Leukemia (2000) 14, 21762181 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.

com/leu

BIO-TECHNICAL METHODS SECTION (BTS)

BTS
Leukemia

Detection of hTERT protein by ow cytometry

ASM Ali1, R Chopra1,2, J Robertson1 and NG Testa1
Departments of 1Experimental Haematology and 2Medical Oncology, Paterson Institute for Cancer Research, Manchester, UK

Telomerase is a telomere-specic DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and are unable to isolate specic cell sub-populations. This report describes the development and validation of a cellular, immunouorescence-based ow cytometry assay that allows detection of intranuclear hTERT while maintaining identiable cell population characteristics. The assay was shown to be both sensitive to changes in telomerase expression and was semi-quantitative. In both cell line differentiation experiments and in primary cells, a good correlation existed between hTERT expression measured by ow cytometry and telomerase activity detected by the telomeric repeat amplication protocol (TRAP). The method developed offers a quick, simple and reproducible cellular-based assay for hTERT expression. This assay will provide a useful, new tool for future investigations, facilitating the analysis of hTERT expression in mixed cell populations. Leukemia (2000) 14, 21762181. Keywords: ow cytometry; hTERT protein; immunouorescence; telomerase

Introduction Eukaryotic chromosomes terminate with specialised structures known as telomeres.1 These consist of tandemly repeated DNA sequences that protect the ends of chromosomes and are important in maintaining their stability.2 Because DNA polymerases cannot completely replicate a linear DNA molecule, telomeric DNA at the end of a chromosome may be lost with each cell division.3 Critical shortening of telomeres is associated with cellular senescence.4 Telomerase is a unique cellular reverse transcriptase involved in telomere maintenance.5,6 Its activity is detectable in the majority of cancers7 as well as germline tissues8 and proliferating somatic cells.9 In cancer, telomerase activation is proposed as one mechanism by which cells can bypass senescence. Structurally, telomerase consists of protein and RNA components. One protein component, hTERT, is the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity.10 Current methods in use for

detection of telomerase activity and hTERT expression are primarily molecular-based techniques. These are well established and sensitive methods for the measurement of mean telomerase activity in a whole population of cells. Such methods cannot, however, measure telomerase activity in cell sub-populations. A telomerase/hTERT negative sub-population of cells will therefore not be identied within a large population of positive cells. Conversely, because the TRAP assay is highly sensitive, the activity in a small highly positive population mixed with a telomerase negative population may be interpreted as the entire population exhibiting a medium to low degree of expression. A further disadvantage of existing methods is that cell lysis is necessary for assay of telomerase or hTERT mRNA, and therefore, it is no longer possible to carry out any further or concurrent analysis on the same set of cells. In human leukaemias, telomerase upregulation has been shown to occur during disease progression.11,12 It has still to be claried whether telomerase activity in the development of cancer follows a pattern of reactivation where telomerase is reactivated in previously telomerase negative cells as a result of critical telomere shortening; or if it follows a pattern of expansion and retention where cells with active telomerase are there from the outset and undergo positive selection during the progression of the disease.13,14 However, because current methods cannot isolate cell populations it has not been possible to investigate this satisfactorily. To facilitate such investigations, a method that can measure hTERT in subpopulations is necessary. We have developed such a method for the detection of intranuclear hTERT. We have validated the measurements in both human HL60 cells undergoing dynamic changes in telomerase during differentiation with dimethyl sulfoxide (DMSO), as well as in primary human haemopoietic cells.

Materials and methods

HL60 cell culture and induced differentiation

HL60 cells, derived from a human promyelocytic leukaemia cell line, were cultured in T75 culture asks (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) in 25 ml of L-glutaminefree RPMI-1640 medium (GIBCO Life Technologies, Paisley, UK) containing 10% fetal calf serum and 2% L-glutamine and streptomycin and benzylpenicillin. Flasks were incubated in a 37C environment with 5% CO2.

Correspondence: R Chopra, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 4BX, UK; Fax: (0161) 446 3940 Received 13 March 2000; accepted 23 August 2000

Bio-technical methods section (BTS) ASM Ali et al

HL60 cells were induced to differentiate with DMSO (1.25% v/v) using standard methods.15 This allowed validation of the sensitivity of the assay because it has been shown by previous authors that telomerase activity declines with induced differentiation.16,17 DMSO differentiation of HL60 cells was undertaken for 6 days and was conrmed by morphological analysis of cytospin preparations. After this period, differentiated cells were dened as those which were approaching the metamyelocyte stage: cells that had lost their nucleolus, showed a decreased nuclear:cytoplasmic ratio of approximately 1:1 with evidence of nuclear lobulation, less basophilia and fewer cytoplasmic granules compared to the promyelocytes.

