Evelyne Ségal-Bendirdjian

Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms

Sensitivity of tumor cells to anticancer therapy depends on the ability of the drug to induce apoptosis. However, multiple signaling pathways control this induction and thus determine this sensitivity. We report here that staurosporine, a well known inducer of apoptosis in a wide range of cell lines, displays distinct ability to trigger apoptosis in two different L1210 sublines (termed L1210/S and L1210/0). Staurosporine treatment resulted in an early cell death (within 3 h) in L1210/S cells, while in L1210/0 cells, death occurred only after 12 h. In both instances, death occurred by apoptosis. A broad spectrum caspase inhibitor, Z-VAD-fmk, blocked early apoptosis in L1210/S cells but did not confer any protection on late apoptosis in L1210/0 cells. Protection by Z-VAD-fmk observed in L1210/S cells was not lasting and unmasked a secondary process of cell death that also exhibited characteristics of apoptosis. Thus, staurosporine induces apoptotic cell death through at least two redundant parallel pathways. These two pathways normally coexist in L1210/S cells. However, the early cell death mechanism depending on caspase activation disguises the late caspase-independent apoptotic process. Staurosporine-induced apoptosis in L1210/0 cells develops only by the caspase-independent mechanism due to a general defect in caspase activation.

Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths

The involvement of telomerase in cellular immortalization and senescence has often been assessed by means of telomerase expression at the RNA level and quantification of telomerase activity by the telomeric repeat amplification protocol assay. However, these methods either neglected the existence of various telomerase splice variants, or ignored the nonconventional functions of telomerase independent of its ability to elongate and maintain telomere length. Immunodetection of telomerase is now being recognized as a necessary approach to precisely elucidate its roles in oncogenesis and senescence. A few antibodies directed against the catalytic subunit of the human telomerase (hTERT) are currently used but their specificity is not always demonstrated. A survey of the literature showed inconsistencies and led us to comparatively re-evaluate the most frequently used antibodies. Surprisingly, mass spectrometry, two-dimensional gel analysis and immunofluorescent experiments revealed that the most frequently used hTERT immunoprobe, a mouse monoclonal antibody that was claimed to be directed against an hTERT protein epitope, in fact recognizes nucleolin rather than telomerase. Our findings have interesting implications regarding the biology of nucleolin and telomerase in the context of pathophysiological investigations recently carried out.

Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation

Human telomerase, a cellular reverse transcriptase (hTERT), is a nuclear ribonucleoprotein enzyme complex that catalyzes the synthesis and extension of telomeric DNA. This enzyme is specifically activated in most malignant tumors but is usually inactive in normal somatic cells, suggesting that telomerase plays an important role in cellular immortalization and tumorigenesis. Terminal maturation of tumor cells has been associated with the repression of telomerase activity. Using maturation-sensitive and -resistant NB4 cell lines, we analyzed the pattern of telomerase expression during the therapeutic treatment of acute promyelocytic leukemia (APL) by retinoids. Two pathways leading to the down-regulation of hTERT and telomerase activity were identified. The first pathway results in a rapid down-regulation of telomerase that is associated with retinoic acid receptor (RAR)-dependent maturation of NB4 cells. Furthermore, during NB4 cell maturation, obtained independently of RAR by retinoic X receptor (RXR)-specific agonists (rexinoids), no change in telomerase activity was observed, suggesting that hTERT regulation requires a specific signaling and occurs autonomously. A second pathway of hTERT regulation, identified in the RAR-responsive, maturation-resistant NB4-R1 cell line, results in a down-regulation of telomerase that develops slowly during two weeks of all-trans retinoic acid (ATRA) treatment. This pathway leads to telomere shortening, growth arrest, and cell death, all events that are overcome by ectopic expression of hTERT. These findings demonstrate a clear and full dissociation between the process of tumor cell maturation and the regulation of hTERT mRNA expression and telomerase activity by retinoids. We propose telomerase expression as an efficient and selective target of retinoids in the therapy of tumors.

Death receptor signaling regulatory function for telomerase: hTERT abolishes TRAIL-induced apoptosis, independently of telomere maintenance.

Human telomerase has been implicated in cell immortalization and cancer. Recent works suggest that telomerase confers additional function required for tumorigenesis that does not depend on its ability to maintain telomeres. This new action may influence tumor therapy outcomes by yet unraveled mechanisms. Here, we show that overexpression of the catalytic subunit of telomerase (hTERT) protects a maturation-resistant acute promyelocytic leukemia (APL) cell line from apoptosis induced by the tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (TRAIL) and not from apoptosis induced by chemotherapeutic drugs such as etoposide or cisplatin. Conversely, in these cells, TRAIL-induced cell death is magnified by all-trans retinoic acid (ATRA) treatment, independently of telomerase activity on telomeres. Of note, this response is subordinated neither to maturation nor to telomere shortening. This work underlines that retinoids and death receptor signaling cross-talks offer new perspectives for antitumor therapy.

Antitumor trans-N-heterocyclic carbene-amine-Pt(II) complexes: synthesis of dinuclear species and exploratory investigations of DNA binding and cytotoxicity mechanisms.

A series of bimetallic [(NHC)PtX2]2(diamine) complexes have been prepared as a new chemotype for potential anticancer agents. These complexes display an uncommon set of structural features as far as they combine two bifunctional, trans-configured platinum centers. They display cytotoxic activities in the micromolar range on many cancerous cell lines and do not cross-react with cisplatin in A2780/DDP cell lines. They bind slowly to double-stranded DNAs, giving monoadducts as the major products. Pathways for cellular toxicity have been investigated for both mono- and bimetallic trans-(NHC)PtX2(amine) complexes. It has been highlighted that, unlike cisplatin, these complexes do not induce cell cycle arrest. They trigger apoptosis in A2780 cells by a pathway involving translocation of apoptosis-inducing factor and caspase 12 to the nucleus. Moreover, bimetallic complexes may induce necrosis.

