Telomerase Activity and Telomere Length in Acute and Chronic Leukemia, Pre- and Post-Ex Vivo Culture1

[CANCER RESEARCH 60, 610–617, February 1, 2000] Telomerase Activity and Telomere Length in Acute and Chronic Leukemia, Pre- and Post-ex Vivo Culture1

Monika Engelhardt,2 Karen Mackenzie, Pamela Drullinsky, Richard T. Silver, and Malcolm A. S. Moore James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center [M. E., K. M., P. D., M. A. S. M.]; and New York Presbyterian Hospital- Weill Medical College of Cornell University, [R. T. S.] New York, New York 10021

ABSTRACT has been observed in dividing somatic cells, eventually leading to cell senescence when telomeres become critically short (1–4). Telomerase We studied telomerase regulation and telomere length in hematopoietic is a ribonucleoprotein which adds telomeric repeats, using an RNA progenitor cells from peripheral blood and bone marrow from patients -؉ subunit as a template. Telomerase expression thereby prevents telo with acute and chronic leukemia and myeloproliferative diseases. CD34 meric shortening during cell division and allows cells to bypass cells from a total of 93 patients with either acute myeloid leukemia (AML; 3 chronic lymphocytic replicative senescence (5). Although high TA is typically found in ,(21 ؍ chronic myeloid leukemia (CML; n ,(25 ؍ n or myelodys- tumor cells of various origin (6–12), borderline TA has also been ,(16 ؍ polycythemia vera (PV; n ,(18 ؍ leukemia (CLL; n were analyzed before and in 19 patients detected in human primitive hematopoietic cells and in unstimulated (13 ؍ plastic syndromes (MDS; n after ex vivo expansion in the presence of multiple cytokines (kit ligand, lymphocytes where TA increases significantly with cytokine-induced interleukin-3, interleukin-6, and granulocyte colony-stimulating factor ex vivo expansion, cell proliferation, and cell cycle activation (13, 14). plus erythropoietin). Compared with hematopoietic progenitor cells from Although previous studies have demonstrated expression of TA asso- telomerase activity (TA) was increased 2- to ciated with most human solid tumors and hematological malignancies ,(108 ؍ normal donors (n 5-fold in chronic phase (CP)-CML, CLL, PV, and MDS. In AML, accel- (5–12), no study to date has investigated telomerase regulation and erated phase (AP) and blastic phase (BP)-CML, basal TA was 10- to ؉ telomere changes in acute and chronic leukemia before and after ex 50-fold higher than normal. TA of CP-CML CD34 cells was up-regulated within 72 h of ex vivo culture, peaked after 1 week, and decreased vivo culture in response to cytokine stimulation. Normal and neoplas- below detection after 2 weeks. In contrast, TA in AP/BP-CML and AML tic hematopoietic cells can be expanded ex vivo and differentiated into CD34؉ cells was down-regulated after 1 week of culture and decreased specific lineages with the use of exogenous growth factors, drugs, and further thereafter. The expansion potential of CD34؉ cells from patients differentiating agents (15–18). This has allowed the elucidation of -with leukemia was considerably decreased compared with CD34؉ cells important growth factors and adhesion molecules that are differen from normal donors. The average expansion of cells from leukemic indi- tially regulated during normal and leukemic cell growth, the identi- viduals was 6.5-, 2.3-, 0.6-, and 0.2-fold in weeks 1, 2, 3, and 4, respectively, fication of normal versus leukemic primitive progenitor cells, the whereas expansion of normal cells was 5- to 15-fold higher. In serial partial detection of defects at the leukemic stem cell level, and the expansion culture, a median telomeric loss of 0.7 kbp was observed during selective expansion of normal versus leukemic hematopoietic cells in 3–4 weeks of expansion. Our results demonstrate that up-regulation of -؉ ex vivo culture (15–21). In contrast to normal progenitor cells, clono telomerase is similar in CD34 cells from CP-CML, CLL, PV, and MDS patients and in normal hematopoietic cells during the first week of culture, genic leukemic cells may have a limited ability to proliferate and whereas in AML and AP/BP-CML, telomerase is high at baseline and differentiate, abnormalities in apoptotic pathways that enable persist- down-regulated during expansion culture. High levels of telomerase in ence of the leukemic clone and may express TA, which promotes leukemic progenitors at baseline may be a feature of both the malignant longevity (16–25). In this study, we analyzed whether TA and TRF in phenotype and rapid cycling. Telomerase down-regulation during culture BM and PB samples from adult patients with acute and chronic of leukemic cells may be due to the decreased expansion potential or leukemia and MPD differ from healthy donor specimens and whether repression of normal hematopoiesis, or in AML it may be due to the TA and TRF measurements performed on leukemic samples show partial differentiation of AML cells, shown previously to be associated significant changes before and after ex vivo culture. The hypothesis of with loss of TA. Telomere shortening during ex vivo expansion correlated the study was that telomeres would be stable under ex vivo expansion with low levels of TA, particularly in chronic leukemic and MDS progen- conditions in AML (because telomerase would be high, whereas in itors where telomerase was insufficient to protect against telomere bp loss during intense proliferation. CML and MDS, telomeres should progressively shorten because telomerase levels are not high enough to stabilize telomeres) and to use CLL CD34ϩ cells as controls for normal stem cells. INTRODUCTION Human telomeres are specialized chromosomal end structures com- MATERIALS AND METHODS posed of G-rich simple repeat sequences that are important for the Patients and Cell Specimens. Specimens were collected from PB and BM function and genome integrity (1). Because conventional DNA poly- of 93 leukemia patients with either AML (n ϭ 25), CML (n ϭ 21), CLL merases cannot fully replicate the extreme ends of linear chromo- (n ϭ 18), PV (n ϭ 16), or MDS (n ϭ 13) at the time of diagnosis. For ex vivo somes, each cell division results in DNA loss. Telomere shortening culture studies, 19 patient samples (8 CML, 5 AML, 4 PV, and 2 CLL) were used. According to the French-American-British classification, 3 of 25 AML Received 6/4/99; accepted 12/2/99. were classified as M0, 5 as M1, 8 as M2, 1 as M3, 5 as M5, 1 as M7, and 2 The costs of publication of this article were defrayed in part by the payment of page had transformed from MDS to AML. In CML, of 21 patients, 13 were in CP charges. This article must therefore be hereby marked advertisement in accordance with and 8 were in AP or BP. In CLL, of 18 patients, 14 had stable disease, whereas 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant U9 CA 67842-01, the Gar Reichman Fund of the Cancer Research Institute (New York), the Byrne Fund (New York), and the Martin Himmel Fund 3 The abbreviations used are: TA, telomerase activity; TRF, telomere restriction (New York) (all to M.A.S.M.). Support was also provided by the United Leukemia Fund, fragments; BM, bone marrow; PB, peripheral blood; MPD, myelo-proliferative disease; Cancer Resaerch and Treatment Fund (New York) (to R.T.S.), and Grant 95/319/1-1 from TRAP, telomeric repeat amplification protocol; kbp, kilobase pairs; bp, base pairs; AML, Deutsche Forschungsgemeinschaft (Bonn) (to M.E.). acute myeloid leukemia; CML, chronic myeloid leukemia; CLL, chronic lymphocytic 2 To whom requests for reprints should be addressed, at University of Freiburg, leukemia; PV, polycythemia vera; MDS, myelodysplastic syndromes; CP, chronic phase; Department of Hematology/Oncology, Hugstetterstr. 55, 79106 Freiburg, Germany. AP, accelerated phase; BP, blastic phase; RA, refractory anemia; RAEB, RA with excess Phone: 49 0761 270 3406; Fax: 49 0761 270 3206; E-mail: [email protected] of blasts; CB, cord blood; MNC, mononuclear cells; TPG, total product generated; CFU, freiburg.de. colony-forming unit. 610

