Functional Analysis of Cell Cycle Regulators Lkb1 and Mat1 ... · Mat1 resulting in reproductive...

Helsinki University Biomedical Dissertation No.(15)

Functional Analysis of Cell Cycle Regulators Lkb1and Mat1 in Genetically Engineered Mice

Derrick J. Rossi

Molecular and Cancer Research ProgramHaartman Institute & Biomedicum Helsinki

University of HelsinkiFinland

Academic DissertationTo be publicly discussed with the permission of the

Medical Faculty of The University of Helsinki,On September 26th at 12 noon in Lecture Room 2, Biomedicum Helsinki

HELSINKI 2002

Derrick J. Rossi

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Supervised by: Dr. Tomi P.Mäkelä, M.D., Ph.D.Molecular and Cancer Biology ProgramHaartman Institute & Biomedicum HelsinkiUniversity of HelsinkiHelsinki, Finland

Reviewed by: Dr. Seppo Vainio, Ph.D.Department of BiochemistryBiocenter OuluUniversity of OuluOulu, Finland

Dr. Juha Partanen, Ph.D.Institute of BiotechnologyUniversity of HelsinkiHelsinki, Finland

Official Opponent: Dr. Rene Medema, Ph.D.Department of Molecular BiologyNetherlands Cancer InstituteAmsterdam, The Netherlands.

ISBN 952-10-0660-9ISBN 952-10-0661-7ISSN 1457-8433Multi Print OyHelsinki 2002

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

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The White Whale swam before him as the monomaniac incarnation of all those

malicious agencies which some deep men feel eating in them, till they are left living

on with half a heart and half a lung. That intangible malignity which has been from

the beginning; to whose dominion even the modern Christians ascribe one-half of

the worlds; which the ancient Ophites of the east reverenced in their statue

devil—Ahab did not fall down and worship it like them; but deliriously transferring

its idea to the abhorred white whale, he pitted himself, all mutilated, against it. All

that most maddens and torments; all that stirs up the lees of things; all truth with

malice in it; all that cracks the sinews and cakes the brain; all the subtle

demonisms of life and thought; all evil, to crazy Ahab, were visibly personified, and

made practically assailable in Moby Dick. He piled upon the whale’s white hump

the sum of all the general rage and hate felt by his whole race from Adam down;

and then, as if his chest had been a mortar, he burst his hot heart’s shell upon it.

Herman Melville, Moby Dick

For Agnes and Fred

Derrick J. Rossi

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CONTENTSABBREVIATIONS ................................................................................................................................6

LIST OF ORIGINAL PUBLICATIONS .................................................................................................7

ABSTRACT ..........................................................................................................................................8

REVIEW OF THE LITERATURE........................................................................................................11

1 THE CELL CYCLE ...........................................................................................................................111.1 Cdks are Mediators of the Cell Cycle ..................................................................................111.2 Cellular Targets of Cdks ......................................................................................................121.3 Cdk Regulation.....................................................................................................................131.4 Cdk activating kinases (CAKs).............................................................................................14

2 AT THE CROSSROADS OF THE CELL CYCLE, DNA REPAIR AND TRANSCRIPTION...............................152.1 Transcription factor IIH (TFIIH) ............................................................................................152.2 Substrates of TFIIH kinase ..................................................................................................162.3 Ménage à trois (Mat1)..........................................................................................................17

3 CANCER AND INHERITED CANCER SUSCEPTIBILITY SYNDROMES ......................................................173.1 Oncogenes...........................................................................................................................193.2 Tumor Suppressors: Caretakers, Gatekeepers and Landscapers ......................................20

4 PEUTZ-JEGHERS SYNDROME ..........................................................................................................224.1 Increased cancer risk in PJS ...............................................................................................234.2 Mutations of LKB1 underlie PJS ..........................................................................................244.3 Biallelic inactivation of LKB1 in tumorigenesis? ..................................................................254.4 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) ............................................29PCR Genotyping (I, II, III, IV) ................................................................................................................29Protein Analysis (I, II, III, IV) .................................................................................................................30Immunofluoresence and Immunohistochemistry (I, II, III, IV)................................................................32Blastocyst Outgrowths (I)......................................................................................................................32Microinjection (I)....................................................................................................................................32BrdU labeling (I, II) ................................................................................................................................32Transmission Electron Microscopy (II)..................................................................................................33TUNEL labeling (III) ..............................................................................................................................33Whole-mount Immunostaining (III)........................................................................................................33Whole-mount in situ Hybridization (III) ..................................................................................................33VEGF Concentration Determination (III) ...............................................................................................33Histology (II, III, IV) ...............................................................................................................................34In situ Hybridization (III, IV)...................................................................................................................34Laser Micro-dissection (IV) ...................................................................................................................34Human Peutz Jeghers Syndrome Tumor Samples (IV)........................................................................34

RESULTS AND DISCUSSION...........................................................................................................35

Targeted disruption of Mat1 Leads to Embryonic Lethality (I) ..............................................................35Mat1 is required for viability in mitotic but not post-mitotic embryonic lineages (I) ...............................35Mat1 specifically regulates the steady state levels of Cdk7 and cyclin H (I).........................................36Mat1 modulates RNA polymerase II CTD phosphorylation (I) ..............................................................36Transcription and translation of an exogenous construct in Mat1-/- cells (I) ..........................................37Mat1 is required for S phase entry in trophoblast giant cells (I)............................................................37Generation of Mat1 loxP conditional (flox) mice (II) ..............................................................................37Mat1 is required for the viability of mitotic germ cells (II) ......................................................................38Targeted disruption of Mat1 in myelinated Schwann cells (II) ..............................................................38


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Remyelination by non-myelinated Schwann cells (II) ...........................................................................39Targeted disruption of Lkb1 Leads to Midgestation Embryonic Lethality (III) .......................................39Embryonic and Extraembryonic Vascular Defects in Lkb1-/- Mice (III) ..................................................40Deregulation of VEGF in Lkb1-/- tissues and cultured cells (III) ............................................................41Lkb1 functions as is a tumor suppressor in mice (IV) ...........................................................................41Polyposis in Lkb1+/- mice models Peutz-Jeghers syndrome (IV) ..........................................................42No evidence for genetic or epigenetic inactivation of Lkb1 in Lkb1+/--associated polyps (IV)...............42Haploinsufficiency of Lkb1 in polyposis of Lkb1+/- mice (IV)..................................................................43Induction of cyclooxygenase-2 in murine and PJS polyps (IV).............................................................44

CONCLUDING REMARKS AND FUTURE DIRECTIONS ............................................................... 45

ACKNOWLEDGEMENTS.................................................................................................................. 48

REFERENCES................................................................................................................................... 50

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ABBREVIATIONS

Akt protein kinase BATP adenosine triphosphateBMP bone morphogenetic proteinCAK Cdk-activating kinaseCdk cyclin dependent kinaseCGH comparative genomic hybridizationCKI Cdk-inhibitorCOX-2 cyclooxygenase-2CTD carboxy terminal domainC-terminus carboxy terminusDNA deoxyribonucleic acidDMEM Dulbecco´s modified Eagle´s mediumE embryonic dayECL enhanced chemiluminescenceES cell embryonic stem cellFCS fetal calf serumFITC fluorescein isothiocyanateflk-1 fetal liver kinase 1 (VEGF receptor 2)flt-1 fms-like tyrosine kinase 1 (VEGF receptor 1)Ink4A-D Inhibitors of Cdk4/6LOH loss of heterozygosityMEF mouse embryonic fibroblastsmRNA messenger ribonucleic acidN-terminus amino terminusNPAT nuclear protein mapped to the AT locusneo neomycinNER nucleotide excision repairpar-4 partition gene-4 (C. elegans)PCR polymerase chain reactionPBS phosphate buffered salinePFA paraformaldehydePJS Peutz-Jeghers syndromepol II RNA polymerase IIRb retinoblastoma susceptibility proteinSDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresisTEM transmission electron microscopyTFIIH transcription factor IIHTGF transforming growth factortk thymidine kinaseTUNEL terminal deoxynucleotidyl transferase–mediated dUTP nick end-labelingVEGF vascular endothelial growth factorXeek1 Xenopus egg and embryo kinase-1


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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original publications.

I Rossi, D.J., Londesborough, A., Korsisaari, N., Pihlak, A., Lehtonen, E.,Henkemeyer, M., Mäkelä, T.P.. Inability to enter S phase and defective RNApolymerase II CTD phosphorylation in mice lacking Mat1. EMBO J. 20,2844-2856, 2001

II Korsisaari, N.*, Rossi, D.J.*, Paetau, A., Charnay, P., Henkemeyer, M.,Mäkelä, T.P. Conditional ablation of the Mat1 subunit of TFIIH in Schwanncells provides evidence that Mat1 is not required for general transcription. J.of Cell Sci., In press

III Ylikorkala, A.*, Rossi, D.J.*, Korsisaari, N., Luukko, K., Alitalo, K.,Henkemeyer, M., Mäkelä, T.P.. Vascular Abnormalities and Deregulation ofVEGF in Lkb1-Deficient Mice. Science 293:1323-6, 2001

IV Rossi, D.J.*, Ylikorkala, A.*, Korsisaari, N., Salovaara, R., Luukko, K.,Launonen, V., Henkemeyer, M., Ristimäki, A., Aaltonen, L.A., Mäkelä, T.P.Induction of Cyclooxygenase-2 in a mouse model of Peutz-Jegherspolyposis. Proc. Natl. Acad. Sci. USA 99, 12327-12332

*) Equal contribution

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ABSTRACT

Cell division lies at the foundation of the biology of all organisms. Single cellorganisms employ cell division as a means to propagate while multi-cellularorganisms require an enormous amount of cellular proliferation during embryonicdevelopment in order to establish the body plan and expand cell numbers for organand tissue building. Cell proliferation is equally important in the adult organism as itis required to maintain organ and tissue homeostasis by replacing cells that areeither lost or die.

The basic scheme of cell proliferation is rooted in a multi-step process whereby anexternal mitogenic growth signal acts upon a quiescent cell to invoke a cascade ofmolecular events resulting in a proliferative state. Mitogenic signals are received bybinding to transmembrane receptors which then transmit signals through thecytoplasm utilizing a wide variety of signal transduction molecules. Growth signalsultimately converge in the nucleus where a subset of molecules that coordinate celldivision are activated. In this study my colleagues and I have experimentallyaddressed the function of two distinct regulators of the cell proliferation: Mat1 andLkb1.

Cell cycle progression is mediated in part by the coordinated activity of cyclin-dependent kinases (Cdks). Cdks are themselves regulated by several mechanismsincluding an activating phosphorylation that is required for full Cdk activity. Theenzymes that catalyze this activation are known as Cdk-activating kinases orCAKs. In mammals, CAK activity appears to be a function of a trimeric complexcomprised of Cdk7, cyclin H and Mat1. Intriguingly, the Cdk7-cyclin H-Mat1 trimeralso provides the kinase activity of the 9-subunit basal transcription factor TFIIH,where it is involved in the phosphorylation of the C-terminal domain (CTD) of thelarge subunit of RNA polymerase II (pol II).

In order to address the role of Mat1 in vivo, we have generated several targetedalleles of Mat1 by homologous recombination in ES cells. We report that Mat1 isrequired for murine embryonic development as disruption of Mat1 leads to peri-implantation lethality. In culture, early Mat1-/- embryos gave rise to viable post-mitotic trophoblast giant cells while mitotic lineages failed to proliferate and survive.In contrast to wild-type trophoblast giant cells, Mat1-/- cells were defective inendoreduplication and became arrested in the endocycle. Additionally, Mat1-/- cellsexhibited defects in phosphorylation of the C-terminal domain (CTD) of RNApolymerase II. These results provide evidence for the requirement of murine Mat1both in cell cycle progression and in modulating CTD phosphorylation.

