SEARCH

SEARCH BY CITATION

Keywords:

  • IGF-1;
  • regulation;
  • telomerase;
  • replicative potential;
  • cord blood cell

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Isolation of cord blood MNC

Human umbilical cord blood was obtained, with written informed consent of their parents, from the placentae of normal full-term infants after delivery. The protocol was approved by the Ethics Committee of the University of Hong Kong. The samples were collected in a heparinized flask. MNC were isolated from whole blood by centrifugation using Ficoll-Hypaque gradients (Pharmacia Biotech, Uppsala, Sweden). The cord blood MNC at the interface were collected, washed three times, and resuspended at a density of 1 × 106 cells/ml in a serum- and hormone-free medium, Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 Ham (DME/F-12), which did not contain insulin, insulin-like growth factor 1 or other hormones (Sigma Chemical Co., St Louis, Mo., U.S.A.), and supplemented with 50 i.u./ml penicillin and 50 μg/ml streptomycin. Cell viability as determined by trypan blue exclusion was >99%.

Culture and activation of cord blood MNC in vitro

1 × 106 MNC were cultured in the presence or absence of phytohaemagglutinin (PHA; 1 μg/ml, Sigma, St Louis, Mo., U.S.A.), with and without IGF-1 (100 ng/ml, R&D Systems, Minneapolis, U.S.A.), and incubated for varying periods at 37°C in a humidified atmosphere containing 5% CO2. For telomerase assays and RNA-PCR analysis, cells were collected at defined times after stimulation. After incubation for an appropriate time, cells were isolated by centrifugation and then stored at −80°C until further processing.

Proliferation assay

Cell proliferation was assayed by [3H]thymidine (TdR) incorporation. 2 × 105 cells were cultured in triplicate samples in U-bottomed 96-well microtitre plates for 1, 2, 3, 4 or 5 d at 37°C in a 5% CO2 atmosphere. [3H]TdR (Amersham, Buckinghamshire, U.K.) was added to a final concentration of 18.5 kBq/well during the last 6 h of culture. Cells were harvested on a fibre-glass filter and counted by liquid scintillation counting.

PCR-based telomerase assay

Telomerase activity was determined by the telomeric repeats amplification protocol (TRAP) as previously described (Chiu et al, 1996; Igarashi & Sakaguchi, 1997). All precautions against RNAse contamination were observed. Cells were lysed with 20 μl of cold CHAPS lysis buffer (10 mm Tris-HCl, pH 7.5; 1 mm MgCl2; 1 mm EGTA; 0.1 mm PMSF; 5 mmβ-mercaptomethanol; 0.5% w/v CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulphomate); 10% glycerol) per 106 cells. The cell lysate was kept on ice for 30 min and centrifuged at 12 000 g for 30 min at 4°C. The supernatant was carefully removed and the protein concentration was determined using Coomassie Protein Assay Reagent (Pierce Chemical Co., Rockford, Ill.). To minimize the effect of telomerase inhibitors which could have been present in the sample extract, telomerase activity from each sample was assayed at four concentrations (3, 0.3, 0.03 and 0.003 μg), and the protein amount determined (0.3 and 0.03 μg) to optimize the TRAP assay.

Assay tubes were prepared by sequestering 0.05 μg of CX primer (5′-[CCCTTA]3CCCTAA-3′) under a wax barrier (AmpliwaxTM, Perkin-Elmer, Foster City, Calif.). 2 μl of each appropriately diluted CHAPS cell extract was layered over the wax barrier in 50 μl of reaction mixture, which was composed of TRAP buffer (20 mm Tris-HCl pH 8.3; 1.5 mm MgCl2; 63 mm KCl; 0.005% Tween 20; 1 mm EGTA; 0.1 mg/ml BSA), 50 μm of dNTPs, 10 ng of TS primer (5′-AATCCG-TCGAGCAGAGTT-3′), 4 u Taq polymerase (Gibco-BRL, Gaithersburg, Md., U.S.A.), 148 kBq of [α-32P]dCTP (Amersham) in DEPC H2O. The tube was incubated for 30 min at 23°C before PCR amplification (30 cycles of 94°C/30 s, 50°C/30 s, 72°C/45 s). Aliquots (15 μl) of radiolabelled PCR products were separated on 15% non-denaturing PAGE gel in 0.6 × TBE. A RNAse control was set up with each cell extract by preincubating with 0.5 μg of DNAse-free RNAse for 15 min at room temperature to confirm the specificity of the telomerase products.

