Oncogene (2002) 21, 3270 ± 3273 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

Replicative of hematopoietic stem cells during serial transplantation: does shortening play a role?

Richard C Allsopp*,1 and Irving L Weissman1

1Department of Pathology, Stanford University School of Medicine, Stanford, California, CA 94305, USA

Hematopoietic stem cells (HSC) have a ®nite prolif- Shamblott et al., 2001), and most advanced tumor cells erative lifespan, based upon the limited number of times in (Kim et al., 1994), carries out the they can be serially transplanted in mice. have complete replication of telomeres. The genetic ablation been shown to shorten during the division of many or mutation of the essential RNA component of normal somatic cells in humans, and the attrition of telomerase (Singer and Gottschling, 1994; Ahmed et telomeres has been shown to ultimately cause replicative al., 1998; Niida et al., 1998) or inhibition of telomerase senescence in vitro for a number of di€erent cell by small molecules (Herbert et al., 1999) in these cells strains. Whereas most human cell types have little to no results in the gradual reduction of the average telomere detectable levels of telomerase activity, hematopoietic length as these cells divide. cells, including HSC, express low to moderate levels of In adult humans, most cells, including, but not telomerase, and yet telomeres shorten considerably limited to, ®broblasts (HDF) (Kim et al., 1994), during replicative aging of these cells. Here we consider neurons (Kruk et al., 1996), kidney cells (Counter et the role telomerase may play in the hematopoietic system al., 1992), endothelial cells (Kim et al., 1994), retinal as well as the e€ect that over-expression of telomerase pigment epithelial cells (Bodnar et al., 1998) and human reverse transcriptase may have on the replicative mammary epithelial cells (HMEC) (Stampfer et al., capacity of hematopoietic stem cells during transplanta- 1997) have no detectable telomerase, and, for those tion. cells that are mitotically active, the telomeres shorten Oncogene (2002) 21, 3270 ± 3273. DOI: 10.1038/sj/ with each (Harley et al., 1990; Chang and onc/1205314 Harley, 1995; Bodnar et al., 1998; Romanov et al., 2001) (Figure 1). The average rate of telomere short- Keywords: telomere; hematopoietic stem cell; senes- ening in these cells (Harley et al., 1990; Counter et al., cence; transplantation 1992; Bodnar et al., 1998; Yang et al., 1999), and in mouse cells that lack functional telomerase (Niida et al., 1998), is 50 ± 150 base pairs/population doubling (bp/pd). The reason telomeres shorten at this rate is not entirely known, but is likely due to the apparent Telomere shortening and senescence in the absence of inability of the normal DNA replication machinery to telomerase completely replicate the telomeric sequences at the very end of each telomere (Levy et al., 1992; Lingner et al., Nearly all normal eukaryotic cells require telomerase, 1995). Another potential contributing factor is the an enzymatic complex composed of RNA and protein accumulation of single-strand breaks, caused by (Greider and Blackburn, 1987), for the complete oxidative damage, in the telomeric DNA (von Zglinicki replication of telomeres, genetic elements which et al., 1995). When these cells are cultured in vitro, provide a protective cap at the ends of telomere shortening continues until the cells reach (McClintock, 1941) (Figure 1). Telomerase is a replicative senescence, or the Hay¯ick limit, the point specialized reverse transcriptase, the RNA component at which cells can no longer proliferate even in the of which contains a short motif with a sequence presence of mitogens (Hay¯ick and Moorhead, 1961). identical to the telomeric DNA sequence, thereby Recently, in several independent studies, researchers allowing the synthesis of new telomeric DNA onto have introduced, using gene expression constructs, the ends of chromosomes (McEachern et al., 2000) telomerase reverse transcriptase (TERT), the catalytic (Figure 1). In immortal cell populations, including component of telomerase into HDF, retinal pigment lower eukaryotes such as yeast (Cohn and Blackburn, epithelial cells, and endothelial cells in vitro, and in all 1995) and ciliates (Greider and Blackburn, 1985), cases, telomerase activity was restored, telomere length murine embryonic stem cells (Niida et al., 1998), germ was maintained during culture, and replicative senes- cells (Allsopp et al., 1992; Mantell and Greider, 1994; cence was postponed inde®nitely (Bodnar et al., 1998; Yang et al., 1999; Ramirez et al., 2001). These observations show that telomere shortening can, in *Correspondence: R Allsopp; E-mail: [email protected] some cells, cause replicative senescence. Telomere shortening in transplanted hematopoietic stem cells RC Allsopp and IL Weissman 3271 Telomere shortening and senescence in the presence of telomerase