To further characterize Mat1 in adult mitotic and post-mitotic lineages, a Cre/loxPstrategy was utilized to conditionally target Mat1 in the adult germline and in


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myelinated Schwann cells. This was achieved by crossing conditional Mat1animals to mice expressing the Cre recombinase under the control of theendogenous Krox-20 promoter. We found that, much like embryonic mitoticlineages, the germ cell lineage was rapidly rendered non-viable upon disruption ofMat1 resulting in reproductive infertility of the targeted animals. Post-mitoticSchwann cells on the other hand were fully able to complete the normal process ofmyelination in the absence of Mat1. However, with time, the post-mitotic Schwanncells also succumbed to Mat1 disruption resulting in a progressive neuromuscularatrophy in the targeted animals. Interestingly, analysis of this phenotype suggestedthat non-myelinated Schwann cells are capable of remyelinating demyelinatedaxons in this system.

Evidence that the serine-threonine kinase LKB1 is involved in regulating someaspect of cell proliferation has its origin in the identification of LKB1 as a tumorsuppressor gene mutated in Peutz-Jeghers syndrome (PJS). PJS is a rareinherited disease in which patients develop gastrointestinal hamartomatous polypsin early adulthood, exhibit mucocutaneous pigmentation on the skin and oralmucosa, and have an increased risk of developing cancers in later life. Inactivatingmutations of LKB1 underlie the majority of Peutz-Jeghers families although themechanisms by which LKB1 mutation predisposes to tumor formation and cancerdevelopment are unknown.

We have attempted to address the function of murine Lkb1 in vivo by geneticallyengineering mice with targeted Lkb1 alleles. We found that homozygous disruptionof Lkb1 led to embryonic lethality at mid-gestation indicating that Lkb1 is essentialfor murine development. Closer examination of embryonic development revealedthat Lkb1-/- conceptuses exhibit severe defects in both embryonic and extra-embryonic vasculature which was coupled with a tissue specific-deregulation ofVEGF. Moreover, when Lkb1-/- cells were cultured, they exhibited increasedexpression of VEGF suggesting that loss of Lkb1 may confer an increasedangiogenic potential on some cell types. This finding could therefore provide arationale to explain why loss of LKB1 in human PJS patients predisposes to tumordevelopment and cancer.

As Lkb1 heterozygous animals represent the genetic equivalent of human PJSpatients with germline LKB1 mutations, we therefore wanted to determine if Lkb1+/-

animals exhibited any of the phenotypes characteristic of PJS. As they aged, weobserved that these mice develop gastrointestinal polyps that were histologicallyindistinguishable from polyps resected from PJS patients. These results establishthat murine Lkb1 functions as a tumor suppressor and that Lkb1+/- mice modelhuman PJS polyposis. Surprisingly, we found that biallelic inactivation of Lkb1 wasnot associated with polyp formation indicating that Lkb1 can be haploinsufficient fortumor suppression. Analysis of the molecular mechanisms underlying Lkb1+/-

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polyposis revealed that cyclooxygenase-2 (COX-2) was highly upregulated inmurine polyps. Subsequent examination of a large series of human PJS polypsrevealed that COX-2 was also highly upregulated in the majority of these polypssuggesting that COX-2 inhibitors could be used to lower the tumor burden in PJSpatients.


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REVIEW OF THE LITERATURE

1 The Cell Cycle

Most somatic cells that are actively cycling pass through four distinct phases of thecell cycle. During the G1 phase, protein and RNAs are synthesized and the cellmonitors its environment, and its own size for signals for progression. S phasemarks the period of the cell cycle in which the DNA comprising the genome isreplicated. G2 denotes the phase of the cycle between S phase and mitosis inwhich the cell ensures that the DNA has been properly replicated and readies fordivision. M phase demarcates the mitotic phase of the cell cycle whereby thereplicated chromosomes are segregated into separate nuclei followed by celldivision.

Although the details vary depending on cell type, the cell cycle is comprised of theset of processes needed to produce a pair of genetically identical daughter cells.To do this, the cell must first faithfully replicate its genome, and, for the vastmajority of cells, double its mass and duplicate all of its cytoplasmic organellesbefore division. In certain types of specialized cells however, DNA synthesis occursby a process known as endoreduplication whereby successive rounds of G and Sphases proceed without intervening mitosis (Barlow et al., 1972). As aconsequence, endocycling cells acquire vast quantities of DNA in their nucleithereby becoming polyploid (Varmuza et al., 1988). Passage through both themitotic cell cycle and the endocycle relies on the coordinated activity of a largenumber of molecules to ensure fidelity. When the mechanisms that govern the cellcycle become deregulated, the consequences on the cell may be manifested in celldeath, uncontrolled growth or aberrant differentiation.

1.1 Cdks are Mediators of the Cell Cycle

Progression through the cell cycle is mediated by the sequential activation ofcyclin-dependent kinases (Cdks) (Figure 1). Cdks function by phosphorylatingcellular targets which in turn coordinate passage through the cell cycle. Firstidentified by genetic analysis of the yeast cell cycle, and in studies on frog andmarine invertebrate embryos, Cdks have since been recognized to lie at the heartof the cell cycle in all eukaryotic organisms (Nurse, 1990; Hunt, 1991; Norbury andNurse, 1992).

Cdks are heterodimeric protein complexes comprised of a catalytic kinase domainand a regulatory cyclin subunit. The kinase subunit of Cdks conforms to the basicprotein kinase structural motif consisting of a β-sheet-rich amino terminal lobe andan α-helix-rich carboxy terminal lobe with the catalytic kinase domain lying between

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the two (Pavletich, 1999). To date, at least nine different Cdks have been identifiedin mammalian cells (Cdk1-9). The cyclin subunits of Cdks are required for Cdkcatalytic activity and appear to play a role in dictating the specificity of Cdk activity(Roberts, 1999). At least 16 different cyclins have been identified in mammaliancells (Johnson and Walker, 1999).

Figure 1. Cdk Activity Through the Mammalian Cell CycleThe kinase activity of the indicated Cdk/cyclin pairs throughout the different phases of the cell cycleis approximated by the width of the graded bands. Adapted from (Lodish, 2000).

1.2 Cellular Targets of Cdks

Despite their central role in cell cycle progression surprisingly little is known abouthow the activity of Cdks modulate the function of their biological targets. Twonotable exceptions are the retinoblastoma susceptibility protein family (RB), andthe nuclear protein mapped to the AT locus (NPAT).

Rb is a nuclear phosphoprotein that functions to arrest cells in the G1 phase of thecell cycle (Weinberg, 1995; Harbour and Dean, 2000). One of the ways that Rbexerts this suppressive effect is by binding to and sequestering E2F transcriptionfactors thereby rendering them unable to transactivate genes critical for cell cycleprogression (Dyson, 1998). In its hypophosphorylated form, Rb has a high affinityfor E2F transcription factors that it binds to through a central ‘pocket’ domain. Upon


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phosphorylation by Cdk complexes Rb looses its affinity for E2F thereby liberatinga free pool of E2F which is then able to transactivate target gene promoters andadvance the cell cycle. Interestingly, inactivation of Rb by Cdks appears to be asequential process as evidence shows that Rb is first phosphorylated by Cdk4-cyclin D and Cdk6-cyclin D, and subsequently by Cdk2-cyclin E thereby regulatingRb activity in a coordinated, step-wise fashion (Lundberg and Weinberg, 1998;Harbour et al., 1999; Zhang et al., 2000).

As histone proteins constitute nearly half of the mass the chromatin, histonebiosynthesis during S phase is critical for the advancement of the cell cycle (Ewen,2000). Originally identified as an interacting protein of Cdk2-cyclin E by a proteininteraction screen, NPAT was demonstrated to be phosphorylated by Cdk2-cyclin E and to accelerate the progression from G1 to S phase when ectopicallyexpressed (Zhao et al., 1998). These findings suggested that the interaction ofNPAT with Cdk2-cyclin E had relevance for cell cycle progression. Subsequentstudies demonstrated that the phosphorylation of NPAT by Cdk2-cyclin E isinvolved in NPAT’s ability to positively regulate the transcription of histones H2B,H4 and H3 in a S phase dependent manner (Ma et al., 2000; Zhao et al., 2000).These reports have thereby provided evidence on how the activity of Cdk2-cyclin Ecoordinates one of the larger scale periodic cell cycle industries that take place inmammalian cells: that of histone synthesis.

1.3 Cdk Regulation

Due to their central role in coordinating cell cycle progression, Cdks themselvesare subject to many levels of regulation in response to both intracellular andextracellular signals. Since Cdks are catalytically inactive in the absence of theirrequisite positive regulatory cyclin subunit, the first level of Cdk regulation is cyclinavailability. Most cyclins are unstable and undergo periodic synthesis anddestruction thereby providing the preeminent mechanism by which Cdk activity isregulated (Ekholm and Reed, 2000). Structural analysis has revealed that cyclinspositively regulate Cdk activity upon binding by inducing a conformational changein the Cdk that allows substrate binding and catalysis (Jeffrey et al., 1995).

A further level of Cdk regulation is provided by the action of Cdk-inhibitorymolecules (CKIs). Two families of CKIs have been identified in mammalian cells:the INK4 proteins (inhibitors of Cdk4), which include p16INK4A, p15INK4B p18 INK4C, andp19INK4D, and the Cip/Kip family, which include p21CIP1, p27KIP1 and p57KIP2. TheINK4 family of inhibitors bind directly to Cdk4 and Cdk6 while the Cip/Kip family ofinhibitors bind to Cdk-cyclin heterodimers (Sherr and Roberts, 1995). Theregulation of cell cycle progression by CKIs took on a new level of complexity withthe discovery that while Cip/Kip proteins inhibit the activity of Cdk2-cyclin E and

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Cdk2-cyclin A complexes, they also act as positive regulators of Cdk4/6-cyclin Dcomplexes (Sherr and Roberts, 1999).

Cdks are also tightly regulated at the level of post-translational modification. Whenin complex with their cognate cyclin, Cdks are rendered sensitive tophosphorylation on two adjacent residues (Tyr15 and Thr14) whose phospho-formsinhibit Cdk activity by inducing conformational changes in the catalytic domain(Morgan, 1997). These inhibitory phosphorylations are in turn removed by theCdc25 family of dual-specificity phosphatases (Nilsson and Hoffmann, 2000).

In addition to cyclin binding, most Cdks require phosphorylation on a conservedresidue to become fully active. This phosphorylation, which was first described inyeast (Gould et al., 1991), modifies a conserved threonine in the T-loop to induce aconformational change that facilitates substrate binding (Russo et al., 1996). Thekinases that catalyze this phosphorylation are known as Cdk-activating kinases(CAKs).

1.4 Cdk activating kinases (CAKs)

The first biochemical purification of a CAK activity was achieved in fractionationexperiments in which an isolated kinase activity was shown to be capable ofphosphorylating and activating Cdk1 (Solomon et al., 1992). Subsequentidentification of the proteins comprising this CAK activity revealed a trimericcomplex comprised of Cdk7, cyclin H and Mat1 (Solomon et al., 1993; Tassan et

al., 1994). In vitro, the mammalian Cdk7-cyclin H-Mat1 trimer has been shown tophosphorylate and activate Cdk1, Cdk2, Cdk3, Cdk4, and Cdk6 when in complexwith their cognate cyclin partners (Kaldis, 1999).

Perhaps contrary to expectations, the CAK activity in the budding yeastSaccharomyces cerevisiae was found to be provided by a monomeric kinasenamed Cak1/Civ1 with surprisingly little homology to mammalian CAK (Espinoza et

al., 1996; Kaldis et al., 1996; Thuret et al., 1996). This finding fueled a debatewherein it was suggested that a similar single subunit CAK might also be active inmammalian cells and that the CAK activity provided by Cdk7-cyclin H-Mat1 mightactually represent in vitro artifact. The CAK debate was further complicated when itwas discovered that in the fission yeast Schizosaccharomyces pombe, both theCdk7 related Mcs6 kinase complex as well as a single subunit kinase Csk1possessed CAK activity in vivo (Hermand et al., 1998; Lee et al., 1999; Hermand et

al., 2001). To date, the best evidence that Cdk7-cyclin H-Mat1 functions as a CAKin metazoan species comes from experiments with the Drosophila Cdk7 homologwhich demonstrated that Cdk7 kinase activity was required for the proper activationof mitotic Cdk-cyclins in vivo (Larochelle et al., 1998).


