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Activation of Telomerase in Human LymphocytesandHematopoietic Progenitor Cells' KeikoHiyama,**+YukoHirai,*SeishiKyoizumi,* EisoHiyama,§ Mieczyslaw A. Piatyszek,"Jerry Shinichi Ishioka,*and Michio Yamakido*
Mitoshi Akiyama,*
W. Shay,"
to be shortened with aging. Even candidate human stem cells with the CD34'CD38'"" phenotype demonstrate telomere shortening upon proliferation in vivo and in vitro (7, 8). Thesc findings have been interpreted as being causedby the lack of telomerase activity, a ribonucleoprotein enzyme that uses its own integral RNA as a template for synthesis on the 'ITAGGG telomere rcpcats to compensate for the normal loss of telomeric repcats during cell divisions (9). However,therehasbeen n o convincing evidence exof telomere length in blood plaining the wide range of variance cells between individuals. Until recently, becauseof the low sensitivityof the conventional telomerase assay, telomerase has been considered to exist only in immortal cells (10-12) or germ-line cells, but not in normal somatic cells (1). Recently, a highly sensitive PCR-based method for detection of telomerase activity, named the telomeric repeat am(13). andwe plificationprotocol(TRAP)',hasbeendcveloped (14) and others (15) found that PBMCs from healthy individuals have a very low level of telomerase activity. If this telomerase activity represents rare hematopoictic stem cells that are sufficient to compensate for the loss of telomeric repeats in the stem cells during cell divisions, the phenomenon of telomere shortening in blood cells with aging cannot be easily explained. To clarify the he extreme ends of human chromosomes are protected by mechanism of telomere shorteningin blood cells during aging and simplehexamerictandemlyrepeated('ITAGGG),seto identify the cells that are responsible for the weak telomerase quences, called telomeres (1-3). The length of telomeric activity detected in whole PBMCs, we applied the TRAP assay in repcats in blood cclls (4) a s well as in fibroblasts ( 5 , 6 ) is reported PBMCs from individuals of a wide range of ages and investigated the fraction of PBMCs and bone marrow cells that have telomerase activity. 'Second Department of Internal Medicine, Hiroshima University School of Medicine. Hiroshima, Japan; 'Department of Genetics and *Department of Radiobi-
This is the first report describing up-regulation of telomerase activity in human normal cells. Telomerase, a ribonucleoprotein enzyme, has been thought to be involved in maintaining telomere length stability in germline andmostcancercells, butnot in normal cells. However, in thepresentstudy, we demonstratethat telomerase activityis detectable atlow levels in normal human T and B cells,increasesby in vitro mitogenic stimulation, increases in hematopoietic progenitor cells upontheir proliferation anddifferentiation,anddecreases with aging. Understanding the regulation of telomerase activity in normal cellsmay provide important insights not only into the mechanisms of normal cellular senescence but also into the mechanisms of telomerase activity deregulation as part of cancer develThe Journal of Immunology, 1995, 155: opment. 371 1-371 5.
T
ology, Radiation Effects Research Foundation, Hiroshima, japan; 'Department of
General Medicine, Hiroshima University School oi Medicine, Hiroshima, lapan; .The University of Texas Southwestern Medical Center at Dallas, Department of Cell Biology and Neuroscience, Dallas, TX 75235 Received ior publication luly 1 1 , 100.5. Accepted f o r publication August 16, 1995.
