telomerase inhibitor BIBR1532 Selective cytotoxicity ...

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Prepublished online October 26, 2004; doi:10.1182/blood-2003-12-4322

Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532 Hesham El-Daly, Miriam Kull, Stefan Zimmermann, Milena Pantic, Cornelius F Waller and Uwe M Martens

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Blood First Edition Paper, prepublished online October 26, 2004; DOI 10.1182/blood-2003-12-4322

SELECTIVE CYTOTOXICITY AND TELOMERE DAMAGE IN LEUKEMIA CELLS USING THE TELOMERASE INHIBITOR BIBR1532

Hesham El-Daly, Miriam Kull, Stefan Zimmermann, Milena Pantic, Cornelius F Waller, and Uwe M Martens

Freiburg University Medical Center, Department of Hematology/Oncology, Freiburg, Germany Albert-Ludwigs-University, Department of Biology, Freiburg, Germany

Short title of Running head: Telomerase inhibition in leukemia Keywords : telomerase, telomere, TRF2, telomere dysfunction, leukemia Abstract word count: 201 Text word count: 5514 Scientific heading: Neoplasia

Corresponding author: Dr. Uwe Martens Freiburg University Medical Center Department of Hematology/Oncology Hugstetterstr.55 D-79106 Freiburg, Germany Phone: +49-761-270-3406 Fax

: +49-761-270-3206

Email: [email protected]

Copyright © 2004 American Society of Hematology


2

ABSTRACT

Telomerase represents an attractive target for a mechanism-based therapeutic approach because its activation has been associated with unlimited proliferation in most cancer cells. Recently, a non-nucleosidic small molecule inhibitor, BIBR1532, has been identified which is highly selective for inhibition of telomerase resulting in delayed growth arrest of tumor cells. Here we examined the effects of BIBR1532 in different leukemia cell lines as well as in primary cells from patients with AML and CLL in short term culture assays. We observed a dose dependent direct cytotoxicity in concentrations ranging from 30 to 80 µM. Interestingly, cell death was not dependent on the catalytic activity of telomerase but was delayed in cells with very long telomeres. We observed time-dependent individual telomere erosion, which was associated with loss of TRF2 and increased phosphorylation of p53. Importantly, the proliferative capacity of normal CD34+ cells from cord blood and leukapheresis samples was not affected by treatment with BIBR1532. We conclude that using this class of telomerase inhibitor at higher concentrations exerts a direct cytotoxic effect on malignant cells of the hematopoietic system, which appears to derive from direct damage of the structure of individual telomeres and which must be dissected from telomerase suppressed overall telomere shortening.


3

INTRODUCTION Maintenance of telomeres is essential for unlimited cellular proliferation and confers immortality in cancer cells.1,2 Telomeres in human cells consist of repetitive double-stranded repeats of the sequence TTAGGG, which terminate in a single-stranded 3' extension overhang of the G rich strand.3 Their major function is to cap the ends of chromosomes and to provide genetic stability. This capping function is mediated by a special architecture in which the 3' overhang participates with telomere binding proteins in a large loop structure called T-loop.4 In addition, a number of proteins constitute the mammalian telomere and serve to regulate the telomere structure.5 The telomeric repeat binding factors TRF1 and TRF2 bind to doublestranded telomeric DNA and are involved in telomere length regulation and telomere protection.6,7 Since TRF2 assists in formation of the T-loop, it is thought to play a major role in stability of the telomere structure.8 In fact, loss of TRF2 has been associated with induction of apoptosis and senescence in human cells.9 Telomerase is a ribonucleoprotein enzyme, which synthesizes telomere repeats de novo.10 In human cells, the telomerase holoenzyme consists of a high-molecular weight complex with a template-containing RNA subunit,11 hTR, and protein components including the catalytic subunit human telomerase reverse transcriptase,12 hTERT. In most normal somatic cells telomerase activity is absent and telomere repeats are lost with cell division and with ageing. Telomere attrition beyond a certain threshold is assumed to uncap chromosome ends which subsequently induces DNA damage13 and onset of replicative senescence.14,15 In contrast, about 80-90% of cancer cells have detectable telomerase activity, which leads to stabilization of telomeres and unlimited growth potential.16 Likewise, telomerase activity has been detected in the majority of acute and chronic leukemias, particularly with disease progression.17 Interestingly, low levels of activity have also been found in activated lymphocytes18 and