Mononuclear and neutrophil cell separation

All fresh samples were collected in vacutainers containing EDTA. The blood was diluted 1:1 with phosphate-buffered saline (PBS) and the mononuclear cells (MNC) and neutrophils were isolated from all samples by density gradient centrifugation on FicollHypaque (Lymphoprep, 1.077 g/ml, Nycomed Pharma, Oslo, Norway). Briey, the mononuclear cells were collected and washed with PBS containing 1% fetal calf serum. The red blood cells within the sample of neutrophils were removed by the addition of red cell lysis solution (Ortho Diagnostic Systems, Raritan, NJ, USA) for 15 min. The cells were then centrifuged at 400 g for 5 min and re-suspended in PBS (80% purity).

10 mmol/l Tris-HCl (pH 7.5), 1 mmol/l MgCl2, 1 mmol/l EGTA, 0.1 mmol/l Benzamidine, 5 mmol/l -mercaptoethanol, and 1 ng/ml leupeptine. The TRAP assay was performed as described previously by Kim et al7 with the use of the TRAPeze kit (Oncor, Gaithersburg, MD, USA). Briey this was done as follows: 2 l of test extract was added to a 48 l reaction mixture containing 5 l of 10 TRAP buffer (200 mmol/l Tris-HCl (pH 8.3), 15 mmol/l MgCl2, 630 mmol/l KCl, 0.5% Tween 20, 10 mmol/l EGTA); 1 l of 50 dNTP mix (2.5 mmol/l each of dATP, dTTp, dGTP and dCTP); 2 l of 32P-labelled TS primer, 1 l of TRAP primer mix, 0.4 l of Taq polymerase and 38.6 l of PCR grade water at 30C for 30 min. The products were amplied by 30 cycles at 94C for 30 s and 48C for 30 s each and were separated by electrophoresis on a 12.5% polyacrylamide gel. A manufacturer-supplied positive control, negative control (lysis buffer) and quantication template (TSR8) were also run on each gel. The gel was dried for 1 h at 80C and then exposed on a Molecular Dynamics Storm 860 Phosphorimager (Molecular Dynamics, Sunnyville, CA, USA) and quantied using Image QuaNT software according to the manufacturers instructions provided with the TRAPeze kit.

2177

Immunouorescent staining for CD 11b

Staining of the CD11b cell differentiation marker was carried out according to the manufacturers guide lines as follows: cultured cells were suspended in cold wash buffer (PBS supplemented with 1% FCS and 1% human AB serum to block non-specic Fc-mediated binding) at a concentration of 2 107/ml. Fifty microliter aliquots of cell suspension (1 106 cells) were then placed in 5 ml ow cytometry tubes and to this 50 l of diluted antibody was added (either 30455X phycoerythrin-conjugated mouse anti-human CD11b antibody or 33815X Mouse IgG1, isotype control (both from BD PharMingen, San Diego, CA, USA)). Antibody was left to incubate for 30 min and the cells were then re-suspended and centrifuged at 400 g in 1 ml of wash buffer twice. The nal suspension was then re-suspended in 200 l of wash buffer for ow cytometry analysis.

T cell separation
To isolate T cells, magnetic activated cell sorting was carried out by with the use of a midi-MACS isolation kit and CD3 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturers guidelines. Enriched samples of CD3+ T cells were obtained with 8085% purity.

TRAP assay
For telomerase activity, cells were stored in CHAPS lysis buffer (1 105 cells in 200 l) at 70C for analysis by the TRAP assay. The lysis buffer contained 0.5% CHAPS (3-[(3cholamidopropyl) dimethylammonio]-1-propane-sulfonate),

Figure 1 Flow cytometry detection of hTERT expression in HL60 cells. A forward and side scatter density plot of HL60 cells in suspension showing the gated region is shown in (a). (b) shows the optimal result obtained with 2% paraformaldehyde xation and Triton-X100 permeabilisation followed by gating. The control antibody is represented by the shaded histogram and hTERT by the open histogram. Mean cell uorescence was calculated by subtracting the geometric mean uorescence of the sample with added control antibody from the geometric mean uorescence of the sample with hTERT antibody added.
Leukemia

Bio-technical methods section (BTS) ASM Ali et al

2178

Figure 2 Detection of changes in hTERT protein expression (open histograms) in HL60cells by the developed assay. (a) shows the gradual reduction of hTERT expression during the rst 3 days of DMSO-induced differentiation followed by a rapid decline on day 4 and thereafter remaining low. The control cells, which remained undifferentiated, showed positive hTERT expression throughout the 6 day period as shown in (b).

