¹INSERM U496, Centre G Hayem, Hôpital Saint-Louis, Paris, France

²Université Es-Sénia, Faculté des Sciences, Département de Biologie, Oran, Algérie

Correspondence to: E Ségal-Bendirdjian, INSERM U496, Centre G Hayem, Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75010, Paris, France; Fax: 33 (0)1 42-40-95-57

Human telomerase, a cellular reverse transcriptase specifically activated in most malignant tumors and usually inactive in normal somatic cells, plays an important role in immortalization and tumorigenesis. Early reports have indicated that terminal differentiation of various cells is associated with a rapid inhibition of telomerase activity, preceded by a down-regulation of telomerase reverse transcriptase (hTERT) mRNA. Recently, we have shown that telomerase can be repressed by all-trans retinoic acid (ATRA) independently of terminal maturation during long-term ATRA treatment of the maturation-resistant promyelocytic leukemia cell line (NB4-R1), leading to shortening of telomeres and cell death, events overcome by ectopic hTERT expression. Here, we report the isolation of a variant of NB4-R1 cells (NB4-R1^SFD), which bypasses this death step, because of a re-activated telomerase, despite the continuous presence of ATRA. While unresponsive to a long-term maturation independent regulation of telomerase by ATRA, these cells retain a functional pathway of telomerase down-regulation associated with retinoid-induced maturation. These findings reinforce the notion that two distinct pathways of telomerase regulation by retinoids co-exist in APL cells. Noteworthy, we show that the slow developing mechanism, that causes death of maturation-resistant cells, is subjected to a new type of retinoid-resistance as yet not understood.

Leukemia (2002) 16, 826-832. DOI: 10.1038/sj/leu/2402470

retinoids; differentiation; resistance; APL; telomerase; telomeres; NB4-R1^SFD

Introduction

Human telomerase, a multicomponent ribonucleoprotein enzyme that extends chromosome ends with (TTAGGG)n telomeric repeat sequences^1,2,3 is usually inactive in normal somatic cells but is specifically activated in most malignant tumours. Therefore, this enzyme is a challenging target for drug development. It is composed of a template RNA component (hTR)⁴ and two proteins, telomerase-associated protein-1 (TP1),⁵ and telomerase reverse transcriptase (hTERT), which is thought to be the enzyme catalytic subunit.^6,7 The factors and mechanisms involved in the regulation of telomerase are presently poorly understood (reviewed in ^{Ref. 8}). There is mounting evidence that telomerase activity is critically regulated by steroid hormones in their target tissues.^9,10 Although telomerase becomes activated during neoplastic transformation, its activity decreases during differentiation of various immortal cells in response to pharmacological agents, including retinoids.^{11,12,13,14,15,16,17}

Retinoids are signalling molecules with important roles in growth and differentiation of a variety of normal adult and embryonic tissues,¹⁸ and have potent antiproliferative effects on many malignant cell types.¹⁹ Their actions are mediated by two classes of ligand-dependent transactivation factors belonging to the nuclear superfamily: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs).²⁰ Their biological effects led to the hypothesis that retinoids might be useful agents in the treatment of human malignancy and could exert cancer preventive activities.²¹ Hence, retinoids have been evaluated as possible therapies for a variety of human cancers including leukemia, skin cancers, cervical cancer, and neuroblastoma.^19,22 Among myeloid leukemias, acute promyelocytic leukemia (APL) was found to be specifically sensitive to all-trans retinoic acid (ATRA) that induces remission of APL patients by stimulating the differentiation of the leukemia cells.^23,24 Although almost all APL patients respond to retinoic acid (RA) therapy, resistance to this agent often develops. Several mechanisms have been proposed to account for the ATRA-resistant phenotype, including enhanced metabolism of ATRA, increased expression of the cellular retinoic acid binding protein II, and genetic defects associated with nuclear receptors or their downstream mediators which may alter retinoid signalling.^{25,26,27,28,29,30}

