Increased leukocyte survival and accelerated onset of lymphoma in the absence of MCL-1 S159-phosphorylation.

The antiapoptotic BCL-2 protein MCL-1, which opposes mitochondrial outer membrane permeabilization, was shown to have a crucial role in the survival of hematopoietic cells. We have previously shown that, upon loss of phosphatidylinositol 3-kinase signaling, S159 of MCL-1 is phosphorylated by glycogen synthase kinase-3 (GSK-3), earmarking MCL-1 for enhanced ubiquitylation and degradation. In this study, we introduced MCL-1(wt) or the phosphorylation-deficient mutant MCL-1(S159A) in mouse BM cells, followed by adoptive transfer to recipient mice. Mice expressing MCL-1(S159A) exhibited significantly elevated white blood cell and lymphocyte counts, whereas no effect was observed on the distribution of T and B lymphocyte subsets or the numbers of monocytes, red blood cells or platelets. Expression of MCL-1(S159A) in Eµ-Myc transgenic bone marrow significantly accelerated the onset of disease, and these mice displayed increased spleen weights compared with Eµ-Myc/MCL-1(wt) mice. Our data demonstrate that the absence of MCL-1 S159 phosphorylation provides a survival advantage for hematopoietic cells in vivo and facilitates oncogenesis.

[Mammary metastasis of extramammary cancers: current knowledge and diagnostic difficulties]. / Métastases mammaires de cancers d'origine extra-mammaire: état des lieux et difficultés diagnostiques.

OBJECTIVES: Intramammary metastasis (IM) of non-breast cancers are infrequent. The purpose of this review of literature was to update the knowledge and explain diagnostic errors. PATIENTS AND METHODS: A review of literature with PubMed was used to select 54 articles published in English between 2003 and 2012. RESULTS: Melanoma (138 cases, 29.8%) were more frequently responsible of 463 cases of MIM, followed by lung, gynecological, gastrointestinal and hematologic cancers. IM occur mainly in women (92.2%), around 50 years, and are metachronous (84.2%). Clinically, they are usually round, painless, without skin retraction and associated with adenopathies (33%). In imaging, they are frequently single. Diagnosis, sometimes evoked by morphological study of the tumor, is confirmed by immunohistochemistry. Systemic metastasis are common, involving a shorter survival. DISCUSSION: The prevalence of primitive cancers is not alone responsible for the frequency of IM, as we can observe it with melanoma. The "seed and soil" hypothesis of Paget may explain it: some tumor cells grow preferentially in selected organs. Vascularity of the breast also seems to be an important factor. Clinically, IM can be confused with benign tumors. However, a history of cancer and multiple lesions should raise the suspicion of malignancy. In imaging, signs are non-specific. The morphological characteristics do not confirm the diagnosis with certainty. Immuno-histochemistry is fundamental, as the comparison with the histology of primary tumor. Support is mainly palliative.

[Intravenous midazolam-ketamine anaesthesia for closed reduction of forearm fractures in children: impact of additional axillary plexus anaesthesia]. / Intravenöse Midazolam-Ketamin-Anästhesie zur geschlossenen Reposition der Vorderarmfraktur bei Kindern: Bringt eine zusätzliche axilläre Plexusblockade Vorteile?

BACKGROUND: The aim of this study was to compare ketamine requirements in children undergoing closed reduction of forearm fractures under midazolam-ketamine anaesthesia with or without axillary plexus anaesthesia. METHODS: With hospital ethical committee approval, we retrospectively analyzed the records of children who received midazolam-ketamine anaesthesia in the years 2000-2001 (group A) and midazolam-ketamine anaesthesia combined with axillary plexus anaesthesia in the years 2002-2004 (group B) for closed reduction of forearm fractures. Requirements for ketamine and postoperative analgesics were noted. Groups were compared with the Mann-Whitney U-test or T-test and the chi2-test (p<0.05). RESULTS: A total of 455 children (group A 225/group B 230) were included in this study. The total amounts of ketamine were not statistically different between the two groups (p=0.154). However, ketamine requirements became less if the time interval between start of axillary plexus anaesthesia and start of intervention became more than 15 min (p<0.05). Patients in group B requested fewer analgesics in the postoperative period (p<0.01). CONCLUSIONS: In the clinical routine of an emergency department the combination of midazolam-ketamine anaesthesia with axillary plexus anesthesia for closed reduction of forearm fractures in children did not result in lower requirements of ketamine.

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.