Results

Optimal staining method

Optimal staining was achieved by the following protocol: 5 106 HL60 cells were taken from a ask, centrifuged at 200 g for 10 min, and re-suspended for washing in 10 ml of wash buffer (PBS supplemented with 1% fetal calf serum). The cells were again centrifuged at 200 g for 10 min. The cells were then xed in 1 ml of 2% paraformaldehyde and incubated on ice for 30 min. The cells were then washed again in 10 ml of wash buffer and re-suspended in 3ml of 0.5% TritonX100. After permeabilisation, cells were washed once more in 10 ml of wash buffer and re-suspended in wash buffer at a concentration of 2 107 per ml. Aliquots of the cell suspension (50 l) were put into 5 ml ow cytometry tubes and to these 50 l of diluted antibody was added (either primary SCLeukemia

7214 goat anti-hTERT antibody, primary SC-6370 goat antiTP1 antibody (TP1 is another protein component of telomerase) or SC-2028 normal IgG control antibody (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA)) and left to incubate for 40 min at room temperature. A manufacturers blocking peptide was also used as an additional control in some experiments. The suspension was then washed twice by addition of 1 ml of wash buffer and centrifuging at 800 g and nally re-suspended in 100 l of wash buffer. A secondary diluted SC-2024 donkey anti-goat IgG uorescein isothiocyanate (FITC)-conjugated antibody (Santa Cruz Biotechnology) was added and left to incubate at room temperature for 30 min. The suspension was then washed twice again (in the same way as after the addition of primary antibody). The sample was then re-suspended in 500 l of wash buffer and taken for analysis on the ow cytometer (FACS Vantage/FACScan, Becton Dickinson, San Jose, CA, USA).

Bio-technical methods section (BTS) ASM Ali et al

Gating was carried out as shown in Figure 1a. Mean cell uorescence was calculated by subtracting the geometric mean uorescence of the sample with added control antibody from the geometric mean uorescence of the sample with hTERT antibody added (Figure 1b).

2179

hTERT protein expression in differentiating HL60 cells

The change in hTERT protein expression of the differentiating HL60 as detected by the developed assay is shown in Figure 2a. It can be seen that hTERT protein expression in the differentiating HL60 cells declined steeply on day 4 of culture and remained low for the nal 2 days. The controls that did not have DMSO added to them showed positive expression throughout the 6 day period (Figure 2b). As a further control, the expression of TP1, another protein component of telomerase, was also assayed using this method and did not change during the 6 day study period (data not shown). When hTERT protein expression was very low a slight negative shift was seen. This was due to a small degree of positive activity exhibited by the control normal IgG antibody used (conrmed by the use of manufacturers blocking peptide (Figure 3).

TRAP analysis
The PCR-based TRAP assay conrms the association between the expression of hTERT protein as shown by the ow cytometry assay and differentiation. It is clear that telomerase activity reduces through the 3 day period and again is virtually lost by day 4 (Figure 4a). The data were also quantied using a Phosphorimager according to the manufacturers protocol and these data can be shown to correlate well with the mean cell uorescence data from the ow cytometry assay (Figure 4b and c). Statistical analysis (Pearsons product moment correlation) comparing the quantied data from the two assays across the three experiments showed a signicant correlation (1% signicance level, r2 = 0.66).

Figure 4 Validation of ow cytometry method for hTERT detection by comparison with conventional TRAP assay of telomerase activity in differentiating HL60 cells. (a) shows a representative gel conrming loss of detectable telomerase activity by day 4 of differentiation, compared with maintenance of telomerase activity in undifferentiated controls. Quantitative comparison between ow cytometry method and TRAP assay is shown in (b). (Due to large data range, Phosphorimager units are shown on a log scale for ease of comparison. Data shown quanties the gel shown in (a)) The signicant correlation of the two methods is shown in (c).

Figure 3 Comparison of control antibody with manufacturers blocking peptide. Conrmation that the negative shift seen in situations of very low expression of hTERT is due to a small degree of positive activity exhibited by the control antibody (shaded) by use of the manufacturers blocking antibody (faint line). Note that hTERT (dark line) is positive compared to the blocking peptide.

