Indicators of replicative damage in equine tendon fibroblast monolayers

BMC Veterinary Research

Janet C Patterson-Kane¹  Hannah Cornell¹  David L Becker²  Livia B Henderson¹  Tina Rich¹ 

[1] Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK;Department of Cell and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

关键词: Comet assay;    GammaH2AX protein;    Replicative stress;    DNA repair;    DNA damage;    Polyploidy;    Cell culture;    Horses;    Tendon;    Superficial digital flexor tendon;   

Others  :  1119458
DOI  :  10.1186/1746-6148-9-180

 received in 2013-03-27, accepted in 2013-09-10,  发布年份 2013

【 摘 要 】

Background

Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it.

Results

We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents.

Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress.

Conclusions

Researchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled.

【 授权许可】

   
2013 Rich et al.; licensee BioMed Central Ltd.

【 图 表 】

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