Identification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN.

The tumour suppressor gene PTEN (also called MMAC1 or TEP1) is somatically mutated in a variety of cancer types [1] [2] [3] [4]. In addition, germline mutation of PTEN is responsible for two dominantly inherited, related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome [4]. PTEN encodes a dual-specificity phosphatase that inhibits cell spreading and migration partly by inhibiting integrin-mediated signalling [5] [6] [7]. Furthermore, PTEN regulates the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring [8]. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase [9]. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signalling cascade.

Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, ß and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates.

The specific activation of gadd45 following UVB radiation requires the POU family gene product N-oct3 in human melanoma cells.

Here we report the specific regulation of gadd45 expression in human melanoma cell lines following UVB radiation. This solar wavelength is likely to be involved in melanoma aetiology. We have previously shown that gadd45 expression is strongly enhanced in a p53-independent manner following UVB irradiation, unlike the other p53 target genes studied. Furthermore, gadd45 is specifically activated in melanocytes since its induction in response to UVB, is not observed in other skin cells such as keratinocytes or fibroblasts. To investigate this particular regulation of gadd45, we analysed the UVB-induced response of different gadd45 promoter regions. Thus, a minimal promoter region of 50 bp length, responsible for gadd45 activation in melanoma cell lines following UVB irradiation, was determined. In electrophoretic mobility shift assays (EMSAs), we showed that this region (-106/-56) of the gadd45 promoter which contains two identical octamers, binds the POU family gene products oct-1 and N-oct3. Given the specific expression pattern of N-oct3 in melanocyte, we invalidated the expression of this transcription factor in melanoma cells: such an abrogation of N-oct3 protein expression in melanoma cells impeded gadd45 UVB-response. Thus the response of melanocyte to UVB may use an original and previously undescribed pathway.

Identification of functional domains involved in BTG1 cell localization.

We have previously shown that BTG1 stimulates myoblast differentiation. In addition, this protein displays a major nuclear localization in confluent myoblasts, decreasing during the early steps of differentiation, and is essentially detected in the cytoplasm of mature myotubes. To identify the domains involved in the cellular trafficking of BTG1, we observed the localization of several BTG1 sequences fused to betaGalactosidase. The highly conserved B box among all members of the BTG family induces a significant nuclear localization of the betaGal moiety, enhanced by presence of the BTG1 carboxy-terminal sequence. In addition, a functional Nuclear Export Signal (NES) overlaps the B box. Moreover, presence of the first 43 NH(2)-terminal amino acids reduced the nuclear localization of each chimeric protein tested. Last, the BTG1 amino-terminal domain bears an LxxLL motif favouring nuclear accumulation, and another region encompassing the A box inhibiting nuclear localization. In contrast to a BTG1 mutant exclusively localized in the cytoplasm, transient expression of a mutant displaying a nuclear localization enhanced myoblasts withdrawal from the cell cycle and terminal differentiation, thus mimicking the myogenic influence of BTG1. In conclusion, several regions of BTG1 are implicated in its cellular localization, and BTG1 myogenic activity is induced at the nuclear level.

Estimation of the allele number in a natural population by the method of isofemale lines.

The number of alleles present in a natural population of unknown structure is estimated using a sequential sampling procedure applied to isofemale lines. Two questions are raised: how many individuals per isofemale line must be assayed and how many isofemale lines must be sampled to get an adequate sample to estimate the number of alleles, at a given risk, of the natural population? On the one hand, we show that when wild females are inseminated once, only two individuals per line are required. On the other hand, the number of isofemale lines that must be sampled depends on the risk chosen of losing an allele, on the number of alleles present in the population and on their drawing probabilities. When the population structure is known, an accurate answer can be provided. For an unknown population structure, one general sequential sampling previously described by J. Rouault and P. Capy is proposed to estimate the number of alleles in the population from data on isofemale lines.

Cloning of the mouse BTG3 gene and definition of a new gene family (the BTG family) involved in the negative control of the cell cycle.

It is well known that loss of tumor suppressor genes and more generally of antiproliferative genes plays a key role in the development of most tumors. We report here the cloning of the mouse BTG3 gene and show that its human counterpart maps on chromosome 21. This evolutionarily conserved gene codes for a 30 kDa protein and is expressed in most adult murine and human tissues analyzed. However, we demonstrate that its expression is cell cycle dependent and peaks at the end of the G1 phase. This gene is homologous to the human BTG1, BTG2 and TOB genes which were demonstrated to act as inhibitors of cell proliferation. Its description allowed us to define better this seven gene family (the BTG gene family) at the structural level and to speculate about its physiological role in normal and tumoral cells. This family is mainly characterized by the presence of two conserved domains (BTG boxes A and B) of as yet undetermined function which are separated by a non-conserved 20-25 amino acid sequence.

