Over-expression of hTERT in CHO K1 results in decreased apoptosis and reduced serum dependency

Abstract

The enzyme telomerase plays a crucial role in cellular proliferation. By adding hexameric repeats to the chromosome ends, it prevents telomeric loss and, thus entry into senescence of limited life span cells. It is unclear, however, what would be the effect of over-expressing telomerase in an immortalised cell line, characterised by unlimited life span and high levels of apoptosis under sub-optimal growth conditions. In order to address this question, we have transfected the immortal cell line CHO K1 with the human telomerase reverse transcriptase (hTERT) catalytic subunit. Differences in the growth profile and apoptosis levels between the cells over-expressing hTERT (Telo) and the cells containing mock vector were found under standard growth conditions. Similarly, the Telo cells showed lower levels of apoptosis, greater attachment tendency and higher viable cell density under serum-deprived conditions compared to the control cell line, suggesting a major role for hTERT over-expression in stressed cultures. Using a mouse cDNA microarray, the collagen type III and V genes were shown to have at least a 10-fold higher expression in the Telo cells than the control cells, suggesting a role of hTERT in the cell attachment pathways.

Introduction

Telomerase, also known as telomere terminal transferase, is a reverse transcriptase-like enzyme that uses an RNA template to synthesise and maintain telomeric sequences by adding TTAGGG sequences to the end of the existing chromosomes called telomeres. The function of telomeres (reviewed in Neidle and Parkinson, 2003) is to maintain the structural integrity of a chromosome, to ensure complete replication of the extreme ends of chromosome termini and to have a role in establishing the three-dimensional architecture of the nucleus and/or chromosome pairing during cell division (Hodes, 1999). Moreover, the length of the telomere mediated by telomerase and all the proteins associated with the telomere can be visualised as a mitotic clock that determines the life span of the cell.

Telomerase is composed of two major subunits, an RNA component that serves as template for the synthesis of telomeric repeats (TR) which is essential for telomerase activity (Chen et al., 2000, Prescott and Blackburn, 2000), and a catalytic subunit called telomerase reverse transcriptase (TERT) that has been implicated as a rate limiting step for telomerase activity. Since its ectopic expression in mortal human cells induces telomere elongation and continuous cell proliferation (Shay, 1999, Counter et al., 1998, Wen et al., 1998, Murasawa et al., 2002, Nisato et al., 2004, Chan and Blackburn, 2004). Telomerase activity is present in foetal tissue, germ cells and also in tumour cells but is repressed in most somatic tissue during development (Morales et al., 1999).

The role of telomerase in preventing apoptosis and immortalising primary cells without inducing malignant phenotypic effect has been well documented (Wright and Shay, 2000, Shay and Wright, 2000, Harrington et al., 1997, Kilian et al., 1997, Ren et al., 2001, Meyerson et al., 1997, Nakamura et al., 1997, Nakayama et al., 1998, Serakinci and Koch, 1999, Holt and Shay, 1999, Simonsen et al., 2002, Shi et al., 2002). However, there is no report thus far regarding the effect of telomerase over-expression in immortalised cell lines, such as CHO K1 cells characterised by unlimited life span and high apoptosis levels when cells are stressed in sub-optimal conditions. To address this question we produced stable CHO K1 cell clones expressing the hTERT gene and measured their response to sub-optimal environmental conditions.

The scale up and optimisation of animal cell cultures to meet production demands has been problematic, due to the low productivity and instability of the cell lines used. Apoptotic death is a major problem limiting prolonged high cell viability particularly in high cell density culture. Systematic approach to cell culture development and optimisation could be achieved through metabolic engineering of cell lines to improve their survivability, growth and productivity (Mercille and Massie, 1994, Singh et al., 1994, Singh et al., 1996, Cotter and Al-Rubeai, 1995, Singh and Al-Rubeai, 1998, Dickson, 1998, Fussenegger and Bailey, 1998, Tey and Al-Rubeai, 2004). On the basis of this cellular improvement novel strategies for optimisation of cell culture should be possible. These strategies include the development of methods to improve adaptation to suspension and serum free culture of mammalian cells.

We discovered that the expression of hTERT in CHO K1 cells markedly increased their resistance to serum-deprivation induced apoptosis and allowed the serum dependent cell line to survive, attach and divide in un-supplemented basal medium. Additionally, this study provides evidence of a new type of interrelation between telomerase and collagen genes, which indicate that telomerase may help cell survival through a mechanism which involves the activation of the fibrous proteins that control cell shape and differentiation.