Low levels of telomerase activity are present in a few human cell types and tissues, including keratinocytes (Yasumoto et al., 1996), the basal layer of the epidermis (Harle-Bachor and Boukamp, 1996), and hematopoietic cells (Broccoli et al., 1995; Hiyama et al., 1995). Like other human somatic cells grown in vitro, telomere length continuously decreases in keratinocytes, at a rate of *50 ± 100 bp/pd until replicative senescence is reached (Stoppler et al., 1997). Also, ectopic expression of TERT in keratinocytes grown on feeder layers allows increased telomerase activity and prevention of replicative senes- cence (Ramirez et al., 2001). Telomerase is readily detectable in lymphocytes (Broccoli et al., 1995), and even higher in progenitor cells (Hiyama et al., 1995; Chiu et al., 1996) and hematopoietic stem cells (HSC) (Morrison et al., 1996). Despite this, telomeres undergo extensive shortening during proliferation of lymphocytes in vitro (Guerrini et al., 1993; Vaziri et al., 1993; Bodnar et al., 1996; Rufer et al., 2001) and in peripheral blood leukocytes during aging in vivo (Hastie et al., 1990; Vaziri et al., 1993). Moreover, the rate of telomere shortening for T lymphocytes cultured in vitro (Vaziri et al., 1993; Rufer et al., 2001) is comparable to that observed for other human cell strains that lack telomerase (50 ± 100 bp/ pd). Thus telomerase is readily detectable in the hematopoietic system but is unable to carry out its function of complete telomere replication (see Figure 1). At present, the reason for the presence of telomerase in these cells is poorly understood. Lymphocytes have a limited lifespan when cultured in vitro (E€ros, 1998), and replicative senescence is prevented and telomere length stabilized in T lymphocytes that over-express TERT (Hooijberg et al., 2000; Rufer et al., 2001). Thus one possible explanation for the presence of telomerase in hematopoietic cells is to partially counter what would otherwise be a relatively rapid rate of telomere

telomere replication is carried out by telomerase. For simplicity, only the essential RNA component (TR) and catalytic component of telomerase, telomerase reverse transcriptase (TERT), are shown. Telomerase may or may not remain associated with the telomere once replication of the telomere is completed. (ii) In most normal somatic cells in adult humans, telomerase is absent. As a result, replication of the end of the telomere cannot be completed and the average telomere length is slightly reduced (unreplicated telomeric DNA indicated by hatched box) after every round of cell division. The RNA component of telomerase is expressed in these cells, allowing full reconstitution of Figure 1 Structure and replication of telomeric DNA in human telomerase upon ectopic expression of TERT. (iii) There are also cells. (a) Telomeric DNA for humans, and other vertebrates, is a few mitotically active types, including hematopoie- composed of tandem repeats of TTAGGG (solid box) that can tic cells, in adult humans that express telomerase, yet, for vary in length from less than 500 base pairs (bp) in some human unknown reasons, complete replication of the telomere does not tumor lines (Counter et al., 1992) to over 100 kbp in some inbred occur. Ectopic expression of TERT in these cells also allows the mice strains (Kipling and Cooke, 1990). Capping the very end of complete replication of the telomere (Hooijberg et al., 2000; Rufer the telomere is a 3' overhang that is 150 ± 200 bp in length et al., 2001). This suggests that telomeres may shorten during (Wright et al., 1997). The overhang is capable of folding back division of these cells because of the presence of a limiting (indicated by arrow) and stably interacting with internal telomeric suppressor of telomerase activity, or competition between re- sequences to form a protective loop at the end of the telomere assembly of telomeric chromatin and the protective loop, and (Grith et al., 1999). (b) (i) In immortal cell populations, extension of the telomere by telomerase

