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2001

Autonomous rexinoid death signaling is suppressed by converging signaling pathways in immature leukemia cells.

Author(s) : Benoit G, Flexor M, Besancon F, Altucci L, Rossin A, Hillion J, Balajthy Z, Legres L, Segal-Bendirdjian E, Gronemeyer H, Lanotte M,
Journal : Mol Endocrinol
2001
On their own, retinoid X receptor (RXR)-selective ligands (rexinoids) are silentin retinoic acid receptor (RAR)-RXR heterodimers, and no selective rexinoid program has been described as yet in cellular systems. We report here on the rexinoid signaling capacity that triggers apoptosis of immature promyelocytic NB4 cells as a default pathway in the absence of survival factors. Rexinoid-induced apoptosis displays all features of bona fide programmed cell death and is inhibited by RXR, but not RAR antagonists. Several types of survival signals block rexinoid-induced apoptosis. RARalpha agonists switch the cellular responsetoward differentiation and induce the expression of antiapoptosis factors. Activation of the protein kinase A pathway in the presence of rexinoid agonists induces maturation and blocks immature cell apoptosis. Addition of nonretinoid serum factors also blocks cell death but does not induce cell differentiation. Rexinoid-induced apoptosis is linked to neither the presence nor stability of the promyelocytic leukemia-RARalpha fusion protein and operates also in non-acute promyelocytic leukemia cells. Together our results support a model according to which rexinoids activate in certain leukemia cells a default death pathway onto which several other signaling paradigms converge. This pathway is entirely distinct from that triggered by RAR agonists, which control cell maturation and postmaturation apoptosis.

Bond cleavage in the excited state of acyl phosphene oxides: Insight on the role of conformation by model calculations: A concept

Author(s) : Spichty M, Turro N, Rist G, Birbaum J, Dietliker K, Wolf J, Gescheidt G,
Journal : Journal of Photochemistry and Photobiology A: Chemistry
2001
Rotation profiles along the C(O)-P(O) bond of model system 1 were calculated for the S0 S1 and T1 state on the B3LYP/6-31G∗ and RCIS/6-31+G∗ level of theory. Moreover, the cleavage of the bond was followed in the T1 state. The computations reveal that the efficiency of the α cleavage reaction may depend on the different rotation barriers of the molecule in the electronic states attained during the photochemical pathway. It can be anticipated that specific solvation which may influence the conformational mobility and the lifetimes in the S1 and T1 states is likely to play a important role for solvent effects.

Carbanions Substituted by Transition Metals: Synthesis, Structure, and Configurational Restrictions of a Lithium Titanium Phosphonate.

Author(s) : Muller J, Kulicke K, Neuburger M, Spichty M,
Journal : Angew Chem Int Ed Engl
2001

Conformational dynamics in a methacrylate-derived radical: A computational and EPR study

Author(s) : Spichty M, Giese B, Matsumoto A, Fischer H, Gescheidt G,
Journal : Macromolecules
2001
Dynamic phenomena are established for the [D3]methyl (2SR,3RS)-2-methyl-4,4,4-trichlorobutanoate-2-yl radical (1). This is revealed by EPR spectroscopy and quantum chemical calculations. The minimum conformations B and D of 1 possess planar radical centers and are sepearated by an energy barrier of 12.0 ± 1.8 kJ·mol-1 (experimental; calculated, 11.0 kJ·mol-1).

Exploring (novel) gene expression during retinoid-induced maturation and cell death of acute promyelocytic leukemia.

Author(s) : Benoit G, Tong J, Balajthy Z, Lanotte M,
Journal : Semin Hematol
2001
During recent years, reports have shown that biological responses of acute promyelocytic leukemia (APL) cells to retinoids are more complex than initially envisioned. PML-RARalpha chimeric protein disturbs various biological processes such as cell proliferation, differentiation, and apoptosis. The distinct biological programs that regulate these processes stem from specific transcriptional activation of distinct (but overlapping) sets of genes. These programs are sometimes mutually exclusive and depend on whether the signals are delivered by RAR or RXR agonists. Furthermore, evidence that retinoid nuclear signaling by retinoid, on its own, is not enough to trigger these cellular responses is rapidly accumulating. Indeed, work with NB4 cells show that the fate of APL cells treated by retinoid depends on complex signaling cross-talk. Elucidation of the sequence of events and cascades of transcriptional regulationnecessary for APL cell maturation will be an additional tool with which to further improve therapy by retinoids. In this task, the classical techniques used to analyze gene expression have proved time consuming, and their yield has been limited. Global analyses of the APL cell transcriptome are needed. We review thetechnical approaches currently available (differential display, complementary DNA microarrays), to identify novel genes involved in the determination of cell fate.

Fission yeast Bub1 is essential in setting up the meiotic pattern of chromosome segregation.