The costs of publication of this article were deirayed in part by the payment of page charges. This article must therefore he hereby marked adverlisernenf in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported by research grants from the lapanese Ministry of Education, Science and Culture. JapaneseMinistry of Health and Weliare, the Ceron Corporation (Menlo Park, CA), and National Institutes of Health Grant AG07992. This publication is bawl upon research performed at the Radiation Effects Research Foundation (RERF) in Hiroshima, and the Second Department of Internal Medicine, Hiroshima University Srhool of Medicine, Japan. RERF is a private, nonproiit foundation funded equally by the Japanese Ministry of Health and Welfare and the United States Department of Energy through the National Academy of Sciences. * Address correspondence and reprint requests to Dr. Keiko Hiyama, The Second Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan Copyright 0 1905 by The American Association of Immunologists
Materials and Methods Samples
Peripheral blood samples were obtained from a total of 124 Japanese individuals ranging from 0 to 86 years old, including 108 healthy adult volunteers and 16 child patients. Among the healthy volunteers,89 individuals who were more than 40 years of age were part of a control group for the Adult Health Study Program (16) in Radiation Effects Research Foundation,Hiroshima,Japan. The child patients wereadmitted to Hiroshima University Hospital with inguinal hernia or other minor diseases without malignancy, infection, or general administration of drugs. With informed consent, blood was drawn for clinical examinations and the remaining portion was usedfor this study. PBMCswereisolated by Ficoll-Hypaque density-gradientcentrifugation from heparinized blood, washedtwicc with
' Abbreviations used in this paper: TRAP, telomeric repeat amplification protocol; PE, phycoerythrin; C-CSF, granulocyte colony stimulating factor; MNC. mononuclear cell. 0022-1 767/95/$02.00
371 2
TELOMERASE ACTIVITY IN BLOOD CELLS Control
aging. Telomerase assays were carried out using extracts corresponding to 3 X lo', IO4, and 3 X 10' PBMCs (and 10' PBMCs for the 1 -year-old child) equivalents for seven individuals with (+) or without (-) RNasepretreatment of theextract.Using diluted extracts of 3 X 10' PBMCs equivalent without RNase pretreatment, many childrenstill showed 6-bptelomeraseladdersextendingfrom 40bp, whereas most adults did not. Lysis buffer and extractsfromtelomerase-positiveimmortal 10 and 100 cells were used as controls.
9
3 3 Y
W Y
3 Y
PBS containing 2.5% FCS (Life Technologies, Grand Island, NY), and samples of 10" cells were stored in a 1.5-ml tube at -XO°C until use. For analysis of lymphocyte subsets, isolated PBMCs were stained with FITC-labeled anti-CD3 and phycoerythrin (PE)-labeled anti-CD25 (IL-2 receptor) mABs. Three lymphocyte subsets, CD3-CD25- ,CD3'CD25-, and CD3'CD25+, were obtained by FACStar sorting (Becton Dickinson lmmunocytometry Systems, San Jose, CA), and stored at -80°C. For analysis of stimulated T cells, isolated PBMCs werecultured in GIT medium (Wako. Osaka, Japan) containing 1:1600 of PHA (Difco, Detroit, MI), 2 ng/ml of recombinant human IL-2 (a gift from Takcda Chemical Industries, Osaka, Japan) and 10% heat-inactivated FCS for I wk. For the analysis of stimulated B cells. isolated PBMCs were cultured with 25 pg/ml of PWM (Life Technologies) for 1 wk. Cultured cells were stained with FITC-anti-CD3 and PE-tlnti-CD19 mABs and CD3 -CDI9' B-cell fraction was collected using FACStar sorting. For analysis of hematopoietic progenitor cells. aliquots of bone marrow cells obtained from a I-year-old boy with hepatoblastoma without metastasis to bone marrow and PBMCs obtained from two non-Hodgkin's lymphoma patients (57 and 62 years old, respectively) who had received 1 week's administration of granulocyte-colony stimulating factor (G-CSF) after chemotherapy were used. These cclls were collcctcd for autologous blood stem cell transplantation and aliquots of them were sorted by FITCanti-CD34 and PE-anti-CD3X mABs. Total bone marrow mononuclear cells from the I-year-old boy and total bone marrow cells from an ;~utopsied female patient without malignancy were also analyzed. All blood s:mples were obtained with informcd consent, and all mAbs used for the present study were purchased from Becton Dickinson Immunocytometry Systems.
