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Originally published online as doi:10.1189/jlb.1207848 on February 14, 2008

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(Journal of Leukocyte Biology. 2008;83:1240-1248.)
© 2008 by Society for Leukocyte Biology

Multistep regulation of telomerase during differentiation of HL60 cells

Osamu Yamada*,{dagger},1, Kohji Ozaki{ddagger}, Mayuka Nakatake{dagger}, Masaharu Akiyama§, Kiyotaka Kawauchi|| and Rumiko Matsuoka{ddagger}

Departments of
* Hematology and
|| Medicine,
{dagger} Medical Research Institute,
{ddagger} IREIIMS, Tokyo Women’s Medical University, Tokyo, Japan; and
§ Department of Pediatrics, Jikei University School of Medicine, Tokyo, Japan

1Correspondence: Tokyo Women’s Medical University, Medical Research Institute and Department of Hematology, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. E-mail: yamadao{at}lab.twmu.ac.jp


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ABSTRACT
 
Using three different differentiation agents (1{alpha}, 25 dihydroxyvitamin D3, all-trans-retinoic acid, and Am80), down-regulation of telomerase activity was found to be a common response during the monocytic or granulocytic differentiation of human acute myeloblastic leukemia cell line 60 (HL60) cells. Rapid down-regulation of telomerase transcription occurred during early differentiation of HL60 cells prior to G1 arrest. Akt kinase activity was suppressed after 6 h of differentiation along with inhibition of telomerase activity, and the extent of the suppression that occurred while maintaining telomerase protein expression suggested the post-translational regulation of telomerase activity. Recombinant Akt dose-dependently increased telomerase activity, and telomerase was inhibited at the transcriptional and post-translational levels by LY294002, suggesting that PI-3K/Akt is one of the key signaling proteins involved in telomerase regulation. Each of the three differentiation agents caused a significant increase of signaling proteins (including Akt) at 3 days after the initiation of differentiation. Changes of acetyl-histone H4, which regulates transcription of the telomerase gene, were observed before the activation of Akt. This finding suggests that epigenetic control of telomerase transcription occurs before activation of Akt during the late stage of differentiation. These results indicate that telomerase activity is regulated by at least two mechanisms during granulocytic and monocytic differentiation, with one mechanism being transcriptional and the other being post-translational.

Key Words: Akt kinase • PI-3K • hTERT


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INTRODUCTION
 
Telomerase is a ribonucleoprotein RT that adds hexameric telomere sequences (TTAGGG) to the ends of chromosomes [1 ]. Telomerase consists of a RNA component and a catalytic subunit with human telomerase RT (hTERT) [2 , 3 ]. Telomerase activity is correlated with hTERT expression [4 5 6 ], suggesting that hTERT is a key regulator of this enzyme. Telomerase is activated in immature somatic cells and is suppressed in differentiated cells [7 ], but the mechanism by which telomerase activity is regulated during cell differentiation remains unclear.

Several mechanisms that may be involved in the regulation of hTERT have been suggested [8 ]. The hTERT promoter contains various binding sites for transcription factors, including activators (c-Myc, Sp1, and STAT3) and repressors (Mad1, WT1, and p53), suggesting that regulation of hTERT expression may occur at the transcriptional level [9 10 11 12 13 ]. Studies of protein phosphorylation have also indicated that regulation of telomerase and up-regulation of telomerase activity in T lymphocytes are dependent on protein kinase C [14 15 16 ]. We have already reported that down-regulation of telomerase activity occurs during the myeloid or erythroid differentiation of hematopoietic cells [17 ], and telomerase activity increases transiently during megakaryocytic differentiation induced by 12-O-tetradecanoyl phorbol 13-acetate [18 ]. These findings suggest that the mechanisms regulating hTERT are more complex than a simple on/off switch and that more than one mechanism of telomerase regulation may operate during cell differentiation.

In the present study, we investigated the changes of telomerase activity during monocytic or granulocytic differentiation to determine whether there was a lineage-specific difference of telomerase regulation.


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MATERIALS AND METHODS
 
Cell lines
HL60, a human acute myeloblastic leukemia cell line, was obtained from the American Type Culture Collection (Manassas, VA, USA) and was maintained in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS, penicillin (100 u/ml), streptomycin (100 ìg/ml), and 2 mM L-glutamine at 37°C in a humidified atmosphere of 5% CO2 in air. When cell starvation was required, HL60 cells were cultured in RPMI-1640 medium containing 0.1% FBS.