By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

Donor stromal cells from human blood engraft in NOD/SCID mice.

Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants. In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34(+) peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells). Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow. Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34(+) PBCs or CBCs, with mean frequencies between 0.6% and 2.4%. In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%). Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-alpha (fibroblast specific) within the bone marrow and spleen of transplanted mice. Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR. Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation. These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice. (Blood. 2000;96:3971-3978)

Interactions in the transcriptional regulation exerted by Stat5 and by members of the steroid hormone receptor family.

The pathways which connect extracellular signals with the regulation of the activity of transcription factors are being investigated in molecular detail. Extensive progress has been made in the description of the mode of action of steroid hormones and of cytokines. Steroid hormones associate intracellularly with latent receptor molecules, cause the dissociation of masking proteins, the dimerization of receptors, and their binding to specific hormone response elements in the promoters of target genes. Cytokines also activate latent transcription factors (Stats--signal transducers and activators of transcription), but act through an enzymatic mechanism. Tyrosine kinases associated with the transmembrane cytokine receptors phosphorylate Stat molecules. The phosphorylated monomers dimerize and assume specific DNA binding ability. Both classes of transcription factors bind to different response elements and regulate different target genes and both signals, cytokines and steroid hormones, can affect growth differentiation and homeostasis of different cell types. Here, we describe that Stat5, a molecule activated by several essential cytokines, functionally interacts with members of the steroid receptor family. We find that glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor synergize with Stat5 in the induction of the transcription from the beta-casein gene promoter. The estrogen receptor diminishes Stat5 mediated induction and the androgen receptor has no effect. Conversely, Stat5 negatively interferes with glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor induced transcription from the MMTV LTR and the estrogen receptor induced transcription from an ERE-containing promoter.

p300/CREB-binding protein enhances the prolactin-mediated transcriptional induction through direct interaction with the transactivation domain of Stat5, but does not participate in the Stat5-mediated suppression of the glucocorticoid response.

Stat5 was discovered as a PRL-induced member of the Stat (signal transducer and activator of transcription) family. Its induction by many other cytokines and interleukins suggests that Stat5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. Cell type- and promoter-specific functions of Stat5 are most likely modulated by the interaction with other transcription factors. We recently showed cross-talk between Stat5 and the glucocorticoid receptor. The activated glucocorticoid receptor forms a complex with Stat5 and enhances Stat5-mediated transcriptional induction. Conversely, Stat5 diminishes the induction of glucocorticoid-responsive genes. Here, we investigated the role of p300/CBP(CREB-binding protein), a transcriptional coactivator of several groups of transcription factors, in Stat5-mediated transactivation and in the cross-talk between Stat5 and the glucocorticoid receptor. p300/ CBP acts as a coactivator of Stat5. Its ectopic expression enhances PRL-induced Stat5-mediated transcriptional activation. Consistent with this observation, we find that the adenovirus E1A protein, which binds to p300/CBP, suppresses Stat5-induced transcriptional activation. This inhibition requires the Stat5 transactivation domain and the p300/CBP binding site of E1A. Coimmunoprecipitation and mammalian two-hybrid assays demonstrate a direct interaction between the carboxyl-terminal transactivation domain of Stat5 and p300/CBP. p300/CBP also positively interacts with the glucocorticoid receptor and enhances glucocorticoid receptor-dependent transcriptional activation of the mouse mammary tumor virus-long terminal repeat promoter. Overexpression of p300/CBP does not counteract the Stat5-mediated inhibition of glucocorticoid receptor-dependent transactivation, i.e. the repression of the glucocorticoid response by Stat5 is not a consequence of competition for limiting amounts of p300/CBP.

Cytokine receptor-independent, constitutively active variants of STAT5.

STAT (signal transducers and activators of transcription) proteins are dual function proteins, which participate in cytokine-mediated signal transduction events at the cell surface and transcriptional regulation in the nucleus. We have exploited insights into the activation mechanism of STAT factors to derive constitutively active variants. Chimeric genes encoding fusion proteins of STAT5 and the kinase domain of JAK2 have been derived. The functional properties of the fusion proteins have been investigated in transiently transfected COS cells or in HeLa cells stably transfected with STAT5-JAK2 gene constructs regulated by a tetracycline-sensitive promoter. The STAT5-JAK2 proteins exhibit tyrosine kinase activity and are phosphorylated on tyrosine. The molecules are activated through an intramolecular or a cross-phosphorylation reaction and exhibit constitutive, STAT5-specific DNA binding activity. The transactivation potentials of three constitutively activated STAT5-JAK2 variants comprising different transactivation domains (TADs) derived from STAT5, STAT6, and VP16 were compared. The chimeric molecule containing the STAT5 TAD had no or only a very low, the molecule with the STAT6 TAD a medium, and the molecule with the VP16 TAD a very high transactivation potential. Transcription from STAT5-responsive gene promoter regions of the beta-casein, oncostatin M, and the cytokine-inducible Src homology 2 domain-containing protein genes was observed. These chimeric STAT molecules allow the study of the function of STAT5 independent of cytokine receptors and the activation of other signal transduction pathways.

