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Senescence was originally described from the observation of the limited ability of normal cells to grow in culture, and may be generated by telomere erosion, accumulation of DNA damages, oxidative stress and modulation of oncogenes or tumor suppressor genes. Senescence corresponds to a cellular response aiming to control tumor progression by limiting cell proliferation and thus constitutes an anticancer barrier. Senescence is observed in pre-malignant tumor stages and disappears from malignant tumors. Agents used in standard chemotherapy also have the potential to induce senescence, which may partly explain their therapeutic activities. It is possible to restore senescence in tumors using targeted therapies that triggers telomere dysfunction or reactivates suppressor genes functions, which are essential for the onset of senescence.

Transcription factors of the signal transducer and activator of transcription (STAT) family are required for cellular responses to multiple signalling molecules. After ligand binding-induced activation of cognate receptors, STAT proteins are phosphorylated, hetero- or homodimerize, and enter the nucleus. STAT dimers bind to specific DNA elements and alter the transcriptional activity of the signal-responsive genes. We report the cloning and developmental pattern of expression of XSTAT5, a Xenopus laevis member of the STAT family, closely related to the mammalian STAT5A and STAT5B. XSTAT5 is expressed maternally and zygotically. With the onset of neurulation, XSTAT5 RNA are clearly localized in the anterior neural plate and subsequently in the neural structures of the developing eye, the pineal gland and the cement gland anlage. At late tailbud stages, a faint expression is detected in a ventral location that might correspond to the ventral blood islands.

Genes encoding fibroblast growth factors (FGFs) are expressed in early Xenopus neurulae in the prospective midbrain-hindbrain boundary (MHB) region of the neural plate. These expression domains overlap those of XWnt-1 and XEn-2, raising the question of the role of FGF signalling in the regulation of these genes, and more generally about the function of FGF during Xenopus midbrain development. We report that explants from the prospective MHB grafted into the anterior neural plate in midneurula stage embryos induce XWnt-1 expression and, at a lower frequency, XEn-2 expression in the vicinity of the graft. Such a process is likely to involve FGF signalling. Implantation of FGF4- or FGF8-soaked beads in the prospective forebrain at neurula and tailbud stages causes the up-regulation of XWnt-1 and XEn-2 in the dorsal and lateral region of the anterior midbrain. This effect is not relayed by endogenous FGF genes since exogenous FGFs inhibit the expression of endogenous XFGF3 or XFGF8. However, consequences of grafting MHB or implanting FGF4 or FGF8 beads on tadpole brain development are different. MHB grafts induce ectopic mesencephalic structures, strongly suggesting that a region homologous to the isthmic organizer of amniotes is specified as early as the midneurula stage. In contrast, exogenous FGFs do not cause the formation of ectopic mesencephalic structures but an overgrowth of mesencephalon and diencephalon. We propose that FGF signals from the prospective MHB play a crucial role in the spatial regulation of XWnt-1 and XEn-2 expression in the posterior midbrain, but that the full organizing activity of the MHB involves other factors in combination with FGF., (Copyright 1998 Elsevier Science Ireland Ltd. All Rights Reserved)

The mechanisms involved in the maintenance or loss of the asymmetric distribution of phospholipids in the cell plasma membrane remain mysterious. In the yeast Saccharomyces cerevisiae, the transmembrane migration of certain phospholipids is controlled by transcription regulators of various ATP-binding cassette (ABC) transporters. The P-glycoprotein membrane transporters encoded by the multidrug resistance (MDR) genes, members of the ABC protein family, act as lipid translocases in mammalian cells. We report here the lack of expression of MDR genes in lymphoblasts derived from the B cells of a patient with an inherited Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications. From microsatellite analysis of 7q21.1 and functional assessment, the most likely explanation accounting for Scott phenotype is a mutation in an unlinked gene coding for a regulatory protein necessary for the expression of MDR genes. Because phosphatidylserine externalization is also one of the hallmarks of cells undergoing apoptosis, these observations are suggestive of a relationship between basic processes such as multidrug transport, apoptosis and procoagulant phospholipid exposure.

A cDNA encoding a novel Xenopus fibroblast growth factor receptor, XFGFR4B, has been cloned. XFGFR4B mRNA is detected throughout embryogenesis. However, from the late gastrula stage on, XFGFR4B transcripts are expressed in two defined areas of the anterior neural plate. Interestingly, these two regions are fated to become parts of the retina, midbrain, hindbrain, and otic vesicle, all of which continue to express XFGFR4B mRNA in tailbud stage embryos and early tadpoles. Expression of XFGFR4B mRNA can be maintained at neurula stage in isolated blastula ectoderm in response to mesodermal induction by activin or neural induction by noggin, suggesting that XFGFR4B expression might be regulated by early cell interactions. Distribution of XFGFR4B mRNA suggests that XFGFR4B might serve an important function during patterning of neuroepithelium.