Oncogene Telomere shortening in transplanted hematopoietic stem cells RC Allsopp and IL Weissman 3272 shortening and a deleteriously brief replicative lifespan et al., 1997). Third, we have recently shown that for these cells. However, activation of T lymphocytes in telomere length decreases in both donor-derived cells vitro is immediately followed by a brief period in which and HSC after two rounds of transplantation of HSC telomerase activity is increased and telomere length is in mice (Allsopp et al., 2001). We have now developed transiently stabilized (Bodnar et al., 1996; Rufer et al., a transgenic mouse strain that over-expresses murine 2001). Furthermore, we have recently demonstrated TERT throughout the hematopoietic system, including that activation of donor-derived murine T lymphocytes HSC, to directly assess whether maintenance of from secondary HSC transplant recipients, which have telomere length during HSC division can extend the relatively short telomeres (Allsopp et al., 2001), results capacity for serial transplantation. We have not in an increase in telomerase activity and a observed any telomere shortening in donor-derived of telomere length to approximately the same size cells after three rounds of serial transplantation of observed for resting T lymphocytes from adult mice HSC from these mice, and are continuing with (our unpublished observations). While it remains to be subsequent rounds of transplantation to assess whether shown whether mitogenic activation of other hemato- replicative capacity is extended in these cells (our poietic cells can lead to an increase in telomerase unpublished observations). activity and telomere length stabilization, a perhaps If extension of replicative lifespan via telomerase more attractive hypothesis for the function of telomer- gene therapy is warranted, it will be important to ase in some hematopoietic cells is to allow a transient consider the safety issues. Given the high frequency of maintenance or regeneration of telomere length, and telomerase activation in tumors, and the recent therefore an extension of replicative lifespan, speci®- demonstration that human ®broblasts and epithelial cally in cells that have a long-term replicative demand. cells, into which the SV40 T antigen and H-ras Experiments involving inhibition of telomerase or expression constructs have been introduced, can be genetic ablation of essential components in cultured made fully tumorigenic upon activation of telomerase human hematopoietic cells should help elucidate the by TERT over-expression (Hahn et al., 1999), it will be role of telomerase in these cells. important to assess the long-term e€ect of over- expression of TERT in HSC before any clinical studies are considered. The e€ect of immortalization of normal Does telomere shortening limit the replicative capacity of somatic cells via TERT over-expression on the long- hematopoietic stem cells? term genomic stability is also an important considera- tion. After extensive growth of HDF immortalized Hematopoietic stem cells can only be serially trans- with TERT, recent studies have not detected aberrant planted 5 ± 7 times in mice, indicating that HSC have a growth in vitro or any indication of tumor formation ®nite replicative capacity (Harrison et al., 1978; when injected into nude mice (Jiang et al., 1999; Vaziri Harrison and Astle, 1982). However, HSC also under- et al., 1999), although a low frequency of chromosomal go continuous self-renewal and di€erentiation to translocations were transiently observed during growth provide a continuous supply of hematopoietic cells of some TERT-immortalized HDF clones (Vaziri et al., throughout the organismal lifespan (Cheshier et al., 1999). To prevent any predisposition to cancer and 1999), and therefore must have a substantial replicative maintain long-term genomic stability, it may be capacity. In bone marrow or HSC transplant recipi- necessary to utilize an inducible TERT expression ents, the continuous and prompt supply of blood cells construct which can be turned on and o€, to allow post-transplant is essential, thus the question as to regeneration of telomere length at timed intervals. It whether telomere shortening limits the replicative may also eventually be possible to develop pharma- capacity of HSC is of general clinical interest. ceutical methods to increase telomerase activity in A number of studies have demonstrated that HSC, once more is known regarding how telomerase telomeres do shorten during replicative aging of HSC. activity is regulated in the hematopoietic system. First, telomere length was found to be shorter for candidate HSC from the marrow of two adult donors Acknowledgments compared to candidate HSC from fetal cord blood WewouldliketothankAntonioCozzioforhelpful (Vaziri et al., 1994). Second, an accelerated decrease in comments. We would like to thank the National Institutes telomere length has been observed for donor-derived of Health (grant #CA 86065 (ILW)) and the Irvington cells from bone marrow transplant recipients (Notaro Institute for Immunological Research for their support.

