Very low oxygen concentration (0.1%) reveals two FDCP-Mix cell subpopulations that differ by their cell cycling, differentiation and p27KIP1 expression.

Oxygen (O(2)) concentrations in bone marrow vary from 4% in capillaries to <0.1% in subendosteum, in which hematopoietic stem cells reside in specific niches. Culture at low O(2) concentrations (3, 1 and 0.1%) influences hematopoietic stem and progenitor cells survival, proliferation and differentiation, depending on their level of differentiation. Culture of human CD34(+) cells at low O(2) concentrations (O(2) ≤3%) maintains stem cell engraftment potential better than at 20% O(2) (NOD/Scid xenograft model). In contrast, progenitors disappear from cultures at/or <1% O(2) concentrations. A very low O(2) concentration (0.1%) induces CD34(+) quiescence in G(0). The exploration of molecules and mechanisms involved in hematopoietic stem and progenitor cells' quiescence and differentiation related to low O(2) concentrations is unfeasible with primary CD34(+) cells. Therefore, we performed it using murine hematopoietic nonleukemic factor-dependent cell Paterson (FDCP)-Mix progenitor cell line. The culture of the FDCP-Mix line at 0.1% O(2) induced in parallel G(0) quiescence and granulo-monocytic differentiation of most cells, whereas a minority of undifferentiated self-renewing cells remained in active cell cycle. Hypoxia also induced hypophosphorylation of pRb and increased the expression of p27(KIP1), the two proteins that have a major role in the control of G(0) and G(1) to S-phase transition.

Bcl2, a transcriptional target of p38alpha, is critical for neuronal commitment of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF) cytokine. LIF starvation leads to cell commitment, and part of the ES-derived differentiated cells die by apoptosis together with caspase3-cleavage and p38alpha activation. Inhibition of p38 activity by chemical compounds (PD169316 and SB203580), along with LIF withdrawal, leads to different outcomes on cell apoptosis, giving the opportunity to study the influence of apoptosis on cell differentiation. By gene profiling studies on ES-derived differentiated cells treated or not with these inhibitors, we have characterized the common and specific set of genes modulated by each inhibitor. We have also identified key genes that might account for their different survival effects. In addition, we have demonstrated that some genes, similarly regulated by both inhibitors (upregulated as Bcl2, Id2, Cd24a or downregulated as Nodal), are bona fide p38alpha targets involved in neurogenesis and found a correlation with their expression profiles and the onset of neuronal differentiation triggered upon retinoic acid treatment. We also showed, in an embryoid body differentiation protocol, that overexpression of EGFP (enhanced green fluorescent protein)-BCL2 fusion protein and repression of p38alpha are essential to increase formation of TUJ1-positive neuronal cell networks along with an increase in Map2-expressing cells.

Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.

A p38 inhibitor allows to dissociate differentiation and apoptotic processes triggered upon LIF withdrawal in mouse embryonic stem cells.

Mouse embryonic stem cells remain pluripotent when maintained in the presence of leukemia inhibitory factor (LIF). Upon LIF withdrawal, most cells differentiate into various lineages, while some die by apoptosis within 3 days. We have analyzed the activation pattern of the mitogen-activated protein kinase (MAPK) families and characterized the expression profile of selected genes modulated during differentiation or apoptosis. We show that p38 MAPKs are activated first, during the apoptotic crisis, while extracellular-regulated kinases and c-Jun N-terminal kinases are induced after the apoptotic crisis in differentiated cells. However, by using both p38 kinase inhibitors (PD169316 and SB203580) and a p38alpha(-/-) cell line, we demonstrate that p38alpha activation is rather a consequence than a cause of apoptosis. We thus reveal novel properties of PD169316, which induces cell survival without impairing cell differentiation, and identify PD169316-sensitive targets like the fibroblast growth factor-5, Brachyury and bcl-2 genes. Finally, we demonstrate that overexpression of the PD169316 - regulated bcl-2 gene prevents LIF withdrawal - induced cell death.

The 'PINIT' motif, of a newly identified conserved domain of the PIAS protein family, is essential for nuclear retention of PIAS3L.

PIAS proteins, cytokine-dependent STAT-associated repressors, exhibit intrinsic E3-type SUMO ligase activities and form a family of transcriptional modulators. Three conserved domains have been identified so far in this protein family, the SAP box, the MIZ-Zn finger/RING module and the acidic C-terminal domain, which are essential for protein interactions, DNA binding or SUMO ligase activity. We have identified a novel conserved domain of 180 residues in PIAS proteins and shown that its 'PINIT' motif as well as other conserved motifs (in the SAP box and in the RING domain) are independently involved in nuclear retention of PIAS3L, the long form of PIAS3, that we have characterized in mouse embryonic stem cells.

