Functional Analysis of Cell Cycle Regulators Lkb1 and Mat1 in Genetically Engineered Mice

.........................................................................................................................................8 REVIEW OF THE LITERATURE........................................................................................................11 1 THE CELL CYCLE ...........................................................................................................................11 1.1 Cdks are Mediators of the Cell Cycle ..................................................................................11 1.2 Cellular Targets of Cdks ......................................................................................................12 1.3 Cdk Regulation.....................................................................................................................13 1.4 Cdk activating kinases (CAKs).............................................................................................14 2 AT THE CROSSROADS OF THE CELL CYCLE, DNA REPAIR AND TRANSCRIPTION...............................15 2.1 Transcription factor IIH (TFIIH) ............................................................................................15 2.2 Substrates of TFIIH kinase ..................................................................................................16 2.3 Ménage à trois (Mat1)..........................................................................................................17 3 CANCER AND INHERITED CANCER SUSCEPTIBILITY SYNDROMES ......................................................17 3.1 Oncogenes...........................................................................................................................19 3.2 Tumor Suppressors: Caretakers, Gatekeepers and Landscapers ......................................20 4 PEUTZ-JEGHERS SYNDROME ..........................................................................................................22 4.1 Increased cancer risk in PJS ...............................................................................................23 4.2 Mutations of LKB1 underlie PJS ..........................................................................................24 4.3 Biallelic inactivation of LKB1 in tumorigenesis? ..................................................................25 4.4 Evidence for LKB1 Function ................................................................................................26 AIMS OF THE STUDY........................................................................................................................28 MATERIALS AND METHODS...........................................................................................................29 Generation of Genetically Engineered Mat1 (I, II) and Lkb1 Mice (III, IV) ............................................29 PCR Genotyping (I, II, III, IV) ................................................................................................................29 Protein Analysis (I, II, III, IV) .................................................................................................................30 Immunofluoresence and Immunohistochemistry (I, II, III, IV)................................................................32 Blastocyst Outgrowths (I)......................................................................................................................32 Microinjection (I)....................................................................................................................................32 BrdU labeling (I, II) ................................................................................................................................32 Transmission Electron Microscopy (II)..................................................................................................33 TUNEL labeling (III) ..............................................................................................................................33 Whole-mount Immunostaining (III)........................................................................................................33 Whole-mount in situ Hybridization (III) ..................................................................................................33 VEGF Concentration Determination (III) ...............................................................................................33 Histology (II, III, IV) ...............................................................................................................................34 In situ Hybridization (III, IV)...................................................................................................................34 Laser Micro-dissection (IV) ...................................................................................................................34 Human Peutz Jeghers Syndrome Tumor Samples (IV)........................................................................34 RESULTS AND DISCUSSION...........................................................................................................35 Targeted disruption of Mat1 Leads to Embryonic Lethality (I) ..............................................................35 Mat1 is required for viability in mitotic but not post-mitotic embryonic lineages (I) ...............................35 Mat1 specifically regulates the steady state levels of Cdk7 and cyclin H (I).........................................36 Mat1 modulates RNA polymerase II CTD phosphorylation (I) ..............................................................36 Transcription and translation of an exogenous construct in Mat1 cells (I) ..........................................37 Mat1 is required for S phase entry in trophoblast giant cells (I)............................................................37 Generation of Mat1 loxP conditional (flox) mice (II) ..............................................................................37 Mat1 is required for the viability of mitotic germ cells (II) ......................................................................38 Targeted disruption of Mat1 in myelinated Schwann cells (II) ..............................................................38 Functional Analysis of Cell Cycle Regulators Lkb1 and Mat1 in Genetically Engineered Mice 5 Remyelination by non-myelinated Schwann cells (II) ...........................................................................39 Targeted disruption of Lkb1 Leads to Midgestation Embryonic Lethality (III) .......................................39 Embryonic and Extraembryonic Vascular Defects in Lkb1 Mice (III) ..................................................40 Deregulation of VEGF in Lkb1 tissues and cultured cells (III) ............................................................41 Lkb1 functions as is a tumor suppressor in mice (IV) ...........................................................................41 Polyposis in Lkb1 mice models Peutz-Jeghers syndrome (IV) ..........................................................42 No evidence for genetic or epigenetic inactivation of Lkb1 in Lkb1-associated polyps (IV)...............42 Haploinsufficiency of Lkb1 in polyposis of Lkb1 mice (IV)..................................................................43 Induction of cyclooxygenase-2 in murine and PJS polyps (IV).............................................................44 CONCLUDING REMARKS AND FUTURE DIRECTIONS ............................................................... 45 ACKNOWLEDGEMENTS.................................................................................................................. 48 REFERENCES................................................................................................................................... 50