Table 1A Patient demographics Dulbecco’s medium containing 0.36% agarose (FMC Bioproducts, Rockland, Median patient age ME), 20% FCS, and cytokines (K36EG) (13). After 14 days of incubation, [years (range)] No. Male Female CFU-granulocyte-macrophage was scored using an inverted microscope. Total 55 (13–81) 93 48 45 TRAP Assay. The assay which incorporates an internal PCR control (des- ignated telomerase substrate-nontelomerase) was performed as described pre- AML 49 (24–74) 25 11 14 viously (5, 26–28). In brief, two ␮g of protein extract were assayed in reaction CML 50 (44–81) 21 13 8 tubes containing 50 ␮l of the TRAP reaction mixture. For each assay, a CLL 66 (38–81) 18 14 4 PV 60 (18–71) 16 5 11 negative control and 0.1 amol of the quantification standard olinucleotide R8 MDS 55 (30–69) 13 5 8 were used. The amount of TA for each reaction was calculated as published (9, 10, 25–28), expressing the final quantitation as TPG. One unit of TPG was defined as 0.001 amol (or 600 molecules) of telomerase substrate primers extended by telomerase present in the extract with at least three telomeric Table 1B Analyses performed based upon disease type, number of patients, and disease status repeats. The assay was in the linear range from 0.001 amol (1 TPG) to 1 amol (1000 TPG) of R8. This range extended over three logs of our target protein No. during Total No. at course of concentration (data not shown). All results were determined from at least three no. diagnosis disease to six independent TRAP assays. Total 101 54 47 TRF Assay. DNA isolation and TRF analyses were performed as described (3, 4, 13, 25). In brief, genomic DNA was digested with MSP I and RSA I AML 29 25 4a (Boehringer Mannheim, Indianapolis, IN); electrophoresis was performed in CML 21 7 14 0.5% agarose gels; and gels were depurinated, denatured, neutralized, trans- CLL 18 6 12 ϫ PV 16 3 13 ferred to a nylon membrane using 20 SSC, dried for 1 h, and hybridized with a Ј MDS 17 13 4 a5-end labeled telomeric probe (TTAGGG)3 (Genset, La Jolla, CA). Telo- a n ϭ 8 patients with serial specimens obtained at diagnosis and during the course of meric smears were visualized by exposing the membranes to imaging plates their disease. with mean and peak TRF lengths analyzed as reported (13, 25, 29, 30). Statistics. Comparisons among groups were made with standard statistical 4 had transformed to high-grade non-Hodgkin’s lymphoma. In MDS, three tests. Results are expressed as median values except when stated otherwise. patients had RA, five had RA with sideroblasts, and five had RAEB. In each Statistical significance of the data obtained was analyzed by the Wilcoxon rank of four AML and four MDS patients, serial specimens were obtained through- sum test and the Student t test. out their disease course. All PV patients had chronic phase disease. Patients were treated according to various chemotherapy regimens. CLL patients re- RESULTS ceived 2-CDA, fludarabine, or no therapy. CML patients were treated with hydroxyurea or IFN-␣, and PV patients were treated with IFN-␣. The MDS Patient Characteristics. Table 1A summarizes the clinical patient patients were treated with folinic acid, retinoid acid, or supportive care only, characteristics. The numbers of male and female patients were com- and in RAEB and AML, patients were treated with induction chemotherapy parable in groups as well as in subgroups of patients. PB and BM treatment. For direct comparison, both BM and PB specimens were collected specimens were collected at diagnosis and during the course of the from eight patients. Patients with follow-up samples collected for TRAP and disease (Table 1B). A total of 54 patients were analyzed at diagnosis, TRF analysis were also subjected to a clinical remission inquiry performing 47 during the course of their disease, and 19 before and after ex vivo