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H Neel

Publications and source records attributed to H Neel.

16 recordsLinked to original sources
PubMed2001

MDM2: life without p53.

The MDM2 protein suppresses the ability of p53 to inhibit cellular proliferation or to induce cell death. This property underlies the oncogenic potential of MDM2, which is overexpressed in various human tumours. However, MDM2 also has p53-independent activities, which we focus on here. Similar to other oncogenes, surveillance pathways might counteract the deleterious effects of deregulated MDM2 expression. These pathways need to be inactivated for MDM2 oncogenic activity, which targets p53 but also other proteins.

3T3 Cells↗
PubMed2001

Preferential expression of Mdm2 oncogene during the development of neural crest and its derivatives in mouse early embryogenesis.

The Mdm2 oncoprotein acts as the principal negative regulator of p53 activities and is essential for its control during mouse early development, at least before implantation. We analyzed Mdm2 expression between 7.5 and 9 days post-coitum (dpc) by whole-mount in situ hybridization and report here a novel expression pattern during neural crest development. At 7.5 dpc Mdm2 becomes preferentially expressed at the top of the neural folds. Between 8 and 9 dpc, this preferential expression is also observed in neural crest cells migrating from the closing brain towards craniofacial regions and the first three branchial arches. It persists in the craniofacial mesenchyme and the first branchial arch in 9 dpc embryos. Migrating neural crest cells in the tail region are also preferentially labeled at this stage. At day 9.5 Mdm2 becomes more ubiquitously expressed throughout the embryo as reported before.

Animals↗
PubMed2000

The Mdm2 gene of zebrafish (Danio rerio): preferential expression during development of neural and muscular tissues, and absence of tumor formation after overexpression of its cDNA during early embryogenesis.

The Mdm2 protein is most probably the main negative cellular regulator of the p53 tumor-suppressor protein. It was found to be overexpressed in a great number of human tumors and is considered as a potential target for anti-tumor therapies. Mdm2 is an essential gene in mice, yet its role in normal development and tissue differentiation is unknown. In order to study the role of this important protein in an evolutionary perspective, we cloned an Mdm2 cDNA from the fish Danio rerio and analyzed its expression pattern as well as the phenotypic consequences of its overexpression. The main functional domains as well as the interaction between Mdm2 and p53 are conserved in zebrafish. Moreover, we show here that the gene is expressed specifically during early development in neural and muscular tissues. Surprisingly, microinjection of Mdm2 mRNA in two-cell-stage embryos led to inhibition of cellular convergence during gastrulation. The clones derived from Mdm2 microinjected blastomeres were significantly smaller than those derived from control microinjections, and, in contrast to what was observed in Xenopus, did not develop tumors. Our results suggest that Mdm2 expression may be important during the differentiation of neural and muscular tissues of zebrafish. They also point to important differences between phyla in the susceptibility to tumor formation.

Amino Acid Sequence↗
PubMed1997

Mdm2: keeping p53 under control.

The Mdm2 gene is overexpressed in several human tumors. The oncogenic potential of Mdm2 is partially explained by the inhibition of the activity of the tumor suppressor protein p53. Determination of the three-dimensional structure of complexes between Mdm2 and the N-terminal p53 peptide provided a molecular basis for the inhibition of the transcriptional function of p53 by Mdm2. More dramatically, p53 is targeted by Mdm2 for rapid degradation. The Mdm2 gene itself is activated by p53, which gives the opportunity for feed-back control of p53 activity. Keeping p53 under control is most likely the major task of Mdm2 during early development. Recently, evidence was provided for an alternative, p53-independent function of Mdm2.

Amino Acid Sequence↗
PubMed1997

MDM-2 protein is expressed in different layers of normal human skin.

MDM-2 is one of the target genes of the p53 tumor suppressor protein. Its best characterized function is found in the inhibition of p53's ability to modulate transcription. Deregulated expression of MDM-2 could thus at least partially substitute for p53 mutation in the process of tumorigenesis. We show here that MDM-2 is highly expressed in biopsies of normal human skin or in vitro reconstituted human skin. The protein is detected in the nucleus of keratinocytes throughout the different layers of the epidermis and in reconstituted skin as early as the two to three cell layer stage. The 90 kiloDalton (kD) protein is one of the major forms detected in Western blot experiments. MDM-2 is detected in skin reconstituted from keratinocytes in which p53 is inactivated by mutation or degradation by E6 protein, providing evidence that MDM-2 expression in the skin can occur in the absence of wild type p53. Moreover, we found no correlation between the p53 status and MDM-2 expression levels in a series of basal and squamous cell carcinomas or Bowen diseases. Our data provide first evidence for the expression of MDM-2 in a differentiated adult tissue.

Blotting, Western↗
PubMed1995

Regulation of pre-mRNA processing by src.