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2 At the Crossroads of the Cell Cycle, DNA Repair andTranscription

Shortly after their identification as Cdk activators, Cdk7-cyclin H-Mat1 - along withthe homologous Kin28-Ccl1-Tfb3 complex of S. cerevisiae - were identified to becomponents of the basal transcription factor TFIIH (Feaver et al., 1994; Serizawaet al., 1995; Shiekhattar et al., 1995). This finding was intriguing in its suggestionthat one kinase activity might be involved in mediating three critical cellularprocesses; cell cycle progression, basal transcription and transcription couplednucleotide excision repair (NER). At the same time however, these discoveriesadded fuel to the debate surrounding the physiological role of Cdk7-cyclin H-Mat1,as it became apparent that the actual in vivo capacities of homologous kinasesfrom different species exist. Notably, the Kin28-Ccl1-Tfb3 complex of S. cerevisiae

was shown to function only in TFIIH-mediated transcription and not in Cdkactivation (Cismowski et al., 1995; Valay et al., 1995).

2.1 Transcription factor IIH (TFIIH)

The basal transcription factor TFIIH is known to function in RNA polymerase II(pol II) mediated transcription where, in concert with at least five other basaltranscription factors (TFIIB, TFIID, TFIIE and TFIIF), it is critically involved intranscription initiation (Roeder, 1996). During transcription initiation TFIIH has beenshown to catalyze the ATP-dependent formation of the open complex as well asbeing involved in efficient promoter escape by suppression of early pol II elongationintermediates (Dvir et al., 2001). TFIIH is also known to function in transcription-coupled nucleotide excision repair (Citterio et al., 2000).

Structure/function studies have indicated that TFIIH can be resolved into severalfunctional subcomplexes; a core TFIIH complex comprised of five subunits (p44,p52, p62, p34 and XPB), and the kinase complex of Cdk7, cyclin H and Mat1 (Egly,2001). The remaining XPD subunit can be found associated with either thecore TFIIH or with the kinase subunit and seems to be the means by which thekinase subunit associates with the core to form the nine subunit TFIIH holoenzyme(Busso et al., 2000).

Recently, 3-dimensional models of mammalian and yeast TFIIH have beendetermined with the use of electron microscopy images of purified TFIIHsubcomplexes (Chang and Kornberg, 2000; Schultz et al., 2000). These modelssuggest that the core subunits of TFIIH form a ring-like structure, which encircle acentral cavity large enough to accommodate a double stranded DNA molecule. The

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kinase subunit appears to contact the ring structure on one discreet surface to forma protruding bulge (Schultz et al., 2000).

In addition to the kinase activity provided by Cdk7-cyclin H-Mat1, TFIIH alsocontains two additional catalytic subunits, XPD and XPB, both of which are ATP-dependent DNA helicases. Of these, XPB appears to have a critical role both inpromoter melting at transcription initiation, and in DNA repair (Egly, 2001).Mutations disabling XPB helicase activity abolish both of these functions (Tirode et

al., 1999). XPD on the other hand seems to be less critical for promoter clearanceas demonstrated by systems in which mutations that disrupt XPD helicase activityare still capable of in vitro transcription albeit at sub-optimal levels (Tirode et al.,1999).

The role of TFIIH kinase in NER is still in debate with some studies suggesting thatkinase activity negatively regulates NER (Araujo et al., 2000) while others havesuggested that TFIIH kinase activity plays no role in this aspect of TFIIH function(Svejstrup et al., 1995). The importance of both TFIIH helicases in NER however isunderscored by the fact that mutations in XPD or XPB can lead to the human DNArepair deficiency syndromes Xeroderma pigmentosum, Cockayne syndrome andtrichothiodystrophy (Egly, 2001; Lehmann, 2001).

2.2 Substrates of TFIIH kinase

The most extensively studied substrate of TFIIH kinase activity is the heptapeptiderepeat sequence (consensus YSPTSPS) which comprises the C-terminal domain(CTD) of the large subunit of RNA polymerase II (Rpb1). In vitro, TFIIH kinaseactivity is primarily directed at serine 5 (Ser-5) of the heptapeptide repeat (Gebaraet al., 1997; Sun et al., 1998; Trigon et al., 1998), although there is some evidencethat serine at position 2 (Ser-2) might also be phosphorylated (Watanabe et al.,2000).

Accumulating evidence suggests that the primary role of CTD phosphorylationduring pol II-mediated transcription is in the recruitment of proteins necessary forproper processing of the nascent transcript. Numerous transcript maturationproteins such as polyadenylation factors (McCracken et al., 1997b), mRNA cappingfactors (McCracken et al., 1997a) and splicing factors (Misteli and Spector, 1999)have all been shown to associate with phosphorylated forms of the CTD.

Numerous other proteins have emerged which have the potential of beingphysiologically relevant substrates of TFIIH kinase activity. These include p53 (Koet al., 1997), the POU domains of the Oct factors (Inamoto et al., 1997), retinoicacid receptor alpha (Rochette-Egly et al., 1997), and estrogen receptor alpha(Chen et al ., 2000). It seems likely however that phosphorylation of at least some


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of these proteins may be catalyzed by the Cdk7-cyclin H-Mat1 trimer outside of thecontext of TFIIH.

2.3 Ménage à trois (Mat1)

The initial observations that Mat1 co-purified with Cdk7 and cyclin H instoichiometric amounts marked the first example of a third protein partner positivelyimpinging upon the sanctity of the Cdk-cyclin binary complex (Devault et al., 1995;Fisher et al., 1995; Tassan et al., 1995). However, the roles of Mat1 in the trimericCdk7-cyclin H-Mat1 kinase, and as a subunit of TFIIH are only beginning to beunderstood.

Originally, Mat1 was shown to enhance cyclin H and Cdk7 complex formation inthe absence of Cdk7 T-loop phosphorylation and was therefore suggested to act asan assembly factor for the CAK kinase (Devault et al., 1995; Fisher et al., 1995;Tassan et al., 1995). A higher order function for Mat1 in protein complex assemblywas revealed by structure/function studies which suggests that Mat1 physicallymediates the interaction between Cdk7-cyclin H-Mat1 and core TFIIH throughcontact with the XPD helicase (Busso et al., 2000; Sandrock and Egly, 2001).

Several studies have suggested a role for Mat1 in the control of substratespecificity whereby Mat1 shifts the kinase activity of dimeric Cdk7-cyclin H fromCdk substrates towards CTD substrates (Inamoto et al., 1997; Rossignol et al.,1997; Yankulov and Bentley, 1997). These observations are consistent with afundamental role for Mat1 in linking the Cdk7-cyclin H-Mat1 trimer to core TFIIH.

The primary amino acid sequence of Mat1 reveals the presence of two conservedstructural domains: a canonical RING finger domain and a coiled-coil domain.Structure/function studies have suggested that the N-terminally located RINGfinger domain of Mat1 is crucial for transcriptional activation and is involved in CTDphosphorylation (Busso et al., 2000). The central coiled-coil domain appears tomediate the contact of Mat1 with TFIIH through interaction with XPD (Busso et al.,2000). The C-terminal sequences Mat1 appear to be sufficient to mediate theassembly of Cdk7-cyclin H-Mat1 trimers (Tassan et al., 1995; Busso et al., 2000).

3 Cancer and Inherited Cancer Susceptibility Syndromes

During the last century, cancer has emerged as one of the leading causes ofmortality worldwide. In particular, a dramatic rise in cancer incidence has beenobserved in developed nations (Doll, 1978). This is almost certainly due in part tothe fact that as health care improves, so too do people live longer and thus thelikelihood of developing cancer over a lifetime is increased. Indeed, a striking link

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between advanced age and increased incidence of cancer has clearly emerged(Piantanelli, 1988) (Figure 2). It has been proposed that cancer incidence mayincrease with age as a result of the combined effects of mutation load, epigeneticregulation, telomere dysfunction and altered stromal milieu (DePinho, 2000).

Figure 2. Cancer incidence as a function of age.Incidence of invasive cancer plotted against age ranges of men and women. Adapted from(Piantanelli, 1988).

Although aging is clearly an important determinant of cancer risk, epidemiologicalstudies have shown that a wide variety of external factors such as smoking, diet,and infectious pathogens, along with a myriad of factors attributable to theworkplace and the environment, can also result in an increased cancer risk (Peto,2001). No one example better illustrates this point more succinctly than thedramatically elevated risk of developing lung carcinoma that accompanies longterm cigarette smoking (Doll and Hill, 1950; Wynder and Graham, 1950). It is nowknown that many of the agents that elevate cancer risk contain components thatincur DNA damage and result in genetic lesions (Loechler, 1996). Such mutationscontribute to cancer development by a variety of mechanisms such as disabling theregular growth control of the cell (loss-of-function mutations), or by selectively


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facilitating the growth of cancerous cells or their precursors (gain-of-functionmutations).

Well before the actual molecular mechanisms had been revealed, clinicalobservation and histopathological analysis had shown that cancer developsthrough a multi-step process (Figure 3) (Foulds, 1954; Stenback, 1980). A greatdeal of experimental evidence from human and animal systems has subsequentlydemonstrated that the process by which normal cells develop into malignantcancerous cells involves the successive acquisition of genetic alterations(Vogelstein et al., 1988; Vogelstein and Kinzler, 1993).

Although there are over 100 distinct types of cancer and subtypes of tumorsdescribed in humans, it has been proposed that most (and perhaps all) result fromalterations in six essential aspects of cell physiology: self-sufficiency in growthsignals, insensitivity to growth inhibitory signals, evasion of apoptosis, limitlessreplicative potential, sustained angiogenesis, and tissue invasion and metastasis(Hanahan and Weinberg, 2000). As each of these capabilities is acquired, a keyphysiological barrier to cancer development is breached, and the cell is furthered inits progression to malignancy. A vast body of literature characterizing the precisegenetic alterations underlying the development of many cancer types has emergedwith the bulk of the culpability falling onto two categories of genetic lesion: thosethat activate oncogenes, and those that inactivate tumor suppressor genes.

Figure 3. Multi-Step Progression to Cancer.Histopathological changes observed in tissues progressing from a normal to a metastatic cancerstate. Adapted from (Vogelstein and Kinzler, 1993).

3.1 Oncogenes

Originally identified in retroviruses, oncogenes were first shown to have thecapacity to induce malignant transformation in cultured cells (Bishop et al., 1973;Varmus et al., 1973). Subsequently, homologous counterparts of retroviraloncogenes were discovered to be normally encoded in the genomes of higherorganisms (Stehelin et al., 1976; Hampe et al., 1982). These cellular genes, termedproto-oncogenes, are now known to play wide ranging roles in cell signaling,growth control and differentiation (Blume-Jensen and Hunter, 2001). Mutations thatalter the normal cellular properties of proto-oncogenes underlie the oncogenicpotential that these genes exhibit in cancer development (Parada et al., 1982;

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Tabin et al., 1982; Varmus, 1984). Oncogenic mutations can be manifested in avariety of ways such as overexpression, gene amplification, fusion proteinsresulting from chromosomal translocations, and gain-of-function point mutations(Bishop, 1991).

Of the signaling cascades susceptible to oncogenic transformation, none is aswidely studied as those that emanate from the Ras proto-oncogene. Ras is a small(21 kD) membrane bound monomeric G protein that activates numerous signalingpathways through a variety of different effector molecules such as PI 3-kinase andRaf (McCormick, 1999). Mutations that affect the conformation of Ras to favor itsactive (GTP bound) form lie at the foundation of the oncogenic potential of Ras.Such oncogenic Ras variants are thereby uncoupled from upstream regulators, andare thus able to constitutively manufacture mitogenic and survival signals for thecell (Ayllon and Rebollo, 2000). By doing so, oncogenic Ras provides a degree ofself-sufficiency in terms of mitogenic signals while at the same time contributing tothe ability of the cell to evade apoptosis. The frequency of oncogenic Rasmutations has been found to be very high in human cancer development withapproximately 25% of all human cancers and up to 50% of human coloncarcinomas exhibiting Ras mutations (Vogelstein et al., 1988). These numbers area testament of the magnitude of the growth/survival advantage that oncogenic Rasimparts onto malignant cells.

