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Expression data from three human cancer cell lines (PC-3, SK-OV-3, WM793B) exposed to experimental cycling and chronic hypoxa in vitro

ABSTRACT: One of the most important features of tumor microenvironment, imposing adverse effect on patient prognosis, is low oxygen tension. There are two types of hypoxia that may occur within tumor mass: chronic and cycling. Preliminary studies point at cycling hypoxia as being more relevant in induction of aggressive phenotype of tumor cells and radioresistance though little is known about the molecular mechanism of this phenomenon. Analysis of gene expression profile of human prostate (PC-3), ovarian (SK-OV-3) and melanoma (WM793B) cancer cells to expermental cycling (interchanging conditions of 1% and 21% oxygen) or chronic (1% oxygen) for 72 hours. Gene expression profiles were analyzed using U133 Plus 2.0 Array (Affymetrix) oligonucleotide microarrays. Data analysis revealed that globally gene expression profiles induced by the two types of hypoxia are similar and they strongly depend on the cell type.However, cycling hypoxia changes expression of lower number of genes in comparison to chronic one ( 3767 vs. 5954 probesets (p<0.001)) and to lower extent (lower fold changes). Analysis of hypoxia-regulated gene lists obtained using Random Variance Model t-test identified 253 probe sets (FC>2, p<0.001) common to all three cell lines, though no universal (changed throughout all analyzed cell lines) genes specifically influanced only by cycling hypoxia was selected. On the other hand, we identified such genes within particular one or two cell lines. Among them those related with EGF pathway seemed to be overrepresented (i.e. EPHA2, AREG, and HBEGF) and together with PLAU and IL-8 were mostly validated by Q-PCR.

ORGANISM(S): Homo sapiens

PROVIDER: GSE53012 | GEO | 2013/12/06

SECONDARY ACCESSION(S): PRJNA230676

REPOSITORIES: GEO

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Project description:One of the most important features of tumor microenvironment, imposing adverse effect on patient prognosis, is low oxygen tension. There are two types of hypoxia that may occur within tumor mass: chronic and cycling. Preliminary studies point at cycling hypoxia as being more relevant in induction of aggressive phenotype of tumor cells and radioresistance though little is known about the molecular mechanism of this phenomenon. Analysis of gene expression profile of human prostate (PC-3), ovarian (SK-OV-3) and melanoma (WM793B) cancer cells to expermental cycling (interchanging conditions of 1% and 21% oxygen) or chronic (1% oxygen) for 72 hours. Gene expression profiles were analyzed using U133 Plus 2.0 Array (Affymetrix) oligonucleotide microarrays. Data analysis revealed that globally gene expression profiles induced by the two types of hypoxia are similar and they strongly depend on the cell type.However, cycling hypoxia changes expression of lower number of genes in comparison to chronic one ( 3767 vs. 5954 probesets (p<0.001)) and to lower extent (lower fold changes). Analysis of hypoxia-regulated gene lists obtained using Random Variance Model t-test identified 253 probe sets (FC>2, p<0.001) common to all three cell lines, though no universal (changed throughout all analyzed cell lines) genes specifically influanced only by cycling hypoxia was selected. On the other hand, we identified such genes within particular one or two cell lines. Among them those related with EGF pathway seemed to be overrepresented (i.e. EPHA2, AREG, and HBEGF) and together with PLAU and IL-8 were mostly validated by Q-PCR. We investigated transcriptional activity of prostate and ovarian cancer cells as well as melanoma cells cultured for 72h under chronic hypoxic (nominal 1% oxygen; 3 experimental samples for each cell type), cycling hypoxia (interchanging periods of nominal 1% and 21% oxygen; 3 samples for each line) and control conditions (21% oxygen; 3 samples for each cell lines).

