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.

Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts.

The expression of the retinoblastoma susceptibility (RB-1) gene was investigated in highly proliferating mouse embryonic stem (ES) cells and in slowly proliferating mouse embryonic fibroblasts. The RB protein was expressed at the same level in these two cell types. Mainly hyperphosphorylated RB was detected in exponentially-growing ES cells. Embryonic fibroblasts and embryonic stem cells were synchronized by colcemid block followed by mitotic shake-off. In embryonic fibroblasts, DNA replication started 10-15 h after exit from mitosis and RB was transiently dephosphorylated during the G1 phase as previously described. In ES cells, DNA replication started 2 h after release from the colcemid block but virtually no hypophosphorylated RB was observed after the release. Instead, there was a dramatic decrease in the total RB protein level between exit from mitosis and entry into S phase. These observations were made by using two different monoclonal antibodies, both in immunoblotting and immunoprecipitation experiments. Absence of hypophosphorylated RB and cell cycle-dependent change in total RB protein level may be relevant to the high proliferation rate and to the tumorigenic nature of mouse embryonic stem cells.

Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells.

The expression of E and D-type cyclins, Cyclin-Dependent Kinase (CDK) 2 and 4, as well as CDK inhibitors p21Cip1 and p27Kip1 were examined during in vitro differentiation of mouse embryonic stem (ES) cells. ES cells cultured in presence of Differentiation Inhibitory Activity/Leukemia Inhibitory Factor (DIA/LIF) express very low levels of cyclin E/CDK2 complexes, p21Cip1 and p27Kip1 CDK inhibitors, while cyclin D/CDK4-associated kinase activity is undetectable. Withdrawal of DIA/LIF, which induces differentiation, results in the progressive up-regulation of all. Up-regulation of D cyclins occurs through an increase in the steady-state levels of mRNA, concomitantly with the activation of Brachyury and Goosecoid, two early markers of mesoderm differentiation. Similarly, cells from the epiblast of the early postimplantation mouse embryo do not express any cyclin D/CDK4 complexes. These are progressively upregulated at gastrulation and early organogenesis. DIA/LIF-stimulated ES cells are not growth-arrested by overexpression of p16Ink4a, a specific inhibitor of CDK4 and CDK6. We propose that the G1/S transition may be regulated by a minimal mechanism in mouse embryonic stem cells. Induction of differentiation triggers the establishment of a more sophisticated mechanism involving both cyclin D/CDK4- and CDK inhibitor-associated control of G1-phase progression.

Effect of nerve growth factor on the expression of cell cycle regulatory proteins in PC12 cells: dissection of the neurotrophic response from the anti-mitogenic response.

PC12 cells treated with nerve growth factor (NGF) undergo a G1 block and differentiate. Expression of selected cell cycle regulatory proteins was studied under culture conditions which permit observation of a differentiation response independently from a mitogenic or anti-mitogenic response. The expression of all cell cycle regulatory proteins studied is modulated by NGF addition to exponentially-growing cultures in the presence of serum. While levels of most of these proteins decrease, accumulation of cyclin D1 and the cyclin-dependent kinase inhibitor p21 Cip1/WAF1 is observed. Cyclin D1 associated kinase activity is inhibited, correlating with an increase in p21 protein. PC12 cells, synchronized by serum starvation, undergo morphological and functional differentiation in the presence of NGF. Neither cyclin D1 nor p21 are present in such cultures, nor is their expression upregulated by NGF, indicating that they are not required for this process. Removal of serum from differentiated PC12 cells results in loss of these proteins, but has no effect on differentiation or the nonproliferative state in presence of NGF. Together, the results indicate that cyclin D1 and p21 are not necessary for differentiation per se, nor are they required for maintenance of the differentiated state in the absence of serum.

Cell-cycle kinetics of neocortical precursors are influenced by embryonic thalamic axons.

Thalamic afferents are known to exert a control over the differentiation of cortical areas at late stages of development. Here, we show that thalamic afferents also influence early stages of corticogenesis at the level of the ventricular zone. Using an in vitro approach, we show that embryonic day 14 mouse thalamic axons release a diffusable factor that promotes the proliferation of cortical precursors over a restricted developmental window. The thalamic mitogenic effect on cortical precursors (1) shortens the total cell-cycle duration via a reduction of the G(1) phase; (2) facilitates the G(1)/S transition leading to an increase in proliferative divisions; (3) is significantly reduced by antibodies directed against bFGF; and (4) influences the proliferation of both glial and neuronal precursors and does not preclude the action of signals that induce differentiation in these two lineages. We have related these in vitro findings to the in vivo condition: the organotypic culture of cortical explants in which anatomical thalamocortical innervation is preserved shows significantly increased proliferation rates compared with cortical explants devoid of subcortical afferents. These results are in line with a number of studies at subcortical levels showing the control of neurogenesis via afferent fibers in both vertebrates and invertebrates. Specifically, they indicate the mechanisms whereby embryonic thalamic afferents contribute to the known early regionalization of the ventricular zone, which plays a major role in the specification of neocortical areas.

Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee.

A 5500 base-pair fragment including the beta-globin gene downstream from codon 122 and about 4000 base-pairs of its 5' flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5' extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.

Identification of transcripts initiated from an internal promoter in the c-erbA alpha locus that encode inhibitors of retinoic acid receptor-alpha and triiodothyronine receptor activities.

The thyroid hormone receptor-coding locus, c-erbA alpha, generates several mRNAs originating from a single primary transcript that undergoes alternative splicing. We have identified for the first time two new transcripts, called TRdelta alpha1 and TRdelta alpha2 [mRNA for isoform alpha1 and alpha2 of the T3 receptor (TR), respectively], whose transcription is initiated from an internal promoter located within intron 7 of the c-erbA alpha gene. These two new transcripts exhibit tissue-specific patterns of expression in the mouse. These two patterns are in sharp contrast with the expression patterns of the full-length transcripts generated from the c-erbA alpha locus. TR alpha1 and TRdelta alpha2 mRNAs encode N-terminally truncated isoforms of T3R alpha1 and T3R alpha2, respectively. The protein product of TRdelta alpha1 antagonizes the transcriptional activation elicited by T3 and retinoic acid. This protein inhibits the ligand-induced activating functions of T3R alpha1 and 9-cis-retinoic acid receptor-alpha but does not affect the retinoic acid-dependent activating function of retinoic acid receptor-alpha. We predict that these truncated proteins may work as down-regulators of transcriptional activity of nuclear hormone receptors in vivo.

[Multiple pregnancies. II. Epidemiology, clinical aspects]. / Les grossesses multifoetales. II. Epidémiologie, aspects cliniques.

It used to be rare for multiple pregnancies to occur but we have seen a spectacular rise in them in France between 1970 and 1986. Triplet deliveries increased threefold. The authors analyse a personal series of 23 pregnancies (19 triplets, 3 quadruplets and 1 quintuplet pregnancy). Sixteen of these 23 were medically induced. The main complications that have been observed were: threatened premature delivery in 86%, high blood pressure in 34.7%, anaemia in 50%, and urinary tract infections in 30.4%, 6.8% of the babies had congenital malformations. Reviewing the literature has made it possible to discern the epidemiological factors causing multi-fetal pregnancies: family history, high female fertility, maternal age, ethnic factors, hormonal contraception etc... At present it is medically assisted reproduction that is the big supplier of multi-fetal pregnancies in developed countries. We have reviews of several maternal as well as fetal complications: the ovarian hyperstimulation syndrome, extra-uterine pregnancy, hypertension, anaemia, spontaneous abortion, prematurity, intra-uterine growth retardation and malformations.

[Is it necessary to specify the HELLP syndrome?]. / Faut-il individualiser le HELLP Syndrome?

In 1982 Weinstein described the Hellp-Syndrome in pre-eclampsia. Two personal case histories have led the authors to discuss the need to specify this syndrome. Apart from the classical elements of pre-eclampsia, which are: arterial hypertension, proteinuria and oedema, there are bowel symptoms in 86% of cases and on the biological level there is thrombocytopenia and a rise in the transaminases as well as a haemolytic anaemia. Some cases of rupture of the liver have been described. Treatment is that of pre-eclampsia. This involves treatment with cortisone and sometimes with heparin. Delivery of the fetus is, however, the only really effective treatment. The characteristic three elements of the Hellp-Syndrome seem to be linked to disseminated intravascular coagulation and have long been considered very serious factors in pre-eclampsia. On the other hand it does seem to be useful for the clinician to know about the Hellp-syndrome in order to estimate the seriousness of a case where there are vasculo-renal elements in the syndrome and in order to avoid diagnosis mistakes in cases where the symptoms are often deceptive.

[Bourneville's tuberous sclerosis. Prenatal diagnosis of a case]. / La sclérose tubéreuse de Bourneville. Diagnositc prénatal à propos d'un cas.

The discovery of a tumour of the heart on fetal ultrasound is a rare event. 60% of heart tumours in the fetus are rhabdomyomas. 50% of rhabdomyomas are associated with Bourneville's tuberous sclerosis. The inherited element of the effect of the gene is variable but often severe (75% of patients die before the age of 25 and 60-80% are disabled). Tuberous sclerosis Bourneville is a multifocal pathological condition of which only the presence of rhabdomyomata in the heart make it possible to diagnose the condition antenatally. Monitoring the fetus consists of looking carefully for the complications that can occur and of planning the health care of the newborn including the need to consider surgical intervention at birth.

