Author: Benjamin Price, Camille Girards, Grace Mitchell
Research Article
Correlation of Cyclins A, E and Topoisomerase IIα With Centrosome Amplification and Genomic Instability
Camille Girards1*, Benjamin Price2 and Grace Mitchell2
1Department of Microbiology, University of Bordeaux, Bordeaux, France
2Department of Biochemistry, Imperial College London, London, UK
Published: 16 July 2014
Abstract
Background: Accurate cell cycle progression relies on the coordinated action of regulatory proteins like cyclins and essential enzymes like Topoisomerase IIα (Topo IIα). Deregulation of Cyclin E, Cyclin A and Topo IIα is common in cancer and associated with uncontrolled proliferation. Centrosome amplification, the presence of more than two centrosomes, is another hallmark of malignancy, leading to chromosome mis-segregation and genomic instability. Flow cytometry is widely used to assess cell cycle distribution, particularly the S-Phase Fraction (SPF), a prognostic marker in oncology. However, the reliability of SPF determination can be compromised in aneuploid or genetically unstable tumors.
Objective: This study aimed to investigate the correlation between the expression levels of Cyclin A, Cyclin E, Topo IIα, the frequency of centrosome amplification and markers of genomic instability in a panel of human cell lines. Furthermore, we assessed how these molecular and cytological alterations impact the determination of the S-phase fraction by standard flow cytometry.
Methods: A panel of human cell lines, including normal diploid fibroblasts and cancer cell lines with varying degrees of genomic instability (e.g., diploid colorectal cancer HCT116, near-diploid breast cancer MCF-7, highly aneuploid breast cancer MDA-MB-231), were analyzed. Protein expression levels of Cyclin A, Cyclin E and Topo IIα were quantified by Western blotting. Centrosome numbers were assessed by immunofluorescence staining for γ-tubulin. Genomic instability was evaluated using Fluorescence InSitu Hybridization (FISH) for chromosome enumeration (aneuploidy) and analysis of micronuclei frequency. Cell cycle distribution, including the S-phase fraction, was determined by univariate flow cytometry (DNA content analysis) and bivariate flow cytometry (DNA content vs. BrdU incorporation).
Results: Cancer cell lines, particularly those with high genomic instability (MDA-MB-231), exhibited significantly higher expression levels of Cyclin E, Cyclin A and Topo IIα compared to normal fibroblasts and low-instability cancer cells (HCT116, MCF-7). A strong positive correlation was observed between the combined high expression of these proteins and the frequency of cells displaying centrosome amplification (>2 centrosomes/cell) (P<0.01). Furthermore, high expression levels and centrosome amplification correlated significantly with increased aneuploidy and micronuclei formation (P<0.01). Standard univariate flow cytometry analysis yielded significantly higher and more variable SPF values in the highly unstable cell lines compared to bivariate BrdU analysis. Discrepancies between univariate SPF and BrdU-incorporation S-phase were most pronounced in cells with high Cyclin/Topo IIα expression and frequent centrosome amplification. These cells often displayed broad, poorly resolved DNA histograms, making accurate deconvolution of S-phase difficult.
Conclusion: Our findings demonstrate a significant correlation between the overexpression of key cell cycle regulators (Cyclin A, Cyclin E) and Topo IIα, the occurrence of centrosome amplification and the extent of genomic instability in human cell lines. These interconnected aberrations contribute to complex DNA content profiles that confound standard univariate flow cytometric determination of the S-phase fraction. Caution is warranted when interpreting SPF values derived solely from DNA content histograms in tumors exhibiting markers of high genomic instability and deregulation of these cell cycle proteins.
Keywords: Cyclin A; Cyclin E; Topoisomerase IIα; Centrosome Amplification; Genomic Instability; Aneuploidy; S-phase Fraction; Flow Cytometry; Cell Cycle.
