[Lucy's fatal childbirth? About materno-foetal mortality in the ancient times]. / L'accouchement tragique de Lucy ? À propos de la mortalité maternofœtale de la préhistoire à l'époque médiévale.

OBJECTIVES: Skeletal remains of pregnant woman whit fetus still in the pelvic region are scarce in the archaeological record. We aimed to review the different cases of maternal and fetal death in the ancient times. METHODS: A review of literature using Medline database and Google about mortality during pregnancy in Prehistory, Antiquity and middle age. The following key words were used: ancient times; paleopathology; immature fetus; medieval; pregnancy; mummies; Antiquity; maternal mortality. RESULTS: Thirty articles were found and we added one personal unpublished case. There were 64 female skeletons with mainly infectious abnormalities (10 dental abscesses and 2 pneumoniae) followed by traumatic lesions (2 frontal fractures and 1 femur luxation). There were 48 fetal remains and 3 twins. We noted 8 obstructed labors (3 breech presentations, 4 transverse lies and one possible shoulder dystocia). CONCLUSIONS: The fact that there were only few cases of maternal deaths with fetal remains raises the questions of the cause of death and the relationship between death and obstetric disorders. Beside the underestimation of these archaecological cases, the reasons of both fetal and maternal death must be looking for among several diseases or anomalies of both or of one of them, related with poor environmental conditions (such as malnutrition and high morbidity from infections) and lack of care the mother and fetus need.

[Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire]. / Vers une maîtrise industrielle du clonage des lymphocytes B humains pour la fabrication des anticorps monoclonaux issus du répertoire humain.

Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

Signalling profile and antitumour activity of the novel Hsp90 inhibitor NVP-AUY922 in multiple myeloma.

We as well as others have recently shown that Hsp90 is overexpressed in multiple myeloma (MM) and critically contributes to tumour cell survival. Pharmacologic blockade of Hsp90 has consistently been found to induce MM cell death. However, most data have been obtained with MM cell lines whereas knowledge about the molecular effects of pharmacologic Hsp90 blockade in primary tumour cells is limited. Furthermore, these investigations have so far focused on geldanamycin derivatives. We analysed the biochemical effects of a novel diarylisoxazole-based Hsp90 inhibitor (NVP-AUY922) on signalling pathways and cell death in a large set of primary MM tumour samples and in MM cell lines. Treated cells displayed the molecular signature and pharmacodynamic properties for abrogation of Hsp90 function, such as downregulation of multiple survival pathways and strong upregulation of Hsp70. NVP-AUY922 treatment efficiently induced MM cell apoptosis and revealed both sensitive and resistant subgroups. Sensitivity was not correlated with TP53 mutation or Hsp70 induction levels and stromal cells from the bone marrow microenvironment were unable to abrogate NVP-AUY922-induced apoptosis of MM cells. Thus, NVP-AUY922 may be a promising drug for treatment of MM and clinical studies are warranted.

Coupling of the antennapedia third helix to a potent antagonist of the p53/hdm2 protein-protein interaction.

The use of cell-membrane translocating sequences for intracellular delivery of peptides can be a powerful approach to validate drug discovery targets in cellular settings. To accomplish this, a protocol has been implemented to couple the antennapedia third helix (residues 43-58) to a potent antagonist of the p53/hdm2 protein-protein interaction without affecting its in vitro inhibitory activity.

The role of tetramerization in p53 function.

The tumour suppressor gene p53 is extensively studied for its importance in cancer. In its active conformation, p53 is tetrameric and one domain - the tetramerization domain - permits the oligomerization of this protein. Until recently, little attention was given to this domain because, in contrast to the DNA-binding domain, it is not often mutated in cancer. However, various experimental studies have shown evidence that the tetramerization domain is essential for DNA binding, protein-protein interactions, post-translational modifications, and p53 degradation. Moreover, single mutations in the tetramerization domain can inactivate the wild-type protein in a manner similar to that seen with mutations in the DNA-binding domain. Interestingly, the phenotype of several tetramerization domain mutants differs from that observed with DNA-binding domain mutants. In this review, current knowledge about the importance of the tetramerization domain to the function of p53 will be summarized.

Targeting p53 in cancer.