3.2 Tumor Suppressors: Caretakers, Gatekeepers and Landscapers

Although the vast majority of cancers develop sporadically without a familial basis,a small percentage of cancers, estimated to represent up to 10% of cases, areattributable to inherited components (Ponder, 2001). Familial cancers fall into twogeneral categories. The first of these are ascribed to multiple genetic loci, andpredispose to cancer under the influence of combinations of alleles from numerousloci. These so called, multi-factorial familial cancers do not display Mendelianinheritance and the likelihood of individuals at risk of developing cancer is usuallyweak to moderate. The second class of familial cancers, which account for only 1-2% of all cancers, are usually attributable to a single predisposing locus. Thesemalignancies, known as inherited cancer syndromes, are inherited in dominantMendelian fashion and are often associated with developmental defects and non-neoplastic phenotypes such as benign tumor formation. The relative risk ofdeveloping malignant cancers in inherited cancer syndromes is often very high withlifetime risk assessments of up to 80% for some of these syndromes (Ponder,2001). Inherited cancer syndromes are associated with mutation of a class ofgenes known as tumor suppressors. Although inherited cancer syndromes arequite rare, the tumor suppressors underlying these syndromes are frequently found


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to be somatically mutated in sporadic cancers pointing to the vital role thesemolecules normally play in growth control and differentiation. Tumor suppressorshave been classified into three groups: gatekeepers, caretakers, and landscapers(Kinzler and Vogelstein, 1997; Kinzler and Vogelstein, 1998).

The term ‘gatekeeper’ has been given to genes whose encoded products directlyregulate the growth of tumors through inhibition of cellular proliferation andpromotion of cell death. Inactivation of these genes is rate-limiting for tumordevelopment and it has been suggested that both the maternal and paternal copiesof the gatekeeper must be inactivated in order to promote tumorigenesis (Knudson,1996). It has been proposed that different cell types employ only one (or a few)gatekeepers as the central cellular ‘brake’ thereby providing a rationale for thespecificity of varied tumor types observed in patients (Kinzler and Vogelstein,1997). Thus inactivation of Rb, VHL, NF1, or APC leads to tumors of the retina(Lee et al., 1987), kidney (Latif et al., 1993), Schwann cells (Cawthon et al., 1990;Viskochil et al., 1990; Wallace et al., 1990) and colon (Kinzler et al., 1991; Nishishoet al., 1991) respectively.

A second category of tumor suppressor genes is the so-called ‘caretakers’ whoseencoded products are involved in mediating DNA repair. Caretaker subgroupsinclude proteins involved in; mismatch repair e.g. MSH2/3/6 and MLH1 (Kolodnerand Marsischky, 1999), nucleotide excision repair e.g. XPD and XPB (Hoeijmakers,2001; Svejstrup, 2002), and double-strand break repair e.g. ATM and BRCA1/2(Khanna and Jackson, 2001). In contrast to gatekeepers whose inactivation directlypromotes tumorigenesis, inactivation of caretaker genes indirectly promotesneoplasia by rendering the genome unstable. As a consequence, the mutation rateof all genes is increased including those whose products are involved in tumorprogression (such as gatekeepers and oncogenes). Caretaker inactivationtherefore increases the probability, and accelerates the rate, of neoplastictransformation.

The third class of tumor suppressor genes, termed ‘landscapers’, are perhaps theleast well characterized due to the fact that the underlying genetic defect does notreside in the neoplastic cell population itself but rather in the supporting stromal cellpopulation (Kinzler and Vogelstein, 1998). Mutations in landscaper genes lead tochanges in the tumor microenvironment that facilitate the induction, selection andexpansion of neoplastic populations (Liotta and Kohn, 2001). Indeed, accumulatingevidence suggests that stromal cells can transform adjacent tissues in the absenceof pre-existing tumor cells by inciting phenotypic and genomic changes in adjacentpre-neoplastic epithelial cells (Tlsty and Hein, 2001).

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4 Peutz-Jeghers syndrome

The first clinical description of Peutz-Jeghers syndrome (PJS) was published in1921 by J.L.A. Peutz (Peutz, 1921), and was followed some 18 years later with thepublication of an article by H. Jeghers (Jeghers et al ., 1949). The defining featureof PJS is a combination of gastrointestinal hamartomatous polyposis coupled withabnormal melanin pigmentation on the skin and mucous membranes (Figure 4).PJS segregates in autosomal dominant fashion with an estimated incidence ofbetween 1:8300 to 1:29000 live births (Mallory and Stough, 1987). The Mendeliansegregation of clinical features in PJS families suggests a complete penetrance forPJS (Amos et al., 1997; Hemminki et al., 1997).

The melanin ‘spots’ that are the external hallmark of PJS patients, usually appearin infancy, reach a maximum at puberty, and tend to fade with age. Persistence ofthe pigmentation on the buccal mucosa, however, is common (Jeghers et al.,1949). The lips and oral mucosa are the most common locations for the spots (94%of cases), although they are also frequently found on the hands (74% of cases) andfeet (62% of cases) of PJS patients (Utsunomiya et al., 1975). Histologically, thePJS melanin spots display a basal layer hyperpigmentation that is associated withan increased number of melanocytes (Stratakis et al., 1998). This histologicalfeature distinguishes PJS melanin spots from common freckles, which arepigmented as a result of melanin donation to adjacent keratinocytes.

PJS is typically diagnosed in early adolescence usually as a result of clinicalcomplications that arise from the polyposis which include bleeding (81% of cases),and intussusception and obstruction (86% of cases) (Vasen, 2000). Polyps developalong the full length of the GI tract in PJS patients with the highest frequency foundin the small intestine (Utsunomiya et al., 1975; Burdick and Prior, 1982; Foley et

al., 1988). As a consequence, a large proportion of PJS patients undergonumerous repeat laparotomies to remove symptomatic polyps (Figure 4) (Vasen,2000). Histologically, the vast majority of PJS polyps are hamartomatous inappearance although mixed histologies with adenomatous and hyperplasticcomponents are not uncommon (Hemminki, 1999). The classic hamartomatousPJS polyp is pedunculated and frequently cystic, and is comprised of well-differentiated epithelia with an underlying stromal core of connective tissue. Thestroma typically contains an abundant smooth muscle component, tracks of whichradiate from a central stalk that is contiguous with the muscularis mucosa. Thehistology of the smooth muscle component is very characteristic of PJS and isfrequently used in diagnosis to distinguish PJS from other polyposis syndromessuch as juvenile polyposis (Hemminki, 1999).

Prior to 1963, no gastrointestinal cancers had been reported to be associated withPJS leading to the belief that PJS hamartomas were benign and had little or no


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malignant potential. This notion however was rescinded when histologicalexamination of biopsied polyps revealed regions of both dysplasia and carcinomain situ (Tomlinson and Houlston, 1997). Mounting evidence now suggests that PJSpolyps possess malignant potential which is realized through a hamartoma-adenoma-carcinoma sequence (Burdick and Prior, 1982; Perzin and Bridge, 1982;Foley et al., 1988; Spigelman et al., 1989; Hizawa et al., 1993; De Facq et al.,1995; Defago et al., 1996; Hemminki et al., 1997; Gruber et al., 1998; Wang et al.,1999b).

Figure 4: Characteristic features of Peutz-Jeghers syndrome.Typical melanin pigmented macules on the lips and around the mouth of a Peutz-Jeghers patient(left panel). A pedunculated hamartomatous polyp being surgically removed from a Peutz-Jegherspatient (right panel).

4.1 Increased cancer risk in PJS

Although early studies had failed to demonstrate an increased cancer risk in PJSpatients (Dormandy, 1957; Burdick, 1963), systematic longer term follow up oflarger patient groups began to yield evidence to the contrary (Dozois et al ., 1969;Dodds et al., 1972; Utsunomiya et al., 1975). Conclusive evidence that PJS was acancer predisposition syndrome came in 1987 with the publication of a reportdetailing a 12-year follow-up study of 31 PJS patients in which cancer developed in15 of the patients (48%) (Giardiello et al., 1987). These numbers corresponded to arelative cancer risk assessment in these PJS patients 18 fold greater than thegeneral population. Interestingly, of the 15 cancers described, 11 of these were ofextraintestinal origin indicating that the increased cancer susceptibility was notrestricted to the hamartoma-adenoma-carcinoma sequence proposed toaccompany the polyposis.

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Subsequent studies have confirmed the initial findings of Giardiello et al. althoughthe relative cancer risk assessment of different cancer types has variedconsiderably between the different studies (Foley et al., 1988; Spigelman et al.,1989; Boardman et al., 1998; Westerman et al., 1999). In an attempt to try toensure that the variability in risk assessments reported in the literature would nothamper patient management, a meta-analysis was done in which the findings from6 separate studies were collated and assessed (Giardiello et al., 2000). This study,which included data from 210 PJS patients, reported an overall cancer risk 15.2fold greater than the general population and a 93% cumulative risk of developingcancer between the ages of 15 and 64. The relative risk of developing specifictypes of cancer varied greatly with cancer of the small intestine, stomach andpancreas being the highest (Table 1).

Site Observed

Cases

Relative Risk(Observed/Expected)

CumulativeRisk

(Ages 15-64)

Small Intestine 6 520.0 13.0%

Stomach 10 213.0 29.0%

Colon 15 84.0 39.0%

Esophagus 1 57.0 0.5%

Pancreas 6 132.0 36.0%

Lung 5 17.0 15.0%

Testes 1 4.5 9.0%

Breast 11 15.2 54.0%

Uterus 2 16.0 9.0%

Ovary 4 27.0 21.0%

Cervix 3 1.5 10.0%

All Cancer 66 15.2 93.0%

Table 1: Cancer risk assessment in Peutz-Jeghers syndrome.Meta-analysis of cancer risk data from 210 Peutz-Jeghers syndrome patients compiled from sixindependent studies (adapted from Giardiello et al. 2000).

4.2 Mutations of LKB1 underlie PJS

In 1997, the PJS susceptibility locus was localized to chromosome 19p using acombination of linkage analysis and comparative genomic hybridization (CGH)(Hemminki et al ., 1997). This localization was subsequently confirmed and refinedto a 6 centimorgan region of 19p13.3 (Amos et al., 1997; Mehenni et al., 1997;Nakagawa et al., 1998b; Olschwang et al., 1998). Although most PJS families havemapped to 19p13.3, a smaller subset of families have not suggesting the presenceof additional PJS loci (Mehenni et al., 1997; Olschwang et al., 1998; Olschwang et


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al., 2001). The gene(s) corresponding to the minor PJS loci have yet to beidentified.

Shortly after the localization of the PJS susceptibility locus, two groupssimultaneously reported that germline mutations in a putative serine-threoninekinase mapping at 19p13.3 underlie PJS (Hemminki et al., 1998; Jenne et al.,1998). The identified gene, named LKB1, had been previously cloned from ahuman fetal liver library and entered into the GenBank database in 1996 (GenBankaccession number U63333) (Nezu, 1996). Despite this, Jenne et al. made an ill-advised attempt at renaming the gene STK11 – the STK11 name has howeverfailed to garner wide support in the literature and has instead merely contributed toa glut of problematic nomenclature.

To date, the vast majority of the germline mutations in PJS families are truncations,small deletions or point mutations that are predicted to disable LKB1 kinasefunction (Gruber et al., 1998; Hemminki et al., 1998; Jenne et al., 1998; Nakagawaet al., 1998a; Resta et al., 1998; Wang et al., 1999a; Westerman et al., 1999;Boardman et al., 2000; Yoon et al., 2000; Olschwang et al., 2001).

4.3 Biallelic inactivation of LKB1 in tumorigenesis?

By definition, a recessive tumor suppression gene must undergo biallelicinactivation to promote tumorigenesis. This two-hit genetic criterion, originallyproposed by Knudson in describing the etiology of retinoblastoma (Knudson,1971), has adequately described the recessive tumor suppressor function ofnumerous genes including Rb (Dryja et al., 1984) and p53 (Baker et al., 1989). Thequestion about whether LKB1 is a recessive tumor suppressor has been widelyaddressed in the literature with inconsistent results. Initially, Hemminki et al.

reported that of 16 polyps that they examined by CGH, only 5 of these presentedevidence for loss of the distal tip of the short arm of chromosome 19 (19p)(Hemminki et al., 1997). The CGH data was supported in part by PCR amplificationof micro-satellite markers near 19p with loss of heterozygosity (LOH) reported in3/3 of the polyps screened. These results were interpreted to indicate that the PJSsusceptibility gene did indeed adhere to the criteria of being a recessive tumorsuppressor.

