Skip to main content
SpringerLink
Log in

DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

Removal of the somatic DNA methylation pattern from donor cells and remodeling of embryonic status have been suggested as integral processes for successful nuclear transfer (NT) reprogramming. This study has investigated the effects of 5-azacytidine (5-azaC), a DNA methylation inhibitor, on global methylation changes in porcine fetal fibroblasts (PFF); this may improve NT attributable to the potential reprogramming of the methyl groups. PFF in 5th passage cultures were treated with 0, 0.5, 1.0, 2.0, and 3.0 μM 5-azaC for 96 h; 5-azaC inhibited the growth at all tested concentrations. At the higher concentrations of 5-azaC used, cells appeared to exhibit morphological changes and to become apoptotic as observed by TUNEL assay. Thus, cells were negatively affected by 5-azaC. Differences in cellular ploidy were also observed at higher concentrations. Analysis showed no considerable changes in the proportion of cells at the G1-phase of the cell cycle with 5-azaC concentrations. The fractional part of the methylated DNA of these cells was significantly reduced by 5-azaC treatment. Confocal microscopy confirmed the inhibition of methylation levels in PFF with increased concentrations of 5-azaC. Exposure to 5-azaC altered the expression of genes involved in imprinting (IGF2) or pro-apoptosis (BAX), whereas there was a reduction in the expression of the main enzyme responsible for replicating the DNA methylation pattern (DNMT1) and anti-apoptosis (BCL2L1). Therefore, 5-azaC induces a relative reduction in methylation in PFF, and cells treated with 0.5 μM 5-azaC may have enhanced potential for porcine NT.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Beaujean N, Hartshorne G, Cavilla J, Taylor J, Gardner J, Wilmut I, Meehan R, Young LE (2004) Non-conservation of mammalian preimplantation methylation dynamics. Curr Biol 14:R266–R267

    Article  PubMed  CAS  Google Scholar 

  • Bird A (2002) DNA methylation patterns and epigenetic memory. Genes Dev 16:6–21

    Article  PubMed  CAS  Google Scholar 

  • Boquest AC, Day BN, Prather RS (1999) Flow cytometric analysis of cultured porcine fetal fibroblast cells. Biol Reprod 60:1013–1019

    Article  PubMed  CAS  Google Scholar 

  • Boquest AC, Grupen CG, Harrison SJ, Mcllfatrick SM, Ashman RJ, d’Apice AJF, Nottle MB (2002) Production of cloned pigs from cultured fetal fibroblast cells. Biol Reprod 66:1283–1287

    Article  PubMed  CAS  Google Scholar 

  • Bour’chis D, LeBourhis D, Patin D, Niveleau A, Cornizolli P, Renard JP, Viegas-Pequignot E (2001) Delayed and incomplete reprogramming of chromosome methylation patterns in bovine embryos. Curr Biol 11:1542–1546

    Article  CAS  Google Scholar 

  • Brison DR, Schultz RM (1997) Apoptosis during mouse blastocyst ormation: evidence for a role for survival factors including transforming growth factor a. Biol Reprod 56:1088–1096

    Article  PubMed  CAS  Google Scholar 

  • Christman JK (2002) 5-Azacytidine and 5-aza-2-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21:5483–5495

    Article  PubMed  CAS  Google Scholar 

  • Cooper S (1998) On the interpretation of the shortening of the G1-phase by overexpression of cyclins in mammalian cells. Exp Cell Res 238:110–115

    Article  PubMed  CAS  Google Scholar 

  • Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, Reik W (2001) Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci USA 98:13734–13738

    Article  PubMed  CAS  Google Scholar 

  • Dean W, Santos F, Reik W (2003) Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer. Semin Cell Dev Biol 14:93–100

    Article  PubMed  CAS  Google Scholar 

  • Enright BP, Kubota C, Yang X, Tian XC (2003) Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2′-deoxycytidine. Biol Reprod 69:896–901

    Article  PubMed  CAS  Google Scholar 

  • Enright BP, Sung LY, Chang CC, Yang X, Tian XC (2005) Methylation and acetylation characteristics of cloned bovine embryos from donor cells treated with 5-aza-2′-deoxycytidine. Biol Reprod 72:944–948

