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Telomerase may contribute to the capacity for cell replication by compensating for the loss of telomere length. Exploring the use of biological modifiers in increasing cellular replicative potential through telomerase activity may be useful for in vitro expansion of haemopoietic stem cells for transplantation or lymphoid cells for adoptive immunotherapy. In this study we showed for the first time that insulin-like growth factor 1 (IGF-1) modulated telomerase activity in human cord blood mononuclear cells (MNC) and some of the known functional determinants of telomerase activity. We found that cord blood MNC expressed constitutively a low level of telomerase activity and human telomerase reverse transcriptase (hTRT) mRNA, and a high level of human telomerase RNA component (hTR) and telomerase-associated protein-1 (TP1) mRNA. Interestingly, IGF-I alone did not increase the telomerase activity of cord blood MNC but could enhance the PHA-induced increase in telomerase activity. These alterations in telomerase activity were not completely in phase with those of proliferation response. On the other hand, IGF-I did not alter hTRT mRNA expression but enhanced the PHA-induced increase in hTRT whereas TP1 mRNA expression was stimulated by either IGF-I or PHA but showed no additive increase when stimulated by both IGF-1 and PHA. Neither IGF-1 nor PHA altered hTR expression. Finally, the temporal dynamics of hTRT mRNA expression and telomerase activity in cord blood MNC over 5 d in culture were not totally concordant, suggesting that key factors other than hTRT were involved in regulating telomerase activity in cord blood MNC. The modulatory effect of IGF-1 on telomerase activity supports its potential role in increasing replicative potential of cord blood lymphoid cells or haemopoietic stem cells.
Human telomeres, comprising of protein and tandemly repeated DNA sequences (TTAGGG) at the ends of chromosomes, protect chromosomes from illegitimate recombination and degradation (Blackburn, 1991). The length of telomeres decreases with increase in age in vivo and with cell division in vitro in haemopoietic stem cells and lymphocytes (Vaziri et al, 1993, 1994). Such shortening may act as a mitotic clock, regulating the number of divisions a normal cell can undergo. When telomeres are shortened to such a critical point that they may no longer stabilize chromosome ends, most cells exit from the cell cycle and die (Blackburn, 1991).
Some of the key functional determinants of telomerase activity have been unravelled. Human telomerase, an RNA-dependent DNA polymerase, can compensate for the loss of telomere length by synthesizing new telomeric repeats (TTAGGG)n, complimentary to human telomerase RNA component (hTR) (Blackburn, 1992; Feng et al, 1995). Telomerase-associated protein 1 (TP1), one of the telomerase protein subunits, has recently been identified and cloned (Harrington et al, 1997). TP1 exhibited extensive amino acid similarity to the Tetrahymena telomerase protein p80 and was shown to interact specifically with mammalian telomerase RNA component (Harrington et al, 1997). Human telomerase reverse transcriptase (hTRT) has more recently been identified as a putative human telomerase catalytic subunit (Nakamura et al, 1997). It was found that hTRT was expressed in telomerase-positive immortal cell lines and absent in telomerase-negative mortal cell lines (Nakamura et al, 1997).
The previous findings that relatively high levels of telomerase activity are detected in most germline and malignant cells suggest that telomerase activity is required to maintain functional telomeres for unlimited cell proliferation (Rhyu, 1995). However, several groups have reported recently that constitutively detectable levels of telomerase are also expressed in non-malignant cells, including peripheral blood T and B lymphocytes as well as haemopoietic stem cells (Counter et al, 1995; Hiyama et al, 1995). It was also found that telomere length was longer in CD4+ naive T cells than in memory cells (Weng et al, 1995). Telomerase activity can be up-regulated in vitro following activation of lymphocytes with mitogen and stimulation of T lymphocytes through TCR/CD3 complex (Hiyama et al, 1995; Igarashi & Sakaguchi, 1996; Bodnar et al, 1996). It has been demonstrated recently that the expression of telomerase in normal human T lymphocytes is developmentally regulated (Weng et al, 1996). These findings suggest that telomerase may play a permissive role in determining the capacity of lymphoid cells for cell division and clonal expansion.
Therefore exploring the use of biological modifiers in increasing cellular replicative potential through telomerase activity may be useful for in vitro expansion of haemopoietic stem cells for transplantation. However, for cord blood cells, a very important source for haemopoietic stem cell transplantation, relatively little is known about their telomerase expression and regulation. In fact, the mechanisms involved in regulating cellular telomerase activity are not clear. It has been demonstrated that hTR expression is regulated in normal human T cells during lineage development and after activation, indicating that regulation of hTR expression may contribute to the regulation of telomerase activity in normal lymphoid cells (Weng et al, 1997).
