[CANCER RESEARCH 58. 2680-2687, June 15. 1998] Antiapoptotic Effect of Ectopie TAL1/SCL Expression in a Human Leukemic T-Cell Line1 Muriel Bernard, Eric Delabesse, Sophie Novault, Olivier Hermine, and Elizabeth A. Macintyre2 Centre National de la Recherche Scientifique URAI46I ¡M.B., E. D., O. H.. E. A. M.I and Department ttf Hemawlogv ¡E.D.. O. H., E. A. M.], Hospital Necker-Enfanls Malades, 75743 Paris Cedex 15. and Institut Gustave Riaissy. INSERM U362. 94805 Villejuif [S. N.J. France ABSTRACT Aberrant expression of TALI occurs frequently in human T-cell acute lymphohlastic leukemia. The effect of TALI expression in the T-cell lymphoid precursor, however, remains unclear. In the current study, we have developed I \l I stable transfectants in a human immature T-cell lymphoid cell line. Whereas no effect on proliferation, cell culture density, or cell cycle was detected, the transfectants were more resistant than the parental cell line to apoptosis induced by chemotherapeutic agents includ ing etoposide, daunoruhicin, doxorubicin, cytosine arabinoside, metho- trexate and vincristine and also to apoptosis induced by Fas/CD95 cross- linking. This effect was independent of the cytostatic effects of the drugs. The basic domain-deleted transfectants did not demonstrate altered sen sitivity, suggesting that DNA binding was necessary for resistance to apoptosis. The ability to alter the response to a wide range of cell death- inducing stimuli suggests that TALI acts at a late stage of the apoptotic cascade. These data therefore provide direct evidence of an antiapoptotic effect of ectopie TALI expression in response to cytotoxic agents, thus providing insight into its oncogenic function in T-cell acute lymphoblastic leukemia and a novel experimental model to further investigate the un derlying mechanisms. These data also have potential practical significance for cytotoxic therapy of this disorder. INTRODUCTION ALLs3 of both T-cell and B-cell lineage are treated by multiagent chemotherapy using drugs of different classes and modes of action to maximize leukemic toxicity while minimizing resistance and undesir able side effects. Several untitumor agents have been shown to induce in vitro and in vivo apoptosis in susceptible cells (1). The fact that drugs with different targets induce what seems to be a final common pathway to cell death and that certain tumor cells are refractory to drug therapy suggests that cytotoxicity is determined by the ability of the cell to initiate its own death in response to drug-induced pertur bations (1, 2). The expression of certain intrinsic determinants of cell survival/death, such as c-Myc, the Bcl-2 family members, and p53, has been shown to determine the apoptotic potential of a given drug (3). Expression of these proteins by malignant cells is frequently deregulated as a result of genetic abnormalities. Such alterations may therefore initiate or modulate the survival/death processes and may thus be involved, in addition to tumor development/progression, in determining chemosensitivity. Genetic alterations leading to the aberrant expression of TALI (SCL), including chromosomal translocation, site-specific interstitial deletion (SIL-SCL/Tal'1}, or unidentified alterations of the TALI gene, are one of the most common tumor-specific abnormalities in T-ALLs Received 12/23/97; accepted 4/14/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by a grant from the Ligue Nationale Contre le Cancer (June 1995). by Cirant 5026 from the Association de Recherche sur le Cancer, and by the Direction for Clinical Research of the Assistance Publique-Hôpitaux de Paris. 2 To whom requests for reprints should he addressed, at Department of Hematology. Hospital Neckcr-Enfants Malade. 149 rue de Sevres. 75743 Paris, France. Phone: 33-1- 44-49-49-47; Fax: 33-1-45-67-91-56; E-mail: cH7.ubcth.macmlyrc@nck.ap-hop-paris.fr. 1The abbreviations used are: ALL, acute lymphoblastic leukemia; T-ALL, T-cell ALL: b-HLH. basic helix-loop-helix: PI. propidium iodide; 7AAD. 7-t>-amino actinomy- cin; TCR. T-cell receptor. (4). The high incidence of TALI deregulation argues for its probable importance in the development of these leukemias, but the mechanism whereby it may do so remains poorly understood. Overexpression of TALI within the T-cell lineage leads to tumor formation in only a proportion of mice, depending on the context (5-7). Concomitant overexpression of LMOI or LMO2, two genes translocated in T-ALL that interact with TALI, enhances the development of T-cell malig nancies in transgenic mice (4, 8, 9). supporting the hypothesis that additional factors are necessary for the oncogenic potential of TALI in the T-cell lymphoid lineage. The TALI gene encodes a lineage-specific b-HLH transcription factor that binds to specific E box DNA motifs on heterodimerization with ubiquitous b-HLH proteins, including the products of the E2A gene (10). The basic domain is necessary for DNA binding, whereas the helix-loop-helix domain mediates heterodimerization. Formation of E2A/TAL1 heterodimers could alter the regulation of E2A target genes and potentially initiate leukemogenesis, as suggested by the T-cell tumors that develop in mice homozygous for an E2A null mutation (11). The TALI protein is normally expressed in hemato- poietic stem cells, early myeloid precursors, erythroid cells, megakaryocytes, mast cells, basophils, and endothelial cells but not in T-cell lymphoid cells (12, 13). The TALI protein acts as a positive regulator of erythroid differentiation (14) and is essential for the development of primitive hematopoiesis and the generation of all adult hematopoietic lineages, including T-cell lymphocytes (15, 16). The direct effect of aberrant TALI expression in the T-cell lineage, however, remains unclear. A mutant TALI protein, exhibiting loss of heterodimerizing capacity with the b-HLH E2A proteins, induced premature apoptosis on medium depletion in the Jurkat T-cell leuke mic cell line (17). Condorelli et al. (18) showed a proliferative and antiapoptotic effect of ectopie TALI expression in myeloid precur sors. In the current study, we investigated whether the ectopie expres sion of TALI in a human T-cell line could change the effect of several antitumor agents and of Fas/CD95 cross-linking. We have developed stable transfectants in a human T-cell leukemic cell line and demon strated that wild-type TALI expression can protect leukemic cells from apoptosis triggered by several drugs commonly used in the treatment of T-ALL. MATERIALS AND METHODS Cell Culture. MKB1 is a human leukemic T-cell line derived from a case of T-ALL (19) that does not demonstrate apparent TALI gene rearrangement or expression.4 Cells were grown in RPMI Glutamax containing 10% heat- inactivated PCS (Life Technologies, Inc., Cergy Pontoise, France). For cell proliferation analysis, cells were diluted at 2 x IO5 cells/ml in medium containing variable concentrations of FCS. Viable and dead cells were counted by trypan blue exclusion. For long-term proliferation analyses, cells were resuspended at their initial density when their number exceeded 5 X 10s cells/ml. Development of TALI and TALlbb Stable Transfectants. The TALI fragments were isolated by EcoRl digestion from the 2736 SCL/TAL1 vector containing a 1.1-kb TALI cDNA insert that allows the expression of a full- 4 E. Macintyre. unpublished observations. 2680 Research. on November 26, 2021. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from