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Stable XIAP knockdown in HCT116 colon cancer cells

ABSTRACT: X-linked inhibitor of apoptosis (XIAP) is the most potent endogenous caspase inhibitor preventing cell death via caspase-9, -7 and -3 (initiator and executioner caspase pathways). Using short hairpin RNA (shRNA) against XIAP, stably expressed in a parent HCT116 human colon cancer cell line, a series of clones have been developed. XIAP mRNA levels were established by RT-PCR, the four X (XIAP knockdown) clonal cell lines show 82-93% reduction in XIAP mRNA when compared to the four L (luciferase control) cell lines. Immunoblot analysis showed a 67-89% reduction in XIAP protein in X cell lines compared to L. RNA was analysed by microarray and XIAP knockdown was confirmed in 7 probe sets, there was no significant compensation of other IAP family members. XIAP knockdown induced a 2-fold increase in the basal level of apoptosis without modification of caspase 3/7 activity. Finally, XIAP knockdown sensitises cells to radiotherapy by 20%, to recombinant TRAIL by a 3-fold factor, and to paclitaxel and docetaxel by >2 fold factor. Future work should focus on targeted agents such as rhTRAIL in combination with strategies to down regulate XIAP. XIAP antisense is now in clinical development in oncology.

ORGANISM(S): Homo sapiens

PROVIDER: GSE11618 | GEO | 2008/12/01

SECONDARY ACCESSION(S): PRJNA106125

REPOSITORIES: GEO

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Project description:This is the standard model described in the article: Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein. Rehm M, Huber HJ, Dussmann H, Prehn JH. EMBO J. 2006 Sep 20;25(18):4338-49. Epub 2006 Aug 24. PMID:16932741, doi:10.1038/sj.emboj.7601295; Abstract: Activation of effector caspases is a final step during apoptosis. Single-cell imaging studies have demonstrated that this process may occur as a rapid, all-or-none response, triggering a complete substrate cleavage within 15 min. Based on biochemical data from HeLa cells, we have developed a computational model of apoptosome-dependent caspase activation that was sufficient to remodel the rapid kinetics of effector caspase activation observed in vivo. Sensitivity analyses predicted a critical role for caspase-3-dependent feedback signalling and the X-linked-inhibitor-of-apoptosis-protein (XIAP), but a less prominent role for the XIAP antagonist Smac. Single-cell experiments employing a caspase fluorescence resonance energy transfer substrate verified these model predictions qualitatively and quantitatively. XIAP was predicted to control this all-or-none response, with concentrations as high as 0.15 microM enabling, but concentrations >0.30 microM significantly blocking substrate cleavage. Overexpression of XIAP within these threshold concentrations produced cells showing slow effector caspase activation and submaximal substrate cleavage. Our study supports the hypothesis that high levels of XIAP control caspase activation and substrate cleavage, and may promote apoptosis resistance and sublethal caspase activation in vivo. This model is slightly altered from the description in the article. Cytochrome C and SMAC release from the mitochondrion is modelled as simple first order kinetics, giving the same form as the (integrated) equations in the supplement of the article. The apoptosome formation is modelled equally - and independent of the Cytochrome C release. The speed is either limited by the Apaf1 or ProCaspase9 concentration, whichever is higher, symbolised via the parameter with the ID apolim. Also, once the substrate concentration falls below 1 percent, the event Production_Breakdown is triggered, leading to a breakdown of XIAP and procaspase3 production and turning off of the enhanced/proteosomal degradation (degradation rate for reactions 38,39,40,43,44,46,48,50,51 changes from 0.0347 to 0.0058). Originally created by libAntimony v1.3 (using libSBML 3.4.1) This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2012 The BioModels.net Team. For more information see the terms of use. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

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Stable XIAP knockdown in HCT116 colon cancer cells

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