Peptide growth factors are well known to affect cell replication and are likely to be involved in some of the regulatory mechanisms. Insulin-like growth factor 1 (IGF-1) has been shown to be involved in the growth, proliferation and transformation of many cell types. IGF-1 actions mediated through the IGF-1 receptor appear to be sufficient for progression through the cell cycle (Rubin & Baserga, 1995; Baserga et al, 1993). Moreover, there was some evidence which indicated that IGF-1 receptor might play an important role in human cancers and transformed cell lines (Werner & Leroith, 1996). Thus, IGF-1 is a tangible candidate involved in telomerase activation in cell growth and proliferation. In fact IGF-1, as a lymphohaemopoietic cytokine, has been reported to have profound positive effects on immune function, such as promoting pro-B-cell proliferation, differentiation, immunoglobulin production and class switching, and increasing T-cell proliferation (Kooijman et al, 1996). However, it remains to be shown whether telomerase activation is involved in the immune regulation of IGF-1 in cord blood cells.
To address these issues, we investigated the effect of IGF-1 on telomerase activity and the expression of hTR, hTRT and TP1 in cord blood mononuclear cells (MNC) stimulated by PHA, which is a predominantly T-cell stimulating agent (Miller, 1983). This, to the best of our knowledge, is the first report about the effect of IGF-1 on telomerase activation and the expression of hTR, hTRT and TP1 in cord blood MNC.
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- MATERIALS AND METHODS
Telomerase activity is expressed in malignant cells but absent in most normal human somatic cells. However, recent findings demonstrated that low level of telomerase activity was expressed in haemopoietic and lymphoid cells, including haemopoietic progenitors, peripheral blood mononuclear cells (PBMC), T and B lymphocytes (Counter et al, 1995; Hiyama et al, 1995; Igarashi & Sakaguchi, 1997).
Studies of telomere length in haemopoietic stem cells and lymphocytes have demonstrated that the reduction of telomere length occurs progressively with age in vivo and with cell divisions in vitro (Vaziri et al, 1993, 1994). Weng et al (1995) also reported that telomere length was longer in CD4+ naive T cells than that in memory cells. These results led to the hypothesis that telomeres serve as a biological clock of the replicative life-span of haemopoietic stem cells and lymphocytes. Thus, in the absence of telomerase activity, cells would undergo progressive telomere shortening with each successive cell division until the reduction of telomere length to a critical level resulting in exhaustion of the capacity for cell replication. Our findings here indicated that telomerase was also expressed constitutively in normal cord blood MNC and suggested that the low level of telomerase activity is associated with the replicative potential of the cord blood haemopoietic stem cells and lymphoid cells (Fig 1).
Several recent studies have shown that telomerase activity can be manipulated in vitro in normal leucocytes. The stimuli that are known to up-regulate telomerase activity include mitogenic lectins for both T and B cells, anti-CD3 mAb for T cells and anti-IgM Ab plus anti-CD40 monoclonal antibody for B cells (Hiyama et al, 1995; Igarashi & Sakaguchi, 1996; Bodnar et al, 1996; Weng et al, 1996). Telomerase expression was shown to be regulated both developmentally and by TCR-mediated activation in cells of human T-lymphocyte lineage (Weng et al, 1996). Igarrashi & Sakaguchi (1997) reported that telomerase expression was regulated by B-cell antigen receptor (BCR)-mediated activation in peripheral B cells. In our study, significant up-regulation of telomerase activity in cord blood MNC stimulated by PHA was also detected (Fig 2). Hiyama et al (1995) suggested that telomerase induction might play an important role in the repeated clonal expansion of lymphocytes. Our data and recent findings provide further evidence that telomerase induction is linked directly to the lymphocyte activation pathway, perhaps as a compensatory mechanism to offset the shortening of telomeres during periods of rapid clonal expansion.
IGF-1 has been reported to have profound positive effects on immune function, such as regulating proliferation of haemopoietic stem cells and monocyte precursors, promoting pro-B-cell proliferation, differentiation, immunoglobulin production and class switching, and increasing T-cell proliferation (Kooijman et al, 1996). In this study we found that IGF-1 alone could not induce cord blood MNC telomerase activation and proliferation, but IGF-1 could up-regulate PHA-activated cord blood MNC telomerase activation and proliferation (Fig 2B). Our results suggest that the expression of telomerase in cord blood MNC was activation-induced, and that telomerase activation might play an important role in IGF-1-induced T-cell clonal survival and expansion. IGF-1 may have a role in increasing replicative potential by increasing telomerase activity in cord blood MNC.
The degrees of telomerase activation in cord blood MNC at various time points of culture were different when they were stimulated by PHA in the presence or absence of IGF-1 (Fig 1B). Telomerase activity and proliferation were also not regulated in step (Figs 3A and 3B). These data suggest that a different mechanism may be involved in the effect of IGF-1 on telomerase activity and on proliferation of cord blood MNC.
