Christian Gaiddon

P73 FUNCTION IS INHIBITED BY TUMOR-DERIVED P53 MUTANTS IN MAMMALIAN CELLS

ABSTRACT The p53 tumor suppressor protein, found mutated in over 50% of all human tumors, is a sequence-specific transcriptional activator. Recent studies have identified a p53 relative, termed p73. We were interested in determining the relative abilities of wild-type and mutant forms of p53 and p73α and -β isoforms to transactivate various p53-responsive promoters. We show that both p73α and p73β activate the transcription of reporters containing a number of p53-responsive promoters in the p53-null cell line H1299. However, a number of significant differences were observed between p53 and p73 and even between p73α and p73β. Additionally, a Saccharomyces cerevisiae-based reporter assay revealed a broad array of transcriptional transactivation abilities by both p73 isoforms at 37°C. Recent data have shown that p73 can associate with p53 by the yeast two-hybrid assay. When we examined complex formation in transfected mammalian cells, we found that p73α coprecipitates with mutant but not wild-type p53. Since many tumor-derived p53 mutants are capable of inhibiting transactivation by wild-type p53, we tested the effects of two representative hot-spot mutants (R175H and R248W) on p73. By cotransfecting p73α along with either p53 mutant and a p53-responsive reporter, we found that both R175H and R248W reduces the transcriptional activity of p73α. This decrease in transcriptional activity is correlated with the reduced ability of p73α to promote apoptosis in the presence of tumor-derived p53 mutants. Our data suggest the possibility that in some tumor cells, an outcome of the expression of mutant p53 protein may be to interfere with the endogenous p73 protein.

A Subset of Tumor-Derived Mutant Forms of p53 Down-Regulate p63 and p73 through a Direct Interaction with the p53 Core Domain

ABSTRACT The p53 protein is related by sequence homology and function to the products of two other genes, p63 and p73, that each encode several isoforms. We and others have discovered previously that certain tumor-derived mutants of p53 can associate and inhibit transcriptional activation by the α and β isoforms of p73. In this study we have extended these observations to show that in transfected cells a number of mutant p53 proteins could bind and down-regulate several isoforms not only of p73 (p73α, -β, -γ, and -δ) but also of p63 (p63α and -γ; ΔNp63α and -γ). Moreover, a correlation existed between the efficiency of p53 binding and the inhibition of p63 or p73 function. We also found that wild-type p63 and p73 interact efficiently with each other when coexpressed in mammalian cells. The interaction between p53 mutants and p63 or p73 was confirmed in a physiological setting by examining tumor cell lines that endogenously express these proteins. We also demonstrated that purified p53 and p73 proteins interact directly and that the p53 core domain, but not the tetramerization domain, mediates this interaction. Using a monoclonal antibody (PAb240) that recognizes an epitope within the core domain of a subset of p53 mutants, we found a correlation between the ability of p53 proteins to be immunoprecipitated by this antibody and their ability to interact with p73 or p63 in vitro and in transfected cells. Based on these results and those of others, we propose that interactions between the members of the p53 family are likely to be widespread and may account in some cases for the ability of tumor-derived p53 mutants to promote tumorigenesis.

Approaching tumour therapy beyond platinum drugs: Status of the art and perspectives of ruthenium drug candidates

The study of metal complexes for the treatment of cancer diseases has resulted in the identification of some unique properties of ruthenium-based compounds. Among these inorganic-based agents, two of them, namely the ruthenium(III) drugs NAMI-A and KP1019 have undertaken with some success the clinical evaluations of phase I and preliminary phase II trials in patients. Here we highlight the strategies that have led to the discovery of metal-based (NAMI-A and KP1019) and of organometallic (RM175, RAPTA-T, RDC11 and DW1/2) ruthenium-based complexes, and we report their main biological/pharmacological characteristics and expectations for further development.

Immune cell-derived beta-endorphin. Production, release, and control of inflammatory pain in rats.