Analysis of hTERT expression in a mixed cell population

The assay was applied to an articially created mixed population of undifferentiated HL60 cells and DMSO differentiated
Leukemia

Bio-technical methods section (BTS) ASM Ali et al

2180

cells (day 6). Analysis showed the presence of two populations: one population showed signicant positive expression of hTERT, while the second population showed no signicant expression (Figure 5a). The nature of the two populations (undifferentiated and differentiated) was further conrmed by surface staining for the CD11b differentiation marker (Figure 5b) on another sample of cells taken from the same mixture. Concurrent dual staining for CD11b and hTERT was not possible because the xation and permeabilising techniques required to access the intranuclear hTERT antigen interfered with the CD11b antibodys action.

Relative expression of hTERT in various haematological cell types

The assay was applied to primary blood cells obtained from 10 healthy individuals and six patients with B-CLL. Using the geometric mean cell uorescence, it was possible to see the relative expression of the protein in the different cell types, as shown in Figure 6. A clear difference in levels of expression can be seen between the malignant and normal cells. Mononuclear cells (MNC) derived from patients with advanced BFigure 6 Application of the ow cytometry assay to primary human cells showing relative hTERT expression compared with undifferentiated HL60 cells. The highest levels are detected in advanced CLL and HL60 cells, with low expression in early CLL and normal MNC and T cells. Neutrophils show no detectable hTERT expression.

CLL (Binet stages B and C) showed higher hTERT expression than MNC from patients with early B-CLL (Binet stage A). The undifferentiated HL60 cell line showed a level of expression intermediate between early and late CLL. In addition, it could be seen that normal MNC and T cells also exhibited a low level of hTERT expression with only neutrophils showing no detectable hTERT expression.

Discussion We describe an immunouorescence ow cytometry-based assay for measurement and quantication of the hTERT component of telomerase. This method will allow sub-populations of cells to be separated according to hTERT expression prior to further manipulation, which is not possible using current techniques of telomerase detection. The sensitivity of the assay and its ability to detect changes in hTERT expression was established using HL60 cells that had been induced to differentiate with DMSO. Differentiation with DMSO has previously been shown to result in decreased hTERT mRNA expression using RT PCR.18,19 No data on relative protein expression have been published previously. It can be clearly seen that the level of hTERT protein expression decreases after the rst 3 days of differentiation, very sharply on day 4 and little further on days 5 and 6 (Figure 2a). This observation correlates well with the TRAP assay which also shows an almost complete loss of telomerase activity on day 4 (Figure 4a). The correlation between the TRAP assay and hTERT protein expression would suggest that the hTERT antibody used is detecting functional isoforms of hTERT. The TP1 component of telomerase, however, showed no signicant change in expression during differentiation, adding further weight to the proposition that hTERT protein is the catalytic rate-limiting sub-unit of telomerase10,18 and that its expression at both the mRNA and protein level can be used as a diagnostic indicator of telomerase activity. Further validation of sensitivity was obtained by carrying out an analysis of a mixed cell population of differentiated and undifferentiated cells where it could be seen that the assay was able to visualise and clearly identify the difference in hTERT expression between the two populations. Surface staining of

Figure 5 Application of the ow cytometry assay to a mixed cell population shows that the difference in hTERT expression between differentiated and undifferentiated HL60 cells in an articial mix can be clearly identied by the assay (a). (b) shows further conrmation of the presence of two populations (one differentiated and one undifferentiated) via use of the CD11b differentiation marker. Control antibody is represented by the shaded histogram and hTERT by open histogram. The twin peaks represent the two sub-populations the intranuclear nature of the hTERT assay means that control antibody also shows twin peaks but with less separation.
Leukemia

