Margherita Annicchiarico-Petruzzelli

Tissue transglutaminase and apoptosis: Sense and antisense transfection studies with human neuroblastoma cells

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.

Cell Death by Oxidative Stress and Ascorbic Acid Regeneration in Human Neuroectodermal Cell Lines

In this paper, we show that human neuroectodermal cells exposed to 1-5 mM hydrogen peroxide or 10 nM-1 mM ascorbate die by programmed cell death induced by oxidative stress. The cell death by peroxide occurs within 4 h and involves approximately 80% of B-mel melanoma cells, while ascorbate causes cell death of approximately 86% of B-mel cells within 24 h. SK-N-BE(2) neuroblastoma cells are more resistant, 32% and 43% cell death for peroxide and ascorbate, respectively. In all cases, cell death causes hypodiploic DNA staining, evaluated by flow cytometry. Both cell lines can efficiently metabolise ascorbate due to significant levels of NADH-dependent semidehydroascorbate reductase and glutathione-dependent dehydroascorbate reductase. The cell death observed suggests a pro-oxidant, rather than anti-oxidant, role for ascorbic acid at physiological concentrations under these experimental conditions.

MicroRNA-203 contributes to skin re-epithelialization.

Keratinocyte proliferation and migration are crucial steps for the rapid closure of the epidermis during wound healing, but the molecular mechanisms involved in this cellular response remain to be completely elucidated. Here, by in situ hybridization we characterize the expression pattern of miR-203 after the induction of wound in mouse epidermis, showing that its expression is downregulated in the highly proliferating keratinocytes of the ‘migrating tongue’, whereas it is strongly expressed in the differentiating cells of the skin outside the wound. Furthermore, subcutaneous injections of antagomiR-203 in new born mice dorsal skin strengthened, in vivo, the inverse correlation between miR-203 expression and two new target mRNAs: RAN and RAPH1. Our data suggest that miR-203, by controlling the expression of target proteins that are responsible for both keratinocyte proliferation and migration, exerts a specific role in wound re-epithelialization and epidermal homeostasis re-establishment of injured skin.

GLS2 is transcriptionally regulated by p73 and contributes to neuronal differentiation

The amino acid Glutamine is converted into Glutamate by a deamidation reaction catalyzed by the enzyme Glutaminase (GLS). Two isoforms of this enzyme have been described, and the GLS2 isoform is regulated by the tumor suppressor gene p53. Here, we show that the p53 family member TAp73 also drives the expression of GLS2. Specifically, we demonstrate that TAp73 regulates GLS2 during retinoic acid-induced terminal neuronal differentiation of neuroblastoma cells, and overexpression or inhibition of GLS2 modulates neuronal differentiation and intracellular levels of ATP. Moreover, inhibition of GLS activity, by removing Glutamine from the growth medium, impairs in vitro differentiation of cortical neurons. Finally, expression of GLS2 increases during mouse cerebellar development. Although, p73 is dispensable for the in vivo expression of GLS2, TAp73 loss affects GABA and Glutamate levels in cortical neurons. Together, these findings suggest a role for GLS2 acting, at least in part, downstream of p73 in neuronal differentiation and highlight a possible role of p73 in regulating neurotransmitter synthesis.

Retinoids in neuroblastoma therapy: distinct biological properties of 9-cis- and all-trans-retinoic acid

We investigated the potential for 9-cis-retinoic acid in the differentiation therapy of neuroblastoma using an N-type neuroblastoma cell line, SH SY 5Y, as an experimental model. In these cells, 9-cis-retinoic acid is more effective than other isomers at inducing the expression of RAR-beta. An RAR-alpha-specific antagonist inhibited the induction of RAR-beta in response to all-trans-but not to 9-cis-retinoic acid. This indicates that the mechanism of gene induction by 9-cis-retinoic acid differs markedly from all-trans-retinoic acid. 9-cis-retinoic acid is also better than all-trans at producing sustained morphological differentiation and inhibition of proliferation of SH SY 5Y cells. Although N-type neuroblastoma cells are not thought to undergo apoptosis in response to all-trans-retinoic acid, we observed a significant degree of apoptosis in SH SY 5Y cells treated with 9-cis-retinoic acid for 5 days and then cultured in the absence of retinoid, an effect not observed in cells treated with the all-trans isomer. These results suggest that 9-cis- and all-trans-retinoic acid have distinct biological properties and that 9-cis retinoic acid may be clinically effective in neuroblastoma by inducing both differentiation and apoptosis under an appropriate treatment regimen.

p63 the guardian of human reproduction

p63 is a transcriptional factor implicated in cancer and development. The presence in TP63 gene of alternative promoters allows expression of one isoform containing the N-terminal transactivation domain (TA isoform) and one N-terminal truncated isoform (ΔN isoform). Complete ablation of all p63 isoforms produced mice with fatal developmental abnormalities, including lack of epidermal barrier, limbs and other epidermal appendages. Specific TAp63-null mice, although they developed normally, failed to undergo in DNA damage-induced apoptosis during primordial follicle meiotic arrest, suggesting a p63 involvement in maternal reproduction. Recent findings have elucidated the role in DNA damage response of a novel Hominidae p63 isoform, GTAp63, specifically expressed in human spermatic precursors. Thus, these findings suggest a unique strategy of p63 gene, to evolve in order to preserve the species as a guardian of reproduction. Elucidation of the biological basis of p63 function in reproduction may provide novel approaches to the control of human fertility.

