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Doxycycline alters metabolism and proliferation of human cell lines

ABSTRACT: The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells -- including inhibition of the mitochondrial ribosome -- there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation and alter cell cycle progression in vitro. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism. Total RNA was extracted from MCF12A cells treated with either vehicle control or Dox at 1 ug/mL. The experiment was performed in biological triplicate. Microarray results were processed using the RMA method.

ORGANISM(S): Homo sapiens

SUBMITTER: William Sullivan

PROVIDER: E-GEOD-45029 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Doxycycline alters metabolism and proliferation of human cell lines

Project description:The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells -- including inhibition of the mitochondrial ribosome -- there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation and alter cell cycle progression in vitro. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism.

2013-04-22 | GSE45029 | GEO

Tetracyclines Specifically Target the Apicoplast of the Malaria Parasite Plasmodium falciparum

Project description:Tetracyclines are effective but slow-acting antimalarial drugs whose mechanism of action remains uncertain. To characterize the antimalarial mechanism of tetracyclines, we evaluated their stage-specific activities, impacts on parasite transcription, and effects on two predicted organelle targets, the apicoplast and the mitochondrion, in cultured Plasmodium falciparum. Antimalarial effects were much greater after two 48-h life cycles than after one cycle, even if the drugs were removed at the end of the first cycle. Doxycycline-treated parasites appeared morphologically normal until late in the second cycle of treatment but failed to develop into merozoites. Doxycycline specifically impaired the expression of apicoplast genes. Apicoplast morphology initially appeared normal in the presence of doxycycline. However, apicoplasts were abnormal in the progeny of doxycycline-treated parasites, as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogeny. Replication of the nuclear and mitochondrial genomes and mitochondrial morphology appeared normal. Our results demonstrate that tetracyclines specifically block expression of the apicoplast genome, resulting in the distribution of nonfunctional apicoplasts into daughter merozoites. The loss of apicoplast function in the progeny of treated parasites leads to a slow but potent antimalarial effect. Keywords: Plasmodium falciparum treated with Doxycycline

2006-07-11 | GSE5267 | GEO

Tetracyclines Specifically Target the Apicoplast of the Malaria Parasite Plasmodium falciparum

Project description:Tetracyclines are effective but slow-acting antimalarial drugs whose mechanism of action remains uncertain. To characterize the antimalarial mechanism of tetracyclines, we evaluated their stage-specific activities, impacts on parasite transcription, and effects on two predicted organelle targets, the apicoplast and the mitochondrion, in cultured Plasmodium falciparum. Antimalarial effects were much greater after two 48-h life cycles than after one cycle, even if the drugs were removed at the end of the first cycle. Doxycycline-treated parasites appeared morphologically normal until late in the second cycle of treatment but failed to develop into merozoites. Doxycycline specifically impaired the expression of apicoplast genes. Apicoplast morphology initially appeared normal in the presence of doxycycline. However, apicoplasts were abnormal in the progeny of doxycycline-treated parasites, as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogeny. Replication of the nuclear and mitochondrial genomes and mitochondrial morphology appeared normal. Our results demonstrate that tetracyclines specifically block expression of the apicoplast genome, resulting in the distribution of nonfunctional apicoplasts into daughter merozoites. The loss of apicoplast function in the progeny of treated parasites leads to a slow but potent antimalarial effect. We analyzed a series of 12 microarrays covering 55 hours of Plasmodium falciparum treated with doxycycline and 12 microarrays covering the same 55 hours with no doxycycline treatment

2010-07-01 | E-GEOD-5267 | biostudies-arrayexpress

MCF7 tet-off cells treated or not with 80 ng/ml doxycycline for 12d

Project description:Tet-on and tet-off systems are among the most popular vector systems for inducible transgene expression in mammalian cells. In tet-regulated systems the expression of the transgene depends on the presence or absence of the antibiotic tetracycline or its derivative doxycycline, which are added to the cell culture medium in concentrations considered to be far below cytotoxic levels. Therefore, potential effects on the treated cells, which are exerted by the antibiotic itself and unrelated to transgene expression, are often ignored. We examined the influence of low dose doxycycline on the transcriptional profile of two independent clones of MCF7 human breast carcinoma cells, transfected with tet-off regulator and response plasmids but not harboring any transgene. Treatment with 80 ng/ml doxycycline for 12 days markedly altered gene expression in these cells. Many genes associated with interferon-signaling were up-regulated while cell cycle-associated genes were down-regulated, which was also accompanied by a reduction of cell growth. These results highlight the importance of appropriate controls when working with tet-regulated gene expression systems, to allow distinction between the effects of transgene expression and potential “side effects” of the antibiotic used for its regulation.