Telomerase activity was semiquantified by a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). The signal intensity from each lane of the gel was defined as the signal within a rectangular boundary around all the visible bands in the lane. A rectangle of the same size and shape was used for quantifying the signal from each lane on a gel. The signal present in the ‘no extract’ lane was subtracted as background. Extracts from telomerase-positive immortal cells (Hela cells) were used as a standard. Relative telomerase activities were calculated by dividing the signal intensity of each lane by the signal intensity with the standard.

RNA-PCR analysis

Cell total RNA was extracted by using RNeasy Total RNA Kits (QIAGEN Inc., Chatsworth, U.S.A.) following the manufacturer's instructions. The RNA content of the solution was quantified using the optical density (OD) at 260 nm measured on a Genequant spectrophotometer (Pharmacia-Biotech, Cambridge, U.K.). The RNA aliquots were stored at −80°C until analysed. The ratio 260/280 nm was always more than 1.8. RNA was treated with deoxyribonuclease I (Amplification Grade, Gibco-BRL) for digesting single- and double-stranded DNA to oligonucleotides before cDNA synthesis.

cDNA was synthesized from oligo-dT-primer RNA by reverse-transcription (RT) with M-MLV superscript reverse transcriptase (Gibco-BRL). 1 μg of total RNA and 1 μl Oligo(dT)12–18 (Gibco-BRL, 500 μg/ml) were mixed with DEPC H2O in a final volume of 12 μl, the mixture was heated to 70°C for 10 min and quickly chilled on ice. 4 μl of 5 × First Strand Buffer, 2 μl 0.1 m dithiothreitol (DTT), 1 μl 10 mm dNTPs and 1 μl (200 units) of SuperscriptTM (Gibco-BRL) were added to the mixture and incubated for 50 min at 42°C. The reaction was inactivated through heating at 70°C for 15 min. The final cDNA product was stored at −20°C for subsequent cDNA amplification by PCR.

PCR conditions, enabling reliable comparison of hTRT mRNA, TP1 mRNA or hTR expression with GAPDH mRNA expression in different samples, were established by making serial dilutions of template cDNA at constant cycle numbers for each primer pair, in order to identify the range of cDNA sample needed for the linearity of PCR amplification. Reaction mixtures for PCR contained 1 μl of appropriately diluted cDNA, 5 μl 10 × PCR buffer (200 mm Tris-HCl pH 8.4, 500 mm KCl), 1 μl 10 mm dNTPs, 1.5 μl 50 mm MgCl2, 1 μl 10 mm primer mix, 0.25 μl Taq DNA polymerase (5 u/ml, Gibco-BRL) and 40.25 μl distilled water in a final volume of 50 μl. PCR was performed on a thermal cycler (Perkin-Elmer, Foster City, U.S.A.). The cycling profile for GAPDH, hTRT, TP1 and hTR were 94°C for 45 s, 55°C for 45 s, 72°C for 90 s, for 30 cycles with a final extension at 72°C for 5 min.

Primer sequences for the internal control, GAPDH, were 5′-GCAGGGGGGAGCCAAAAGGG-3′ for the upstream primer, and 5′-TGCCAGCCCCAGCGTCAAAG-3′ for the downstream primer (Gibco-BRL). Primer sequences for hTRT mRNA were 5′-CGGAAGAGTGTCTGGAGCAA-3′ for the upstream primer (LT5), 5′-GGATGAAGCGGAGTCTGGA-3′ for the downstream primer (LT6) (Nakamura et al, 1997); for hTR, 5′-TCTAACCCTAACTGAGAAGGGCGTAG-3′ for the upstream primer (F3b), 5′-GTTTGCTCTAGAATGAACGGTGGAAG-3′ for the downstream primer (R3c) (Nakamura et al, 1997); for TP1, 5′-TCAAGCCAAACCTGAATCTGAG-3′ for the upstream primer (TP1.1), 5′-CCCGAGTGAATCTTTCTACGC-3′ for the downstream primer (TP1.2) (Nakamura et al, 1997).