4 hematopoietic stem cells19 which appear to confer extended proliferative capacity but which do not prevent overall telomere shortening.20

Since most cancer cells are reliant on telomerase for their survival, telomerase has become an attractive target for the development of new cancer therapeutics.21 Indeed, during the recent years a variety of different classes of telomerase inhibitors, which block the catalytic activity of the enzyme or the access of telomerase to the telomere, have been evaluated.22-24 ‘Proof of principle’ for validation of telomerase inhibition as therapeutic concept was demonstrated in human tumor cells using dominant-negative mutant forms of hTERT.25,26 In these experiments, telomerase activity was abolished which was associated with continuous telomere shortening leading to senescence or apoptosis. It is assumed that not direct inhibition of telomerase induces growth arrest but subsequent loss of telomere function, which derives from uncapping of individual short telomeres.27 A challenge for potential clinical application of pharmaceutically useful telomerase inhibitors is its therapeutic window. Since the antiproliferative effect is dependent on the telomere length of a given tumor cell, clinical response will be delayed until chromosomes run out of their telomeres. This will require a series of cell divisions to become apparent, and treatment may have to be given continuously for weeks to months, potentially in conjunction with other modalities. Recently a novel structural class of non-peptidic, non-nucleosidic inhibitors of human telomerase has been described28 that are specific in inhibition of the catalytic activity of telomerase.

One example of this class of compounds, designated BIBR1532 {2-[(E)-3-

naphtalen-2-yl-but-2-enoylamino]-benzoic acid}, inhibits the in vitro processivity of telomerase in a dose dependent manner, with half-maximal inhibitory concentrations (IC50) of 93 nM.29 The selectivity of BIBR1532 was assessed in a panel of DNA and RNA polymerases, showing that none of these enzymes were inhibited in concentrations up to 100 µM.


5

In this work, we investigated the activity of BIBR1532 in normal and malignant hematopoietic cells. In addition to its known role in telomerase inhibition we provide evidence that high dose BIBR1532 has a direct cytotoxic effect in leukemia cells but not in normal hematopoietic stem cells. The mechanism that induces immediate rather than delayed growth arrest is most likely due to direct damage to the telomere structure, which possibly reflects a novel approach for cancer therapy.


6

PATIENTS, MATERIALS AND METHODS Reagents and antibodies. BIBR 1532 was synthesized by Boehringer Ingelheim (Biberach, Germany) and prepared as described previously.28 A stock solution of BIBR1532 at a concentration of 1 mM was prepared by dissolving the compound in sterile DMSO and stored at –20°C until use. The TRF2, p53, phospho p53, ß-actin antibodies were purchased from Biocarta, (Hamburg, Germany). Cells and cell lines. The JVM13 cell line represents an immortalized B-cell line from a patient with prolymphocytic leukemia, HL-60 is an AML cell line, Jurkat is a T cell lymphoma cell line and Nalm1 was derived from a patient with CML in blastic phase. All cell lines were purchased from DFMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Cells were cultured in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) supplemented by 10% heat inactivated fetal calf serum (Invitrogen, Karlsruhe, Germany), 2 mM L-glutamine, and 1% penicillin/streptomycin (complete medium) in a humidified 5% CO2 atmosphere. Human diploid fibroblast cells (HK1 line) were cultured as described.30 In addition to telomerase negative wild-type cells, immortalized HK-1 cells were used expressing ectopic hTERT.31,32 Patient population and isolation of leukemia cells. EDTA venous blood samples were collected from consenting CLL and AML patients and separated over Ficoll-hypaque (Biochrome, Berlin, Germany). For the CLL samples the mononuclear cell fraction was washed twice and adjusted to 1-3 x 107/ml. Cells were cultured in 3 ml medium using 6 well plates, and the following concentrations of BIBR1532 were used: 10 µM, 30 µM, 50 µM, and 80 µM. Likewise, corresponding concentrations of DMSO were used as another negative control in addition to the negative control (no inhibitor). Cultures were maintained for a minimum of 14 days and every 4 days aliquots were taken (volume replaced by medium) for different assays. Cord blood and leukapharesis products following stem cell mobilization


