SEARCH

SEARCH BY CITATION

Keywords:

  • telomerase;
  • AML;
  • CD34+;
  • vitamin D3;
  • clinical response

Abstract

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

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)2D3, and vitamin D3 analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D3-sensitive HL-60 cells, whereas vitamin D3 insensitive K562 cells did not change its activity. This down-regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34 cells > leukaemic CD34+ cells > normal CD34 cells > normal CD34+ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S-phase, cytotoxicity to cytosine arabinoside and percentage of CD34+ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.

Cancer is thought to be caused by multiple mutations of proto-oncogenes or tumour suppressor genes which confer a growth advantage to the tumour cells ( Vogelstein & Kinzler, 1993). Another important feature of cancer cells is their characteristic unlimited proliferative capacity ( Harley et al, 1994 ).

Recent studies have shown that telomeres and telomerase might be important in the regulation of cell mortality ( Rhyu et al, 1995 ). Telomeres are the specialized structure at the end of eukaryotic chromosomes that are believed to protect the chromosomal ends from degradation and prevent them from illegitimate fusion and recombination ( Blackburn, 1991). In humans, telomeres consist of tandem repeats of the sequence of TTAGGG hexamers ( Blackburn, 1991; de Lange et al, 1990 ). Telomeric DNA is lost during each cell division due to the inability of the lagging strand of DNA to fully replicate the end of linear chromosomes, referred to as the end replication problem ( Watson, 1972; Levy et al, 1992 ). Telomerase is a ribonucleoprotein which synthesizes telomeric DNA onto chromosomal ends and therefore is able to compensate for the loss of terminal telomeric DNA ( Greider & Blackburn, 1985; Morin, 1989). In normal human somatic cells expressing low or undetectable telomerase activity, a progressive shortening of the telomeres is observed every time somatic cells divide both in vitro and in vivo due to the end replication problem, contributing to replicative cell senescence ( Wright & Shay, 1992; Allsopp et al, 1992 ). This telomeric shortening, in the absence of telomerase, acts as a mitotic clock in determining the remaining replicative capacity of a cell ( Shay et al, 1996 ). In contrast to somatic cells, germline cells maintain the length of telomere through an indefinite number of cell divisions by the expression of telomerase ( Counter et al, 1992 ; Mantell & Greider, 1994).

Transformed cells with viral oncogenes continue to lose telomeric DNA during cell divisions and eventually reach crisis ( Counter et al, 1994a ). At crisis, telomeres are critically shortened and genomic instability is marked, leading to cell death ( Harley et al, 1990 ). However, immortal cells escape this crisis and express telomerase activity with a stable telomeric length ( Counter et al, 1992 ). Therefore telomerase activation can play a critical role in cell immortalization ( Harley et al, 1994 ).

Telomerase activity in human tumour tissue was first demonstrated in metastatic ovarian carcinoma with short stabilized telomeres ( Counter et al, 1994b ). A variety of human tumours have shown short telomeres despite detectable telomerase activity ( Kim et al, 1994 ), suggesting that telomerase activation may contribute to late progression in tumourigenesis ( Counter et al, 1994b , 1995). Until recently, telomerase activity was detected in approximately 85–90% of human primary tumours ( Shay et al, 1996 ; de Lange, 1994).

Unlike other somatic tissues, peripheral blood (PB) and bone marrow (BM) leucocytes from normal donors expressed low levels of telomerase activity ( Counter et al, 1995 ). In addition, committed haemopoietic progenitor cells were telomerase positive, whereas primitive haemopoietic progenitor stem cells were telomerase negative ( Hiyama et al, 1995 ). Telomerase activation in haemopoietic malignancy, including acute myelogenous leukaemia (AML), has also been examined recently ( Nilsson et al, 1994 ; Counter et al, 1995 ; Broccoli et al, 1995 ; Zhang et al, 1996 ), showing that leukaemic blasts often revealed telomerase activity. However, very few investigations have been performed on telomerase activity in AML and the relationship of telomerase activity with clinical outcome. Also, telomerase activity in CD34+ cells from AML blasts has not been reported. Here, using a sensitive polymerase chain reaction (PCR)-based telomerase assay (TRAP assay), we investigated the expression of telomerase in AML cell lines, leukaemic blasts from de novo AML patients, and CD34+ cells isolated from normal bone marrow mononuclear (BMNC) cells and AML blasts. In addition, expression change of telomerase activity was accompanied with the induction of cyclin-dependent kinase inhibitor (CDKI), p21, following treatment of AML cells with differentiating agents, and telomerase activity was compared to clinical and biological parameters in AML patients.