[Multiple pregnancies. III. Therapeutic, psychologic and social aspects]. / Les grossesses multifoetales. III. Aspects thérapeutiques, psychologiques et sociaux.

The authors analyse a series of 23 multiple pregnancies (19 triplet pregnancies, 3 quadruplets and 1 quintuplet). The first objective is to fight prematurity. Over and above all use of drugs as tocolytics (beta-mimetic drugs and progesterone) should routinely be advised and as soon as there is any threat of premature labour hospitalisation is needed. Twenty one of the 23 patients had prophylactic cerclage (Shirodkar's stitch). In 77% of the cases respiratory distress in the newborn was avoided by using cortico-therapy. Vaginal delivery can be carried out under certain conditions in triplet pregnancies. If certain precautions are taken there does not seem to be any immediate difference in the post delivery period of these children if they are born vaginally or by caesarean. Perinatal mortality is raised (at 5.6% for triplets and 58.3% for quadruplets). The psychological implications of these pregnancies are important. Problems appear as soon as the diagnosis is made and continue for years afterwards. On the social level, help given by the social services are usually inadequate. If the couples belong to the National Association for Mutual Aid of Parents of Children of Multiple Births, a system of mutual support is available. We recommend that these pregnancies should be looked after by several disciplines. These consist not only of obstetricians, paediatricians, anaesthetists, those who resuscitate together, but also psychologists, dietitians, social workers, community workers and physiotherapists.

[Embryonal stem cells do not undergo cell cycle arrest upon exposure to damaging factors]. / Embrional'nye stvolovye kletki ne ostanavlivaiutsia v kletochnom tsikle pri deistvie povrezhdaiushchikh faktorov.

As shown recently (Malashicheva et al., 2000), embryonic teratocarcinoma F9 mouse cells do not stop on the G1/S border after the treatment with agents causing G1 arrest in normal fibroblast cells. Since after a prolonged cultivation in vitro F9 cells could lose some properties characteristic of the stem cells, we studied here the capability of mouse ES cells to undergo cell cycle blocks following gamma-irradiation, adriamycin and PALA treatment as well as upon cultivation in the presence of nocodazol, an inhibitor of spindle assembly. The results obtained show that ES cells, similarly as their tumorigenic derivative F9 cells, do not demonstrate any delay on the G1/S boundary of the cell cycle. Moreover, nocodazol treatment for 48 h leads to accumulation of polyploid cells. Immunoblot experiments reveal a low level of p21/Waf1 expression both in F9 and in ES cells. Interestingly, the content of p21/Waf1 has been found to increase after cell treatment with proteasome inhibitor lactacystin, implying that p21/Waf1 level is regulated by proteasomal degradation. Thus, the p21/Waf1--dependent mechanisms of cell cycle control (checkpoint control) do not function properly in embryonic stem cells.

Generation of rabbit pluripotent stem cell lines.

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.

Permissiveness to murine leukemia, virus expression during preimplantation and early postimplantation mouse development.

Permissiveness to Moloney Murine Leukemia Virus (MoMuLV) expression was examined during preimplantation and early postimplantation development of the mouse embryo. Blastocysts and 8th, 9th and 10th day postimplantation embryos were infected in vitro with a MoMuLV-based retroviral vector expressing the lacZ gene driven off an internal rat beta-actin promoter. Beta-galactosidase-positive cells were identified in all embryonic tissues including inner cell mass, epiblast, mesoderm, endoderm and definitive ectoderm. In contrast, embryos infected with a MoMuLV-based vector expressing the lacZ gene driven off the viral LTR showed beta-galactosidase-positive cells only in mesoderm and definitive ectoderm. We conclude that permissiveness to transcriptional activity of the LTR is acquired immediately upon differentiation of epiblast during gastrulation of the mouse embryo.

In vitro differentiation of embryonic stem cells into glial cells and functional neurons.

Mouse embryonic stem cells were induced to differentiate in culture with retinoic acid. Putative precursors of neurons and glial cells (nestin-positive cells) were clearly identified as early as three days after the onset of differentiation. At day 6, neuron-like cells could be clearly identified, either as isolated cells or as cellular networks. Some of these cells were positive for astrocyte- or oligodendrocyte-specific antigens (GFAP or O4 antigens, respectively). Other cells were positive for neuron-specific antigens (cytoskeleton proteins MAP2, MAP5 and NF200, as well as synaptophysin). Some neuronal-like cells were also positive for acetylcholinesterase activity or glutamic acid decarboxylase expression, indicating that ES cells could differentiate into GABAergic and possibly cholinergic neurons. Electrophysiological analyses performed in voltage clamp conditions showed that cell membranes contained voltage-dependent channels. Overshooting action potentials could be triggered by current injection. Taken together, these data provide evidence that embryonic stem cells can differentiate first into neuron-glia progenitors, and later into glial cells and functional neurons, in vitro. This technique provides an unique system to study early steps of neuronal differentiation in vitro.

