Sandrine Dabernat

Differential expression of nm23 genes in adult mouse dorsal root ganglia

Nm23 has been identified as a gene family encoding different isoforms of nucleoside diphosphate kinase (NDPK). This protein is a key enzyme in nucleotide metabolism and has been shown to play important roles in various cellular functions. In the present study, we have investigated the expression of three isotypes in mouse dorsal root ganglia. In situ hybridization and reverse transcriptase‐polymerase chain reaction analysis demonstrated high levels of nm23‐M1, ‐M2, and ‐M3 mRNA expression in peripheral nervous tissue. Moreover, in situ hybridization also displayed a specific nuclear localization for nm23‐M2 mRNA. Immunohistochemistry with light and electron microscopy on isoform‐specific antibodies revealed a differential subcellular distribution of NDPK isoforms. Isoform A was mainly cytosolic, showing only partial association with organelles. In contrast, isoform B was also found in the nucleus, which is in agreement with its proposed role as a transcription factor. The results also indicate a preferential association of isoform C with endoplasmic reticulum and plasma membranes in neuronal cells. Furthermore, isoform C appeared to combine with other NDPK isoforms as demonstrated by double‐labeling evidence by electron microscopy and might be responsible for binding NDPK oligomers to membranes. Thus, isoform C may be considered as a protein of importance for maintaining intracellular pools of GTP in the vicinity of membranes and, hence, for transmembrane signaling. The results indicate a high expression of NDPK isoforms, not only in the central but also in the peripheral nervous system. Their different subcellular compartmentalization suggests that they have isoform‐specific roles in neuronal cell physiology. J. Comp. Neurol. 444:306–323, 2002.

Characterization of the nm23-M2, nm23-M3 and nm23-M4 mouse genes: comparison with their human orthologs

The nm23 gene family is thought to be involved in physiopathological processes such as growth, differentiation and cancer promotion, progression or metastasis. We report here the mouse nm23-M3 and nm23-M4 complementary DNA sequences and the genomic cloning, characterization and tissue expression pattern of the nm23-M2, nm23-M3 and nm23-M4 genes, in comparison with their human and rat orthologs and with the human nm23-H1 and mouse nm23-M1 genes. The organization and structure of the members of this gene family are remarkably similar in human and rodents. Accordingly, the striking similarities between the human and mouse nm23 genes enable the use of mouse transgenic and knock-out models for studying the role of nucleoside diphosphate kinase isoforms in human physiopathology.

Organization and expression of mouse nm23-M1 gene. Comparison with nm23-M2 expression.

Nm23 is a gene family encoding different isoforms of the nucleotide diphosphate kinase (NDPK), an enzyme involved in the synthesis of nucleoside triphosphates. In the present study, the organization and expression of the nm23-M1 gene encoding the mouse NDPKA isoform are described. This gene is about 10kb long and composed of five exons. The organization and the exon-intron boundaries are strictly conserved as compared to the human and rat related genes. The gene promoter region did not exhibit any consensus TATA box, SP1 binding element or Inr sequence. By contrast, TCF-1/LEF-1 binding elements and Pit-1 consensus sequence were present. Northern blotting and in situ hybridization methods were carried out in adult and 18.5 days post-coitum (dpc) mouse embryo, respectively. They showed tissue-specific expression of nm23-M1 transcripts, despite housekeeping gene promoter features. The strongest signals were detected in the nervous system, sensory organs and embryonic thymus. In contrast nm23-M2 mRNA was shown to be more widely expressed.The relationship between nm23-M1 gene tissue-specific expression and the putative binding element of the promoter region is discussed.

Preventing Pluripotent Cell Teratoma in Regenerative Medicine Applied to Hematology Disorders

Iatrogenic tumorigenesis is a major limitation for the use of human induced pluripotent stem cells (hiPSCs) in hematology. The teratoma risk comes from the persistence of hiPSCs in differentiated cell populations. Our goal was to evaluate the best system to purge residual hiPSCs before graft without compromising hematopoietic repopulation capability. Teratoma risk after systemic injection of hiPSCs expressing the reporter gene luciferase was assessed for the first time. Teratoma formation in immune‐deficient mice was tracked by in vivo bioimaging. We observed that systemic injection of hiPSCs produced multisite teratoma as soon as 5 weeks after injection. To eliminate hiPSCs before grafting, we tested the embryonic‐specific expression of suicide genes under the control of the pmiR‐302/367 promoter. This promoter was highly active in hiPSCs but not in differentiated cells. The gene/prodrug inducible Caspase‐9 (iCaspase‐9)/AP20187 was more efficient and rapid than thymidine kinase/ganciclovir, fully specific, and without bystander effect. We observed that iCaspase‐9‐expressing hiPSCs died in a dose‐dependent manner with AP20187, without reaching full eradication in vitro. Unexpectedly, nonspecific toxicity of AP20187 on iCaspase‐9‐negative hiPSCs and on CD34+ cells was evidenced in vitro. This toxic effect strongly impaired CD34+‐derived human hematopoiesis in adoptive transfers. Survivin inhibition is an alternative to the suicide gene approach because hiPSCs fully rely on survivin for survival. Survivin inhibitor YM155 was more efficient than AP20187/iCaspase‐9 for killing hiPSCs, without toxicity on CD34+ cells, in vitro and in adoptive transfers. hiPSC purge by survivin inhibitor fully eradicated teratoma formation in immune‐deficient mice. This will be useful to improve the safety management for hiPSC‐based medicine. Stem Cells Translational Medicine 2017;6:382–393

Proteomic analysis of NME1/NDPK A null mouse liver: evidence for a post-translational regulation of annexin IV and EF-1Bα

NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography–tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1−/− mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1−/− hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.

Validation de la mesure d’un paramètre critique en culture d’embryons humains : le pH

In vitro human embryos culture depends largely on the atmospheric conditions within the incubators of the laboratory. The pH of culture media, an indirect reflection of the CO2 content inside these incubators, is a critical parameter. Collaboration between the biochemistry and reproductive biology departments enabled the automated measurement of the pH in the culture medium on a blood gas analyzer. This method has been validated and evaluated. It is applicable in all laboratories whatever the medium and the conditions of culture. It allows strict monitoring of this parameter for the optimization of the culture conditions necessary to improve the results of in vitro fertilization attempts.