MATERIALS AND METHODS

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

Patients

BM biopsy specimens from patients with de novo diagnosis of AML between December 1994 and April 1996 were analysed. Informed consent for all studies was obtained from each patient. The diagnosis of AML was based on the morphologic and cytochemical criteria of the French–American–British (FAB) Cooperative Working Group ( Bennett et al, 1985 ). Clinical information about these patients was obtained by chart review. There were 27 AML samples from three patients classified as M1, four as M2, two as M3, 11 as M4, three as M5, and four as mixed leukaemia. The mean age of the AML patients was 40.1 years, ranging from 17 years to 73 years; 17 AML patients were male and 10 were female. All the AML patients in this study were treated with daunorubicin and cytosine arabinoside (Ara-C) as induction chemotherapy. Complete remission (CR) status was determined 3 weeks after induction chemotherapy. When the BM cellularity was not normocellular, an additional 2 weeks were needed to evaluate CR. CR was defined as < 5% blast cells in a normocellular BM with PB counts in the normal range.

Cells and cell lines

AML cell lines were cultured in tissue flasks in RPMI 1640 medium (GIBCO-BRL, Gaithersburg, Md., U.S.A.) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, Utah, U.S.A.), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma Chemical Co., St Louis, Mo., U.S.A.). Aspirates of BM and PB were obtained after informed consent from bone marrow transplantation (BMT) donors and healthy volunteers, respectively. Before chemotherapy, leukaemic blasts were harvested from BM aspirates of consenting initially diagnosed AML patients or their PB when the blast counts were high (> 85%) at presentation. The mononuclear cells were isolated by centrifugation on Ficoll-Hypaque gradients (1.077 g/cm3), washed twice in phosphate-buffered saline (PBS), and suspended in RPMI 1640 containing 10% FBS.

Vitamin D3 compounds

The vitamin D3 compounds used in this experiment were EB1089, KH1060 and 1,25(OH)2D3 as a reference agent, and were synthesized by Leo Pharmaceutical Products, Ballerup, Denmark. Each was dissolved in absolute ethanol at 1 × 10−3m as a stock solution; these were stored at −20°C and protected from light. Dilutions of the stock solution were made in RPMI 1640 without FBS. The maximum concentration of ethanol in the culture (0.1%) did not influence clonal growth of cell lines.

Induction of differentiation

Differentiation of cell lines was assessed by the ability of the cells to express superoxide as measured by reduction of nitroblue tetrazolium and the diffuse brown staining after exposure to α-naphthyl acetate esterase ( Jung et al, 1994 ).

Western blotting

Cells were washed in PBS, suspended in lysis buffer containing 50 m m Tris (pH 7.5), 1% NP-40, 2 m m EDTA, 10 m m NaCl, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1 m m phenylmethylsulphonyl fluoride, and placed on ice for 30 min. After centrifugation at 15 000 g for 1 h at 4°C, the supernatant was collected. Whole lysate (100 μg) was resolved by 12% sodium dodecyl sulphate polyacrylamide gel, transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, Calif., U.S.A.) by electroblotting, and probed with anti-p21 and anti-p27 monoclonal antibody (Oncogene Science, Uniondale, N.Y., U.S.A.). The blot was developed by using the ECL kit (Amersham, Arlington Heights, Ill., U.S.A.).

Purification of CD34+cells

The purification of CD34+cells was performed from BMNC cells using an immunomagnetic procedure as described previously ( Lee et al, 1996 ). Briefly, nonadherent mononuclear cells were incubated with a mouse anti-human CD34 antibody, My-10 (Becton Dickinson, Mountain View, Calif., U.S.A.) for 30 min at 4°C. They were washed with PBS, and the sensitized cells were rosetted with sterile Dynal paramagnetic microspheres coated with sheep anti-mouse IgG1(Fc) antibody (SAM beads; Baxter-Fenwall, Santa Ana, Calif., U.S.A.), at 2 cells/bead, in the chamber under slow (~ 4 rpm) rotation for 30 min at 4°C. The cell concentration was maintained between 1 and 5 × 107/ml in RPMI 1640 containing 1% of human serum albumin (RPMI/HSA). Sensitized cells with attached beads were collected and washed three times with RPMI/HSA using the magnetic cell separator (ISOLEX-50; Baxter Health Corp., Deerfield, Ill., U.S.A.). After the final washing, beads were detached from CD34+ cells by incubation with chymopapain (Boots Company Inc., Lincolnshire, Ill., U.S.A.) in RPMI/HSA for 15 min at 37°C. Released CD34+ cells were collected by centrifugation, and the purity of CD34+ cells was determined by flow cytometric procedure using fluorescein isothiocyanate (FITC)-conjugated anti-HPCA2 (Beckton Dickinson) which recognizes a chymopapain-resistant epitope of the CD34 antigen. The purity of the cells isolated by this method was reproducibly > 90% as determined by flow cytometric analysis.