    Article  PubMed  CAS  Google Scholar 

  • Fairburn HR, Young LE, Hendrich BD (2002) Epigenetic reprogramming: how now, cloned cow? Curr Biol 12:R68–R70

    Article  PubMed  CAS  Google Scholar 

  • Haaf T, Schmidt M (1989) 5-Azadeoxycytidine induced under condensation in the giant X chromosomes of Microtus agrestis. Chromosoma 98:93–98

    Article  PubMed  CAS  Google Scholar 

  • Hu JF, Nguyen PH, Pharn NV, Vu TH, Hoffman AR (1997) Modulation of Igf2 genomic imprinting in mice induced by 5-azacytidine, an inhibitor of DNA methylation. Mol Endocrinol 11:1891–1898

    Article  PubMed  CAS  Google Scholar 

  • Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM 3rd, Biniszkiewicz D, Yanagimachi R, Jaenisch R (2001) Epigenetic instability in ES cells and cloned mice. Science 293:95–97

    Article  PubMed  CAS  Google Scholar 

  • Jablonka E, Goitein R, Marcus M, Cedar H (1985) DNA hypomethylation causes an increase in DNase-I sensitivity and an advance in the time of replication of the entire inactive chromosome. Chromosoma 93:152–156

    Article  PubMed  CAS  Google Scholar 

  • Jackson-Grusby L, Beard C, Possemato R, Tudor M, Fambrough D, Csankovszki G, Dausman J, Lee P, Wilson C, Lander E, Jaenisch R (2001) Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat Genet 27:31–39

    Article  PubMed  CAS  Google Scholar 

  • Jones KL, Hill J, Shin TY, Lui L, Westhusin M (2001) DNA hypomethylation of karyoplasts for bovine nuclear transfer. Mol Reprod Dev 60:208–213

    Article  PubMed  CAS  Google Scholar 

  • Kang YK, Park JS, Choi YH, Chung AS, Lee KK, Han YM (2001a) Aberrant methylation of donor genome in cloned bovine embryos. Nat Genet 28:173–177

    Article  PubMed  CAS  Google Scholar 

  • Kang YK, Koo DB, Park JS, Choi YH, Kim SU, Chang WK, Lee KK, Han YM (2001b) Typical demethylation events in cloned pig embryos. Clues on species-specific differences in epigenetic reprogramming of a cloned donor genome. J Biol Chem 276:39980–39984

    Article  PubMed  CAS  Google Scholar 

  • Kang YK, Park JS, Koo DB, Choi YH, Kim SU, Lee KK, Han YM (2002) Limited demethylation leaves mosaic-type methylation states in bovine pre-implantation embryos. EMBO J 21:1092–1100

    Article  PubMed  CAS  Google Scholar 

  • Kharroubi AE, Piras G, Stewart CL (2001) DNA demethylation reactivates a subset of imprinted genes in uniparental mouse embryonic fibroblasts. J Biol Chem 276:8674–8680

    Article  PubMed  Google Scholar 

  • King WA, Linares T, Gustavsson I, Bane AA (1979) Method for preparation of chromosomes from bovine zygotes and blastocysts. Vet Sci Commun 3:51–56

    Article  Google Scholar 

  • Li E (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3:662–673

    Article  PubMed  CAS  Google Scholar 

  • Mayer W, Niveleau A, Walter J, Fundele R, Haaf T (2000) Demethylation of the zygotic paternal genome. Nature 403:501–502

    Article  PubMed  CAS  Google Scholar 

  • Memili E, First NL (2000) Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote 8:87–96

    Article  PubMed  CAS  Google Scholar 

  • Morgan HD, Santos F, Green K, Dean W, Reik W (2005) Epigenetic reprogramming in mammals. Hum Mol Gen 14:R47–R58

    Article  PubMed  CAS  Google Scholar 

  • Ohgane J, Wakayama T, Kogo Y, Seneda S, Hattori N, Tanaka S, Yanagimachi R, Shiota K (2001) DNA methylation variation in cloned mice. Genesis 30:45–50

    Article  PubMed  CAS  Google Scholar 

  • Oswald J, Engemann S, Lane N, Mayer W, Olek A, Fundele R, Dean W, Reik W, Walter J (2000) Active demethylation of the parental genome in the mouse zygote. Curr Biol 10:475–478