Chemicals and antibodies
1{alpha}, 25 Dihydroxyvitamin D3 (VD3) and all-trans-retinoic acid (ATRA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Am80 (tamibarotene) is a retinoic acid receptor (RAR)-{alpha}β-selective retinoid that does not bind with or activate the RAR-{gamma} receptor or the retinoid X receptor (RXR). It was synthesized by the introduction of heteroatoms into an ATRA-like structure and was kindly provided by Toko Pharmaceutical Co. (Tokyo, Japan) [19 ]. LY294002 was purchased from Calbiochem (La Jolla, CA, USA) and reconstituted in DMSO. Propidium iodide (PI) staining solution was obtained from Wako Pure Chemicals Industries (Osaka, Japan). Rabbit Akt, phospho (p)-Akt (Ser473), p70S6Kinase (S6K), and p-S6K (Thr421/Ser424) antisera were obtained from Cell Signaling (Beverly, MA, USA). Dr. Peter J. Houghton (St. Jude Children’s Research Hospital, Memphis, TN, USA) kindly provided rabbit antibody directed against mammalian target of rapamycin (mTOR), and rabbit antibody against p-mTOR (Ser2448) was obtained from Cell Signaling. Polyclonal rabbit antibodies directed against hTERT were purchased from Calbiochem (La Jolla, CA, USA) and Abcam (Cambridge, UK), and rabbit β-actin antiserum was purchased from Sigma Chemical Co.

Cell culture and induction of differentiation
HL60 cells were induced to differentiate by exposure to 1–4 x 10–8 M ATRA, 1–10 x 10–9 M VD3, or 1–10 x 10–9 M Am80 for 1–9 days. Differentiation was assessed by the detection of immunophenotypic changes in addition to the identification of morphological changes by light microscopy. The optimum concentration of each inducer was selected from the results of preliminary experiments. Cell viability was assessed by trypan blue dye exclusion.

Immunophenotyping
Samples were immunophenotyped using a panel of mAb and flow cytometry, as reported previously [18 ]. The monocyte fraction was determined as the percentage of CD14+ cells, and differentiation to mature granulocytes was assessed by the shift from CD11b– CD14– cells to CD11b+ CD14– cells.

Cell-cycle analysis
Cells were harvested at the indicated times, washed twice in PBS, and fixed in 70% ethanol at 4°C. The fixed cells were treated with RNase for 30 min at 37°C, stained with PI, and analyzed with an EPICS-XL flow cytometer (Beckman Coulter Electronics Inc., Hialeah, FL, USA). Then, the cycle distribution was determined with software for cell-cycle analysis (WinCycle).

RT-PCR
Expression of hTERT and β-actin was detected by RT-PCR, as reported previously [17 ]. Total RNA was isolated using Isogen (Nippongene, Tokyo, Japan), after which cDNA was synthesized with an Advantage RT-for-PCR kit (Clontech, Palo Alto, CA, USA). The resulting cDNA (25 ng) was subjected to PCR amplification, and the products were visualized by agarose gel electrophoresis and staining of the gels with ethidium bromide. The relative concentrations of the PCR products were determined by comparing the ratio of the product in each lane to β-actin. The PCR primers used were 5'-CCTCTGTGCTGGGCCTGGACGATA-3' and 5'-ACGGCTGGAGGTCTGTCAAGGTAG-3' for hTERT as well as 5'-CTTCTACAATGAGCTGCGTG-3' and 5'-TCATGAGGTAGTCAGTCAGG-3' for β-actin.

Telomerase assay and quantification of enzyme activity
Telomerase activity was measured by the telomere repeat amplification protocol (TRAP), as described elsewhere [20 ]. PCR products were resolved by electrophoresis on 12.5% polyacrylamide gel and visualized by staining with SYBR Green DNA stain (BMA, Rockland, ME, USA). For quantification of the telomerase activity in each sample, enzyme activity was expressed in arbitrary units using the following formula: [total peak area for a sample/peak area of internal telomerase assay standard (ITAS) in the sample]/(total peak area for the positive control/peak area of ITAS in the positive control), as reported previously [21 ]. ITAS was used as the internal control [22 ]. To investigate the effect of Akt on telomerase, we added recombinant Akt to cell lysates. The lysates obtained from starved HL60 cells were incubated with or without recombinant Akt (Upstate Biotechnology, Lake Placid, NY, USA) in a reaction buffer consisting of 20 mM HEPES/NaOH (pH 7.5), 0.03% Triton X-100, 100 mM CaCl2, and Akt lipid activator (Upstate Biotechnology) for 10 min at 30°C. Then, aliquots of the pretreated mixture were used for the TRAP assay.