TRAP assay

Cells were washed in ice-cold wash buffer (10 m m HEPES-KOH, pH 7.5, 1.5 m m MgCl2, 10 m m KCl, 1 m m dithiothreitol), then lysed in 200 μl of ice-cold CHAPS lysis buffer (10 m m Tris-HCl, pH 7.5, 1 m m MgCl2, 1 m m EGTA, 0.1 m m phenylmethylsulphonyl fluoride, 5 m mβ-mercaptoethanol, 0.5% CHAPS, 10% glycerol), incubated on ice for 30 min and centrifuged at 12 000 g for 30 min at 4°C. The supernatant was stored at −80°C. The concentration of protein in each extract was measured using the BCA protein assay kit (Pierce Chemical Co., Rockford, Ill., U.S.A.). To determine the optimal protein amount for TRAP assay, telomerase positive extract was analysed by serial diution: 6 μg, 2 μg and 0.6 μg of protein. The TRAP assay was performed as previously described ( Kim et al, 1994 ). Briefly, assay tubes contained 0.1 μg of CX primer (5′-{CCCTTA}3CCCTAA-3′) at the bottom sequestered by a wax barrier (AmpliwaxTM; Perkin-Elmer, Foster City, Calif., U.S.A.). Protein extracts were assayed in 50 μl of reaction mixture containing 20 m m Tris-HCl (pH 8.3), 1.5 m m MgCl2, 63 m m KCl, 0.005% Tween-20, 1 m m EGTA, 50 μm dNTPs, 0.1 μg of TS primer (5′-AATCCGTCGAGCAGAGTT-3′), 1 μg of T4 gene 32 protein (USB, Cleveland, Ohio, U.S.A.), bovine serum albumin (0.1 mg/ml), [α-32P]deoxycytidine triphosphate, 2 units of Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany). After 30 min of incubation at room temperature for extension of oligonucleotide TS primer by telomerase, tubes were transferred to a thermal cycler (Perkin-Elmer 9600). The reaction mixture was heated at 95°C for 5 min for inactivation of telomerase and then subjected to 27 PCR cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 1.5 min. The PCR product was electrophoresed on a 10% non-denaturing acrylamide gel.

Quantitative analysis of telomerase activity

To quantitate the relative telomerase activity of leukaemic blasts in AML patients, the initial TRAP reaction was carried out using TRAP-ezeTM Telomerase Detection Kit (ONCOR, Gaithersburg, Md., U.S.A.) according to the manufacturer's instruction. The quantitative value of telomerase activity using this method was expressed as TPG (Total Product Generated) unit. The assay had a linear range of 1–300 TPG, which was equivalent to telomerase activity from approximately 30–10 000 control cells.

Cytotoxicity assay

In vitro drug cytotoxicity assay was performed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) dye colourimetric assay ( Campling et al, 1988 ). After adding 50 μl of serial diluted Ara-C to the test wells, 150 μl of cell suspension was plated in 96-well microtitre plates (Nunc, Denmark) at 2 × 105 cells per well. After exposure to the drug for 4 d, 50 μl of MTT (Sigma) solution (2 mg/ml in PBS) was added to each well and the plates were incubated for additional 4 h at 37°C. MTT solution in medium was aspirated off. To achieve solubilization of the formazan crystal formed in viable cells, 200 μl of dimethylsulphoxide (Sigma) was added to each well. The plates were shaken for 30 min at room temperature and the absorbance was read immediately at a wavelength of 540 nm on a scanning multiwell spectrophotometer (Titertek Multiscan MC; Flow Laboratories, U.S.A.). The IC50 value was defined as the drug concentration required to inhibit cell growth by 50%.

Cell cycle analysis

Cell cycle distribution was determined by staining DNA with propidium iodide (PI) (Sigma) as previously described ( Powell et al, 1990 ). To quantitate the amount of DNA per cell, 1 × 106 cells were incubated at 37°C for 1 h with 10 μm of bromodeoxyuridine (BrdU) (Sigma). Cells then were washed in PBS and fixed in 70% ethanol. After incubation of cells with 1 ml of 5 m HCl containing Triton X-100 (Fisher Scientific, Fair Lawn, N.J., U.S.A.) for 30 min at room temperature followed by two washes with PBS, cells were incubated with 20 μl of anti-BrdU for 30 min at room temperature. After two washes with PBS, cells were incubated with 1 μg of FITC-goat-anti-mouse IgG (CALTAC Lab, San Francisco, Calif., U.S.A.) for 30 min at room temperature. Cells were again washed with PBS, then incubated with 1 μg of PI. The percentage of cells in different phases of the cell cycle was measured with FACStar flow cytometer (Becton Dickinson, San Jose, Calif., U.S.A.), analysed using Becton Dickinson software (Lysis II, Cellfit).

Flow cytometric determination of leukaemic CD34+ cells

Flow cytometric evaluation for CD34 antigen expression was performed in leukaemic blasts from AML patients. Cells (5 × 105) were incubated for 30 min at 4°C with FITC-conjugated anti-CD34, FITC-conjugated anti-CD45 monoclonal antibody and phycoerythrin-conjugated mouse IgG1 antibody (Becton Dickinson) as an isotype control. After two washes with cold PBS, cells were resuspended in PBS containing 2% paraformaldehyde. Flow cytometry was performed on FACStar flow cytometer using Lysis II software.

