Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Background. Radiation therapy is an important treatment regimen for malignant disease. Radiation therapy uses ionizing radiation to induce DNA damage in tumor cells in order to kill them. Tumor cells are more sensitive than normal cells, since they have an increased proliferation rate and often lack the ability to properly repair the induced damage. Radiation can be delivered by an external source outside the body, by brachytherapy delivered inside the patient near the tumor, or systemically by injection into the blood stream. When delivered systemically, the radiation is administered as radioisotope alone or conjugated to antibodies targeting tumor antigens (radioimmunotherapy). Radiotherapy (RT) usually is administered using high doses, causing necrotic cell death. Low doses of radiation (by RT or RIT) have been observed to induce different types of cell deaths, like apoptosis, mitotic catastrophe or senescence.Aims. We wanted to elucidate the molecular and cellular events responsible for the induction of cell death in cells of different origin and p53 status. We also wanted to identify the kinetics behind gene expression alterations induced in response to irradiation and correlate these to cell death specific molecular and cellular events. In the end this research aims to identify key regulators of the main radiation induced cell death modalities in order to improve our understanding and potentially use this knowledge to increase treatment efficacy of radiation therapy. Methods. Four different cell lines were used in these studies to elucidate the role of p53 status cell origin in radiation induced cell death. HeLa Hep2 tumor cells have been used previously in our group in several RIT and RT studies. During these studies we observed morphological alterations in shrinking tumors that were typical for mitotic catastrophe. This led to studies on the underlying mechanisms causing these aberrations. Isogenic solid tumor cell lines HCT116 p53 +/+ and HCT116 p53 -/- were included to further elucidate the role of p53, and also to study senescence, one of the main outcomes in irradiated tumor cells. MOLT-4 was finally included to compare these finding to classical apoptosis. Gene expression analysis was done using Illumina bead chip arrays, and pathway analysis was performed using MetaCore (Thomson Reuters). Results. In paper I, II, and III, transient G2/M arrests were observed in HeLa Hep2 and HCT116 p53 -/- cells following irradiation. The lack of p53 in these cells caused checkpoint adaptation due to an unscheduled accumulation of genes promoting mitosis. Anaphase bridges were observedivin HeLa Hep2 cells, as a consequence of premature mitotic entry with unrepaired DNA damage. Centrosome amplification, as well as deregulation of genes involved in centrosome amplification and clustering was observed in both cell lines. We observed changes in expression of several genes responsible for maintaining the spindle assembly checkpoint (SAC) arrest. A prolonged SAC arrest has been shown to be important for execution of mitotic catastrophe. SAC activation was followed by mitotic slippage and a subsequent failure of cytokinesis. We observed multipolar mitoses (both cell lines), multiple- and micronuclei (HeLa Hep2, paper I), and an increased frequency of tetraploid cells (HeLa Hep2 and HCT116 p53 -/- cells). A fraction of HeLa Hep2 cells also displayed apoptotic features, including caspase activation and DNA fragmentation (paper I). These findings indicate that mitotic catastrophe and the activation of a delayed type of apoptosis are involved in cell death following RIT.HCT116 p53 +/+ cells induced both G1 and G2 arrest following irradiation (paper III). Gene expression analysis revealed significantly decreased expression of genes responsible for cell cycle progression (pronounced decrease compared to HeLa Hep2 and HCT116 p53 -/-), especially mitotic genes. The prolonged arrest transitioned into senescence starting 3 days following irradiation and peaked after 7 days. Several genes associated with SASP were upregulated in the same time frame as senescence was induced, further supporting the fact that senescence is the main radiation induced response in HCT116 p53 +/+ cells.MOLT-4 cells, similar to HCT116 p53 +/+ cells, induced both G1 and G2 arrests in response to irradiation (paper IV). Morphological studies revealed apoptotic features like shrunken cells with condensed DNA. Caspase assays showed increased activity of caspases -3, -8, and -9. Gene expression analysis confirmed an increased expression of genes important for both extrinsic (FAS and TRAIL) and intrinsic (BAX) apoptosis. Furthermore, changed expression also included genes involved in cell cycle checkpoints and their regulation and genes important for T-cell activation/proliferation. Conclusions. RIT is successfully used to treat lymphoma, but treatment of solid tumors with RIT is still difficult. This thesis elucidates cellular alterations characteristic for the 3 main radiation death modalities, i.e. mitotic catastrophe, senescence and apoptosis. Furthermore, cell death specific traits are correlated to alterations in gene expression. Treatment efficacy can potentially be improved by finding key cell death mediators to inhibit in combination with radiation. / Bakgrund. Strålbehandling används för att bota eller lindra symptomen av cancer och består av joniserande strålning vars syfte är att skada DNAt i cellerna vilket leder till att de dör. Tumörceller är känsligare för strålning än normala celler eftersom de delar sig i snabbare takt och ofta saknar förmågan att reparera skadorna som uppstår. Det finns flera typer av strålbehandling: extern strålbehandling, d.v.s. när strålkällan är placerad utanför kroppen, brachyterapi, när strålkällan placeras i en kapsel inuti kroppen, eller systemisk strålning, där en radioisotop injiceras, antingen själv eller kopplad