Staff :
Group leader : ROBERTO TESTI
Universita' degli Studi di ROMA "Tor Vergata"
MEDICINE AND SURGERY
DIPARTIMENTO DI MEDICINA SPERIMENTALE
1. DANIELA BARILA' UNIVERSITA' DI ROMA TOR VERGATA DIPARTIMENTO DI MEDICINA SPERIMENTALE
2.PAOLO SBRACCIA UNIVERSITA'DI ROMA TOR VERGATA DIPARTIMENTO DI MEDICINA INTERNA
cDNAs corresponding to selected disease-associated
variants will be cloned in frame with a cDNA coding for a trackable fluorescence
protein, to allow detection in transfected cells. The fusion proteins will be
transiently expressed in appropriate cell lines and the espression levels evaluated
by fuorescence microscopy. At the same time relevant cDNAs will be modified
with a small tag sequence suitable for immunofluorescence and confocal microscopy
as well as for detailed biochemical studies. Appropriate cell lines will be
transiently transfected with the tagged cDNAs and the expression levels and
molecular size evaluated by western blot analysis.
Once the selected cDNA have been checked for the appropriate expression and
expected molecular size of the product, functional studies will be undertaken.
The possible ability of the disease-associated cDNAs to directly trigger cell
death will be investigated by transient expression in appropriate cell lines
or primary cell cultures. Transfection approaches (electroporation, liposome-mediated,
adenovirus-mediated) will depend on the particular cell to be transfected. Expression
of the apropriate gene product will be checked by fuorescence microscopy and
western blot analysis in each individual cellular system utilized. The usage
of the fluorescent or tagged proteins will depend on the particular experimental
set up. Induction of apoptotic cell death will be detected by microscopic inspection
of transfected (fluorescent) cells at different times from the transfection.
Upregulation of death receptors and/or their ligands will be evaluated by FACS
and western blot analysis. DNA fragmentation and hypodiploidity in transfected
cells will be investigated by FACS analysis of nuclei. Activation of the caspases
proteolytic cascade in transfected cells will be evaluated by immunostaining
of activated caspases products or by colorimetric assays and western blot analysis,
depending on the transfection efficiency in each individual cellular system
utilized. Similarly, mitochondrial changes in transfected cells, including induction
of mitochondrial permeability transition, release of cytochrome c and activation
of caspase9 will be evaluated by FACS analysis and western blot analysis.
To functionally map the level of initiation of the apoptotic program by the
disease-associated variants, the corresponding cDNAs will be expressed in cell
lines overexpressing known inhibitors of the apoptotic program such as FLIPs,
IAPs, HSPs and protective bcl2 family members. This will be achieved by transiently
co-expressing protective cDNAs along with the variants, or by taking advantage
of available cell lines stably overexpressing the protective products. The inability
of the disease-associated variant to induce death in "protected" cell
lines will provide indications on the topology of action of the variant.
Alternatively, disease-associated variants might negatively regulate the apoptotic
program. This possibility will be investigated by exposing cell lines or primary
cell cultures, where the disease-associated variants have been expressed, to
known inducers of apoptosis, such as death receptors ligands and ceramides,
or major stressors such as UVB radiations. The ability of the variants to confer
resistance to the above mentioned death stimuli will be evaluated by microscpic
inspection of transfected (fluorescent) cells. Variants which consistently provide
protections in transient transfections will be stably expressed in appropriate
cell lines. Stable transfectants will be valuable tools for further studies.
Analysis of the expression of death receptors/ligands, specific caspases activation,
mitochondrial changes and DNA fragmentation, as above described, will allow
to functionally map the level of interference on the apoptotic program by each
individual variant. Moreover, the ability of the variants to induce the downregulation
of known cellular protective molecules will be investigated by western blot
analysis in stably transfected cells. Finally, detailed confocal microscopic
analysis of tagged variants in stably transfected cells will provide informations
on the subcellular localization and traffic of the variant product.