Statistical analysis

For statistical analysis, telomerase activity levels in AML samples were divided into CR positive and negative. Comparison of CR status with the levels of telomerase activity was evaluated by Wilcoxon rank sum test. The correlation between telomerase activity and biological parameters was determined by linear regression.

RESULTS

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

Telomerase activity in normal BMNC cells and AML cell lines

Five normal BMNC cells isolated from BMT donors were assayed for telomerase activity using TRAP assay. To achieve optimal telomerase activity, TRAP assay was initially carried out with three concentrations at 6 μg, 2 μg and 0.6 μg of protein, respectively; telomerase activity was the most evident at 0.6 μg (data not shown). Therefore TRAP assay was performed using 0.6 μg of protein in all of the experiments. All normal individuals had weak, but detectable 6 base pair DNA ladders which were interfered with by pretreatment of the extracts with RNase as well as heat inactivation (data not shown), showing that these signals were telomerase-specific. Assaying of extracts from nine AML cell lines (myeloblastic for KCL22 and ML1, promyelocytic for HL-60 and NB4, erythroid for HEL and TF1, myelomonocytic for U937, megakaryoblastic for MEGO1, and myelogenous for KG1) and chronic myelogenous leukaemic K562 cells revealed high levels of telomerase activity in all cell lines compared to normal BMNC cells, although different levels of activity were noted among cell lines (Fig 1).

image

Figure 1. Fig 1. Telomerase activity in myeloid leukaemic cell lines. Telomerase assay was carried out using extracts containing 0.6 μg of protein from myeloid leukaemic cell lines with (+) or without (−) heat inactivation by TRAP assay, as described in Materials and Methods.

Download figure to PowerPoint

Effect of vitamin D3 analogues on telomerase activity

Effect of differentiating agents on telomerase activity was evaluated in HL-60 and K562 cell lines which was responsive and not responsive to 1,25(OH)2D3, respectively. Treatment of 1,25(OH)2D3 and two different vitamin D3 analogues, EB1089 and KH1060, for 3 d induced differentiation of HL-60 cells in a dose-dependent fashion. However, treatment of K562 cells with these analogues did not reveal any evidence of differentiation induction (data not shown). Telomerase activity by treatment with 10−8m of each of vitamin D3 compounds for 3 d was markedly downregulated in vitamin D3-sensitive HL-60 cells, whereas HL-60 cells cultured in the absence of vitamin D3 showed significant telomerase activity (Fig 2A). In contrast, treatment of vitamin D3 insensitive K562 cells with 10−8m of 1,25(OH)2D3, EB1089 and KH1060, for 3 d did not result in a change of telomerase activity compared to control (Fig 2A). To observe the association of change in telomerase activity by vitamin D3 compound with the induction of cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 protein, HL-60 cells were treated with 10−8m of EB1089 for 3 d. The p21 protein was induced 24 h after treatment with EB1089 and continued to be expressed until 72 h (Fig 2B), whereas the p27 protein level was unchanged at all time points (data not shown).

image

Figure 2. 1 monoclonal antibody.

Download figure to PowerPoint

Telomerase activity in CD34+ cells from normal BMNC cells and AML cells

Table 1. Table I. Telomerase activity and clinicobiological parameters in AML patients. CR: complete remission; ND: not determined.* The percentage of CD34+ cell population in leukaemic blasts.† Total Product Generated unit. 1–300 TPG is equivalent to telomerase activity from approximately 30–10 000 control cells.‡ The percentage of cell population at S-phase in cell cycle.§ Drug concentration required to inhibit cell growth by 50%. Thumbnail image of
image

Figure 3. 4+ cells.

Download figure to PowerPoint

Telomerase activity in AML patients

Telomerase activity in the leukaemic cells of 27 patients with AML was examined. To quantitate the relative telomerase activity, we used the TRAPezeTM Telomerase Detection Kit. Using this method, incorporation of 36 base pair internal positive control makes it possible to quantitate telomerase activity more accurately and to identify false negative samples that contain Taq polymerase inhibitor. The relative level of telomerase activity was expressed by TPG (Total Product Generated) unit. The assay had a linear range of 1–300 TPG, which was equivalent to telomerase activity from approximately 30–10 000 control cells, indicating that higher TPG showed higher telomerase activity. Fig 4 shows representative results from six AML patients. Telomerase activity was detected in 27/27 (100%) AML patients ( Table I and Fig 4). However, heterogenous levels of telomerase activity were observed between patients. Based on these experimental results, we then compared telomerase activity with clinical response to induction chemotherapy. 25 samples out of 27 AML patients were evaluable, as two patients refused induction chemotherapy. Unexpectedly, AML patients with higher telomerase activity showed a higher rate of CR (P < 0.001) ( Table I and Fig 5). However, there was no relationship between the levels of telomerase activity and patient's age (data not shown).

image

Figure 4. Fig 4. Heterogeneity of telomerase activity in AML patients. AML blasts were isolated by Ficoll-Hypaque gradient. Protein extracts from AML blasts were examined for telomerase activity using TRAPezeTM Telomerase Detection Kit with (+) or without (−) heat inactivation. The representative AML patients with CR following chemotherapy (+) and without CR following chemotherapy (−) are indicated by their telomerase activity. Internal standard (36 bp) was included in this assay for semiquantitation of telomerase activity.