Author(s) : Bernard P, Maure J, Javerzat J,
Journal : Nat Cell Biol
2001
In meiosis, sister-chromatids move to the same spindle pole during the first division (MI) and to opposite poles during the second division (MII). This requires that MI sister kinetochores are co-orientated and form an apparent single functional unit that only interacts with microtubules from one pole, and that sister-chromatids remain associated through their centromeres until anaphase II. Here we investigate the function of Bub1 and Mad2, which are components of the mitotic-spindle checkpoint, on chromosome segregation during meiosis. Both proteins are required to prevent the occurrence of non-disjunction events in MI,which is consistent with recent findings that components of the mitotic-spindle checkpoint also operate during meiosis. However, Bub1 has several functions thatare not shared with Mad2. When the bub1 gene is deleted, sister chromatids oftenmove to opposite spindle poles during MI, indicating that sister kinetochores are disunited. Furthermore, the cohesin Rec8 is never retained at centromeres at anaphase I and sister-chromatid cohesion is lost. Our results show that Bub1, besides its functions in monitoring chromosome attachment, is essential for two other significant aspects of MI - unification of sister kinetochores and retention of centromeric cohesion.

fringe and Notch specify polar cell fate during Drosophila oogenesis.

Author(s) : Grammont M, Irvine K,
Journal : Development
2001
fringe encodes a glycosyltransferase that modulates the ability of the Notch receptor to be activated by its ligands. We describe studies of fringe function during early stages of Drosophila oogenesis. Animals mutant for hypomorphic alleles of fringe contain follicles with an incorrect number of germline cells, which are separated by abnormally long and disorganized stalks. Analysis of clones of somatic cells mutant for a null allele of fringe localizes the requirement for fringe in follicle formation to the polar cells, and demonstrates that fringe is required for polar cell fate. Clones of cells mutant for Notch also lack polar cells and the requirement for Notch in follicle formation appears to map to the polar cells. Ectopic expression of fringe or of an activated form of Notch can generate an extra polar cell. Our results indicate that fringe plays a key role in positioning Notch activation during early oogenesis, and establisha function for the polar cells in separating germline cysts into individual follicles.

Granulocytic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid metabolites.

Author(s) : Idres N, Benoit G, Flexor M, Lanotte M, Chabot G,
Journal : Cancer Res
2001
The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid(18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRAand all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required todifferentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Lineage restriction of the RARalpha gene expression in myeloid differentiation.

Author(s) : Zhu J, Heyworth C, Glasow A, Huang Q, Petrie K, Lanotte M, Benoit G, Gallagher R, Waxman S, Enver T, Zelent A,
Journal : Blood
2001
To better understand the role of retinoids in myelopoiesis, expression of the retinoid receptor genes (retinoic acid receptors [RARs] and retinoid X receptors[RXRs]) were examined during differentiation of factor-dependent cell-Paterson (FDCP)-mixA4 murine progenitor cells. The major receptor expressed in undifferentiated A4 cells was RARalpha (primarily the RARalpha1 isoform). Following induction of myelomonocytic differentiation with granulocyte and granulocyte-macrophage colony-stimulating factors, a dramatic increase in RARalpha expression (particularly the RARalpha2 isoform) was seen. In contrast, expression of both RARalpha isoforms was rapidly extinguished upon induction of erythroid differentiation with erythropoeitin (EPO). A modest induction of RXRalpha expression was seen, particularly during differentiation in the myelomonocytic lineage. Low expression levels of RARgamma2 and RXRbeta remained unchanged, irrespective of differentiation pathway. Consistent with the gene expression patterns, RARalpha agonists and antagonists stimulated myelomonocyticand erythroid differentiation of FDCP-mixA4 cells, respectively. Taken together,these results suggest that erythropoiesis and granulopoiesis require diminished and enhanced RARalpha activities, respectively, which at physiological all-trans-retinoic acid (RA) concentrations may be accomplished by reciprocal effects of EPO and myelomonocytic growth factors on its expression. This hypothesis is corroborated by data showing that RA, which positively regulates RARalpha2 expression, can exert inhibitory effects on erythroid differentiation.

Long-distance charge transport through DNA. An extended hopping model

Author(s) : Giese B, Spichty M, Wessely S,
Journal : Pure and Applied Chemistry
2001
Long-distance transfer of a positive charge through DNA can be described by a hopping model. In double strands where the (A:T)n bridges between the guanines are short (n 3), the charge hops only between guanines, and each hopping step depends strongly upon the guanine to guanine distances. In strands where the (A:T)n sequences between the guanines are rather long (n 4), also the adenines act as charge carriers. To predict the yields of the H2O-trapping products one has to take into account not only the charge-transfer rates but also the rates of H2O-trapping reactions.

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.

Author(s) : Benoit G, Roussel M, Pendino F, Segal-Bendirdjian E, Lanotte M,
Journal : Oncogene
2001
Despite intensive molecular biology investigations over the past 10 years, and an important breakthrough on how PML-RARalpha, the fusion protein resulting from t(15;17), can alter RARalpha 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-retinoicacid (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 effectivemedicines work.

Requirement of heterochromatin for cohesion at centromeres.

