Inactivation of promoter 1B of APC causes partial gene silencing: evidence for a significant role of the promoter in regulation and causative of familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Two promoters, 1A and 1B, have been recognized in APC, and 1B is thought to have a minor role in the regulation of the gene. We have identified a novel deletion encompassing half of this promoter in the largest family (Family 1) of the Swedish Polyposis Registry. The mutation leads to an imbalance in allele-specific expression of APC, and transcription from promoter 1B was highly impaired in both normal colorectal mucosa and blood from mutation carriers. To establish the significance of promoter 1B in normal colorectal mucosa (from controls), expression levels of specific transcripts from each of the promoters, 1A and 1B, were examined, and the expression from 1B was significantly higher compared with 1A. Significant amounts of transcripts generated from promoter 1B were also determined in a panel of 20 various normal tissues examined. In FAP-related tumors, the APC germline mutation is proposed to dictate the second hit. Mutations leaving two or three out of seven 20-amino-acid repeats in the central domain of APC intact seem to be required for tumorigenesis. We examined adenomas from mutation carriers in Family 1 for second hits in the entire gene without any findings, however, loss of the residual expression of the deleterious allele was observed. Three major conclusions of significant importance in relation to the function of APC can be drawn from this study; (i) germline inactivation of promoter 1B is disease causing in FAP; (ii) expression of transcripts from promoter 1B is generated at considerable higher levels compared with 1A, demonstrating a hitherto unknown importance of 1B; (iii) adenoma formation in FAP, caused by impaired function of promoter 1B, does not require homozygous inactivation of APC allowing for alternative genetic models as basis for adenoma formation

Caffeine as a tool for investigating the integration of Cdc25 phosphorylation, activity and ubiquitin-dependent degradation in Schizosaccharomyces pombe

The evolutionarily conserved Cdc25 phosphatase is an essential protein that removes inhibitory phosphorylation moieties on the mitotic regulator Cdc2. Together with the Wee1 kinase, a negative regulator of Cdc2 activity, Cdc25 is thus a central regulator of cell cycle progression in Schizosaccharomyces pombe. The expression and activity of Cdc25 is dependent on the activity of the Target of Rapamycin Complex 1 (TORC1). TORC1 inhibition leads to the activation of Cdc25 and repression of Wee1, leading to advanced entry into mitosis. Withdrawal of nitrogen leads to rapid Cdc25 degradation via the ubiquitin- dependent degradation pathway by the Pub1 E3- ligase. Caffeine is believed to mediate the override of DNA damage checkpoint signalling, by inhibiting the activity of the ataxia telangiectasia mutated (ATM)/Rad3 homologues. This model remains controversial, as TORC1 appears to be the preferred target of caffeine in vivo. Recent studies suggest that caffeine induces DNA damage checkpoint override by inducing the nuclear accumulation of Cdc25 in S. pombe. Caffeine may thus modulate Cdc25 activity and stability via inhibition of TORC1. A clearer understanding of the mechanisms by which caffeine stabilises Cdc25, may provide novel insights into how TORC1 and DNA damage signalling is integrated