In order to investigate the biological significance of the action of retinoids in regulating telomerase activity, we benefited from a model of APL, the NB4 cell line responsive to retinoid-induced maturation and two resistant variants, NB4-R1 and NB4-R2.^31,32,33,34 NB4-R1 cells do respond to ATRA but do not undergo terminal maturation. Therefore, this cell line represents a useful tool to identify genes whose regulation by ATRA occurs independently of maturation. The maturation defect in these cells can be rescued by retinoid and cAMP cross-talk.³⁴ No such cooperation exists in the NB4-R2 cells, which are clearly unresponsive to ATRA, because of a mutation of Gln411 to an in-phase stop codon in the ligand binding domain of PML-RAR.³⁰

Using these cell lines, two levels of regulation of telomerase by retinoids have been identified.³⁵ The first pathway results in a rapid down-regulation of telomerase associated with ATRA-induced granulocytic maturation of NB4 cells. The second pathway identified in the RAR-responsive, maturation-resistant NB4-R1 cell line, results in a down-regulation of telomerase that develops slowly during 10 days of ATRA treatment. This pathway leads to telomere shortening and cell death by 3 weeks, all events that are overcome by ectopic expression of hTERT, demonstrating that telomerase could represent an efficient target of retinoids in maturation-resistant cells.

The purpose of our study was to investigate a possible mechanism of resistance to this death signalling pathway. We characterized a new type of retinoid-resistant cells isolated from the NB4-R1 cell line during prolonged culture in the presence of ATRA. Nevertheless, when treated with a combination of ATRA and cAMP, this new cell line exhibited normal granulocytic maturation, accompanied with a down-regulation of telomerase activity and hTERT mRNA as in the parental cell line. Here, we provide evidence that the mechanism whereby this new variant overcomes the death step is the re-expression of telomerase activity. These findings reinforce the notion that two distinct pathways of telomerase regulation by retinoids co-exist.

Materials and methods

Reagents

All-trans retinoic acid (ATRA), and 8-(4-chlorophenyl- thio)adenosine cyclic 3',5'-monophosphate (8-CPT-cAMP) were purchased from Sigma (St Louis, MO, USA).

Cell lines and cell culture

The human promyelocytic leukemia cell line NB4, its resistant subclone NB4-R1,^31,32,33,34 and the ectopic hTERT expressing subline NB4-R1/hTERT³⁵ were cultured as previously described in RPMI 1640 medium (Invitrogen, Cergy-Pontoise France) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (50 IU/ml), streptomycin (50 g/ml), L-glutamine (2 mM) and sodium bicarbonate (70 mg/l), and incubated at 37°C at 5% CO₂ atmosphere. For the treatments, cells were seeded at a density of 1-2 ´ 10⁵ cells/ml in the medium supplemented with the various agents ATRA and 8-CPT-cAMP, used at 1 M and 200 M, respectively. Cells were grown in the dark and harvested at various time points for analysis. Every 2 days, cell density was determined using a Coulter counter and proliferation was represented in population doublings (PD). Differentiation was evaluated by morphology with May-Grünwald-Giemsa on cytocentrifuge slide preparation and by nitroblue tetrazolium (NBT; Sigma) as described previously.³³

Telomerase activity assay

Telomerase activity (TA) was measured by a modified version of the standard TRAP method,³⁶ the telomerase polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) kit (TRAPeze Elisa telomerase detection kit, Qbiogene, Illkirch, France) according to the manufacturer's instructions with 1 g of protein extract, during 22 PCR cycles. TA was expressed as absorbance values (OD) measured using a microtiter reader at 450 nm with a reference wavelength of 620 nm. All assays were performed in triplicate and a dilution series of control telomerase extracts was always examined in parallel to give a titration curve for normalizing experimental variations. In selected experiments, results from treated cells were expressed as relative telomerase activity, attributing a 100% activity to the control extract of each cell line.

Telomere length analysis

Telomere length was measured using a non-radioactive chemiluminescent assay developed by Roche Diagnostics (Meylan, France). In brief, genomic DNA was isolated and digested with HinfI and RsaI, fractionalized on a 0.8% agarose gel and processed by Southern blotting and chemiluminescent detection. The average telomere length can be determined by comparing the signals relative to a molecular weight standard.

