Quantitative Analysis of Circulating Mitochondrial DNA in Plasma

The transcriptional regulation of several dozen genes in response to low oxygen tension is mediated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric protein composed of two subunits, HIF-1α and HIF-1β. In the HIF-1α-deficient human leukemic cell line, Z-33, exposed to mild (8% O2) or severe (1% O2) hypoxia, we found significant upregulation of two related heterogenous nuclear ribonucleoproteins, RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), which are highly conserved cold stress proteins with RNA-binding properties. Hypoxia also induced upregulation of RBM3 and CIRP in the murine HIF-1β-deficient cell line, Hepa-1 c4. In various HIF-1 competent cells, RBM3 and CIRP were induced by moderate hypothermia (32°C) but hypothermia was ineffective in increasing HIF-1α or vascular endothelial growth factor (VEGF), a known HIF-1 target. In contrast, iron chelators induced VEGF but not RBM3 or CIRP. The RBM3 and CIRP mRNA increase after hypoxia was inhibited by actinomycin-D, and in vitro nuclear run-on assays demonstrated specific increases in RBM3 and CIRP mRNA after hypoxia, which suggests that regulation takes place at the level of gene transcription. Hypoxia-induced RBM3 or CIRP transcription was inhibited by the respiratory chain inhibitors NaN3 and cyanide in a dose-dependent fashion. However, cells depleted of mitochondria were still able to upregulate RBM3 and CIRP in response to hypoxia. Thus, RBM3 and CIRP are adaptatively expressed in response to hypoxia by a mechanism that involves neither HIF-1 nor mitochondria.

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“…Mitochondrial DNA was quantitated by real-time PCR according to a previously reported method (Chiu et al, 2003), β-globin was used as control.…”

Section: Dna Analysismentioning

confidence: 99%

Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism

Wellmann

Bührer

Moderegger

et al. 2004

Journal of Cell Science

219

162

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“…Similarly, both down-and upregulation of cfmtDNA have been reported in cancer patients (8,9 ). Whether these discrepancies can be attributed to different sample-processing protocols (10 ) or are indicative of alternative/additive mechanisms of cf-DNA release requires further investigation.…”

mentioning

confidence: 97%

Short-Term Treadmill Running as a Model for Studying Cell-Free DNA Kinetics In Vivo

et al. 2011

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BACKGROUND Increased plasma concentrations of cell-free DNA (cf-DNA) are considered a hallmark of various clinical conditions. Despite intensive research in this field, limited data are available concerning the time course of release and clearance of cf-DNA in vivo. METHODS We extracted cf-DNA from plasma samples taken before and immediately after a 10-km cross-country run, and from samples taken before, immediately after, and 30 min after exhaustive short-term treadmill exercise. The contribution of nuclear (nDNA) and mitochondrial DNA (mtDNA) was measured by quantitative real-time PCR. The incremental treadmill exercise setup was exploited to delineate the precise sequencing and timing of cf-nDNA, lactate, and high-mobility group box 1 protein (HMGB1) release during the exercise and recovery phases. RESULTS Postexercise plasma cf-nDNA concentrations in cross-country and treadmill runners were significantly increased, by 7.6-fold and 9.9-fold, respectively (P < 0.001). cf-nDNA concentrations were not correlated with age, sex, or body mass index. Plasma concentrations of cf-nDNA and HMGB1 in postexercise samples of treadmill runners were significantly correlated (r = 0.84; P = 0.004). cf-mtDNA concentrations were not affected by treadmill exercise. Time-course analyses demonstrated that cf-nDNA is released within minutes after the onset of exercise and is rapidly cleared from the circulation after the cessation of exercise. Nearly congruent kinetics for cf-nDNA, lactate, and HMGB1 were observed during the exercise phase. CONCLUSIONS A single bout of exhaustive short-term treadmill exercise constitutes a versatile model system suitable for addressing basic questions about cf-DNA biology.

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“…Митохон-дриальную ДНК выделяли из плазмы крови, очищен-ной от тромбоцитов [3]. Количественный анализ свободно циркулирующей мтДНК осуществляли ме-тодом ПЦР в реальном времени (амплификатор «DT lite», ДНК-технология, Россия) с использованием ре-акционной смеси, содержащей SYBR Green (Maxima …”

Section: материалы и методыunclassified

Mitochondrial Dysfunction at Atherosclerosis and Myocardial Infarction: Molecular and Cytochemical Cell-Markers

Судаков¹,

Судаков²,

Klimenkov³

et al. 2017

Бюллетень Восточно-Сибирского Научного Центра Сибирского Отделения Российской Академии Медицинских Наук

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ВВЕДЕНИЕНа сегодняшний день инфаркт миокарда (одно из осложнений дислипопротеидемии и атеросклероза коронарных сосудов) является основной причиной смертности населения индустриально развитых стран. Совершенствование технологий диагностики данных заболеваний во многом предопределяет эф-фективность методов их профилактики и лечения [6]. Важным фактором развития дислипопротеидемии является избыточное накопление в клетках печени липидных капель, способных повреждать органеллы, в первую очередь митохондрии [5]. Известна ключе-вая роль дисфункции митохондрий кардиомиоцитов в развитии повреждений миокарда после воздействия ишемии/реперфузии [2]. Тем не менее, в настоящее время не выявлены биомаркеры, связующие со-стояние митохондрий и процессы гибели клетки при инфаркте миокарда и атеросклерозе. Одним из перспективных показателей разрушения, стрессиро-вания клеток и, возможно, состояния митохондрий является свободно циркулирующая митохондриаль-ная ДНК (мтДНК) крови [1,4,7], которую относят к «молекулярным паттернам риска» (danger associated molecular patterns, DAMPs) [8]. Таким образом, высо-кую актуальность для разработки диагностических и лечебных технологий представляет изучение данных цитологических и молекулярных показателей на ран-них сроках развития дислипопротеидемии, а также в динамике острого ишемического повреждения мио-карда, что явилось целью настоящего исследования. МАТЕРИАЛЫ И МЕТОДЫДислипопротеидемию вызывали атерогенной диетой (350 мг холестерина на 1 кг веса животного

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Quantitative Analysis of Circulating Mitochondrial DNA in Plasma

Cited by 181 publications

References 28 publications

Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism

Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism

Short-Term Treadmill Running as a Model for Studying Cell-Free DNA Kinetics In Vivo

Mitochondrial Dysfunction at Atherosclerosis and Myocardial Infarction: Molecular and Cytochemical Cell-Markers

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