To semiquantify the amounts of hTRT mRNA, TP1 mRNA and hTR expression, the signal intensity of hTRT, TP1 and hTR production was compared with that of GAPDH product amplified from that same cDNA sample in separate reactions. The products were separated on 2% agarose gel, stained with ethidium bromide, and then quantitative analysis by Fluorimager obtained from Molecular Dynamics (Sunnyvale, Calif.) for each of the fragments. The density of GAPDH of each sample was set at 100%. The value for hTRT mRNA, TP1 mRNA and hTR was the percentage of the density of GAPDH in the same RNA sample.

Statistical methods

To determine differences between two groups the Wilcoxon matched pairs signed rank sum test was used.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Constitutive telomerase activity in cord blood MNC

To assess the constitutive expression of telomerase in normal cord blood MNC we examined telomerase activity in both fresh cord blood MNC and cultured cord blood MNC. The telomerase activity of fresh cord blood MNC was maintained at a level of about 25% of that of Hela cells (Fig 1). In unstimulated cord blood MNC cultured for 1–5 d, the telomerase activity was still maintained at a level of 20–30% of that of Hela cells (Fig 1). Similar results were also observed in unstimulated cord blood MNC from 16 infants after 3 d of culture (Fig 2).

image

Figure 1. . Telomerase activities in cord blood MNC at different times of culture. Cord blood MNC (1 × 106/ml) were incubated for different time periods with IGF-1 (100 ng/ml) and/or PHA (1 μg/ml). (A) The telomerase assay was carried out using cell extracts containing 0.3 μg protein per reaction by TRAP assay, as described in Materials and Methods. Extracts from Hela cells were used as a standard. (B) Relative telomerase activity was evaluated by PhosphorImager as described in Materials and Methods. Results shown are representative of two different cases.

Download figure to PowerPoint

image

Figure 2. . Effect of IGF-1 on telomerase activities and proliferative responses in cord blood MNC. Cord blood MNC (1 × 106/ml) were incubated for 3 d with IGF-1 (100 ng/ml) and/or PHA (1 μg/ml). The telomerase assay was carried out using cell extracts containing 0.3 μg and 0.03 μg protein per reaction by TRAP assay, as described in Materials and Methods. Cell extracts were pre-treated with (+) or without (−) RNase. Extracts from Hela cells were used as standard. The control lane (C) has no cell extract added. RNase pre-treatment of extracts from cord blood MNC abrogated the generation of TRAP products (A). Relative telomerase activity and [3H]TdR incorporation were measured as described in Materials and Methods. Results of proliferative responses and relative telomerase activities shown in panel B are of 16 different cases (mean ± SD, n = 16). For proliferative response, there was no difference between ‘medium’ and ‘IGF-1’ (P > 0.05); the difference between ‘IGF-1’ and ‘PHA’ was significant (P = 0.0001), as was the difference between ‘PHA’ and ‘IGF-1+PHA’ (P = 0.0002). For telomerase activity there was also no difference between ‘medium’ and ‘IGF-1’ (P > 0.05); the difference between ‘IGF-1’ and ‘PHA’ was significant (P < 0.0001), as was the difference between ‘PHA’ and ‘IGF-1 + PHA’ (P = 0.0002).

Download figure to PowerPoint

Regulation of the telomerase activity in cord blood MNC by IGF-1

IGF-1 alone could not induce the telomerase activity in cord blood MNC over 5 d of culture (Fig 1). PHA alone could significantly up-regulate the telomerase activity in cord blood MNC during the same period (Fig 1). IGF-1 could further increase the telomerase activity in PHA-activated cord blood MNC throughout day 1 to day 4 of culture, reaching a peak on day 3 (Fig 1). We further examined the effect of IGF-1 on the telomerase activities in PHA-activated cord blood MNC from 16 infants after 3 d of culture, which demonstrated a significant increase (Fig 2).

