Quest for a stable Cu-ligand complex with a high catalytic activity to produce reactive oxygen species.

Metal ion-catalyzed overproduction of reactive oxygen species (ROS) is believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defense to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can efficiently catalyze the production of ROS in the presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in the biological environment, and high ROS catalysis is of interest for applications as antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. Two of them with mixed arm length showed a higher catalytic activity in the oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity toward Escherichia coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L-based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data are discussed.

The unfolded protein response-glutathione metabolism axis: A novel target of a cycloruthenated complexes bypassing tumor resistance mechanisms.

Platinum-based drugs remain the reference treatment for gastric cancer (GC). However, the frequency of resistance, due to mutations in TP53 or alterations in the energy and redox metabolisms, impairs the efficacy of current treatments, highlighting the need for alternative therapeutic options. Here, we show that a cycloruthenated compound targeting the redox metabolism, RDC11, induces higher cytotoxicity than oxaliplatin in GC cells and is more potent in reducing tumor growth in vivo. Detailed investigations into the mode of action of RDC11 indicated that it targets the glutathione (GSH) metabolism, which is an important drug resistance mechanism. We demonstrate that cycloruthenated complexes regulate the expression of enzymes of the transsulfuration pathway via the Unfolded Protein Response (UPR) and its effector ATF4. Furthermore, RDC11 induces the expression of SLC7A11 encoding for the cystine/glutamate antiporter xCT. These effects lead to a lower cellular GSH content and elevated oxygen reactive species production, causing the activation of a caspase-independent apoptosis. Altogether, this study provides the first evidence that cycloruthenated complexes target the GSH metabolism, neutralizing thereby a major resistance mechanism towards platinum-based chemotherapies and anticancer immune response.

Altering relative metal-binding affinities in multifunctional Metallochaperones for mutant p53 reactivation.

The p53 protein plays a major role in cancer prevention, and over 50% of cancer diagnoses can be attributed to p53 malfunction. p53 incorporates a structural Zn site that is required for proper protein folding and function, and in many cases point mutations can result in loss of the Zn2+ ion, destabilization of the tertiary structure, and eventual amyloid aggregation. Herein, we report a series of compounds designed to act as small molecule stabilizers of mutant p53, and feature Zn-binding fragments to chaperone Zn2+ to the metal depleted site and restore wild-type (WT) function. Many Zn metallochaperones (ZMCs) have been shown to generate intracellular reactive oxygen species (ROS), likely by chelating redox-active metals such as Fe2+/3+ and Cu+/2+ and undergoing associated Fenton chemistry. High levels of ROS can result in off-target effects and general toxicity, and thus, careful tuning of ligand Zn2+ affinity, in comparison to the affinity for other endogenous metals, is important for selective mutant p53 targeting. In this work we show that by using carboxylate donors in place of pyridine we can change the relative Zn2+/Cu2+ binding ability in a series of ligands, and we investigate the impact of donor group changes on metallochaperone activity and overall cytotoxicity in two mutant p53 cancer cell lines (NUGC3 and SKGT2).

Multifunctional metallochaperone modifications for targeting subsite cavities in mutant p53-Y220C.

The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.

Ultrasound and Transcriptomics Identify a Differential Impact of Cisplatin and Histone Deacetylation on Tumor Structure and Microenvironment in a Patient-Derived In Vivo Model of Gastric Cancer.

