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Genome-wide analysis in human colorectal cancer cells reveals ischemia-mediated expression of motility genes via DNA hypomethylation.

Skowronski K, Skowronki K, Andrews J, Rodenhiser DI, Coomber BL - PLoS ONE (2014)

Bottom Line: DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability.Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61.Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences; Ontario Veterinary College; University of Guelph; Guelph, ON, Canada.

ABSTRACT
DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumours, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes.

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SNP 6.0 array of HCT116 cells to visualize copy number variation.Numbers (and X/Y) correspond to chromosomes, and MT represents mitochondrial DNA. Results were compared to a dataset from Affymetrix containing 270 mixed population samples from the International HapMap Project [29]. Regions in grey indicate no change in copy number, red indicates a copy number gain, and blue a copy number loss. This array was performed in duplicate.
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pone-0103243-g001: SNP 6.0 array of HCT116 cells to visualize copy number variation.Numbers (and X/Y) correspond to chromosomes, and MT represents mitochondrial DNA. Results were compared to a dataset from Affymetrix containing 270 mixed population samples from the International HapMap Project [29]. Regions in grey indicate no change in copy number, red indicates a copy number gain, and blue a copy number loss. This array was performed in duplicate.

Mentions: The copy number of genes in the HCT116 cells was assessed with the Affymetrix SNP 6.0 array to determine if, and to what extent, the cells we used had drifted/mutated in culture compared to the original karyotype of this cell line. When compared to a pooled normal human karyotype from the International HapMap project database [29], the cells lacked extensive chromosomal abnormalities (Figure 1). We did observe regions on 8q, 10q, 16q, and 17q that displayed regional chromosomal gains (indicated in red), as previously reported by others [30]. Very few complete loss of copies were seen (blue), except for the Y chromosome which has been reported to be absent in 50–100% of HCT116 cells [31]. Thus, in comparison with other cancer cell lines, HCT116 have a relatively normal and stable karyotype with minimal chromosomal instability [30].


Genome-wide analysis in human colorectal cancer cells reveals ischemia-mediated expression of motility genes via DNA hypomethylation.

Skowronski K, Skowronki K, Andrews J, Rodenhiser DI, Coomber BL - PLoS ONE (2014)

SNP 6.0 array of HCT116 cells to visualize copy number variation.Numbers (and X/Y) correspond to chromosomes, and MT represents mitochondrial DNA. Results were compared to a dataset from Affymetrix containing 270 mixed population samples from the International HapMap Project [29]. Regions in grey indicate no change in copy number, red indicates a copy number gain, and blue a copy number loss. This array was performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117527&req=5

pone-0103243-g001: SNP 6.0 array of HCT116 cells to visualize copy number variation.Numbers (and X/Y) correspond to chromosomes, and MT represents mitochondrial DNA. Results were compared to a dataset from Affymetrix containing 270 mixed population samples from the International HapMap Project [29]. Regions in grey indicate no change in copy number, red indicates a copy number gain, and blue a copy number loss. This array was performed in duplicate.
Mentions: The copy number of genes in the HCT116 cells was assessed with the Affymetrix SNP 6.0 array to determine if, and to what extent, the cells we used had drifted/mutated in culture compared to the original karyotype of this cell line. When compared to a pooled normal human karyotype from the International HapMap project database [29], the cells lacked extensive chromosomal abnormalities (Figure 1). We did observe regions on 8q, 10q, 16q, and 17q that displayed regional chromosomal gains (indicated in red), as previously reported by others [30]. Very few complete loss of copies were seen (blue), except for the Y chromosome which has been reported to be absent in 50–100% of HCT116 cells [31]. Thus, in comparison with other cancer cell lines, HCT116 have a relatively normal and stable karyotype with minimal chromosomal instability [30].

Bottom Line: DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability.Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61.Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences; Ontario Veterinary College; University of Guelph; Guelph, ON, Canada.

ABSTRACT
DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumours, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes.

Show MeSH
Related in: MedlinePlus