Transcription profiling of E. coli dam and mutS mutants exposed to cisplatin
ABSTRACT: We used Affymetrix microarrays to determine the cisplatin-induced gene expression changes in E. coli deficient in dam and mismatch repair (dam, dam mutS, and mutS mutant strains). E. coli deficient in dam are hypersensitive to cisplatin. However introducing an additional mutation in mismatch repair (i.e., a mutation in mutS or mutL) abrogates this sensitivity and essentially restores wildtype levels of resistance. Experiment Overall Design: Overnight cultures were diluted 1000-fold and grown in Luria-Bertani (LB) medium until the cells reached exponential growth as determined by OD600. The exponentially growing cells were resuspended in M9 minimal medium at a cell density of 2 X 108 cells/ml in a volume of 15 ml and treated with 150 uM cisplatin for 2 hours at 37°C. Following treatment cultures were resuspended in 15 ml LB and allowed to recover for 90 minutes at 37°C. OD600 readings were taken after the recovery period, when RNA isolation began. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) according to the manufacture’s protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20mM 3-[N-morpholino]propanesulfonic acid, 5mM sodium acetate, 1mM ethylenadiaminetetraacetic acide (EDTA)) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure.
Project description:We determined the global gene expression profiles of wildtype, dam, dam mutS, and mutS mutant E. coli strains. Experiment Overall Design: Overnight cultures were diluted 1000-fold with fresh LB medium and cultured further. Log phase cultures were diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which they were resuspended in LB broth for 90 minutes. This treatment served as a mock treatment for cultures incubated with a chemical (cisplatin) in a parallel study (Robbins et al., unpublished results). Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturer’s protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.
Project description:A2780 ovarian cancer cells and a cisplatin resistant derivate of A2780 cells, obtained from ECACC, UK, No. 93112519 [A2780] and No. 93112517 [A2780 cis] were seeded out in T-75 flasks at a density of 2x106 for A2780sens and 3x106 for A2780cis cells in 15 ml of medium and preincubated overnight. Medium was removed and 15 ml fresh medium (37°C) with different concentrations of cisplatin, liposomal cisplatin or empty liposomes were added and incubated for 72 h at 37°C in a 5% CO2 incubator. In case of A2780sens cells, 1.72 µM cisplatin (IC50 concentration) and in case of A2780cis cells 8.94 µM cisplatin (IC50 concentration) were added. Liposomal formulations contained equal cisplatin concentrations. Empty liposomes were added in the same concentration as the liposomal cisplatin, to analyze the impact of liposomal lipids (A2780sens: 0.80µmol lipid, A2780cis: 4.15 µmol lipid). After incubation, medium was removed and cells were washed thrice with 10 ml PBS. 1 ml RLT-buffer was added and cells lysates were stored at -80°C until RNA extraction.
Project description:Gene expression of P. aerruginosa changes after short-term exposure to ciprofloxacin at sub-inhibitory concentrations but the effect of long-term exposure which select for the most fitted subpopulations is not known. We used microarrays to investigate the changes in gene expression of P.aeruginosa PAO1 and mutator (Δ mutS) after long-term evolution (94 daily passages) in LB in the presence and absence of ciprofloxacin Three different colonies from the ancestral populations of PAO1 and mutator (Δ mutS) as well as from the evolved populations (day 94) of each of the three lineages (A;B;C) in the presence or absence of ciprofloxacin at a concentration of 0.05 µg/ml were used for overnight cultures in LB and total RNA was extracted at OD600nm=1 and hybridized on P. aeruginosa Affymetrix chip.
