Modulation of molecular mechanisms involved in protein synthesis machinery as a new tool for the control of cell proliferation.

In the past years, the attention of scientists has focused mainly on the study of the genetic information and alterations that regulate eukaryotic cell proliferation and that lead to neoplastic transformation. All therapeutic strategies against cancer are, to date, directed at DNA either with cytotoxic drugs or gene therapy. Little or no interest has been aroused by protein synthesis mechanisms. However, an increasing body of data is emerging about the involvement of translational processes and factors in control of cell proliferation, indicating that protein synthesis can be an additional target for anticancer strategies. In this paper we review the novel insights on the biochemical and molecular events leading to protein biosynthesis and we describe their involvement in cell proliferation and tumorigenesis. A possible mechanistic explanation is given by the interactions that occur between protein synthesis machinery and the proliferative signal transduction pathways and that are therefore suitable targets for indirect modulation of protein synthesis. We briefly describe the molecular tools used to block protein synthesis and the attempts made at increasing their efficacy. Finally, we propose a new multimodal strategy against cancer based on the simultaneous intervention on protein synthesis and signal transduction.     

Identification of a Novel Gene Signature of ES Cells Self-Renewal Fluctuation through System-Wide Analysis

Embryonic Stem cells (ESCs) can be differentiated into ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. In regular culture conditions, ESCs'' self-renewal is maintained through molecules that inhibit spontaneous differentiation enabling long-term cellular expansion. This undifferentiating condition is characterized by multiple metastable states that fluctuate between self-renewal and differentiation balance. Here, we aim to characterize the high-pluripotent ESC metastate marked by the expression of Zscan4 through a supervised machine learning framework based on an ensemble of support vector machine (SVM) classifiers. Our study revealed a leukaemia inhibitor factor (Lif) dependent not-canonical pluripotency signature ({"type":"entrez-nucleotide","attrs":{"text":"AF067063","term_id":"3171143"}}AF067063, {"type":"entrez-nucleotide","attrs":{"text":"BC061212","term_id":"38173992"}}BC061212, Dub1, Eif1a, Gm12794, Gm13871, Gm4340, Gm4850, Tcstv1/3, and Zfp352), that specifically marks Zscan4 ESCs'' fluctuation. This novel ESC metastate is enhanced by high-pluripotency culture conditions obtained through Extracellular signal Regulated-Kinase (ERK) and Glycogen synthase kinase-3 (Gsk-3) signaling inhibition (2i). Significantly, we reported that the conditional ablation of the novel ESC metastate marked by the expression of Gm12794 is required for ESCs self-renewal maintenance. In conclusion, we extend the comprehension of ESCs biology through the identification of a novel molecular signature associated to pluripotency programming.     

Retinoic Acid Specifically Enhances Embryonic Stem Cell Metastate Marked by Zscan4

Pluripotency confers Embryonic Stem Cells (ESCs) the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. Although the majority of ESCs divide without losing pluripotency, it has become evident that ESCs culture consists of multiple cell populations with different degrees of potency that are spontaneously induced in regular ESC culture conditions. Zscan4, a key pluripotency factor, marks ESC subpopulation that is referred to as high-level of pluripotency metastate. Here, we report that in ESC cultures treated with retinoic acid (RA), Zscan4 ESCs metastate is strongly enhanced. In particular, we found that induction of Zscan4 metastate is mediated via RA receptors (RAR-alpha, RAR-beta, and RAR-gamma), and it is dependent on phosphoinositide-3-kinase (PI3K) signaling. Remarkably, Zscan4 metastate induced by RA lacks canonical pluripotency genes Oct3/4 and Nanog but retained both self-renewal and pluripotency capabilities. Finally we demonstrated that the conditional ablation of Zscan4 subpopulation is dispensable for both endoderm and mesoderm but is required for ectoderm lineage. In conclusion, our research provides new insights about the role of RA signaling during ESCs high pluripotency metastate fluctuation.     

