XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta

Lectures
TOXCAT METHOD: APPLICATION IN MOLECULar ONCOLOGY
Martin Benej 1 and Martina Poturnajová 2
1
Department of Molecular Biology UK Bratislava,
2
Cancer Research Institute SAS Bratislava
Understanding the molecular mechanisms of cancer onset is one of the goals of contemporary
cancer research. Individual approach to each disease is of crucial importance
in this issue. Moreover, not only each disease, but also each patient carrying causative
mutation in his/her genome requires individual approach. Thus, a need of a whole set
of methods for characterization of causative mutations has arisen. ToxCAT is a method
simulating the natural lipid bilayer environment, enabling the study of transmembrane
(TM) domain interactions in vitro. The method is based on introduction of chimaeric constructs
containing a TM domain of interest into periplasmic region of E.coli MM39 strain.
Interactions of the chimerae result in expression of chloramphenicol acetyltransferase
(CAT) reporter gene. The quantity of CAT expression corresponds with the strength of TM
domain association. We illustrate the ToxCAT method application on the specific model
of our interest – Medullary thyroid carcinoma (MTC). MTC is caused predominantly by
single point mutations in six RET proto-oncogene exons. RET protein, the product of the
RET gene is a tyrosine kinase cell-surface receptor. Mutations in extracellular domain of
the receptor result in dimerization of the RET protein with other mutant RET molecule,
thus enabling ligand-independent permanent activation of the RET receptor kinase. For
this interaction, the strength of transmembrane domain oligomerization of the two RET
molecules is responsible. We focus on the impact of RET TM domain mutations of Slovak
MTC patients on the strength of TM domain oligomerization, to investigate a possible
correlation with the age of onset and aggressiveness of the disease.
46 XXII. Biochemistry Congress, Martin