The reasons behind the poor efficacy of transition metal-based chemotherapies (e.g., cisplatin) or targeted therapies (e.g., histone deacetylase inhibitors, HDACi) on gastric cancer (GC) remain elusive and recent studies suggested that the tumor microenvironment could contribute to the resistance. Hence, our objective was to gain information on the impact of cisplatin and the pan-HDACi SAHA (suberanilohydroxamic acid) on the tumor substructure and microenvironment of GC, by establishing patient-derived xenografts of GC and a combination of ultrasound, immunohistochemistry, and transcriptomics to analyze. The tumors responded partially to SAHA and cisplatin. An ultrasound gave more accurate tumor measures than a caliper. Importantly, an ultrasound allowed a noninvasive real-time access to the tumor substructure, showing differences between cisplatin and SAHA. These differences were confirmed by immunohistochemistry and transcriptomic analyses of the tumor microenvironment, identifying specific cell type signatures and transcription factor activation. For instance, cisplatin induced an "epithelial cell like" signature while SAHA favored a "mesenchymal cell like" one. Altogether, an ultrasound allowed a precise follow-up of the tumor progression while enabling a noninvasive real-time access to the tumor substructure. Combined with transcriptomics, our results underline the different intra-tumoral structural changes caused by both drugs that impact differently on the tumor microenvironment.

Gold(III) to Ruthenium(III) Metal Exchange in Dithiocarbamato Complexes Tunes Their Biological Mode of Action for Cytotoxicity in Cancer Cells.

Malignant tumors have affected the human being since the pharaoh period, but in the last century the incidence of this disease has increased due to a large number of risk factors, including deleterious lifestyle habits (i.e., smoking) and the higher longevity. Many efforts have been spent in the last decades on achieving an early stage diagnosis of cancer, and more effective cures, leading to a decline in age-standardized cancer mortality rates. In the last years, our research groups have developed new metal-based complexes, with the aim to obtain a better selectivity for cancer cells and less side effects than the clinically established reference drug cisplatin. This work is focused on four novel Au(III) and Ru(III) complexes that share the piperidine dithiocarbamato (pipe-DTC) as the ligand, in a different molar ratio. The compounds [AuCl2(pipeDTC)], [Au(pipeDTC)2]Cl, [Ru(pipeDTC)3] and ß-[Ru2(pipeDTC)5] have been synthesized and fully characterized by several chemical analyses. We have then investigated their biological properties in two different cell lines, namely, AGS (gastric adenocarcinoma) and HCT116 (colon carcinomas), showing significant differences among the four compounds. First, the two gold-based compounds and ß-[Ru2(pipeDTC)5] display IC50 in the µM range, significantly lower than cisplatin. Second, we showed that [AuCl2(pipeDTC)] and ß-[Ru2(pipeDTC)5]Cl drive different molecular mechanisms. The first was able to induce the protein level of the DNA damage response factor p53 and the autophagy protein p62, in contrast to the second that induced the ATF4 protein level, but repressed p62 expression. This study highlights that the biological activity of different complexes bringing the same organic ligand depends on the electronic and structural properties of the metal, which are able to fine tune the biological properties, giving us precious information that can help to design more selective anticancer drugs.

HDAC4 Levels Control Sensibility toward Cisplatin in Gastric Cancer via the p53-p73/BIK Pathway.

Gastric cancer (GC) remains a health issue due to the low efficiency of therapies, such as cisplatin. This unsatisfactory situation highlights the necessity of finding factors impacting GC sensibility to therapies. We analyzed the cisplatin pangenomic response in cancer cells and found HDAC4 as a major epigenetic regulator being inhibited. HDAC4 mRNA repression was partly mediated by the cisplatin-induced expression of miR-140. At a functional level, HDAC4 inhibition favored cisplatin cytotoxicity and reduced tumor growth. Inversely, overexpression of HDAC4 inhibits cisplatin cytotoxicity. Importantly, HDAC4 expression was found to be elevated in gastric tumors compared to healthy tissues, and in particular in specific molecular subgroups. Furthermore, mutations in HDAC4 correlate with good prognosis. Pathway analysis of genes whose expression in patients correlated strongly with HDAC4 highlighted DNA damage, p53 stabilization, and apoptosis as processes downregulated by HDAC4. This was further confirmed by silencing of HDAC4, which favored cisplatin-induced apoptosis characterized by cleavage of caspase 3 and induction of proapoptotic genes, such as BIK, in part via a p53-dependent mechanism. Altogether, these results reveal HDAC4 as a resistance factor for cisplatin in GC cells that impacts on patients' survival.