standard remission analyses (BM morphology, histology, FACS, cytogenetics, etc.). Specimens were also collected from 108 normal healthy donors [PB culture. Patients from which specimens were obtained at diagnosis (n ϭ 78), CB (n ϭ 21), and BM (n ϭ 9)] for comparative analysis. The study had received no prior therapy, whereas those samples from patients was performed in accordance with local institution-approved regulations. obtained during the course of their disease had received Յ6 chemo- Specimens from patients and donors were collected and analyzed between therapy cycles in 34 cases and Ͼ6 chemotherapy cycles in 13 cases. April 1995 and April 1997. TA and Telomere Length in Healthy Donors. Table 2 summa- Cell Source and Separation of CD34؉ Cells and Subsets. BM and PB rizes the results obtained from healthy donors (n ϭ 108). Median TA specimens from healthy donors and patients were obtained after gaining was higher in CD34ϩ cells than in MNC and higher in PB and BM informed written consent. CB, which was otherwise to be discarded, was compared with CB cells (Table 2). TA in MNC and CD34ϩ cells exempt from the consent process according to the policy of the Institutional using PB and BM was low and decreased in intensity with age Review Board of Research Associates of the New York University Medical (TA ϭ 4.21 Ϫ 0.0482 ϫ A; and TA ϭ 8.2 Ϫ 0.065 ϫ A; Center. Heparinized MNC from healthy donors and patients were separated by MNC CD34ϩ Ficoll-Paque (Pharmacia, Uppsala, Sweden), CD34ϩ cells were selected with where TA is telomerase activity in TPG and A is age in years), as immunomagnetic beads (Dynal, Oslo, Norway) (13) and used for suspension previously reported (25). Because Southern blot TRF analysis is culture assay, progenitor assay (colony assay), TRAP, and TRF analysis. resolved as a characteristic smear of cellular DNA due to interchro- Suspension Culture Assay (Delta Culture). CD34ϩ cells were cultured at mosomal and intercellular heterogeneity, both mean and peak TRF ϩ 4 ϫ 104 cells per ml in Iscove’s modified Dulbecco’s medium plus 20% FCS values were analyzed. Mean and peak TRFs in CD34 cells were supplemented with gentamicin and monothioglycerol in the presence of a similar with 7.8 and 8.1 kbp, respectively (Table 2). Telomere lengths five-factor cytokine cocktail of kit ligand (20 ng/ml), interleukin-3 (50 ng/ml), of CB CD34ϩ cells were longer than from PB and BM, in accordance interleukin-6 (100 units/ml), erythropoietin (6 units/ml), and granulocyte colony-stimulating factor (1000 units/ml; K36EG), as previously reported (13). The CD34ϩ cell recovery after 1 week of culture was similar for patient and Table 2 TA and TRF in healthy donors [median (range)] healthy donor specimens with a median of 2.21%. However, the highest All Cord Peripheral Bone ϩ CD34 cell yield (5.6%) was obtained from AML samples; next were CML, donors blood blood marrow PV, and CLL specimens with 1.7, 1.6 and 0.9%, respectively. Cell viability Number 108 21 78 9 was assessed using the trypan blue exclusion assay. After 7 days of culture, Age (years) 35 (0–92) 0 46.5 (17–92) 25 (19–53) ϫ 3 TA (TPG) in MNC 1 (0–6) 0 2 (0–6) 4 (1–5) cells were recovered, plated at 2 10 cells/milliliter in agarose for CFU ϩ assay, and repassaged at the starting concentration of 4 ϫ 104 cells/milliliter. TA (TPG) in CD34 3 (0–15) 2 (0–5) 6 (0–15) 10.5 (3–13) cells Weekly passages and colony assays were performed for 3–4 weeks until no Mean TRF (kbp) 7.8 (5.1–13.6) 10 (8.5–13.6) 7.3 (5.1–9.9) 7.3 (7–8.9) further cellular expansion was observed. Peak TRF (kbp) 8.1 (5.5–13) 10.3 (9–13) 7.5 (5.5–9) 8.1 (7.3–9.1) Colony Assay. CD34ϩ cells (1 ϫ 103/ml) and ex vivo expanded CD34ϩ TRF loss in culture 1.1 (0.4–2.9) 1.1 (0.4–2.9) 1 (0.4–1.9) 1 (0.4–1.4) cells (2 ϫ 103–4 ϫ 104/ml) were cultured in triplicate in Iscove’s modified (kbp) 611

