Differentiation of immortal cells inhibits telomerase activity

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USA. Vol. 92, pp. 12343-12346, December 1995. Cell Biology. Differentiation of immortal cells inhibits telomerase activity. (cancer/telomeres/ribonucleoprotein).
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12343-12346, December 1995 Cell Biology

Differentiation of immortal cells inhibits telomerase activity (cancer/telomeres/ribonucleoprotein)

HARSH W. SHARMA*, JOHN A. SOKOLOSKIt, JOSE R. PEREZ*, JEAN YVES MALTESE*, ALAN C. SARTORELLIt, C. A. STEINt, GWEN NICHOLSt, ZAHANGIR KHALEDt, NITIN T. TELANG§, AND RAMASWAMY NARAYANAN*¶ *Division of Oncology, Roche Research Center, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110; tDepartment of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520; tDepartment of Medicine, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032; and §Strang Cornell Cancer Research Laboratory, 510 East 73rd Street, New York, NY 10021

Communicated by John J. Burns, Roche Institute of Molecular Biology, Nutley, NJ, September 18, 1995

ABSTRACT Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, downregulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

Thus, terminally differentiated cells would be expected to lose or repress telomerase activity. To gain further insight into the function(s) of telomerase, it is important to elucidate the mechanisms that regulate its expression and activity. Since only rare somatic cells express telomerase activity and few assays for telomerase activity are available, this is a formidable task. As an initial step, we examined telomerase activity in immortalized cell lines that have the ability to differentiate into more mature cells. The present report demonstrates that the induction of differentiation is associated with the loss of telomerase activity and that immortalized, differentiation-competent cells are a useful system for study of the mechanism(s) regulating telomerase activity.

MATERIALS AND METHODS Cell Lines. The cell lines used were HL-60 human promyelocytic leukemia cells (12), K-562 human erythroid leukemia cells (13), F9 murine teratocarcinoma cells (14), CCE-24 murine embryonal stem cells (15), SW480 human colon carcinoma cells (16), and 293 immortal human kidney cells (8). All cell lines were maintained as described in the indicated references. Chemicals and Growth Factors. 12-O-Tetradecanoylphorbol 13-acetate (TPA), all-trans-retinoic acid (RA), sodium butyrate, and dimethyl sulfoxide (DMSO) were obtained from Sigma. 1,25-Dihydroxyvitamin D3 (vitamin D3) was obtained from Hoffmann-La Roche. Leukemia inhibitory factor (LIF) was obtained from Upstate Biotechnology. Telomerase Assay. Cells were treated with the inducers of differentiation for various lengths of time, and total cellular extracts were made according to the protocol of Kim et al. (8). The methods we utilized for nuclear and cytoplasmic extracts and for performing the electrophoretic mobility shift assay for NF-KB and Spl have been described (15). Telomerase activity was measured by the PCR-based TRAP assay as described (8, 17), and the products were separated by 10% PAGE, dried, and autoradiographed. For each TRAP assay, the following three controls were included: (i) omitting nontelomeric primer, TS (8), (ii) 1 ,ug of CHAPS extract from 293 cells, and (iii) RNase A (200 ,ug/ml) pretreated 293 CHAPS extract. The basal level of telomerase activity (ladder formation) was measured by serial dilution (10 fold) of the protein extracts from each cell line, and an appropriate range of protein concentrations was selected that produced a linear response.

The ends of eukaryotic chromosomes, called telomeres, consist of an array of tandem repeats of the hexanucleotide 5'TTAGGG-3'. It is currently assumed that telomeres were evolved to protect the ends of chromosomes against exonucleases and ligases, to prevent the activation of DNA-damage checkpoints, and to counter the loss of terminal DNA segments that occurs when linear DNA is replicated (for a review, see refs. 1-6). At least the last-mentioned function depends upon a ribonucleoprotein enzyme called telomerase, which uses RNA as a template to add the hexanucleotide to the ends of replicating chromosomes (2). The telomerase activity has been detected in human cell cytosolic extracts by a primer extension assay (7) and more recently by a very sensitive assay (telomeric repeat amplification protocol; TRAP), which is based upon PCR amplification of the initial telomerase product using detergent {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPS}-disrupted total cell lysates (8). Telomerase is found in the developing Xenopus up to the neurula stage (9). In humans it is present in fetal and adult testes and in ovarian follicles (8) and in certain somatic tissues, albeit at very low levels (10, 11). In the majority of somatic cell types the enzyme is either not expressed or is repressed (8). These findings suggest that telomerase is active in germ cells and in rare somatic cells with a high proliferative potential.

Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate; DMSO, dimethyl sulfoxide; ES, embryonal stem; LIF, leukemia inhibitory factor; RA, all-trans-retinoic acid; TPA, 12-0-tetradecanoylphorbol 13-acetate; TRAP, telomeric repeat amplification protocol; vitamin D3, 1,25-dihydroxyvitamin D3. ITo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Each experiment was repeated two to five independent times with cells at various passages. Induction of Differentiation. HL-60 cells were induced to differentiate by exposure to either 1.3% DMSO, 1 ,uM RA, or 100 ng of vitamin D3 per ml for 7 days or to 200 ng of TPA per ml for 3 days. Differentiation was assessed by the attainment of nitroblue tetrazolium positivity and of CD11b (M01) integrin expression as described (12). K-562 cells were treated with 1 mM sodium butyrate for 4-6 days, and the extent of differentiation was monitored by benzidine staining (18). F9 teratocarcinoma cells were treated with 1 ,tM RA for 3-12 days, and differentiation was assessed by microscopic observation of changes in cellular morphology (14). The murine embryonal stem (ES) cells were induced to mature by withdrawal of the differentiation-inhibitory agent LIF for 3-12 days. The state of differentiation was assessed microscopically by changes in cellular morphology as well as by monitoring the cytokine expression profiles (15).

RESULTS Loss of Telomerase Activity in Differentiated Leukemia Cells. HL-60 cells cultured in the absence of inducing agents had significant telomerase activity. Differentiation of HL-60 cells to granulocyte-like cells occurred in the presence of 1.3% DMSO or 1 /.LM RA; these cells ceased to grow and became both nitroblue tetrazolium and M01 (CD11b) positive. These phenotypic changes were accompanied by a marked decrease in telomerase activity (Fig. 1A). A similar reduction of telomerase activity was also observed after the differentiation of HL-60 cells into mature monocyte-like cells by exposure to either 100 ng of vitamin D3 per ml for 7 days or 200 ng of TPA per ml for 3 days (Fig. 1A). Treatment of HL-60 cells with TPA for 1 day or with DMSO, RA, or vitamin D3 for up to 3 days did not result in a change in telomerase activity. On the other hand, at day 5 of treatment with DMSO, RA, or vitamin D3, the inhibition of telomerase activity was identical to that on day 7. The inhibition of telomerase activity correlated with induction of growth arrest, the attainment of a differentiated state, and the blockage of cells at the G, phase of the cell cycle (data not shown). These findings in HL-60 cells were extended to K-562, an erythroid leukemia cell line. Treatment of K-562 cells with 1 mM sodium butyrate for 6 days resulted in a pronounced inhibition of telomerase activity (Fig. 1B), whereas exposure to sodium butyrate for 4 days caused a smaller decrease in telomerase activity, in a manner that correlated with the extent of differentiation. Sodium butyrate did not directly inhibit telomerase activity at a concentration of 1 mM. These results indicate that the terminal differentiation of leukemia cells is associated with the downregulation of telomerase activity. Loss of Telomerase Activity in Differentiating Murine Embryonal Cells. We next tested telomerase activity in murine embryonal cell systems (ES cells and F9 teratocarcinoma cells) before and after the induction of differentiation by withdrawal of LIF and treatment with RA, respectively (Fig. 2). In the ES cell system, telomerase activity was completely abolished upon attainment of a differentiated state at 12 days (Fig. 2A). Shorter periods of withdrawal of LIF (up to 6 days) had no effect on telomerase activity. Analogous results were obtained with F9 teratocarcinoma cells treated with RA (Fig. 2A). Morphological changes of ES and F9 cells indicative of a differentiated state with parietal endodermal properties are shown in Fig. 2B. Loss of Telomerase Activity by Differentiation Inducers Is Cell Mediated. We next addressed whether inhibition of telomerase activity by the diverse chemical agents used in this study resulted from a direct inhibition of telomerase activity (Fig. 3). Extracts from 293 cells were pretreated with inducing agents and analyzed for telomerase activity. None of the agents