IGF-1 plays a critical role in G1 and S phase of the cell cycle. It cannot on its own stimulate entry into G1 phase, but is required for maintaining G1 and entry into S phase in many cell types, including mitogen-stimulated human PBMC (Rubin & Baserga, 1995; Baserga et al, 1993; Reiss et al, 1992). These phenomena are consistent with our observation that IGF-1 alone could not induce the cord blood MNC proliferative responses and telomerase activities in the absence of PHA. Our results showed that the telomerase activity was maintained at a high level even when the proliferation had rapidly declined to a low level on day 4 in PHA-activated cord blood MNC with IGF-1 treatment (Fig 3B). Therefore the telomerase activity of cord blood MNC was still maintained at a high level when most of the cord blood MNC had already exited from S phase.
A previous study demonstrated that telomerase was regulated in G1 phase as normal human T cells enter the cell cycle (Buchkovich & Greider, 1996). However, considering the fact that IGF-1 is a major growth factor present in fetal bovine serum (FBS) (Correa et al, 1994), and there was 20% FBS in their culture medium (Buchkovich & Greider, 1996), the telomerase regulation in G1 phase might have been partly due to IGF-1 activity. Using serum- and hormone-free medium as in our study could help to elucidate the regulation of telomerase more clearly in vitro.
The telomerase nucleoprotein complex consists of multiple components. One component of telomerase, the telomerase RNA component, has been cloned in mice and humans (Feng et al, 1995; Blasco et al, 1995). However, the quantitative relationship between RNA component expression and telomerase activity has been controversial. In the mouse, it has been reported that up-regulation of telomerase RNA expression occurred in parallel with telomerase activity during tumourigenesis (Blasco et al, 1996). In the human it has also been reported that telomerase RNA component (hTR) was regulated in a manner that paralleled the regulation of telomerase activity during T-cell development and activation (Buchkovich & Greider, 1996; Weng et al, 1997). However, when the level of hTR was compared with telomerase activity in immortal cell lines and tumour tissues there was no apparent correlation between the levels of hTR expression and telomerase activity (Avilion et al, 1996). Our results here demonstrated that the hTR expression of cord blood MNC remained constant when telomerase activity was up-regulated by PHA or IGF-1 plus PHA, consistent with the absence of correlation between hTR expression and telomerase activity observed in human peripheral blood T cells (Buchkovich & Greider, 1996; Weng et al, 1997).
Another component of telomerase, the telomerase-associated protein 1 (TP1), has also been identified and cloned recently, and was shown to interact specifically with hTR (Harrington et al, 1997). Our results here indicated that IGF-1 alone could increase TP1 mRNA significantly, but could not increase further TP1 mRNA expression in addition to the increase of telomerase activity in PHA-activated cord blood MNC (Figs 4B and 4D). Our results also demonstrated that steady-state TP1 mRNA level alone was not directly associated with increase of telomerase activity in cord blood MNC since IGF-1 could increase TP1 mRNA but not telomerase activity (Fig 4B). On the contrary, TP1 mRNA expression was maintained at a high level when telomerase activity of cord blood MNC stimulated by PHA or IGF-1 plus PHA declined rapidly from day 3 to day 5 (Figs 4C and 4D). In fact, at least in HL60 cells, down-regulation of telomerase has been shown to be loosely associated with up-regulation of TP1 (Reichman et al, 1997). Taken together, it remains speculative whether a high level of TP1 mRNA might be related with down-regulation of telomerase activity in cord blood MNC. IGF-1 may have a multi-level effect on the telomerase nucleoprotein complex in that, on its own, IGF-1 could increase TP-1, but with PHA it could further increase telomerase activity, perhaps through increased hTRT expression.
Human telomerase catalytic subunit gene (hTRT) has recently been cloned and shown to be a critical determinant of enzyme activity of telomerase (Nakamura et al, 1997). Furthermore, expression of hTRT in telomerase-negative human normal fibroblast cells transduced with gene constructs encoding hTRT could induce telomerase activity (Nakayam et al, 1998). In addition, a study of 20 cancerous and 19 non-cancerous liver tissues revealed good correlation between telomerase activities and the levels of hTRT expression in most cases (Nakayam et al, 1998), further supporting hTRT as the catalytic subunit of human telomerase. Our results here showed that IGF-1 up-regulated hTRT mRNA expression as well as telomerase activity in PHA-activated cord blood MNC (Figs 4D and 5). Therefore hTRT mRNA showed correlation with telomerase activity in cord blood MNC as well. However, Nakayam et al (1998) reported in the same study that telomerase activity and hTRT expression levels showed no correlation in some cases. Our study also showed that the dynamics of hTRT mRNA expression was not completely in phase with that of telomerase activity in cord blood MNC stimulated by PHA or PHA plus IGF-1. This suggests that factors other than hTRT might also have a role in regulating telomerase activity.
The effects of IGF-1 on telomerase activity, hTR, hTRT mRNA and TP1 mRNA in cord blood MNC reported here suggest that IGF-1 may have a role in increasing the replicative potential of cord blood lymphoid cells or haemopoietic stem cells. The best way to induce telomerase activity in these cells by IGF-1 and its underlying mechanism need to be further defined in purified cell populations and this may be useful for in vitro expansion of these cells for transplantation or adoptive immunotherapy.