Relationship between the telomerase activity and proliferative response in cord blood MNC

IGF-1 alone could not induce proliferative response of cord blood MNC (Fig 2B). However, up-regulation of both the telomerase activity and the proliferative response was detected in cord blood MNC stimulated with PHA alone (Fig 2B). IGF-1 could further increase both the telomerase activity and the proliferative response of PHA-activated cord blood MNC (Fig 2B). Analysis of the dynamics of telomerase activity and proliferation of cord blood MNC in response to PHA (Fig 3A) over the 5 d of culture, showed that the telomerase activity increased in phase with proliferation in the first 2 d and was maintained at a high level for 2 more days before decline, while proliferation in response to PHA dropped from day 2 onwards (Fig 3A). In contrast, the proliferation in response to PHA plus IGF-1 dropped from day 3 onwards, whereas the telomerase activity was maintained at high level until day 4 before rapid decline (Fig 3B).

image

Figure 3. . Relationship between telomerase activity and proliferative response in cord blood MNC. Cord blood MNC (1 × 106/ml) were incubated for different time periods with IGF-1 (100 ng/ml) and/or PHA (1 μg/ml). (A) Relative telomerase activity and proliferative response in cord blood MNC stimulated by PHA (1 μg/ml) alone. Relative telomerase activities were measured by TRAP assay and proliferative responses were measured by [3H]TdR incorporation, as described in Materials and Methods. (B) Relative telomerase activity and proliferative response in cord blood MNC stimulated by PHA (1 μg/ml) plus IGF-1 (100 ng/ml). Results shown are representative of two different cases.

Download figure to PowerPoint

Constitutive hTR, hTRT mRNA and TP1 mRNA expression in cord blood MNC

To assess the constitutive expression of hTR, hTRT mRNA and TP1 mRNA in normal cord blood MNC, we examined hTR, hTRT mRNA and TP1 mRNA in both fresh cord blood MNC and cultured cord blood MNC. Fresh cord blood MNC from four infants expressed relatively high levels of hTR and TP1 mRNA but a low level of hTRT mRNA (Fig 4A). Similar results were also found in unstimulated cord blood MNC cultured for 1–5 d (Fig 4A).

image

Figure 4. . Telomerase activity, hTRT mRNA, TP1 mRNA and hTR expression in cord blood MNC at different times of culture. Cord blood MNC (1 × 106/ml) were incubated with IGF-1 (100 ng/ml) and/or PHA (1 μg/ml). RNA isolation and semiquantitative RNA-PCR were described in Materials and Methods. Amplification products of hTRT, TP1, hTR and GAPDH cDNA were separated on 2% agarose gel (A) and evaluated by Fluorimager (B, C, D) as described in Materials and Methods. Panel B indicates results from cord blood MNC stimulated by IGF-1 alone. Panel C indicates results from cord blood MNC stimulated by PHA alone. Panel D indicates results from cord blood MNC stimulated by IGF-1 plus PHA. Results shown are representative of four different cases.

Download figure to PowerPoint

Regulation of hTRT mRNA, TP1 mRNA and hTR expression in cord blood MNC by IGF-1

IGF-1 alone could not induce hTRT mRNA and hTR expression, but it could induce TP1 mRNA expression in cord blood MNC from four infants over the 5 d of culture (Fig 4B). PHA also could not induce hTR expression, but it could induce the expression of TP1 mRNA and hTRT mRNA in cord blood MNC from four infants over the 5 d of culture (Fig 4C). IGF-1 could further increase hTRT mRNA expression in PHA-activated cord blood MNC over the 5 d of culture (Fig 4D). However, up-regulation of hTR and TP1 mRNA expression was not found in cord blood MNC stimulated by IGF-1 plus PHA (Fig 4D) compared with that in cord blood MNC stimulated by IGF-1 alone (Fig 4B) or PHA alone (Fig 4C). IGF-1 was to shown to up-regulate significantly hTRT mRNA expression in PHA-activated cord blood MNC from 16 infants after 12 h of culture (Fig 5).

image

Figure 5. . Effect of IGF-1 on hTRT mRNA in cord blood MNC. Cord blood MNC were incubated for 12 h with IGF-1 (100 ng/ml) and/or PHA (1 μg/ml). RNA isolation and semiquantitative RNA-PCR were described in Materials and Methods. Result shown is individual value of 16 different cases. The difference between ‘PHA’ and ‘IGF-1 + PHA’ was significant (P = 0.011).