References

Ahmed S, Sheng H, Niu L and Henderson E. (1998). Bodnar AG, Kim NW, E€ros RB and Chiu CP. (1996). Exp. Genetics, 150, 643 ± 650. Cell. Res., 228, 58 ± 64. Allsopp RC, Cheshier S and Weissman IL. (2001). J. Exp. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Med., 193, 917 ± 924. Morin GB, Harley CB, Shay JW, Lichtsteiner S and Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai Wright WE. (1998). Science, 279, 349 ± 352. EV, Futcher AB, Greider CW and Harley CB. (1992). Broccoli D, Young JW and de Lange T. (1995). Proc. Natl. Proc. Natl. Acad. Sci. USA, 89, 10114 ± 10118. Acad.Sci.USA,92, 9082 ± 9086.

Oncogene Telomere shortening in transplanted hematopoietic stem cells RC Allsopp and IL Weissman 3273 Chang E and Harley CB. (1995). Proc. Natl. Acad. Sci. USA, Lingner J, Cooper JP and Cech TR. (1995). Science, 269, 92, 11190 ± 11194. 1533 ± 1534. Cheshier SH, Morrison SJ, Liao X and Weissman IL. (1999). Mantell LL and Greider CW. (1994). EMBO J., 13, 3211 ± Proc. Natl. Acad. Sci. USA, 96, 3120 ± 3125. 3217. Chiu CP, Dragowska W, Kim NW, Vaziri H, Yui J, Thomas McClintock B. (1941). Genetics, 26, 234 ± 282. TE, Harley CB and Lansdorp PM. (1996). Stem Cells, 14, McEachern MJ, Krauskopf A and Blackburn EH. (2000). 239 ± 248. Annu. Rev. Genet., 34, 331 ± 358. Cohn M and Blackburn EH. (1995). Science, 269, 396 ± 400. Morrison SJ, Prowse KR, Ho P and Weissman IL. (1996). Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Immunity, 5, 207 ± 216. Greider CW, Harley CB and Bacchetti S. (1992). EMBO Niida H, Matsumoto T, Satoh H, Shiwa M, Tokutake Y, J., 11, 1921 ± 1929. Furuichi Y and Shinkai Y. (1998). Nat. Genet., 19, 203 ± E€ros RB. (1998). Am.J.Hum.Genet.,62, 1003 ± 1007. 206. Greider CW and Blackburn EH. (1985). Cell, 43, 405 ± 413. Notaro R, Cimmino A, Tabarini D, Rotoli B and Luzzatto Greider CW and Blackburn EH. (1987). Cell, 51, 887 ± 898. L. (1997). Proc. Natl. Acad. Sci. USA, 94, 13782 ± 13785. Grith JD, Comeau L, Rosenfeld S, Stansel RM, Bianchi A, Ramirez RD, Morales CP, Herbert BS, Rohde JM, Passons Moss H and de Lange T. (1999). Cell, 97, 503 ± 514. C, Shay JW and Wright WE. (2001). Genes Dev., 15, 398 ± Guerrini AM, Camponeschi B, Ascenzioni F, Piccolella E 403. and Donini P. (1993). Hum. Mol. Genet., 2, 455 ± 460. Romanov SR, Kozakiewicz BK, Holst CR, Stampfer MR, Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Haupt LM and Tlsty TD. (2001). Nature, 409, 633 ± 637. Brooks MW and Weinberg RA. (1999). Nature, 400, 464 ± Rufer N, Migliaccio M, Antonchuk J, Humphries RK, 468. Roosnek E and Lansdorp PM. (2001). Blood, 98, 597 ± Harle-Bachor C and Boukamp P. (1996). Proc. Natl. Acad. 603. Sci. USA, 93, 6476 ± 6481. Shamblott MJ, Axelman J, Little®eld JW, Blumenthal PD, Harley CB, Futcher AB and Greider CW. (1990). Nature, Huggins GR, Cui Y, Cheng L and Gearhart JD. (2001). 