Elsevier

Experimental Gerontology

Volume 37, Issues 8–9, 9 August 2002, Pages 1005-1014
Experimental Gerontology

Demethylation of classical satellite 2 and 3 DNA with chromosomal instability in senescent human fibroblasts

https://doi.org/10.1016/S0531-5565(02)00061-XGet rights and content

Abstract

Demethylation of genomic 5-methylcytosine is reported in aged human tissues and senesced human cells, although it is not understood to what extent this phenomenon contributes to replicative senescence. We examined methylation status of satellite 2 and 3 sequences during passages of normal human fibroblasts. These sequences are abundant in the juxtacentromeric heterochromatin of human chromosomes 1, 9 and 16, and heavily methylated in tissues of normal individuals. The decrease in DNA methylation level was two times faster in satellite 3 DNA than in satellite 2 and total DNA. Then we monitored appearance of micronuclei during the passages since they are indicative of heterochromatin decondensation or chromosome breakage. Concomitant with the DNA demethylation, micronuclei containing the heterochromatin of chromosomes 1, 9 or 16, appeared specifically. These results suggest that demethylation of heterochromatin has a role in replicative senescence through chromosome instability.

Introduction

Normal human cells have a limited replicative potential in culture and stop dividing irreversibly, a phenomenon termed cellular senescence (Hayflick, 1965). Senescent cells are morphologically altered and express specific genes called senescence-associated genes (Campisi, 1997). The numbers of cell division depend on cell types and genetic backgrounds of donor cells under standard culture conditions (Stanulis-Praeger, 1987). Limitation of proliferative potential is thought to arise by an intrinsic mechanism that counts the number of cell division. Telomere shortening is proposed to be one candidate for such a mechanism (Harley, 1991). However, this hypothesis is not in agreement with the several observations. Mouse primary cells senesce much faster than human cells despite mouse cells having much longer telomeres (Holliday, 1996). In addition, there is little correlation between proliferative potential and telomerase activity or telomere length in stable clones expressing introduced telomerase (Franco et al., 2001, Ouellette et al., 2000).

Gradual demethylation of 5-methylcyosine bases in DNA is thought to be another mechanism to count cell division (Hoal-van Helden and van Helden, 1989, Wilson and Jones, 1983). In human cells, 60–90% of the cytosine residues in CpG dinucleotides are methylated. Methylation of CpG islands is associated with condensation of chromatins enriched in hypoacetylated histones, thereby leading to transcriptional silencing (Henikoff, 2000, Razin, 1998). On the other hand, DNA methylation is shown to be essential for normal development, X chromosome inactivation, and genome imprinting (Constancia et al., 1998). The level of DNA methylation is shown to decrease in normal mammalian cells during passages (Fairweather et al., 1987, Wilson and Jones, 1983) and in tissues during ageing (Singhal et al., 1987, Wilson et al., 1987). Potent inhibitors of DNA methyltransferase, 5-azacytidine (aza-C) and 5-azadeoxycytidine (aza-dC), respectively, are shown to lead to shortening of in vitro lifespan (Fairweather et al., 1987, Holliday, 1986) and premature senescence followed by reduction in DNA methylation in normal human fibroblasts (Young and Smith, 2001). The premature senescence was not observed in p21-deficient fibroblasts, suggesting that it occurs by activation of cell cycle checkpoint pathway in response to DNA damage or lesions due to DNA demethylation, rather than transcriptional activation of particular genes by DNA demethylation.

On the other hand, it is well known that a low dose of aza-C or aza-dC, dramatically inhibits condensation of the constitutive heterochromatins in human cells, especially in the pericentromeric regions of chromosomes 1, 9 and 16 and the q arm of chromosome Y in human lymphocytes (Haaf and Schmid, 2000). These regions are abundant in the classical satellite 2 and 3 sequences (Tagarro et al., 1994), and most prominently stained with antibody against 5-methylcytosine in metaphase spreads (Lubit et al., 1976). Further, demethylation of the satellite sequences is shown to cause chromosome breakage and rearrangements followed by decondensation of the heterochromatins (Hernandez et al., 1997, Kokalj-Vokac et al., 1993). Therefore, regarding cellular senescence, one of the specific targets for DNA demethylation may be the classical satellite DNA that constitutes heterochromatin. To date, decreases in DNA methylation in the satellite sequences during aging in vitro and in vivo have been reported in mice and bovines (Hornsby et al., 1992, Howlett et al., 1989), but not in humans.

The aim of this study is to find a role of DNA demethylation in replicative senescence in normal human fibroblasts. We focused on methylation status of the classical satellite 2 and 3 sequences and micronuclei containing these sequences. Although DNA demethylation was not specific to the satellite sequences, we found that micronuclei containing the constitutive heterochromatin of chromosomes 1, 9 or 16 preferentially appeared in human fibroblasts undergoing replicative senescence and premature senescence mediated by aza-C treatment. Thus, demethylation of heterochromatin is suggested to assist replicative senescence through chromosome instability.

Section snippets

Materials

Aza-C, distamycin A, 6-diamidino-2-phenylindole (DAPI), Taq DNA polymerase, and human placenta DNA (type XIII) were obtained from Sigma. Restriction endonucleases and T4 polynucleotide kinase were purchased from New England Biolabs. Tetramethylrhodamine-5-dUTP was from Roche Diagnostics. [γ-32P]ATP and [α-32P]dCTP (3000 Ci/mmol) were obtained from ICN and HAS, respectively. The reagents used were of reagent grade.

Cell culture

Human embryonic lung fibroblast line (TIG-7) was obtained from the Japanese Cancer

Inhibitors of DNA methyltransferase induce premature senescence in TIG-7 cells

Recently, it has been reported that aza-dC induces premature senescence with demethylation of genomic 5-methylcytosine in human fibroblasts (Young and Smith, 2001). We used aza-C, another DNA methyltransferase inhibitor. When normal human fibroblasts (TIG-7 cells) were cultured in the presence of 1 μM aza-C, the cells stopped growing within 1 week (data not shown). These cells became flat and enlarged in shape and strongly induced senescence-associated β-galactosidase (Dimri et al., 1995) (Fig. 1

Discussion

Demethylation of genomic 5-methylcytosine has been reported in aged tissues and senescent human cells. It is, however, unknown whether demethylation in a specific locus or chromosomal region has some relevance to aging or cellular senescence. In this study, we monitored methylation levels in the classical satellite 2 and 3 sequences, which form constitutive heterochromatin, during passages of normal human fibroblasts. The demethylation seemed to proceed in any regions of the genomic DNA during

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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