Bifunctional ligand design for modulating mutant p53 aggregation in cancer.

Protein misfolding and aggregation contributes to the development of a wide range of diseases. In cancer, over 50% of diagnoses are attributed to p53 malfunction due to missense mutations, many of which result in protein misfolding and accelerated aggregation. p53 mutations also frequently result in alteration or loss of zinc at the DNA-binding site, which increases aggregation via nucleation with zinc-bound p53. Herein, we designed two novel bifunctional ligands, LI and LH , to modulate mutant p53 aggregation and restore zinc binding using a metallochaperone approach. Interestingly, only the incorporation of iodine function in LI resulted in modulation of mutant p53 aggregation, both in recombinant and cellular environments. Native mass spectrometry shows a protein-ligand interaction for LI , as opposed to LH , which is hypothesized to lead to the distinct difference in the p53 aggregation profile for the two ligands. Incorporation of a di-2-picolylamine binding unit into the ligand design provided efficient intracellular zinc uptake, resulting in metallochaperone capability for both LI and LH . The ability of LI to reduce mutant p53 aggregation results in increased restoration of p53 transcriptional function and mediates both caspase-dependent and -independent cell death pathways. We further demonstrate that LI exhibits minimal toxicity in non-cancerous organoids, and that it is well tolerated in mice. These results demonstrate that iodination of our ligand framework restores p53 function by interacting with and inhibiting mutant p53 aggregation and highlights LI as a suitable candidate for comprehensive in vivo anticancer preclinical evaluations.

Multifunctional Compounds for Activation of the p53-Y220C Mutant in Cancer.

The p53 protein plays a major role in cancer prevention, and over 50 % of cancer diagnoses can be attributed to p53 malfunction. The common p53 mutation Y220C causes local protein unfolding, aggregation, and can result in a loss of Zn in the DNA-binding domain. Structural analysis has shown that this mutant creates a surface site that can be stabilized using small molecules, and herein a multifunctional approach to restore function to p53-Y220C is reported. A series of compounds has been designed that contain iodinated phenols aimed for interaction and stabilization of the p53-Y220C surface cavity, and Zn-binding fragments for metallochaperone activity. Their Zn-binding affinity was characterized using spectroscopic methods and demonstrate the ability of compounds L4 and L5 to increase intracellular levels of Zn2+ in a p53-Y220C-mutant cell line. The in vitro cytotoxicity of our compounds was initially screened by the National Cancer Institute (NCI-60), followed by testing in three stomach cancer cell lines with varying p53 status', including AGS (WTp53), MKN1 (V143A), and NUGC3 (Y220C). Our most promising ligand, L5, is nearly 3-fold more cytotoxic than cisplatin in a large number of cell lines. The impressive cytotoxicity of L5 is further maintained in a NUGC3 3D spheroid model. L5 also induces Y220C-specific apoptosis in a cleaved caspase-3 assay, reduces levels of unfolded mutant p53, and recovers p53 transcriptional function in the NUGC3 cell line. These results show that these multifunctional scaffolds have the potential to restore wild-type function in mutant p53-Y220C.

Rollover Cyclometalated Bipyridine Platinum Complexes as Potent Anticancer Agents: Impact of the Ancillary Ligands on the Mode of Action.

Platinum-based anticancer coordination compounds are widely used in the treatment of many tumor types, where they are very effective but also cause severe side effects. Organoplatinum compounds are significantly less investigated than the analogous coordination compounds. We report here rollover cyclometalated Pt compounds based on 2,2'-bipyridine which are demonstrated to be potent antitumor agents both in vitro and in vivo. Variation of the co-ligands on the Pt(2,2'-bipyridine) backbone resulted in the establishment of structure-activity relationships. They showed that the biological activity was in general inversely correlated with the reaction kinetics to biomolecules as shown for amino acids, proteins, and DNA. The less stable compounds caused higher reactivity with biomolecules and were shown to induce p53-dependent DNA damage. In contrast, the presence of bulky PTA and PPh3 ligands was demonstrated to cause lower reactivity and increased antineoplastic activity. Such compounds were devoid of DNA-damaging activity and induced ATF4, a component of the endoplasmic reticulum (ER) stress pathway. The lead complex inhibited tumor growth similar to oxaliplatin while showing no signs of toxicity in test mice. Therefore, we demonstrated that it is possible to fine-tune rollover-cyclometalated Pt(II) compounds to target different cancer pathways and be a means to overcome the side effects associated with cisplatin and analogous compounds in cancer chemotherapy.

