Biomedical subjects

Simona Ronzoni

Publications and source records attributed to Simona Ronzoni.

8 recordsLinked to original sources

PubMed2006

Recruitment of the histone methyltransferase SUV39H1 and its role in the oncogenic properties of the leukemia-associated PML-retinoic acid receptor fusion protein.

Leukemia-associated fusion proteins establish aberrant transcriptional programs, which result in the block of hematopoietic differentiation, a prominent feature of the leukemic phenotype. The dissection of the mechanisms of deregulated transcription by leukemia fusion proteins is therefore critical for the design of tailored antileukemic strategies, aimed at reestablishing the differentiation program of leukemic cells. The acute promyelocytic leukemia (APL)-associated fusion protein PML-retinoic acid receptor (RAR) behaves as an aberrant transcriptional repressor, due to its ability to induce chromatin modifications (histone deacetylation and DNA methylation) and silencing of PML-RAR target genes. Here, we indicate that the ultimate result of PML-RAR action is to impose a heterochromatin-like structure on its target genes, thereby establishing a permanent transcriptional silencing. This effect is mediated by the previously described association of PML-RAR with chromatin-modifying enzymes (histone deacetylases and DNA methyltransferases) and by recruitment of the histone methyltransferase SUV39H1, responsible for trimethylation of lysine 9 of histone H3.

Cell Differentiation↗

PubMed2005

New method to detect histone acetylation levels by flow cytometry.

BACKGROUND: Reversible histone acetylation affects chromatin structural organization, thus regulating gene expression and other nuclear events. Levels of histone acetylation are tightly modulated in normal cells, and alterations of their regulating mechanisms have been shown to be involved in tumorigenesis. METHODS: We developed a new flow cytometric technique for detection of histone acetylation, based on a specific monoclonal antibody that recognizes acetylated histone tails. Bivariate analysis for histone acetylation levels and DNA were performed to study modulation of chromatin organization during the cell cycle and after induction of histone hyperacetylation by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Histone acetylation and transcription levels were monitored during differentiation induced by retinoic acid alone or in combination with TSA. Blood samples from patients were analyzed with the described protocol to monitor the effects of HDAC inhibitors in vivo and validate the developed protocol for clinical usage. RESULTS: Flow cytometric detection of acetylation status can successfully detect modifications induced by HDAC inhibitor treatment in vivo as demonstrated by analysis of various blood samples from patients treated with valproic acid. Changes in acetylation levels during the cell cycle demonstrated a reproducible increase in histone acetylation during the replication phase that was subsequently decreased at the G2M entrance, thus paralleling the behavior of DNA replication and transcriptional activity. CONCLUSIONS: Multiparameter analysis of histone acetylation and expression of molecular markers, DNA ploidy, and/or cell cycle kinetics can provide a quick and statistically reliable tool for the diagnosis and evaluation of treatment efficacy in clinical trials using HDAC inhibitors.

Acetylation↗

PubMed2004

Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway.

Histone deacetylases (HDACs) regulate transcription and specific cellular functions, such as tumor suppression by p53, and are frequently altered in cancer. Inhibitors of HDACs (HDACIs) possess antitumor activity and are well tolerated, supporting the idea that their use might develop as a specific strategy for cancer treatment. The molecular basis for their selective antitumor activity is, however, unknown. We investigated the effects of HDACIs on leukemias expressing the PML-RAR or AML1-ETO oncoproteins, known to initiate leukemogenesis through deregulation of HDACs. Here we report that: (i) HDACIs induce apoptosis of leukemic blasts, although oncogene expression is not sufficient to confer HDACI sensitivity to normal cells; (ii) apoptosis is p53 independent and depends, both in vitro and in vivo, upon activation of the death receptor pathway (TRAIL and Fas signaling pathways); (iii) TRAIL, DR5, FasL and Fas are upregulated by HDACIs in the leukemic cells, but not in normal hematopoietic progenitors. These results show that sensitivity to HDACIs in leukemias is a property of the fully transformed phenotype and depends on activation of a specific death pathway.

Animals↗

PubMed2004

Impairment of p53 acetylation, stability and function by an oncogenic transcription factor.

Mutations of p53 are remarkably rare in acute promyelocytic leukemias (APLs). Here, we demonstrate that the APL-associated fusion proteins PML-RAR and PLZF-RAR directly inhibit p53, allowing leukemic blasts to evade p53-dependent cancer surveillance pathways. PML-RAR causes deacetylation and degradation of p53, resulting in repression of p53 transcriptional activity, and protection from p53-dependent responses to genotoxic stress. These phenomena are dependent on the expression of wild-type PML, acting as a bridge between p53 and PML-RAR. Recruitment of histone deacetylase (HDAC) to p53 and inhibition of p53 activity were abrogated by conditions that either inactivate HDACs or trigger HDAC release from the fusion protein, implicating recruitment of HDAC by PML-RAR as the mechanism underlying p53 inhibition.

