Abstract
The discovery of regulatory elements that can direct tissue specific
expression to adipocytes has made possible experiments to study the function of
endogenous genes in fat metabolism and to assess the effect of these genes in other
metabolic pathways. It has also opened up the possibility of regulating the fatness of
laboratory and domestic animals by directing targeted expression of cytotoxic genes
to this tissue.
The present dissertation describes the use of a novel system to achieve
specific cell ablation in fat tissue. The method is based on the use of E.coli
nitroreductase (NTR) enzyme that activates certain nitro compounds into cytotoxic
DNA interstrand cross-linking agents. This system was assessed first in vitro, in a
preadipocyte cell line (3T3L1). Clones of cells that expressed NTR were
successfully killed after treatment with CB1954. It was confirmed that the
mechanism of cell killing involved is apoptosis and the presence of a cell-permeable
metabolite that is released to the medium triggering a bystander effect was observed.
This prodrug system was also assessed in vivo, for which transgenic mice
were generated expressing NTR specifically in adipose tissue under the control of
the aP2 promoter. Upon CB1954 treatment, transgenic mice showed extensive cell
depletion in different fat deposits, which was directly correlated to both the dose of
prodrug and the levels of NTR expressed. The present model provides a new
inducible approach to manipulate the number of adipocytes at different stages of the
mouse development and provides a new system for the study of fat metabolism
especially in abnormal conditions such as obesity and its modulation through the
manipulation of the target cell population.
Also reported are preliminary experiments to assess a novel system of
ablation mediated by the murine adapter molecule RAIDD. Stable cell lines were
generated to overexpress RAIDD after differentiation. A range of phenotypes was
observed with these clones from a complete blockage of the differentiation to the
killing of cells that escape the blockage. The present results suggest a new
developmental role for this gene and strongly encourage further experimentation to
confirm this effect in an experimental animal model.
