Franck Toledo

Regulating the p53 pathway: in vitro hypotheses, in vivo veritas

Mutations in TP53, the gene that encodes the tumour suppressor p53, are found in 50% of human cancers, and increased levels of its negative regulators MDM2 and MDM4 (also known as MDMX) downregulate p53 function in many of the rest. Understanding p53 regulation remains a crucial goal to design broadly applicable anticancer strategies based on this pathway. This Review of in vitro studies, human tumour data and recent mouse models shows that p53 post-translational modifications have modulatory roles, and MDM2 and MDM4 have more profound roles for regulating p53. Importantly, MDM4 emerges as an independent target for drug development, as its inactivation is crucial for full p53 activation.

Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4

1 Laboratory For Molecular Cancer Biology, Flanders Interuniversity Institute for Biotechnology (VIB), University of Ghent, Technologiepark, 927, Ghent B9052, Belgium 2 Salk Institute for Biological Studies, Gene Expression Laboratory, La Jolla, CA 92037, USA 3 Gene Expression and Diseases Unit, Institut Pasteur, Paris, France 4 The University of Texas Graduate School of Biomedical Sciences and department of Molecular Genetics, Section of Cancer Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA * Corresponding author: J-C Marine, Laboratory For Molecular Cancer Biology, VIB, Technologiepark, 927, Ghent B-9052, Belgium. Tel: þ 32-93-313-640; Fax: þ 32-93-313-516; E-mail: chris.marine@dmbr.ugent.be

A New Role for Hypoxia in Tumor Progression: Induction of Fragile Site Triggering Genomic Rearrangements and Formation of Complex DMs and HSRs

Genome rearrangements including gene amplification are frequent properties of tumor cells, but how they are related to the tumor microenvironment is unknown. Here, we report direct evidence for a causal relationship between hypoxia, induction of fragile sites, and gene amplification. Recently, we showed that breaks at fragile sites initiate intrachromosomal amplification. We demonstrate here that hypoxia is a potent fragile site inducer and that, like fragile sites inducing drugs, it drives fusion of double minutes (DMs) and their targeted reintegration into chromosomal fragile sites, generating homogeneously staining regions (HSRs). This pathway operates efficiently for DMs bearing different sequences, suggesting a model of hypoxia-driven formation of the HSRs containing nonsyntenic sequences frequently observed in solid tumors.

Co-amplified markers alternate in megabase long chromosomal inverted repeats and cluster independently in interphase nuclei at early steps of mammalian gene amplification.

Two‐colour in situ hybridization with probes for two co‐amplified markers located several megabases apart on chromosome 1 has been used to analyse early stages of adenylate deaminase 2 (AMPD2) gene amplification in Chinese hamster cells. In the amplified chromosomal structures, the distribution of hybridization spots identifies megabase‐long inverted repeats. Their organization is remarkably well accounted for if breakage‐fusion‐bridge cycles involving sister chromatids drive the amplification process at these early stages. During interphase the markers often segregate into distinct nuclear domains. Many nuclei have bulges or release micronuclei, carrying several copies of one or both markers. These observations indicate that the amplified units destabilize the nuclear organization and eventually lead to DNA breakage during interphase. We propose a model in which interphase breakage has a role in the progression of gene amplification.

Mouse Mutants Reveal that Putative Protein Interaction Sites in the p53 Proline-Rich Domain Are Dispensable for Tumor Suppression