Download figure to PowerPoint

image

Figure 5. Fig 5. Correlation of telomerase activity with clinical response to induction chemotherapy. Telomerase activity was expressed as TPG unit using TRAPezeTM Telomerase Detection Kit. CR(+): AML patients with complete remission. CR(−): AML patients without complete remission. Comparison of CR status with telomerase activity was evaluated by Wilcoxon rank sum test. P < 0.001.

Download figure to PowerPoint

Relationship between telomerase activity and biological parameters in AML

To determine whether telomerase activity was associated with biological parameters in AML blast cells, we investigated the relationship between telomerase activity and biological parameters such as percentage of cell population at S-phase, cytotoxicity against Ara-C, and percentage of CD34+cell population in leukaemic blasts from AML patients. As shown Fig 6, the levels of telomerase activity of AML patients did not demonstrate any differences in distribution of their biological parameters. In addition, no correlation was seen among these biological parameters and CR rate of AML patients (data not shown).

image

Figure 6. Fig 6. Correlation of telomerase activity with biological parameters in AML patients. Telomerase activity was expressed as TPG units using TRAPezeTM Telomerase Detection Kit. Correlation of telomerase activity with percentage of cell population at S-phase (A), with percentage of CD34+ cell population in leukaemic blasts (B), and with cytotoxicity against Ara-C (C). No correlation was observed between telomerase activity and these biological parameters. IC50: drug concentration to inhibit cell growth by 50%.

Download figure to PowerPoint

DISCUSSION

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

Most human primary tumours express telomerase activity, whereas most normal somatic cells lack this activity, suggesting that telomerase may be a new marker of malignant tumours ( Shay et al, 1996 ). However, unlike other somatic tissues, PB, cord blood and BM leucocytes including T and B cells from normal donors were found to express low levels of telomerase activity ( Counter et al, 1995 ; Hiyama et al, 1995 ). In the present study we also confirmed this finding, showing that normal BMNC cells expressed very low telomerase activity. Among AML cell lines, HL-60, U937 and HEL cells were reported to contain telomerase activity ( Kim et al, 1994 ). We extended the assay of telomerase activity to additional AML cell lines. All AML cell lines examined demonstrated higher levels of telomerase activity, suggesting that expression of telomerase is common in AML cell lines.

A large body of knowledge indicates that telomerase activity is inhibited during the terminal differentiation of human tumour cells ( Sharma et al, 1995 ; Albanell et al, 1996 ; Bestilny et al, 1996 Savoysky et al, 1996 ; Zhang et al, 1996 ). Biologically active vitamin D3 compound, 1,25(OH)2D3, is able to induce human promyelocyte leukaemia cell line HL-60 cells to differentiate into macrophage-like cells in vitro ( Koeffler, 1983). We also investigated changes in telomerase activity following treatment of HL-60 cells with 1,25(OH)2D3 and two different analogues, EB1089 and KH1060, for 3 d. These differentiating agents inhibited telomerase activity in treated HL-60 cells compared to untreated HL-60 cells, whereas the level of telomerase activity was not changed in vitamin D3 insensitive K562 cells with these vitamin D3 compounds. Down-regulation of telomerase activity in treated HL-60 cells might be simply the consequence of the differentiated status because macrophage/monocyte revealed very weak telomerase activity compared to HL-60 cells. However, it has been reported that inhibition of telomerase activity is an early event of the differentiation process in HL-60 cells and NB4 cells rather than its consequence ( Albanell et al, 1996 ; Savoysky et al, 1996 ), suggesting that telomerase may be a regulated enzyme during induction of differentiation in human tumour cells. Therefore it is likely that phenotypic differentiation of HL-60 cells is linked to the regulation of telomerase activity.

Several groups have demonstrated that expression of CDKI, p21, is up-regulated during the differentiation of a number of cell types in vitro ( Jiang et al, 1994 ; Steinman et al, 1994 ; Macleod et al, 1995 ; Zhang et al; 1995 ). We found that p21 protein was induced within at least 24 h after treatment of HL-60 cells with 10−8m of vitamin D3 analogue, EB1089, for 3 d and continued to be expressed at 48 and 72 h. Although we did not examine time-dependent inhibition of telomerase activity with EB1089, this observation is consistent with data showing that 1,25(OH)2D3 and all-trans retinoic acid induced an early increase of p21 mRNA within a few hours, immediately followed by a time-dependent inhibition of telomerase activity ( Savoysky et al, 1996 ). Thus, telomerase inhibition during differentiation of HL-60 cells might be associated with induction of p21 protein.