TRAP assay Cells for telomerase activity were processed as previously described (1 3, 14, 17). Each blood samplewas treated with ice-cold lysis buffer (IO" PBMCs1100 @I: 0.5% CHAPS, 1 0 mM Tris-HCI (pH 7.5). 1 mMMgCI,, 1 mM EGTA, 10% glycerol, S mM bet;l-mercaptoethanol, 0 . 1 mM AEBSF), incubated on ice for 30 min, centrifuged a t 12,000 g for 20 min at4"C, and the supernatant was stored at -80°C. The TRAP assay was performed as follows: the 1-3 pI aliquots of extracts were assayed in SO pl o f reaction mixture containing 50 p M each o f dNTP, 0 . 1 p g of TS primer (S'-AATCCGTCGAGCAGAGIT-3'), 0.5 p M of T4 gene 32 protein (USB, Cleveland, OH), 1.50 kBq of [a."P]dCTP, and 2 units of Taq DNA polymerase (Wako, Osaka, Japan) in a 0.5-ml HotStart SO tube (Molecular BioProducts, San Diego, CA) which contained 0. I p g of CX primer (S'CCCTTACCCTTACCCTTACCCTAA-3') sequestered at the bottom by a wax barrier. After 30 min of incubation at room temperature. the reaction mixture was heated at 90OC for 90 s and then it was subjected to 31 PCR cycles of 94OC for 40 s, 50°C for 40 s, and 72°C for 45 s. The PCR product was electrophoresed on a 10% acrylamide gel. We classified a sample as telomerase positive when the telomerase specific 6-bp DNA ladder was
3
observed after an overnight exposure, and whose intensity was equivalent to orstronger than that obtained using 1 0 cells of an immortalized human cell line.
Statistical analysis For statistical analysis, 124 individuals without serious diseases were divided into two groups accordingto telomerasc activity: positive o r negative when using a cellular extract derived from 10'' PBMCs equivalcnt/assay ( I O X the standard amount used for detecting telomerase activity in immortal cells; Ref. 13). Age groups included 0 to 1 0 years, 20 t o 39 years, 40 to 5 9 years. 60 to 79 years, and more than X 0 years old, respectively. The positive ratio in each age group was comp;lrcd using the 2 test or Fisher's exact test. where appropriate. Differences were considered statistically significant at p < 0.0.5.
Results and Discussion Telomerase activity in PBMCs with aging
Using an extract derived from 3 X I O " PBMCs equivalent/assay, most butnot all individuals had a detectable 6-bp telomerase ladder. The telomerase activity signal was generally stronger in childrenthanin older individuals(Fig. l). Thespecificity of these telomerase signals was confirmed by the observation thatthe telomerase ladders were abolished by the pretreatment of extracts with RNase. The validityof these telomerase signalswas also confirmed by previous experiments demonstratingthat the telomerase signal was dependent on both CX and TS primers and was not produccd by contamination of genomic DNA (14). When a 1 / 1 0 aliquot of that amount (3 X I O 3 PBMCs equivalentkassay) was used, the telomerase ladder was not observed in most older donors. Among the 124 individuals, 1 0 0 showed telomerase activity using 3 X I O " PBMCs (80.6%) and 55 showed telomerase activity using 10" PBMCs (44.4%)).The percentage of telomerase-positive individuals decreased with age until age 60 (Fig. 2). The positive ratio among 0 to IY-year-old individualsusingextracts derived from I O " PBMCs equivalent per assay was significantly higher than those from groups between the ages of 20 to 39 ( p = 0.02); 40 to 5 0 ( p < 0.0001); 60 to 79 ( p = 0.0003); and from individuals older than 80 ( p = 0.0006) years, respectively. The positive ratio among 20- t o 39-year-old individuals using extracts derived from 1 0 " PBMCs equivalent per assay was also significantlyhigher than those among the individuals older than 40 ( p = 0.04). For patients under the age of 19, telomerase activity was often detected using