Download figure to PowerPoint

Relationship among telomerase activity, hTR, hTRT mRNA and TP1 mRNA expression in cord blood MNC

The dynamics of telomerase activity, hTR, hTRT mRNA and TP1 mRNA expression in cord blood MNC over the 5 d of culture are shown in 4Figs 44B, 4C and 44D. The dynamics of hTRT mRNA expression was not in phase with that of telomerase activity in cord blood MNC stimulated with PHA or PHA plus IGF-1 (Figs 4C and 4D). The telomerase activity increased upto day 3 and then fell dramatically, whereas the hTRT mRNA expression peaked at around day 1, maintained the level until day 3, and then decreased gradually (Figs 4C and 4D). The dynamics of TP1 mRNA expression was similar to that of telomerase activity in cord blood MNC induced by PHA or PHA plus IGF-1 over the 5 d of culture (Figs 4C and 4D). However, IGF-1 did not further increase TP1 mRNA expression of PHA-activated cord blood MNC as that of telomerase activity (Figs 4C and 4D). The expression of hTR in cord blood MNC remained constant when the telomerase activity and hTRT mRNA expression were up-regulated by PHA or IGF-1 plus PHA (Figs 4C and 4D).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We thank Dr C. P. Chiu (Geron Company, Cupertino, Calif.) for advice in the initial establishment of the TRAP assay; we also thank Miss Y. L. Wong for her technical assistance. This work was supported by the Hong Kong Research Grant Council.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
  • 1
    Avilion, A.A., Piatyszek, M.A., Gupta, J., Shay, J,W., Bacchatti, S. & Greider, C.W. (1996) Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Research, 56, 645650.
  • 2
    Baserga, R., Porcu, P. & Sell, C. (1993) Oncogenes, growth factors and control of cell cycle. Cancer Surveys, 16, 201213.
  • 3
    Blackburn, E.H. (1991) Structure and function of telomeres. Nature, 350, 569573.
  • 4
    Blackburn, E.H. (1992) Telomerases. Annual Review of Biochemistry, 61, 113129.
  • 5
    Blasco, M.A., Funk, W., Villeponteau, B. & Greider, C.W. (1995) Functional characterization and developmental regulation of mouse telomerase RNA. Science, 269, 12671270.
  • 6
    Blasco, M.A., Rizen, M., Greider, C.W. & Hanahan, D. (1996) Differential regulation of telomerase activity and telomerase RNA during multi-stage tumorigenesis. Nature Genetics, 12, 200204.
  • 7
    Bodnar, H., Kim, N.W., Effros, R.S. & Chiu, C.P. (1996) Mechanism of telomerase induction during T cell activation. Experimental Cell Research, 228, 5864.
  • 8
    Buchkovich, K.J. & Greider, C.W. (1996) Telomerase regulation during entry into the cell cycle in normal human T cells. Molecular Biology of the Cell, 7, 14431454.
  • 9
    Chiu, C.P., Dragowska, W., Kim, N.W., Vaziri, H., Yui, J., Thomas, T.E., Harley, C.B. & Lansdorp, P.M. (1996) Differential expression of telomerase activity in hematopoietic progenitor from adult human bone marrow. Stem Cells, 14, 239248.
  • 10
    Correa, P.N., Eskinazi, D. & Axelrad, A.A. (1994) Circulating erythroid progenitors in polycythemia vera are hypersensitive to insulin-like growth factor-1 in vitro: studies in an improved serum-free medium. Blood, 83, 99112.
  • 11
    Counter, C.M., Gupta, J., Harley, C.B., Leber, B. & Bacchetti, S. (1995) Telomerase activity in normal leukocytes and in hematologic malignancies. Blood, 85, 23152320.
  • 12
    Feng, J., Funk, W.D., Wang, S.S., Weinrich, S.L., Avilion, A.A., Chiu, C.P., Adams, R.R., Chang, E., Allshopp, R.C., Yu, J., Le, S., West, M.D., Harley, C.B., Andrews, W.H., Greider, C.W. & Villeponteau, B. (1995) The RNA component of human telomerase. Science, 269, 12361241.
  • 13
    Harrington, L., McPhail, T., Mar, V., Zhou, W., Oulton, R., Bass, M.B., Arruda, I. & Robinson, M.O. (1997) A mammalian telomerase-associated protein. Science, 275, 973977.DOI: 10.1126/science.275.5302.973
