3 days and staining with anti-phospho-H2AX antibodies. Distortion of the GFP-CENP-T-LacI containing chromosome is clearly seen after removal of IPTG, as well as co-localization of DNA damage signal with GFP-CENP-T-LacI. Right panel shows nuclear morphology after removal of IPTG and growth in 30 μM blebbistatin, indicating reduction in nuclear abnormalities after inhibition of cytokinesis. Boxes indicate the location of the GFP signal. Scale bar shows 5 μm. b Quantification of the number of 3T3-lacO cells with irregular nuclear morphology (including nuclear protrusions, multi-lobed nuclei, and separated nuclei connected by chromatin bridges) in the presence of the indicated LacI fusion protein and following removal of IPTG for 3 or 7 days, or removal for 3 days, then re-addition for 4 days. c Quantification of the number of 3T3-lacO cells with phospo-H2AX signals co-localized with GFP foci in immunofluorescence images after growth in the indicated conditions. For b and c; n ≥ 100 cells in three independent experiments. Error bars show standard deviation. d Shows representative images of LacO (green) containing chromosomes that also have an active endogenous centromere in the indicated conditions, as detected by CENP-C immunofluorescence staining in red. Arrows indicate CENP-C-specific staining. Scale bar shows 1 μm. Graph shows the number of 3T3-LacO cells containing the lacO array in the indicated conditions

Fig. 2

Induction of an ectopic kinetochore causes genomic rearrangements. 3T3-lacO cells expressing GFP-LacI or GFP-CENP-T-ΔC-LacI were grown in the absence of IPTG for 3 days and then prepared for FISH analysis using probes against chromosome 3 (pink) and LacO sequence (green). Images show representative metaphase spreads after FISH. Note that nonspecific background signal from chromosome 3 probes is also visible on centromeres. Scale bar shows 10 μm. Insets show examples of chromosome rearrangements. Graph shows quantification of the number of chromosomes containing chromosome 3 sequences. N > 15 cells per treatment

Fig. 3

Dicentric chromosome induction leads to global genomic rearrangement. a Spectral karyotype (SKY) analysis of 3T3-lacO cells expressing GFP-LacI or GFP-CENP-T-ΔC-LacI at a time point 3 days after removal of IPTG. Representative images of chromosomes involved in translocations are pseudo-colored. b Quantification of the rates of chromosome translocations in a

Fig. 4

Induction of an ectopic kinetochore causes cellular transformation. a 3T3 cells with or without an integrated lacO array and expressing GFP-LacI or GFP-CENP-T-ΔC-LacI were grown for 4 days in the presence or absence of IPTG as indicated. Cells were then plated in triplicate in soft agarose in the presence of IPTG. After 4 weeks of growth, 200 cells were counted per replicate, and the percentage of cells forming colonies was scored. Clonal cell lines expressing (high) or (low) levels of the fusion protein were tested for each condition as indicated. Primary mouse embryonic fibroblasts (MEFs) and HeLa cells were used as negative and positive controls, respectively. Asterisk indicates significant difference as determined by T test, P < 0.001. b 3T3-lacO cells expressing GFP-LacI or GFP-CENP-T-ΔC-LacI were grown for 14 days in the presence or absence of IPTG as indicated. Cells were then seeded into the upper chamber of a basement membrane-coated Boyden’s chamber and migration across the membrane was assayed at the indicated time points by staining with crystal violet. N = 20 fields. Error bars show standard deviation. Right hand panel shows images of lower membrane staining from a representative experiment