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2000 American Association for Cancer Research. TELOMERASE AND TELOMERES IN ACUTE AND CHRONIC LEUKEMIAS with previous studies (2, 13). Telomere length in MNC cells declined with age (TL ϭ 10.052 Ϫ 0.054 ϫ A[r ϭϪ0.6; p Ͻ 0.05], where TL is the TRF in kbp and A is age in years) (Fig. 1A). CD34ϩ cells were a median of 0.2 kbp longer than MNC with 8.7 versus 8.5 kbp TRFs, respectively (n ϭ 11; n ϭ 6 CB, n ϭ 5 PB); however, no significant telomere length difference in CD34ϩ cells and MNC was observed (Fig. 1B). In ex vivo culture, CB (n ϭ 12), PB (n ϭ 20), and BM (n ϭ 7) CD34ϩ cells were found to lose 1–2 kbp over a 4-week period, with a comparable TRF loss in all three cell sources (Table 2). TA and Telomere Length in Leukemia Patients. Fig. 2 summarizes TA and telomere length in leukemia and MPD patients as well as in healthy donors. Increased TA and short telomeres were defined as such when TA in patients was higher than normal [Ͼ(mean Ϫ 2 ϫ SD)] and when their TRF lengths were shorter than normal relative to their age-matched healthy donors [i.e., Ͻ(mean Ϫ 2 ϫ SD)]. Median TA in all patients was increased 7.7-fold, and median telomeres were 1.2 kbp shorter compared with age-matched healthy donors (p Ͻ 0.0001). Median TA in CD34ϩ cells from PV and CLL was similar to normal CD34ϩ cells, moderately increased in CML and MDS (p Ͻ 0.05), and increased 18-fold in AML specimens (Fig. 2A). Telomere length, particularly in AML and CML, was significantly shorter (p Ͻ 0.01), decreased to a lesser extend in PV and MDS (p Ͻ 0.05), and was not significantly different for CLL patients who had similar telomere lengths as age-matched healthy donors (Fig. 2B). Again, we found no significant telomere length difference in CD34ϩ and MNC from CML patients; however, telomeres of CD34ϩ cells were a median of 0.4 kbp longer than those of MNC with 4.8 versus 4.4 kbp, respectively (n ϭ 8) (Fig. 2C). TA and Telomere Length at Diagnosis and during the Course of Disease. TA in all patients was significantly increased in diagnostic specimens with 15 TPG (range: 0–55.3) compared with 2.4 TPG (range: 0–14.3) in follow-up specimens (p Ͻ 0.0001). This was true for all subgroups of patients (PV, 2.8 versus 2 TPG; CLL, 5 versus 2.6 TPG; CML, 15.1 versus 1 TPG; AML, 32 versus 2.16 TPG) except for MDS (7 versus 7.3 TPG) (Fig. 3A). In serial samples from AML patients taken at diagnosis and after courses of induction chemotherapy (n ϭ 4), telomerase decreased 14.8-fold after chemotherapy, correlating with the reduction of leukemic cells and the attainment of normal cells in BM and PB. In serial specimens of four MDS patients,