Western blot analysis
Proteins containing equal amounts of sample buffer were subjected to electrophoresis on polyacrylamide gel and then were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA), which was incubated with the primary antibodies, followed by reaction with HRP-conjugated anti-rabbit IgG. Immunoreactive bands were visualized with ECL Plus detection reagent according to the manufacturer’s protocol (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).

In vitro kinase assay
HL60 cells were cultured for 5 days in the presence or absence of VD3, and then equal amounts of cell lysates (containing 200 µg total protein) were incubated overnight at 4°C with 20 µL immobilized Akt antibody. Immobilized beads were washed with lysis buffer and then with kinase buffer, as reported previously [23 ]. Next, the beads were mixed with 1 µg glucogen synthase kinase 3 (GSK-3) fusion protein (Cell Signaling) in the presence of 200 µM ATP, and the kinase reaction was allowed to proceed at 30°C for 30 min. Samples were subjected to SDS-PAGE and transferred to a PVDF membrane, after which immunoblotting was performed overnight at 4°C with rabbit anti-p-GSK-3{alpha}/β (Ser21/9).

Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed as described previously with some modifications [24 , 25 ]. Cells were fixed in formaldehyde for 10 min at 25°C and then were resuspended in lysis buffer [1% SDS, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin]. The lysates were subjected to sonication, and the resulting supernatants were diluted with dilution buffer [50 mM Tris-HCl (pH 8.0), 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin], followed by preclearing with salmon sperm DNA/protein G-Agarose slurry at 4°C. Then each precleared solution was incubated with acetyl-histone H4 antibody, trimethyl-histone H3 antibody, or normal rabbit serum overnight at 4°C. Immune complexes were detected by incubation with salmon sperm DNA/protein G-Agarose slurry at 4°C. After washing the pellets, DNA elution buffer [10 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM EDTA, and 0.5% SDS] was added, and heating was done at 65°C, followed by treatment with 4 µg RNase at 37°C and then 10 µg proteinase K at 55°C. DNA was purified, and PCR was performed to amplify the hTERT promoter region (sense 5'-TTCGACCTCTCTCCGCTGGG-3', antisense 5'-TTCCCACGTGCGCAGCAGGA-3').


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RESULTS
 
Effect of each differentiation agent on HL60 cells
HL60 cells were plated at 5 x 104/well and cultured in the presence of VD3, Am80, or ATRA for the indicated times (Fig. 1 ). The cells were also treated with ethanol as a control. Cell counts and cell viability were determined on a daily basis by trypan blue dye exclusion. The optimum concentration and culture time for each reagent were found to be 5 nM and 5 days for VD3 or Am80 as well as 20 nM and 5 days for ATRA. Although VD3 induced HL60 cells to differentiate into CD11b+ CD14+ monocytes/macrophages, Am80 and ATRA induced differentiation to CD11b+ CD14– granulocytes [19 , 26 , 27 ]. Representative data from three independent experiments are displayed in Table 1 . More than 93% of HL60 cells acquired expression of CD11b without CD14 after incubation in the presence of 2 x 10–8 M ATRA or 5 x 10–9 M Am80, and treatment with 5 x 10–9 M VD3 resulted in >98% CD14 positivity. Maturation of the cells was confirmed by examining their morphology.


Figure 1
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  		Figure 1. Effect of the three reagents on HL60 cells, which were plated at 5 x 104/well in the presence of VD3 (A), Am80 (B), or ATRA (C) at several different concentrations for the indicated times. HL60 cells were also treated with ethanol as a control. The daily cell count and viability were determined by trypan blue dye exclusion. The optimum concentration and culture period for each reagent were determined to be 5 nM and 5 days for VD3 or Am80 and 20 nM and 5 days for ATRA.