7 were separated over Ficoll-hypaque and washed. CD34+ cells were purified by positive selection using a MACS progenitor enrichment kit according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany) and adjusted to 0.5-1 x 105/ml. Cells were cultured in serum free medium (CellGro , CellGenix, Freiburg, Germany) supplemented with SCF 300 ng/ml, Flt-3 L 300 ng/ml, IL-3 100 ng/ml, IL-6 10 ng/ml (each provided by CellGenix, Freiburg, Germany), and G-CSF 10 ng/ml (Amgen, Thousand Oaks, CA). Cells were maintained for 21 days in culture at 37°C and 5% CO2, medium was changed at 7 days interval, at which the cells were counted, re-seeded at 3-6 x 106/ml and the rest was harvested for different assays. Colony forming assays. CD34+ cells from cord blood or normal bone marrow were plated at 1 x 103 /ml in serum free methylcellulose medium supplemented with cytokines (MethoCult GF H4434, Stemcell Technologies Inc., Vancouver, Canada) with different concentrations of BIBR1532 as mentioned before. BFU-E and CFU-GM were counted by day 14 of culture. Measurement of cell death. Determination of CLL cell viability in this study is based on the analysis of mitochondrial transmembrane potential by 3,3' dihexyloxacarbocyanine iodine (DiOC6-- Molecular Probes, Eugene OR) and cell membrane permeability to propidium iodide (PI- Molecular Probes, Eugene OR), as previously described.33,34 For comparison, CLL cell viability was also examined using the different relative size and granularity (forward scatter and side scatter) characteristics of vital and dead cells. Trypan blue exclusion was also used to determine the number of viable cells. Western blot analysis. Protein was extracted with RIBA lysis buffer. An equal amount of protein (10-50 µg) of the samples was separated on 10% SDS-polyacrylamide gel and transferred onto transfer membrane (Immobilon-P, Millipore corporation, Bedford MA). After blocking (in 3% non-fat dry milk, 3% BSA in PBS), the membranes were incubated with primary and secondary antibodies and the bands were detected by the chemiluminescence method according to the manufacturer recommendations (Amersham Biosciences, UK).


8 Telomere length analysis using flow-FISH. To determine mean telomere length in individual cells, cells were hybridised in situ with a fluorescent telomere-specific peptide nucleic acid (PNA) probe as described recently.31,35 Telomere length was expressed in Telomere Fluorescence Units (TFUTRF) based on calibration experiments using TRF length analysis by Southern blotting. Telomerase activity measurements. Telomerase activity of cell populations was determined using the TeloTaGGG PCR ELISAPLUS kit (Roche, Mannheim, Germany) according to the manufacturer’s protocol. Heat-treated cellular lysates (85°C for 10 min) were used as negative controls for each sample. Samples are considered as telomerase-positive if the difference in absorbance is higher than twofold background activity. WST-1 assays. 0.5–5 x 104 cells were plated as triplicates in complete RPMI 1640 medium with various concentrations of BIBR1532. After 24-72 hours tetrazolium WST-1 (Roche, Mannheim, Germany) was added, which is transformed into formazan by mitochondrial reductase-systems and increase of number of viable cells results in increase of activity of mitochondrial dehydrogenases leading to increase of formazan dye formed, which was quantified by ELISA reader after 2, 3, and 4 hours incubation. Fluorescence in situ hybridization and quantitative image analysis. Metaphase chromosomes from JVM 13 cells were prepared after 2 h treatment with 0.1 µg/ml colcemid (Invitrogen, Karlsruhe, Germany) followed by hypotonic solution in 75 mM KCl (SigmaAldrich Steinheim, Germany) according to standard methods. The number of analyzed metaphases was between 8 and 15. Individual telomere length was analyzed by quantitative fluorescence in situ hybridization (Q-FISH) as described previously.15,28 Digital images of metaphase spreads were recorded with a digital camera (Sensys, Photometric) on an Axioplan II fluorescence microscope (Zeiss, Jena, Germany) using the Vysis workstation QUIPS (Vysis, Downers Grove, IL).


