RICERCA ITALIANA     

STUDY OF THE EXPRESSION, ACTIVITY AND INTRACELLULAR COMPARTIMENTALIZATION OF SIGNALLING PROTEINS IN TUMOUR CELLS EXPOSED OR NOT TO BIOLOGICAL AND CYTOTOXIC AGENTS: A GENOMIC AND PROTEOMIC APPROACH.

Seconda Università degli Studi di Napoli

Abstract

The medical therapy of neoplasms is presently based on the use of conventional cytotoxic agents that mainly act through the induction of an irreversible and lethal damage of tumour cell DNA. Beside the lethal hit to DNA, one of the mechanisms of action of conventional chemotherapy is the triggering of apoptotic program in cancer cells. However, tumour cells can develop mechanisms of escape from the apoptosis involving regulatory loops of the mitogenic signalling and of the cell cycle. To-day it is emerging the need of finding newly synthesized molecules able to inhibit the proliferation of tumour cells and with anticancer activity in vivo. Moreover, it is becoming even more evident that, for the clinical application, it is necessary the definitioon of the effects of these agents on the intracellular genomic and proteic expression profile. Therefore our project is intended to detect new anti-tumor molecules and to define their mechanism of action through the study of their effects on signal transduction pathway, proteic and genomic expression profile in human tumour cells.

Principal Investigator

Ettore BISMUTO Seconda Università degli Studi di NAPOLI

Research Objectives

The work of the Bismuto research unit will develop along two directions: the first one utilizing FLIM microscopy as main investigation technique, and oriented to the intracellular localization of fluorescent ras-FGP (ras chimeric protein with Fluorescent Green Protein)in normal and tumoral cells, treated or not with proper molecules supposed potential drugs; the second one oriented to in vitro structural and functional featuring of the fluorescent ras constructs and of its complexes with other cellular proteins involved in the transduction pathway. Such studies will avail in particular of time-resolved fluorescence techniques and fluorescence correlation spectroscopy provided with two-photon excitation, which allows to observe interactions as well as dynamic and diffusion processes at single molecule level.
The maín goal of Beninati project is the identification of an effective mechanism, to reduce the growth of cancer cell and their metastatic potential by inductíon of cell differentiation. We have enough evidences to believe that this target can be reached by an indirect influence on the metabolism of polyamines, obtained inducing Tgase activity in cancer cells. Since it is addressed only toward cell wíth a negligible Tgase activity, the novelty of thís approach is the improvement of the selectivity of the intervention. Indeed, cancer cells are characterízed by a very low Tgase activíty, usually unsuitable for the índuction of cell differentiation; the treatment >>>

First Results

Bismuto
- Construction of plasmids in eukaryotic vectors for the transfection of molecular constructs between ras and GFP in human normal and tumour cells before and after the treatment with molecules of biological interest: 5 months.
- Characterization of proteic expression in normal and cancer cells after appropriate treatments: 2 months.
- Expression of the constructs in E.Coli and successive extraction and purification: 2 months.

Beninati
1. Identification and synthesis of intracellular Tgase activators (Tg-activators). This part of the program will be performed in collaboration with the laboratory of Dr. J.E.Folk, National Institutes of Health, Oral and Pharyngeal Cancer Branch, NIDR, Bethesda, MD,USA. The synthesis procedures are based on experimental protocols of Dr. Folk. The modified molecules are methylxanthines and retinoic acid analogues. It will be estimated the rate of Tgase activation in vitro and in vivo , with the discrimination between protein activation and gene expression. Determination of the parameters, affected by the Tg-activators, correlated with cell growth, differentiation and apoptosis (p53, c-myc and bcl-2 expression and FACS analysis of the cell cycle). Investigation of the action of Tg-activators on normal (primary culture of melanocytes) and neoplastic cells (human and murine melanoma cells). Preparation of plasmids in order to increase the expression of Tgase on experimental animals (with the >>>

Timescale

24 months

National and international background

From the pioneering work with acute transforming retroviruses to the current post-genomic era, ras genes have always been at the leading edge of signal transduction and molecular oncology (1,2). Yet, a complete understanding of ras function and dysfunction — mainly in human cancer — is still to come (3). The knowledge of such cellular mechanisms is fundamental for developping efficacious drugs for the treatment of different tumor types (4). The Ras proteins are members of a large superfamily of low molecular-weight GTP-binding proteins (5). In vitro purified Ras possess low rated GTP-ase activity due to which they slowly convert the GTP molecule bound to them. The GDP may afterwards be exchanged to GTP again. However, these processes are catalyzed and regulated inside the cell by means of different effector proteins (guanine nucleotide exchange factors GEFs e GTPases activating proteins GAPs). Proteins belonging to the Ras family monitor cell growth processes and play a role in apoptosis and senescence processes (6,7). They exist in either activated and inhibited mutated forms in different human tumors. The normal function of Ras proteins requires post-translational modifications whose main aim is to localize them to the correct subcellular compartment — principally the inner face of the plasma membrane, for the sake of efficiently interacting with the proper molecules of the signal transduction pathway (2,8).
The aim of this research unit's project is to state the >>>