That biallelic inactivation of LKB1 would be rate-limiting for polyp formation hashowever been called into question in subsequent studies with some groupsreporting that LOH of micro-satellite markers mapping near LKB1 is frequentlyobserved in PJS polyps (Gruber et al., 1998; Miyaki et al., 2000), while others havediscovered that LOH of LKB1 may be more rare (Wang et al., 1999b; Entius et al.,2001). It is likely that these disparate results are attributable to technical difficulties

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arising from contaminating tissues and/or PCR artifact. In an attempt to addressthe issue of LKB1 biallelic inactivation using an alternative technique, Rowan et al.examined expression of LKB1 in a large series of hamartomatous PJS polyps by insitu hybridization (Rowan et al., 2000). This study reported loss of LKB1 mRNAexpression in only 1 out of 16 polyps examined, thereby casting further doubt onwhether LOH of LKB1 is an obligate initiating event in PJS polyposis. Interestingly,recent studies have provided evidence suggesting that LOH of LKB1 may be amore consistent feature of advanced PJS malignancies as opposed tohamartomatous polyp formation (Su et al., 1999; Entius et al., 2001).

4.4 Evidence for LKB1 Function

A high degree of sequence homology between genes from different species isoften an indicator of a functional homology shared by the encoded proteins. Todate, putative LKB1 homologues have been identified in the frog Xenopus laevis,the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, andthe mouse Mus musculus. In Xenopus, a protein which shares 84% overall identitywith LKB1, called Xeek1, is highly expressed in the oocyte and fertilized egg butdisappears during gastrulation (Su et al., 1996). Despite the high degree ofsequence identity, the restricted expression pattern of Xeek1 makes it unlikely thatit represents a full functional homologue of LKB1 since the mouse Lkb1 gene(which encodes a protein with 90% identity to human LKB1) is expressedubiquitously throughout murine development (Luukko et al., 1999). The putativehomologue in C. elegans, par-4, shares only 26% identity with LKB1 and isinvolved in establishing asymmetries in early embryonic development that areessential for determining the subsequent developmental fate of daughter cells(Watts et al., 2000).

The primary sequence of the LKB1 gene predicted that it would encode a proteinwith serine-threonine kinase activity (Hemminki et al., 1998; Jenne et al., 1998).This prediction was validated with the demonstration of an autocatalytic kinaseactivity (Ylikorkala et al., 1999). Subsequent work has demonstrated that theautocatalytic activity is principally directed at two threonine residues, Thr336 andThr366 (Sapkota et al., 2002). In addition to its autocatalytic activity, LKB1 has alsobeen shown to phosphorylate p53 in vitro although whether this phosphorylation isphysiologically relevant is unknown (Sapkota et al., 2001). Interestingly, anindependent group demonstrated that LKB1 physically interacts with p53 andfunctions to regulate specific p53-dependent apoptosis pathways (Karuman et al.,2001). These results suggest that a deficiency in apoptosis may be involved in thedevelopment of PJS polyps and malignancy.


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Though no physiological substrates have yet been identified for LKB1, a recentpaper detailing the interaction of LKB1 with a LIP1 (LKB1-interacting protein 1) hassuggested that LKB1 may have functional links to TGFβ or BMP signaling as LIP1is able to mediate a LKB1-LIP1-SMAD4 ternary complex (Smith et al., 2001). ThatLKB1 may be linked to TGFβ or BMP signaling is particularly intriguing consideringthat mutations of both SMAD4 (Howe et al., 1998) and BMPR1A (Howe et al.,2001) are known to give rise to subsets of patients with juvenile polyposissyndrome, a disease which is clinically very similar to PJS.

In addition to the cancer predisposition and polyposis phenotypes of PJS patients,functional evidence that LKB1 is involved in regulating some aspect of cell cycleprogression was provided by Tiainen et al. who showed that reintroduction of LKB1into tumor cell lines that had reduced LKB1 expression and activity resulted in a G1cell cycle arrest (Tiainen et al., 1999). An additional piece of evidence that LKB1controls some aspect of cell cycle regulation comes from a report detailing theinteraction of LKB1 with the cell cycle/chromatin remodeling molecule Brg1,wherein LKB1 was reported to be required for Brg1-mediated growth arrest inSW13 cells (Marignani et al., 2001).

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AIMS OF THE STUDY

This study was undertaken to study the function of Mat1 and Lkb1 in vivo. Theapproach taken involved the generation and characterization of mice withgenetically engineered mutations in the Mat1 and Lkb1 genes. My collaboratorsand I reasoned that such an approach would permit the examination of multipleaspects of Mat1 and Lkb1 function both during embryonic development, and in theadult. In this study we have generated and utilized these animals to address thefollowing:

1. The requirement of Mat1 and Lkb1 during murine embryonicdevelopment.

2. The involvement of Mat1 in the mediation of cell cycle progression and asa modulator of CTD phosphorylation.

3. The differential requirement of Mat1 in mitotic and post-mitotic lineages.

4. The role of Lkb1 in the development of a murine polyposis syndromemodeling Peutz-Jeghers syndrome.

5. The molecular biology underlying murine Lkb1-associated polyposis inorder to identify potential targets for therapy for Peutz-Jeghers syndrome


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MATERIALS AND METHODS

A detailed description of the materials and methods are provided in the originalpublications.

Generation of Genetically Engineered Mat1 (I, II) and Lkb1 Mice (III, IV)

Genomic sequences spanning the Mat1 and Lkb1 loci were cloned and subjectedto restriction mapping and sequencing. For Mat1, one exon encoding the 3’ half ofthe RING finger domain was targeted for ablation utilizing a loxP conditionaltargeting strategy (Figure 5). To generate the null allele of Mat1 (Study I), the loxPconditional target construct was first transformed into a bacterial strain expressingthe Cre recombinase before introduction into ES cells (Buchholz et al ., 1996). ForLkb1, two independent strategies were used to target exons 2 through 7 forablation (Figure 6, panels A and F). All target constructs were linearized andelectroporated into ES cells. Individual ES cell clones (3700 for Mat1, and 3800 forLkb1) were isolated, expanded, frozen and DNAs extracted from these cells werescreened for homologous recombination by Southern blotting. ES cell clones wereconfirmed to be correctly targeted (13 for Mat1, 5 for Lkb1), and cells fromindividual lines were subsequently injected into C57/BL6 blastocysts which werethen surgically implanted into the uterine horns of pseudopregnant females.Chimeric animals were identified by coat color and several males for each strategywere subsequently identified to be transmitting the targeted allele through thegermline by Southern blotting and PCR genotyping. Experimental and control micewere maintained on several heterogeneous genetic backgrounds

PCR Genotyping (I, II, III, IV)

Tail clips from ear-marked animals (3-4 weeks old) were incubated overnight at55oC in 200 µl of buffer containing 50 mM KCl, 0.1mM Tris pH 8.3, 0.2 mM MgCl2,0.1% gelatin, 0.45% IPEGAL CA-630, 0.45% Tween 20 and 1 mg/ml proteinase K.The following day the proteinase K was inactivated by heating the tubes for 100o Cfor 10 minutes followed by PCR using 1 µl of tail prep as a template. PCR reactionswere performed in a thermocycler by heating the reaction at 95o C for 5 minutesfollowed by 35 cycles of 95oC for 50 seconds, 58oC for 50 seconds, 72oC for 50seconds. Reaction products were run on 1.5% agarose gels.

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Figure 5: Generation of a Mat1 loxP conditional targeting vector.The endogenous Mat1 locus spanning the targeted exon (black tab) is schematized at the top of thefigure. Cloning steps in the construction of the vector are indicated with arrows. Restriction sites areabbreviated as follows: B, BamH1; S, Sal1; C, Cla1; H, HindIII; N, Not1; K, Kpn1; ScI, SacI; ScII,SacII; and Xb, Xba1. Introduced sites are in red. The parental pDelboy vector was constructed byD.J. Rossi and is available upon request.

Protein Analysis (I, II, III, IV)

Protein lysates from whole tissue samples were made by dissecting tissues fromsacrificed animals and snap-freezing the samples in liquid nitrogen followed bypulverization with a liquid nitrogen chilled tissue homogenizer. Lysates from tissueculture were prepared directly from PBS washed tissue culture plates on ice. Alllysates were prepared in ELB lysis buffer and protein concentrations weredetermined by spectrophotometry. For Western blotting analysis, samples wereseparated on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes,and immunoblotted followed by detection with ECL reagents. Kinase assays wereperformed according to standard protocols.


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Figure 6. Generation of Lkb1 mutant mice.A Strategy 1 for disrupting exons 2-7 of the murine Lkb1. B Southern blotting with 5' probe. CSouthern blotting with a 3' probe. D PCR genotyping of targeted animals. E PCR genotyping of yolksac DNAs isolated from E9.5 conceptuses. F Strategy 2 for disrupting exons 2-7 of the murine Lkb1.lox P sites are indicated with open triangles. G Southern blotting with 5' probe. H Southern blottingwith 3' probe. I PCR genotyping of targeted animals. J PCR genotyping of yolk sac DNAs isolatedfrom E9.5 conceptuses. Neo, Neomycin selection cassette; TK, thymidine kinase selection cassette;B, BamHI; H, HindIII; R1, EcoRI; ScI, SacI.

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Immunofluoresence and Immunohistochemistry (I, II, III, IV)

Immunofluoresence was performed variably depending upon the antibodies used.Cell culture or freshly dissected tissue samples were fixed in either 3.5% PFA,Ethanol or methanol followed by a permeabilization with either 0.5% Triton X-100 inPBS or with cold methanol. Samples were blocked in 10% FCS in PBS. Primaryand secondary antibodies were left on the samples for varying amounts of timedepending on the experiment. DNA was visualized by staining with Hoechst 33342.Each step was followed by 3 to 4 washes with PBS with a final rinse in waterbefore mounting. Samples on coverslips were mounted onto glass slides forepifluorescence microscopy. Immunohistochemistry of paraffin embedded tissueswas performed according to standard protocols following epitope unmasking bymicrowaving samples at full power for 5 minutes in 10 mM sodium citrate buffer.

Blastocyst Outgrowths (I)

Mat1 heterozygous animals were intercrossed and plugged females weresacrificed 2.5 days post-coital. Morula stage embryos (16-32 cell) were thenflushed from the oviducts and maintained in culture for 48 h. Expanded blastocystswere then transferred to micro-well plates seeded with coverslips and maintained inculture for up to 7 days.

Microinjection (I)

Trophoblast giant cells from blastocyst outgrowths were microinjected with 25 ng/µlof pEGFP-N2 (Clontech) and 0.1 mg/ml Texas Red dextran tracer dye. Cells wereinjected for 0.5 seconds under 120 hPa pressure using an Eppendorf microinjectorand transjector and a Zeiss Axiovert microscope. After injection, cells were washedtwice with media, returned to the incubator and analyzed 24 h later by fluorescencemicroscopy.

BrdU labeling (I, II)

For BrdU labeling of blastocysts and outgrowths, cells were cultured in thepresence of 10 µM BrdU for 16 and 46 hours, respectively. Cells were then fixed in4% PFA, washed with PBS, and treated with 0.5N HCl for 30 minutes followed byimmunofluorescence. For labeling of sciatic nerves, experimental animals wereintraperitoneally injected once a day for 10 days with 50 µg/g body weight of BrdUin 0.9% NaCl, 7 mM NaOH solution. On day 11 sciatic nerves were collected andcryosectioned before immunodetection of BrdU.


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Transmission Electron Microscopy (II)

Sciatic nerves were fixed in 1% PFA, 0.5% glutaraldehyde for 2 hours, treated with1% osmium tetroxide (OsO4), dehydrated in ethanol, and embedded in epoxy resin.Ultra-thin sections (60-90 nm) were cut and stained with uranyl acetate and leadcitrate followed by transmission electron microscopy.