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  		Table 1. Expression of Differentiation Marker

Changes of the cell cycle during cell differentiation
HL60 cells were cultured with VD3, Am80, or ATRA for the indicated times, and the DNA content was analyzed by flow cytometry. As can be seen in Figure 2 , during monocytic and granulocytic differentiation of HL60 cells, the cell population in the G1 phase increased to 74–78% after 3 days of treatment with each differentiation inducer. The expression of CD14 and CD11b mRNA, indicating monocytic and granulocytic differentiation, respectively, was up-regulated after 6 h of incubation, and DNA synthesis was still ongoing.


Figure 2
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  		Figure 2. Changes of the cell cycle and expression of CD14 and CD11b mRNA during cell differentiation. (A) HL60 cells were cultured with VD3, Am80, or ATRA for the indicated times, and the DNA content was analyzed by flow cytometry (EPICS-XL). The percentage of cells in each phase of the cell cycle is shown (G1, S, G2). Each experiment was performed three times, and representative results are shown. (B) Changes of CD14, C11b, and β-actin mRNA expression during the differentiation of HL60 cells. (β-actin was the internal control.) Each experiment was performed three times, and representative results are shown. NC, negative control.

Changes of telomerase activity and hTERT expression during differentiation of HL60 cells
To determine the effect of cell differentiation on telomerase activity, mRNA, and protein and to assess whether the changes of these parameters were specific to a particular pathway of differentiation, HL60 cells were induced to differentiate into the monocytic lineage by VD3 and into neutrophils by Am80 or ATRA (Fig. 3 ). Telomerase activity decreased soon after the induction of differentiation and declined to 66% of the control value after 24 h of VD3 treatment. In addition, telomerase activity decreased to 78% after 6 h and to 48% after 24 h of ATRA treatment. Expression of hTERT mRNA decreased to 84% after 6 h and to 62% after 24 h of VD3 treatment, and it decreased to 70% after 6 h and to 30% after 24 h of ATRA treatment. The level of hTERT protein remained stable for 6 h in the presence of all inducers but decreased to 84% after 24 h of incubation with VD3 or to 76% with ATRA treatment. In Am80-treated cells, telomerase activity and the expression of hTERT mRNA and protein showed similar but less-prominent changes than in ATRA-treated cells. The difference may have arisen, as Am80 is a RAR-{alpha}/β-selective retinoid that does not bind with and activate RAR-{gamma} or RXRs, unlike ATRA. These results indicated that rapid down-regulation of telomerase transcription occurs during early differentiation of HL60 cells prior to G1 arrest. Differences of protein expression, the timing of telomerase inhibition, and the extent of its suppression may reflect the differential, post-translational regulation of telomerase activity. Neither extracts from differentiated cells nor the addition of these differentiation inducers inhibited telomerase activity in HL60 cell extracts. The loss of this activity was also independent of differentiation-induced apoptotic cell death, which was assessed by using a hemocytometer (data not shown).


Figure 3
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  		Figure 3. Changes of telomerase activity and hTERT expression during differentiation of HL60 cells. (A, B) HL60 cells were incubated with VD3, Am80, or ATRA for the indicated times. Then, expression of hTERT and β-actin mRNA as well as hTERT protein was determined. (β-actin was the internal control, and relative expression is shown.) WB, Western blot. (C) HL60 cells were incubated with VD3, Am80, or ATRA for the indicated times. Then, cell extracts (0.1 µg) were assayed, and relative telomerase activity was determined. Data represent the mean ± SD of three independent experiments. IC, Internal control.

Increased phosphorylation of Akt after differentiation of HL60 cells
We extended our investigation to the mechanisms underlying the signaling processes during monocytic and granulocytic differentiation. The phosphorylation of Akt, which is known to be associated with activation of Akt kinase activity, was examined. As is shown in Figure 4 , the low level of constitutive Akt phosphorylation was increased by exposure to any of the differentiation inducers. Similarly, phosphorylation of the two other downstream components of the Akt signaling pathway that were investigated in this study (mTOR and p70S6K) also showed an increase.


Figure 4
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  		Figure 4. Changes of signaling proteins during differentiation of HL60 cells, which were incubated with VD3, Am80, or ATRA for the indicated times, and the expression of signaling proteins was determined by Western blotting. (β-actin was the loading control.) The experiments were repeated, and similar results were obtained; representative data are shown.

