Adriana Oliva

Genes modulated by histone acetylation as new effectors of butyrate activity

A wealth of evidence correlates the chemopreventive activity of a fiber‐rich diet with the production of butyrate. In order to identify the genes transcriptionally modulated by the molecule, we analyzed the expression profile of butyrate‐treated colon cancer cells by means of cDNA expression arrays. Moreover, the effect of trichostatin A, a specific histone deacetylase inhibitor, was studied. A superimposable group of 23 genes out of 588 investigated is modulated by both butyrate and trichostatin A. Among them, a major target was tob‐1, a gene involved in the control of cell cycle. tob‐1 is also up‐regulated by butyrate in a neuroblastoma‐derived cell line, and its overexpression in the colon cells caused growth arrest. Our findings represent an extensive analysis of genes modulated by butyrate and identify completely new effectors of its biological activities.

Resveratrol: From Basic Science to the Clinic

Plants produce an extraordinary array of low-molecular-mass natural products endowed with biological activity. Among these molecules, resveratrol (3,5,4’-trihydroxystilbene) has been identified as an inhibitor of carcinogenesis with a pleiotropic mode of action. Extensive Literature on its anticancer activity, performed in cellular models, suggests a potential antiproliferative and apoptogenic use of the stilbene. Similarly, studies on implanted cancers and chemical-induced tumors confirm a potential chemotherapeutical interest of the compound. Moreover, recent intriguing studies have demonstrated, in mice, that the negative effects (insulin resistance and hyperglycemia) of a high-fat diet might be prevented by resveratrol treatment. Despite these promising observations, only few clinical trials have been performed on the compound due to the scarce interest of pharmaceutical industry. We suggest that resveratrol might be considered an interesting compound in association with more specific target-oriented drugs.

p27Kip1 Metabolism: A Fascinating Labyrinth

The progression through the phases of cell division cycle is regulated by different cyclins and cyclin-dependent kinases (CDKs) complexes. Due to their key function, the activity of cyclin/CDK complexes is controlled by several mechanisms, including the inhibition by a number of proteins collectively defined CDK inhibitors or CKIs. Among the CKIs, p27Kip1 represents a protein of central activity for the control of several phenotypes, including proliferation, differentiation and malignant transformation. p27Kip1 belongs to the growing family of “natively unfolded”, “in¬trinsically disordered” or “intrinsically unstructured” proteins. The disorder proteins present a very large number of possible conformations that, after the binding, converge to a well-defined structure with an extraordinary affinity for the target. As matter of fact, the absence of a preexisting folding strongly facilitates p27Kip1 interaction with a number of targets. Until recently, p27Kip1 has been solely viewed as a nuclear protein with the function of modulating cyclin–CDK activity and hence, cell-cycle progression. However, exhaustive studies have now demonstrated that the protein plays additional roles outside of the nucleus, including, particularly, the control of cell motility. Thus, the cellular localization is of fundamental importance in p27Kip1 function. Accordingly, at least two different mechanisms of degradation, occurring either in the nucleus or in the cytosol, have been observed. Convincing evidences have demonstrated that p27Kip1 is a phosphoprotein showing at least 6-8 phosphorylatable residues. However, the precise functional roles of the phosphorylations and the identification of the kinases responsible for the post-synthetic modifications are still debated. In this brief review, we will report the Literature data that connect the post-synthetic modifications of p27Kip1 with its function, localization and metabolism. The picture that emerges demonstrates that several of the pieces of the CKI metabolism are still nebulous.

Biocompatibility studies on glass ionomer cements by primary cultures of human osteoblasts.

Glass ionomer cements (GICs) are materials largely employed in the dental field that have been considered recently as cements in orthopaedic surgery for their proven osteogenic features. The aim of this study was to compare the response of cultured human osteoblastic cells to a number of commercial glass ionomer cements in order to provide indications useful for the further development of formulations that have potential for use as cements or implants in repair and replacement of bone tissue. The GICs tested were: Ketac-Fil Aplicap, lonocem lonocap 1,0, GC Fuji II, GC Fuji II LC and Vitremer 3M. Several features such as plating efficiency, adhesion and morphology of the cells were studied, as well as the only specific biochemical parameter of osteoblastic phenotype, namely osteocalcin production. In addition, the colonisation of materials by osteoblastic cells was verified by means of scanning electron microscopy. Altogether, the results obtained indicate that four of the five glass ionomer cements tested are biocompatible, showing vital cells adhering to the materials, proliferating and expressing the biochemical markers of osteoblastic phenotype, whereas Vitremer 3M, although currently employed in the dental field, exhibits a great cytotoxicity toward the cells. The adverse reaction of this GIC can be attributed to the leaching of at least two components of the polyacidic phase evidenced by protonic magnetic resonance analysis (PMR), namely 2-hydroxyethylmethacrylate (HEMA), and an unidentified acidic species. The addition of pure HEMA at the same concentrations found by means of PMR to cultures of osteoblastic cells resulted in a complete cell death. Our results also show that in vitro methods employing primary cultures of human cells specific to the implant sites of prostheses are appropriate and suitable tools for evaluating biocompatibility of materials. Furthermore, this kind of approach can provide indications useful in the design of novel materials as well as in improving the characteristics of the formulations already available.

p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation: evidence of a decreased proteasome-dependent degradation.

Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27Kip1 protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation. p27Kip1 build-up was observable as an early phenomenon (12–24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27Kip1 immunoreactive bands, which probably represent ubiquitinated forms of the protein. Finally, in vitro experiments demonstrated that extracts prepared from retinoic-treated LAN-5 cells degraded recombinant p27Kip1 at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27Kip1 levels by down-regulating the ubiquitin-proteasome p27Kip1 degrading pathway.

Behaviour of human osteoblasts cultured on bioactive glass coatings

Two new formulations of bioactive glasses were used as coatings on titanium alloy (TiAl6V4) implants for prosthetic applications in the orthopaedic field. The biocompatibility of these bioglasses, as well as their osteoconductive properties, were assessed by employing primary cultures of human osteoblasts. A nonbioactive glass, the titanium alloy and polystyrene surface were used as controls. The results obtained demonstrated that the two bioglasses elicited a rapid and strong proliferative response by osteoblasts, which spread, formed a close layer and then expressed the specific osteoblastic marker i.e. osteocalcin. In comparison, cells grew on the nonbioactive glass to a much minor extent, similar to that of polystyrene control, showing individual cellular elements not forming a compact sheet, but expressed levels of osteocalcin clearly higher than both the polystyrene control and the two bioglasses. Finally, a very low proliferative rate of osteoblasts and the synthesis of hardly detectable osteocalcin amounts were observed with the titanium alloy. In conclusion, our studies indicate that the new bioactive glasses are effective in stimulating osteoblast growth and differentiation.

Preparation and characterisation of composites based on biodegradable polymers for “in vivo” application

Abstract Poly( l -lactic) acid (PLLA), polycaprolactone (PCL), three different copolymers based on poly( l -lactic) acid and polyglycolic acid (PLLA-co-PGA), and their composites with hydroxyapatite obtained from bovine bone (ossein), were tested in order to have information on the thermal, morphological, mechanical and biochemical properties in view of their use as biocompatible/biodegradable materials. Ossein, which is essentially a biological hydroxyapatite, was found to improve the modulus and increase the hydrophilicity of the polymeric substrate. In addition, the size of the ossein particles was found to be critical for the improvement of mechanical properties. Finally, preliminary results on the in vitro biocompatibility of selected blends carried out by using primary cultures of human osteoblasts showed that the presence of hydroxyapatite stimulates a more positive cellular response.

Biochemical Characterization of p16INK4- and p18-containing Complexes in Human Cell Lines

The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16INK4 and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16INK4 gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16INK4 and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16INK4 or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16INK4 bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16INK4 protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G1 → S transition.

p57(Kip2) and cancer: time for a critical appraisal.

p57Kip2 is a cyclin-dependent kinase inhibitor belonging to the Cip/Kip family, which also includes p21Cip1 and p27Kip1. So far, p57Kip2 is the least-studied Cip/Kip protein, and for a long time its relevance has been related mainly to its unique role in embryogenesis. Moreover, genetic and molecular studies on animal models and patients with Beckwith-Wiedemann syndrome have shown that alterations in CDKN1C (the p57Kip2 encoding gene) have functional relevance in the pathogenesis of this disease. Recently, a number of investigations have identified and characterized heretofore unexpected roles for p57Kip2. The protein appears to be critically involved in initial steps of cell and tissue differentiation, and particularly in neuronal development and erythropoiesis. Intriguingly, p27Kip1, the Cip/Kip member that is most homologous to p57Kip2, is primarily involved in the process of cell cycle exit. p57Kip2 also plays a critical role in controlling cytoskeletal organization and cell migration through its interaction with LIMK-1. Furthermore, p57Kip2 appears to modulate genome expression. Finally, accumulating evidence indicates that p57Kip2 protein is frequently downregulated in different types of human epithelial and nonepithelial cancers as a consequence of genetic and epigenetic events. In summary, the emerging picture is that several aspects of p57Kip2s functions are only poorly clarified. This review represents an appraisal of the data available on the p57Kip2 gene and protein structure, and its role in human physiology and pathology. We particularly focus our attention on p57Kip2 changes in cancers and pharmacological approaches for modulating p57Kip2 levels. Mol Cancer Res; 9(10); 1269–84. ©2011 AACR.

Targeting p27Kip1 protein: its relevance in the therapy of human cancer

Introduction: Cell division cycle progression is achieved by a sequential and stringently concerted activation of a family of serine–threonine kinases, namely the cyclin-dependent kinases (CDKs). p27Kip1 is a pivotal CDK inhibitor and a tight modulator of CDK-dependent phenotypes. Thus, p27Kip1 plays a fundamental role in key cellular processes such as proliferation, differentiation, apoptosis, substrate adhesion and motility. Intriguingly, when p27Kip1 is localized in the nucleus, it acts as an antiproliferative protein, while, in the cytosol, p27Kip1 promotes cytoskeleton remodeling and might positively influence metastatization. Downregulation of p27Kip1 nuclear level or its cytosolic mislocalization are consistently correlated with poor prognosis of numerous types of human epithelial and non-epithelial cancers. Areas covered: This review illustrates the basic structural features of p27Kip1 protein, its metabolism and alterations in human malignancies, along with describing anticancer strategies based on targeting p27Kip1. Expert opinion: Given the role of p27Kip1 in the control of cell proliferation and its decreased level observed in malignancies with poor outcome, drugs able to handle the protein levels and localization might represent an important goal for novel specific and effective anticancer strategies. Although no convincing proofs have been reported, putative negative consequences of p27Kip1 targeting might be also conceivable.