ABSTRACT The stability and activity of tumor suppressor p53 are tightly regulated and partially depend on the p53 proline-rich domain (PRD). We recently analyzed mice expressing p53 with a deletion of the PRD (p53ΔP). p53ΔP, a weak transactivator hypersensitive to Mdm2-mediated degradation, is unable to suppress oncogene-induced tumors. This phenotype could result from the loss of two motifs: Pin1 sites proposed to influence p53 stabilization and PXXP motifs proposed to mediate protein interactions. We investigated the importance of these motifs by generating mice encoding point mutations in the PRD. p53TTAA contains mutations suppressing all putative Pin1 sites in the PRD, while p53AXXA lacks PXXP motifs but retains one intact Pin1 site. Both mutant proteins accumulated in response to DNA damage, although the accumulation of p53TTAA was partially impaired. Importantly, p53TTAA and p53AXXA are efficient transactivators and potent suppressors of oncogene-induced tumors. Thus, Pin1 sites in the PRD may modulate p53 stability but do not significantly affect function. In addition, PXXP motifs are not essential, but structure dictated by the presence of prolines, PXXXXP motifs that may mediate protein interactions, and/or the length of this region appears to be functionally significant. These results may explain why the sequence of the p53 PRD is so variable in evolution.

Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.

Gene Amplification Mechanisms: The Role of Fragile Sites

We studied the early stages of gene amplification in a Chinese hamster cell line and identified two distinct amplification mechanisms, both relying on an unequal segregation of gene copies at mitosis. In some cases, a sequence containing the selected gene is looped out, generating an acentric circular molecule, and amplification proceeds through unequal segregation of such extrachromosomal elements in successive cell cycles. In other cases, the accumulation of intrachromosomally amplified copies is driven by cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere, which leads to bridge formation and further break in mitosis (BFB cycles). We showed that some clastogenic drugs specifically trigger the intrachromosomal amplification pathway and strictly correlated this induction of BFB cycles to the ability of these drugs to activate fragile sites. In three model systems, we also established, that the location of centromeric and telomeric fragile sites relative to the selected genes determines the size and sequence content of the early amplicons.

RMCE-ASAP: a gene targeting method for ES and somatic cells to accelerate phenotype analyses

In recent years, tremendous insight has been gained on p53 regulation by targeting mutations at the p53 locus using homologous recombination in ES cells to generate mutant mice. Although informative, this approach is inefficient, slow and expensive. To facilitate targeting at the p53 locus, we developed an improved Recombinase-Mediated Cassette Exchange (RMCE) method. Our approach enables efficient targeting in ES cells to facilitate the production of mutant mice. But more importantly, the approach was Adapted for targeting in Somatic cells to Accelerate Phenotyping (RMCE-ASAP). We provide proof-of-concept for this at the p53 locus, by showing efficient targeting in fibroblasts, and rapid phenotypic read-out of a recessive mutation after a single exchange. RMCE-ASAP combines inverted heterologous recombinase target sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and the power of mouse genetics. These general principles should make RMCE-ASAP applicable to any locus.

The evolution of the amplified adenylate deaminase 2 domains in Chinese hamster cells suggests the sequential operation of different mechanisms of DNA amplification.

Fluorescent in situ hybridization was used to localize the adenylate deaminase 2 (AMPD2) genes and flanking sequences on the chromosomes of the Chinese hamster line GMA32 and to study the distribution of additional copies of these genetic sequences in amplified mutants selected at several early stages of the amplification process. The synteny of AMPD2 genes and MDR1 genes, located on chromosomes 1, was demonstrated; in GMA32 the existence of a rearrangement positioning the two AMPD2 genes at different distances from the telomeres was disclosed. Using this structural marker, we showed that the amplified copies distribute along only one of the chromosomes 1. Their organization in different cells of clonal mutant populations at a very early stage of amplification was extremely heterogeneous; classes of organization could be recognized however. Their quantitative distribution at this stage and in cells which went through 10 more division cycles suggests an evolution pathway common to the mutant clones under study: as a rule, tandems of few units of identical and very large size (47 Mb) appear to be the first detected product of amplification; this organization is progressively overtaken by structures with more units of reduced and irregular size, while, in a growing number of cells, clusters of much shorter units can be observed. The nature of segregative amplification mechanisms operating in these processes and the possible involvement of replicative ones are discussed.

Replication Initiation In Mammalian Cells: Changing Preferences

No abstract yet available.