As a parallel approach to the understanding of the possible interference of
the expression of disease-associated variants in the death program, RNAs from
cell lines both transiently and stably transfected with selected disease-associated
variants will be probed on DNA microarrays for apoptosis-associated gene expression
analysis. This will allow the identification of apoptosis-associated genes whose
expression is modulated by the variant. Products of variant-modulated genes
of particular interest will be analyzed in further detail both biochemically
and functionally in variant-transfected cells.
Together these informations will provide a molecular framework to functionally
evaluate the impact of the expression of disease-associated variants on life
or death decisions of human cells.
As for type 2 diabetes, once we have identified
differentially expressed cDNAs in muscle and adipose tissue, a series of studies
will be performed to characterize the proteins encoded by the isolated cDNAs.
Depending on the nature of the isolated proteins (known vs. unknown; secreted
or cellular) different experiments will be performed. We will focus on the role
of these proteins in insulin signaling and their possible implication in insulin
resistance. From the subphenotypes, tissue will be obtained to establish; 1)
primary cultures from cells of particular interest (myotubes, preadipocytes,
vascular cells) for functional studies in cell cultures and; 2) tissue biopsies
of muscle and adipose tissue for studies of gene expression levels and intracellular
signaling molecules. These studies will be addressed by classical biochemical
and cellular approaches. In the case of "cellular" proteins, membrane
or cytosolic, its possible role will be approached by expression of the protein
in insulin-responsive cell lines such as 3T3L1 pre-adipocytes or L6 myoblasts
or other nonclassical insulin-responsive cell lines such as endothelial cells
or smooth muscle cells. We will study the effect of the protein on metabolism,
growth/differentiation and apoptosis. Then, in an attempt to better understand
the metabolic significance of the altered expression of the genes in the different
phenotypes, transgenic and/or knockout animal models will be generated (see
research line 2, WP3).
As for IBD, intestinal mucosal samples will be obtained from surgical specimens of patients with CD, UC and non-IBD controls. Both involved and uninvolved areas will be tested. LPMC will be isolated by DTT-EDTA-Collagenase and Percoll gradient. EDTA and whole blood samples will be obtained from the same patients for DNA-extraction and isolation of autologous PBMC. LPMC and the autologous PBMC will be cultured with and without methylprednisolone (from 1 to 10 ug/ml) or anti-TNFa (from 1 to 10 ug/ml) for 24 hours. Transcripts for IL12Rb1 and IL12Rb2, IL-12/p40 and IL-12/p35 will be examined by southern blotting and expression levels compared with DNA-sequence variations of the human IL-12p40 promoter as also to the "in vivo" responsiveness to corticosteroids and anti-TNFa.
Amount (ML) 185
Source(s) MURST; CNR; AIRC; Min Sanità
1) Rippo M.R., F. Malisan, L. Ravagnan, B. Tomassini, I. Condo', P. Costantini,
S. A. Susin, A. Rufini, M. Todaro, G. Kroemer and R. Testi. 2000. GD3 ganglioside
directly targets mitochondria in a bcl-2-controlled fashion. FASEB Journal 14:2047-2054.
2) De Maria, R., M.R. Rippo, E.H. Schuchman, and R. Testi. 1998. Acidic Sphingomyelinase (ASM) Is Necessary for Fas-induced GD3 Ganglioside Accumulation and Efficient Apoptosis of Lymphoid Cells. Journal of Experimental Medicine 187: 897-902.
3) Papoff G., G. Stassi, R. De Maria, C. Giordano, A. Galluzzo, M. Bagnasco, G. Ruberti, and R. Testi. 1998. Constitutive Expression of FasL in Thyrocytes. Science 279:2015a.
4) De Maria R. and R. Testi. 1998. Fas-FasL interactions: a common pathogenetic mechanism in organ-specific autoimmunity. Immunology Today 19:121-125.
5) De Maria R., M.L. Lenti, F. Malisan, F. d'Agostino, B. Tomassini, A. Zeuner, M.R. Rippo and R. Testi. 1997. Requirement for GD3 ganglioside in CD95- and ceramide-induced cell death. Science 277:1652-1655.