RT-PCR analysis of hTERT expression

Expression of hTERT mRNA was analyzed by semi-quantitative reverse transcription (RT)-PCR amplification. Total cellular RNA was collected from samples using TRIzol reagent (Invitrogen, France). RNA yields and purity were determined spectrophotometrically at 260-280 nm and by visualization on 1% agarose gel stained with ethidium bromide (0.5 g/ml). RT-PCRs were carried out with 1 g of mRNA by using the 'Reverse Transcription System' (Promega, Charbonnières, France) according to the manufacturer's instructions with oligo(dT)₁₅ primers. Four microliters of the first-strand cDNA reaction were amplified in a 50 l reaction mixture containing 50 pmol of the primers TERT-2164S (5'-GCCTGA GCTGTACTTTGTCAA-3') and TERT-2620A (5'-CGCAAACAGCAGCTTGTTCTCCATGTC-3') with an initial heating at 94°C for 5 min, followed by 35 cycles of 95°C for 25 s, 68°C for 50 s, and 72°C for 50 s. GAPDH (glyceraldhehyde-3-phosphate dehydrogenase) mRNA used as an external standard was amplified from the same cDNA reaction mixture, during 21 cycles, using the primers K136 (5'-CTCAGACACCATGGGGAAGGTGA-3') and K137 (5'-ATGATCTTGAGGCTGTTGTCATA-3') as already described.³⁵ The exponential phase of amplification was previously determined by both serial dilution of RT reaction mixture for each cDNA template and variation in the number of cycles. A possible contamination of the samples with genomic DNA was checked in all RT-PCR by performing a mock RT-PCR from the same RNA, omitting the reverse transcriptase during the RT reaction. Negative controls (ie amplification performed without nucleic acid) were also included in order to detect possible cross-contamination. Amplified products were electrophoresed on a 2% high resolution agarose gel (Metaphore Agarose, Tebu, France) and stained with ethidium bromide (0.5 g/ml), visualized in UV light and photographed.

Detection of PML-RAR protein by immunoblot

After washing twice in PBS, 2 ´ 10⁶ cells were lysed by adding 100 l of boiling Laemmli solution (64.7 mM dithiothreitol, 2% SDS, 0.01% bromophenol blue, 10% glycerol, Tris-HCl 250 mM, pH 6.8) and disrupted with a pestle. Samples were then boiled for 5 min and insoluble material removed by centrifugation at 12 000 r.p.m. for 5 min. Protein amount was quantified by Coomassie Blue staining. Protein extracts were loaded on 8% SDS-polyacrylamide gel, electrophoresed, and blotted on to PVDF membranes (Millipore, Roissy, France). After transfer, membranes were blocked with 5% unfatted dry milk in PBS 0.1% Tween 20, then incubated overnight at 4°C with a rabbit polyclonal antibody raised against RAR (kindly provided by Dr Chambon, IGBMC, Illkirch, France). Membranes were incubated with horseradish peroxidase (HRP)-coupled anti-rabbit antibody (Jackson Laboratories) for 45 min at room temperature. Each step was followed by three 10 min washes in PBS-Tween. Detection was performed as described in the ECL kit (Amersham, Saclay, France).

Results

NB4-R1^SFD cell line overcomes the death step induced by long-term ATRA treatment

