Epigenetic clock analysis of human fibroblasts in vitro: effects of hypoxia, donor age, and expression of hTERT and SV40 largeT.

Mieko Matsuyama,Shigemi Matsuyama,Steve Horvath,David J Wuwong

doi:10.18632/aging.101955

Mieko Matsuyama, Shigemi Matsuyama + Show 2 more

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https://doi.org/10.18632/aging.101955

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Abstract

Aging is associated with a genome-wide change of DNA methylation (DNAm). "DNAm age" is defined as the predicted chronological age by the age estimator based on DNAm. The estimator is called the epigenetic clock. The molecular mechanism underlining the epigenetic clock is still unknown. Here, we evaluated the effects of hypoxia and two immortalization factors, hTERT and SV40-LargeT (LT), on the DNAm age of human fibroblasts in vitro. We detected the cell division-associated progression of DNAm age after >10 population doublings. Moreover, the progression of DNAm age was slower under hypoxia (1% oxygen) compared to normoxia (21% oxygen), suggesting that oxygen levels determine the speed of the epigenetic aging. We show that the speed of cell division-associated DNAm age progression depends on the chronological age of the cell donor. hTERT expression did not arrest cell division-associated progression of DNAm age in most cells. SV40LT expression produced inconsistent effects, including rejuvenation of DNAm age. Our results show that a) oxygen and the targets of SV40LT (e.g. p53) modulate epigenetic aging rates and b) the chronological age of donor cells determines the speed of mitosis-associated DNAm age progression in daughter cells.

Highlights

There are two fundamental questions regarding the mechanism of aging
As previously established [17], in normoxia, the cell division speed declined after PD30 (Figure 1, black symbols), and the cell size increased (Figure 2A and BB)
The present study demonstrated that (1) hypoxia slows down the progression of DNAm age, (2) cell divisionassociated DNAm age progression depends on the chronological age of the cell donor, (3) SV40LT immortalization induces abnormal progression of DNAm age, and (4) hTERT expression does not arrest progression of DNAmAge

Summary

Introduction

There are two fundamental questions regarding the mechanism of aging. The first question is what kind of time-dependent changes (chemical and/or biological changes) drive aging. The present study investigates these two questions by focusing on the epigenetic aging of primary cultured human cells. Most of the previously reported age-dependent DNAm changes were tissue- or cell type-specific (Reviewed in [1]). The pan-tissue age estimator was developed based on the DNAm levels of 353 CpG sites [3]. In 2018, another age estimator was developed that predicts the chronological age of skin and blood cells more accurately than the pan-tissue age estimator [6]. This new age estimator uses the DNAm levels of 391 CpG sites (60 CpG sites are overlapped with the pan-tissue age estimator) [6]. The DNAm age functions as a biomarker to predict the risk of age-associated diseases as accelerated DNAm age has been observed in a variety of conditions, www.aging-us.com including obesity [7], HIV infection [8], Down syndrome [9], Parkinson's disease [10], Werner syndrome [11], and menopause [12]

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