Nucleotide sequence of the delta-beta-globin intergenic segment in the macaque: structure and evolutionary rates in higher primates.

A 5600-base-pair (bp) fragment including the beta-globin gene and about 4000 bp of its 5' flanking sequence was cloned from the DNA of Macaca cynomolgus (an Old World monkey), and the 5' flanking region was sequenced. Comparison with human, chimpanzee, mouse, rabbit, and Xenopus orthologous sequences reveals a tandemly repeated sequence called RS4 at the same position (about 500 bp 5' from the transcription start of the adult beta-globin gene) in all six species. We suggest that a tandemly repeated sequence has been maintained by functional constraints since the divergence between amphibians and reptiles. Excluding tandemly repeated sequences as well as about 400 nucleotides upstream from the cap site, the average base substitution frequencies among human, chimpanzee, and macaque intergenic sequences were calculated. They appear to be strongly correlated with the delta T50 values measured between the corresponding nuclear DNAs. They are also similar to base substitution frequencies calculated by Chang and Slightom (1984) at the pseudo-eta-globin locus. Thus, exclusion of sequences involved in specific modes of variation might allow the use of intergenic sequences for the accurate calculation of genetic distances. Using a time scale based on the dating of the Atlantic split, we estimate the base substitution rate of primate noncoding DNA to be 1.0 X 10(-9) substitution/site/year.

Nucleotide sequence of the beta-globin genes in gorilla and macaque: the origin of nucleotide polymorphisms in human.

Part of the beta-globin genes of Macaca cynomolgus and Gorilla gorilla has been cloned and sequenced. Ten putatively neutral nucleotide polymorphisms have been described at the beta-globin locus in humans. They are associated in seven combinations, which define seven different haplotypes of the beta-globin gene: four major frameworks--1, 2, 3, and 3--and three minor frameworks, which we term KI1, KA1, and OR1. The nucleotide sequences of these frameworks are compared with those of homologous sequences in chimpanzee, colobus, macaque, and gorilla. This comparison provides strong evidence that framework 2 was the earliest framework in the human lineage. From framework 2, a rooted parsimonious tree for the six other frameworks is constructed. This phylogenetic tree is discussed in terms of the evolution of nucleotide polymorphisms as well as in terms of genetic affinities between human populations. For each position at which there is base difference in comparing human, gorilla, and chimpanzee beta-globin genes, the phyletic lineage where the corresponding substitution occurred has been identified using the maximum parsimony procedure. The data provide evidence that polymorphisms may represent a significant component of differences between closely related species. If so, nucleotide polymorphisms may strongly bias estimates of small evolutionary distances.

Mouse embryonic stem cells with aberrant transforming growth factor beta signalling exhibit impaired differentiation in vitro and in vivo.

Embryonic stem (ES) cells are resistant to transforming growth factor beta (TGF beta). We have shown previously that they lack type-II binding receptors (T beta RII) and in this respect resemble the inner cell mass and ectoderm cells of mouse embryos 4.5-7.5 days post coitum (dpc); they do however express type-I (alk-5) signalling receptors. Here we show that in contrast to several tumour cell lines, stable transfection of wtT beta RII is not sufficient for ES cells to become biologically sensitive to TGF beta. We analysed the expression of several down-stream molecules known to be involved in TGF beta signalling (Smads) and TGF beta-mediated cell cycle regulation (cyclins D) during the differentiation of control and wtT beta RII-expressing ES cells and showed that upregulation of these molecules correlated with (i) an increase in plasminogen activator inhibitor-1 (PAI-1) synthesis and (ii) growth inhibition, following addition of TGF beta 1. These TGF beta responses were reduced in an ES cell line expressing a dominant negative (truncated) T beta RII (delta T beta RII). The differentiation pattern of control and wtT beta RII-expressing ES cells was indistinguishable in monolayer culture and as embryoid bodies, but in delta T beta RII ES cells, the capacity to form mesodermal derivatives in monolayer cultures in response to the addition of retinoic acid (RA) and removal of leukemia inhibitory factor (LIF) was lost, and only endoderm-like cells formed. The T beta RII and delta T beta RII ES cells were, however, both distinguishable from control ES cells when allowed to differentiate in chimaeric embryos following aggregation with morula-stage hosts. Conceptuses containing mutant cells, recovered from pseudopregnant females at the equivalent of 9.5 dpc, exhibited highly defective yolk sac development; most strikingly, no blood vessels were present and in addition the yolk sacs with derivatives of ES cells containing wtT beta RII were blistered and lacked haematopoietic cells. The implications for understanding TGF beta signalling in early mouse development are discussed.

An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives.

We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the beta-galactosidase/neo(R) fusion protein and the Cre-ER(T2) (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygro(R) protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4'hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4'hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.