9 Statistical analysis. Data analysis was performed using Microsoft Excel with WinSTAT module and Microcal Origin software. Results are shown as means ± SE of values obtained in independent experiments. Students t-test was used to determine statistical significance.


10

RESULTS Antiproliferative effect of BIBR1532 in leukemia cell lines. The telomerase inhibitor BIBR1532 has been shown to inhibit telomerase activity in several human cancer cell lines resulting in progressive shortening and subsequent dysfunction of telomeres when used at concentration of 10 µM.28 Here we analyzed short term effects of BIBR1532 using concentrations between 10 and 80 µM in the CLL cell line, JVM13. Unlike a delayed effect that was dependent on substantial telomere erosion, a direct antiproliferative effect was observed in a dose depending range using inhibitor concentrations above 20 µM (Fig. 1A). With treatment of 80 µM BIBR1532 we found more than 80% decrease in viability of JVM13 cells within 9 days (Fig. 1B, C). In addition, increasing concentrations of BIBR1532 showed a dose dependent inhibition of proliferation using the WST-1 proliferation assay which translated in a IC50 value of 52 µM. Similar results were obtained for other leukemia cell lines such as Nalm-1, HL-60 and Jurkat (data not shown). Thus, increasing the doses of BIBR1532 in cell cultures above the concentration that has been reported to induce telomere shortening does result in a direct antiproliferative effect in contrast to delayed growth arrest.

Antiproliferative effect of BIBR1532 on primary leukemia cells. In order to explore the antiproliferative effect of BIBR1532 on primary leukemia cells in vitro, we treated peripheral blood cells from patients with chronic lymphocytic leukemia (CLL) in short term cultures as previously described.34 Samples from 20 different CLL patients were used in these experiments (Table1). The mean telomere length for patients with early stage CLL, RAI 0-II (n=12), was found to be 7.0 ± 0.6 kb, whereas telomeres were relatively shorter in those with late stage CLL, RAI III-IV (n=7) (6.3 ± 1.2 kb) although the difference was not significant (p=0.1). Interestingly, only 33% of patients samples with early


11 stage CLL exhibited telomerase activity, while in those with late stage CLL, 57% were telomerase positive.

A 1000

Relative cell number

Neg. control

800

10 µM 30 µM

600

50 µM

400

80 µM DMSO

200 0 0

3

6

9

Culture Time (Days)

100 80 60 40 20 0 0

3

6

Culture Time (Days)

9

Proliferation (% of controls)

Viability (% of controls)

B

C 100 80 60 40 20 0

0

8

24

48

72

Culture Time (Hours)

Figure 1. Antiproliferative effect of BIBR1532 in JVM13 leukemia cell line. (A) JVM13 cells were cultured for 9 days in RPMI medium supplemented with increasing concentrations of BIBR1532. Cells were counted at indicated time points using trypan blue exclusion assay. (B) The viability of JVM13 cells treated with 80 µM was determined at indicated time points using FSC/SSC pattern of FACS analysis. A gradual decrease in viability was observed with cells cultured in the presence of 80 µM BIBR1532. Values represent means ± SEM from triplicates. (C) Proliferation of JVM13 cells was determined using WST-1 assay.43 Bars represent the proliferation of cells cultured in the presence of 80 µM BIBR1532 as percentage of controls at indicated timepoints. Values are means ± SEM from triplicates.