Monomeric Ti(iv)-based complexes incorporating luminescent nitrogen ligands: synthesis, structural characterization, emission spectroscopy and cytotoxic activities.

This manuscript describes the synthesis of a series of neutral titanium(iv) monomeric complexes constructed around a TiO4N2 core. The two nitrogen atoms that compose the coordination sphere of the metallic center belong to 2,2'-bipyrimidine ligands homo-disubstituted in the 4 and 4' positions by methyl (2a), phenylvinyl (2b), naphthylvinyl (2c) or anthrylvinyl (2d) groups. The crystal structures of these complexes named [Ti(1)2(2a)], [Ti(1)2(2b)], [Ti(1)2(2c)] and [Ti(1)2(2d)] (where 1 is a 2,2'-biphenolato ligand substituted in the 6 and 6' positions by phenyl groups) are reported. The hydrolytic stability of the four complexes is evaluated by monitoring the evolution of the free 2a-d signals by 1H NMR spectroscopy. For the conditions tested (6 mM, DMSO-d6/D2O: 8/1), a rather good stability with t1/2 ranging from 180 to 300 min is determined for the complexes. In the presence of an acid (DCl), the hydrolysis of [Ti(1)2(2a)] is faster than without an acid. The cytotoxic activity against gastric cancer cells of the titanium-based compounds and the free disubstituted 2,2'-bipyrimidine ligands is tested, showing IC50 ranging from 6.2 ± 1.2 µM to 274 ± 56 µM. The fluorescence studies of the ligands 2a-d, and the complexes [Ti(1)2(2a-d)] reveal an important fluorescence loss of the ligands 2c and 2d upon coordination with the Ti(1)2 fragment. Frontier orbitals obtained by DFT calculations permit us to explain this fluorescence quenching.

Impact of cyclometalated ruthenium(II) complexes on lactate dehydrogenase activity and cytotoxicity in gastric and colon cancer cells.

Lactate dehydrogenase (LDH) is a redox enzyme often overexpressed in cancer cells allowing their survival in stressful metabolic tumor environment. Ruthenium(II) complexes have been shown to impact on the activity of purified horseradish peroxidase and glucose oxidase but the physiological relevance remains unclear. In this study we investigated how ruthenium complexes impact on the activity of LDH in vitro and in cancer cells and performed a comparative study using polypyridine ruthenium(II) complex [Ru(bpy)3]2+ (1) and its structurally related cyclometalated 2-phenylpyridinato counterpart [Ru(phpy)(bpy)2]+ (2) (bpy=2,2'-bipyridine, phpyH=2-phenylpyridine). We show that the cytotoxicity in gastric and colon cancer cells induced by 2 is significantly higher compared to 1. The kinetic inhibition mechanisms on purified LDH and the corresponding inhibition constants Ki or i0.5 values were calculated. Though complexes 1 and 2 are structurally very similar (one Ru-C bond in 2 replaces one Ru-N bond in 1), their inhibition modes are different. Cyclometalated complex 2 behaves exclusively as a non-competitive inhibitor of LDH from rabbit muscle (LDHrm), strongly suggesting that 2 does not interact with LDH in the vicinities of either lactate/pyruvate or NAD+/NADH binding sites. Sites of interaction of 1 and 2 with LDHrm were revealed theoretically through computational molecular docking. Inhibition of LDH activity by 2 was confirmed in cancer cells. Altogether, these results revealed an inhibition of LDH activity by ruthenium complex through a direct interaction structurally tuned by a Ru-C bond.