BACKGROUND: Changes in gene expression in response to external signals provide a key mechanisms for the regulation of higher eukaryotic cell functions. The importance of transcriptional control in the response of cells to growth factors and cytokines has been extensively documented, but gene expression has also been shown to be controlled at other levels, such as the stability of mRNA in the cytoplasm, its localization and translation. By contrast to transcriptional control, little is known of the contribution of pre-mRNA nuclear processing to the regulation of gene expression, as most of our knowledge of pre-mRNA processing in vivo is indirect, being inferred from comparisons of transcription rates and levels of mRNA accumulation. RESULTS: In this study, we have used as a model the well-characterized maturation pathway of transcripts of the cytokine, tumour necrosis factor beta (TNF beta). We have used the murine TNF beta gene as a reporter for pre-mRNA processing, using a co-transfection approach to investigate whether overproduction of proteins involved in signal transduction influences the processing of TNF beta transcripts. Although transfection of both activated ras and src genes led to an increase in RNA accumulation in the nuclear and cytoplasmic compartments, as expected from their transactivation of the TNF beta expression vector, only src induced a modification of RNA processing. Comparison of several modes of src activation indicated that two distinct effects of src on pre-mRNA processing can be coupled: one involves slowing down splicing and the other allows the export of partially spliced transcripts. These effects can be observed not only on the three introns of TNF beta but also on transcripts from a beta globin expression vector. DISCUSSION: We have characterized how the processing of transcripts of TNF beta and beta globin is regulated by the signal transduction pathway that includes the Src protein, establishing that external signals have the capacity to regulate gene expression at a post-transcriptional level within the nucleus. Src seems to act on a general mechanism of splicing and/or mRNA transport, but its biologically relevant targets are likely to be restricted to genes for which either alternative processing pathways are in competition, or the kinetics of splicing is critical. This regulation could reflect a modulation by Src of the activity of components of the splicing and transport machineries, but could also involve RNA-binding proteins, which have been shown to interact with Src.

3T3 Cells↗
PubMed1994

Infection with a Kirsten-retrovirus can induce a multiplicity of tumorigenic phenotypes in the interleukin-3-dependent FDC-P1 cells.

The identification of ras oncogenes in both human and animal tumors as well as in preleukemic and precancerous lesions suggests that activated ras genes participate in neoplastic development, yet the precise role of ras oncogenes in leukemogenesis is not clear. To assess the functional role of ras genes in tumorigenesis, we introduced with a retroviral vector either a wild-type (Gly-12) or a mutant (Val-12) Kirsten ras cDNA into the cells of a factor-dependent myeloid cell line, FDC-P1. FDC-P1 cells are nontumorigenic and their proliferation is dependent on either interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The Ki-Val 12-infected FDC-P1 cell population is still strictly IL-3-dependent but has acquired the ability to survive up to 72 hours in the absence of growth factor and to form tumors in nude mice. These tumors are easily established into cell lines that are clonal and show a multiplicity of phenotypes with respect to their growth factor dependence. These results suggest that, in contrast with the overexpression of a normal Ki-ras, Ki-ras oncogene can efficiently promote the tumorigenic conversion of FDC-P1 cells. However, the clonality of the tumors as well as the distinct phenotypes indicates that other genetic events are required for tumorigenicity. Therefore, in FDC-P1 cells, an activated ras gene acts as a dominant oncogene through the induction of tumor progression. Finally, in this simple experimental system we observed a multiplicity of tumorigenic phenotypes which are reminiscent of those observed in patients with acute myeloid leukemia.

Animals↗
PubMed1993

In vivo cooperation between introns during pre-mRNA processing.

In higher eukaryotes the large number of introns present in most genes implies that the pre-mRNA processing machinery should be efficient and accurate. Although this could be achieved at the level of each intron, an attractive alternative would be that interactions between introns improve the performance of this machinery. In this study we tested this hypothesis by comparing the processing of transcripts of the tumor necrosis factor beta gene, which differ only by their number of introns. We took advantage of the ordered splicing of the three introns present in this gene to design constructs that should generate, as primary transcripts, molecules that are normally produced by splicing. We established that the apparent splicing rate of intron 3 is increased 2.5- and 3.5-fold by the presence of one or two other introns on the primary transcript, respectively. Similarly, the apparent splicing rate of intron 2 is increased by the presence of intron 1. As these effects involve the splice sites of the upstream intron, these observations support the existence of cooperative interactions between introns during pre-mRNA processing.

3T3 Cells↗
PubMed1989

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.

Animals↗
PubMed1985

Density of granulomonocytic colony-forming cells (GM-CFC's) in myelofibrosis.

The circulating GM-CFC density-distribution profile was studied in 19 cases of myelofibrosis with myeloid metaplasia. The distribution profile for the population studied appeared to be similar to that of normal bone marrow GM-CFC's but shifted toward lower densities in comparison with normal circulating GM-CFC's. For individual patients, it appeared that the greater the circulating blood GM-CFC's concentration, the lower the mean density. It appears likely that the premature release into blood of the GM-CFC's in myelofibrosis is a property of the neoplastic haematopoietic stem cell clone at the core of the disease.

Blood Cell Count↗
PubMed1985

Human granulocyte colony growth: differences between serum-free and serum-dependent cultures.