12 Table 1. Summary of CLL samples.

Pt code

TL

TA

RAI

Viability index

001TA 002GB 003ZW 004LW

Neg. Pos. Neg. Pos. Neg. Pos. Pos.

n.a

005GP 006GK 007TK

7.4 7.1 5.4 5.9 6.9 7.2 6.6

IV III II 0 II IV

25 97 1 9 2 1 2

008RH 009SH 010IK 011HH 012CG 013SE 014TP 015BJ 016VG 017FL 018DM 019WA 020MH

6.9 6.9 6.9 6.5 5.7 8.5 7.4 7.8 7.7 7.3 5.4 5.2 6.2

Neg. Pos. Neg. Neg. Pos. Neg. Neg. Neg. Neg. Neg. Pos. Neg. Pos.

II II II II IV IV II I I II IV IV 0

34 1 4 31 80 89 1 1 98 2 8 5 1

Indicated is the classification for the patients according to RAI staging system. Telomerase activity (TA) was determined using the TRAP (PCR ELISAPLUS) assay, the mean telomere length (TL) was measured using flowFISH. The response to 80 µM BIBR1532 is expressed as viability index representing the viable cells as percentage of controls after 2 weeks of cell culture. All experimental conditions were performed in duplicates. n.a. = not available.

The cytotoxic effect of BIBR1532 on the CLL samples was examined using the DioC6/PI assay and FACS analysis of forward/side scatter (Fig. 2A, B). Of the 20 samples tested, 16 showed more than 50% loss of viability with 80 µM BIBR1532 within 14 days of culture, whereas 14 samples showed more than 80% loss of viability in during that time period compared to DMSO treated and negative controls (Table 1). Similar to the analysis on leukemic cell lines, the IC50 value was found to be in the range of 50 µM (53 ± 3.8). Notably,


13 four samples responded poorly to the treatment with BIBR1532 demonstrating less than 50% loss of viability. Three of the four non-responders had progressive disease, and one of them (002GB) had a long history of different treatment modalities and was refractory to chemotherapy upon time point of analysis. The cytotoxic effect was not dependent on telomerase activity as measured by TRAP assay, because telomerase negative samples responded as well. Furthermore, the response did not appear to be influenced by differences in overall telomere length since samples with relatively long telomeres such as 014TP (7.4 kb) or 015BJ (7.8 kb), respectively, showed marked reduction in viability as did samples with short telomeres like 003ZW (5.4 kb) and 019WA (5.2 kb). The mean telomere length did not change significantly within the culture period of 14 days as measured by flow-FISH (Fig. 2C). We also investigated the antiproliferative effect of BIBR1532 for acute myeloid leukemia (AML). Mononuclear bone marrow cells from 9 different patients (Table 2) were cultured with increasing concentrations of BIBR1532 for 48 and 72 hours, respectively. Again, concentrations above 20 µM resulted in a direct antiproliferative effect (Fig. 3). Eight out of 9 AML samples showed more than 70% inhibition of proliferation with 80 µM BIBR1532. One sample from a patient with secondary AML responded poorly to the treatment, showing less than 50% inhibition of proliferation. The IC50 value was 56 ± 5 µM (Fig. 3A). As described for CLL cells, the cytotoxic effect of high dose BIBR1532 was not dependent on the presence of telomerase activity. This supports the hypothesis of an additional cytotoxic mechanism.