Author(s) : Bernard P, Maure J, Partridge J, Genier S, Javerzat J, Allshire R,
Journal : Science
2001
Centromeres are heterochromatic in many organisms, but the mitotic function of this silent chromatin remains unknown. During cell division, newly replicated sister chromatids must cohere until anaphase when Scc1/Rad21-mediated cohesion is destroyed. In metazoans, chromosome arm cohesins dissociate during prophase, leaving centromeres as the only linkage before anaphase. It is not known what distinguishes centromere cohesion from arm cohesion. Fission yeast Swi6 (a Heterochromatin protein 1 counterpart) is a component of silent heterochromatin.Here we show that this heterochromatin is specifically required for cohesion between sister centromeres. Swi6 is required for association of Rad21-cohesin with centromeres but not along chromosome arms and, thus, acts to distinguish centromere from arm cohesion. Therefore, one function of centromeric heterochromatin is to attract cohesin, thereby ensuring sister centromere cohesion and proper chromosome segregation.

The v-ErbA oncoprotein quenches the activity of an erythroid-specific enhancer.

Author(s) : Braliou G, Ciana P, Klaassen W, Gandrillon O, Stunnenberg H,
Journal : Oncogene
2001
v-ErbA is a mutated variant of thyroid hormone receptor (TRalpha/NR1A1) borne bythe Avian Erythroblastosis virus causing erythroleukemia. TRalpha is known to activate transcription of specific genes in the presence of its cognate ligand, T3 hormone, while in its absence it represses it. v-ErbA is unable to bind ligand, and hence is thought to contribute to leukemogenesis by actively repressing erythroid-specific genes such as the carbonic anhydrase II gene (CA II). In the prevailing model, v-ErbA occludes liganded TR from binding to its cognate elements and constitutively interacts with the corepressors NCoR/SMRT. We previously identified a v-ErbA responsive element (VRE) within a DNase I hypersensitive region (HS2) located in the second intron of the CA II gene. We now show that HS2 fulfils all the requirements for a genuine enhancer that functions independent of its orientation and position with a profound erythroid-specific activity in normal erythroid progenitors (T2ECs) and in leukemic erythroid cell lines. We find that the HS2 enhancer activity is governed by two adjacent GATA-factor binding sites. v-ErbA as well as unliganded TR prevent HS2 activity by nullifying the positive function of factors bound to GATA-sites. However, v-ErbA, in contrast to TR, does not convey active repression to silence the transcriptional activity intrinsic to a heterologous tk promoter.We propose that depending on the sequence and context of the binding site, v-ErbA contributes to leukemogenesis by occluding liganded TR as well as unliganded TR thereby preventing activation or repression, respectively.

Toucan protein is essential for the assembly of syncytial mitotic spindles in Drosophila melanogaster.

Author(s) : Debec A, Grammont M, Berson G, Dastugue B, Sullivan W, Couderc J,
Journal : Genesis
2001
The toc gene of Drosophila melanogaster encodes a 235-kD polypeptide with a coiled-coil domain, which is highly expressed during oogenesis (Grammont et al.,1997, 2000). We now report the localization of the Toucan protein during early embryonic development. The Toucan protein is present only during the syncytial stages and is associated with the nuclear envelope and the cytoskeletal structures of the syncytial embryo. In anaphase A, Toucan is concentrated at thespindle poles near the minus end of microtubules. This microtubule association is very dynamic during the nuclear cell cycle. Mutant embryos lacking the Toucan protein are blocked in a metaphase-like state. They display abnormal and nonfunctional spindles, characterized by broad poles, detachment of the centrosomes, and failure of migration of the chromosomes. These results stronglysuggest that Toucan represents a factor essential for the assembly and the function of the syncytial mitotic spindles.

Two-step process for photoreceptor formation in Drosophila.

Author(s) : Mollereau B, Dominguez M, Webel R, Colley N, Keung B, de Celis J, Desplan C,
Journal : Nature
2001
The formation of photoreceptor cells (PRCs) in Drosophila serves as a paradigm for understanding neuronal determination and differentiation. During larval stages, a precise series of sequential inductive processes leads to the recruitment of eight distinct PRCs (R1-R8). But, final photoreceptor differentiation, including rhabdomere morphogenesis and opsin expression, is completed four days later, during pupal development. It is thought that photoreceptor cell fate is irreversibly established during larval development, when each photoreceptor expresses a particular set of transcriptional regulatorsand sends its projection to different layers of the optic lobes. Here, we show that the spalt (sal) gene complex encodes two transcription factors that are required late in pupation for photoreceptor differentiation. In the absence of the sal complex, rhabdomere morphology and expression of opsin genes in the inner PRCs R7 and R8 are changed to become identical to those of outer R1-R6 PRCs. However, these cells maintain their normal projections to the medulla part of the optic lobe, and not to the lamina where outer PRCs project. These data indicate that photoreceptor differentiation occurs as a two-step process. First, during larval development, the photoreceptor neurons become committed and send their axonal projections to their targets in the brain. Second, terminal differentiation is executed during pupal development and the photoreceptors adopt their final cellular properties.