2011-05-20 | GSE13926 | GEO

RNA-seq of human melanoma cell line A375 treated with the tetracycline analogs Col-3 or doxycycline against untreated controls

Project description:Apart from their antibacterial activity, tetracyclines have demonstrated diverse and beneficial “off-target” biological activities in human disease, spurring interest in their development as non-antimicrobial therapeutics in diseases such as cancer. To better define biological pathways modified by the tetracyclines in human cancer, we sought to determine differential gene expression in the A375 cancer cell line treated with the tetracyclines, Col-3 and doxycycline, relative to control (vehicle-treated) cells. A375 cells were treated with either 10 μM Col-3, 10 μM doxycycline, or vehicle (DMSO) for 6 h, and changes to gene expression were assessed by RNA-Seq.

2018-09-01 | E-MTAB-7126 | biostudies-arrayexpress

Tetracyclines Modify Translation By Targeting Key Human rRNA Substructures

Project description:Mortison JD, Schenone M, Myers JA, Zhang Z, Chen L, Ciarlo C, Comer E, Natchiar SK, Carr SA, Klaholz BP, Myers AG. Cell Chem Bio 2018. Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-Seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

2018-08-15 | MSV000082819 | MassIVE

EWS/ATF1 activates Fos and induces soft tissue sarcomas from neural crest-derived cells [Agilent].

Project description:Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12; 22) translocation, leading to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying how EWS/ATF1 is involved in the development of CCSs. In addition, the cells of origin for CCSs remain to be determined. We generated EWS/ATF1-inducible mice, and examined the effects of EWS/ATF1 expression in adult cells. We show that the forced expression of EWS/ATF1 results in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembles that of CCSs and EWS/ATF1-induced tumor cells express CCS-markers, such as S100, Sox10, and Mitf. A lineage tracing experiment revealed that such sarcomas are derived from neural crest-lineage cells. Finally, we found that EWS/ATF1 directly induces Fos in an ERK-independent manner, and demonstrated that the increased Fos expression is important for the active cell proliferation in not only EWS/ATF1-induced sarcomas, but also in human CCSs. Our results indicate that FOS, as well as EWS/ATF1 itself, could be a promising therapeutic target for the treatment of EWS/ATF1-related sarcomas. Tumor cell lines were established from soft tissue sarcomas in EWS/ATF1-induced mice. Because these cell lines contain doxycycline-inducible EWS/ATF1 alleles, EWS/ATF1 expression can be regulated by the different concentrations of doxycycline. Tumor cell lines were exposed to different concentrations of doxycycline, and total RNAs were isolated at 3 and 48 hours after the doxycycline exposure.

2013-04-04 | E-GEOD-41122 | biostudies-arrayexpress

CEBPD suppresses motility-associated gene signatures and reduces pancreatic ductal adenocarcinoma cell migration

Project description:To investigate the effects of CEBPD on pancreatic ductal cell tumorigenicity, we established doxycycline-inducible MIA PaCa-2 cells for CEBPD over-expression, as well as non-control cells with an empty inducible construct. RNAseq was performed on these cells in the presence and absence of doxycycline and we showed that CEBPD activates gene signatures associated with cell motility.

2022-09-26 | GSE214028 | GEO

Effect of ATG7 reconstitution in AGY571 cells

Project description:AGY571 cells engineered to express Dox-inducible GFP or ATG7 were treated with 500 ng/mL or 1 ug/mL Doxycycline for 48 hours. Assessing the effects of ATG7 reexpression in B cell lymphoma.

2016-09-08 | ST000460 | MetabolomicsWorkbench

Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4 (expression)

Project description:Temporal analysis of B cell activation in vitro using CD40L and IL-2/4/5 cytokines in wild type Irf4+/+ B cells or in mutant Irf4-/- B cells harboring a tet-inducible allele of Irf4. IRF4 expression was restored, or not, in the Irf4-/- background by culturing in the presence of low or high concentrations of doxycycline. The results provide insight in the role of IRF4 expression levels in coordinating different programs of B cell differentiation. Resting mature peripheral primary B cells were enriched from the spleens of Irf4+/+ or Irf4-inducible mice on the Irf4-/- background. We sought to compare gene expression profiles of wild type and Irf4 mutant B cells in response to mitogens that promote the differentiation of the B cells into plasma cells and cells that have undergone class switch recombination. Day 0 samples represent RNA analysis of unstimulated cells, whereas Day 1 and Day 3 samples represent analysis of stimulated cells in culture. For stimulation, cells were cultured with insect cell purified CD40L and IL-2/4/5 cytokines for the indicated days. In the case of doxycycline-mediated rescue of IRF4 expression, doxycycline was added at predefined low and high concentrations that yield low or high numbers of plasma cells, respectively (see Molecular Systems Biology 7:495). Each replicate represents analysis of B cells from an individual mouse. Of the three replicates in each group, two were performed in parallel and one was performed at a different time. All cells were lysed using Trizol and total RNA was purified according to manufacturer's suggestions. The high quality of the RNA was confirmed using Agilent Bioanalyzer 2100 system. 250ng of RNA was then processed into biotinylated cRNA according to standard procedures and used to hybridize to Affymetrix 430 2.0 arrays. Signal intensities were normalized using the D-Chip algorithm (PM-only model) and the output was used to quantify differential gene expression between groups.

2013-05-04 | E-GEOD-46606 | biostudies-arrayexpress

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