14

Viability (% of controls)

A

B

120

Negative control 89%

DMSO control 89%

50 µM BIBR 35%

80 µM BIBR 2%

100 80

Neg. conrol DMSO

60

80 µm

40 20 0 0

4

10

14

Culture Time (Days)

C Negative control

80 µM

8,5kbp

8.5 kb

8.7 kb

Figure 2. Antiproliferative effect of BIBR1532 in primary CLL cells. (A) Primary CLL cells were cultured in complete RPMI for 14 days supplemented with 80 µM BIBR1532. As previously reported34, viability was determined using the DiOC6/PI assay at indicated time points and expressed as percentage of the controls. Values are means ± SEM (n = 20). (B) FSC/SSC pattern is shown for a representative CLL patient sample measured by day 14 using FACS analysis. Gated cells represent the viable cells at the end of culture. (C) Analysis of the mean telomere length was measured using flow-FISH technique.35 Representative telomere fluorescence histograms of one CLL patient sample are demonstrated following treatment of BIBR1532 and of DMSO (control cells) for 14 days. Telomere fluorescence intensity was calculated by subtracting the mean background fluorescence (white) from the corresponding mean fluorescence provided by the telomere specific probe (grey).


15 Table 2. Summary of AML patient samples. Pt Code

TA

TL

CD34 %

Blast % in BM

FAB

Karyotype

Inhibition of Proliferation

101

Pos.

6.1

Pos.

90%

M2

ND

>70%

102

Pos.

ND

Pos.

75%

M4

Normal

>70%

103

Pos.

7.3

Neg.

75%

M5

Monosomy 12,16,17

>70%

104

Neg.

7.6

Neg.

50%

ND

Normal

>70%

105

Neg.

ND

ND

94%

M2

Complex

>70%

106

Pos.

6.3

Neg.

85%

M4/2ryAML

Complex

70%

108

ND

ND

Pos.

80%

M2/MDS

Normal

>70%

109

ND

ND

Pos.

75%

M5

Normal

>70%

Telomerase activity (TA) was determined using TRAP assay, Telomere length (TL) was analyzed using flowFISH. The response to 80 µM BIBR1532 is expressed as percentage of proliferation inhibition of the controls within 72 hours of culture. ND = not detected

BIBR1532 does not affect the proliferative capacity of normal hematopoietic progenitor cells. Since some normal cells, including those with stem cell-like properties, retain the ability to activate telomerase physiologically, inhibition of the enzyme may potentially have detrimental effects on their proliferative capacity. Therefore, we investigated the effects of BIBR1532 on hematopoietic, non-malignant, progenitor cells. CD34+ cells from cord blood (n=7) and leukapharesis products (n=5) were cultured for 21 days in a serum free medium supplemented with cytokines and with increasing concentrations of BIBR1532. Cells were harvested periodically for cell count, telomere length measurement, and viability assays. We did not observe any negative effect of BIBR1532 on viability or cell proliferation using concentrations up to 120 µM as determined by Trypan blue exclusion assay and the DioC6/PI assay (Fig. 4A, B).


16

B Proliferation (% of controls)

Proliferation (% of controls)

A

100

100 80 60 40 20 0 Neg PL 10

20 30 40 50 60

70 80

80 60 40 20 0

BIBR1532 (µM)

0

48

72

Culture Time (Hours)

Figure 3. Antiproliferative effects of BIBR1532 in primary AML blasts. (A) Mononuclear bone marrow cells from AML patients (n=9; see table 3) were plated in 96 well plates with indicated concentrations of BIBR1532. WST-1 reagent was added after 72 hours for the determination of proliferation, which is expressed as a percentage of the controls ± SDM. Experiments were performed in triplicates. (B) BIBR1532 (80 µM) induced a gradual decrease of proliferation within 72 hours (>80%). Grey bars represent means of percentage of proliferation measured by WST-1 assay at the indicated time points ± SDM (n = 9).

In addition, no significant differences in the mean telomere length were measured by flowFISH at different time points of suspension culture compared to control cultures with DMSO and without any treatment (Fig. 4C). Finally, we investigated the effects of BIBR1532 in clonogenic assays. These were initiated from suspension culture cells by day 10. Again, no inhibition of colony formation was observed using increasing concentrations of BIBR1532 (Table 3).