Localized inflammation of a rats hindpaw elicits an accumulation of beta-endorphin-(END) containing immune cells. We investigated the production, release, and antinociceptive effects of lymphocyte-derived END in relation to cell trafficking. In normal animals, END and proopiomelanocortin mRNA were less abundant in circulating lymphocytes than in those residing in lymph nodes (LN), suggesting that a finite cell population produces END and homes to LN. Inflammation increased proopiomelanocortin mRNA in cells from noninflamed and inflamed LN. However, END content was increased only in inflamed paw tissue and noninflamed LN-immune cells. Accordingly, corticotropin-releasing factor and IL-1beta released significantly more END from noninflamed than from inflamed LN-immune cells. This secretion was receptor specific, calcium dependent, and mimicked by potassium, consistent with vesicular release. Finally, both agents, injected into the inflamed paw, induced analgesia which was blocked by the co-administration of antiserum against END. Together, these findings suggest that END-producing lymphocytes home to inflamed tissue where they secrete END to reduce pain. Afterwards they migrate to the regional LN, depleted of the peptide. Consistent with this notion, immunofluorescence studies of cell suspensions revealed that END is contained predominantly within memory-type T cells. Thus, the immune system is important for the control of inflammatory pain. This has implications for the understanding of pain in immunosuppressed conditions like cancer or AIDS.

REF-1 REGULATES THE TRANSACTIVATION AND PRO-APOPTOTIC FUNCTIONS OF P53 IN VIVO

Ref‐1 is a multifunctional protein that stimulates DNA binding by a number of transcription factors and serves as the abasic (A/P) endonuclease in base excision repair. Ref‐1 was discovered to be a potent activator of p53 DNA binding in vitro. To address the physiological significance of the effects of Ref‐1 on p53, we have analyzed its role in regulating p53 function in vivo. We found that Ref‐1 over‐expression enhances the ability of p53 to transactivate a number of p53 target promoters and increases the ability of p53 to stimulate endogenous p21 and cyclin G expression. Additionally, it was observed that Ref‐1 associates with p53 in vivo and in vitro. Importantly, downregulation of Ref‐1 (by antisense) causes a marked reduction in p53 induction of p21 mRNA and protein, as well as diminished ability of p53 to transactivate the p21 and Bax promoters. Moreover, Ref‐1 levels are correlated with the extent of apoptosis induced by p53. Finally, we observed that Ref‐1 cooperates with a DNA‐damaging compound, camptothecin, to stimulate the transcriptional activity of p53. Together these data indicate that Ref‐1 is a key cellular regulator of p53.

A Ruthenium-Containing Organometallic Compound Reduces Tumor Growth through Induction of the Endoplasmic Reticulum Stress Gene CHOP

Cisplatin-derived anticancer therapy has been used for three decades despite its side effects. Other types of organometallic complexes, namely, some ruthenium-derived compounds (RDC), which would display cytotoxicity through different modes of action, might represent alternative therapeutic agents. We have studied both in vitro and in vivo the biological properties of RDC11, one of the most active compounds of a new class of RDCs that contain a covalent bond between the ruthenium atom and a carbon. We showed that RDC11 inhibited the growth of various tumors implanted in mice more efficiently than cisplatin. Importantly, in striking contrast with cisplatin, RDC11 did not cause severe side effects on the liver, kidneys, or the neuronal sensory system. We analyzed the mode of action of RDC11 and showed that RDC11 interacted poorly with DNA and induced only limited DNA damages compared with cisplatin, suggesting alternative transduction pathways. Indeed, we found that target genes of the endoplasmic reticulum stress pathway, such as Bip, XBP1, PDI, and CHOP, were activated in RDC11-treated cells. Induction of the transcription factor CHOP, a crucial mediator of endoplasmic reticulum stress apoptosis, was also confirmed in tumors treated with RDC11. Activation of CHOP led to the expression of several of its target genes, including proapoptotic genes. In addition, the silencing of CHOP by RNA interference significantly reduced the cytotoxicity of RDC11. Altogether, our results led us to conclude that RDC11 acts by an atypical pathway involving CHOP and endoplasmic reticulum stress, and thus might provide an interesting alternative for anticancer therapy.

