Defects in Tricarboxylic Acid Cycle Enzymes Fumarate Hydratase and Succinate Dehydrogenase in Cancer

............................................................................................................... 9 1 REVIEW OF THE LITERATURE ................................................................... 11 1.1 CANCER IS A GENETIC DISEASE...................................................................... 11 1.1.1 Oncogenes ............................................................................................... 11 1.1.2 Tumour suppressor genes......................................................................... 12 1.1.3 Epigenetic and environmental factors....................................................... 13 1.1.4 Cancer is a multistep process ................................................................... 14 1.1.5 Tumour stem cells ................................................................................... 15 1.2 RENAL CELL CARCINOMA.................................................................................. 16 1.2.1 Hereditary cancer syndromes with predisposition to renal cell carcinoma 16 1.2.1.1 Hereditary leiomyomatosis and renal cell cancer............................................16 1.2.1.2 Other hereditary syndromes with predisposition to renal cell carcinoma.........18 1.3 UTERINE LEIOMYOSARCOMA ........................................................................ 19 1.4 TRICARBOXYLIC ACID CYCLE........................................................................ 20 1.4.1 The role of succinate dehydrogenase and fumarate hydratase in TCAC.... 21 1.4.2 Paragangliomas and pheochromocytomas result from defective SDH ...... 23 1.4.3 HLRCC and FH deficiency are consequences of defective FH................. 24 1.4.4 Tricarboxylic acid cycle and cancer ......................................................... 26 2 AIMS OF THE STUDY...................................................................................... 28 3. MATERIALS AND METHODS........................................................................ 29 3.1 PATIENT MATERIALS ..................................................................................... 29 3.1.1 Renal cell carcinomas (I) ......................................................................... 29 3.1.2 Uterine leiomyosarcomas (II)................................................................... 30 3.1.3 Patient material to study putative FH mutation related phenotypes (III) ... 31 3.1.4 Patient and cell line material for mutation analyses of FHv (IV) .............. 31 3.1.5 Other materials used in study IV .............................................................. 32 3.2 ANALYSIS METHODS ...................................................................................... 32 3.2.1 Mutation analyses of SDH (I) .................................................................. 32 3.2.2 Mutation analyses of FH (II, III, IV) ........................................................ 33 3.2.3 Analysis of allelic imbalance (AI) (II)...................................................... 34 3.2.4 FH enzyme activity assay (II, IV) ............................................................ 34 3.2.5 In silico analyses (II, IV).......................................................................... 34 3.2.6 Analysis of expression of FHv (IV).......................................................... 35 3.2.6.1 Analysis of mRNA expression by quantitative RT-PCR ....................................35 3.2.6.2 Analyses of expression and localization of FHv in cell lines ............................35 3.2.6.3 Cell culturing and stress experiments..............................................................35 3.2.6.4 Western blot analyses .....................................................................................36