17 Table 3. Clonogenic assay for cord blood samples in the presence of BIBR1532.

BFU-E

CFU-GM

Total count

128 ± 111

13 ± 8

138 ± 117

DMSO

126 ± 82

24 ± 14

148 ± 90

10µm

114 ± 98

12 ± 8

125 ± 105

30µm

133 ± 78

24 ± 13

155 ± 86

50µm

120 ± 67

25 ± 13

144 ± 75

80µm

132 ± 82

22 ± 11

152 ± 89

Neg control

CD34+ cord blood cells (n=7) were harvested from suspension culture by day 10, cultured in serum-free methylcellulose medium supplemented with cytokines (SCF, GM-CSF, G-CSF, IL-3, IL-6, and EPO) and with increasing concentrations of BIBR1532 for 14 days. Experiments for each individual specimen were performed in triplicates. BFU-E: Burst forming unit erythroid, CFU-GM: colony forming unit granulocyte/monocyte. Note: There is no significant difference observed in number or type of colonies at day 14 for those cultured with up to 80 µM BIBR1532 compared to the controls.

Figure 4 (next page). Proliferation and viability of normal hematopoietic progenitor cells. (A) CD34+ cells enriched from cord blood samples (n=7) were cultured in the presence of BIBR1532 in serumfree medium supplemented with cytokines (SCF, FLT, IL-3, IL-6, and G-CSF). At indicated time intervals viable cells were counted using Trypan blue exclusion assay. Note that cell expansion was only moderately reduced at drug concentrations of 160 µM. Experiments were performed in duplicates. (B) Viability was measured using FSC/SSC pattern and DiOC6/PI staining at indicated time points. FACS diagrams represent the non-treated control cells, the DMSO treated cells (not shown), and the cells cultured with 80 µM BIBR1532. There was no decrease in cell viability of cells cultured under presence of 80 µM BIBR1532 compared to negative and DMSO controls at any time point of culture.


18 (C) The mean telomere length was measured in peripheral blood stem cells (CD34+ leukapheresis sample) following treatment with BIBR1532. Representative telomere fluorescence histograms analyzed at indicated timepoints are shown, which illustrate no substantial difference in the mean telomere length of treated compared to untreated cells.

A 10000

Fold change

1000 Negative DMSO 80 µm 120 µm 160 µm

100 10 1 0

5

10

15

20

25

30

Culture Time (days)

B 90%

84%

80%

93%

85%

80%

Negative Control

BIBR1532 80 µM

Day 6

Day 14

Day 29

C 8.3 kb

8.1 kb

8.0 kb

8.5 kb

7.9 kb

8.3 kb

Negative control

BIBR1532 80µM

Day 8

Day 22

Day 32


19 High dose BIBR1532 induces rapid telomere dysfunction and apoptosis. Because higher concentrations of BIBR1532 directly induced growth arrest in AML and CLL cells without detectable changes in overall telomere length as measured by flow-FISH, we thought to analyze individual telomere length dynamics in JVM13 cells using the Q-FISH technique which is more sensitive to identify subtle changes in telomere dynamics. Although it is a major challenge to generate metaphase spreads from cells entering cell cycle exit, we were able to obtain 8 to 15 metaphases from this cell line following incubation with 80 µM of BIBR1532 for different time points. In contrast to the flow-FISH analysis, a small but significant reduction of the mean telomere fluorescence (p100 PDs) which was associated with substantial telomere shortening. Here, the antiproliferative effect occured within 72 hours when leukemia cells were exposed to the drug at higher concentrations (30-80 µM). The IC50 value for this direct effect was found to be in the range of 50 to 60 µM. Interestingly, this cytotoxicity was