TUNEL labeling (III)

Terminal deoxytransferase-mediated deoxy-uridine nick end-labeling analysis wasperformed using the In Situ Cell Death Detection Kit (Roche) according tomanufacturer’s instructions.

Whole-mount Immunostaining (III)

For whole-mount immunostainings, E8.5 or E9.5 embryos were fixed in 4%paraformaldehyde, bleached for 5 hours in 5% H2O2-95% methanol, and blockedovernight (4OC) in PBS containing 3% milk and 0.1% Triton X-100. After anovernight (4OC) incubation with primary antibodies (PECAM-1, Pharmingen;Smooth muscle actin, Sigma), the embryos were treated with peroxidase-conjugated secondary antibodies, and developed in 3,3´diaminobenzidinetetrahydrochloride (Sigma).

Whole-mount in situ Hybridization (III)

Embryos were fixed in 4% PFA, bleached in 7% H2O2/93% methanol, treated with5 µg/ml proteinase K and post-fixed in 4% PFA/0.2% glutaraldehyde. Digoxygenin-UTP-labeled anti-sense probes were hybridized overnight at 63OC followed byRNaseA/RNaseT1 treatment and blocking in 10% goat serum. Signal was thenvisualized using an alkaline phosphatase conjugated anti-digoxigenin antibody anddetection with BM purple.

VEGF Concentration Determination (III)

For the VEGF analysis, 25,000 MEFs from individual embryos at passage 3 wereplated on 24-well plates and cultured in normoxic (21% O2) or hypoxic (1% O2 )conditions for 24 hours. Conditioned medium was removed and VEGFconcentration was analyzed by enzyme-linked immunosorbent assay (ELISA)(R&D Systems).

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Histology (II, III, IV)

Tissues were fixed over night in 4% paraformaldehyde (PFA) followed bydehydration through an ethanol/xylene series and embedded in paraffin. Sampleswere sectioned at 7 µm and mounted on glass slides. Histological sections werethen deparaffinized, rehydrated and stained with hematoxylin eosin (HE), Herovici’sstain or immunostained and analyzed by light microscopy.

In situ Hybridization (III, IV)

Hybridization was carried out using sense and anti-sense probes made by in vitro

transcription in the presence of 35S-UTP. In situ hybridizations were performedessentially according to standard protocols (Wilkinson et al., 1990) with somemodifications (Luukko et al., 1996).

Laser Micro-dissection (IV)

PFA-fixed, paraffin embedded polyp and control tissues were sectioned at 3 µmonto LPC-membrane coated glass slides (P.A.L.M. Mikrolaser Technologie) anddissected using a Robot-Microbeam laser micro-dissector (P.A.L.M.).

Human Peutz Jeghers Syndrome Tumor Samples (IV)

Polyps from Peutz-Jeghers syndrome patients were surgically resected and fixed in10% formalin. Histological sectioning and staining was done as described abovefor murine tissue samples.


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RESULTS AND DISCUSSION

Targeted disruption of Mat1 Leads to Embryonic Lethality (I)

To study the function of Mat1 in vivo, sequences encoding most of the RING fingerdomain of Mat1 were targeted for disruption in ES cells with the aim of generating anull allele. Targeted ES cell clones were then used to generate chimeric animals,several of which were subsequently found to transmit the targeted allele in thegermline. Western blot analysis on lysates from targeted ES cells using a Mat1antibody revealed no evidence of truncated Mat1 proteins suggesting that thetargeted allele is a true null.

Mat1 heterozygous (Mat1+/-) intercrosses were set and analysis of F1 littersindicated that while both Mat1+/+ and Mat1+/- animals were observed at expectedMendelian frequencies, no homozygote null animals (Mat1-/-) were obtained. Thisresult indicated that disruption of Mat1 leads to embryonic lethality. Analysis ofembryos at various stages of embryonic development indicated that the lethalityassociated with Mat1 disruption occurred shortly after implantation yet beforegastrulation.

As the Mat1 homologue in S. cerevisiae (rig2/tfb3) is an essential gene (Faye et al.,1997) it was therefore surprising that early mammalian embryogenesis wouldproceed in a genetically Mat1 null background. We therefore examined whethersurvival of Mat1-/- embryos to the implantation stage of development might be dueto maternally provided Mat1 by immunostaining pre-implantation embryos (at 8-cell, 16-cell, and blastocyst stages) for Mat1 expression. We observed diminishingMat1 immunoreactivity in Mat1-/- embryos such that by the blastocyst stage, Mat1signal was barely observable.

Taken together, these results indicate that disruption of murine Mat1 leads to peri-implantation lethality and that the timing of the lethality likely reflects the depletionof maternal Mat1 protein below threshold levels required to sustain an essentialfunction.

Mat1 is required for viability in mitotic but not post-mitotic embryoniclineages (I)

To examine more directly the role of Mat1 in cell proliferation and differentiation wefollowed the development of pre-implantation embryos derived from Mat1+/-

intercrosses in blastocyst outgrowth cultures. We found that while both Mat1+/- andMat1+/+ outgrowths established both mitotic inner cell mass (ICM) cells and post-mitotic trophoblast giant cells, the Mat1-/- embryos only gave rise to post-mitotictrophoblast giant cells. This result indicated that Mat1 was required for the viability

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of mitotic embryonic lineages but not post-mitotic embryonic lineages. Moreover,these results allowed us to address the function of Mat1 beyond embryonic lethalityby taking advantage of the blastocyst outgrowth system as a means of generatinga population of Mat1-/- trophoblast giant cells for further analysis. It should be notedthat as Mat1+/- and Mat1+/+ outgrowths are phenocopies of each other and wereboth used as controls in subsequent experiments, they are therefore collectivelyreferred to as Mat1wt.

Mat1 specifically regulates the steady state levels of Cdk7 and cyclin H (I)

As Mat1 has been implicated in stabilizing the Cdk7-cyclin H-Mat1 trimer (Devaultet al., 1995; Fisher et al., 1995; Tassan et al., 1995), we therefore wanted toaddress whether or not the expression of Cdk7 and cyclin H was affected in Mat1-/-

cells. We therefore immunostained outgrown trophoblast cells and found thatimmunoreactivity of both Cdk7 and cyclin H was consistently diminished in theMat1-/- cells compared to control cells. In order to determine whether this result wasspecific to Mat1 loss or instead a manifestation of a more general transcriptionalderegulation, we immunostained for expression of several additional proteinsincluding PCNA, p53, Cdk2, cyclin E, cyclin D1, Cdk6 and the large subunit of RNApolymerase II. We found that the expression of all of these proteins wascomparable in both Mat1-/- and Mat1wt cells. These results indicate that the loss ofmammalian Mat1 does not globally deregulate transcription or translation andsuggest that Mat1 specifically regulates the steady state levels of Cdk7 andcyclin H in vivo.

Mat1 modulates RNA polymerase II CTD phosphorylation (I)

As many studies have implicated the CTD of the large subunit of RNA polymeraseII as a physiological substrate of TFIIH kinase activity, we therefore wanted toanalyze the phosphorylation status of the CTD in Mat1-/- cells. This wasaccomplished by immunofluorescence analysis utilizing monoclonal antibodiesrecognizing specific phosphoepitopes of pol II (Thompson et al., 1989; Patturajanet al., 1998). These experiments indicated that while total pol II expression wasunaffected by the loss of Mat1, phosphorylation at serine 2 (Ser-2) and serine 5(Ser-5) of the heptapeptide repeat of the CTD was diminished in the Mat1-/- cells.These results provide evidence that Mat1 modulates the phosphorylation of pol II invivo either by phosphorylating the CTD directly or by regulating the activity of otherCTD kinases.


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Transcription and translation of an exogenous construct in Mat1-/- cells (I)

In order to more directly assay the ability of Mat1-/- cells to engage in de novo

transcription and translation, an exogenous CMV promoter-driven greenfluorescent protein (GFP) expression plasmid was microinjected into outgrowthtrophoblast cells and assayed for GFP expression the following day. Regardless ofgenotype, all microinjected cells exhibited strong expression of the GFP proteinindicating that Mat1-/- cells were comparable to control cells in their ability toengage in de novo transcription and translation of the exogenous construct.

Mat1 is required for S phase entry in trophoblast giant cells (I)

We noted that the Mat1-/- trophoblast cells in our outgrowth experiments wereconsistently smaller in size and stained less intensely for DNA than control cells.As trophoblast giant cells normally undergo DNA synthesis by endoreduplication(Barlow et al., 1972), our observations suggested that the Mat1-/- cells might bedefective in this process. In order to examine this possibility in detail, the DNAcontent of outgrown cells was quantified by measuring fluorescence intensity ofDNA (Hoechst 33342) stained nuclei. This analysis revealed that the Mat1-/-

trophoblast cells had undergone markedly less DNA synthesis than had controls.Blastocysts and outgrowths were then BrdU labeled to identify cells undergoingDNA synthesis. We found that while both Mat1-/- and Mat1wt blastocysts showed nodifference in DNA synthesis only wild type trophoblasts were actively engaged inDNA synthesis while the Mat1-/- outgrowths were arrested. These results indicatedthat coincident with depletion of maternal Mat1 stores, Mat1-/- cells were unable toenter S phase and were arrested in the endocycle thereby demonstrating anessential role for Mat1 in endocycle progression. Combined with our datademonstrating the transcriptional integrity of the Mat1-/- trophoblast cells, wepropose that the observed endocycle arrest may be due to defects in Cdkactivation.

Generation of Mat1 loxP conditional (flox) mice (II)

To study the function of Mat1 in a tissue specific manner in vivo, a vector designedto conditionally disrupt Mat1 in a loxP/Cre-recombinase-dependent manner wasconstructed and introduced into ES cells (Figure 5). Correctly targeted ES cellclones were then used to generate mice bearing the conditionally targeted allele(Mat1flox). The Mat1flox/flox mice were found to be aphenotypic indicating that theconditional allele had no overt hypomorphic effects. The functionality of the Mat1flox

allele was then tested in vivo by crossing Mat1flox/flox mice to a PGK driven Cre-deleter strain (Lallemand et al., 1998) resulting in the recombined allele beingobserved in all offspring (Mat1+/-). To generate Mat1-/- cell populations, Mat1flox/-

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animals were crossed to Krox-20-Cre knock-in mice (KCN) in which the Crerecombinase is expressed under the control of the endogenous Krox-20 promoter(Voiculescu et al., 2000). The use of this system enabled us to target Mat1 fortissue specific ablation in the mitotic cells of male germ lineage upon sexualmaturation, and in post-mitotic myelinating Schwann cells of the peripheral nervoussystem (Topilko et al., 1994; Voiculescu et al., 2000).

Mat1 is required for the viability of mitotic germ cells (II)

Mat1 was targeted for ablation in the mitotic cells of the male germ lineage bycrossing Mat1flox/- animals with the Krox-20-Cre strain. In the seminiferous tubulesof the testis of mutant animals at 6 weeks post-natal, pycnotic germ cells werereadily observed. Moreover, an underrepresentation or absence of cellscorresponding to the different stages of spermatid differentiation was also noted.These observations were indicative of spermatogonia and spermatocyte cell deathand suggested a requirement of Mat1 for germ lineage viability. This assertion wasconfirmed by examination of the testis of mutant animals at 14 weeks post-natal,which revealed that they were entirely devoid of all germ cells and their derivatives.These results demonstrate that Mat1 is required to maintain the viability of themitotic germ cells.

Targeted disruption of Mat1 in myelinated Schwann cells (II)

Although myelinated Schwann cells are post-mitotic they are nonetheless highlytranscriptionally active particularly during the first month postnatal during whichtime great quantities of transcripts encoding myelin structural components aregenerated (Stahl et al., 1990). By disrupting Mat1 in the Schwann cells at the onsetof myelination we were therefore able to address the role of Mat1 in an activetranscriptional context unencumbered by the demands of cell cycle progression.We found that the histology of Mat1-deficient myelinated Schwann cells wascomparable to controls through the first two months of age. Moreover, the Mat1-deficient cells were found to express several myelination markers at wild typelevels. These data indicated that Mat1-deficient myelinated Schwann cells wereable to assume a differentiated mature myelinated phenotype suggesting that Mat1was not required for the transcription underlying myelination.

