Novel CARD11 Mutations in Human Cutaneous Squamous Cell Carcinoma Lead to Aberrant NF-κB Regulation.

NF-κB signaling plays a crucial role in regulating proliferation and differentiation in the epidermis. Alterations in the NF-κB pathway can lead to skin pathologies with a significant burden to human health such as psoriasis and cutaneous squamous cell carcinoma (cSCC). Caspase recruitment domain (CARD)-containing scaffold proteins are key regulators of NF-κB signaling by providing a link between membrane receptors and NF-κB transcriptional subunits. Mutations in the CARD family member, CARD14, have been identified in patients with the inflammatory skin diseases psoriasis and pityriasis rubra pilaris. Here, we describe that the gene coding for another CARD scaffold protein, CARD11, is mutated in more than 38% of 111 cSCCs, and show that novel variants outside of the coiled-coil domain lead to constitutively activated NF-κB signaling. CARD11 protein expression was detectable in normal skin and increased in all cSCCs tested. CARD11 mRNA levels were comparable with CARD14 in normal skin and CARD11 mRNA was increased in cSCC. In addition, we identified CARD11 mutations in peritumoral and sun-exposed skin, suggesting that CARD11-mediated alterations in NF-κB signaling may be an early event in the development of cSCC.

Real-time GUS analysis using Q-PCR instrumentation.

The development of new technology within biological sciences has resulted in a number of real-time PCR instruments that have become essential tools within molecular biology. This equipment has facilitated high throughput analysis of samples and optimal information gathering of completed PCR reactions for example estimating the copy number of a gene of interest that is inserted into particular genomes. Real-time PCR instruments frequently come with optional filter sets, e.g. the ALEXA filter set which has parameters in common with excitation and emission wavelengths of sodium methyl umbelliferone (NaMU) widely used in beta-glucuronidase reporter gene assays. Using these filter sets it has been possible to quantify and measure gus A activity of Ulmus procera SR4 in real-time removing the necessity for aliquots of reactions to be stopped by pipetting into carbonate buffer for each time point. The introduction of real-time GUS analysis leads to faster, more accurate and reproducible assays with reduced potential for pipetting errors, requires fewer manipulations and encourages high throughput analysis of inter-individual gene expression variation.