The loss of p53 function is a very important event in cancer. The absence of a functional p53 in tumours favours cancer development, and it has been suggested that it also influences the efficacy of both chemotherapy and radiotherapy. The p53 pathway is therefore the subject of intense research, both in academia and in industry, to identify new compounds targeting tumours with an altered p53. Different strategies are currently under investigation. They can be divided into two groups: biological and chemical strategies. The biological strategies utilise the immune system to eliminate the tumours overexpressing p53 mutants. Alternatively they use vectors delivering a functional p53 gene in tumours or engineered viruses, which selectively target p53-defective cells. The chemical strategies are based on the design of compounds that stabilise or activate the p53 mutant proteins, inhibit the interaction between p53 and hdm2, or act on the p53-binding chaperone proteins. In this review, the current progress in these different approaches will be analysed.

p53 transdominance but no gain of function in mouse brain tumor model.

Although p53 mutations in tumors typically result in loss of transactivation of p53 target genes some mutants display gain-of-function activity. The latter has important implications for the design of rational cancer therapy. We previously described a germ-line p53 mutation (deletion of codon 236, Y236delta) associated with a familial brain tumor syndrome. To determine whether this tissue-specific tumor predisposition reflects a gain-of-function activity of Y236delta or an effect of genetic background we have developed a mouse brain tumor model. Primary neuroectodermal cells deficient for p53 (+/- or -/-) and transduced with Y236delta using a retroviral vector were transplanted into the brain of adult wild-type mice. This neurografting paradigm circumvents the problem of early lethal tumors at extracerebral sites associated with germ-line p53 deficiency. Brain tumors arising in this mouse model were highly invasive, reflecting an important feature of the human disease. Tumors arose from p53+/- cells only when transduced with Y236delta. In keeping with in vitro data showing that Y236delta has dominant-negative activity, these tumors retained the endogenous wild-type p53 allele but accumulated high levels of Y236delta. However, the presence of Y236delta in transplanted p53-/- cells had no effect on the tumor frequency, 15% versus 27% without the mutant. In conclusion, Y236delta is transdominant but exerts no gain-of-function activity mediating a more penetrant tumor phenotype.

A small synthetic peptide, which inhibits the p53-hdm2 interaction, stimulates the p53 pathway in tumour cell lines.

The hdm2 protein negatively regulates p53 tumour suppressor activity. Upon binding to p53, hdm2 stimulates p53 degradation and inhibits its transcriptional activity. Moreover, the hdm2 protein is overexpressed in various tumours inactivating p53. We report here that an octamer synthetic peptide derived from p53 inhibits the p53-hdm2 interaction in vitro. In cellular assays, this untagged peptide penetrates tumour cells and induces the accumulation of p53. The accumulation of p53 leads to its activation. Two gene products transcriptionally regulated by p53, p21Waf1/Cip1 and hdm2, are induced in the presence of the peptide. When used with tumour cells that overexpress hdm2, the peptide induces the death of these tumour cells by apoptosis. The mode of action of this peptide differs from that of DNA-damaging agents (e.g. cisplatin) in that it does not induce p53 phosphorylation on serine 15. This work validates with a low molecular mass molecule our current knowledge on the regulation of the p53 pathway by the hdm2 protein. It also shows that inhibitors of the p53-hdm2 interaction are very attractive candidates for the activation of the p53 pathway in tumours expressing wild-type p53.

Mutations at position 277 modify the DNA-binding specificity of human p53 in vitro.

p53 regulates the expression of different genes that contain in their promoter a DNA sequence with two copies of the 10-base motif Pu(1)Pu(2)Pu(3)C(4)(A/T)(5)(T/A)(6)G(7)Py(8)Py(9)Py(10). This sequence is degenerated, and thymine or cytidine is found equally at position 3 or 8. These two bases make contact with cysteine-277 of the human p53. An in vitro study was carried out to determine whether p53 could be mutated at position 277 so that it binds preferentially to a sequence containing thymine or cytidine. Various mutant proteins were created and their DNA-binding specificity was determined by gel shift assay. Two of them show an altered specificity. The Cys277Ser protein binds preferentially to cytidine-containing sequences while the Cys277Ala mutant has a preference for thymine-containing sequences. This specificity is presumably achieved because an alanine residue at position 277 interacts with the thymine via hydrophobic interactions and a serine makes a hydrogen bond with the cytidine but not with the thymine.