The ribosomal S6 kinases, cAMP-responsive element-binding, and STAT3 proteins are regulated by different leukemia inhibitory factor signaling pathways in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

Role of suppressors of cytokine signaling (Socs) in leukemia inhibitory factor (LIF) -dependent embryonic stem cell survival.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF withdrawal results in progressive ES cell differentiation. Here we show that during this differentiation process, part of the cells undergo apoptosis concomitant with an activation of the p38 MAP kinase. To gain insight into events mediated by LIF in ES cells, the expression of potential candidate genes was analyzed in the absence or presence of this cytokine by using a semiquantitative RT-PCR assay. We focused on early response genes and on a new type of cytokine repressors (the Socs proteins), some of which exhibit anti-apoptotic properties. We found that expression of c-Fos, c-Jun, and JunB was induced upon LIF treatment whereas that of JunD, the tyrosine phosphatase ESP, and the components of the LIF receptor remained unaffected. Expression of Socs-3, but not Socs-1 or Socs-2, was stimulated in the presence of LIF. Finally, uncontrolled overexpression of Socs-1 and Socs-3 led to repression of LIF-dependent transcription and severely reduced cell viability, suggesting that the disturbance of a well balanced Socs protein content has adverse effects on cell survival.

Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions.

The Lasp-1 gene, which has been localized to the q12-q21 region of human chromosome 17, is amplified and overexpressed in human breast cancers. In addition to the previously reported LIM and SH3 domains of Lasp-1, we report here the identification of an actin-binding domain in the core of the protein. This domain is functional as we demonstrate that Lasp-1 binds actin in vivo and in vitro. In addition, confocal analysis of the Lasp-1 subcellular distribution shows that the protein is colocalized with actin at peripheral cell extensions in individual epithelial cancer cells and in transformed fibroblastic cells. Moreover, Lasp-1 is tyrosine phosphorylated in fibroblast cell lines transformed by a constitutively active form of c-Src (c-SrcY527F). Altogether, our results show that Lasp-1 defines a new type of actin-binding protein and suggest that the protein may play a role in a signaling pathway involved in the organization of the cytoskeleton.

Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells.

STAT transcription factors are induced by a number of growth factors and cytokines. Within minutes of induction, the STAT proteins are phosphorylated on tyrosine and serine residues and translocated to the nucleus, where they bind to their DNA targets. The leukemia inhibitory factor (LIF) mediates pleiotropic and sometimes opposite effects both in vivo and in cultured cells. It is known, for example, to prevent differentiation of embryonic stem (ES) cells in vitro. To get insights into LIF-regulated signaling in ES cells, we have analyzed protein-binding and transcriptional properties of STAT recognition sites in ES cells cultivated in the presence and in the absence of LIF. We have detected a specific LIF-regulated DNA-binding activity implicating the STAT3 protein. We show that STAT3 phosphorylation is essential for this LIF-dependent DNA-binding activity. The possibility that ERK2 or a closely related protein kinase, whose activity is modulated in a LIF-dependent manner, contributes to this phosphorylation is discussed. Finally, we show that the multimerized STAT3-binding DNA element confers LIF responsiveness to a minimal thymidine kinase promoter. This, together with our observation that overexpression of STAT3 dominant-negative mutants abrogates this LIF responsiveness, clearly indicates that STAT3 is involved in LIF-regulated transcriptional events in ES cells. Finally, stable expression of such a dominant negative mutant of STAT3 induces morphological differentiation of ES cells despite continuous LIF supply. Our results suggest that STAT3 is a critical target of the LIF signaling pathway, which maintains pluripotent cell proliferation.

Binding in vitro of phosphotyrosine-containing proteins to pp60c-src SH2 domain does not correlate with CEF transformation.

We have previously described pp60c-src SH2 mutants that are host-range-dependent for cell transformation; most of these mutants can transform CEF cells but not NIH3T3 cells, and other transform NIH3T3 cells more efficiently than CEF (Hirai and Varmus, 1990c). In an attempt to understand the molecular basis of these phenotypes, we analysed the ability of mutant SH2 domains in GST fusion proteins to bind to tyrosine phosphorylated proteins in lysates from CEF and NIH3T3 cells. The relative affinity of mutated versions of the SH2 domain for phosphotyrosine-containing proteins from CEF and NIH3T3 cells was compared with the relative ability of the mutant Src proteins to transform the two cell types. While the affinity of the SH2 domain for phosphotyrosine-containing proteins was closely correlated with transformation in NIH3T3 cells, there was no correlation between phosphotyrosine binding and transformation of CEF cells, and none of the host range mutant SH2 domains showed significant differences in their ability to bind phosphotyrosine-containing proteins from lysates from either cell type. In addition, the SH3 domain was shown to augment the capacity of mutant SH2 domain to bind phosphotyrosine-containing proteins.

Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.

The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity.

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.

EIa-mediated transactivation of the adenovirus EIIa early promoter is restricted in undifferentiated F9 cells.

The murine embryonal carcinoma (EC) F9 cells express an adenovirus EIa-like activity which is abolished after differentiation of these cells. We have examined the ability of the adenovirus EIa gene products to transactivate the viral early EIIa (EIIaE) promoter in this cell system. Surprisingly, as shown both by infection and transfection experiments, the EIIaE promoter was refractory to viral EIa-mediated activation in the undifferentiated F9 EC cells, EIa-responsiveness being recovered only after differentiation of these cells. This EC-cell-specific restriction did not correspond to a lack of EIa gene expression, nor to a deficiency of any of the major transcription factors involved in EIIaE basal promoter activity. In these differentiated F9 cells, EIIaE promoter activation correlates with a modification of E2F resulting in its ability to cooperatively interact with the promoter. Our observation that such a modification neither occurs in uninfected F9 EC cells (Jansen-Durr et al., 1989, EMBO J., 8, 3365), nor at early times after infection of these cells suggests that a viral product is required together with the EIa-like activity to induce this modification of E2F. The implication of an EIV gene product in this effect is discussed.

Differential activation of the E2F transcription factor by the adenovirus EIa and EIV products in F9 cells.

Expression studies of the early EIIa transcription unit (EIIaE) of the adenovirus EIa-deletion mutant dl312 in murine embryonal carcinoma stem cells suggested that these cells contain an activity that substitutes for the viral EIa. To further characterize this cellular EIa-like activity, we analyzed expression of the EIIaE promoter as well as the binding activity of the cognate E2F transcription factor after infection of F9 embryonal carcinoma cells and their differentiated derivatives with wild-type adenovirus, EIa (dl312), or EIV (dl808) deletion mutants. We show that, in contrast to the viral EIa proteins that transactivate the EIIaE promoter in F9 cells only after differentiation, the viral EIV products activate the EIIaE promoter most efficiently in undifferentiated F9 cells. We also show that the EIV products induce a specific modification of the E2F transcription factor leading to its cooperative binding to the EIIaE promoter. Although the EIa-dependent transactivation of EIIaE in differentiated cells is also in part mediated by E2F, it does not by itself correlate with the simultaneous binding of two E2F molecules. In these cells E2F dimer binding only occurs as a secondary effect of EIa that also stimulates EIV expression. Our results suggest that EIa and EIV act through separate pathways, inversely regulated during cell differentiation, with the so-called "EIa-like" activity contributing to this modulation.

Cooperative binding of two E2F molecules to an Ela-responsive promoter is triggered by the adenovirus Ela, but not by a cellular Ela-like activity.

The binding of the cellular E2F transcription factor to the central EIa-responsive element of the adenovirus EIIa early promoter (EIIaE) was compared in extracts of HeLa cells which had been infected with either wild-type adenovirus or the EIa-deficient mutant dl312. No quantitative differences in the E2F-binding activity were detected as a function of EIa gene expression. However, complexes formed by the E2F factor in the presence of EIa were qualitatively different from those formed on the same sequence element in the absence of EIa. Specifically, the formation of complexes containing two E2F molecules is favoured by EIa, probably through the induction of protein-protein interactions. Protein binding to EIIaE promoter in extracts from non-infected F9 embryonal carcinoma cells, prepared before and after in vitro differentiation of these cells was also analysed. The higher expression of EIIaE in undifferentiated cells, which was originally attributed to a cellular EIa-like function, may be correlated with the increased binding activity of a murine E2F-like protein which does not, however, result in the simultaneous occupation of both E2F sites on the EIIaE promoter, suggesting that the viral EIa and the presumptive cellular EIa-like functions trans-activate the EIIaE promoter through different pathways.

Replication-induced stimulation of the major late promoter of adenovirus is correlated to the binding of a factor to sequences in the first intron.