Increased Akt kinase activity during differentiation of HL60 cells
To determine whether the phosphorylation of Akt was associated with activation of its kinase activity, an in vitro kinase assay was performed. When Akt was immunoprecipitated from HL60 cells that had been treated with VD3 for various times, it showed a sustained increase of in vitro kinase activity for its recombinant GSK peptide substrate (Fig. 5 ).


Figure 5
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  		Figure 5. Differentiation-induced activation of Akt kinase. HL60 cells were cultured for the indicated time in the presence of VD3. Akt kinase activity was measured by an in vitro kinase assay with GSK-3 as described in Materials and Methods. A representative band and the relative kinase activity are shown. Data represent the mean ± SD of three independent experiments.

Effect of LY294002 and recombinant Akt on telomerase activity
To investigate whether Akt activates telomerase, we performed the TRAP assay after treating cell lysates with recombinant Akt. After HL60 cells were starved for 4 days without serum, lysates were obtained. Pretreatment with 100 or 200 ng recombinant Akt enhanced telomerase activity by 1.1-fold and 1.6-fold, respectively (Fig. 6A ). In addition, cells were starved for 2 days without FBS and then incubated with DMSO or 10 µM LY294002 for 5 min, after which, the cell extracts were assayed for telomerase activity, which was suppressed after short-term exposure to LY294002 (Fig. 6B) , and transcription of hTERT was also suppressed in the presence of LY294002 (Fig. 6C) . These results suggested that activation of telomerase involves transcriptional or post-translational regulation via the pathway that includes PI-3K/Akt in HL60 cells.


Figure 6
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  		Figure 6. Effect of recombinant Akt and a PI-3K inhibitor on telomerase activity. (A) Cells were starved for 4 days, and cell extracts were assayed for telomerase activity, with or without the addition of recombinant Akt. A representative ladder indicating telomerase activity (upper) and the relative telomerase activity (lower) are shown. A significant increase of telomerase activity was observed after the addition of recombinant Akt. (B) Cells were starved for 2 days and then incubated with DMSO or 10 µM LY294002 for 5 min, after which, the cell extract was assayed for telomerase activity. A representative telomerase ladder and relative telomerase activity are displayed. (C) Cells were incubated with 10 µM LY294002 for the indicated times, and then the expression of hTERT and β-actin mRNA was determined. (β-actin was the internal control, and relative expression is shown.) Values represent the mean ± SD of three independent experiments, and representative data are shown.

Epigenetic control of telomerase transcription during differentiation of HL60 cells
To further investigate the transcriptional regulation of telomerase, the ChIP assay was performed. After cross-linking, protein-DNA complexes were immunoprecipitated with acetyl-histone H4 and trimethyl-histone H3 antibodies. Then, cross-linking was reversed, DNA was extracted, and PCR amplification was done targeting the hTERT promoter. It was found that acetyl-histone H4 underwent deacetylation during differentiation, and trimethyl-histone H3 remained stable during differentiation (Fig. 7 ).


Figure 7
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  		Figure 7. Epigenetic control of telomerase transcription during differentiation of HL60 cells, which were incubated with or without differentiation inducers. After cross-linking, protein-DNA complexes were immunoprecipitated with antibodies directed against acetyl-histone H4 or trimethyl-histone H3. Purified DNA was amplified by PCR to assess binding of the hTERT promoter (upper band). The imunoprecipitant from the same number of cells as used in the ChIP assay was also subjected to Western blotting to confirm the results of immunoprecipitation (lower band). Each experiment was performed three times, and representative data are shown. NRS, normal rabbit serum; I.B., immunoblot.


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DISCUSSION
 
Telomerase is a key enzyme involved in the regulation of cellular senescence and immortality, but the biological mechanisms underlying the control of telomerase activity during cell differentiation are still not well understood. It has been proposed that telomerase activity is regulated by altering the transcription of hTERT, by post-transcriptional, alternative splicing of hTERT, by structural changes of the telomerase holoenzyme, and by changes in the localization of hTERT [14 , 28 29 30 ]. In fact, there may be multiple mechanisms of telomerase regulation during cell differentiation. In this study, HL60 cells were induced to differentiate into the granulocytic lineage with ATRA or Am80 and into the monocytic lineage with VD3. Am80 was used to determine whether there was a difference between RAR-dependent and RXR-dependent induction of telomerase. In both cases, telomerase activity declined within 24 h, and the expression of CD14 and CD11b mRNA was up-regulated after 6 h of incubation, and DNA synthesis was still ongoing. Telomerase activity is thought to vary during the cell cycle, and its activation has been reported to show linkage with the S-phase [31 , 32 ]. Considering that the time required for suppression of telomerase activity in our study was shorter than reported previously [33 , 34 ], early commitment to differentiation may influence the enzyme activity of telomerase and lead to its inactivation.

