http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10550 Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIPL as the levels of FLIPL were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIPL, decreased the amount of the E3 ligase Itch associated with FLIPL, and reduced FLIPL ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIPL and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIPL and thereby TRAIL-induced apoptosis in melanoma cells. 2012-03-27T04:20:05.287Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9452 Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, BimS, remains largely unappreciated. Here, we show that inhibition of the mutant B-RAFᵛ⁶⁰⁰ᴱ triggers preferential splicing to produce BimS, which is particularly important in induction of apoptosis in B-RAFᵛ⁶⁰⁰ᴱ melanoma cells. Although the specific B-RAFᵛ⁶⁰⁰ᴱ inhibitor PLX4720 upregulates all three major isoforms of Bim, BimEL, BimL, and BimS, at the protein and mRNA levels in B-RAFᵛ⁶⁰⁰ᴱ melanoma cells, the increase in the ratios of BimS mRNA to BimEL and BimL mRNA indicates that it favours BimS splicing. Consistently, enforced expression of B-RAFᵛ⁶⁰⁰ᴱ in wild-type B-RAF melanoma cells and melanocytes inhibits BimS expression. The splicing factor SRp55 appears necessary for the increase in BimS splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of BimS and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in BimS splicing is also mirrored in freshly isolated B-RAFᵛ⁶⁰⁰ᴱ melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAFᵛ⁶⁰⁰ᴱ inhibitors. 2012-01-30T05:11:45.732Z ]]>