Maria Visalli

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.

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.

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.

MSX1 and TGF-β3 are novel target genes functionally regulated by FOXE1

FOXE1 mutations cause the Bamforth-Lazarus syndrome characterized by thyroid and craniofacial defects. Although a pioneer activity of FOXE1 in thyroid development has been reported, FOXE1 regulation in other contexts remains unexplored. We pointed to: (i) a role of FOXE1 in controlling the expression of MSX1 and TGF-β3 relevant in craniofacial development and (ii) a causative part of FOXE1 mutations or mice Foxe1(-/-) genotype in the pathogenesis of cleft palate in the Bamforth-Lazarus syndrome. The MSX1 and TGF-β3 up-regulation in response to FOXE1 at both transcriptional and translational levels and the recruitment of FOXE1 to specific binding motifs, together with the transactivation of the promoters of these genes, indicate that MSX1 and TGF-β3 are direct FOXE1 targets. Moreover, we showed that all the known forkhead-domain mutations, but not the polyalanine-stretch polymorphisms, affect the FOXE1 ability to bind to and transactivate MSX1 and TGF-β3 promoters. In 14-day Foxe1(-/-) mice embryos, Tgf-β3 and Msx1 mRNAs were almost absent in palatal shelves compared with Foxe1(+/-) embryos. Our findings give new insights into the genetic mechanisms underlying the Bamforth-Lazarus syndrome-associated facial defects.

Class I-specific histone deacetylase inhibitor MS-275 overrides TRAIL-resistance in melanoma cells by downregulating c-FLIP.

Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) has selective killing effect toward malignant cells; however some human melanomas are intrinsically resistant. In this study, we have shown that class I-specific histone deacetylase inhibitor (HDACi) MS-275 can synergize with TRAIL to induce apoptosis in TRAIL-resistant cell lines and to enhance susceptibility of sensitive cells. Conversely, class II-selective HDACi MC1575 has shown no effect on the resistance of melanoma cells and was able exclusively to increase TRAIL-induced cell death in responsive cells. Both the HDACis variably increased DR4, DR5, and procaspase 8 expression, regardless whether cells were TRAIL-sensitive or TRAIL-resistant. However, only MS-275 markedly decreased the expression levels of both the long and short c-FLIP isoforms. RNAi-mediated c-FLIP silencing resulted in caspase 8-dependent apoptosis in survivor cells which was comparable to that observed following MS-275 treatment. Accordingly, enforced expression of ectopic c-FLIP has abolished the cooperative induction of apoptosis by the combination of MS-275 and TRAIL. These data indicate that c-FLIP is a critical regulator of death ligand sensitivity in melanoma. Inhibition of class I HDAC isoenzymes 1, 2 and 3 has resulted to be functionally important for c-FLIP downregulation by MS-275. In contrast, knockdown of class II HDACs has had no effect on c-FLIP expression, thus explaining the dual incapacity of MC1575 to inhibit c-FLIP expression and sensitize cells resistant to TRAIL. The data reported here suggest that MS-275 represents a promising therapeutic approach in combination with TRAIL for treatment of cutaneous and uveal melanoma due to its ability to reduce c-FLIP expression.

Determination of polyamines in human saliva by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines (spermine, spermidine and putrescine) in human saliva was developed. This method is based on pre-column derivatization with o-phthaldialdehyde (OPA). The derivatives were separated on a Nucleosil ODS column (250x4.6 mm I.D.; 5 microm). The gradient elution was performed with two mobile phases A (water) and B (methanol) at a flow rate of 0.8 ml/min. The column eluate was monitored by fluorescence detection (excitation, 360 nm; emission, 510 nm). The within- and between-assay coefficients of variation for all the compounds were below 5%. The detection limits for spermine, spermidine and putrescine were 0.04, 0.05 and 0.06 nmol/ml, respectively. The recovery was greater than 90%. Our analytical technique requires neither preliminary extraction with an organic solvent, nor long multi-step procedures. For saliva samples, this is a simple, rapid and highly reproducible method that can be easily applied to the routine determination of salivary polyamines, whose levels increase early in several pathological conditions.

Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.

Class II-specific histone deacetylase inhibitors MC1568 and MC1575 suppress IL-8 expression in human melanoma cells.

Here, we explored the effects of the novel class II‐specific histone deacetylase inhibitors (HDACis) MC1568 and MC1575 on interleukin‐8 (IL‐8) expression and cell proliferation in cutaneous melanoma cell line GR‐M and uveal melanoma cell line OCM‐3 upon stimulation with phorbol 12‐myristate 13‐acetate (PMA). We found that PMA upregulated IL‐8 transcription via the AP‐1 binding site and identified c‐Jun as the transcription factor involved in this eventS. MC1568 and MC1575 inhibited IL‐8 levels and cell proliferation in either unstimulated or PMA‐stimulated melanoma cells. They acted by suppressing (i) c‐Jun binding to the IL‐8 promoter, (ii) recruitment of histones 3 and 4, RNA polymerase II and TFIIB to the c‐Jun promoter, and (iii) c‐Jun expression. Our findings provide new insights into mechanisms underlying anti‐tumoral activities of class II‐specific HDACis in human melanoma and suggest that they may constitute a novel therapeutic strategy for improving the treatment of this cancer.

Pseudomonas aeruginosa Induces Interleukin-8 (IL-8) Gene Expression in Human Conjunctiva through the Recruitment of Both RelA and CCAAT/Enhancer-binding Protein β to the IL-8 Promoter

The purpose of this study was to identify the Pseudomonas aeruginosa-activated signaling pathway leading to interleukin (IL)-8 gene expression and protein synthesis by human conjunctival epithelium. IL-8 protein and mRNA were determined by enzyme-linked immunosorbent assay and reverse transcription-PCR, respectively. Activation of MAPKs and NF-κB was analyzed by Western blotting using phosphospecific antibodies. We used transfection with wild-type or mutated IL-8 promoters and cotransfection with transcription factor overexpressing plasmids or small interfering RNAs. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) were performed for in vitro and in vivo protein-DNA binding studies, respectively. P. aeruginosa increased IL-8 expression at the transcriptional level by phosphorylating CCAAT/enhancer-binding protein β (C/EBPβ) via p38MAPK and activating NF-κB. The simultaneous involvement of RelA and C/EBPβ and the integrity of the corresponding consensus sites were required, whereas c-Jun was involved only in basal IL-8 expression. Re-ChIP experiments showed that RelA and C/EBPβ act together at the IL-8 promoter level upon P. aeruginosa infection. Taken together, our results suggest that P. aeruginosa induces IL-8 promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBPβ and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription.

Cellular Mechanisms of Oxidative Stress and Action in Melanoma

Most melanomas occur on the skin, but a small percentage of these life-threatening cancers affect other parts of the body, such as the eye and mucous membranes, including the mouth. Given that most melanomas are caused by ultraviolet radiation (UV) exposure, close attention has been paid to the impact of oxidative stress on these tumors. The possibility that key epigenetic enzymes cannot act on a DNA altered by oxidative stress has opened new perspectives. Therefore, much attention has been paid to the alteration of DNA methylation by oxidative stress. We review the current evidence about (i) the role of oxidative stress in melanoma initiation and progression; (ii) the mechanisms by which ROS influence the DNA methylation pattern of transformed melanocytes; (iii) the transformative potential of oxidative stress-induced changes in global and/or local gene methylation and expression; (iv) the employment of this epimutation as a biomarker for melanoma diagnosis, prognosis, and drug resistance evaluation; (v) the impact of this new knowledge in clinical practice for melanoma treatment.