Specific DNA binding of Stat5, but not of glucocorticoid receptor, is required for their functional cooperation in the regulation of gene transcription.

Prolactin and glucocorticoid hormone are signals which regulate the transcription of milk protein genes in mammary epithelial cells. We have investigated the molecular mechanisms by which these hormones cooperate in the induction of transcription. Both hormones activate latent transcription factors in the cytoplasm of mammary epithelial cells. Prolactin exerts its effect through binding to the extracellular domain of the prolactin receptor and through receptor dimerization. This leads to the activation of a protein tyrosine kinase (Jak2), which is noncovalently associated with the cytoplasmic domain of the prolactin receptor. Jak2 phosphorylates the signal transducer and transcription activator (Stat5) which causes its dimerization and nuclear translocation where Stat5 specifically binds to sequence elements in the promoter regions of milk protein genes. In comparison, the glucocorticoid receptor is activated by a lipophilic steroid ligand in the cytoplasm which causes allosteric changes in the molecule, dimerization, and nuclear localization. It has been demonstrated that Stat5 and the glucocorticoid receptor form a molecular complex which cooperates in the induction of transcription of the beta-casein gene. We have defined the DNA sequence requirements for this cooperative mechanism and have delimited the functional domains in Stat5 and the glucocorticoid receptor that are necessary for the functional interaction. We find that the Stat5 response element (Stat5RE) within the beta-casein gene promoter is sufficient to elicit the cooperative action of Stat5 and the glucocorticoid receptor on transcription. Activation of Stat5 through phosphorylation of tyrosine 694 is an absolute prerequisite for transcription. Deletion of the transactivation domain of Stat5 results in a molecule which cannot mediate transactivation by itself but can still cooperate with the glucocorticoid receptor. Mutated variants of the glucocorticoid receptor with a nonfunctional DNA binding domain or a DNA binding domain contributed by the estrogen receptor are still able to cooperate with Stat5 in transcriptional induction. Deletion of the ligand binding domain of the glucocorticoid receptor does not impede cooperation with Stat5, whereas deletion of the AF-1 transactivation domain does prevent cooperation. Our results indicate that the glucocorticoid receptor acts as a ligand-dependent coactivator of Stat5 independently of its DNA binding function.

Comparison of the transactivation domains of Stat5 and Stat6 in lymphoid cells and mammary epithelial cells.

Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. Stat5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates Stat5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of Stat5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of Stat5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function.

Functional interactions between Stat5 and the glucocorticoid receptor.

Signal transduction pathways enable extracellular signals to activate latent transcription factors in the cytoplasm of cells. Dimerization, nuclear localization and binding to specific DNA sequences result in the induction of gene transcription by these proteins. These events are necessary for the functioning of the JAK/STAT pathway and of the glucocorticoid-receptor pathway. In the former, the protein Stat5, which is a member of a family of signal transducers and activators of transcription, is activated by cytokines, hormones and growth factors. These polypeptide ligands bind at the outside of the cell to specific transmembrane receptors and activate intracellular Janus protein tyrosine kinases (JAKs) to tyrosine-phosphorylate STAT proteins; interaction with the SH2 domain of the dimerization partner then confers the ability to bind to DNA at the STAT-response element and induce transcription. In the glucocorticoid-receptor pathway, the receptor interacts with its steroid hormone ligand in the cytoplasm, undergoes an allosteric change that enables the hormone receptor complex to bind to specific DNA-response elements (glucocorticoid response elements, or GRE) and modulate transcription. Although these pathways appear to be unrelated, we show here that the glucocorticoid receptor can act as a transcriptional co-activator for Stat5 and enhance Stat5-dependent transcription. Stat5 forms a complex with the glucocorticoid receptor which binds to DNA independently of the GRE. This complex formation between Stat5 and the glucocorticoid receptor diminishes the glucocorticoid response of a GRE-containing promoter.