There are at least two well-characterized mechanism of resistance to Topo I and II inhibitors: modifications of intracellular accumulation and reduced formation of cleavable complexes. Limited drug accumulation is usually due to P-glycoproteinMDR or to Multidrug Resistance associated Protein (MRP). Reduction of Topo I (or II) cleavable complexes not related to drug transport can either be due to decreased enzyme levels or enzyme mutations. For Topo II inhibitors, differential expression of the Topo II isoforms alpha and beta and changes in Topo II phosphorylation may also contribute to resistance. For dual Topo I and II inhibitors, resistance mechanisms are more complex to analyze but may also involve dual Topo I and II alterations. Finally, in a given cell line, several mechanisms are commonly associated in pleiotropic resistance to Topo inhibitors.

Taxotere (RP 56976, NSC 628503), an analog of taxol, is an inhibitor of depolymerisation of microtubules and is currently in Phase I clinical trials. Comparisons of the cytotoxicities of Taxotere and taxol have been studied on several murine (P388, SVras) and human cell lines (Calc18, HCT116, T24, N417, KB). Taxotere was found more potent than taxol (1.3-12 fold), a result which could be explained by its higher affinity than taxol for microtubules. In agreement with its postulated mechanism of action, Taxotere is more cytotoxic on proliferating than on non proliferating N417 cells and does not inhibit cellular DNA, RNA and protein synthesis. Taxotere gives partial cross resistance on P-glycoprotein resistant P388/DOX cell line, in contrast to taxol which gives a complete cross resistance. On the other hand, no cross resistances were observed on Calc18/AM and P388/CPT5 cell lines, bearing modified activities of topoisomerase II and topoisomerase I, respectively. These results underline the higher cytotoxic activity of Taxotere compared to taxol, and the lack of cross resistance of that class of agent with the topoisomerase I and II-related multidrug resistance phenotypes.

We have used amphibian gastrulation as a model system to study the action of the extracellular matrix (ECM) glycoprotein tenascin on mesodermal cell migration. Tenascin function was assayed in vitro during spreading of isolated cells from the dorsal marginal zone (DMZ) and during cell migration from DMZ explants. Plastic coated with bovine fibronectin or gastrula ECM was used as a substratum. In both cases, tenascin added to the medium inhibited spreading and migration of mesodermal cells. In addition, a substratum coated with a mixture of fibronectin and tenascin was found to prevent mesodermal cell migration. Tenascin was also microinjected into the blastocoel cavity of living embryos at the late blastula stage. This led to a complete arrest of gastrulation in more than 80% of the cases. Scanning electron microscopy of fractures from arrested gastrulae showed that mesodermal cell migration was blocked. Similar injection experiments carried out at the middle gastrula stage demonstrated that tenascin is able to inhibit cell migration after cells have already contacted the ECM. Mesodermal cell migration in the presence of tenascin could be restored in vitro and in vivo by the monoclonal antibody mAb Tn68 which is known to mask a cell binding site of the molecule. Finally, tenascin microinjected into the blastocoel of blastula or gastrula stage embryos bound within 15 min to the ECM fibrils at all the stages studied. Our results show that exogenous tenascin can be incorporated into embryonic ECM and interferes in vivo with the interactions of cells with a fibronectin-rich matrix.

Several antitumor drugs including DNA intercalative and non intercalative agents induce in vitro and in vivo double-stranded DNA breaks by stabilization of a topoisomerase II-DNA complex. In order to locate cleavage sites in an actively transcribed oncogene, N417 cells, originating from a human small cell lung carcinoma and containing 45-50 copies of c-myc oncogene, were treated with mAMSA, 9 hydroxyellipticine and VM 26. The presence of DNA lesions in c-myc was investigated by Southern blot hybridization with a human c-myc probe. In addition to normal bands, DNA patterns of drug treated-cells revealed the presence of new bands most likely corresponding to topoisomerase II-mediated cleavage as these bands were not found in untreated control DNA and in DNA treated with oAMSA, a biologically inactive stereoisomer of mAMSA. Major cleavage sites induced by drugs in the N417 cell c-myc locus were located in the 5' end of the c-myc exon 1 closely to some DNAse I hypersensitive sites which are assumed to reflect an activity of the gene. Therefore our data suggest that TopoII-mediated drug activity correlates with gene activity.

[125I] Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, [125I] iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.

Most experimental data clearly suggest that antitumor agents including DNA intercalative molecules (acridine derivatives, ellipticine and derivatives), or non intercalative ones (epipodophyllotoxines), exert their cytotoxic activity by stabilizing DNA-Topoisomerase II complexes. This phenomenon can be revealed by the presence of DNA breaks upon protein denaturing treatment. In this work, BD-40, an ellipticine analog has been shown to interact in vivo with Topoisomerase II. Moreover, cleavage sites generated by the drug treatment in human proto-oncogene c-myc appear to be mostly localized in the 5' end of the gene locus, which contains regulatory elements. Some of these sites are in a striking correspondence with DNAse I hypersensitive sites, which reportedly reflect the state of activity of genes.