Diana Teti

Estrogens and Progesterone Promote Persistent CCND1 Gene Activation during G1 by Inducing Transcriptional Derepression via c-Jun/c-Fos/Estrogen Receptor (Progesterone Receptor) Complex Assembly to a Distal Regulatory Element and Recruitment of Cyclin D1 to Its Own Gene Promoter

ABSTRACT Transcriptional activation of the cyclin D1 gene (CCND1) plays a pivotal role in G1-phase progression, which is thereby controlled by multiple regulatory factors, including nuclear receptors (NRs). Appropriate CCND1 gene activity is essential for normal development and physiology of the mammary gland, where it is regulated by ovarian steroids through a mechanism(s) that is not fully elucidated. We report here that CCND1 promoter activation by estrogens in human breast cancer cells is mediated by recruitment of a c-Jun/c-Fos/estrogen receptor α complex to the tetradecanoyl phorbol acetate-responsive element of the gene, together with Oct-1 to a site immediately adjacent. This process coincides with the release from the same DNA region of a transcriptional repressor complex including Yin-Yang 1 (YY1) and histone deacetylase 1 and is sufficient to induce the assembly of the basal transcription machinery on the promoter and to lead to initial cyclin D1 accumulation in the cell. Later on in estrogen stimulation, the cyclin D1/Cdk4 holoenzyme associates with the CCND1 promoter, where E2F and pRb can also be found, contributing to the long-lasting gene enhancement required to drive G1-phase completion. Interestingly, progesterone triggers similar regulatory events through its own NRs, suggesting that the gene regulation cascade described here represents a crossroad for the transcriptional control of G1-phase progression by different classes of NRs.

Analysis of polyamines as markers of (patho)physiological conditions

The aliphatic polyamines, putrescine, spermidine and spermine, are normal cell constituents that play important roles in cell proliferation and differentiation. The equilibrium between cellular uptake and release and the balanced activities of biosynthetic and catabolic enzymes of polyamines are essential for normal homeostasis in the proliferation and functions of cells and tissues. However, the intracellular polyamine content increases in hyperplastic or neoplastic growth. Although the involvement of polyamines in physiological and pathological cell proliferation and differentiation has been well established, the role they play is quite different in relation to cell systems and animal models and is dependent on inducer agents and stimuli. Also, the experimental procedures used to deplete polyamines have been shown to influence the cell responses. In this paper, the assay methods currently in use for polyamines are reviewed and compared with respect to sensitivity, reproducibility and applicability to routine analysis. The relevance of polyamine metabolism and the uptake/release process in many physiological and pathological processes is highlighted, and the cellular polyamine pathways are discussed in relation to the possible diagnostic and therapeutic significance of these mediators.

Alemtuzumab therapy for refractory idiopathic hypereosinophilic syndrome with abnormal T cells: a case report.

A diagnosis of idiopathic hypereosinophilic syndrome was made in a 68-year-old woman in 2002. She was treated with hydroxycarbamide, interferon-a and imatinib (400 mg/d) without evidence of response. In November 2003 she was admitted to our department with anaemia, fever and several painful pruritic skin lesions (top). Immunophenotypic analysis of circulating lymphocytes showed an abnormal CD4 subset characterized by absent surface expression of the CD3 antigen (abnormal T cells have a CD3CD4 CD8 phenotype). Serum interleukin-5 (IL-5) concentration was 280 pg/mol (normal 10 pg/mol). Twodimensional Doppler echocardiography showed endocardial thickening; the left ventricular ejection fraction (LVEF) was 48%. A skin biopsy demonstrated a dermal infiltrate consisting primarily of eosinophils (bottom left). A diagnostic work-up including radiological evaluation failed to reveal an overt lymphoproliferative disorder. As a consequence of the previous demonstration that CD52 is expressed on human eosinophils, the patient was administered subcutaneous alemtuzumab in increasing doses of up to 30 mg weekly. Once commenced on alemtuzumab, her pyrexia settled and the cutaneous lesions started to resolve (bottom right), the LVEF improved to 61% and serum IL-5 concentration fell to 9 pg/mol. Currently, 6 months after the revised diagnosis, the patient is well with an eosinophil count of 0.3 · 10/l on alemtuzumab (30 mg) every 3 weeks.