In contrast to most of the somatic cells lacking telomerase activity, a low level of activity was found in human haemopoietic stem cells and their progenitors ( Counter et al, 1995 ; Broccoli et al, 1995 ). It has been reported that low levels of telomerase activity were detected in the primitive cells (CD34+CD71loCD45RAlo) and the more mature cells (CD34), whereas high activity was present in the early progenitors (CD34+CD71+) ( Chiu et al, 1996 ). Hiyama et al (1995 ) found that telomerase activity in committed progenitor cells (CD34+CD38+) was positive, but not in primitive progenitor cells (CD34+CD38−/lo). Although haemopoietic cells were not subfractionated in this current study, we exhibited that telomerase activity of CD34+ cells isolated from normal BMNC cells was negligible and lower than that of CD34 cells. Telomerase activity of CD34 cells was weak, and almost the same as the basal level of normal BMNC cells. Because CD34+fraction comprise approximately 1% of the BMNC cells and its telomerase activity was negligible, the telomerase activity of normal BMNC cells might be largely reflected by CD34 cells. In a separate study, no telomerase activity was detected in myeloid progenitor cells (CD34+CD33) and mature myeloid cells (CD34CD33+CD13+) isolated from normal BMNC cells ( Zhang et al, 1996 ). Taken together, it is likely that telomerase activity is down-regulated upon proliferation and differentiation. Although CD34 antigen is generally not detectably expressed in solid tumours, leukaemic blasts of AML express CD34 antigen ( Krause et al, 1996 ). However, no data has been available about telomerase activity in leukaemic CD34+ blasts. In this regard, we investigated telomerase activity of leukaemic CD34+ cells isolated from AML patient. CD34+ and CD34 leukaemic blasts had higher telomerase activity than their normal counterparts, and telomerase activity of CD34 leukaemic blasts was higher compared to CD34+ leukaemic blasts. Considering that AML blasts have more proliferative capacity than normal haemopoietic cells, it is conceivable that the level of telomerase activity responds to the proliferative activity of the cell population.

A few investigators have studied telomerase activity in AML patients. Counter et al (1995 ) and Broccoli et al (1995 ) have detected telomerase activity in 7/7 AML and in 2/2 de novo AML samples, respectively. This small population of AML samples encouraged us to pursue our investigation on telomerase activity using an extended number of patients with AML. We detected telomerase activity in 27/27 (100%) AML patients. In contrast, Nilsson et al (1994 ) reported no telomerase activity in four AML patients. However, it is unlikely that telomerase activity was actually not expressed in those AML patients, because an amplification step was not included in their assay process. Very recently, a study of telomerase activity utilizing a large number of AML samples has been performed, which showed that there was detectable telomerase activity in 41 (73%) of 56 AML patients ( Zhang et al, 1996 ). Taken together, it appears that telomerase activity is expressed in most human AML blasts, as it is in most human solid tumours. Although, in our series, detectable telomerase activity was observed in all AML examined, the level of activity was very heterogenous among the patients. Therefore we compared the level of telomerase activity with clinical response to induction chemotherapy. Interestingly, the CR rate of AML patients with higher telomerase activity was higher than those patients with lower telomerase activity (P < 0.001). We are unable to explain why the higher telomerase activity was associated with the higher CR rate. To the contrary, Zhang et al (1996 ) reported that very high telomerase activity was often associated with resistance to therapy, although the differences were not statistically significant. This different result might be caused by different methodology in the evaluation of the quantitation of telomerase activity. In addition, they defined the relative telomerase activity arbitrarily to estimate the relationship of telomerase activity with clinical response to therapy. However, we evaluated a small heterogenous group of AML patients; further examination utilizing a significant number of patients with homogenous AML subtypes is necessary to determine whether telomerase activity is associated with clinical response in AML.

Zhu et al (1996 ) reported that the highest telomerase activity was detected in S-phase cells. However, we found that no association between telomerase activity and percentage of cell population at S-phase in leukaemic blasts was observed in each AML patient. It is generally believed that telomerase activity does not vary throughout all phases of the cell cycle ( Mantell & Greider, 1994; Holt et al, 1996 ; Weng et al, 1996 ). In addition, our result showed that the cytotoxic effect of S-phase-specific agent, Ara-C, on leukaemic blasts, and percentage of CD34+ leukaemic cell population were not correlated with telomerase activity. However, further evaluation will be required on the relationship between telomerase activity and these biological parameters in AML because of the limited number of AML patients examined in this study.

In the present study we demonstrated that telomerase activity was inhibited during the differentiation of HL-60 cells, which might be associated with induction of p21 protein. High frequency of telomerase activation in AML patients indicated that telomerase is likely to play an important role in leukaemogenesis. Although no obvious association between telomerase activity and biological parameters including percentage of S-phase, cytotoxicity to Ara-C and percentage of CD34+ cell population was observed, its activation might be associated with clinical response. However, further examination will be needed to investigate whether telomerase activity is involved in the evolution of AML, and if it is associated with clinicobiological parameters, because our sample was small in size and heterogenous.