Fig. 2. A, compared with healthy donors, median TA was highly increased in AML, ,ء .moderately increased in CML and MDS, and fairly low in PV and CLL patients p Ͻ 0.05. B depicts short telomeres, particularly in AML and CML, to a lesser extent in PV and MDS, and with similar length as expected from age-matched healthy donors in CLL patients. C, representative Southern blot analysis demonstrating the TRF difference of CD34ϩ and MNC in CML.

TA increased with disease progression. Particularly in one patient who progressed from RA to RAEB, TA increased 11-fold, and in progression from RAEB to overt AML, TA increased 18-fold (Fig. 3B). No significant difference in telomere lengths could be demonstrated in patient specimens at diagnosis and follow-up, with 5.7 (range: 3.2– 7.7) versus 5.6 kbp (range: 3.4–8.2), respectively (p ϭ 0.833). A trend toward telomere shortening was observed in subgroups of patients with PV, CLL, and CML, in which telomeres tended to shorten as the disease continued, with 5.9 versus 5.7, 6.6 versus 6.1, and 5.1 versus 4.7 kbp, respectively. In contrast, longer telomeres were found in AML and MDS patients after induction chemotherapy (6.6 and 6.1

Fig. 1. A, Telomere length in healthy donors declined with age, accounting for a bp loss kbp) compared with those found in diagnostic specimens (5 and 5.9 of 54/year. B, small TRF length difference of CD34ϩ and MNC from healthy donors. kbp). Most likely this was due to the loss of the leukemic clone (with 612