371 3
The Journal of Immunology Table I. Telomerase activity in fractionated and/or stimulated blood cells 100
30,000 cells I assay Sample
E3 10,000 cells I assay 80
60
PBMCs Sorted with anti-CD3 and antiLCD25 mABs 36 y Total PBMCs CD3 -/CD25 CD3 'ICD25CD3+/CD25+ Culture with PHA and IL2" 51 y before culture after culture 61 y before culture after culture 73 y before culture after culture Culture with PWM" 27 y before culture, total before culture, CD3-CD19+ after culture, total after culture, CD3 CD19 ' Hematopoietic progenitor cells Sorted with antiLCD34 and anti-CD38 mABs MNCs 1 y Total in BM CD34'38 ~/"'.*\' of BM CD34+38' of EM PBMCs of 57 y CD34+38+ 62 y CD34+38' PBMCs of PBMCs of CD34 t38"10W Total cells in BM 53 y BM (6 p g of protein) ~
40
20
n "
0
19 (n = 16)
20
-
39
(n = 19)
40
- 59
(n =32)
60
-
79
(n = 4 2 )
80 - (Y) (n = 15)
Relationship between the detection ratios of positive telomerase activity in PBMCs and aging. Using extracts corresponding to 3 X I O 4 and lo4 PBMCs respectively (30 X and 10 X the standard amount used for detecting telomerase activity in immortal cells; Ref. 13), telomerase activity was detected i n all (100%) and i n 15 (93.8%) individuals among 0 to 19-year-olds; in 17 (89.5%) and 11 (57.9%) individuals among the 20- to 39-year-olds; in 25 (78.1%) and 9 (28.1%) individuals among the 40- to 59-year-olds; in 31 (73.8%) and 15 (35.7%) individuals among the 60- to 79-year-olds; and 1 1 (73.3%) and 5 (33.3%) among individuals older than 80 years old. The positive ratio of telomerase activity decreases with aging until 60 years old, but not in more elderly individuals.
FIGURE 2.
extracts derived from 3 X IO3 PBMCs equivalent, and for seven children, it wasdetected using only lo3 PBMCs (43.8%). For adults, only one individual (a 26-year-old woman) showed telomerase activity using this number of cells (0.9%, p < 0.0001). Telomerase activity in fractionated or stimulated PBMCs and bone marrow cells
We determined the relative telomerase activity in samples by estimating the cell numbers required to obtain the telomerase ladder of equivalent intensity to that obtained using 10 cells of a telomerase-expressing immortalized human mammary epithelial cell line (Table 1). Thus, when telomerase activity could be detected using lo4 cells, the activity was estimated as 0.1% of that of immortal cells. Each sample was examined in duplicate or triplicate by the TRAP assay on different occasions and the reproducibility was confirmed (data not shown). When PBMCs were fractionated with anti-CD3 and anti-CD25 mABs, each fraction of CD3 CD25-, CD3+CD25-, and CD3+CD25+ had comparable telomerase activity (Fig. 3A). This finding indicates that the telomerase activity in blood cells is not derived from only a small fraction with a specific phenotype, such as hematopoietic stem cells, but from both T cells, activated and nonactivated, and non-T cells. Although the decrease of telomeraseactivity in PBMCs was observed with aging in vivo, when PBMCs from three older individuals were stimulated by a 1-wk culture with PHA and IL2 in vitro, telomerase activity elevated 300- to 1000-fold or more (Fig. 3B). Since it is not likelythata small population of cells with telomerase activity could have proliferatedto account for this large increase within 1 wk, this result indicates that the telomerase activity in each, or at least a significant fraction of T cells, increased by mitogenic stimulation. While the telomerase activity in the B cell fraction with the CD3-CD19+ phenotype was not detected using 3 X lo4 cells per assay, it wasdetectedusing 1000
Telomerase Activity ("4"
0.1 0.3 0.1 0.3