Acknowledgements

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

This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Cancer Research Centre at Seoul National University, Seoul, Korea.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
  • 1
    Albanell, J., Han, W., Mellado, B., Gunawardane, R., Scher, H.I., Dmitrovsky, E., Moore, M.A.S. (1996) Telomerase activity is repressed during differentiation of maturation-sensitive but not resistant human tumor cell lines. Cancer Research, 56, 1503 1508.
  • 2
    Allsopp, R.C., Varizi, H., Patterson, C., Goldstein, S., Younglai, E.V., Futcher, B.A., Greider, C.W., Harley, C.B. (1992) Telomere length predicts replicative capacity of human fibroblasts. Proceedings of the National Academy of Sciences of the United States of America, 89, 10114 10118.
  • 3
    Bennett, J.M., Catovsky, D., Daniel, M.T., Flandrin, G., Galton, D.A.G., Gralnick, H.R., Sultan, C. (1985) Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French–American–British (FAB) Cooperative Group. Annals of Internal Medicine, 33, 451 458.
  • 4
    Bestilny, L.J., Brown, C.B., Miura, Y., Robertson, L.D., Riabowol, K.T. (1996) Selective inhibition of telomerase activity during terminal differentiation of immortal cell lines. Cancer Research, 56, 3796 3802.
  • 5
    Blackburn, E.H. (1991) Structure and function of telomeres. Nature, 350, 569 573.
  • 6
    Broccoli, D., Young, J.W., De Lange, T. (1995) Telomerase activity in normal and malignant hematopoietic cells. Proceedings of the National Academy of Sciences of the United States of America, 92, 9082 9086.
  • 7
    Campling, B.G., Pym, J., Galbraith, P.R., Cole, S.P.C. (1988) Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells. Leukemia Research, 12, 823 831.
  • 8
    Chui, 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 progenitors from adult human bone marrow. Stem Cells, 14, 239 248.
  • 9
    Counter, C.M., Avilion, A.A., LeFeuvre, C.E., Stewart, N.G., Greider, C.W., Harley, C.B., Bacchetti, S. (1992) Telomere shortening associated with chromosome instability in immortal cells which express telomerase activity. EMBO Journal, 11, 1921 1929.
  • 10
    Counter, C.M., Botelho, F., Harley, C.B., Bacchetti, S. (1994 a) Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes. Journal of Virology, 68, 3410 3414.
  • 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, 2315 2320.
  • 12
    Counter, C.M., Hirte, H.W., Bacchetti, S., Harley, C.B. (1994 b) Telomerase activity in human ovarian carcinoma. Proceedings of the National Academy of Sciences of the United States of America, 91, 2900 2904.
  • 13
    De Lange, T. (1994) Activation of telomerase in a human tumor. Proceedings of the National Academy of Sciences of the United States of America, 91, 2882 2885.
  • 14
    De Lange, T., Shiue, L., Myers, R.M., Cox, D.R., Naylor, S.L., Killery, A.M., Varmus, H.E. (1990) Structure and variability of human chromosome ends. Molecular and Cellular Biology, 10, 518 527.
  • 15
    Greider, C.W. & Blackburn, E.H. (1985) Identification of a specific telomere terminal extrachromosomal RNA genes in Tetrahymena extracts. Cell, 43, 405 413.
  • 16
    Harley, C.B., Futcher, A.B., Greider, C.W. (1990) Telomeres shorten during aging of human fibroblasts. Nature, 345, 458 460.
  • 17
    Harley, C.B., Kim, N.W., Prowse, K.R., Weinrich, S.L., Hirsch, K.S., West, M.D., Bacchetti, S., Hirte, H.W., Counter, C.M., Greider, C.W., Piatyszek, M.A., Wright, W.E., Shay, J.W. (1994) Telomerase, cell immortality, and cancer. Cold Spring Harbor Symposia on Quantitative Biology, 59, 307 315.
  • 18
    Hiyama, K., Hirai, 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 hematopoietic progenitor cells. Journal of Immunology, 155, 3711 3715.
  • 19
    Holt, S.E., Wright, W.E., Shay, J.W. (1996) Regulation of telomerase activity in immortal cell lines. Molecular and Cellular Biology, 16, 2932 2939.
  • 20
    Jiang, H., Lin, J., Su, Z.Z., Collart, F.R., Huberman, E., Fisher, P.B. (1994) Induction of differentiation in human promyelocytic HL-60 cells activates p21, WAF1/CIP1, expression in the absence of p53. Oncogene, 9, 3397 3406.
  • 21