Functional analyses of a unique p53 germline mutant (Y236delta) associated with a familial brain tumor syndrome.

We have evaluated the functional properties of the unique p53 mutant Y236delta (deletion of codon 236) that gave rise to apparent cell-type specific tumor development. Four family members carrying this mutation in the germline developed early onset brain tumors, as previously reported. Deletion of residue Y236, which is tightly packed in an evolutionary conserved hydrophobic pocket, results in a protein with a mutant conformation according to immunoprecipitation with the conformation-sensitive antibodies PAb240 and PAb1620. The Y236delta mutant lacks specific DNA binding to the p53-responsive element in the WAF1-promoter, and functional analysis in Saos-2 cells revealed inability to transactivate the p53-responsive elements in the WAF1-promoter and the RGC sequence. The mutant has retained a functional oligomerization domain, a key element mediating the dominant negative effect, and inhibits DNA binding of wild-type p53. In addition, transactivation of endogenous wild-type p53 in LoVo cells was inhibited upon transfection of the mutant in a dose-dependent manner. Thus, in vitro and in vivo data suggest the loss of important tumor-suppressing functions and demonstrate a dominant negative effect of this unique p53 mutant that is associated with an unusual clustering of familial brain tumors.

Influence of the N-terminal region on the oligomerisation between human and Xenopus laevis p53.

The p53 proteins from human, murine and Xenopus laevis are tetrameric. Previous work has shown that both human and murine p53 can form heterotetramers. However, despite a very high level of sequence homology at their tetramerisation domain, the human and the X. laevis p53 do not form heterooligomers. It is shown here that when inserted in the human p53 protein, the X. laevis tetramerisation domain is able to oligomerise with the human one. This indicates that the inability of X. laevis p53 to heterooligomerise with human p53 is due to another structural difference. The use of N and C-truncated X. laevis p53 proteins reveals that the deletion of the N terminus favours from the heterooligomerisation between the human and the X. laevis p53. The oligomerisation of the X. laevis p53 with the human protein is also enhanced when the N terminus of the X. laevis p53 is replaced by the human one. Altogether these data suggest that the N terminus of p53 influences the oligomerisation.

Cellular characterisation of p53 mutants with a single missense mutation in the beta-strand 326-333 and correlation of their cellular activities with in vitro properties.

Recent evidence has shown that missense mutations in the p53 tetramerisation domain can inactivate the protein. However, most of these studies have been done only in vitro or concern proteins whose physico-chemical properties have not been fully investigated. Alanine mutants of the beta-strand 326-333 from the tetramerisation domain have been characterized in vitro and studied for their thermodynamic stability. They therefore offer a unique opportunity to establish a correlation between in vitro and cellular activities of proteins with a mutated tetramerisation domain. The eight mutant proteins resulting from the mutation of the eight residues of the beta-strand 326-333 to alanine were analysed for their ability to stimulate transcription, to inhibit the growth of Saos-2 cells and to repress the promoter of the multidrug resistance gene 1. The experimental results show a perfect correlation between in vitro and cellular data. The Leu330Ala and Ile332Ala proteins are inactive, the Phe328Ala protein has a moderate activity. The Glu326Ala, Tyr327Ala, Thr329Ala, Gln331Ala and Arg333Ala proteins show activity similar to that of wild-type protein. This work is based on an exhaustive analysis of p53 mutants both in vitro and in cells and shows that mutations in the tetramerisation domain might be of importance in cancer development since they inactivate the p53 protein.

p53 mutants without a functional tetramerisation domain are not oncogenic.

p53 is altered in about 50 % of cancers. Most of the p53 mutants have lost the wild-type tumour suppressor activity but show oncogenic properties. The majority of the p53 alterations are missense mutations of residues located in its DNA binding domain (DBD). Only a few mutations concern residues in its tetramerisation domain (TD). However, the study of mutant proteins identified in tumors that do not form tetramers has shown that they have lost the wild-type activity like most of the p53 DBD mutants. Here, we show that two of such mutant proteins, Arg342Pro and Leu344Pro are not dominant negative and do not stimulate the expression of a reporter gene under the control of the multi-drug resistance gene-1 (MDR-1). This suggests that to be oncogenic, p53 mutants need to form tetramers. Accordingly, the dominant negative effect and the ability of a tetrameric mutant protein, Asp281Gly, to stimulate the MDR-1 promoter are abolished when its TD is rendered non-functional by the mutation of leucine 344 to a proline residue. These results suggest that mutations in the TD, are less selected in tumors than mutations in the DBD because they do not lead to oncogenic proteins.