We have previously shown that retinoids may exert an antitumoral response independently of cell maturation through a down-regulation of telomerase ultimately leading to cell death.³⁵ To investigate a possible mechanism of resistance to this death signalling pathway, we wanted to know whether 'resistant' cells could be 'saved from death' during long-term ATRA treatment. Many attempts failed. Interestingly, in one experiment, after prolonged culture of NB4-R1 cells under the selective presence of ATRA, a population of cells emerged from the culture by 20 days, regained viability and returned to normal growth by day 35 (Figure 1a). These NB4-R1 cells 'saved from death' were given the provisional name 'NB4-R1^SFD'. Examination of telomerase activity at each passage (Figure 1b) revealed that survival and return to growth correlated with an ongoing re-activation of telomerase activity despite the continued presence of ATRA, suggesting a strong selection for telomerase activity in maintaining viability of these cells. We investigated whether the reactivated telomerase acts on its normal chromosomal substrate to regulate telomere lengths (Figure 1c). The mean telomere length of ATRA-treated NB4-R1 cells was reduced by 21 days of treatment, ie after about 10 days of culture with low level of telomerase activity. However, by day 39 telomere shortening ceased and, in NB4-R1^SFD cells, by day 112 telomere length was slightly but significantly increased. These results demonstrate that the re-activation of telomerase in these cells leads to a stabilization and homogenization of the telomere length. Even though telomeres did not return to their initial length at that time, this result indicates that the generation of this new variant did not result from the selection of an alternative lengthening of telomeres (ALT) mechanism, a telomerase-independent recombination-based pathway involved in some human-immortalized and cancer-derived cells to maintain telomere length.^37,38 The unresponsiveness of these cells to ATRA-induced cell death during a prolonged treatment was stable and was retained even after a 4 month culture of these cells in the absence of ATRA (Figure 2), indicating that this rescue was not secondary to a drug adaptation.

Altogether these results established that these cells constitute a new permanent NB4-R1 subline, that overcomes the long-term ATRA-induced death step because of a re-activation of telomerase despite the continuous action of ATRA. This new cell line was then investigated in terms of regulation of telomerase and differentiation.

Long-term regulation of hTERT by ATRA is defective in NB4-R1^SFD cells while functional in NB4-R1/hTERT cells

Although this new variant of the NB4-R1 cell line (NB4-R1^SFD) resembles the ectopic hTERT expressing NB4-R1/hTERT subline previously described³⁵ in that (1) it expresses a high level of telomerase activity, (2) it overcomes the death step, and (3) it proliferates despite continued ATRA treatment, NB4-R1^SFD results from a re-activation of endogenous telomerase expression in the presence of ATRA, while the NB4-R1/hTERT cell line was engineered by retroviral infection of a cell population to constitutively express ectopic telomerase.

The effect of long-term ATRA treatment on endogenous expression of hTERT was compared in both cell lines by semi-quantitative RT-PCR. To discriminate between ectopic and endogenous hTERT mRNA in the NB4-R1/hTERT cells, we took advantage of the presence of alternative splicing within the reverse transcriptase domain in a PCR assay using primers TERT-2164S and TERT-2620A.³⁹ In NB4-R1/hTERT cells, both the endogenous full-length transcript and the ectopic hTERT mRNA will migrate at the same position (457 bp). Thus, the endogenous hTERT mRNA will be represented only by the alternative splicing variant at 275 bp. We have previously shown that ATRA-induced differentiation leads to a rapid down-regulation of all hTERT mRNA variants.³⁵ Figure 3 shows that, as previously shown, long-term ATRA treatment of NB4-R1 cells induced a down-regulation of hTERT mRNA variants (lane 2). As expected, the re-activation of telomerase activity in NB4-R1^SFD cells is accompanied by the re-expression of hTERT mRNA (lane 3). That this re-expression is maintained despite the continuous presence of ATRA indicates a defect in the long-term regulation of hTERT by this agent. In contrast, in NB4-R1/hTERT cells, despite the high constitutive expression of ectopic hTERT, long-term ATRA treatment is associated with a down-regulation of endogenous hTERT mRNA at 275 bp (lane 6). This result shows that in this cell line long-term regulation of the endogenous hTERT by ATRA remains functional. The low level of the 275 bp band in NB4-R1/hTERT control cells (lane 5) compared to that in NB4-R1 control cells (lane 1) was merely explained by a more efficient amplification of the over-expressed full length transcript (457 bp PCR product) due to the ectopic hTERT (data not shown). As levels of hTERT mRNA (compare lanes 1 and 5) and telomerase activity (data not shown) were similar in both NB4-R1^SFD and NB4-R1 cells , it seems very unlikely that the resistance of NB4-R1^SFD cells could result from a hTERT gene amplification.⁴⁰

Altogether, these results show, first that a stable expression of hTERT determines long-term survival to prolonged retinoid treatment; second that, in contrast to NB4-R1/hTERT cells, the new selected NB4-R1^SFD cell line has acquired a defect in the long-term regulation of telomerase by ATRA.