differences were also due to differences in the number of leukemic cells in patient specimens, e.g., that patients who required more therapy had more leukemic cells and, therefore, that this added to the effects of the chemotherapy on the results. Nevertheless, it was of interest that CD34ϩ cells from CLL patients progressively shortened from 6.6 to 6.1 and 5.9 kbp, whereas CD34ϩ cells from CML, PV, MDS, and AML patients initially increased from 6.1 to 6.7 kbp and then decreased to 5 kbp with 0, Ͻ6, or Ͼ6 chemotherapy cycles, respectively. TA and Telomere Length in Patients with Stable and Acceler- ated Disease Course. Patients with stable versus accelerated disease where defined as a group with CP versus AP and BP disease and were defined in MDS/AML as a patient group with RA/RAS (RA with sideroblasts) versus RAEB/AML disease. In CLL, patients with stage RAI I–III disease and patients with transformed aggressive lymphocytic lymphoma (Richter’s syndrome) were subdivided. TA was significantly lower in all patients with stable compared with accelerated disease, with 2.8 TPG (range: 0–14.3) compared with 21 TPG (range: 2–41.5), respectively (p Ͻ 0.0001). This was also true for subgroups of CLL, CML, and MDS/AML, with 1.9 versus 31.5, 2.3 versus 5, and 4.95 versus 15 TPG, respectively. Telomeres in patients with stable disease were 5.9 kbp (range: 4.2–8.6) compared with 5 kbp (range: 3.2–7.6) in patients with progressive disease (p ϭ 0.091), 6.5 versus 6.4 kbp in CLL, 5.6 versus 4.6 kbp in CML, and 5.9 versus 5.1 kbp in AML/MDS. TA and Telomeres in PB Compared with BM AML and MDS Specimens. BM specimens obtained from AML and MDS patients at diagnosis had slightly elevated TA (1.6-fold higher) and shorter telomeres (4.9 kbp) compared with PB specimens (5.2 kbp) from the same patient. TA was identical in BM and PB specimens from patients in complete remission, displaying a low or undetectable TA with a median of 1.4 TPG. Telomeres in remission patients (with a median age of 46 years) were 6.94 kbp in BM and 6.9 in PB, which was 0.6 kbp shorter compared with age-matched healthy donor controls. TA, Telomere Dynamics, and Cell Expansion under ex Vivo Culture Conditions. Like TA kinetics observed in healthy donors (Fig. 4A), telomerase was up-regulated within 72 h of ex vivo culture of CD34ϩ cells from CP-CML, PV, and CLL patients (Fig. 4B). TA in these cultured cells peaked after 1 week and decreased below detection after 2 weeks of culture (Fig. 4A). In contrast, in AP/BP- CML and AML patients, telomerase in CD34ϩ cells was down- regulated after 1 week of expansion and decreased further thereafter (Figs. 4, A and B, and 5A). Telomerase up-regulation in CP-CML, PV, and CLL patients was lower than that observed in normal hematopoietic cells, whereas in AP/BP-CML and AML, telomerase was elevated Fig. 3. A, TA in MPD and leukemia patients was increased at diagnosis compared with at baseline and highly exceeded levels found in healthy donors (Fig. follow-up specimens. B depicts a TRAP assay from one MDS patient progressing from ϩ RA to RAEB and overt AML. C, telomere length analysis failed to demonstrate a 5A). The expansion potential of CD34 cells from patient samples significant difference at diagnosis and follow-up. However, a trend in PV, CLL, and CML was considerably decreased compared with normal hematopoiesis was to shorten from diagnosis to follow-up, whereas in AML and MDS, longer telomeres were observed after induction chemotherapy compared with diagnostic specimens. (Fig. 5B). The cell expansion in healthy donors in weeks 1 to 4 was 36-, 37-, 3.5-, and 1.2-fold, respectively, whereas in patients, the expansion potential was significantly reduced with 6.5-, 2.3-, 0.6-, and shorter telomeres) and the emergence of normal hematopoietic cells 0.2-fold, respectively. The expansion potential was also reduced in (with longer telomeres) after induction therapy (Fig. 3C). each patient subpopulation, as shown in Fig. 5B. The cell expansion Telomeres in Patients with <6 and >6 Chemotherapy Cycles. potential of CML samples was higher compared with patients with Patients who received moderate pretreatment (Ͻ6 chemotherapy cy- AML, PV, or CLL (Fig. 5B). Lower amounts of TA in weeks 2 to 4 cles) had significantly longer telomeres than patients who received of culture were associated with telomere erosion during ex vivo extensive pretreatment (Ͼ6 chemotherapy cycles), with a median of culture. During 3 to 4 weeks of culture, a median telomeric loss of 0.7 6.4 (range: 4.3–8.6) versus 5.0 kbp (range: 3.2–7.6), respectively kbp was detected in patient samples compared with 1.1 kbp in healthy (p Ͻ 0.0001). Patients with no pretreatment had telomeres of 6.5 kbp, donors (Fig. 5C). Higher TA in AML and AP/BP-CML specimens suggesting that the initial chemotherapy cycles both eradicated the correlated with a reduced telomere loss compared with lower TA and leukemic clone and shortened the telomeres of normal hematopoietic a greater TRF loss in CLL and PV patients (r ϭϪ0.61) (Fig. 5D). cells (with the latter phenomenon predominating with successive Clonogenic Results. Cell clusters and normal-sized colonies were chemotherapy). However, it cannot be ruled out that telomere length observed mainly in AML and BP/AP-CML. Only normal-sized col- 613

