TY - JOUR
T1 - The acquisition of resistance to TNFα in breast cancer cells is associated with constitutive activation of autophagy as revealed by a transcriptome analysis using a custom microarray
AU - Moussay, Etienne
AU - Kaoma, Tony
AU - Baginska, Joanna
AU - Muller, Arnaud
AU - Van Moer, Kris
AU - Nicot, Nathalie
AU - Nazarov, Petr V.
AU - Vallar, Laurent
AU - Chouaib, Salem
AU - Berchem, Guy
AU - Janji, Bassam
N1 - Funding Information:
The quality of microarraytring and scaling of log-rat%s was cio distrPontrolled ibutionsOTC. prPG Cimers wUAG Aere ET-3' (F5'-GGJorA TTwardGG TU) anSd 3GT CJ'-TCTG TC VIgG peroxU(JacidaFkson ImmunoResearch 111-035-se-conjugated goat anti-rabbit by distributions of log-ratio values of the GCA CTT TCT GTG GAC AT-3' 144) and goat anti-mouse IgG (Jackson good-quality spots, their total number of (Reverse). LC3 primers were 5'-AGC ImmunoResearch 115-035-146). Protein per array, average correlation with other AGC ATC CAA CCA AAA TC-3' bands were revealed by enhanced che-arrays and spatial homogeneity. To find (Forward) and 3'-CTG TGT CCG TTC miluminescence ECL (GE Healthcare, genes with statistically significant regula-ACC AAC AG-3' (Reverse). BCL2 prim-RPN2135V1). tion in each class (MCF-7 vs. 1001 and ers were 5'-ATC GCC CTG TGG ATG Indirect immunofluorescence. Cells 1001 + TNFα vs. 1001) and between these ACT GA-3' (Forward) and 3'-GGG CCG were fixed with 4% paraformaldehyde, two classes, the empirical Bayes method,40 TAC AGT TCC ACA AA-3' (Reverse). detergent permeabilized with 0.4% Triton implemented in R/Bioconductor’s limma P62 primers were 5'-CAG CTT CTG X-100 and stained with primary rabbit package (R-code available at http://www. CTG CAG CCC CG-3' (Forward) and monoclonal anti-LC3B antibody (Cell bioinformatics.lu), was used. Raw data 3'-TCC TCA TCG CGG TAG TGC Signaling, 3868) and secondary Alexa have been submitted to the EMBL-EBI GC-3' (Reverse). Five nanograms of Fluor 488-coupled rabbit anti IgG anti-ArrayExpress repository under the refer-cDNA were used in a 25 μl PCR reac-body. Cells were analyzed with a Zeiss ence E-MEXP-3074 and can be visualized tion mixture containing 12.5 μl SYBR® laser scanning confocal microscope at the website at http://www.ebi.ac.uk/ Green PCR MasterMix and 300 nM of (LSM-510-Meta). arrayexpress/. Genes with adjusted p-value each primer. PCR amplifications were lower than 0.05 were considered as sig-performed on ABI 7300 Real-Time PCR nificantly regulated. Microarray Data System (40 cycles at 95°C for 15 sec and This work was supported by grants from mining was performed using the data-60°C for 60 sec). Values were normalized the Luxembourg Ministry of Culture, bases from Ingenuity Pathway Analysis to 28S rRNA and were processed by using Higher Education and Research (REC-system (Ingenuity® Systems, IPA® 8) and the 2-ΔΔCtcalculation method. LHCE-2009 0201) to Bassam Janji, the MetaCoreTM (MetaCoreTM version 6.5 Cholesterol staining. MCF-7 and Télévie (N° 7.4552.05) and the National build 27009, GeneGo Inc., MI). 1001 cells were grown on cover slips in Research Fund (FNR) of Luxembourg to Validation of autophagy-microarray complete medium until reaching 70–80% Etienne Moussay. Joanna Baginska is a data with commercial whole genome of confluence. After two washes with PBS, recipient of an AFR grant from the FNR. microarray data. RNA from MCF-7 cells were fixed in 4% paraformaldehyde The authors thank Sandrine Pierson
PY - 2011/7
Y1 - 2011/7