  • 14
    Hiyama, K., Hivai, Y., Kyoizumi, S., Akiyama, M., Hiyama, E., Piatyszek, M.A., Shay, J.W., Ishioka, S. & Yamakido, M. (1995) Activation of telomerase in human lymphocytes and hemopoietic progenitor cells. Journal of Immunology, 155, 37113715.
  • 15
    Igarashi, H. & Sakaguchi, N. (1996) Telomerase activity is induced by the stimulation to antigen receptor in human peripheral lymphocytes. Biochemical and Biophysical Research Communications, 219, 649655.DOI: 10.1006/bbrc.1996.0288
  • 16
    Igarashi, H. & Sakaguchi, N. (1997) Telomerase activity is induced in human peripheral B lymphocyte by the stimulation to antigen receptor. Blood, 89, 12991307.
  • 17
    Kooijman, R., Hooghe-Peters, E.L. & Hooghe, R. (1996) Prolactin, growth hormone, and insulin-like growth factor-1 in the immune system. Advances in Immunology, 63, 377454.
  • 18
    Miller, K. (1983) The stimulation of human B and T lymphocytes by various lectins. Immunobiology, 165, 132146.
  • 19
    Nakamura, T.M., Morin, G.B., Chapman, K.B., Weinrich, S.L., Andrews, W.H, , Lingner, J., Harley, C.B. & Cech, T.R. (1997) Telomerase catalytic subunit homologs from fission yeast and human. Science, 277, 955959.DOI: 10.1126/science.277.5328.955
  • 20
    Nakayama, J.-I., Tahara, H., Tahara, E., Saito, M., Ito, K., Nakamura, H., Nakanishi, T., Tahara, E., Ide, T. & Ishikawa, F. (1998) Telomerase activation by hTRT in human normal fibroblast and hepatocellular carcinomas. Nature Genetics, 18, 6568.DOI: 10.1038/ng0198-65
  • 21
    Reichman, T.W., Albanell, J., Wang, X., Moore, M.A. & Studzinski, G.P. (1997) Downregulation of telomerase activity in HL60 cells by differentiating agents is accompanied by increased expression of telomerase-associated protein. Journal of Cellular Biochemistry, 67, 1323.DOI: 10.1002/(SICI)1097-4644(19971001)67:1<13::AID-JCB2>3.3.CO;2-5
  • 22
    Reiss, K., Porcu, P., Sell, C., Pietrzkowski, Z. & Baserga, R. (1992) The insulin-like growth factor 1 receptor is required for the proliferation of hemopoietic cells. Oncogene, 7, 22432248.
  • 23
    Rhyu, M.S. (1995) Telomeres, telomerase and immortality. Journal of the National Cancer Institute, 87, 884894.
  • 24
    Rubin, R. & Baserga, R. (1995) Insulin-like growth factor. I. Its role in cell proliferation, apoptosis, and tumorigenicity. Laboratory Investigation, 73, 311339.
  • 25
    Vaziri, H., Dragowska, W., Allsopp, R.C., Thomas, T.E., Harley, C.B. & Lansdorp, P.M. (1994) Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proceedings of the National Academy of Sciences of the United States of America, 91, 98579860.
  • 26
    Vaziri, H., Schachter, F., Uchida, I., Wei, L., Zhu, X., Effros, R., Cohen, D. & Harley, C.B. (1993) Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. American Journal of Human Genetics, 52, 661667.
  • 27
    Weng, N.P., Levine, B.L., June, C.H. & Hodes, R. (1995) Human naive and memory T lymphocytes differ in telomeric length and replicative potential. Proceedings of the National Academy of Sciences of the United States of America, 92, 1109111094.
  • 28
    Weng, N.P., Levine, B.L., June, C.H. & Hodes, R.J. (1996) Regulated expression of telomerase activity in human T lymphocyte development and activation. Journal of Experimental Medicine, 183, 24712479.DOI: 10.1084/jem.183.6.2471
  • 29
    Weng, N.P., Levine, B.L., June, C.H. & Hodes, R.J. (1997) Regulation of telomerase RNA template expression in human T lymphocyte development and activation. Journal of Immunology, 158, 32153220.
  • 30
    Werner, H. & Leroith, D. (1996) The role of the insulin-like growth factor system in human cancer. Advances in Cancer Research, 68, 183221.