Acetylation↗

PubMed2003

Lipopolysaccharide or whole bacteria block the conversion of inflammatory monocytes into dendritic cells in vivo.

Monocytes can develop into dendritic cells (DCs) that migrate to lymph nodes (LNs) and present antigens to T cells. However, we find that this differentiation is blocked when monocytes accumulate subcutaneously in response to bacteria or lipopolysaccharide (LPS). The inhibition of DC differentiation is mediated by the bacteria and in conjunction with inflammatory cells recruited at the site of injection. Inhibition of migratory DC development was reversed in Toll-like receptor (TLR)4-mutated mice when LPS, but not whole bacteria, was injected, suggesting that TLR4 is one but not the only mediator of the inhibition. The block imposed by bacteria was partly relieved by the absence of interleukin (IL)-12 p40, but not by individual absence of several cytokines involved in DC differentiation or in inflammation, i.e., IL-6, IL-10, IL-12 p35, and interferon gamma. Consistent with the inability of monocytes to yield migrating DCs, and the finding that other DCs had limited access to particulate or bacterial antigens, these antigens were weakly presented to T cells in the draining LN. These results illustrate that bacteria-associated signals can have a negative regulatory role on adaptive immunity and that local innate responses for containment of infectious bacteria can at least initially supersede development of adaptive responses.

Animals↗

PubMed2003

Kit regulatory elements required for expression in developing hematopoietic and germ cell lineages.

The Kit (White) gene encodes the transmembrane receptor of stem cell factor/Kit ligand (KL) and is essential for the normal development/maintenance of pluripotent primordial germ cells (PGCs), hematopoietic stem cells (HSCs), melanoblasts, and some of their descendants. The molecular basis for the transcriptional regulation of Kit during development of these important cell types is unknown. We investigated Kit regulation in hematopoietic cells and PGCs. We identified 6 DNase I hypersensitive sites (HS1-HS6) within the promoter and first intron of the mouse Kit gene and developed mouse lines expressing transgenic green fluorescent protein (GFP) under the control of these regulatory elements. A construct driven by the Kit promoter and including all 6 HS sites is highly expressed during mouse development in Kit+ cells including PGCs and hematopoietic progenitors (erythroid blast-forming units and mixed colony-forming units). In contrast, the Kit promoter alone (comprising HS1) is sufficient to drive low-level GFP expression in PGCs, but unable to function in hematopoietic cells. Hematopoietic expression further requires the addition of the intronproximal HS2 fragment; HS2 also greatly potentiates the activity in PGCs. Thus, HS2 acts as an enhancer integrating transcriptional signals common to 2 developmentally unrelated stem cell/progenitor lineages. Optimal hematopoietic expression further requires HS3-HS6.

Animals↗

PubMed2002

PML-RAR induces promyelocytic leukemias with high efficiency following retroviral gene transfer into purified murine hematopoietic progenitors.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations resulting in fusion proteins of the retinoic acid receptor (RAR). Here, we report a novel murine model system for APL, based on the transduction of purified murine hematopoietic progenitors (lin(-)) using high-titer retroviral vectors encoding promyelocytic leukemia-RAR (PML-RAR), and the green fluorescent protein (GFP) as a marker. PML-RAR-expressing lin(-) cells were impaired in their ability to undergo terminal myeloid differentiation and showed increased proliferative potential in vitro. Inoculation of transduced lin(-) cells into syngeneic, irradiated mice resulted in the development of retinoic acid-sensitive promyelocytic leukemias at high frequency (> 80%) and short latency (approximately 4 months). Morphologic and immunophenotypic analysis revealed no gross abnormalities of the preleukemic bone marrows. However, hematopoietic progenitors from PML-RAR preleukemic mice showed a severe impairment in their ability to undergo myeloid differentiation in vitro. This result, together with the monoclonality or oligoclonality of the leukemic blasts, supports a "multiple-hit" model, where the fusion protein causes a "preleukemic" phase, and leukemia occurs after additional genetic lesions. This model system faithfully reproduces the main characteristics of human APL and represents a versatile tool for the in vitro and in vivo study of mechanisms of leukemogenesis and the design of protocols for differentiation treatment.

Animals↗

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↗