    Jung, S.J., Lee, Y.Y., Pakkala, S., De Vos, S., Elstner, E., Norman, A.W., Green, J., Uskokovic, M., Koeffler, H.P. (1994) 1,25(OH)2-16ene-vitamin D3 is a potent antileukemic agent with low potential to cause hypercalcemia. Leukemia Research, 18, 453 463.
  • 22
    Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L.C., Coviello, G.M., Wright, W.E., Weinrich, S.L., Shay, J.W. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science, 266, 2011 2015.
  • 23
    Koeffler, H.P. (1983) Induction of differentiation of human acute myelogenous leukemia cells: therapeutic implications. Blood, 62, 709 721.
  • 24
    Krause, D.S., Fackler, M.J., Civin, C.I., May, W.S. (1996) CD34: structure, biology, and clinical utility. Blood, 87, 1 13.
  • 25
    Lee, Y.Y., Kim, E.S., Seol, J.G., Kim, B.K., Binderup, L., Elstner, E., Park, D.J., Koeffler, H.P. (1996) Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts. Leukemia, 10, 1751 1757.
  • 26
    Levy, M.Z., Allsopp, R.C., Futcher, A.B., Greider, C.W., Harley, C.B. (1992) Telomere end-replication problem and cell aging. Journal of Molecular Biology, 225, 951 960.
  • 27
    Macleod, K.F., Sherry, N., Hannon, G., Beach, G., Tokino, T., Kinzler, K.W., Vogelstein, B., Jacks, T. (1995) p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes and Development, 9, 935 944.
  • 28
    Mantell, L.L. & Greider, C.W. (1994) Telomerase activity in germline and embryonic cells of Xenopus. EMBO Journal, 13, 3211 3217.
  • 29
    Morin, G.B. (1989) The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesies TTAGGG repeats. Cell, 59, 521 529.
  • 30
    Nilsson, P., Mehle, C., Remes, K., Roos, G. (1994) Telomerase activity in vivo in human malignant hematopoietic cells. Oncogene, 9, 3043 3048.
  • 31
    Powell, B.L., Gregory, B.W., Kute, T.E., Morgan, T.M., Lyerly, E.S., Capizzi, R.L. (1990) Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent. Cytometry, 11, 438 441.
  • 32
    Rhyu, M.S. (1995) Telomeres, telomerase, and immortality. Journal of the National Cancer Institute, 87, 884 894.
  • 33
    Savoysky, E., Yoshida, K., Ohtomo, T., Yamaguchi, Y., Akamatsu, K.I., Yamazaki, T., Yoshida, S., Tsuchiya, M. (1996) Down-regulation of telomerase activity is an early event in the differentiation of HL60 cells. Biochemical and Biophysical Research Communications, 226, 329 334.
  • 34
    Sharma, H.W., Sokoloski, J.A., Perez, J.R., Maltese, J.Y., Sartorelli, A.C., Stein, C.A., Nichols, G., Khaled, Z., Telang, N.T., Narayanan, R. (1995) Differentiation of immortal cells inhibits telomerase activity. Proceedings of the National Academy of Sciences of the United States of America, 92, 12343 12346.
  • 35
    Shay, J.W., Werbin, H., Wright, W.E. (1996) Telomeres and telomerase in human leukemias. Leukemia, 10, 1255 1261.
  • 36
    Steinman, R.A., Hoffman, B., Iro, A., Guillouf, C., Liebermann, D.A., El-Houseini, M.E. (1994) Induction of p21 (WAF1/CIP1) during differentiation. Oncogene, 9, 3389 3396.
  • 37
    Vogelstein, B. & Kinzler, K. (1993) The multistep nature of cancer. Trends in Genetics, 9, 138 141.
  • 38
    Watson, J.D. (1972) Origin of concatemeric T7 DNA. Nature New Biology, 239, 197 201.
  • 39
    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, 2471 2479.
  • 40
    Wright, E.W. & Shay, J.W. (1992) Telomere positional effects and the regulation of cellular senescence. Trends in Genetics, 8, 193 197.
  • 41
    Zhang, W., Grasso, L., McCain, C.D., Gambel, A.M., Cha, Y., Travali, S., Deisseroth, A.B., Mercer, W.E. (1995) p53-independent induction of WAF1/Cip1 in human leukemia cells is correlated with growth arrest accompanying monocyte/macrophage differentiation. Cancer Research, 55, 668 674.
  • 42
    Zhang, W., Piatyszek, M.A., Kobayashi, T., Estey, E., Andreeff, M., Deisseroth, A.B., Wright, W.E., Shay, J.W. (1996) Telomerase activity in human acute myelogenous leukemia inhibition of telomerase activity by differentiation-inducing agents. Clinical Cancer Research, 2, 799 803.
  • 43
    Zhu, X., Kumar, R., Mandal, M., Sharma, N., Sharma, H.W., Dhingra, U., Sokoloski, J.A., Hsiao, R., Narayanan, R. (1996) Cell cycle-dependent modulation of telomerase activity in tumor cells. Proceedings of the National Academy of Sciences of the United States of America, 93, 6091 6095.