Chromosomal instability in cells with elevated Mad-2 expression. FISH analysis of IMR90 human fibroblast cells infected with E1A, shRB or Mad2 (top right, bottom left, bottom right, respectively) and stained with centromeric probes for chromosomes 1 and 17 (red and green) and DAPI (blue).

Aneuploidy is a hallmark of many human cancers, although its causal involvement in cellular transformation remains a matter of debate. Mutations debilitating the Rb pathway are commonly observed in cancers, and result in derailed cell-cycle regulation as a consequence of unchecked activation of the E2F family of transcription factors. How this is linked to the characteristic genomic instability of such cancers has remained unclear. Now, Hernando et al. report in Nature (430, 797–802; 2004) that the spindle checkpoint protein Mad2 is a transcriptional target of E2F, and that elevated Mad2 expression contributes to aneuploidy.

The spindle checkpoint senses aberrant kinetochore microtubule attachment and inhibits chromatid separation, cytokinesis and mitotic exit by inhibiting the ubiquitin ligase APC/C. Experimental inactivation of two checkpoint components, Mad2 and Bub3, results in chromosome instability in mice. However, these genes are rarely targeted in spontaneous tumours.

Hernando et al. observed constitutive Mad2 expression in fibroblasts with compromised Rb (knockout mouse embryonic fibroblasts, RNAi against Rb or E1A expressing cells). Correlating with E2F activity during the cell cycle, Mad2 is not expressed in normal quiescent cells and is induced in S phase, peaking in G2/M. Furthermore, constitutive Mad2 expression is observed in carcinoma and neuroblastoma cell lines, as well as primary human retinoblastomas, bladder carcinomas and neuroblastomas with deregulated E2F activity, where it correlates with poor patient prognosis. Consistent with previous global microarray and ChIP results, Hernando et al. found that the Mad2 promoter binds to — and is trans-activated by — E2F-1 in cells.

In order to address the somewhat paradoxical notion that overexpression of a spindle checkpoint protein may be causally involved in aneuploidy, Hernando et al. surveyed the genomes of fibroblasts with increased Mad2 levels by laser-scanning cytometry, fluorescence in situ hybridization and metaphase spreads. Frequent aneuploidy was observed in fibroblasts with constitutive Mad2 expression. This went hand-in-hand with delayed cytokinesis. Importantly, RNAi-mediated reduction of Mad2 in cells with defective Rb reduced the appearance of aneuploidy.

The E2F family of transcription factors function at the core of cell-cycle regulation, and their function is frequently derailed in cancers. Hernando et al. have uncovered that an unexpected corollary of this is super-induction of the spindle checkpoint protein Mad2 and, consequently, chromosome mis-segregation and aneuploidy — one of the major hallmarks of cancer. Although this data has uncovered a direct link from the regulation of cell-cycle progression to genomic instability, implying that aneuploidy is an early event in cancer, the question of chicken and egg stands. Nevertheless, as in life, the old adage that there can be too much of a good thing seems to apply also to the cell cycle.