Project description:Dictyostelium discoideum is a useful model for studying mechanisms of cisplatin drug sensitivity. Our previous findings, that mutations in sphingolipid-metabolism genes confer cisplatin resistance in D. discoideum and in human cells, raised interest in the resistance mechanisms and their implications for cisplatin chemotherapy. Here we used expression microarrays to monitor physiological changes and to identify pathways that are affected by cisplatin treatment of D. discoideum. We found over 400 genes whose regulation was altered by cisplatin treatment of wild type cells, including groups of genes that participate in cell proliferation and in nucleotide and protein metabolism. These findings show that the cisplatin response is orderly and multifaceted. Transcriptional profiling of two isogenic cisplatin-resistant mutants, impaired in different sphingolipid metabolism steps, showed that the effect of cisplatin treatment was greater than the effect of the mutations, indicating that cisplatin-resistance in the mutants is due to specific abilities to overcome the drug effects rather than to general drug insensitivity. Nevertheless, the mutants exhibited significantly different responses to cisplatin compared to the parent and over 200 genes accounted for that difference. We mutated some of the cisplatin-response genes and found that the mutants had altered drug sensitivity. These findings reveal the power of this system to identify pathways and genes that are affected by drugs and mutations. Our data illustrate how modeling complex cellular responses to drugs in genetically stable and tractable systems can uncover new targets with the potential for improving chemotherapy. Keywords: Dictyostelium discoideum, cisplatin treatment, transcriptional profiles, physiological changes Overall design: Exponentially growing cells (2x10E6 cells/ml) were divided into twelve 50 ml aliquots in 500 ml flasks. The cells were shaken for 30 min, 300 µM cisplatin was added to 6 cultures (PT buffer was added to the 6 controls), and the cells were shaken for 3 hr at 22C in the dark. The cells were washed with phosphate buffered saline (PBS) and resuspended in 1.0 ml TRIzol reagent (Life Technologies, Gaithersburg, MD) and RNA was extracted. For technical variation, RNA samples from each experiment were hybridized to at least 5 arrays (technical replication). For biological variation, each treatment (with or without cisplatin) was repeated twice for each mutant and 6 times for the wild type (biological replication). We analyzed both wild type and 2 mutant strains that have a higher resistance to cisplatin than wild type.
Project description:To investigate the difference of mRNA and lncRNA profiling between cisplatin-resistance and regular T24 bladder cancer cells, T24 cells were treated with a gradual increment (4, 8, 16, 32, 64 ug/ml cisplatin) with a discontinuous period until cells recover. 10^6 cells T24R and T24 cells were harvested for RNA-seq.
Project description:Gene level analysis of lactating day 10-11 16h Sprague-Dawley dams compared against age matched virgins. Lactation is a time of significantly increased energy demands imposed by the suckling young that requires a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules to meet the dietary needs of both the offspring and the dam. We have shown an increase in the size and hydrophobicity of the bile acid pool during lactation , implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the level of mRNA, we utilized an exon array and calculated gene level summaries to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Affymetrix Rat Exon 1.0ST arrays were used to determine expression of genes in the livers and small intestines of day 10-11 lactating Sprague Dawley rats and compared against virgins of comparable age.
Project description:Ixr1 is a transcriptional factor from Saccharomyces cerevisae with high affinity to cisplatin-DNA adducts through their two HMG-box DNA binding domains. Its transcriptional regulation is essential in the cytotoxicity caused by cisplatin, although the molecular mechanisms supporting this function are not understood. We present a transcriptome analysis discriminating between RNA changes induced by cisplatin which are dependent or independent of the Ixr1 function. Overall design: The S. cerevisiae strain W303 (MATa ade2-1 can1-100 leu2-3,112 trp1-100 ura3- 52) and its derivative W303-Δsky1 previously described (Rodríguez- Lombardero et al., 2012) have been used in these analyses. Biological replicates of cultures and treatments were run in triplicate. The yeast cells were pre-cultured over night in 10mL of complete synthetic medium (SD) prepared as previously described (Zitomer and Hall, 1976). The following day the cells were inoculated at initial OD600 of 0.4 in 70 mL SD and grown in 250 mL Erlenmeyer flasks at 30 ºC and with agitation at 250 rpm. When cells reached OD600 of 0.6, the cultures from each strain were divided in two aliquots of 25 mL (control and cisplatin treatment). A stock solution of cisplatin 6 mM in dimethyl sulfoxide (DMSO) was prepared and the drug was added to the treated cultures at a final concentration of 600 microM. An equivalent volume of DMSO was added to the control cultures. The treatment was done at 30ºC and with agitation at 250 rpm during four hours in darkness. RNA was extracted from a number of cells corresponding to OD600 of 3 with the AurumTM Total RNA Mini Kit (Bio-Rad) and following the manufacturer’s instructions. The RIN parameter (RNA Integrity Number) evaluated with the 2100 was near to the value 9 in all the samples.