345, 458 ± 460. Proc. Natl. Acad. Sci. USA, 98, 113 ± 118. Harrison DE and Astle CM. (1982). J. Exp. Med., 156, Singer MS and Gottschling DE. (1994). Science, 266, 404 ± 1767 ± 1779. 409. Harrison DE, Astle CM and Delaittre JA. (1978). J. Exp. Stampfer MR, Bodnar A, Garbe J, Wong M, Pan A, Med., 147, 1526 ± 1531. Villeponteau B and Yaswen P. (1997). Mol. Biol. Cell, 8, Hastie ND, Dempster M, Dunlop MG, Thompson AM, 2391 ± 2405. Green DK and Allshire RC. (1990). Nature, 346, 866 ± 868. Stoppler H, Hartmann DP, Sherman L and Schlegel R. Hay¯ick L and Moorhead PS. (1961). Exp. Cell Res., 25, (1997). J. Biol. Chem., 272, 13332 ± 13337. 585 ± 621. Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley HerbertB,PittsAE,BakerSI,HamiltonSE,WrightWE, CB and Lansdorp PM. (1994). Proc. Natl. Acad. Sci. USA, Shay JW and Corey DR. (1999). Proc. Natl. Acad. Sci. 91, 9857 ± 9860. USA, 96, 14276 ± 14281. Vaziri H, Schachter F, Uchida I, Wei L, Zhu X, E€ros R, Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Cohen D and Harley CB. (1993). Am.J.Hum.Genet.,52, Piatyszek MA, Shay JW, Ishioka S and Yamakido M. 661 ± 667. (1995). J. Immunol., 155, 3711 ± 3715. Vaziri H, Squire JA, Pandita TK, Bradley G, Kuba RM, Hooijberg E, Ruizendaal JJ, Snijders PJ, Kueter EW, Zhang H, Gulyas S, Hill RP, Nolan GP and Benchimol S. Walboomers JM and Spits H. (2000). J. Immunol., 165, (1999). Mol. Cell. Biol., 19, 2373 ± 2379. 4239 ± 4245. von Zglinicki T, Saretzki G, Docke W and Lotze C. (1995). Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage Exp. Cell. Res., 220, 186 ± 193. M,BeecheM,BodnarAG,WahlGM,TlstyTDandChiu Wright WE, Tesmer VM, Hu€man KE, Levene SD and Shay CP. (1999). Nat. Genet., 21, 111 ± 114. JW. (1997). Genes Dev., 11, 2801 ± 2809. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, YangJ,ChangE,CherryAM,BangsCD,OeiY,BodnarA, Ho PL, Coviello GM, Wright WE, Weinrich SL and Shay Bronstein A, Chiu CP and Herron GS. (1999). J. Biol. JW. (1994). Science, 266, 2011 ± 2015. Chem., 274, 26141 ± 26148. Kipling D and Cooke HJ. (1990). Nature, 347, 400 ± 402. YasumotoS,KunimuraC,KikuchiK,TaharaH,OhjiH, Kruk PA, Balajee AS, Rao KS and Bohr VA. (1996). Yamamoto H, Ide T and Utakoji T. (1996). Oncogene, 13, Biochem. Biophys. Res. Commun., 224, 487 ± 492. 433 ± 439. Levy MZ, Allsopp RC, Futcher AB, Greider CW and Harley CB. (1992). J. Mol. Biol., 225, 951 ± 960.

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