At around three months of age however, the mutant animals began to exhibit avariety of symptoms including gait abnormalities, ataxia and muscular atrophy inthe hind limbs suggestive of a neuromuscular neuropathy. Examination of sciaticnerves from mutant animals before symptomatic onset revealed that they werenormal while animals beyond the age of symptomatic onset showed multiple signsof disease pathology. These observations provided evidence that myelinated


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Schwann cells succumb to Mat1 loss at around three months post-natal resulting ina severe demyelination of peripheral axons followed by remyelination.

Taken together, these results suggest that Mat1 may be involved in regulating asubset of genes required for maintaining Schwann cell integrity rather thanmediating an essential function in RNA polymerase II transcriptional activity.

Remyelination by non-myelinated Schwann cells (II)

The extent of remyelination that we observed in the mutant sciatic nerve suggestedthat a proliferative response might underlie the remyelination phenotype. To testthis possibility, in vivo BrdU labeling experiments were done with mutant andcontrol animals followed by double immunofluorescence labeling (BrdU andKrox-20) on sciatic nerve sections to reveal proliferation in the Schwann lineage.These results indicated that the remyelination in mutant sciatic nerves wascoincident with proliferation of the Schwann cell lineage.

The myelinated Schwann lineage of the peripheral nervous system has beenshown to have a great capacity for regeneration in response to injury and bothmyelinated and non-myelinated Schwann cells have been implicated in theremyelination process (Griffin et al., 1990; Messing et al., 1992; Stoll and Muller,1999). As the insult leading to demyelination in our model is the result of thegenetically programmed death of the myelinated Schwann lineage, this suggeststhat the non-myelinating Schwann cell pool must therefore underlie theremyelination observed in our mutant animals.

Targeted disruption of Lkb1 Leads to Midgestation Embryonic Lethality (III)

To study the function of Lkb1 in vivo, sequences encoding exons 2-7 of the murineLkb1 gene were disrupted by homologous recombination in ES cells. Targeted EScell clones were then used to generate chimeric animals, which were subsequentlyfound to transmit the targeted allele in the germline. No live Lkb1 homozygote nullanimals (Lkb1-/-) were recovered from Lkb1 heterozygous (Lkb1+/-) intercrossesindicating that disruption of Lkb1 leads to embryonic lethality. Analysis of embryosthroughout embryonic development revealed that Lkb1-/- embryos beyond E8.5 hadmultiple developmental abnormalities including a failure of the embryo to turn, adefect in neural tube closure, and a hypoplastic or absent first branchial arch. Noviable embryos were recovered past E11.0, indicating that Lkb1 is essential forembryonic development.

The integrity of various developmental lineages in E8.5 and E9.5 Lkb1-/- embryoswas examined by whole-mount in situ hybridization. The expression of themesodermal marker brachyury (Wilkinson et al., 1990) showed that although the

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notochord developed along the full length of the anterior/posterior axis in themutant embryos, it was misaligned and contorted. This was accompanied bydefective somitogenesis, which resulted in dysmorphic protrusive somites, whichfailed to express engrailed 1 (Joyner and Martin, 1987) at E9.5. No pronouncedchanges in the expression of Wnt3A, Fgf8 or Krox-20 were noted, suggestingnormal development of the mesoderm of the tail bud, forebrain and primitive streak,and normal segmentation of the hindbrain.

Embryonic and Extraembryonic Vascular Defects in Lkb1-/- Mice (III)

It was noted that Lkb1-/- embryos at E9.25 had a translucent appearance,suggesting the possibility of vascular defects. We therefore immunostained E8.5and E9.5 embryos with an antibody against platelet endothelial cell adhesionmolecule-1 (PECAM-1) to visualize the endothelial cells of the embryonicvasculature. These experiments revealed a number of vascular defects in themutant embryos including defective development of the aorta which was thin anddiscontinuous and whose intersomitic branches terminated prematurely in themesenchyme. Abnormalities were also observed in the vascular smooth musclecells as revealed by immunostaining with and antibody against smooth muscleactin suggesting that defects in vascular support cell development may contributeto the vascular defects observed in the Lkb1-/- embryos.

In addition to embryonic vascular defects, we also noted defects in theextraembryonic vasculature as the yolk sacs of mutant E9.5 conceptuses failed todevelop large vitelline vessels or an extensive capillary network. In contrast tocontrols, the mutant yolk sac developed only rudimentary vessels, which wereoften congested by pools of nucleated embryonic blood cells. The vitelline arterywas completely atretic in Lkb1-/- yolk sacs, effectively disconnecting the embryofrom the yolk sac (vitelline) circulation. We also observed that the placentas ofmutants at E9.5 were small and hemorrhagic. Although chorio-allantoic fusion wasnoted in mutant conceptuses, invasion of embryonic blood vessels into theplacenta did not occur. In situ hybridization with probes for the VEGF receptors flk-

1 (Yamaguchi et al., 1993) and flt-1 (Dumont et al., 1995) which delineate migratingand maturing embryonic vessels in the placenta (respectively) confirmed that thefetal allantoic blood vessels did not migrate into the placenta. As a consequence, amature labyrinth layer did not develop in the mutant conceptuses.

Taken together, these results suggest that the Lkb1-/- embryos likely die atmidgestation due to the inability of the embryonic and extraembryonic vasculatureto develop properly in the absence of Lkb1.


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Deregulation of VEGF in Lkb1-/- tissues and cultured cells (III)

As vascular endothelial growth factor (VEGF) is known to be critical for embryonicvascular development (Carmeliet et al., 1996; Ferrara et al., 1996), we thereforedid in situ hybridization experiments on mutant and control conceptuses with aprobe for VEGF. We found that expression of VEGF mRNA was markedlydiminished in the trophoblast giant cells of the mutant placenta, while the Lkb1-/-

embryos expressed elevated levels of VEGF. These results suggested that VEGF

was deregulated in both the embryonic and extraembryonic compartments ofmutant conceptuses.

To investigate whether Lkb1 directly regulated VEGF expression in cultured cells,we isolated primary mouse embryonic fibroblasts (MEFs) from mutant and controlembryos and assayed for expression of VEGF. This analysis revealed thatexpression of VEGF was significantly elevated in Lkb1-/- MEF cultures under bothstimulated (hypoxia) and non-stimulated conditions. These results indicated thatloss of Lkb1 leads to increased basal and induced expression of VEGF infibroblasts. Moreover, these results suggest a rationale for the increased risk ofcancer incidence associated with Peutz-Jeghers syndrome (Giardiello et al., 1987)by demonstrating that loss of Lkb1 can confer an increased angiogenic potential incertain cell types through up-regulation of VEGF.

Lkb1 functions as is a tumor suppressor in mice (IV)

Inactivating germline mutations of LKB1 lead to Peutz-Jeghers syndrome (PJS)(Hemminki et al., 1998; Jenne et al., 1998). As Lkb1 heterozygous mice representthe genetic equivalent of human Peutz-Jeghers patients, we therefore wanted toexamine whether Lkb1+/- mice were prone to a disease spectrum similar to thatfound in PJS patients. To this end, cohorts of Lkb1+/- and control Lkb1+/+ mice wereestablished and monitored. We found that the Lkb1+/- mice had markedly reducedlife spans with ∼80% dying by 16 months of age. It was noted that beyond the ageof 7 months, many of the Lkb1+/- mice presented grossly distended abdomenssuggesting gastrointestinal obstruction. At necropsy, macroscopic gastrointestinalpolyps were identified in 100% of the Lkb1+/- animals over 6 months of age with upto 40 polyps scored in individual animals. The vast majority of the polyps werelocalized to the glandular stomach. Moreover, very large pyloric polyps (20-30 mmin diameter representing up to 25% of the total weight of the animal) were oftenfound protruding into the duodenum thereby rendering it grossly distended andobstructed. Based on these observations, the high mortality associated with Lkb1

heterozygosity was likely due to gastrointestinal occlusion. Taken together, theseresults indicate that Lkb1 functions as a suppressor of polyposis in mice.

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Polyposis in Lkb1+/- mice models Peutz-Jeghers syndrome (IV)

Histological examination of polyps from Lkb1+/- animals revealed that all werehamartomas with well-differentiated glandular epithelium and normal laminapropria. Strikingly, all polyps had an extensive, well-developed smooth musclecomponent, which provided a latticed framework between pockets of glandularepithelia. Histological comparison of polyps derived from Lkb1+/- mice to polypsresected from human PJS patients revealed a striking similarity. Particularly, thehistology of the smooth muscle component, the most widely used criteria forestablishing diagnosis of PJS, was indistinguishable from the human polyps. Itshould be noted however that while the murine Lkb1+/- polyps were found to bepredominantly gastric, polyps in PJS patients are found more evenly distributedalong the full length of the digestive tract with the highest frequency in the smallintestine (Utsunomiya et al., 1975).

In addition to the characteristic hamartomas that develop in Peutz-Jegherspatients, PJS is also a cancer predisposition syndrome with patients exhibiting anincreased risk of developing cancers of both gastrointestinal and extraintestinalorigin in later life (Giardiello et al., 1987; Boardman et al., 1998). We howeverfound no evidence of an increased cancer incidence in Lkb1+/- mice compared tocontrol animals although we were unable to establish whether this held true in olderLkb1+/- mice due to the compromised longevity of Lkb1+/- mice resulting from thepolyposis phenotype. Unlike human PJS patients, abnormal pigmentation of theoral mucosa in Lkb1+/- mice was not observed.

Taken together, these results indicate that while Lkb1+/- mice model PJS polyposis,the phenotype of these animals does not recapitulate all of the clinical aspects ofPeutz-Jeghers syndrome.

No evidence for genetic or epigenetic inactivation of Lkb1 inLkb1+/--associated polyps (IV)

Establishing that a gene functions as a tumor suppressor has traditionally relied onthe demonstration that both alleles of the putative tumor suppressor are inactivatedin order to promote tumorigenesis (Knudson, 1971). Studies addressing this issuein human PJS polyposis have yielded inconsistent results with some groupsreporting that loss of heterozygosity (LOH) of LKB1 often accompanies polypformation (Gruber et al., 1998; Hemminki et al., 1998; Miyaki et al., 2000), whileothers have reported that biallelic inactivation of LKB1 may be more rare (Wang et

al., 1999b; Entius et al., 2001). Thus, the question of whether or not LOH of LKB1

is an obligate initiating event in human PJS polyposis has remained unresolved. Toaddress this issue in our murine model, DNAs extracted directly from polyps orfrom laser micro-dissected polyp samples (to isolate exclusively epithelial or


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stromal cell DNAs) were genotyped for wild type and mutant alleles. This analysisindicated that both alleles were comparably amplified in all tumors analyzed.Moreover, when the copy number of the wild type allele was assayed from polypand control DNAs by real-time PCR we found no statistical difference in the copynumber of the wild type allele in the polyps compared to controls.

It has been suggested that transcriptional silencing by promoter hypermethylationmay be an alternative mechanism by which LKB1 can be inactivated in PJSpatients that do not possess identifiable genetic second hits (Esteller et al., 2000).In order to examine whether epigenetic inactivation might lead to biallelicinactivation of Lkb1 in murine Lkb1+/- polyposis, we assayed for Lkb1 mRNAexpression by in situ hybridization in a number of polyps. This analysisdemonstrated that Lkb1 mRNA levels were maintained throughout the entirety of allpolyps examined at levels comparable to those observed in the adjacentunaffected gastric epithelia and that Lkb1 was expression was retained in both thestromal and epithelial cells of the polyp.

Taken together these results indicated that neither LOH of the wild type Lkb1 locus,or epigenetic silencing of Lkb1 transcription were features of either of the majorcellular components comprising the polyps.

