CDC25A targeting by miR-483-3p decreases CCND-CDK4/6 assembly and contributes to cell cycle arrest.

Disruption of contact inhibition and serum afflux that occur after a tissue injury activate cell cycle, which then stops when confluence is reached again. Although the events involved in cell cycle entry have been widely documented, those managing cell cycle exit have remained so far ill defined. We have identified that the final stage of wound closure is preceded in keratinocytes by a strong accumulation of miR-483-3p, which acts as a mandatory signal triggering cell cycle arrest when confluence is reached. Blocking miR-483-3p accumulation strongly delays cell cycle exit, maintains cells into a proliferative state and retards their differentiation program. Using two models of cell cycle synchronization (i.e. mechanical injury and serum addition), we show that an ectopic upregulation of miR-483-3p blocks cell cycle progression in early G1 phase. This arrest results from a direct targeting of the CDC25A phosphatase by miR-483-3p, which can be impeded using an anti-miRNA against miR-483-3p or a protector that blocks the complex formation between miR-483-3p and the 3'-untranslated region (UTR) of CDC25A transcript. We show that the miRNA-induced silencing of CDC25A increases the tyrosine phosphorylation status of CDK4/6 cyclin-dependent kinases which, in turn, abolishes CDK4/6 capacity to associate with D-type cyclins. This prevents CDK4/6 kinases' activation, impairs downstream events such as cyclin E stimulation and sequesters cells in early G1. We propose this new regulatory process of cyclin-CDK association as a general mechanism coupling miRNA-mediated CDC25A invalidation to CDK post-transcriptional modifications and cell cycle control.

Id3 is a novel regulator of p27kip1 mRNA in early G1 phase and is required for cell-cycle progression.

P27kip is a key inhibitory protein of the cell-cycle progression, which is rapidly downregulated in early G1 phase by a post-translational mechanism involving the proteosomal degradation. In this study, using a wounding model that induces cell-cycle entry of human dermal fibroblasts, we demonstrate that p27mRNA is downregulated when cells progress into the G1 phase, and then it returns to its basal level when cells approach the S phase. By using a quantitative polymerase chain reaction screening we identified inhibitors of differentiation (Id3), a bHLH transcriptional repressor, as a candidate mediator accounting for p27 mRNA decrease. Id3 silencing, using an small interfering RNA approach, reversed the injury mediated p27 downregulation demonstrating that Id3 is involved in the transcriptional repression of p27. Reporter gene experiments and a chromatin immunoprecipitation assay showed that Id3 likely exerts its repressive action through ELK1 inhibition. By inhibiting early p27 downregulation, Id3 depletion blocked (i) the G1-phase progression as assessed by the inhibition of pRb phosphorylation and p130 degradation and (ii) the G1/S transition as observed by the inhibition of cyclin A induction, demonstrating that p27 mRNA decrease is required for cell proliferation. Apart from its effect on the early p27 diminution, Id3 appears also involved in the control of the steady-state level of p27 at the G1/S boundary. In conclusion, this study identifies a novel mechanism of p27 regulation which besides p27 protein degradation also implicates a transcriptional mechanism mediated by Id3.

Increased MAPK signaling during in vitro muscle wounding.

Regeneration of skeletal muscle upon injury is a complex process, involving activation of satellite cells, followed by migration, fusion, and regeneration of damaged myofibers. Previous work concerning the role of the mitogen activated protein (MAP) kinase signaling pathways in muscle injury comes primarily from studies using chemically induced wounding. The purpose of this study was to test the hypothesis that physical injury to skeletal muscle cells in vitro activates the MAP kinase signaling pathways. We demonstrate that extracellular signal regulated kinases (ERKs) 1, 2, and p38 are rapidly and transiently activated in response to injury in C2C12 cells, and are primarily localized to cells adjacent to the wound bed. Culture medium from wounded cells is able to stimulate activation of p38 but not ERK in unwounded cells. These results suggest that both ERK and p38 are involved in the response of muscle cells to physical injury in culture, and reflect what is seen in whole tissues in vivo.