Differential growth of N- and S-type human neuroblastoma cells xenografted into SCID mice. Correlation with apoptosis

This study concerns the role of apoptosis in the growth of human neuroblastomas transplanted into immunodeficient SCID mice. Human neuroblastoma cell lines may consist of one or more distinct phenotypes including the neural ‘N‐type’ and flat substrate‐adherent ‘S‐type’. A differential phenotype‐specific proliferation was apparent among S‐ and N‐type cell clones transplanted into SCID mice when compared with the wild‐type SK‐N‐BE(2) cell line. This differential growth capacity of the tumours was correlated with spontaneous apoptosis. Another SK‐N‐BE(2)‐derived cell line (TGA), displaying high levels of apoptosis upon stable transfection with the full length ‘tissue’ transglutaminase (tTG) cDNA, was unable to induce tumour development when xenografted into SCID mice. To support these observations, the expression of apoptosis‐related genes (i.e., bcl‐2, p53, and tTG) in the various neuroblastomas was also investigated.

Expression and down-regulation by retinoic acid of IGF binding protein-2 and -4 in medium from human neuroblastoma cells

Insulin‐like growth factors (IGFs) regulate the autocrine/paracrine growth of neuroblastomas. The IGFs bind to specific binding proteins (IGFBPs) which modulate their biological activity. We investigated, by Western ligand blotting (WLB), the presence of IGFBPs and their possible modulation by retinoic acid (RA), IGF‐I, IGF‐II and truncated Des(1‐3)IGF‐l in conditioned medium (CM) of the human neuroblastoma SK‐N‐BE(2) cell line. We demonstrated the presence of two IGFBPs, with MW 37 kDa and 25 kDa. Following immunoprecipitation, they turned out to be IGFBP‐2 and ‐4, respectively. The RA‐induced differentiation in SK‐N‐BE(2) cells was accompanied by a marked reduction of the intensity of both IGFBP bands after 48 h (32% and 24% of control, respectively) and 72 h (2% and 0% of control, respectively) incubation. The addition of exogenous IGFs, which did not induce cell differentiation, did not change the IGFBP pattern significantly, except for the truncated form of IGF‐I, which induced a marked decrease in both the 37 kDa and 25kDa bands after 72 h incubation (45% and 18% of control, respectively). These findings suggest that IGFBPs have a role in RA‐induced differentiation in human neuroblastoma cells.

Impact of Mediterranean diet on metabolic syndrome, cancer and longevity

Obesity symbolizes a major public health problem. Overweight and obesity are associated to the occurrence of the metabolic syndrome and to adipose tissue dysfunction. The adipose tissue is metabolically active and an endocrine organ, whose dysregulation causes a low-grade inflammatory state and ectopic fat depositions. The Mediterranean Diet represents a possible therapy for metabolic syndrome, preventing adiposopathy or “sick fat” formation. The Mediterranean Diet exerts protective effects in elderly subjects with and without baseline of chronic diseases. Recent studies have demonstrated a relationship between cancer and obesity. In the US, diet represents amount 30-35% of death causes related to cancer. Currently, the cancer is the second cause of death after cardiovascular diseases worldwide. Furthermore, populations living in the Mediterranean area have a decreased incidence of cancer compared with populations living in Northern Europe or the US, likely due to healthier dietary habits. The bioactive food components have a potential preventive action on cancer. The aims of this review are to evaluate the impact of Mediterranean Diet on onset, progression and regression of metabolic syndrome, cancer and on longevity.

Vascular ageing and endothelial cell senescence: Molecular mechanisms of physiology and diseases

Ageing leads to a progressive deterioration of structure and function of all organs over the time. During this process endothelial cells undergo senescence and manifest significant changes in their properties, resulting in impairment of the vascular functionality and neo-angiogenic capability. This ageing-dependent impairment of endothelial functions is considered a key factor contributing to vascular dysfunctions, which is responsible of several age-related diseases of the vascular system and other organs. Several mechanisms have been described to control ageing-related endothelial cell senescence including microRNAs, mitochondrial dysfunction and micro environmental stressors, such as hypoxia. In this review, we attempt to summarize the recent literature in the field, discussing the major mechanisms involved in endothelial cell senescence. We also underline key molecular aspects of ageing-associated vascular dysfunction in the attempt to highlight potential innovative therapeutic targets to delay the onset of age-related diseases.