NB4-R1^SFD cells remain sensitive to ATRA signalling and preserve a functional maturation-dependent regulation of telomerase

As already shown for ATRA-treated NB4-R1 cells,⁴¹ the PML-RAR protein is rapidly degraded upon ATRA treatment of NB4-R1^SFD cells (Figure 4) indicating that the resistance of these cells to the 'death signal' did not result from a complete blockade to ATRA signalling. Note that PML-RAR degradation also occurred in ATRA-treated NB4-R1/hTERT cells, which express ectopic hTERT and retain viability.

It is noteworthy that NB4-R1 cells do respond to ATRA and become competent to undergo terminal maturation which can be achieved by cAMP-elevating agents such as 8-CPT-cAMP.³⁴ The maturation response of the NB4-R1^SFD cell subline to these agents was analyzed. The combination of ATRA and 8-CPT-cAMP treatment triggered the full maturation of these cells (Figure 5a, 95% of NBT-positive cells), associated with a decline of the endogenous hTERT mRNA (Figure 5b) and a down-regulation of telomerase activity (Figure 5c). This result indicates that this cell subline is not altered in its ability to mature rapidly following cooperative-ATRA-cAMP stimulation. Interestingly, in NB4-R1/hTERT cells, despite the constitutive expression of ectopic hTERT and a stable telomerase activity, terminal maturation is also observed (92% of NBT-positive cells) and associated with a down-regulation of endogenous hTERT mRNA as shown by the analysis of the expression of the alternate splice transcript.

Altogether, these results demonstrate that neither a defect in ATRA-induced long-term regulation of hTERT (in NB4-R1^SFD cells) nor an ectopic hTERT expression (in NB4-R1/hTERT cells) can hamper granulocytic maturation.

Discussion

In the present work, we report the isolation of a new variant cell line that overcomes the cell death step induced by long-term ATRA treatment (Figure 6). This cell line, named NB4-R1^SFD, was characterized by a steady expression of hTERT in the continuous presence of ATRA. This deregulated expression of the telomerase was not due to a general alteration in RA signalling since these cells do respond to ATRA as demonstrated by their ability to degrade PML-RAR after treatment by ATRA and to differentiate in the presence of ATRA and 8-CPT-cAMP in combination. This new variant is the result of a specific defect in the signalling pathway leading to long-term regulation of telomerase by retinoids. Interestingly, even though NB4-R1/hTERT cells show the same response to long-term ATRA treatment as NB4-R1^SFD cells, the endogenous hTERT remains regulated by ATRA during long-term treatment despite a stable telomerase activity due to the ectopic expression of hTERT.

That both NB4-R1^SFD and NB4-R1/hTERT cell lines exhibited normal granulocytic maturation when treated with a combination of RA and 8-CPT-cAMP and endogenous hTERT was down-regulated during this event, demonstrates that (1) a forced expression of telomerase activity neither prevents nor alters granulocytic maturation; (2) reinforces the notion that two distinct pathways for telomerase regulation by retinoids co-exist in APL cells. One is associated with maturation, the other operates independently of this event. That only this latter pathway is defective in the NB4-R1^SFD cells demonstrates that the selection of this new variant is not the consequence of a general defect in telomerase regulation.

Our results have broader implications. First, it is likely that similar defects in telomerase regulation may exist in other tumor cells, and may explain their as yet unelucidated resistance to the antiproliferative effects of retinoids; second, the re-activation of telomerase after an initial step of down-regulation resembles what is observed during tumorigenesis when telomerase is re-activated; third, our results provide the first evidence that besides classical retinoid resistance mechanisms, which reportedly account for failure of ATRA therapy, a new kind of resistance could emerge from telomerase-based therapies. For all these reasons, the NB4-R1^SFD new variant will provide an efficient tool to gain further insights into several important issues on both the molecular biology of telomerase regulation and ultimately the evaluation of the therapeutic potential of retinoids as anti-telomerase agents for cancer treatment, through targeting of hTERT by a hormonal pathway. Taken together, these prospects indicate that it is important to elucidate the molecular basis for the resistance of NB4-R1^SFD to long-term ATRA treatment.