Mitogen-Activated Protein Kinases and NF-κB Are Involved in TNF-α Responses to Group B Streptococci

TNF-α is a mediator of lethality in experimental infections by group B streptococcus (GBS), an important human pathogen. Little is known of signal transduction pathways involved in GBS-induced TNF-α production. Here we investigate the role of mitogen-activated protein kinases (MAPKs) and NF-κB in TNF-α production by human monocytes stimulated with GBS or LPS, used as a positive control. Western blot analysis of cell lysates indicates that extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun N-terminal kinase MAPKs, as well as IκBα, became phosphorylated, and hence activated, in both LPS- and GBS-stimulated monocytes. The kinetics of these phosphorylation events, as well as those of TNF-α production, were delayed by 30–60 min in GBS-stimulated, relative to LPS-stimulated, monocytes. Selective inhibitors of ERK 1/2 (PD98059 or U0126), p38 (SB203580), or NF-κB (caffeic acid phenetyl ester (CAPE)) could all significantly reduce TNF-α production, although none of the inhibitors used alone was able to completely prevent TNF-α release. However, this was completely blocked by combinations of the inhibitors, including PD98059-SB203580, PD98059-CAPE, or SB203580-CAPE combinations, in both LPS- and GBS-stimulated monocytes. In conclusion, our data indicate that the simultaneous activation of multiple pathways, including NF-κB, ERK 1/2, and p38 MAPKs, is required to induce maximal TNF-α production. Accordingly, in septic shock caused by either GBS or Gram-negative bacteria, complete inhibition of TNF-α release may require treatment with drugs or drug combinations capable of inhibiting multiple activation pathways.

Proinflammatory Gene Expression at Chronic Periodontitis and Peri-Implantitis Sites in Patients With or Without Type 2 Diabetes

BACKGROUND Diabetes and periodontal diseases are often associated. Both have highly inflammatory components, but the role played by distinct phlogistic mediators in their pathogenesis is not fully understood and remains controversial. The purpose of this study is to evaluate whether type 2 diabetes alters the expression of inflammatory mediators in sites with chronic periodontitis (CP) or peri-implantitis (P-IM). METHODS The expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and -8, and monocyte chemotactic protein (MCP)-1 plus key CC chemokine receptors (CCR1 through 5) and CXC chemokine receptors (CXCR1 through 3) was quantified by real-time polymerase chain reaction (PCR) in gingival or peri-implant biopsies from 135 patients with well-controlled or poorly controlled diabetes and periodontal disease, 65 patients with periodontal disease but otherwise healthy, and 90 systematically and periodontally healthy subjects. Western blots were performed. RESULTS Relative to controls, in patients without diabetes and patients with well-controlled diabetes, TNF-alpha, CCR5, and CXCR3 expression was exclusively higher in sites with P-IM (P <0.01), whereas IL-6 and -8 were overexpressed in sites with CP and, even more, in sites with P-IM (P <0.01). In patients with poor glycemic control, TNF-alpha, CCR5, and CXCR3 mRNAs were increased in sites with CP (P <0.01). A statistically significant higher IL-6 and -8 expression from patients without diabetes and patients with well-controlled diabetes was observed compared to patients with poorly controlled diabetes. Regardless of metabolic/glycemic status, MCP-1 and CCR2 and 4 were markedly higher in both of the oral pathologies examined (P <0.01). At the protein levels, Western blot experiments confirmed the real-time PCR results. CONCLUSIONS These findings showed that: 1) in subjects without diabetes and patients with well-controlled diabetes, TNF-alpha, CCR5, and CXCR3 may constitute distinctive biomarkers of P-IM; 2) poor glycemic control abolished the differences between CP and P-IM regarding the expression of these mediators; and 3) type 2 diabetes affected the expression of TNF-alpha, IL-6 and -8, CCR5, and CXCR3.

Prostaglandin E2 induces interleukin-8 gene transcription by activating C/EBP homologous protein in human T lymphocytes

The effect of prostaglandin E2 (PGE2) in regulating the synthesis of the pro-inflammatory chemokine inter-leukin-8 (IL-8) in T lymphocytes is not yet defined, even though it may reduce or enhance IL-8 synthesis in other cell types. Here, we demonstrate that, in human T cells, PGE2 induced IL-8 mRNA transcription through prostaglandin E2 receptors 1- and 4-dependent signal transduction pathways leading to the activation of the transcription factor C/EBP homologous protein (CHOP), never before implicated in IL-8 transcription. Several kinases, including protein kinase C, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and p38 MAPK, were involved in PGE2-induced CHOP activation and IL-8 production. The transactivation of the IL-8 promoter by CHOP was NF-κB-independent. Our data suggest that PGE2 acts as a potent pro-inflammatory mediator by inducing IL-8 gene transcription in activated T cells through different signal transduction pathways leading to CHOP activation. These findings show the complexity with which PGE2 regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions.