Identification and molecular analysis of BANP.

BTG3 belongs to a family of structurally related genes whose biochemical functions remain elusive. In order to investigate the mechanism underlying BTG3-mediated functions, we tried to identify BTG3 potential partners. The use of the yeast 'two-hybrid system', with BTG3 as bait, enabled us to isolate BANP (BTG3 Associated Nuclear Protein). Other commonly used protein-binding assays did not confirm this yeast interaction. However, BANP had never been described before, and this prompted us to further characterise this gene. In this paper, we present data on its molecular organization in mouse, then we speculate on the nature of this nuclear protein, and finally we localise BANP on the human chromosome 16q24 subregion; we discuss the fact that frequent loss of heterozygosity within this region has been observed in different tumours.

Sequence analysis reveals that the BTG1 anti-proliferative gene is conserved throughout evolution in its coding and 3' non-coding regions.

The human BTG1 gene (expressing an anti-proliferative function) is an evolutionarily conserved gene homologous to the murine PC3/TIS21 genes. Here, we report the cloning and sequencing of the murine BTG1 coding region and chicken BTG1 cDNA. The putative human and mouse BTG1 proteins are 100% identical; the chicken BTG1 cDNA contains an open reading frame of 170 amino acids with a 91% identity to its human and murine counterparts. The 3'-untranslated region of BTG1 is also highly conserved (82% homology between human and chicken), suggesting that it plays a key role in the regulation of BTG1 expression. These data confirm that BTG1 is phylogenetically highly conserved and that BTG1 and PC3/TIS21 may constitute the first members of a new family of functionally related genes.

In search of a function for the TIS21/PC3/BTG1/TOB family.

The Btg family of anti-proliferative gene products includes Pc 3/Tis 21/Btg 2, Btg 1, Tob, Tob2, Ana/Btg3, Pc3k and others. These proteins are characterized by similarities in their amino-terminal region: the Btg1 homology domain. However, the pleiotropic nature of these family proteins has been observed and no common physiological function among family members was suggested from the history of their identification. Recent progress in the search for Btg family functions has come from the analysis of cell regulation and of cell differentiation. It is now emerging that every member of this family has a potential to regulate cell growth. We would like to propose here to use a nomenclature APRO as a new term for the family.

Apoptosis without decrease of cell DNA content.

Apoptosis of human B cells and murine T and B cells was analyzed by DNA agarose gel electrophoresis, clamped homogeneous electric field, measurement of cell DNA content by flow cytometry, transmission electron microscopy and by UV microscopy. Apoptosis was induced by etoposide (an inhibitor of topoisomerase II), by the calcium ionophore ionomycin or by cross-linking of membrane immunoglobulins (Ig) with anti-Ig-antibodies. Two types of apoptosis could be defined. Apoptosis resulting in small DNA fragments (180-200 base pairs and multiples thereof) was associated with a typical 'ladder' in agarose gel electrophoresis and a decrease in cell DNA content assessed by flow cytometry. Conversely apoptosis with large DNA fragments (100-150 kilobase pairs) was only demonstrated by clamped homogeneous electric field but was not associated with decreased cell DNA content or the observation of DNA ladders. Nuclear condensation without fragmentation was more frequent when apoptosis generated large DNA fragments. The type of apoptosis appears to be an intrinsic property of each cell type.

High gastrin releasing peptide receptor mRNA level is related to tumour dedifferentiation and lymphatic vessel invasion in human colon cancer.

The neuropeptide bombesin stimulates tumour cell proliferation in vitro. Through pharmacological testing, 20-40% of human colorectal tumours have been shown to be equipped with bombesin/gastrin releasing peptide receptor (GRP-R). The aim of the present study was to test whether GRP-R expression is correlated with tumour characteristics and usual prognostic factors in colorectal adenocarcinomas. A sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) method was validated by studying GRP-R mRNA in separated layers of normal colonic wall, and GRP-R mRNA levels (in parallel with binding studies) in colon cancer cell lines LoVo and Caco-2. GRP-R mRNA levels were then determined in 29 surgical tumour specimens and the results compared with tumour histology and, using histochemistry, with the accumulation of p53 protein and a Ki-67 cell proliferation index. The mRNA was not detected in normal colonic epithelium, whereas a distinct signal was observed after amplification in 27/29 (93%) tumour specimens. Estimates of mRNA levels in the 27 positive tumours ranged from 52 to 8000 amol/0.25 microgram total RNA, and were significantly higher in poorly/moderately differentiated tumours (P < 0.05) and in tumours with lymphatic vessel invasion (P < 0.01). There was no relationship with p53 accumulation or to the proliferation index. Our results show that GRP-R mRNA can be detected in most colorectal tumour specimens, and suggest a link between high mRNA levels and both tumour dedifferentiation and lymph vessel invasion, but not proliferation.

