Dataset Information

Identification and characterisation of human apoptosis inducing proteins using cell-based transfection microarrays and expression analysis.

ABSTRACT: BACKGROUND: Cell-based microarrays were first described by Ziauddin and Sabatini in 2001 as a powerful new approach for performing high throughput screens of gene function. An important application of cell-based microarrays is in screening for proteins that modulate gene networks. To this end, cells are grown over the surface of arrays of RNAi or expression reagents. Cells growing in the immediate vicinity of the arrayed reagents are transfected and the arrays can then be scanned for cells showing localised changes in function. Here we describe the construction of a large-scale microarray using expression plasmids containing human genes, its use in screening for genes that induce apoptosis when over-expressed and the characterisation of a number of these genes by following the transcriptional response of cell cultures during their induction of apoptosis. RESULTS: High-density cell-based arrays were successfully fabricated using 1,959 un-tagged open reading frames (ORFs) taken from the Mammalian Gene Collection (MGC) in mammalian expression vectors. The arrays were then used to screen for genes inducing apoptosis in Human Embryonic Kidney (HEK293T) cells. Using this approach, 10 genes were clearly identified and confirmed to induce apoptosis. Some of these genes have previously been linked to apoptosis, others not. The mechanism of action of three of the 10 genes were then characterised further by following the transcriptional events associated with apoptosis induction using expression profiling microarrays. This data demonstrates a clear pro-apoptotic transcriptional response in cells undergoing apoptosis and also suggests the use of common apoptotic pathways regardless of the nature of the over-expressed protein triggering cell death. CONCLUSION: This study reports the design and use of the first truly large-scale cell-based microarrays for over-expression studies. Ten genes were confirmed to induce apoptosis, some of which were not previously known to possess this activity. Transcriptome analysis on three of the 10 genes demonstrated their use of similar pathways to invoke apoptosis.

SUBMITTER: Palmer EL

PROVIDER: S-EPMC1525185 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Identification and characterisation of human apoptosis inducing proteins using cell-based transfection microarrays and expression analysis.

Palmer Ella L EL Miller Andrew D AD Freeman Tom C TC

BMC genomics 20060612

<h4>Background</h4>Cell-based microarrays were first described by Ziauddin and Sabatini in 2001 as a powerful new approach for performing high throughput screens of gene function. An important application of cell-based microarrays is in screening for proteins that modulate gene networks. To this end, cells are grown over the surface of arrays of RNAi or expression reagents. Cells growing in the immediate vicinity of the arrayed reagents are transfected and the arrays can then be scanned for cell ...[more]

PMID: 16768789

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Project description:BACKGROUND:Inducing apoptosis in cancer cells is an important step for the successful treatment of cancer patients. Bcl-2 is an antiapoptotic protein which determines apoptosis by interacting with proapoptotic members of the Bcl-2 family. Exome sequencing has identified Bcl-2 and Bax missense mutations in more than 40 cancer types. However, a little information is available about the functional impact of each Bcl-2 and Bax mutation on the pathogenesis of cancer. METHODS:The mutational data from cancer tissues and cell lines were retrieved from the cBioPortal web resource. The 13 mutated Bcl-2 and wild-type Bax complexes with experimentally verified binding were identified from previous studies wherein, binding for all complexes was reportedly disrupted except one. Several protein-protein docking methods such as ClusPro, HDOCK, PatchDock, FireDock, InterEVDock2 and several mutation prediction methods such as PolyPhen-2, SIFT, and OncoKB have been used to predict the effect of mutation to disrupt the binding between Bcl-2 and Bax. The result obtained was compared with the known experimental data. RESULTS:The protein-protein docking method, ClusPro, employed in the present study confirmed that the binding affinity of 11 out of 13 complexes decreases. Similarly, binding affinity computed for all the 10 wild-type Bcl-2 and mutated Bax complexes agreed with experimentally verified results. CONCLUSION:Several methods like PolyPhen-2, SIFT, and OncoKB have been developed to predict cancer-associated or deleterious mutations, but no method is available to predict apoptosis-inducing mutations. Thus, in this study, we have examined the mutations in Bcl-2 and Bax proteins that disrupt their binding, which is crucial for inducing apoptosis to eradicate cancer. This study suggests that protein-protein docking methods can play a significant role in the identification of hotspot mutations in Bcl-2 or Bax that can disrupt their binding with wild-type partner to induce apoptosis in cancer cells.

Project description:Vitamin D is the strongest known natural anti-proliferative. A large number of studies in a wide spectrum of cancers, including epidemiological, in vitro and animal models, demonstrate that the active form of Vitamin D has anti-cancer benefits, affecting both progression and metastasis. Alike the role in calcium Vitamin D regulation, its anti-proliferative effect is thought to function through the Vitamin D receptor (VDR), although convincing evidence is lacking. Notwithstanding, separation of the calcemic and the anti-proliferative activity of Vitamin D analogues has been a major obstacle in developing new drugs for the treatment of cancer. The work presented attempts to unveil the molecular mechanism behind the anti-proliferative action of Vitamin D using genomic tools. For that purpose four independently developed Vitamin D sensitive/resistant MCF7 cell line pairs were collected. These unique biological replicates enabled us, both to increase the power of our study and to omit the use of Vitamin D. We deem this omission crucial since in the presence of Vitamin D only downstream genes involved in proliferation and cell cycle would be identified rather than causal resistance genes. The use of a variety of genomic techniques including expression, NMD and oligo CGH arrays reveal in the resistant cell lines the 11q13-14 as a region of DNA copy number loss and an altered expression of EGFR signaling pathway genes. Surprisingly, no genes known from calcium Vitamin D regulation were identified, nor did the VDR silencing by RNAi induce resistance to the sensitive cell lines. Keywords: Expression microarray, cell type comparision Several MCF7 breast tumor cell lines independently derived from the human breast cancer cell line, both resistant and sensitive to Vitamin D, were used. The pairs of cell lines (parental-resistant) were developed in different laboratories and using different methodologies; while some cell lines acquired resistance by long exposure to the physiological concentration of Vitamin D (100nM), others were induced to resistant by being exposed to increasing amounts of Vitamin D. A total of 14 microarray expression experiments were carried out, including four biological replicates and 10 technical replicates including dye swaps. In all microarray experiments RNA from the Vitamin D resistant cell line was hybrizided agains RNA from the respsective sensitive/parental cell line.

Dataset Information

Identification and characterisation of human apoptosis inducing proteins using cell-based transfection microarrays and expression analysis.

Publications

Identification and characterisation of human apoptosis inducing proteins using cell-based transfection microarrays and expression analysis.

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