Expression and functional studies of the GDNF family receptor alpha 3 in the pancreas.

The generation of therapeutic ß-cells from human pluripotent stem cells relies on the identification of growth factors that faithfully mimic pancreatic ß-cell development in vitro. In this context, the aim of the study was to determine the expression and function of the glial cell line derived neurotrophic factor receptor alpha 3 (GFRα3) and its ligand artemin (Artn) in islet cell development and function. GFRα3 and Artn expression were characterized by in situ hybridization, immunochemistry, and qRT-PCR. We used GFRα3-deficient mice to study GFRα3 function and generated transgenic mice overexpressing Artn in the embryonic pancreas to study Artn function. We found that GFRα3 is expressed at the surface of a subset of Ngn3-positive endocrine progenitors as well as of embryonic α- and ß-cells, while Artn is found in the pancreatic mesenchyme. Adult ß-cells lack GFRα3 but α-cells express the receptor. GFRα3 was also found in parasympathetic and sympathetic intra-islet neurons as well as in glial cells in the embryonic and adult pancreas. The loss of GFRα3 or overexpression of Artn has no impact on Ngn3 and islet cell formation and maintenance in the embryo. Islet organization and innervation as well as glucose homeostasis is normal in GFRα3-deficient mice suggesting functional redundancy.

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development.

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

The transcription factor c-Myb affects pre-mRNA splicing.

c-Myb is a transcription factor which plays a key role in haematopoietic proliferation and lineage commitment. We raised the question of whether c-Myb may have abilities beyond the extensively studied transcriptional activation function. In this report we show that c-Myb influences alternative pre-mRNA splicing. This was seen by its marked effect on the 5'-splice site selection during E1A alternative splicing, while no effect of c-Myb was observed when reporters for the 3'-splice site selection or for the constitutive splicing process were tested. Moreover, co-immunoprecipitation experiments provided evidence for interactions between c-Myb and distinct components of the splicing apparatus, such as the general splicing factor U2AF(65) and hnRNPA1 involved in the 5'-splice site selection. The effect on 5'-splice site selection was abolished in the oncogenic variant v-Myb. Altogether, these data provide evidence that c-Myb may serve a previously unappreciated role in the coupling between transcription and splicing.

Transcription factors in pancreatic development. Animal models.

Through the analysis of genetically modified mice a hierarchy of transcription factors regulating pancreas specification, endocrine destiny as well as endocrine subtype specification and differentiation has been established. In addition to conventional approaches such as transgenic technologies and gene targeting, recombinase fate mapping in mice has been key in establishing the lineage relationship between progenitor cells and their progeny in understanding pancreas formation. Moreover, the design of specific mouse models to conditionally express transcription factors in different populations of progenitor cells has revealed to what extent transcription factors required for islet cell development are also sufficient to induce endocrine differentiation and the importance of the competence of progenitor cells to respond to the genetic program implemented by these factors. Taking advantage of this basic science knowledge acquired in rodents, immature insulin-producing cells have recently been differentiated in vitro from human embryonic stem cells. Taken together these major advances emphasize the need to gain further in-depth knowledge of the molecular and cellular mechanisms controlling beta-cell differentiation in mice to generate functional beta-cells in the future that could be used for cell therapy in diabetes.

Pancreatic islet progenitor cells in neurogenin 3-yellow fluorescent protein knock-add-on mice.