levels of telomerase were found in CB cells (most likely due to their quiescent nature) where only Ͻ2% of cells are in cell cycle, whereas higher TA was observed in more actively cycling PB and BM cells. In leukemia (especially in diagnostic specimens analyzed in this study) telomerase was highly increased in contrast to normal hematopoietic cells. TA rose with the acuteness and aggressiveness of the leukemic disease: (a) was low in PV and CLL; (b) increased moderately in CML and MDS; and (c) increased significantly in AML patients. Although other investigators have reported less consistent telomerase expression in leukemia with no correlation to biological and clinical parameters (32), our results are in line with previous reports (11, 12, 22–25) and suggest that telomerase plays an important role in the process of multistage leukemogenesis, correlates with disease progression, and decreases to borderline activity with the attainment of complete remission in leukemic patients. Telomeres had progressively shortened, particularly in AML and CML, to a lesser extend in PV and MDS, and least in CLL CD34ϩ cells. Short telomeres in leukemia and MPD support the concept that there is progressive telomere erosion in patients with ongoing or active disease and that telomerase was turned on after extensive

Fig. 4. A, telomerase kinetics during ex vivo culture. Lanes 1–5: TA in healthy donors with up-regulation after 1 week of culture. Lanes 6–8: TA in AP/BP-CML with a substantial decrease during ex vivo culture. Lanes 9–12: same phenomenon but in AML. Lanes 13–16: TA in CLL CD34ϩ with similar telomerase up-regulation as observed in healthy donors. B, TA in CD34ϩ cells from CP-CML displayed a similar up-regulation within 72 h of ex vivo expansion as in healthy donors.

onies were counted, which were less frequent in AML and AP/BP- CML than in CLL and PV specimens (Fig. 6A). As a result, total colony numbers generated after 3 to 4 weeks of culture were significantly lower in AML and AP/BP-CML compared with those of CLL and PV patients (Fig. 6B). Compared with healthy donors, the total cell and progenitor expansion were 1- to 5-log decreased in MPD, PV, and leukemia patients (CML, CLL, and AML) (Fig. 6, C–F). In normal donors, total cells (starting with a seeding concentration of 4 ϫ 104 cells/ml) were highly increased in weeks 1 to 4, reaching the maximum cell expansion in weeks 1 and 2 compared with significantly lower numbers in leukemia (Fig. 6C). Total colony numbers (Fig. 6D), fold cell increase (Fig. 6E), and the fold progenitor expansion (Fig. 6F) were significantly diminished in leukemia patients.

DISCUSSION Previous studies have shown that telomeres within hematopoietic cells and other somatic tissues progressively shorten with age in in vivo and ex vivo culture (1, 2, 4, 13) and that telomeric DNA loss may correlate with genetic instability, the pathogenesis of de novo MDS or leukemia, and disease progression (22–25, 31). However, no study to date has conclusively addressed TA and telomere length changes before and after ex vivo culture in response to cytokine stimulation in acute and chronic myeloid leukemias, myelodysplastic syndromes, Fig. 5. A, similar to TA kinetics observed in healthy donors, telomerase was up- CLL, and PV. Therefore, we sought to determine whether TA and regulated during ex vivo expansion of CD34ϩ cells from CP-CML, PV, and CLL patients, TRF measurements in adult leukemia patients differ from healthy peaked after 1 week, and decreased below detection thereafter. In contrast, in AML, telomerase was down-regulated after 1 week and decreased even further. B, expansion donors and whether both show significant changes before and after ex ϩ ϩ potential of CD34 cells in leukemia was considerably decreased compared with normal vivo culture. We found that TA in MNC and CD34 cells from hematopoiesis. The cell expansion potential in CML patients was higher compared with healthy donors was low and that TA in CD34ϩ cells was increased AML, PV, or CLL and correlated with the amount of TA. C, in serial culture within 3–4 compared with MNC. Therefore, TA appears to be required for weeks of expansion, a median telomeric loss of 0.7 kbp was detected, with a reduced loss ϩ in AML and CML correlating with higher TA compared with a greater loss in PV and maintenance and proliferation potential of CD34 cells. Very low CLL when TA was low (D). 614

Fig. 6. A, the colony number in each week of culture as well as the total colony numbers (B) were significantly lower in CD34ϩ cells from AML and AP/BP-CML compared with CLL, PV, and CP- CML patients. Compared with healthy donors, cell (C) and colony counts (D) were 1–5 log decreased in MPD and leukemia in expansion culture. Cell counts in healthy donors reached the maximum cell expansion in weeks 1 and 2 and decreased thereafter compared with significantly lower numbers in weeks 1–4 in leukemia patients (C). In line with the decreased cell expansion potential, total colony numbers (D), the fold cell increase (E), and the fold progenitor expansion (F) were also significantly diminished in leukemia compared with normal donors.