New pharmacokinetic and pharmacodynamic tools for interferon-alpha (IFN-α) treatment of human cancer

Interferon (IFN-) has been widely used in the treatment of human solid and haematologic malignancies. Although the antitumour activity of IFN- is well recognised at present, no major advances have been achieved in the last few years. Recent findings have provided new information on the molecular mechanisms of the antitumour activity of the cytokine. In fact, IFN- appears to block cell proliferation, at least in part, through the induction of apoptotic effects. This cytokine can also regulate the progression of tumour cells through the different phases of the cell cycle inducing an increase of the expression of the cyclin-dependent kinase inhibitors p21 and p27. However, it must be considered that IFN- is a physiologic molecule with ubiquitously expressed receptors that is likely to activate survival mechanisms in the cell. We have recently identified an epidermal growth factor (EGF) Ras-dependent protective response to the apoptosis induced by IFN- in epidermoid cancer cells. The identification of tissue- and/or tumour-specific survival pathways and their selective targeting might provide a new approach to improve the efficacy of IFN-–based treatment of human cancer. Moreover, new pegylated species of IFN- are now available with a more favourable pharmacokinetic profile. We will review these achievements, and we will specifically address the topic of IFN-–based molecularly targeted combinatory antitumour approaches.     

Evaluation of a multisensor armband in estimating energy expenditure in obese individuals

Objective: To examine the reliability and validity of the SenseWear Pro 2 Armband (SWA; Body Media, Pittsburgh, PA) during rest and exercise compared with indirect calorimetry (IC) in obese individuals. Research Methods and Procedures: Energy expenditure was assessed during rest with the SWA and IC in 142 obese adults (37 men and 105 women, BMI = 42.3 ± 7.0) and in 25 lean and overweight adults (BMI = 25.3 ± 3.2) who were used as a comparison group. Twenty‐nine of the obese adults also participated in three separate short exercise sessions including cycle ergometry, stair stepping, and treadmill walking. Results: The repeatability of SWA estimates in obese subjects was high (r = 0.88, p < 0.001). The SWA generally underestimated the resting energy expenditure (REE) (1811 ± 346 vs. 1880 ± 382 kcal/d) and highly overestimated the energy expenditure during the exercise sessions in obese individuals. REE estimations by SWA were significantly correlated with fat‐free mass (r = 0.88, p < 0.001). Bland‐Altman plots based statistical analysis for the estimated REE, and measured IC showed a low agreement (Total Error > 20% but Systematic Error < 5%) between the two methods in obese subjects, although they showed a high correlation and a very good agreement in lean and overweight patients. Discussion: The SWA is an easy to handle, practical, new portable device for measuring energy expenditure. The accuracy of the SWA appeared to be poor in the obese subjects we examined, especially those with high REE both in rest and exercise. We believe that it is necessary to incorporate new, obesity‐specific algorithms in the relative software.     

Phorbol 12-myristate 13-acetate induces resistance of human melanoma cells to natural-killer-and lymphokine-activated-killer-mediated cytotoxicity

Summary Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.This work has been supported by the Italian Association for Cancer Research (A. I. R. C.) and by Istituto Superiore di Sanità, Italy-USA joint program on New Therapies on Neoplasia.     

Ouabain sensitivity is linked to ras -transformation in human HOS cells

Mouse cells transformed by the retroviral oncogene v-Ki- ras are significantly more sensitive to the toxic effects of 1mM ouabain than are their nontransformed counterparts. We have extended these findings to a human cell line (HOS). HOS cells (ATCC CRL 1543) are relatively resistant to treatment with 1 microM ouabain while KHOS cells (transformed by Kirsten murine sarcoma virus) are extremely sensitive. Two flat revertant cell lines isolated from the KHOS line and lacking the v- ras gene sequences are resistant to ouabain. This effect may be observed morphologically and can also be demonstrated by dye exclusion and plating efficiency tests. In addition, the toxic effects of ouabain may be rapidly and efficiently quantitated in a 51Cr-release assay. This differential lethality may be used to enrich the proportion of non-transformed revertants in populations of mutagen-treated transformed cells.