Orchestration of multiple arrays of signal cross-talk and combinatorial interactions for maturation and cell death: another vision of t(15;17) preleukemic blast and APL-cell maturation

Despite intensive molecular biology investigations over the past 10 years, and an important breakthrough on how PML–RARα, the fusion protein resulting from t(15;17), can alter RARα and PML functions, no definitive views on how leukemia is generated and by what mechanism(s) the normal phenotype is restored, are yet available. ‘Resistances’ to pharmacological levels of all-trans-retinoic acid (ATRA) have been observed in experimental in vivo and in vitro models. In this review, we emphasize the key role played by signal cross-talk for both normal and neoplastic hemopoiesis. After an overview of reported experimental data on APL-cell maturation and apoptosis, we apply our current knowledge on signaling pathways to underline those which might generate signal cross-talks. The design of biological models suitable to decipher the integration of signal cross-talks at the transcriptional level should be our first priority today, to generate some realistic therapeutic approaches After ‘Ten Years of Molecular APL’, we still know very little about how the disease develops and how effective medicines work.

Retinoic acid receptor α and retinoid-X receptor-specific agonists synergistically target telomerase expression and induce tumor cell death

Retinoids modulate growth and differentiation of cancer cells through activation of gene transcription via the nuclear retinoic-acid receptors (RAR) and retinoid-X receptors (RXR). Their use in differentiation therapy of acute promyelocytic leukemia (APL) represents a model concept for reprogramming cancer cells. However, they also regulate antiproliferative genes whose functions do not mechanistically concur to this program. Recently, we have shown that, independently of maturation, a long-term all-trans retinoic acid (ATRA) treatment of the maturation-resistant APL cell line (NB4-LR1) represses telomerase (hTERT), leading to telomere shortening and death. Using retinoid-receptor-specific agonists, we demonstrate herein that cross-talk between RARα and RXR dual-liganded to their respective agonists resulted in strong synergistic downregulation of hTERT and subsequent cell death. Importantly, unlike ATRA, this synergy was obtained at very low agonist concentrations and occurred in other ATRA maturation-resistant APL cells. These findings provide the first demonstration that dual-liganded RXR and RARα signaling should allow efficient targeting of telomerase in differentiation-resistant tumor cells. Such a combination therapy might hold promise in clinic to avoid side effects of ATRA whose administration can indiscriminately activate all RARs. Given the tissue-specific expression of RARs, a tissue-selective therapy targeting telomerase in tumor cells by synthetic agonists can be envisioned.

Functional involvement of RINF, retinoid-inducible nuclear factor (CXXC5), in normal and tumoral human myelopoiesis.

Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach, we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor, retinoid-inducible nuclear factor (RINF), that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34(+) progenitors. Furthermore, short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly, RINF localizes to 5q31.3, a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene.

Retinoid/arsenic combination therapy of promyelocytic leukemia: induction of telomerase-dependent cell death

Acute promyelocytic leukemia (APL) is efficiently treated with a cell differentiation inducer, all-trans retinoic acid (ATRA). However, a significant percentage of patients still develop resistance to this treatment. Recently, arsenic trioxide (As2O3), alone or in combination with ATRA, has been identified as an alternative therapy in patients with both ATRA-sensitive and ATRA-resistant APL. Previous investigations restricted the mechanism of this synergism to the modulation and/or degradation of PML-RARα oncoprotein through distinct pathways. In this study, using several ATRA maturation-resistant APL cell lines, we demonstrate in vitro that the success of ATRA/As2O3 treatment in APL pathology can be explained, at least in part, by a synergistic effect of these two drugs in triggering downregulation of telomerase efficient enough to cause telomere shortening and subsequent cell death. Such long-term low-dose combinatorial therapy strategies, developed also to avoid acute side effects, reinforce the notion that the antitelomerase strategy, based on a combination of active agents, should now be considered and evaluated not only in APL but also in other malignancies.

Epigenetic plasticity of hTERT gene promoter determines retinoid capacity to repress telomerase in maturation-resistant acute promyelocytic leukemia cells

The expression of hTERT gene, encoding the catalytic subunit of telomerase, is a feature of most cancer cells. Changes in the chromatin environment of its promoter and binding of transcriptional factors have been reported in differentiating cells when its transcription is repressed. However, it is not clear whether these changes are directly involved in this repression or only linked to differentiation. In a maturation-resistant acute promyelocytic leukemia (APL) cell line (NB4-LR1), we have previously identified a new pathway of retinoid-induced hTERT repression independent of differentiation. Using a variant of this cell line (NB4-LR1SFD), which resists to this repression, we show that although distinct patterns of histone modifications and transcription factor binding at the proximal domain of hTERT gene promoter could concur to modulate its expression, this region is not sufficient to the on/off switch of hTERT by retinoids. DNA methylation analysis of the hTERT promoter led to the identification of two distinct functional domains, a proximal one, fully unmethylated in both cell lines, and a distal one, significantly methylated in NB4-LR1SFD cells, whose methylation was further re-enforced by retinoid treatment. Interestingly, we showed that the binding to this distal domain of a known hTERT repressor, WT1, was defective only in NB4-LR1SFD cells. We propose that epigenetic modifications targeting this distal region could modulate the binding of hTERT repressors and account either for hTERT reactivation and resistance to retinoid-induced hTERT downregulation.