Haploinsufficiency of Lkb1 in polyposis of Lkb1+/- mice (IV)

Having found no evidence for biallelic inactivation of Lkb1 in the polyps of Lkb1+/-

animals, we speculated that haploinsufficiency of Lkb1 might underlie thedevelopment of murine polyposis. This idea is not without precedent as a numberof tumor suppressor genes have recently been demonstrated to behaploinsufficient for tumor suppression (Fero et al., 1998; Venkatachalam et al.,1998; Wetmore et al., 2000; Inoue et al., 2001).

In order to explore this possibility, Lkb1 protein levels and activity were examinedfrom Lkb1+/+ and Lkb1+/- mouse embryonic fibroblasts. These results indicated thatLkb1 protein expression was significantly reduced in Lkb1+/- cells compared toLkb1+/+ cells and that the reduced expression resulted in a similar decrease in Lkb1kinase activity.

We then assayed for Lkb1 expression and activity from whole tissue lysatesprepared from multiple, independently isolated polyps, adjacent unaffectedstomachs, and stomachs from Lkb1+/+ animals. Similar to what had been observedin MEFs, Lkb1 protein levels and activity in the unaffected stomachs of Lkb1+/-

animals were found to be lower than in wild type stomachs. Moreover, we foundthat Lkb1 levels and activity in the polyps were comparable to those in adjacentunaffected stomach lysates.

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Taken together, these data argue that murine Lkb1 is functionally haploinsufficientfor tumor suppression by providing molecular evidence that Lkb1 heterozygosityleads to a reduction in Lkb1 expression and activity that is sufficient to mediatepolyposis in Lkb1+/- animals in the absence of biallelic inactivation.

Induction of cyclooxygenase-2 in murine and PJS polyps (IV)

Cyclooxygenase-2 (COX-2) has been demonstrated to promote gastrointestinaltumorigenesis in a number of human and murine systems (Gupta and DuBois,2001). Although COX-2 expression has frequently been shown to be upregulated ingastrointestinal tumorigenesis in both mice (Williams et al., 1996) and in humans(Eberhart et al., 1994; van Rees and Ristimäki, 2001), it has not been examined inPeutz-Jeghers syndrome polyposis. We therefore assayed for expression of COX-2 in polyp and stomach lysates to establish whether induction of COX-2 might be afeature of murine Lkb1+/- polyposis. We found that expression of COX-2 was highlyupregulated in majority (75%) of the polyps examined.

COX-2 expression is known to be regulated by several signal transductionpathways including those mediated by p38 MAP kinase, Akt/PKB, JNK kinases,and Erk1/2 kinases. Analysis of activated mediators of these pathways in murineLkb1+/- polyposis revealed that only the Erk1/2 kinases were activated suggestingthat the Ras/Raf-1/MEK/ERK signal transduction pathway is likely to mediate COX-2 induction in murine Lkb1+/- polyposis.

In order to investigate whether the induction of COX-2 that we observed in Lkb1+/-

murine polyps was also a feature of the polyposis associated with PJS patients, alarge number of human polyps (23 polyps from 5 PJS patients) were assayed forCOX-2 expression by immunohistochemistry. Elevated COX-2 expression wasobserved in 70% (16/23) of the polyps examined. In all the samples, COX-2immunoreactivity localized to the cytoplasm of the epithelial cells while thesurrounding stroma was negative apart from restricted sporadic staining. Theseresults indicate that upregulation of COX-2 expression is a common feature of PJSpolyposis.

Inhibition of cyclooxygenase enzymes with non-steroidal anti-inflammatory drugshas proven effective in repressing gastrointestinal tumorigenesis (Thun et al., 1991;Giardiello et al., 1993) In particular, COX-2 has emerged as the central target ofthese therapies as shown by clinical trials utilizing selective COX-2 inhibitors(Steinbach et al., 2000). Our finding that COX-2 expression is upregulated in asignificant percentage of human PJS polyps suggests that COX-2 inhibitors mightbe useful in providing a first therapeutic approach in reducing the tumor burden andneed for surgery in Peutz-Jeghers patients.


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CONCLUDING REMARKS AND FUTURE DIRECTIONS

The control of cell cycle progression is regulated in a myriad of different ways bothduring embryonic development as well as in the adult. Cell cycle regulatorymolecules exert their effects through both autonomous and non-autonomousmechanisms to promote or inhibit cell division. The tenuous balance in maintainingcontrol of the cell cycle is illustrated by the consequences that deregulation of keycell cycle regulators impart on the organism – such as cancer development ordefective embryogenesis. However, it is precisely through the study of such‘mutants’ that insight is gained into the underlying molecular biology of normalgrowth and development as well as in disease initiation and progression.Accordingly, mutational analysis has become the principal tool of thedevelopmental biologist and the cancer biologist alike.

The past 15 years has seen the emergence of gene targeting technologies thathave allowed for the generation of animals with specific alterations in the genome.These technologies rely on the property of mouse embryonic stem (ES) cells toremain pluripotent even after rigorous manipulation in vitro. ES cells can thus begenetically modified at any locus in culture followed by returning these cells to theembryo where they maintain the developmental capacity to contribute to anylineage. Phenotypic analysis of targeted animals has proven a powerful tool ingaining insight into the function of hundreds of genes in vivo. The first generation oftargeted animals was almost exclusively engineered to harbour loss of function,null mutations (knockouts). The limitations of this approach soon became apparent,particularly in cases in which the gene product of a targeted locus is essential forearly embryonic development. The second generation of targeted strategieshowever found a way around these limitations with the use of site-specificrecombination systems to facilitate the control of the engineered mutation bothtemporally (at specified times of embryonic development or in the adult) andspatially (in specified tissues or cell types).

In the work presented in this dissertation, my colleagues and I have takenadvantage of the recent advances in genetic engineering and applied thesetechnologies to study the function of two key regulators of cell proliferation, Mat1and Lkb1. This work has provided valuable insight into the function of these twomolecules in vivo, in addition to opening many doors to future experimentation.

Our work with Mat1 has demonstrated a requirement of Mat1 in both mitoticembryonic and adult lineages. As Mat1 has been suggested to be a mediator ofcell cycle progression as a component of the Cdk-activating enzyme (CAK), it ispossible that the requirement that we see for Mat1 in mitotic lineages primarilyreflects a defect in Cdk-activation. This supposition is supported by our findings

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that Mat1-deficieny results in a cell cycle arrest of endocycling trophoblast giantcells. Interestingly, this issue has a great opportunity of being experimentallyaddressed by manipulating the systems we have generated in this current study.Experimental design could involve taking loxP conditional Mat1 cells into culture,introducing the Cre recombinase to these cells (by retrovirus or adenovirusmediated transfer for example) and then examining the molecular biology of thecell cycle machinery for evidence of defective Cdk-activation.

Unexpectedly, our work with Mat1-deficient trophoblast giant cells and myelinatingSchwann cells has provided evidence that mammalian Mat1 – and indeed TFIIHkinase – is not an absolute regulator of RNA polymerase II mediated transcriptionas had been previously suggested for TFIIH kinase homologues in yeast (Valay et

al., 1995; Valay et al., 1996; Faye et al., 1997; Holstege et al., 1998). Our resultssuggest that either differences in experimental design account for this incongruity,or that hitherto unexplored differences exist in the basal transcriptional apparatusof yeast and mammals. Again, the systems we have generated could bemanipulated to address these issues. For example, gene chip technology foranalyzing genome wide changes in transcription profile could be utilized inconjunction with Mat1-deficient tissues or cultured cells to address whichtranscripts exhibit differential expression in the absence of Mat1.

Our work with Lkb1 has demonstrated a requirement for Lkb1 in murine embryonicdevelopment, which we linked to severe defects in vascular development. Thevascular defects that we noted were coupled to a tissue specific deregulation of theVEGF, a key regulator of angiogenesis. A link between Lkb1 and VEGF wasconfirmed in culture with the discovery that Lkb1-deficient cells expressedmarkedly elevated levels of VEGF. These results establish that Lkb1-deficiencyhas the ability to confer an increased angiogenic potential on certain cell types.These findings have obvious implications for the tumorigenic phenotypes of PJSsince the neovascularization of tumors serves both to satisfy the metabolic needsof the tumor cells, as well as providing a possible route for metastasis (Folkman,1995).

Our results demonstrating that mice heterozygous for a targeted inactivating alleleof Lkb1 develop a polyposis that models PJS provides compelling experimentalevidence of the primary role of LKB1 in the development of PJS. We were able toshow that, contrary to expectations, biallelic inactivation of Lkb1 did not accompanytumorigenesis but rather that haploinsufficiency of Lkb1 was sufficient to mediatethe disease. These findings strongly suggest that PJS polyposis is also likely to bemediated by haploinsufficiency of LKB1.

We have also discovered that, much like gastrointestinal adenoma andadenocarcinoma development, both murine and PJS polyposis are associated with


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induction of Cyclooxygenase-2. This finding has significant clinical implications byidentifying COX-2 as the first potential target for chemoprevention in PJSpolyposis. To test the utility of such therapy, we have initiated experimentsadministering the COX-2 inhibitor celecoxib to Lkb1+/- mice as a first step indetermining whether such treatment will be effective in lowering the tumor burdenin PJS patients. We have also undertaken steps towards initiating clinical trials totest the efficacy of COX-2 inhibition in the reduction of PJS tumor burden.

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ACKNOWLEDGEMENTS

The studies detailed in this dissertation were carried out over a five year periodfrom 1997-2002 at The Haartman Institute, and Biomedicum Helsinki at theUniversity of Helsinki, and the Center for Developmental Biology, University ofTexas Southwestern Medical Center, Dallas. I would like to thank professors EeroSaksela in Helsinki, and Luis Parada at UTSW for providing excellent researchfacilities. I would also like to acknowledge the Helsinki Biomedical Graduate Schoolfor providing financial support as well as for arranging and conducting usefulcourses and seminars.

I wish to express my gratitude to my supervisor Tomi Mäkelä. The depth of hiscontribution to these studies has been widespread and is too expansive to list here.It has indeed been a great learning experience to have worked with such anexceptional and multi-faceted scientist.

I am extremely grateful and indebted to Juha Partanen and Seppo Vainio forcarefully and thoughtfully reviewing the manuscript of this thesis and for providingvaluable and insightful comments and suggestions. I also extend thanks to KariAlitalo and Juha Partanen for their efforts and input on my thesis committee.

I also wish to express my thanks to all the members of the cell cycle lab, past andpresent. In particular, I would like to extend special thanks for the excellenttechnical support that I have received from Birgitte Tjäder, Kirsi Mänttäri, SusannaRäsänen and Sanna Kihlberg. I am also especially indebted to those in the lab withwhom I have collaborated, or am in the midst of actively collaborating with includingArno Pihlak, Pekka Katajisto and Lina Udd. Enormous thanks and kudos go to AnttiYlikorkala for extensive and fruitful collaboration, and for putting to good use asharp, inquiring mind. To Nina Korsisaari I owe the most gratitude. Her unerringefforts in the animal colony, unsurpassed skill at the bench, and tireless work ethichave been an integral part of much of the work presented in this thesis.

It has been my great pleasure to have collaborated with a number of talentedscientists from other labs who have enthusiastically contributed their experienceand expertise to various aspects of my work. I extend my sincerest thanks to LauriAaltonen, Ari Ristimäki and Kari Alitalo. Special thanks to Anou Londesborough,Keijo Luukko, and Reijo Salovarra each of whom has played pivotal roles in variedaspects of the studies detailed herein. I also tip the hat to my old friend andcollaborator Mark Henkemeyer with whom it has always been a pleasure to work(and party) with.


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Beyond the realm of science I wish to express my gratitude to the Finnish FilmArchive in conjunction with the Orion Cinema for providing year after year of qualityfilm programming.

I wish to express my thoughts and thanks to all of my family for their unconditionalsupport and encouragement. Above all I want to thank my mother Agnes and myfather Alfred for everything from A to Z, from then until now, and from now untilthen. Everything I have ever, or will ever accomplish, I owe to you.

Finally I would like to express my deepest gratitude and love to Nina for all of herpatience, love and support. Her companionship has provided the most importantand vital meaning in the midst of this furious scientific whirlwind. Thank you Robot,you have made this all worthwhile.

Helsinki, August 30, 2002

Derrick J. Rossi

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