A novel homozygous nonsense mutation in CAST associated with PLACK syndrome.

Peeling skin syndrome is a heterogeneous group of rare disorders. Peeling skin, leukonychia, acral punctate keratoses, cheilitis and knuckle pads (PLACK syndrome, OMIM616295) is a newly described form of PSS with an autosomal recessive mode of inheritance. We report a 5.5-year-old boy with features of PLACK syndrome. Additionally, he had mild cerebral atrophy and mild muscle involvements. Whole exome sequencing was performed in genomic DNA of this individual and subsequent analysis revealed a homozygous c.544G > T (p.Glu182*) nonsense mutation in the CAST gene encoding calpastatin. Sanger sequencing confirmed this variant and demonstrated that his affected aunt was also homozygous. Real-time qRT-PCR and immunoblot analysis showed reduced calpastatin expression in skin fibroblasts derived from both affected individuals compared to heterozygous family members. In vitro calpastatin activity assays also showed decreased activity in affected individuals. This study further supports a key role for calpastatin in the tight regulation of proteolytic pathways within the skin.

Displacement of Bim by Bmf and Puma rather than increase in Bim level mediates paclitaxel-induced apoptosis in breast cancer cells.

Taxanes exert their antitumor effect through stabilizing microtubule dynamics and initiating G2/M arrest in cancer cells followed by apoptotic cell death. However, the signaling pathways that connect paclitaxel-induced microtubule perturbation to mitochondrial outer membrane permeabilization and cytochrome c release are not well characterized. Here, we show that in breast cancer cells, paclitaxel induces a novel displacement mechanism: prodeath BH3-only proteins Bmf and Puma competitively displace prodeath BH3-only protein Bim from antiapoptotic proteins to activate Bax and Bak and commit the cell to apoptotic death. Bim and either Puma or Bmf are required for paclitaxel toxicity. Although prior mechanisms of apoptosis induced by taxol have focused on changes in Bim levels, we find that an increase is not required for paclitaxel killing of breast cancer cells. Rather, competitive displacement of Bim from antiapoptotic proteins is the important step committing the cell to death. This novel mechanism suggests the potential usage of novel therapies targeted at altering BH3-only protein heterodimerization.

Pramanicin induces apoptosis in Jurkat leukemia cells: a role for JNK, p38 and caspase activation.

Pramanicin is a novel anti-fungal drug with a wide range of potential application against human diseases. It has been previously shown that pramanicin induces cell death and increases calcium levels in vascular endothelial cells. In the present study, we showed that pramanicin induced apoptosis in Jurkat T leukemia cells in a dose- and time-dependent manner. Our data reveal that pramanicin induced the release of cytochrome c and caspase-9 and caspase-3 activation, as evidenced by detection of active caspase fragments and fluorometric caspase assays. Pramanicin also activated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK 1/2) with different time and dose kinetics. Treatment of cells with specific MAP kinase and caspase inhibitors further confirmed the mechanistic involvement of these signalling cascades in pramanicin-induced apoptosis. JNK and p38 pathways acted as pro-apoptotic signalling pathways in pramanicin-induced apoptosis, in which they regulated release of cytochrome c and caspase activation. In contrast the ERK 1/2 pathway exerted a protective effect through inhibition of cytochrome c leakage from mitochondria and caspase activation, which were only observed when lower concentrations of pramanicin were used as apoptosis-inducing agent and which were masked by the intense apoptosis induction by higher concentrations of pramanicin. These results suggest pramanicin as a potential apoptosis-inducing small molecule, which acts through a well-defined JNK- and p38-dependent apoptosis signalling pathway in Jurkat T leukemia cells.