Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse dermal fibroblasts from X linked GFP transgenics - improved dna methylation analysis via enrichment of demethylated cells expressing an X-inactivated GFP transgene

ABSTRACT: The demethylating drug 5-aza-2â-deoxycytidine (5-aza-2dC) is frequently used to investigate the; effect of global DNA demethylation on gene expression in cultured mammalian cells. Here, we utilize a novel method that uses the reactivation of an X-inactivated GFP transgene as a marker to enrich for cells that have undergone drug-induced demethylation. By combining it with microarray gene expression profiling, we demonstrate the methodâs utility in identifying genes activated by global DNA demethylation. Experiment Overall Design: Mice used have an X-linked GFP transgene driven by the chicken Î²-actin promoter and CMV Experiment Overall Design: intermediate early enhancer in a 129/ICR mixed genetic background obtained from The Jackson Experiment Overall Design: Laboratory. Male transgenic animals were bred to wildtype BL6 female mice to obtain females that were heterozygous for the transgene. Dermal fibroblasts were obtained from explant cultures of neonatal female mice from these crosses. The cells were then sorted using FACS to obtain only GFP-nonexpressing cells. These cells represent the population with the GFP transgene on the inactive X chromosome. The GFP-silent cells were treated with the demethylating drug 5-aza-2'-deoxycytidine. This drug does not affect all cells and uses a passive form of demethylation that results in a heterogenous population of unaffected and demethylated cells. A portion of the cells affected by demethylation activate the inactive GFP transgene by demethylating the inactive X chromosome. The GFP-active and GFP-inactive cells were sorted by FACS and assayed by microarray to determine what genes were affected by demethylation. Also the microarray results indicated if sorting for GFP-active cells enriches for cells affected by the drug.

ORGANISM(S): Mus musculus

SUBMITTER: Tammy Vallender

PROVIDER: E-GEOD-4718 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Improved DNA methylation analysis via enrichment of demethylated cells expressing an X-inactivated transgene.

Vallender Tammy W TW Lahn Bruce T BT

BioTechniques 20061001 4

The demethylating drug 5-aza-2'-deoxycytidine (5-aza-2dC) is frequently used to investigate the effect of global DNA demethylation on gene expression in cultured mammalian cells. Here, we describe a method that uses the reactivation of an X-inactivated green fluorescent protein (GFP) transgene as a marker to enrich for cells that have undergone drug-induced demethylation. By combining it with microarray gene expression profiling, we demonstrate the method's utility in identifying genes activated ...[more]

PMID: 17068962

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Project description:Silencing of genes that suppress the malignant phenotype by DNA methylation spurred an interest in the clinical use of epigenetic reprogramming agents. Single therapy is unlikely to be curative in the context of a heterogeneous disease such as Diffuse Large B cell Lymphomas (DLBCL). The combination of DNA demethylating drugs could increase the chance to respond to classical and new treatments. We found that DLBCL cell lines respond heterogeneously to DNA demethylating agents. In sensitive cell lines, 5-aza-2’-deoxycytidine induced a genomic signature similar to that of doxorubicin, the most important drug of the combinatorial chemotherapy regimen for DLBCL treatment. Accordingly, the combination of 5-aza-2’-deoxycytidine and doxorubicin proved to be synergistic in cell killing in vitro and in vivo for DLBCL cell lines individually responsive to these drugs. In doxorubicin resistant cell lines, long-term exposure to low-dose of 5-aza-2’-deoxycytidine induces DNA demethylation and subsequent doxorubicin sensitization in vitro and in vivo. This later effect correlates with SMAD1 demethylation. SMAD1 is epigenetically silenced in doxorubicin-resistant DLBCL cells and DLBCL patients with poor prognostic. In addition, we found that DNA demethylating agents can sensitize primary DLBCL cells to doxorubicin. Primary cells obtained from a DLBCL patient treated with 5-azacytidine shows SMAD1 demethylation and ex vivo sensitization to multiple drugs. Therefore, DNA demethylating drugs can reprogram otherwise resistant DLBCL cells to respond to chemotherapy agents without increasing the toxicity to normal tissues. Our data also indicate that DNA methylation and consequent suppression of SMAD1 expression represent a previously undescribed molecular mechanism of chemoresistance in DLBCL that can be further exploit for therapy. A microarray study using genomic DNA from different DLBCL cell lines before any treatment. Two to four biological replicates by cell line. The HELP data wil be used to find genes hypermethylated in resistant cell lines compared to sensitive cell lines to doxorubicin and other drugs.

Project description:Silencing of genes that suppress the malignant phenotype by DNA methylation spurred an interest in the clinical use of epigenetic reprogramming agents. Single therapy is unlikely to be curative in the context of a heterogeneous disease such as Diffuse Large B cell Lymphomas (DLBCL). The combination of DNA demethylating drugs could increase the chance to respond to classical and new treatments. We found that DLBCL cell lines respond heterogeneously to DNA demethylating agents. In sensitive cell lines, 5-aza-2M-bM-^@M-^Y-deoxycytidine induced a genomic signature similar to that of doxorubicin, the most important drug of the combinatorial chemotherapy regimen for DLBCL treatment. Accordingly, the combination of 5-aza-2M-bM-^@M-^Y-deoxycytidine and doxorubicin proved to be synergistic in cell killing in vitro and in vivo for DLBCL cell lines individually responsive to these drugs. In doxorubicin resistant cell lines, long-term exposure to low-dose of 5-aza-2M-bM-^@M-^Y-deoxycytidine induces DNA demethylation and subsequent doxorubicin sensitization in vitro and in vivo. This later effect correlates with SMAD1 demethylation. SMAD1 is epigenetically silenced in doxorubicin-resistant DLBCL cells and DLBCL patients with poor prognostic. In addition, we found that DNA demethylating agents can sensitize primary DLBCL cells to doxorubicin. Primary cells obtained from a DLBCL patient treated with 5-azacytidine shows SMAD1 demethylation and ex vivo sensitization to multiple drugs. Therefore, DNA demethylating drugs can reprogram otherwise resistant DLBCL cells to respond to chemotherapy agents without increasing the toxicity to normal tissues. Our data also indicate that DNA methylation and consequent suppression of SMAD1 expression represent a previously undescribed molecular mechanism of chemoresistance in DLBCL that can be further exploit for therapy. A microarray study using double-stranded cDNA from different DLBCL cell lines before any treatment. Two biological replicates by cell line. The gene expression will be used to find gene expression signatures between sensitive and resistant cell lines to chemotherapeutics.

Dataset Information

Transcription profiling of mouse dermal fibroblasts from X linked GFP transgenics - improved dna methylation analysis via enrichment of demethylated cells expressing an X-inactivated GFP transgene

Publications

Improved DNA methylation analysis via enrichment of demethylated cells expressing an X-inactivated transgene.

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