Immediate and gradual gene expression changes in telomerase over-expressing fibroblasts

https://doi.org/10.1016/j.bbrc.2010.07.018Get rights and content

Abstract

Most human somatic cells contain no or very low levels of telomerase. The over-expression of the catalytic subunit (hTERT) of human telomerase is a common method to generate cells with a greatly prolonged lifespan. These cells serve as models for cells that are either difficult to cultivate or have a limited lifespan in vitro. In addition, hTERT over-expressing cells are thought to be a useful resource for tissue engineering and regenerative medicine.

While tumour suppressors and cell cycle checkpoints are maintained for an extended period in most hTERT over-expressing cells we found that there is a gradual change in gene expression over a range of 130 population doublings (PD) for the majority of genes analysed. Seven genes were significantly down-regulated with increasing population doublings (PDs), while only two were up-regulated. One gene, stanniocalcin 2, was highly expressed in parental fibroblasts but completely diminished as a consequence of hTERT transgene expression.

These data demonstrate that in hTERT over-expressing cells two different types of expression changes occur: one can be directly associated with hTERT transgene expression itself, while others might occur more gradual and with varying kinetics. These changes should be taken into account when these cells are used as functional models or for regenerative purposes.

Research highlights

► Gene expression changes gradually over time in telomerase over-expressing cells. ► Expression of STC2 is totally diminished in parallel to hTERT over-expression. ► Expression changes occur with a different kinetics in various genes. ► Functional consequences of these changes are not clear yet.

Introduction

hTERT over-expression greatly extends the lifespan of various human cell types such as fibroblasts, skin keratinocytes, vascular endothelial, retinal, urothelial and breast epithelial cells. The main cause for the extended proliferation capacity is telomere maintenance thereby overcoming cellular senescence and the Hayflick limit [1]. These cells are being widely used as models for the functional characterisation of the different primary cell types that they are derived from, since these cells are thought to retain many properties of the original cell type such as physiological function and differentiation capacity [2], [3], [4], [5].

Telomerase over-expressing cells are also regarded as a source for cell transplantation and to create bioengineered tissues for regenerative medicine in order to treat various diseases as well as age-related conditions [6], [7].

At the same time it has been shown that telomerase expression can change the gene expression pattern of cell cycle regulated genes, signalling pathways and confer resistance to apoptosis. Although it seems that the most important cell cycle and tumour suppressors are unperturbed during the initial 50 population doublings after hTERT over-expression [8], [9] changes have been found in expression of tumour suppressors such as p53 and p16 at later time points [10], [11].

The aim of the study was to analyse whether changes in gene expression between parental MRC-5 fibroblasts and their hTERT over-expressing derivatives occur as an immediate effect of hTERT over-expression or rather continuously with time of culture. Here we analysed gene expression changes of 11 genes in non-clonally derived progeny from a retroviral mass transfection of hTERT into MRC-5 fibroblasts over a range of 130 population doublings [12]. These genes had been identified previously as being differentially regulated in young, senescent and telomerase over-expressing fibroblasts and might be part of a retrograde response signature in these cells [12], [13].

We found that expression of most genes changed gradually with increasing population doublings while the disappearance of STC2 expression occurred immediately after transfection and seemed to be directly correlated to the presence of the hTERT transgene.

Section snippets

Cells and culture

MRC-5 cells were purchased from ECACC (London). Senescent cells were generated by serial passaging of MRC-5 fibroblasts reaching senescence at around 50 PD±5. hTERT-over-expressing MRC-5 was generated using retroviral transfection using pLCphTERT (Clontech) into MRC-5 at PD 30 [12]. After initial selection, the transfected mass culture was aliquoted at a PD < 41 and MRC-5/hTERT cells were independently grown to the final PDs (between 41 and 173). In all cultures, main tumour suppressors, p53 and

Results

The aim of our study was to characterise the long term effects of hTERT over-expression on gene expression in fibroblasts over a large range of population doublings. Comparing parental MRC-5 fibroblasts with hTERT over-expressing MRC-5 at high passage (PD 125 ± 10), we recently found significant changes in the expression of various genes related to mitochondrial (dys-)function and retrograde response [12]. To determine whether these changes were due to hTERT over-expression itself or rather

Discussion

The present study analyses gene expression changes in hTERT over-expressing MRC-5 fibroblasts, starting at 11 PDs after retroviral transfection and spanning 130 PDs in total.

We specifically choose independent subcultures for all time points in order to avoid any amplification of randomly appearing changes in subsequent culturing of the same subculture.

Our results demonstrate that expression of six of the analysed genes changed gradually and significantly with increasing culture age: DSG2,

Conclusion

Telomerase over-expression is used to generate material for functional analysis of various cell types as well as a source for regenerative medicine. Although major tumour suppressor genes are functionally maintained for extended periods of culturing [7], [8] the current study demonstrates that that expression of selected genes is not stable and can change substantially during extended culture periods. These changes could be either immediately associated with hTERT transgene expression or occur

Conflict of interest

None of the authors has any conflict of interest.

Acknowledgment

This study was funded by a BBSRC funded grant to the Centre for Integrated Systems Biology for Ageing and Nutrition at the IAH.

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