The finding that Akt kinase activity was suppressed after 6 h of differentiation along with inhibition of telomerase activity and the extent of the suppression that occurred while maintaining protein expression may suggest that there is post-translational regulation of telomerase activity. Some studies have indicated that Akt is required for post-translational modification of hTERT [23 , 35 ]. Short-term incubation of HL60 cells with the PI-3K inhibitor LY294002 suppressed telomerase activity, and pretreatment of cell lysates with recombinant Akt caused dose-dependent enhancement of telomerase activity. These results suggest that Akt may play an important role in the post-translational regulation of telomerase activity during the differentiation of HL60 cells [36 ]. Based on our data, it is difficult to determine whether the effect of Akt was direct or indirect. Taken together with the report that the catalytic subunits of telomerase have a specific amino acid sequence recognized by Akt [37 ], however, our results strongly suggest that Akt acts as a post-translational modulator of telomerase during differentiation of HL60 cells.

When HL60 cells were cultured in the presence of VD3, Am80, or ATRA for 7 days, our findings suggested that these differentiation inducers could potentiate cell survival. We therefore extended our investigation of the mechanisms underlying signaling for monocytic and granulocytic differentiation. We found that phosphorylation of Akt, which is associated with activation of its kinase activity, gradually increased upon exposure to any of the three differentiation inducers examined. The phosphorylation of Akt following induction of differentiation was associated with an increase of Akt kinase activity according to the in vitro kinase assay. Akt activates the transcription of a wide variety of genes, especially those involved in cell proliferation, apoptosis, and cell survival [38 39 40 ]. mTOR is one of the downstream signaling proteins for Akt that regulates cell proliferation and survival by altering ribosomal protein translation and initiation of cap-dependent translation [41 , 42 ]. Expression of mTOR was also increased, which could help to explain resistance to proapoptotic agents that accompanies the differentiation of leukemic cells, as has been demonstrated for several cell lines [43 44 45 46 47 48 ].

Differentiation of HL60 cells into either lineage was associated with an unexpected increase of p-p70S6K compared with the level of phosphorylation of mTOR. It is important to note that p70S6K may also be activated by a mTOR-insensitive signaling pathway. Recent studies have shown that other signaling pathways involving phosphoinositide-dependent protein kinase-1 and MAPK also regulate p70S6K and are likely to be involved in leukemic cell differentiation [49 , 50 ]. These observation could explain the discrepancy between the extent of phosphorylation of p-mTOR and p-p70S6K.

It has been reported that Akt promotes the transcription of hTERT [23 , 51 ]. The observed discrepancy between the increase of p-Akt and decrease of telomerase transcription in the late stage of differentiation suggests that epigenetic regulation of telomerase needs to be taken into consideration [52 53 54 ]. In the present study, a change of acetyl-histone H4 binding to the telomerase gene was observed before there was an increase in the phosphorylation of Akt, which regulates the transcription of telomerase. Removal of acetyl groups by histone deacetylases could lead to condensation of chromatin around the unmodified histones, resulting in the repression of telomerase gene transcription, which might mean that gene silencing occurs before Akt activation during differentiation. In conclusion, our results indicate that telomerase activity is regulated by at least two mechanisms during granulocytic and monocytic differentiation, with one mechanism being transcriptional (including epigenetic regulation) and the other mechanism being post-translational. We did not study primary leukemic cells, and the cell line we used (HL60) is not directly comparable with normal hematopoietic stem cells, but our results could be a first step toward understanding the regulation of telomerase during myeloid cell differentiation.


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ACKNOWLEDGEMENTS
 
This research was supported by the Program for Promoting the Establishment of Strategic Research Centers, Special Coordination Funds for Promoting Science and Technology by Ministry of Education, Culture, Sports, Science and Technology (Japan).

Received December 21, 2007; revised January 16, 2008; accepted January 17, 2008.


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