Bio-technical methods section (BTS) ASM Ali et al

the CD11b differentiation marker gave further conrmation of the differentiation status of the two populations. The analysis of hTERT protein expression by ow cytometry showed the general applicability of the assay and also enabled a relative quantication of telomerase activity in different components of blood. The lack of hTERT expression in neutrophils correlates well with previous ndings which have generally shown that telomerase activity is not present in neutrophils.20,21 The low expression of hTERT protein in normal mononuclear cells and T cells is also consistent with previous ndings of telomerase activity in normal blood leukocytes and may explain the continuous age-related telomere shortening in T cells despite telomerase expression.21 The importance of developing such a sensitive assay is further underlined by the fact that hTERT mRNA expression is not readily detected by Northern blotting.18 The analysis of mononuclear cells (MNC) derived from patients suffering from early and advanced stage B cell chronic lymphocytic leukaemia (B-CLL) shows an increase in hTERT activity according to the stage of the disease. This nding is in agreement with previous studies of CLL, which have suggested that, in NZB mice, telomerase activity correlates with disease stage22 and in humans it is associated with both disease progression and probability of survival.12 To conclude, we have developed a quick, safe and sensitive assay for hTERT protein expression. hTERT expression as measured by this assay correlates well with telomerase activity and the assay allows a degree of relative quantication. The antibodies used in this study were polyclonal, but as monoclonal antibodies to hTERT become commercially available, it should be possible to increase the sensitivity and reliability of the assay even further. It is hoped that the multi-parameter nature of ow cytometry and its ability to identify cellular subpopulations will enable a fuller understanding of the mechanisms of activation of telomerase and hTERT during disease progression and allow concurrent analysis of other cellular proteins which may be involved in its regulation. Acknowledgements ASM Ali is supported by the University of Manchester and the JHG Holt Scholarship and Special Exhibition in Medicine. R Chopra is supported by the Christie Hospital Leukaemia Endowment fund and Chugai Pharmaceuticals. J Robertson is supported by the Leukaemia Research Fund. NG Testa is supported by the Cancer Research Campaign. References
1 Moyzis RK, Buckingham J, Cram L, Dani M, Deaven L, Jones M, Meyne J, Ratliff R, Wu J. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci USA 1988; 85: 66226626. 2 Blackburn EH. Structure and function of telomeres. Nature 1991; 350: 569573. 3 Watson JD. Origin of concatemeric T7 DNA. Nat New Biol 1972; 239: 197201.

4 Levy MZ, Allsopp R, Futcher A, Greider C, Harley C. Telomere end replication problem and cell aging. J Mol Biol 1992; 225: 951960. 5 Greider CW, Blackburn EH. Identication of a specic telomere terminal transferase activity in Tetrahymena extracts. Cell 1985; 43: 405413. 6 Greider CW, Blackburn EH. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specicity. Cell 1987; 51: 887898. 7 Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW. Specic association of human telomerase activity with immortal cells and cancer. Science 1994; 266: 20112015. 8 Harley CB. Telomere loss: mitotic clock or genetic time bomb? Mutat Res 1991; 256: 271282. 9 Broccoli D, Young JW, de Lange T. Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci USA 1995; 92: 90829086. 10 Counter CM, Meyerson M, Eaton EN, Ellisen LW, Caddle SD, Haber DA, Weinberg RA. Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene 1998; 16: 12171222. 11 Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S. Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 1995; 85: 23152320. 12 Bechter OE, Eisterer W, Pall G, Hilbe W, Kuhr T, Thaler J. Telomere length and telomerase activity predict survival in patients with B cell chronic lymphocytic leukemia. Cancer Res 1998; 58: 49184922. 13 Greaves M. Is telomerase activity in cancer due to selection of stem cells and differentiation arrest? Trends Genet 1996; 12: 127128. 14 Engelhardt M, Kumar R, Albanell J, Pettengell R, Han, W., Moore MA. Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood 1997; 90: 182193. 15 Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci USA 1978; 75: 24582462. 16 Xu D, Gruber A, Peterson C, Pisa P. Suppression of telomerase activity in HL60 cells after treatment with differentiating agents. Leukemia 1996; 10: 13541357. 17 Yamada O, Takanashi M, Ujihara M, Mizoguchi H. Down-regulation of telomerase activity is an early event of cellular differentiation without apparent telomeric DNA change. Leukemia Res 1998; 22: 711717. 18 Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, Ziaugra L, Beijersbergen RL, Davidoff MJ, Liu Q, Bacchetti S, Haber DA, Weinberg RA. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 1997; 90: 785795. 19 Ramakrishnan S, Eppenberger U, Mueller H, Shinkai Y, Narayanan R. Expression prole of the putative catalytic subunit of the telomerase gene. Cancer Res 1998; 58: 622625. 20 Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek M, Shay J, Ishioka S, Yamakido M. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995; 155: 37113715. 21 Robertson JD, Gale RE, Wynn RF, Dougal M, Linch DC, Testa NG, Chopra R. Dynamics of telomere shortening in neutrophils and T-lymphocytes during ageing and the relationship to skewed X chromosome inactivation patterns. Br J Haematol 2000; 109: 272279. 22 Peng B, Zhang M, Sun R, Lin YC, Chong SY, Lai H, Stein D, Raveche ES. The correlation of telomerase and IL-10 with leukemia transformation in a mouse model of chronic lymphocytic leukemia (CLL). Leukemia Res 1998; 22: 509516.

2181

Leukemia