Transformation by T-antigen and other oncogenes delays Hsp25 accumulation in heat shocked NIH 3T3 fibroblasts.

We have recently reported that transformation of murine NIH 3T3 cells by v-fos oncogene interfered with Hsp70 and Hsp25 accumulation after heat shock. Here, we have investigated the effect mediated by other oncogenes on the accumulation of these stress proteins. We report that T-antigen transformation of NIH 3T3 cells delayed and reduced the accumulation of Hsp25 after heat shock and decreased the heat-mediated phosphorylation of this protein. This decreased level of Hsp25 correlated with a reduced accumulation of the corresponding mRNA and was related to T-antigen level. In contrast, T-antigen had no effect on the expression of the major stress protein Hsp70 nor did it interfere with the level of Hsp90 or Hsp60. We report also that v-src or Ha-ras oncogenes delayed Hsp25 accumulation after heat shock but that only v-src reduced the heat-induced phosphorylation of this protein. v-src, but not Ha-ras, interfered with Hsp70 expression and none of these oncogenes had an effect on Hsp60 or Hsp90 levels. Taken together, these observations suggest that an altered accumulation of Hsp25 after heat shock is a common characteristic of NIH 3T3 fibroblasts transformed by different oncogenes.

Involvement of the BCL2 Gene in 131 Cases of Non-Hodgkin's B Lymphomas: Analysis of Correlations with Immunological Findings and Cell Cycle.

The t(14;18) chromosomal translocation is widely recognized as a cytogenetic abnormality associated with follicular lymphomas, but estimates of its frequency in this type of lymphoma vary from less than 40% to almost 90% according to the geographic origin of the patients. Using two human genomic probes for major and minor breakpoint cluster regions mapping at chromosome 18q21, we have analysed 131 cases of B non-Hodgkin's lymphomas obtained from France, by the Southern blot technique. The genotypic study was complemented in most cases by immunophenotypic and cell kinetic analyses. The BCL2 gene located at 18q21 band was rearranged in 39 of 56 (70%) follicular lymphomas and in 9 of 74 (12%) diffuse lymphomas; probes for major and minor breakpoint regions detected two thirds and one third of the rearrangements respectively. Regarding the morphologic subtypes of follicular and diffuse lymphomas, no significant differences were observed irrespective of the probe used. Review of the literature showed that comparable results have been obtained previously using both cytogenetic and molecular approaches and our results support the view that the global incidence of the t(14;18)(q32;q21) translocation in follicular lymphomas is about 70% with wide geographic variations. The immunological study provides evidence for a significant correlation of BCL2 rearrangement with surface immunoglobulin gamma isotype expression and with the lack of reactivity of the malignant cells with an antibody against the CD5 cluster. In the cases where cell kinetics was analysed, we did not find any significant difference between the rate of proliferation and BCL2 rearrangement. These data should be compared with previously reported observations made in humans or in transgenic mice and enable us to propose a model accounting for the role of BCL2 in B cell tumorigenesis.

[Notes on the reptiles of Tunisia. V. Biometric characteristics of Mauremys caspica leprosa (Schweigger, 1812) (Reptilia: Emydidae)]. / Notes sur les reptiles de Tunisie. V. - Caractéristiques biométriques de Mauremys caspica leprosa (Schweigger, 1812) (Reptilia: Emydidae).

This morphological study is based on 45 specimens of Mauremys caspica leprosa (Reptilia: Emydidae) sampled in northern and central Tunisia. A first principal component analysis of 9 variables: 8 measurements of the shell and the weight in life, allow to define a factor "size"; a second analysis of these variables divided by the size make obvious a factor "shape". In the plane of these two factors, our specimens may be classified into 6 groups from the hatchling stage to the large adults; in addition, the factor shape express a dysharmony in the growth, beginning with the third group (weight: 300 to 500 g). Among the 8 parameters used, 4 are adequate to characterize the morphology of this turtle.