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K-562 FIG. 1. Downregulation of telomerase activity in differentiated leukemia cells. (A) Increasing amounts of CHAPS extract from HL-60 cells (0.01, 0.1, 1.0, and 3.0 txg of protein), either untreated or treated with 1.3% DMSO, 1 j.LM RA, or 100 ng of vitamin D3 per ml for 7 days or with 200 ng of TPA per ml for 3 days, were analyzed for telomerase activity by the TRAP assay. - TS, control for the TRAP assay, without the nontelomeric primer; + 293, 1 jig of CHAPS extract from 293 cells as a positive control for the TRAP assay; + RNase A, 293 extract pretreated with RNase A (200 jLglml) to inactivate the RNA component of telomerase; VIT-D3, vitamin D3. (B) Increasing amounts of the CHAPS extract from K-562 cells (0.001, 0.01, 0.1, 0.5, and 1.0 gg) from either control (undifferentiated; - Diff.) or 6 day differentiated (+ Diff.) with 1 mM sodium butyrate were analyzed for telomerase activity as in A.

employed inhibited telomerase activity (Fig. 3A). Treatment of 293 cells or SW480 colon carcinoma cells with DMSO or RA (which do not cause differentiation in either of these cell lines) for 7 days did not result in morphological alterations or inhibition of telomerase activity despite growth inhibition (data not shown). Similarly, serum deprivation of 293 cells for 72 hr, which resulted in significant growth inhibitio-n and withdrawal from the cell cycle, did not inhibit telomerase activity (data not shown). Fig. 3B shows that the telomerase activity in diverse cell lines was sensitive to RNase A treatment, thereby establishing the specificity of the measurement of telomerase activity, which is catalyzed by a ribonucleoprotein

complex. The possibility that a heat-labile repressor or inhibitor of telomerase activity was present in differentiated cell extracts was next investigated (Fig. 3C). Various amounts of DMSOdifferentiated HL-60 cell extracts (with or without prior heat inactivation) were preincubated with a fixed amount of a 293 cell extract, and telomerase activity was measured by the TRAP assay. Extracts from fully differentiated HL-60 cells did not significantly inhibit the telomerase activity of 293 cells, suggesting that no repressor molecule(s) was present in differentiated HL-60 cells. The telomerase activity in the fractionated cell lysates (nuclear and cytoplasmic) of differentiated and undifferentiated cells was also measured. Fig. 4A shows that both cyto-

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Proc. Natl. Acad. Sci. USA 92 (1995)

differentiated HL-60 cells, no telomerase activity was detected in either fraction, corroborating the results obtained with total cell lysates (see Fig. 1A). The same cytoplasmic and nuclear extracts of HL-60 cells (both undifferentiated and DMSOdifferentiated cells) showed high levels of the transcription factor NF-KB activity. The cytoplasmic extracts showed a high basal level of NF-KB activity. In contrast, when the same extracts were analyzed for the transcription factor Spl, which is localized in the nucleus and is cell-cycle regulated (19), the differentiated HL-60 cells showed a complete inhibition of Spl activity in the nucleus (Fig. 4B). No Spl activity was detectable in the cytoplasmic extracts. Identical results were obtained with 12-day differentiated F9 teratocarcinoma cells (data not shown).

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DISCUSSION The present study demonstrates that immortalized cell lines that retain the ability to differentiate may be used as models to study the regulation of telomerase activity in normal somatic cells. In two human hematopoietic cell lines and two murine embryonal cell lines, the telomerase activity was lost after terminal differentiation was induced by exposure to DMSO, RA, or sodium butyrate or by the withdrawal of the cytokine LIF from the culture medium. These findings establish a strong link between telomerase activity and the state of differentiation of these cell lines. At present it is not clear whether induction of differentiation abolished telomerase gene expression or inhibited its enzymatic activity. In investigations by others (8), no inhibitors of telomerase activity were detected when extracts of telomerase-negative and telomerase-positive cells were mixed. Correspondingly, in the present study, no inhibitor of telomerase activity was detected in the differentiated cells; moreover, none of the differentiation-inducing agents themselves directly inhibited

FIG. 2. Murine embryonal cell differentiation is associated with the downregulation of telomerase activity. (A) Murine ES cells were differentiated by removal of LIF for 12 days or left undifferentiated by growth in LIF-containing medium; F9 teratocarcinoma cells were differentiated by treatment with 1 ALM RA for 12 days. CHAPS extracts were made from these cells, and increasing amounts of protein (0.01, 0.1, 0.5, and 1.0 jig) were analyzed as described in Fig. 1A. (B) Photomicrographs of undifferentiated and differentiated ES and F9 cells before and after the induction of differentiation. (x30.)

plasmic and nuclear extracts of undifferentiated HL-60 cells had similar levels of telomerase activity. In the DMSO-

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