Fbw7 and p53 cooperatively suppress advanced and chromosomally unstable intestinal cancer.

Colorectal cancer (CRC) remains a major cause of cancer mortality worldwide. Murine models have yielded critical insights into CRC pathogenesis, but they often fail to recapitulate advanced-disease phenotypes, notably metastasis and chromosomal instability (CIN). New models are thus needed to understand disease progression and to develop therapies. We sought to model advanced CRC by inactivating two tumor suppressors that are mutated in human CRCs, the Fbw7 ubiquitin ligase and p53. Here we report that Fbw7 deletion alters differentiation and proliferation in the gut epithelium and stabilizes oncogenic Fbw7 substrates, such as cyclin E and Myc. However, Fbw7 deletion does not cause tumorigenesis in the gut. In contrast, codeletion of both Fbw7 and p53 causes highly penetrant, aggressive, and metastatic adenocarcinomas, and allografts derived from these tumors form highly malignant adenocarcinomas. In vitro evidence indicates that Fbw7 ablation promotes genetic instability that is suppressed by p53, and we show that most Fbw7⁻/⁻; p53⁻/⁻ carcinomas exhibit a CIN⁺ phenotype. We conclude that Fbw7 and p53 synergistically suppress adenocarcinomas that mimic advanced human CRC with respect to histopathology, metastasis, and CIN. This model thus represents a novel tool for studies of advanced CRC as well as carcinogenesis associated with ubiquitin pathway mutations.

Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase.

The SCF(FBW7) ubiquitin ligase degrades proteins involved in cell division, growth, and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations, suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7alpha isoform accounts for almost all Fbw7 activity toward cyclin E, c-Myc, and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle, and this correlates with changes in cyclin E-cyclin-dependent kinase 2 (CDK2)-specific activity, cyclin E autophosphorylation, and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E-CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover, they highlight the utility of adeno-associated virus-mediated gene targeting in functional analyses of complex loci.

MALDI-TOF mass spectrometric identification of dyes and pigments.

We have used MALDI-TOF mass spectrometry to characterize a selection of dyes from the Schweppe dye collection and pigments from the Tate Gallery collection. MALDI-TOF mass spectra of such samples are easily obtained and, through observation of both positive and negative ion spectra, provide a convenient, versatile method for dye characterization and identification. Such pairs of positive and negative ion spectra immediately distinguish between acidic and basic dyes and provide the characteristic mass of either the molecular ion or a simply related fragment ion. This approach is especially useful in situations where very small amounts of analyte are available, as in museum research and forensic analysis. In the case of textile dyes, we have carried out identification on material from single fibers and, with insoluble pigments, have begun to identify components of historically important pastel sticks from submicrogram samples.