Characterization of p53 mutants identified in human tumors with a missense mutation in the tetramerization domain.

p53 is very often mutated in human cancers. The majority of alterations are missense mutations located within the DNA-binding domain of the protein. Many reports have characterized such mutant proteins. Little is known, however, about the properties of proteins that have a missense mutation outside this domain. We investigated here the properties of 8 mutant proteins identified in human tumors as having a missense mutation in the tetramerization domain. The Arg342Gln, Glu349Asp and Gln354Arg proteins behaved like wild-type both in vitro and in cells. Two mutants, Arg342Pro and Leu344Pro, were inactive in all assays. Finally, the 3 mutant proteins Leu330His, Arg337Cys and Arg337Leu, which are inactive in vitro, showed no activity at low expression levels in cells but became active at higher expression levels. Our results reveal new phenotypes for p53 mutants and suggest that sequencing of the p53 gene from patients with tumors should be extended to exons 9 and 10 in clinical investigations.

Crystallization and structure solution of p53 (residues 326-356) by molecular replacement using an NMR model as template.

The molecular replacement method is a powerful technique for crystal structure solution but the use of NMR structures as templates often causes problems. In this work the NMR structure of the p53 tetramerization domain has been used to solve the crystal structure by molecular replacement. Since the rotation- and translation-functions were not sufficiently clear, additional information about the symmetry of the crystal and the protein complex was used to identify correct solutions. The three-dimensional structure of residues 326-356 was subsequently refined to a final R factor of 19.1% at 1.5 A resolution.

In vitro analysis of the dominant negative effect of p53 mutants.

Missense mutations of the p53 tumour suppressor gene induce the formation of proteins with an altered affinity for DNA. These mutant proteins have either a wild-type or a mutant conformation. It has been established that, on association with wild-type protein, molecules with mutant conformation can drive the wild-type p53 into a mutant conformation. It is shown here that mutant proteins with a wild-type conformation can also inactivate wild-type p53 upon oligomerisation. The dominant negative activity of these mutants depends on their ability to bind to DNA. The less a mutant protein binds to DNA, the more it is dominant negative. Their dominant negative activity is also dependent on the DNA-binding site. The binding of wild-type to a low-affinity DNA element is more easily inactivated by a dominant negative mutant than its binding to a high-affinity DNA-binding site.

Molecular characterization of the hdm2-p53 interaction.

A number of viral oncogenes target the tumour suppressor protein p53 and inactivate its function. This is an important step in tumourogenesis. The cellular oncogene hdm2 acts through a similar mechanism. It binds the N terminus of p53, thereby interfering with the ability of p53 transcriptionally to activate genes responsible for growth arrest or apoptosis after genotoxic insults. The disruption of the interaction of the two proteins therefore comprises a promising therapeutic target for treatment of the subset of human cancers in which this pathway is active. In this paper we attempt to characterize the p53-hdm2 interaction biochemically. We analyse the potential of a series of peptide inhibitors, derived from previously described mdm2 binding peptide display phage, to disrupt this interaction in ELISA assays. We conclude that F19, W23 and L26 of p53 are critical contact points for p53 binding to hdm2. Furthermore, we show the potential of the monoclonal antibody 3G5 to interfere with binding of p53 to hdm2 in ELISA assays. Consequently, we define the binding site of 3G5 on hdm2 using overlapping peptides derived from the N terminus of hdm2 and phage display libraries. The result indicates L66, Y67 and E69 on hdm2 as critical binding points for 3G5. In electrophoretic mobility shift assay we demonstrate the formation of hdm2-p53 complexes that can be disrupted in the presence of 3G5 or inhibitory peptides. Finally, we describe the effects of NEM and DTT on the interaction between the two molecules in ELISA assays. All our results are discussed in the light of the recently published crystal structure of the mdm2-p53 complex. A striking correspondence between our findings and the crystal structure is revealed.