Note added in proof

During the process of the review, a new nomenclature has been proposed for the various NB₄ sublines (Roussel MJS, Lanotte M. Maturation-sensitive and -resistant t(15;17) NB₄ cell lines as tool for APL physiopathology: nomenclature of cells and repertory of their known genetic alterations and phenotypes. Oncogene 2001; 20: 7287-7291). According to this nomenclature, NB₄-R₁ is renamed NB₄-LR₁ and NB₄-LR₁/hTERT will replace NB₄-R₁/hTERT.

Acknowledgements

This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Ligue Nationale contre le Cancer and the Association pour la Recherche contre le Cancer (ARC Nos 5384, and 5707). TS and FP were supported by the Coopération Franco-Algérienne, and the Ministère de la Recherche et de la Technologie, respectively. We thank Dr G Chabot (INSERM U496) for critical reading of the manuscript.

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Figure 1 Selection of NB4-R1^SFD cells during long-term treatment of NB4-R1 cells with ATRA. (a) Growth curves showing the cumulative increase in population doubling for control and ATRA-treated (+ATRA) NB4-R1 cells. At time * most of the cells underwent cell death. However, a new population, named NB4-R1^SFD, overcame this cell death step and could be cultured in the continuous presence of ATRA without any alteration of the proliferation. (b) At the indicated times, protein extracts were prepared, and telomerase activity measured. Enzyme activity is expressed as a percentage of that detected in untreated cells. (c) Telomere restriction length of NB4-R1 cell sublines during long-term treatment with 1 M ATRA.

Figure 2 Growth curves of NB4-R1 and NB4-R1^SFD cells showing the cumulative increase in population doubling with time. () NB4-R1 control cells; () NB4-R1 cells treated with ATRA; () NB4-R1^SFD cells were maintained in the continuous presence of ATRA (1 M). NB4-R1^SFD cells were cultured for 4 months in ATRA-free medium (), then transferred in medium containing 1 M ATRA ().

Figure 3 RT-PCR analysis of expression of hTERT in NB4-R1, NB4-R1^SFD, and NB4-R1/hTERT cell lines. NB4-R1 cells were exposed to 1 M ATRA for 6 days (lane 2). NB4-R1^SFD (lane 3) and NB4-R1/hTERT (R1/H) (lane 6) were maintained in the continuous presence of ATRA (1 M). The respective control cells (lanes 1, 4 and 5) were maintained in ATRA-free medium. The full-length hTERT transcript and the alternative splicing variant of 457 and 275 bp, respectively, are indicated. GAPDH expression was used as control for RNA loading and RT efficiency. As a control for total RNA preparations ribosomal bands were evaluated on agarose electrophoresis gel.

Figure 4 Western blot analysis of PML-RAR in NB4-R1 cell lines. The same amounts of protein extracts prepared from cells treated or not treated with ATRA (1 M) for 72 h were electrophoresed, blotted and incubated with an anti-RAR. Level of PML-RAR (140 kDa) is shown.

Figure 5 Maturation and hTERT expression in NB4-R1-derived cell lines. Morphology (a) and expression of telomerase (b) in NB4-R1, NB4-R1^SFD and NB4-R1/hTERT cells after induction of maturation (+) by a 72 h treatment with the combination of ATRA (1 M) and 8-CPT-cAMP (200 M). RT-PCR was performed as in Figure 3 (upper panel). Telomerase activity measured on protein extract is expressed as a percentage of that detected in untreated cells (bottom panel).

Figure 6 Schematic representation of the establishment of NB4-R1^SFD and NB4-R1/hTERT and their distinct responses to RA signalling in terms of growth, maturation, and telomerase regulation. Variations are estimated compared to control NB4-R1 cells: (=) no change; (↘) a strong decrease. The data correspond to experiments described in Figures 1, 3, and 5