Rosanna Bagnulo | PubMed Health

PubMed2006

Tumor-specific hyperactive low-molecular-weight cyclin E isoforms detection and characterization in non-metastatic colorectal tumors.

PURPOSE: Several molecules involved in cancer biology have been studied as potential prognostic markers. Recently, overexpression of cyclin E and its low-molecular-weight (LMW) isoforms has been reported to be the most prominent prognostic marker in breast cancer, surpassing proliferation index, ploidy, and axillary nodal involvement. Furthermore, cyclin E and p53 are considered the main factors controlling the euploid equilibrium in human cells. We investigated the status of cyclin E and p53 in cell lines and tissue samples of colorectal cancer, one of the leading causes of death from a tumor in the Western world. EXPERIMENTAL DESIGN: We analyzed colorectal cancer cells, from established cell lines and patient specimens, to determine the protein levels of cyclin E and p53, and to detect p53 and APC mutations, microsatellite and chromosome instability. In addition, we assessed the presence of cyclin E LMW isoforms and their enzymatic activity. RESULTS: Colorectal cancer cells expressed hyperactive LMW forms both in vitro and in vivo. These tumor-specific isoforms are correlated to genomic instability even in p53-proficient cells, and represented a constant feature in the tumors analyzed. CONCLUSIONS: In colorectal cancer, the formation of cyclin E LMW forms is an early event leading to DNA-damage checkpoint-independent proliferation. Collectively, our results provide evidence that evaluation of LMW forms could represent a novel tool in the molecular characterization of colorectal tumors aimed at identifying sensitive prognostic factors and uncovering subsets of high-risk patients within the traditional categories.

Adenomatous Polyposis Coli Protein↗

PubMed2006

A homozygous frameshift mutation in the ESCO2 gene: evidence of intertissue and interindividual variation in Nmd efficiency.

Roberts syndrome (RS) is a rare disorder characterized by tetraphocomelia and several other clinical features. Cells from RS patients exhibit characteristic premature separation of heterochromatic region of many chromosomes and abnormalities in cell cycle. Mutations in the ESCO2 gene have recently been identified in 20 RS families. We performed mutational analysis of the ESCO2 gene in two fetuses diagnosed with RS and their normal parents. In both fetuses, we identified homozygosity for the c. 745_746delGT mutation, while the non-consanguineous parents were both heterozygous for the same mutation. Considering the position of the mutation identified, we carried out qualitative and quantitative real-time ESCO2 cDNA analysis on RNA isolated from CVS-stromal cells in one fetus, amniocytes in the second fetus, and lymphocytes from the heterozygous parents. The results of this analysis showed that despite the presence of a premature termination codon (PTC) 112 nucleotides upstream of the next exon3-exon4 junction, the mutant ESCO2 mRNA was present in both fetuses, albeit at low levels, indicating a partial resistance to nonsense mediated decay (NMD). Interestingly, when cells derived from the two fetuses were treated with an inhibitor of translation, they revealed the presence of tissue and individual variability in NMD efficiency, despite the identical mutational status. The existence of such a variation in the NMD efficiency could explain the broad intrafamilial and interfamilial variability in the clinical presentation of RS patients, and in other genetic diseases where nonsense mutations are responsible for most of the mutation load. Moreover, considering that a mutated full length mRNA was produced in both fetuses, we used Western blot analysis to demonstrate the absence of the ESCO2-truncated protein in cells derived from both fetuses and in a lymphoblastoid cell line derived from the parents.

Abnormalities, Multiple↗