The basic helix-loop-helix transcription factor Neurogenin 3 (NGN3) controls endocrine cell fate specification in uncommitted pancreatic progenitor cells. Ngn3-deficient mice do not develop any islet cells and are diabetic. All the major islet cell types, including insulin-producing beta-cells, derive from Ngn3-positive endocrine progenitor cells. Therefore, the characterization of this population of immature cells is of particular interest for the development of novel strategies for cell replacement therapies in type 1 diabetes. To explore further the biology of islet progenitor cells we have generated a mouse in which Ngn3-expressing cells are labeled with the enhanced yellow fluorescent protein (EYFP) using a knock-add-on strategy. In this approach, the EYFP cDNA is introduced into the 3'-untranslated region of the proendocrine transcription factor, Neurogenin 3, without deleting any endogenous coding or regulatory sequences. In Ngn3(EYFP/+) and Ngn3(EYFP/EYFP) mice, the EYFP protein is targeted to Ngn3-expressing progenitors in the developing pancreas, and islets develop normally. Islet progenitors can be purified from whole embryonic pancreas by fluorescence-activated cell sorting from Ngn3(EYFP/+) mice and their development can be monitored in real time in pancreas explant cultures. These experiments showed that endocrine progenitors can form de novo and expand, in vitro, in the absence of signals from the surrounding mesenchyme, suggesting that endocrine commitment is a default pathway. The Ngn3(EYFP) mice represent a valuable tool to study islet cell development and neogenesis in normal and diabetic animals as well as for the determination of the conditions to generate beta-cells in vitro.

Multiple functional domains of the oncoproteins Spi-1/PU.1 and TLS are involved in their opposite splicing effects in erythroleukemic cells.

The hematopoietic transcription factor Spi-1/PU.1 is an oncoprotein participating to the malignant transformation of proerythroblasts in the Friend erythroleukemia or in the erythroleukemic process developed in spi-1 transgenic mice. Overexpression of Spi-1 in proerythroblasts blocks their differentiation. We have shown that Spi-1 promotes the use of the proximal 5'-splice site during the E1A pre-mRNA splicing and interferes with the effect of TLS (Translocated in LipoSarcoma) in this splicing assay. TLS was identified from chromosomal translocations in human liposarcoma and acute myeloid leukemia. Here, we determine the function of Spi-1 domains in splicing and in the interference with TLS. In transient transfection assays in erythroid cells, we show that the DNA binding domain cooperates with the transactivation domain or the PEST region of Spi-1 to modify the function of TLS in splicing. Interestingly, the 27 C-terminal amino acids, which determine the DNA binding activity of Spi-1, are necessary for the splicing function of Spi-1 as well as for its ability to interfere with TLS. Finally, we demonstrate that in leukemic proerythroblasts overexpressing Spi-1, TLS has lost its splicing effect. Thus, we hypothesize that oncogenic pathways in proerythroblasts may involve the ability of Spi-1 to alter splicing.

The human neuroendocrine thyrotropin-releasing hormone receptor promoter is activated by the haematopoietic transcription factor c-Myb.

Thyrotropin-releasing hormone (TRH) receptor (TRHR) is a G-protein-coupled receptor playing a crucial role in the anterior pituitary where it controls the synthesis and secretion of thyroid-stimulating hormone and prolactin. Its widespread presence not only in the central nervous system, but also in peripheral tissues, including thymus, indicates other important, but unknown, functions. One hypothesis is that the neuropeptide TRH could play a role in the immune system. We report here that the human TRHR promoter contains 11 putative response elements for the haematopoietic transcription factor c-Myb and is highly Myb-responsive in transfection assays. Analysis of Myb binding to putative response elements revealed one preferred binding site in intron 1 of the receptor gene. Transfection studies of promoter deletions confirmed that this high-affinity element is necessary for efficient Myb-dependent transactivation of reporter plasmids in CV-1 cells. The Myb-dependent activation of the TRHR promoter was strongly suppressed by expression of a dominant negative Myb-Engrailed fusion. In line with these observations, reverse transcriptase PCR analysis of rat tissues showed that the TRHR gene is expressed both in thymocytes and bone marrow. Furthermore, specific, high-affinity TRH agonist binding to cell-surface receptors was demonstrated in thymocytes and a haematopoietic cell line. Our findings imply a novel functional link between the neuroendocrine and the immune systems at the level of promoter regulation.