Project description:Hypoxia has profound and diverse effects on aerobic organisms, disrupting oxidative phosphorylation and activating several protective pathways. Predictions have been made that exposure to mild intermittent hypoxia may be protective against more severe exposure and may extend lifespan. Here we report the lifespan effects of chronic, mild, intermittent hypoxia and short-term survival in acute severe hypoxia in four clones of Daphnia magna originating from either permanent or intermittent habitats. We test the hypothesis that acclimation to chronic mild intermittent hypoxia can extend lifespan through activation of antioxidant and stress-tolerance pathways and increase survival in acute severe hypoxia through activation of oxygen transport and storage proteins and adjustment to carbohydrate metabolism. Unexpectedly, we show that chronic hypoxia extended the lifespan in the two clones originating from intermittent habitats but had the opposite effect in the two clones from permanent habitats, which also showed lower tolerance to acute hypoxia. Exposure to chronic hypoxia did not protect against acute hypoxia; to the contrary, Daphnia from the chronic hypoxia treatment had lower acute hypoxia tolerance than normoxic controls. Few transcripts changed their abundance in response to the chronic hypoxia treatment in any of the clones. After 12 hours of acute hypoxia treatment, the transcriptional response was more pronounced, with numerous protein-coding genes with functionality in oxygen transport, mitochondrial and respiratory metabolism, and gluconeogenesis, showing up-regulation. While clones from intermittent habitats showed somewhat stronger differential expression in response to acute hypoxia than those from permanent habitats, contrary to predictions, there were no significant hypoxia-by-habitat of origin or chronic-by-acute treatment interactions. GO enrichment analysis revealed a possible hypoxia tolerance role by accelerating the molting cycle and regulating neuron survival through up-regulation of cuticular proteins and neurotrophins, respectively.

Project description:Background: Genes upregulated by low oxygen have been suggested as endogenous markers for tumor hypoxia. Yet, most of the genes investigated have shown inconsistent results, which have led to concerns about their ability to be true hypoxia markers. Previous studies have demonstrated that expression of hypoxia induced genes can be affected by extracellular pH (pH e ). Methods: Five different human cell lines (SiHa, FaDu DD, UTSCC5, UTSCC14 and UTSCC15) were exposed to different oxygen concentrations and pH (7.5 or 6.3), and gene expression analyzed with microarray (Affymetrix - Human Genome U133 Plus 2.0 Array). Results: An analysis of two of the cell lines using SAM identified 461 probesets that were able to separate the four groups “ Normal oxygen, normal pH ” , “ Low oxygen, normal pH ” , “ Normal oxygen, low pH ” and “ Low oxygen, low pH ” . From here it was possible to identify a fraction of probesets induced at low oxygen independent of pH in these two cell lines, this fraction included HIG2, NDRG1, PAI1 and RORA. Further verifi cation by qPCR highlighted the necessity of using more cell lines to obtain a robust gene expression profi les. To specifi cally select pH independent hypoxia regulated genes across more cell lines, data for FaDu DD, UTSCC5, UTSCC14 and UTSCC15 were analyzed to identify genes that were induced by hypoxia in each cell line, where the induction was not affected by low pH, and where the gene was not signifi cantly induced by low pH alone. Each cell line had 65 – 122 probesets meeting these criteria. For genes to be considered as target genes (hypoxia inducible pH independent), genes had to be present in three of four cell lines. Conclusion: The result is a robust hypoxia profile unaffected by pH across cell lines consisting of 27 genes. This study demonstrates a way to identify hypoxia markers by microarray, where other factors in the tumor microenvironment are taken into account. Five cell lines, four HNSCC (FaDuDD, UTSCC5, UTSCC14 and UTSCC15) and one cervical (SiHa). Hypoxia was achieved by continually gassing the cells in an airtight chamber for 24 hours with either atmo- spheric air, 5%, 1%, 0.1%, 0.01% or 0% oxygen, sup- plemented with 5% CO 2 and nitrogen, at 37ºC. To simulate acidosis, pH of the medium (without sodium bicarbonate) was buffered with 20 mM tris (Hydroxym- ethyl) aminomethane (TRIS) base (Sigma), 20 mM 2-(N-Morpholino)ethanesulfonic acid (MES) (Sigma) and 0.52 g/l NaHCO 3 (Sigma) and titrated to 6.3 or 7.4. The pH adjusted media was applied immediately before the induction of hypoxia. No replicates.

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Expression data from three human cancer cell lines (PC-3, SK-OV-3, WM793B) exposed to experimental cycling and chronic hypoxa in vitro

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