proliferation (25, 31). Assuming a bp loss of 50/cell division, average MDS patients, longer telomeres were found after induction chemo- population doublings in CLL CD34ϩ cells (used as controls for therapy, most likely due to the loss of the leukemic clone (with shorter normal stem cells) were low but increased 20-fold in PV and MDS telomeres) and the emergence of normal hematopoietic cells (with and 45- to 50-fold in CML and AML. Telomere reduction was longer telomeres). Patients who received moderate doses of chemo- previously demonstrated in acute and chronic leukemia (22, 25, 31), therapy (Ͻ6 cycles) had significantly longer telomeres compared with in MDS (33, 34), and in others such as aplastic anemia (35), IDDM those of patients exposed to Ͼ6 chemotherapy cycles, accounting for (insulin-dependent diabetes mellitus) (36), and scleroderma (37). Te- a telomere length difference of 1.4 kbp (or 28 years of premature lomere shortening in disease states has been linked to chromosome aging). Telomeres in patients in complete remission were a median of abnormalities and disease progression, leading to increased prolifer- 0.6 kbp shorter compared with age-matched healthy donors, account- ation, cell apoptosis, and/or immune destruction (12, 22–25, 30, 33, ing for 12 years of premature aging. Telomere shortening in hemato- 34). Comparison of diagnostic and follow-up samples from leukemic poietic cells has been observed after standard chemotherapy (25), after patients revealed significantly increased TA in diagnostic specimens intensified treatment protocols such as high-dose chemotherapy in compared with specimens obtained after treatment initiation. This was autologous (38) and allogeneic transplantation (39, 40), in cells true for all subgroups of patients except for MDS, most likely because treated with cisplatin (41), and after irradiation (42). Telomere reduc- only one MDS patient progressed to AML, whereas the others had a tion associated with transplantation has been demonstrated to corre- stable disease course. In AML patients, TA decreased after induction late inversely with the number of nucleated cells infused (40), sug- chemotherapy, which correlated with the disappearance of leukemic gesting that the proliferative pressure is less intense with large cells and with the attainment of remission and, conversely, whereby a progenitor cell support. Telomere erosion after high-dose chemother- substantial telomerase increase was observed with MDS progression apy protocols seems likely to occur as a consequence of strong to RAEB and AML. In general, we observed that TA was significantly proliferative stress, possibly accounting for hematopoietic abnormal- lower and that telomeres were longer in patients with stable compared ities such as reduced hematopoietic and stromal cell compartments, with accelerated disease. Telomeres in PV, CLL, and CML showed a impaired CFU and long-term culture-initiating cell (LTC-IC) capac- trend of shortening from diagnosis to follow-up, whereas in AML and ity, poor response of progenitors to growth factors in vivo and ex vivo, 615

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2000 American Association for Cancer Research. TELOMERASE AND TELOMERES IN ACUTE AND CHRONIC LEUKEMIAS and the high incidence of MDS that has been observed after autolo- against telomere bp loss upon proliferation. Alternatively, telomere gous and allogeneic transplantation (43, 44). shortening may reflect the emergence of nonleukemic populations We and others have previously demonstrated TA in normal hema- with low TA which is unable to prevent telomere shortening upon topoietic cells and that TA further increases in ex vivo culture which extensive proliferation (13). Although telomerase may not be the only thereby prevents progressive telomere shortening despite high cell mechanism for maintaining chromosome ends and alternative length- turnover (12, 13). We found that the kinetics of TA in CD34ϩ cells ening of telomeres also functions to elongate telomeres (50), TA from CP-CML, PV, and CLL patients were similar to normal hema- seems vital for cell survival and organ homeostasis in most prolifer- topoiesis, whereas high TA in AP/BP-CML and AML specimens was ating cells through maintenance of telomere structures during cell down-regulated. The amount of telomerase correlated with the cell division. Novel telomerase inhibitors, as recently shown using a expansion potential, in which elevated TA was associated with greater genetic approach (51), should be tested in hematopoietic culture proliferative potential, whereas a decline in proliferation and in- systems with use of normal and malignant hematopoietic cells be- creased cell apoptosis was observed with subsequent telomere erosion cause telomerase dynamics, cell proliferation, and telomere length in weeks 2–4 of expansion when TA was low. C. Eaves and M. changes can be determined. 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Monika Engelhardt, Karen Mackenzie, Pamela Drullinsky, et al.

Cancer Res 2000;60:610-617.

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