Direct cooperation between androgen receptor and E2F1 reveals a common regulation mechanism for androgen-responsive genes in prostate cells.

We have studied the regulation of ATAD2 gene expression by androgens in prostate cells. ATAD2 is a coactivator of the androgen receptor (AR) and the MYC protein. We showed that ATAD2 expression is directly regulated by AR via an AR binding sequence (ARBS) located in the distal enhancer of its regulatory region. The gene is also regulated by the E2F1 transcription factor. Using knockdown and chromatin immunoprecipitation technique approaches, we could demonstrate that AR and E2F1 functionally collaborate and physically interact between each other. From the analysis of chromatin conformation, we conclude that this cooperation results from a chromatin looping over the ATAD2 promoter region between the ARBS and E2F1 binding site in an androgen-dependent manner. Furthermore, we could show that several genes overexpressed in prostate cancer and potentially involved in several aspects of tumor development have an ARBS and an E2F1 binding site in their regulatory regions and exhibit the same mechanism of regulation by both transcription factors as ATAD2.

Prognostic value of miR-16 expression in childhood acute lymphoblastic leukemia relationships to normal and malignant lymphocyte proliferation.

miR-16, a miRNA involved in cell proliferation and apoptosis regulation, may interfere with either oncogenic or tumor-suppressor pathways and is implicated in leukemogenesis. We then explored its expression in 93 childhood acute lymphoblastic leukemia (ALL) cases. A high miR-16 expression was associated with hyperleukocytosis and poor cytogenetic groups. In the whole group and in B-cell ALLs, disease-free survival (DFS) was significantly shorter for miR-16 above quartile 75. In T-cell ALLs, for both DFS and overall survival, a significant trend was found with a survival shortening from the lowest to the highest miR-16 levels. miR-16 expression neither significantly correlated with normal and malignant lymphocyte proliferation nor varied according to lymphocyte differentiation. The prognostic value of miR-16 in childhood ALL highlighted the complexity of miR-16 functions.

Olfactory quality: from descriptor profiles to similarities.

The model described here, which links olfactory semantic profiles to canonical similarities, performs significantly better than the current state of the art, mainly due to the use of an optimization procedure. Application of Tversky's model in this study shows that in olfaction, estimation of similarities is based exclusively on the distinctive elements of the profiles, and does not take into account the common elements. Moreover, the optimum number of descriptors to reckon with seems to lie between 25 and 30.

Variations of male cuticular hydrocarbons with geoclimatic variables: an adaptative mechanism in Drosophila melanogaster?

7-tricosene (7T) and 7-pentacosene (7P) are the major components of cuticular hydrocarbons in Drosophila simulans and D. melanogaster males. A chemical study of 16 isofemale lines of D. melanogaster sampled at the first and eighth generations in laboratory conditions showed the stability of chromatographical profiles. Then a large scale study of male 7T/7P polymorphism was performed with 85 populations of D. melanogaster and 29 of D. simulans collected all over the world. There were significant correlations of the values of the balanced ratio (7T- 7P)/(7T + 7P) with geo-climatic parameters, such as latitude, longitude, mean temperature, temperature range and vapour pressure. Parallel variations were also reported for the homologous linear alkanes (23 and 25 Carbon atoms) but not for the longer branched alkanes (27 and 29 Carbon atoms). No correlation was significant for the D. simulans populations studied. In this species a similar polymorphism of 7T/7P was found but restricted to a few populations from West Equatorial Africa.

Effects of BCL-2 antisense oligodeoxynucleotides on in vitro proliferation and survival of normal marrow progenitors and leukemic cells.

Previous studies have shown that the BCL-2 protooncogene encodes a mitochondrial protein that promotes cell survival by blocking programmed cell death. Bcl-2 protein has been detected in normal immature myeloid cells and in acute myeloid leukemia (AML) cells. To assess its functional role in normal and leukemic hematopoiesis, we performed serum-free cultures of CD34+ normal marrow cells, of bcl-2-positive myeloid lines, and of AML cells in the presence of bcl-2 sense, nonsense, and antisense phosphorothioate oligodeoxynucleotides. In all antisense-treated cultures, we observed (1) an inhibition of bcl-2 protein expression by day 4 to 6 of culture; (2) a decrease in cell survival duration; and (3) a decrease in the number of clonogenic cells present in the culture. Moreover, exposure to chemotherapeutic drugs resulted in more effective killing of AML cells in the presence of antisense oligomers. We conclude that bcl-2 protein is necessary for the survival of myeloid cells in culture, and that it may be implicated in the resistance of AML cells to chemotherapy.