Evidence for a direct correlation between c-Jun NH2 terminal kinase 1 activation, cyclin D2 expression, and G(1)/S phase transition in the murine hybridoma 7TD1 cells.

In this study we show that the addition of fresh culture medium to high-density growth-arrested 7TD1 cells induces a strong and transient stimulation of the c-Jun NH2 terminal kinase activity (Jun kinase/JNK), a marked increase in cyclin D2 expression, the phosphorylation of pRb, and the transition from G(1) to S phase. The stimulation of cyclin D2 expression and the induction of JNK activity appear to be the consequences of the alkalinization of the extracellular medium. Indeed both parameters (i) can be induced, regardless of cell dilution, by the addition of a weak base such as triethylamine, and (ii) are together inhibited by (N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the Na(+)/H(+) exchanger. We provide a strong argument indicating the existence of a direct correlation between JNK1 activation and cyclin D2 stimulation. Indeed, we demonstrate that cyclin D2 expression is blocked by SB 202190, an agent known to inhibit both JNK and p38(MAPK), but not by SB 203580, a specific inhibitor of p38(MAPK). Furthermore, we also observed that DMSO and forskolin, two agents that inhibit the proliferation of 7TD1 cells, inhibit in parallel cyclin D2 and JNK1. Altogether our results suggest that (i) JNK1 participates in the signaling pathway which controls the expression of cyclin D2 and (ii) that the inhibition of JNK1 by DMSO and forskolin could explain, at least in part, the antiproliferative action of these drugs in 7TD1 cells.

Clavicular hypoplasia, zygomatic arch hypoplasia, and micrognathia: a newly defined syndrome.

We report on a 6-year-old boy with a previously undefined syndrome of clavicular hypoplasia, frontonasal malformation, zygomatic arch hypoplasia, micrognathia, and normal intelligence. His condition differs from similar syndromes on the basis of unique facial findings such as microcornea, stellate irises, and a midline maxillary cleft. We present his case, a review of the literature, and propose the acronym CHZAM, for clavicular hypoplasia, zygomatic arch, and micrognathia, to represent this syndrome.

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest.

p27[KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the specific blockade of these cells in early G1 phase reveals the induction of a protein of 23 kDa (p23) specifically recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21[CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a 'caspase-3-like' protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation.

Influenza vaccination among the elderly in Italy.

This article surveys the attitudes and perceptions of a random sample of the elderly population in three regions of Italy on the use and efficacy of influenza vaccine. The data were collected by direct interviews using a standard questionnaire. The results show that vaccination coverage against influenza is inadequate (26-48.6%). The major reasons for nonvaccination were lack of faith in the vaccine and disbelief that influenza is a dangerous illness. These data emphasize the need for a systematic education programme targeted at the elderly and the provision of influenza vaccination, with the increased cooperation of general practitioners.

Chromosome 8, occupational exposures, smoking, and acute nonlymphocytic leukemias: a population-based study.

In a previous epidemiological study on acute myelocytic leukemia (M. M. Crane et al., Cancer Epidemiol. Biomark. Prev., 5: 639-644, 1996), clonal aberrations in chromosome 8 have been reported to be in excess in smokers and in workers exposed to paints. In that study, cytogenetics was performed after therapy. In our report, we describe a population-based survey on nonlymphocytic leukemias in northern Italy, in which 79 patients (acute myelocytic leukemia, myelodysplastic syndromes, or other nonlymphocytic leukemias) were studied before cytotoxic therapy. We found 9 aberrations involving chromosome 8 (six +8, two -8, and one translocation), whereas abnormalities involving chromosomes 5 and 7 occurred with a low frequency compared with previous studies. Aberrations involving chromosome 8 were associated with smoking (odds ratio, 6.3; 95% confidence interval, 0.9-42.3; among smokers of 10 or more cigarettes/day: odds ratio, 14.2; 95% confidence interval, 1.4-142.3); +8 aberrations were found in 1 of 24 nonsmokers and in 5 of 38 smokers. Three +8 aberrations were found in 22 subjects potentially exposed to solvents or polycyclic aromatic hydrocarbons. The low frequency of chromosome 5 and 7 aberrations in our population-based series (compared with other studies) can be attributed to the recruitment before cytotoxic therapies. Aberrations involving chromosome 8 (particularly +8) were associated with smoking habits. Chromosome 8 includes the c-myc oncogene.

Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin D2 inhibition and p21[CIP1] induction.

Dimethylsulfoxide (DMSO) was shown to inhibit the proliferation of several B cell lines including Raji, Daudi, and SKW6-CL4 but the mechanisms involved in this growth arrest are still unclear. We show that in 7TD1 mouse hybridoma cells a DMSO-induced reversible G1 arrest involves inactivation of Rb kinases, cyclin D2/CDK4 and cyclin E/CDK2. This occurs by at least three distinct mechanisms. Inhibition of cyclin D2 neosynthesis leads to a dramatic decrease of cyclinD2/CDK4 complexes. This in turn enables the redistribution of p27[KIP1] from cyclin D2/CDK4 to cyclin E/CDK2 complexes. In addition, the simultaneous accumulation of p21[CIP1] entails increasing association with cyclin D3/CDK4 and cyclin E/CDK2. Thus, p21[CIP1] and p27[KIP1], act in concert to inhibit cyclin E/CDK2 activity which, together with CDK4 inactivation, confers a G1-phase arrest.

Interleukin 6 stimulates a tyrosine kinase activity potentially involved in mouse hybridoma cell growth.

In this report the authors describe the characterization of a cytosolic tyrosine kinase activity (IL-6PTK) stimulated by interleukin 6 (IL-6). IL-6PTK appears 6 h after IL-6 addition and is inhibited by tyrphostin but not genistein. It is active under its phosphorylated form although it is not immunoprecipitated by antiphosphotyrosine antibodies, suggesting that autophosphorylation occurs on residues other than tyrosine. Using the ATP-binding site covalent label, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), two phosphoproteins have been identified of 52 and 59 kDa respectively, that could potentially harbour IL-6PTK activity. The intracellular elevation of cAMP, which inhibits 7TD1 cell proliferation, decreases as the same time IL-6PTK activity suggesting that the cAMP-dependent kinase could act as a negative regulator of this tyrosine kinase species. Taken together the results strongly suggest that a tyrosine kinase (IL-6PTK) might be involved in the cascade of events leading to the proliferation of 7TD1 cells under IL-6 stimulation.

Cyclic AMP stimulates a JunD/Fra-2 AP-1 complex and inhibits the proliferation of interleukin-6-dependent cell lines.

Interleukin-6 (IL-6) is a proinflammatory cytokine which also acts as a growth factor for some murine hybridomas (7TD1) or human myelomas (U266). We demonstrate that elevation of cAMP cellular content inhibits IL-6-stimulated cell growth, by blocking cells mainly in G1 phase. This inhibition is associated with increased expression of the Fos family protein Fra-2. Treatment of cells with 8Br-cAMP results in increased DNA-binding activity of two distinct AP-1 complexes; JunD/Fra-2 and JunB/Fra-2, and also in elevated AP-1 transactivation. When 8Br-cAMP is withdrawn from the medium, cells enter S phase and Fra-2 protein levels and AP-1 DNA-binding activity decrease to their basal value indicating that a temporally correlation exists between the 8Br-cAMP-mediated induction of JunD/Fra-2 AP-1 complex and the 7TD1 and U266 cell growth inhibition.

Two distinct signalling pathways are involved in the control of the biphasic junB transcription induced by interleukin-6 in the B cell hybridoma 7TD1.