Brain-derived neurotrophic factor stimulates AP-1 and cyclic AMP-responsive element dependent transcriptional activity in central nervous system neurons

Abstract: Brain‐derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high‐affinity membrane receptors displaying tyrosine kinase activity (trk). However, the intracellular pathways and genetic mechanisms associated with these receptors are largely unknown. Here we show that BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex as it is associated with the induction of AP1‐dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c‐fos and c‐jun mRNAs after BDNF treatment, resulting from an induction of their promoters. The cis‐acting elements by which BDNF stimulates c‐fos transcription were further studied. We show that BDNF impinges on multiple regulatory elements, including the serum‐responsive element, Fos AP1‐like element, and cyclic AMP (cAMP)‐responsive element (CRE) sequences. The latter was stimulated without any detectable increase in cAMP or Ca2+ levels. To confirm that BDNF induces c‐fos transcription independently of the protein kinase A/cAMP pathway, we transfected a dominant inhibitory mutant of the regulatory subunit of protein kinase A. The overexpression of this mutant does not affect the c‐fos promoter transactivation by BDNF. In summary, we show that BDNF stimulates AP1‐ and CRE‐dependent transcription through a mechanism that is distinct from the cAMP‐ and Ca2+‐dependent pathways in CNS neurons.

Discovery and Investigation of Anticancer Ruthenium–Arene Schiff-Base Complexes via Water-Promoted Combinatorial Three-Component Assembly

The structural diversity of metal scaffolds makes them a viable alternative to traditional organic scaffolds for drug design. Combinatorial chemistry and multicomponent reactions, coupled with high-throughput screening, are useful techniques in drug discovery, but they are rarely used in metal-based drug design. We report the optimization and validation of a new combinatorial, metal-based, three-component assembly reaction for the synthesis of a library of 442 Ru-arene Schiff-base (RAS) complexes. These RAS complexes were synthesized in a one-pot, on-a-plate format using commercially available starting materials under aqueous conditions. The library was screened for their anticancer activity, and several cytotoxic lead compounds were identified. In particular, [(η6-1,3,5-triisopropylbenzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)aniline)]Cl (4) displayed low micromolar IC50 values in ovarian cancers (A2780, A2780cisR), breast cancer (MCF7), and colorectal cancer (HCT116, SW480). The absence of p53 activation or changes in IC50 value between p53+/+ and p53-/- cells suggests that 4 and possibly the other lead compounds may act independently of the p53 tumor suppressor gene frequently mutated in cancer.

Sprouty2 inhibits BDNF-induced signaling and modulates neuronal differentiation and survival

Sprouty (Spry) proteins are ligand-inducible inhibitors of receptor tyrosine kinases-dependent signaling pathways, which control various biological processes, including proliferation, differentiation and survival. Here, we investigated the regulation and the role of Spry2 in cells of the central nervous system (CNS). In primary cultures of immature neurons, the neurotrophic factor BDNF (brain-derived neurotrophic factor) regulates spry2 expression. We identified the transcription factors CREB and SP1 as important regulators of the BDNF activation of the spry2 promoter. In immature neurons, we show that overexpression of wild-type Spry2 blocks neurite formation and neurofilament light chain expression, whereas inhibition of Spry2 by a dominant-negative mutant or small interfering RNA favors sprouting of multiple neurites. In mature neurons that exhibit an extensive neurite network, spry2 expression is sustained by BDNF and is downregulated during neuronal apoptosis. Interestingly, in these differentiated neurons, overexpression of Spry2 induces neuronal cell death, whereas its inhibition favors neuronal survival. Together, our results imply that Spry2 is involved in the development of the CNS by inhibiting both neuronal differentiation and survival through a negative-feedback loop that downregulates neurotrophic factors-driven signaling pathways.

Library of second-generation cycloruthenated compounds and evaluation of their biological properties as potential anticancer drugs: Passing the nanomolar barrier

A library of 32 organoruthenium compounds has been synthesised. Known and novel C-N cyclometalated compounds as well as N-C-N and N-N-C pincer derivatives of this metal have been used in this purpose. Most of the compounds have been tested for their in vitro antitumoral behaviours, good to excellent activities have thus been found. Several of the newly synthesized compounds pass the symbolic barrier of the nanomolar range for their IC(50) indicating a critical improvement. The level of activity is tentatively correlated to physicochemical properties of the compounds such as their Ru(III/II) redox potential and their lipophilicity (log P).