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Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

Pal A, Srivastava T, Sharma MK, Mehndiratta M, Das P, Sinha S, Chattopadhyay P - J. Cell. Mol. Med. (2010)

Bottom Line: Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability.U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration.Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

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(A) Morphological analysis: Effects of prolonged (6 weeks) normoxia (21% O2) and hypoxia (0.1% O2) on the morphology of the U87MG and SaOS2 cells. Characteristic signs of apoptosis like cellular shrinkage, nuclear condensation, blebbing of cell membrane in the form of apoptotic bodies are marked in hypoxic cells. N – Normal, A – Apoptotic bodies, F – Fragmentation, C – Condensation. (B) Cell viability assay: Trypan blue stained cell counting was done for measuring survival in hypoxia and normoxia treated U87MG cells with equal number of seeding (5 × 103 cells) per well. The cells cultured initially in hypoxia and normoxia conditions were continued in the same condition (N or H) or reverted back to normoxia (HR). (C) Caspase-3 cleavage for apoptosis: Western blot for pro-caspase 3 and cleaved caspase-3 in cell extracts. Cleaved caspase was observed in U87MG cells exposed to 0.1% hypoxia at various time-points including 6-week hypoxia.
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fig01: (A) Morphological analysis: Effects of prolonged (6 weeks) normoxia (21% O2) and hypoxia (0.1% O2) on the morphology of the U87MG and SaOS2 cells. Characteristic signs of apoptosis like cellular shrinkage, nuclear condensation, blebbing of cell membrane in the form of apoptotic bodies are marked in hypoxic cells. N – Normal, A – Apoptotic bodies, F – Fragmentation, C – Condensation. (B) Cell viability assay: Trypan blue stained cell counting was done for measuring survival in hypoxia and normoxia treated U87MG cells with equal number of seeding (5 × 103 cells) per well. The cells cultured initially in hypoxia and normoxia conditions were continued in the same condition (N or H) or reverted back to normoxia (HR). (C) Caspase-3 cleavage for apoptosis: Western blot for pro-caspase 3 and cleaved caspase-3 in cell extracts. Cleaved caspase was observed in U87MG cells exposed to 0.1% hypoxia at various time-points including 6-week hypoxia.

Mentions: U87MG cells cultured in 0.1% O2 demonstrated evidence of HIF-1α protein expression (Fig. S1) by 4 hrs. The cells also showed characteristic signs of apoptosis like cellular shrinkage, nuclear condensation and membrane blebbing in the form of apoptotic bodies even at 6 weeks (Fig. 1A). Similar characteristic changes were also observed in SaOS2 cells. The apoptotic feature of cleaved caspase was also seen in U87MG cell lysates at 1, 2 and 3 days of hypoxia and was found to persist till at least 6 weeks of hypoxic treatment (Fig. 1C).


Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

Pal A, Srivastava T, Sharma MK, Mehndiratta M, Das P, Sinha S, Chattopadhyay P - J. Cell. Mol. Med. (2010)

(A) Morphological analysis: Effects of prolonged (6 weeks) normoxia (21% O2) and hypoxia (0.1% O2) on the morphology of the U87MG and SaOS2 cells. Characteristic signs of apoptosis like cellular shrinkage, nuclear condensation, blebbing of cell membrane in the form of apoptotic bodies are marked in hypoxic cells. N – Normal, A – Apoptotic bodies, F – Fragmentation, C – Condensation. (B) Cell viability assay: Trypan blue stained cell counting was done for measuring survival in hypoxia and normoxia treated U87MG cells with equal number of seeding (5 × 103 cells) per well. The cells cultured initially in hypoxia and normoxia conditions were continued in the same condition (N or H) or reverted back to normoxia (HR). (C) Caspase-3 cleavage for apoptosis: Western blot for pro-caspase 3 and cleaved caspase-3 in cell extracts. Cleaved caspase was observed in U87MG cells exposed to 0.1% hypoxia at various time-points including 6-week hypoxia.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4373486&req=5

fig01: (A) Morphological analysis: Effects of prolonged (6 weeks) normoxia (21% O2) and hypoxia (0.1% O2) on the morphology of the U87MG and SaOS2 cells. Characteristic signs of apoptosis like cellular shrinkage, nuclear condensation, blebbing of cell membrane in the form of apoptotic bodies are marked in hypoxic cells. N – Normal, A – Apoptotic bodies, F – Fragmentation, C – Condensation. (B) Cell viability assay: Trypan blue stained cell counting was done for measuring survival in hypoxia and normoxia treated U87MG cells with equal number of seeding (5 × 103 cells) per well. The cells cultured initially in hypoxia and normoxia conditions were continued in the same condition (N or H) or reverted back to normoxia (HR). (C) Caspase-3 cleavage for apoptosis: Western blot for pro-caspase 3 and cleaved caspase-3 in cell extracts. Cleaved caspase was observed in U87MG cells exposed to 0.1% hypoxia at various time-points including 6-week hypoxia.
Mentions: U87MG cells cultured in 0.1% O2 demonstrated evidence of HIF-1α protein expression (Fig. S1) by 4 hrs. The cells also showed characteristic signs of apoptosis like cellular shrinkage, nuclear condensation and membrane blebbing in the form of apoptotic bodies even at 6 weeks (Fig. 1A). Similar characteristic changes were also observed in SaOS2 cells. The apoptotic feature of cleaved caspase was also seen in U87MG cell lysates at 1, 2 and 3 days of hypoxia and was found to persist till at least 6 weeks of hypoxic treatment (Fig. 1C).

Bottom Line: Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability.U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration.Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

Show MeSH
Related in: MedlinePlus