Serum levels of interleukin 1β, interleukin 8 and tumour necrosis factor α as markers of gastric cancer

Abstract Despite the efforts made, a serum marker reliable for the screening and follow-up of patients with gastric cancer has not yet been identified. The aim of this preliminary study was to test the role of pro-inflammatory cytokines interleukin 1β, interleukin 8 and tumour necrosis factor α in patients with gastric cancer and in control groups. The statistical analysis of cytokines serum levels in the group with gastric cancer versus control groups has shown considerable differences (p<0.001) in their mean rates. The results indicate that the cytokines interleukin 1β, interleukin 8 and tumour necrosis factor α might perhaps act as diagnostic markers in patients with gastric cancer. Therefore, it is hypothesized that after more extended trials, their use in the screening and prognostic assessment of these patients could be a possibility.

HDACs class II-selective inhibition alters nuclear receptor-dependent differentiation

Epigenetic deregulation contributes to diseases including cancer, neurodegeneration, osteodystrophy, cardiovascular defects, and obesity. For this reason, several inhibitors for histone deacetylases (HDACs) are being validated as novel anti-cancer drugs in clinical studies and display important anti-proliferative activities. While most inhibitors act on both class I, II, and IV HDACs, evidence is accumulating that class I is directly involved in regulation of cell growth and death, whereas class II members regulate differentiation processes, such as muscle and neuronal differentiation. Here, we show that the novel class II-selective inhibitor MC1568 interferes with the RAR- and peroxisome proliferator-activated receptor γ (PPARγ)-mediated differentiation-inducing signaling pathways. In F9 cells, this inhibitor specifically blocks endodermal differentiation despite not affecting retinoic acid-induced maturation of promyelocytic NB4 cells. In 3T3-L1 cells, MC1568 attenuates PPARγ-induced adipogenesis, while the class I-selective MS275 blocked adipogenesis completely thus revealing a different mode of action and/or target profile of the two classes of HDACs. Using in vivo reporting PPRE-Luc mice, we find that MC1568 impairs PPARγ signaling mostly in the heart and adipose tissues. These results illustrate how HDAC functions can be dissected by selective inhibitors.

FOXE1 is a target for aberrant methylation in cutaneous squamous cell carcinoma.

Background  Several cancer‐related genes are silenced by promoter hypermethylation in skin cancers. However, to date the somatic epigenetic events that occur in cutaneous squamous cell carcinoma (SCC) tumorigenesis have not been well defined.

Synergistic effect of peroxisome proliferator activated receptor-gamma and liver X receptor-alpha in the regulation of inflammation in macrophages.

ABSTRACT Peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) and liver X receptor-&agr; (LXR&agr;) are nuclear ligand-activated transcription factors, which regulate lipid metabolism and inflammation. Murine J774.2 macrophages were stimulated with Escherichia coli lipopolysaccharide (concentration, 10 &mgr;g/mL) with or without the PPAR&ggr; ligand, 15-deoxy-&Dgr;12,14 prostaglandin J2 (15d-PGJ2), or the LXR&agr; ligands, 22(R)-hydroxycholesterol and T0901317 (concentration range, 0.01-10 &mgr;mol/L), alone or in combination. Nitric oxide (NO) metabolites and tumor necrosis factor &agr; production, inducible NO synthase expression, and mitochondrial respiration were measured. When added to the cells as single agents, 15d-PGJ2, 22(R)-hydroxycholesterol, or T0901317 reduced the lipopolysaccharide-induced NO and tumor necrosis factor &agr; production and the inducible NO synthase expression, and partially maintained mitochondrial respiration in a concentration-dependent manner. When added to the cells in combination at suboptimal concentrations, 15d-PGJ2 with 22(R)-hydroxycholesterol, or 15d-PGJ2 with T0901317, exerted anti-inflammatory effects similar to much higher concentrations (10,000-fold to 100,000-fold) of each ligand alone. The anti-inflammatory effects of these ligands, alone or in combination, were associated with reduction of nuclear factor-&kgr;B activation and with enhancement of PPAR&ggr; DNA binding. LXR&agr; expression was upregulated in response to 15d-PGJ2 and to the LXR&agr; ligands when added alone or in combination. Immunoprecipitation experiments revealed that PPAR&ggr; interacted with LXR&agr;. Our data demonstrate that the PPAR&ggr; ligand, 15d-PGJ2, and the LXR&agr; ligands, 22(R)-hydroxycholesterol and T0901317, although binding to different nuclear receptors (i.e., PPAR&ggr; and LXR&agr;, respectively), affect mediator production through common cell signaling events and exert a synergistic potentiation in a combined treatment at suboptimal concentrations. Thus, our data suggest that PPAR&ggr; and LXR&agr; may interact in controlling the inflammatory response in macrophages.