The sequence requirements for transcriptional stimulation of the adenovirus major late promoter (MLP) by the products of the early transcription unit Ela and by the replication of viral DNA were analyzed by in vitro transcription. Sequences upstream of +33 are involved in the moderate Ela-responsiveness of the MLP, while sequences between +33 and +131 are required for its major replication-induced transcriptional activation. Dnase I footprinting experiments delineate a sequence component, extending from +76 to +120, which binds protein(s) only in extracts of cells where viral DNA replication occurred. Taken together, these results suggest that the replication-dependent stimulation of the MLP is mediated by the increased binding of this protein(s).

E1a inducibility of the adenoviral early E2a promoter is determined by specific combinations of sequence elements.

By transient expression analysis in HeLa cells of adenovirus-2 E2a early (E2aE) promoter mutants and hybrid E2aE-beta-globin gene constructs, we demonstrate the existence of three nucleotide (nt) sequence elements involved in the E1a-responsiveness of the E2aE transcription unit: element I, localized within a segment (nt -13 to +62) surrounding the major E2aE cap site (nt + 1); element II (between nt -71 and -29), and element III (between nt -146 and -86). Each element is unable by itself to confer E1a responsiveness. Only combinations of sequences including elements I and II (spanning nt -71 and +62) or II and III (spanning nt -146 and -29) ensure maximal inducibility, for which element II appears of central importance.

Specific cellular proteins bind to critical promoter sequences of the adenovirus early EIIa promoter.

As an approach to the identification of essential factors required for specific expression of the adenovirus type 2 EIIaE early (EIIaE) promoter, an in vitro system was established. Under appropriate conditions, using crude extracts of non-infected HeLa cells, efficient and accurate EIIaE expression has been reproduced. As in vivo, this transcription was strongly dependent upon the integrity of two non-consensus TATA-like elements, T1 and T2, corresponding to the major (EIIaE1) and minor (EIIaE2) start sites, respectively, as well as upon intact upstream elements (A, between -40 and -50 and B, between -70 and -90) common to both overlapping promoters. The implication of specific DNA-binding proteins in the transcriptional effects mediated by these elements was demonstrated by DNAse I footprinting analyses. Both crude nuclear extracts and partially purified fractions confer specific protection against DNAse I digestion to the T1 and B promoter elements defined above, and to a far upstream region (element C, between -110 and -150), which has previously been identified as a weaker promoter element by in vivo transcriptional studies. Separation of the T1 recognition factor from those which bind to the upstream elements B and C by chromatographic fractionation of the extracts has also been achieved.

Genotoxic activities of 2-nitronaphthofurans and related molecules.

The genotoxic activities of 63, 2-nitronaphthofurans and related molecules were examined using two bacterial short-term tests, the Salmonella mammalian microsome assay test or Mutatest, a mutagenesis assay, and/or the SOS Chromotest, an assay for induction of an SOS function in Escherichia coli. Seven compounds were also investigated in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) test, a mammalian gene mutation assay. Our main conclusions are the following: (a) Simple empirical rules relating structure to mutagenic activity in the Mutatest can be derived for some of the compounds. In particular, they account for the extremely high Mutagenic Potency of 7-methoxy-1-methyl-2-nitronaphtho[2,1-b]furan (R7372), approximately 2 X 10(6) mutants/nmol on strain TA100. (b) There is a good quantitative correlation between the Mutagenic Potency in the Salmonella/mammalian microsomes assay and the SOS-inducing potency in the SOS Chromotest. This, and previous evidence, suggests strongly that the 2-nitronaphthofurans derivatives are essentially recA and thus probably umuDC-dependent mutagens. (c) Four out of seven compounds tested in the CHO/HGPRT assay gave responses correlated with the bacterial responses. One of them, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), is among, or is, the strongest mutagen described for mammalian cells. We briefly discuss the practical and theoretical implications of these results.

Sequence-specific trans-activation of the adenovirus EIIa early promoter by the viral EIV transcription unit.

The contribution of adenovirus early genes, other than that of the well-documented EIa immediate early gene, to the transcriptional regulation of the viral EIIa early transcription unit was examined. HeLa cells were transfected with EIIa-containing plasmids and co-transfected with distinct plasmids bearing one of the viral regions EIa, EIII or EIV. Co-transfection with the EIV-recombinants, but not the EIII constructs, stimulated specific transcription from the major EIIaE start site (EIIaE1) by 5- to 15-fold, as concluded from quantitative S1 nuclease analysis of cytoplasmic RNA and in vitro nuclear 'run-on' transcription assays. The extent of the EIV-induced stimulation was similar to that achieved by EIa under identical conditions. However, in contrast to our observations for EIa-mediated stimulation, where no unique EIIaE1 promoter elements were implicated, maximal induction by EIV requires sequences between positions -48 and -19 (with respect to the EIIaE1 start site).