Human granulocyte colony formation has been observed in serum-free methylcellulose cultures with Iscove medium, delipidated bovine serum albumin, iron-saturated transferrin, alpha-thioglycerol, oleylpalmitoyl lecithin, cholesterol, and linoleic acid using serum-free human placental-conditioned medium (SF-HPCM) as the source of colony stimulating factor (CSF). Dose-response curves for SF-HPCM indicated a lower sensitivity to colony-stimulating activity in serum-free cultures than in serum-dependent cultures. Gel filtration of SF-HPCM revealed that CSF fractions with molecular weights in the range of 30 kD are inefficient in serum-free cultures, while fractions with molecular weights in the range of 40 kD stimulate granulocyte colony formation in both types of cultures. These results demonstrate that serum constituents modulate the effects of one of the stimulating factors for granulocyte colony formation, and that serum-free culture conditions are essential for establishing the growth factor requirements of the granulocyte lineage.

Cells, Cultured↗
PubMed1984

Effect of alkylating agents on hematopoiesis in myelofibrosis. 4 case report.

Four patients presenting with myelofibrosis (2 primary myelofibrosis and 2 postpolycythemic myeloid metaplasia) were treated with alkylating agents. For three patients (one treated with busulfan and two treated with chlorambucil) the treatment was a success: the general condition improved, the splenomegaly decreased or disappeared, and the blood picture returned to normal. Moreover, for two cases, a trend towards polycythemia was observed under treatment. For the fourth patient, treated with chlorambucil, there was no improvement: a life-threatening, pancytopenic phase developed at the end of the treatment, but it disappeared after 2 months. For the three successfully treated cases, a redistribution of hematopoiesis from spleen to bone marrow was shown by ferrokinetics, 59Fe scans, and bone marrow biopsies. In addition, in the case treated with busulfan, a decrease in the bone marrow granulopoietic pool at the expense of the erythropoietic one was observed. No redistribution was seen in the patient for whom the treatment was a failure. In this case, the spleen remained the major site of active hematopoiesis. Studies on blood granulomonocytic-colony forming cells (GM-CFC's) helped to discriminate the successfully treated patients from the unsuccessfully treated one. In the successfully treated patients, the GM-CFC concentrations dropped to normal values and increased again within weeks following the treatment interruption; this increase involved mainly high density GM-CFC's (greater than 1.060). In the unsuccessfully treated patient, GM-CFC concentrations decreased only after 5 weeks of intensive treatment. The mean density of the GM-CFC's was 1.064 before treatment, shifted towards 1.060 during the neutropenic phase and returned to 1.064 during the recovery.

Alkylating Agents↗
PubMed1984

Redistribution of granulopoiesis from extramedullary territories to bone marrow in a case of spent polycythemia treated with alkylating agents.

We report a case of splenectomized spent polycythemia, where under the influence of alkylating agents (busulfan or chlorambucil), a true redistribution of granulopoiesis appeared. Before treatment, granulopoiesis was confined to the liver (within the portal spaces and the sinusoids), whereas bone marrow was depleted of any hematopoiesis, being the site of a mutilating fibrosis; the blood concentration of granulomonocytic colony-forming cells (GM-CFCs) was high. After a year of discontinuous treatment an intense granulopoiesis was present in bone marrow, comprising more than 90% of the hematopoietic cells, whereas circulating colony-forming cells were low to nil. This phenomenon was long-lasting, since after another year of treatment, a third bone marrow biopsy revealed the persistence of an active granulopoiesis. During the last year of treatment, the treatment was continuous with chlorambucil. After 6 months a decrease in polymorph and platelet values appeared and treatment had to be interrupted. Death supervened one month after treatment interruption. Histological examination revealed the lack of any intrahepatic hematopoiesis whereas bone marrow granulopoiesis was active but presented gross maturation abnormalities. The results obtained in this case permit discussion of some aspects of the physiopathology of myelofibrosis, and particularly the genesis of extramedullary hematopoiesis in this clonal disease.

Biopsy↗
PubMed1979

Enrichment of murine granulomonocytic progenitors using a continuous linear Ficoll-Isopaque gradient.

Murine bone marrow was fractionated using a Ficoll-Isopaque continuous linear gradient characterized by an osmolarity of 290 mOsm constant throughout the gradient and a physiological pH of 7.4. The cellular peak detected prior to fractionation by means of a 405 nm light beam served as a guide for determining fraction collection. Under these conditions CFC enrichment was observed constantly for densities lower than that of the cellular peak. In 17 experiments the enrichment factor amounted to 3.6 +/- 1.4. Enrichment appeared to be due to both an increase in CFC concentration and an improvement in CFC cloning efficiency. A correlation between the concentration of CFC and that of undifferentiated blasts was observed. The CFC density distribution was dependent on the cell load. For cell loads lower than 25 x 10(6) the modal density of CFC was within the range 1.0615-1.0715. For cell loads higher than 25 x 10(6) there was a shift of the distribution curve towards higher density. Clu-CFC appeared to be denser than CFC. This increase in density may be due to a maturation process from CFC to Clu-FC as a maturation gradient with increasing density was found for the granulocytic series and for erythroblasts.

Animals↗