We have measured the level of junB mRNA in the B hybridoma cell line 7TD1, under interleukin-6 (IL-6) stimulation. IL-6 increases junB mRNA in a biphasic fashion. The first early-induced peak was transient and likely corresponds to the well documented typical junB mRNA, stimulated in response to numerous growth factors, including IL-6. At variance, the second peak which has never been reported previously, lasted several hours. As a consequence of its effect on junB mRNA, IL-6 stimulated, in a biphasic fashion, the nuclear accumulation of the JunB protein. In this study, we demonstrated that IL-6 regulation occurred exclusively at the transcriptional level and that the bimodal increase of junB mRNA and JunB protein can be accounted for by a biphasic stimulation of junB transcription. Furthermore, our data point to two major differences between the mechanism of control of the early and the late IL-6-induced junB transcription waves. First, cycloheximide strongly potentiated the transcription of the second wave, whereas it failed to affect the early-induced burst. Second, tyrphostin, a tyrosine kinase inhibitor, impaired the expression of the first but not the second junB mRNA peak. Conversely, genistein, another tyrosine kinase inhibitor, totally abolished the expression of the second peak of junB mRNA whereas it did not affect the expression of the first peak. Altogether these data indicate that, in 7TD1 cells, IL-6 controls junB transcription in a biphasic fashion by means of two separate transduction pathways.

Chromosome 22 monosomy in a radiation-induced meningioma.

We report for the first time on the cytogenetics of a radiation-induced meningioma, in a patient without neurofibromatosis. Chromosome 22 monosomy was found in more than 50% of the cells examined. These findings seem to suggest that the same kind of chromosomal aberrations are found in both "idiopathic" and radiation-induced meningiomas and that a common etiopathogenetic mechanism might be at work in meningiomas of whatever origin.

Jurkat T cells express a functional neutral endopeptidase activity (CALLA) involved in T cell activation.

We have characterized a T lymphocyte endopeptidase activity that hydrolyses succinyl-alanine-alanine-phenylalanine-paranitroanilide (Suc-Ala-Ala-Phe-pNa). Hydrolysis of this substrate by intact Jurkat T cells was markedly enhanced when exogenous aminopeptidase N was added to the incubation medium. It thus appears that the release of paranitroaniline from Suc-Ala-Ala-Phe-pNA results from the combination of two distinct enzymatic activities: (i) an endopeptidase activity that cleaves the substrate at the alanyl bond and (ii) an aminopeptidase activity that ultimately cleaves the phenylalanyl bond. This cleavage was further confirmed by HPLC analysis. Specific endopeptidase 24.11 inhibitors were shown to inhibit the endopeptidase activity. These features are reminiscent of the characteristics of neutral endopeptidase (NEP, also known as endopeptidase 24.11, CALLA or CD10). Anti-CD10 monoclonal antibodies (mAbs) recognized the CD10+ B cell line Raji, but not Jurkat cells as assessed by FACS analysis. This is probably due to a lack of sensitivity of this method, the level of NEP activity in Jurkat T cells being 3-5% of that measured in B cell lines. Anti-CD10 mAbs immunoprecipitated endopeptidase 24.11 activities in both Jurkat T cells and Raji B cells, demonstrating that T lymphocytes express a CALLA-related endopeptidase. We also demonstrate that T and B cell endopeptidases have the same molecular weight, that T cells express less functional CALLA mRNA than B cells and that there are at least two shorter transcripts (1.8 and 0.8 kb) in both T and B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

[Pentasomy X: a clinical case report]. / Pentasomia X: descrizione di un caso clinico.

Cytogenetic investigations gave evidence of pentasomy X in a 3-year-old female with typical facies and psychomotor retardation. The parents and the grandparents showed a normal karyotype. The clinical symptoms of our case were compared with the other authors, we found a low birth weight, short stature, delayed expressive language, multiple abnormalities of craniofacial skeleton and some minor deformities of the arts. The parental origin of the extra set of X chromosome were determined by the restriction fragment length analysis (RFLPs) using the very polymorphic probes M27beta, L1.28 and St14. These data support the hypothesis of a maternal meiotic double non-disjunction.