26 independent of measured telomerase activity, because 12 out of 20 CLL and 2 out of 6 AML samples were negative in the TRAP assay. Thus, what could be the mechanism that mediates the direct cytotoxic effect of BIBR1532 in contrast to delayed induction of senescence? Our data provide evidence that treatment with concentrations from 30 to 80 µM interfered with the capping function of telomeres. Although flow-FISH was unable to detect changes in the mean telomere length during the short term culture, the more sensitive Q-FISH analysis demonstrated a small but significant loss of the mean telomere length and - more importantly - a time-dependent erosion of individual telomeres. This was associated with a trend towards increased chromosome fusions and loss of TRF2 protein which suggests that the primary function of telomeres was severely compromised. In agreement with this theory was our observation that high dose BIBR1532 had also a delayed antiproliferative effect on normal cells without detectable telomerase activity such as diploid fibroblasts. In these cells, growth arrest did not occur within 72 hours but after 28 days. The lag period was even extended in a subline with overlong telomeres and ectopic expression of hTERT (>100 days). Therefore, a distinct threshold of overall telomere length may exist which exerts a protective function towards treatment with high dose BIBR1532. It is believed that telomeres form a higher-order chromatin structure that physically protects the 3' end from cellular activities.37 The telomere binding protein TRF2 plays a key role in the protective activity of telomeres and loss of function leads to apoptosis and telomere-induced senescence.7 Increasing evidence is accumulating which suggests that the physical presence of telomerase or its transient activity is part of the integrity of the telomere cap.38,39 In fact, even normal, early passage fibroblasts have been described as showing low expression of hTERT, which is upregulated during their transit through S phase. Such periodic expression of hTERT is supposed to partially maintain the 3' telomeric overhang and to facilitate cell proliferation. The molecular effects of telomerase inhibition by different approaches are not completely understood. It has been speculated that ectopic expression of a dominant-negative mutant of


27 hTERT (DN-hTERT) may titrate telomerase or telomere components from the chromosome end, thereby perturbing capping, while the small molecule inhibitor BIBR1532 may simply decrease the catalytic activity of the enzyme without effecting the telomere structure.40 Based on the studies presented here, it is possible that high dose BIBR1532 does not only suppress the catalytic activity of telomerase but may compromise binding of protein partners of the enzyme resulting in telomere dysfunction. Loss of TRF2 may be a direct consequence of such induced telomere damage. In fact, Karlseder et al.41 reported that a change in the status of the telomere complex rather than telomere shortening will induce DNA damage and induction of growth arrest. Furthermore, Masutomi et al.39 described that disruption of telomerase activity in human fibroblast cells restricts cell lifespan by destabilization of the telomeric 3' overhang without changing the rate of overall telomere shortening. Strikingly, hematopoietic progenitor cells seem to be more resistant towards treatment with high dose BIBR1532. Possibly, differences in telomere structure and binding of associated proteins may exist between normal and malignant cells of the hematopoietic system that make latter more vulnerable towards telomerase associated DNA damage. Such difference could be due to the higher size in telomere repeats that are typical for the stem cell compartment. Compatible with this theory is our observation that fibroblast cells with overlong telomeres (>11 kb) were primarily resistant towards this treatment. Finally, we cannot exclude additional mechanisms that cause cell death upon high dose of BIBR1532. Since loss of TRF2 has been also described upon exposure to DNA damage inducing agents such as ionizing radiation

42

, it is possible that high dose BIBR1532 is not

only inducing damage at the telomere locus but also DNA damage elsewhere in the genome. Nevertheless, telomere damaging agents might represent an interesting avenue for novel cancer therapies although they might be more toxic than simple inhibition of the catalytic activity of telomerase. However, from an oncologist's perspective, this form of approach could circumvent the long lag period, which is in our hands not useful for a successful cancer


28 therapy. Importantly, more knowledge about the structure and function of the telomere complex in normal and malignant cells is essential in order to design such telomere directed therapy approaches.


29

ACKNOWLEDGMENTS We greatfully acknowledge the excellent technical assistance from I. Skatulla and the support from Dr. J. Burger providing CLL samples. We are indebted to Prof. Mertelsmann for his continous support. We thank Dr. J. Lingner for critical comments on the manuscript. This work was supported by grants from the European Union (Fifth Framework Program), Sonderforschungsbereich 364 (DFG), Verein zur Leukämieforschung, Freiburg, and from Boehringer Ingelheim Pharma KG, Biberach, Germany.


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