    Article  PubMed  CAS  Google Scholar 

  • Pedone PV, Pikaart MJ, Cerrato F, Vernucci M, Ungaro P, Bruni CB, Riccio A (1999) Role of histone acetylation and DNA methylation in the maintenance of imprinted expression of the H19 and Igf2 genes. FEBS Lett 458:45–50

    Article  PubMed  CAS  Google Scholar 

  • Petti MC, Mandelli F, Zagonel V, De Gregoris C, Merola MC, Latagne R, Gattei V, Fazi P, Monfardini S, Pinto A (1993) Pilot study of 5-aza-2-deoxycytidine (decitabine) in the treatment of poor prognosis acute myelogenous leukemia patients: preliminary results. Leukemia 1(Suppl):36–41

    Google Scholar 

  • Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai YF, Boone J, Walker S, Ayares DL, Colman A, Campbell KH (2000) Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86–90

    Article  PubMed  CAS  Google Scholar 

  • Reik W, Dean W (2001) DNA methylation and mammalian epigenetics. Electrophoresis 22:2838–2843

    Article  PubMed  CAS  Google Scholar 

  • Reik W, Walter J (2001) Genomic imprinting: parental influence on the genome. Nat Rev Genet 2:21–32

    Article  PubMed  CAS  Google Scholar 

  • Santini V, Kantarjian HM, Issa JP (2001) Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med 134:573–586

    PubMed  CAS  Google Scholar 

  • Santos F, Dean W (2004) Epigenetic reprogramming during early development in mammals. Reproduction 127:643–651

    Article  PubMed  CAS  Google Scholar 

  • Santos F, Hendrich B, Reik W, Dean W (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241:172–182

    Article  PubMed  CAS  Google Scholar 

  • Santos F, Zakhartchenko V, Stojkovic M, Peters A, Jenuwein T, Wolf E, Reik W, Dean W (2003) Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol 13:1116–1121

    Article  PubMed  CAS  Google Scholar 

  • Schneider-Stock R, Diab-Assef M, Rohrbeck A, Foltzer-Jourdainne C, Boltze C, Hartig R, Schonfeld P, Roessner A, Gali-Muhtasib H (2005) 5-Azacytidine is a potent inhibitor of DNA methyltransferase 3a and induces apoptosis in HCT-116 colon cancer cells via Gadd45- and p53-dependent mechanisms. J Pharma Exp Ther 312:525–536

    Article  CAS  Google Scholar 

  • Shi W, Zakhartchenko V, Wolf E (2003a) Epigenetic reprogramming in mammalian nuclear transfer. Differentiation 71:91–113

    Article  PubMed  CAS  Google Scholar 

  • Shi W, Hoeflich A, Flaswinkel H, Stojkovic M, Wolf E, Zakhartchenko V (2003b) Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos. Biol Reprod 69:374–380

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Chinju, 660-701, South Korea

    B. Mohana Kumar, H.-F. Jin, J.-G. Kim, H.-J. Song, S. Balasubramanian, S.-Y. Choe & G.-J. Rho

  2. Department of Physical Therapy, Cardiovascular & Metabolic Disease Center, College of Biomedical Science & Engineering, Inje University, Gimhae, 621-749, South Korea

    Y. Hong

  3. Department of Clinics, Madras Veterinary College, Tamilnadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, 600 007, India

    S. Balasubramanian

  4. Department of Obstetrics and Theriogenology, College of Veterinary Medicine, Gyeongsang National University, 900 Gazwa, Chinju, 660-701, South Korea

    G.-J. Rho

Authors
  1. B. Mohana Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H.-F. Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J.-G. Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H.-J. Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Y. Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. S. Balasubramanian
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S.-Y. Choe
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. G.-J. Rho
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G.-J. Rho.

Additional information

The financial support of BioGreen 21 (grant no. 100052004002000) and KOSEF (grant no. R05-2004-000-10702-0) in Korea is gratefully acknowledged.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mohana Kumar, B., Jin, HF., Kim, JG. et al. DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine. Cell Tissue Res 325, 445–454 (2006). https://doi.org/10.1007/s00441-006-0201-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-006-0201-9

Keywords

Search

Navigation