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Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR.

Hypoxia influences many key biological functions. In cancer, it is generally believed that hypoxic condition is generated deep inside the tumor because of the lack of oxygen supply. However, consumption of oxygen by cancer should be one of the key means of regulating oxygen concentration to induce hypoxia but has not been well studied. Here, we provide direct evidence of the mitochondrial role in the induction of intracellular hypoxia. We used Acetylacetonatobis [2-(2′-benzothienyl) pyridinato-kN, kC3’] iridium (III) (BTP), a novel oxygen sensor, to detect intracellular hypoxia in living cells via microscopy. The well-differentiated cancer cell lines, LNCaP and MCF-7, showed intracellular hypoxia without exogenous hypoxia in an open environment. This may be caused by high oxygen consumption, low oxygen diffusion in water, and low oxygen incorporation to the cells. In contrast, the poorly-differentiated cancer cell lines: PC-3 and MDAMB231 exhibited intracellular normoxia by low oxygen consumption. The specific complex I inhibitor, rotenone, and the reduction of mitochondrial DNA (mtDNA) content reduced intracellular hypoxia, indicating that intracellular oxygen concentration is regulated by the consumption of oxygen by mitochondria. HIF-1α was activated in endogenously hypoxic LNCaP and the activation was dependent on mitochondrial respiratory function. Intracellular hypoxic status is regulated by glucose by parabolic dose response. The low concentration of glucose (0.045 mg/ml) induced strongest intracellular hypoxia possibly because of the Crabtree effect. Addition of FCS to the media induced intracellular hypoxia in LNCaP, and this effect was partially mimicked by an androgen analog, R1881, and inhibited by the anti-androgen, flutamide. These results indicate that mitochondrial respiratory function determines intracellular hypoxic status and may regulate oxygen-dependent biological functions.

Intracellular oxygen concentration ([O2]i.c.) regulates key biological functions such as HIF-1α activation, glucose transport, potassium pumps, intracellular calcium concentration, P450 family enzymes and HMGR expression. However, the mechanisms regulating [O2]i.c remain to be defined. Here, we provide direct evidence of the mitochondrion's role in [O2]i.c. regulation. We used bis(2-(2′-benzothienyl)-pyridianto-N,C3’irridium(acetylacetonate) (BTP) to measure [O2]i.c. in living cells via microscopy.5 Well-differentiated cancers, LNCaP and MCF-7 showed intracellular hypoxia without exogenous hypoxia. In contrast, poorly-differentiated cancers exhibited intracellular normoxia, and exogenous hypoxia could induce hypoxia. The specific complex I inhibitor, rotenone, and the reduction of mitochondrial DNA (mtDNA) content increased in [O2]i.c,, indicating that [O2]i.c. is regulated by mitochondrial respiratory activity. [O2]i.c. is additionally regulated by glucose, but the dose-dependent relationship is parabolic. The no glucose condition induced normoxia, and the low concentration glucose condition (0.045mg/ml) induced strong hypoxia. [O2]i.c. was found to be additionally determined by the presence of FCS in the growth medium, and this effect was mimicked by an androgen analog, R1881, suggesting that androgen, glucose concentration and respiratory function are important regulators of [O2]i.c.. Citation Format: Sara Prior, Ara Kim, Seiji Tobita, Toshiyuki Takeuchi, Masahiro Higuchi. Intracellular hypoxia is dominantly determined by mitochondrial respiratory activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1693. doi:10.1158/1538-7445.AM2013-1693