Biomedical subjects

Saverio Minucci

Publications and source records attributed to Saverio Minucci.

25 records · Page 2Linked to original sources

PubMed2002

Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to arsenic trioxide.

Arsenic trioxide induces differentiation and apoptosis of malignant cells in vitro and in vivo, but the mechanisms by which such effects occur have not been elucidated. In the present study we provide evidence that arsenic trioxide induces activation of the small G-protein Rac1 and the alpha and beta isoforms of the p38 mitogen-activated protein (MAP) kinase in several leukemia cell lines. Such activation of Rac1 and p38-isoforms results in downstream engagement of the MAP kinase-activated protein kinase-2 and is enhanced by pre-treatment of cells with ascorbic acid. Interestingly, pharmacological inhibition of p38 potentiates arsenic-dependent apoptosis and suppression of growth of leukemia cell lines, suggesting that this signaling cascade negatively regulates induction of antileukemic responses by arsenic trioxide. Consistent with this, overexpression of a dominant-negative p38 mutant (p38betaAGF) enhances the antiproliferative effects of arsenic trioxide on target cells. To further define the relevance of activation of the Rac1/p38 MAP kinase pathway in the induction of arsenic-dependent antileukemic effects, studies were performed using bone marrows from patients with chronic myelogenous leukemia. Arsenic trioxide suppressed the growth of leukemic myeloid (CFU-GM) progenitors from such patients, whereas concomitant pharmacological inhibition of the p38 pathway enhanced its growth-suppressive effects. Altogether, these data provide evidence for a novel function of the p38 MAP kinase pathway, acting as a negative regulator of arsenic trioxide-induced apoptosis and inhibition of malignant cell growth.

Antioxidants↗

PubMed2002

A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis.

Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not influenced by p66Shc expression. In basal conditions, p66Shc-/- and p53-/- cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2002

Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence.

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.

Animals↗

PubMed2002

PML NBs associate with the hMre11 complex and p53 at sites of irradiation induced DNA damage.

PML nuclear bodies (PML NBs) respond to many cellular stresses including viral infection, heat shock, arsenic and oncogenes and have been implicated in the regulation of p53-dependent replicative senescence and apoptosis. Recently, the hMre11/Rad50/NBS1 repair complex, involved in Double Strand Breaks (DSBs) repair, was found to colocalize within PML NBs, suggesting a role for these nuclear sub-domains in the DNA repair signalling pathway. We report here that in normal human fibroblasts, after ionizing radiation (IR), the PML NBs are modified and recognize sites of DNA breaks (ssDNA breaks and DSBs). Eight to 12 h after radiation PML NBs associate with hMre11 Ionizing Radiation-Induced Foci (IRIF), and subsequently with p53 within discrete foci. The PML, hMre11 and p53 colocalizing structures mark sites of DSBs as identified by immunolocalization with anti phosphorylated histone gamma-H2AX. Furthermore, we demonstrate that ionizing radiation induces the stable association of p53 with hMre11 and PML. These results suggest that the PML NBs are involved in the recognition and/or processing of DNA breaks and possibly in the recruitment of proteins (p53 and hMre11) required for both checkpoint and DNA-repair responses.

Cells, Cultured↗

PubMed2002

Targeting protein inactivation through an oligomerization chain reaction.

A general strategy for inactivation of target proteins is presented, which we have termed "oligomerization chain reaction." This technique is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally. Oligomerization through the CC region of promyelocytic leukemia, and through the natural self-associating domain, triggers the oligomerization chain reaction, leading to formation of large molecular weight complexes and functional inactivation of the target. As a test case, we have chosen the oncosuppressor p53, naturally occurring as a tetramer. Fusion of the CC to p53 leads to formation of stable high molecular weight complexes-as shown by size exclusion chromatography-to which wild-type p53 is recruited with high efficiency. CC-p53 chimeras delocalize wild-type p53 to the cytoplasm and inhibit its transcriptional regulatory properties, resulting in a loss of p53 function. We propose that this strategy may be of general application to self-associating factors and represent a complementary approach to currently used functional inactivation-based strategies.

3T3 Cells↗

PubMed2002

Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor.

DNA methylation of tumor suppressor genes is a frequent mechanism of transcriptional silencing in cancer. The molecular mechanisms underlying the specificity of methylation are unknown. We report here that the leukemia-promoting PML-RAR fusion protein induces gene hypermethylation and silencing by recruiting DNA methyltransferases to target promoters and that hypermethylation contributes to its leukemogenic potential. Retinoic acid treatment induces promoter demethylation, gene reexpression, and reversion of the transformed phenotype. These results establish a mechanistic link between genetic and epigenetic changes during transformation and suggest that hypermethylation contributes to the early steps of carcinogenesis.

Azacitidine↗

PubMed2002

In vitro and in vivo hematopoietic potential of human stem cells residing in muscle tissue.

OBJECTIVE: We studied the in vitro and in vivo hematopoietic potential of human stem cells residing in muscle tissue collected from adults with head and neck cancer. MATERIALS AND METHODS: Adherent muscle cells were cultured in F12 medium with 10% fetal bovine serum and transplanted into immunodeficient mice. RESULTS: On day 12 we obtained a median of 500,000 adherent cells per gram muscle sample. Thy-1, endoglin, HER2/neu, and P1H12 were expressed in the majority of cells. CD34, VEGFR2, c-kit, VCAM-1, and CXCR4 were expressed in 0.5-1.5%, 1-5%, 1-15%, 9-15%, and 30% of cells, respectively. Immunodeficient mice transplanted with fresh muscle cells or less than 500,000 cultured cells showed little or no human engraftment. In mice transplanted with more than 500,000 cultured cells, up to 14% human CD45(+) hematopoietic cells (including myeloid and lymphoid subsets) were detected by flow cytometry. Engraftment was confirmed by polymerase chain reaction, Southern blotting, and DNA sequencing. Liver, muscle, and spleen evaluated for human DNA were positive in the majority of mice showing hematopoietic engraftment in the bone marrow. In vivo hematopoietic engraftment potential was maintained in cultured CD45(-) muscle cells transduced with the green fluorescence protein gene. CONCLUSIONS: Human stem cells residing in muscle tissue can generate multilineage hematopoiesis in immunodeficient mice. Surprisingly, this hematopoietic potential increased in cultured versus fresh cells from muscle tissue.

Adult↗