N2 - While the autophagic process is mainly regulated at the posttranslational level, a growing body of evidence suggests that autophagy might also be regulated at the transcriptional level. The identification of transcription factors involved in the regulation of autophagy genes has provided compelling evidence for such regulation. In this context, a powerful high throughput analysis tool to simultaneously monitor the expression level of autophagy genes is urgently needed. Here we describe setting up the first comprehensive human autophagy database (HADb, available at www.autophagy. lu) and the development of a companion Human Autophagy-dedicated cDNA Microarray which comprises 234 genes involved in or related to autophagy. The autophagy microarray tool used on breast adenocarcinoma MCF-7 cell line allowed the identification of 47 differentially expressed autophagy genes associated with the acquisition of resistance to the cytotoxic effect of TNFα. The autophagy-core machinery genes DRAM (Damage-Regulated Autophagy Modulator), BNIP3L (BCL2/adenovirus E1B 19 kDa interacting protein 3-like), BECN1 (Beclin 1), GABARAP (Gamma-AminoButyric Acid Receptor-Associated Protein) and UVRAG (UV radiation resistance associated gene) were found upregulated in TNF-resistant cells, suggesting a constitutive activation of the autophagy machinery in these cells. More interestingly, we identified NPC1 as the most upregulated genes in TNF-resistant compared to TNF-sensitive MCF-7 cells, suggesting a relation between the intracellular transport of cholesterol, the regulation of autophagy and NPC1 expression in TNF-resistant tumor cells. In conclusion, we describe here new tools that may help investigating autophagy gene regulation in various cellular models and diseases.
AB - While the autophagic process is mainly regulated at the posttranslational level, a growing body of evidence suggests that autophagy might also be regulated at the transcriptional level. The identification of transcription factors involved in the regulation of autophagy genes has provided compelling evidence for such regulation. In this context, a powerful high throughput analysis tool to simultaneously monitor the expression level of autophagy genes is urgently needed. Here we describe setting up the first comprehensive human autophagy database (HADb, available at www.autophagy. lu) and the development of a companion Human Autophagy-dedicated cDNA Microarray which comprises 234 genes involved in or related to autophagy. The autophagy microarray tool used on breast adenocarcinoma MCF-7 cell line allowed the identification of 47 differentially expressed autophagy genes associated with the acquisition of resistance to the cytotoxic effect of TNFα. The autophagy-core machinery genes DRAM (Damage-Regulated Autophagy Modulator), BNIP3L (BCL2/adenovirus E1B 19 kDa interacting protein 3-like), BECN1 (Beclin 1), GABARAP (Gamma-AminoButyric Acid Receptor-Associated Protein) and UVRAG (UV radiation resistance associated gene) were found upregulated in TNF-resistant cells, suggesting a constitutive activation of the autophagy machinery in these cells. More interestingly, we identified NPC1 as the most upregulated genes in TNF-resistant compared to TNF-sensitive MCF-7 cells, suggesting a relation between the intracellular transport of cholesterol, the regulation of autophagy and NPC1 expression in TNF-resistant tumor cells. In conclusion, we describe here new tools that may help investigating autophagy gene regulation in various cellular models and diseases.
KW - Autophagy-related genes and proteins
KW - Database
KW - Microarray
UR - http://www.scopus.com/inward/record.url?scp=79960008323&partnerID=8YFLogxK
UR - https://pubmed.ncbi.nlm.nih.gov/21490427
U2 - 10.4161/auto.7.7.15454
DO - 10.4161/auto.7.7.15454
M3 - Article
AN - SCOPUS:79960008323
SN - 1554-8627
VL - 7
SP - 760
EP - 770
JO - Autophagy
JF - Autophagy
IS - 7
ER -