till en antikropp, då kallad radioimmunoterapi (RIT). Vid extern strålbehandling använder man sig ofta av relativt höga doser av strålning under ett kortare tidsintervall. Dessa celler dör ofta en nekrosliknande död. Med RIT kan man behandla patienterna med lägre doser under en längre tid och strålningen kan riktas specifikt till tumören, vilket minskar risken för bieffekter. Dessa celler dör av andra former av celldöd, apoptos, senescence eller mitotisk katastrof. Apoptos är för många synonymt med programmerad celldöd, och sker till exempel i respons till DNA skada. En apoptotisk cell känns igen på sitt utseende med fragmenterat DNA, nedbrutet cytoskelett och apoptotiska kroppar. Senescence är associerat med cellens åldrande men kan även orsakas av DNA-skador, och är en vanlig form av celldöd hos solida tumörceller med funktionell p53-signalering. Bestrålade solida tumörceller som saknar p53-signalering, antingen på grund av mutationer eller på grund av virusinducerad inaktivering, dör oftast i en helt annan celldöd, kallad mitotisk katastrof. Avsaknad av p53 leder till att en cell som erhållit skador på DNAt inte klarar av att uppehålla cellcykeln länge nog för att reparera skadorna. Inte heller apoptos induceras, eftersom p53 saknas. Detta leder till att cellen kommer att gå in i mitos med skador i sitt DNA som ej hunnit repareras. Celler i mitotisk katastrof har ett väldigt typiskt utseende med multipla kärnor, mikrokärnor (kromosomrester), multipla centrosomer och multipolära mitotiska spindlar. En del celler dör i mitosen medan andra försöker dela sig och kan överleva i flera generationer till, dock med skador på DNA. Målet med denna avhandling var att utreda de molekylära och transkriptionella mekanismerna bakom strålningsinducerad celldöd, och p53s roll i detta. Dessa studier kan så småningom leda till att viktiga regulatoriska proteiner av de strålnigsinducerade celldödsmekanismerna kan identifieras. Specifika inhibitorer riktade mot dessa proteiner kan med ökad kunskap strategiskt användas i kombination med strålning och potentiellt leda till förbättrade behandlingseffekter. Metoder. Vi använde fyra cellinjer med olika bakgrund och p53 status. Vi har tidigare studerat HeLa Hep2 (en solid tumörcellslinje infekterad medviHPV som slår ut funktionen av p53) och sett vid både RT och RIT studier, att cellernas morfologi avviker från klassiks apoptos (stora celler med stora mängder DNA, istället för små celler med lite DNA). Detta ledde till studier av mekanismerna bakom denna avvikande cellmorfologin, som är typisk för mitotisk katastrof. Vi utökade studien med HCT116 p53 +/+ och HCT116 p53 -/- som är identiska så när som på p53, där ena cellinjen saknar denna gen. Detta skulle ge ökad förståelse för p53s roll vid mitotisk katastrof och även visa mekanismerna bakom senescence, en annan vanlig celldödsmekanism i strålade solida tumörceller. Även MOLT-4 inkluderades i studien för att kunna jämföra våra resultat med en cellinje som genomgår klassisk apoptos och är mer känslig för strålning. Resultat. I celler där mitotisk katastrof inducerades efter strålning (HeLa Hep2, HCT116 p53-/-) såg vi ett övergående G2 arrest. Eftersom cellerna inte klarade av att underhålla detta arrest, då de saknar p53, fortsatte de in i nästa fas av cellcykeln, mitos. Detta ledde till att DNA skador kvarstod och en ökad frekvens av anafasbryggor. Dessutom skedde en centrosomamplifiering i dessa celler vilket gav upphov till multipolära mitotiska spindlar och en efterföljande icke fungerande cytokines. Detta gav i sin tur celler med multipla kärnor eller mikrokärnor. En ökad frekvens av tetraploida och polyploidaEn förändrad expression av gener som kunde kopplas till flera av dessa för mitotisk katastrof specifika karaktäristika observerades också. Flera gener associerade med reglering av centrosomen och dess amplifiering, med kontrollen av cellens progression från G2 till M-fasen av cellcykeln, samt involverade i kontrollen av en rätt utförd mitos (SAC) hade en ändrad genexpression som korrelerade väl i tid med de ovan nämda fenotyperna. Caspaser som är viktiga för apoptos visade sig vara aktiva i HeLa Hep2, vilket indikerar att mitotisk katastrof kan leda till fördröjd apoptos. Men en del celler lyckas smita undan från apoptosinduktionen och fortsätter i en ny runda i cellcykeln, och detta kunde ses som en växande population viabla celler med ökad mängd DNA (tetraploida celler).HCT116 p53 +/+ celler som har funktionellt p53 kunde inducera både G1 and G2 arrest och genexpressionen visade att många gener som styr övergången till mitos var nedreglerade och förhindrade detta (till skillnad från HeLa Hep2 och HCT116 p53 -/-, där dessa nivåer var högre). Dessa arrester övergick till senescence 3 dagar efter strålning och många gener kopplade till senescence visade ett ökat uttryck. Vi såg ingen markant ökning av centrosomer eller polyploida celler vilket skiljde sig från HeLa Hep2 och HCT116 p53 -/-. Detta tyder på att senescence skiljer sig markant åt från mitotisk katastrof och att p53 är viktig för induktionen av denna form av celldöd.viiVi såg att MOLT-4, precis som HCT116 p53 +/+, inducerar både G1 and G2 arrest. Denna arrest resulterade dock i ökad expression av gener viktiga för cellcykelarrest och apoptosinduktion, och vi såg även en ökad aktivitet av caspaser. Morfologiska studier visade att strålade MOLT-4 celler ofta var små och hade kondenserat DNA, vilket är typiska kännetecken för apoptos. Strålning av MOLT-4 celler ledde till aktivering av klassisk apoptos, och tidsförloppet var mycket snabbare jämfört med de övriga cellinjerna. Slutsats. RIT är en framgångsrik metod för att behandla hematologiska maligniteter, men solida tumörer svarar fortfarande dåligt på denna form av behandling. Denna avhandling visar på komplexiteten bakom strålningsinducerad celldöd och att det är viktigt att identifiera de reglerande mekanismerna för att kunna förbättra RIT av solida tumörer. Vi visar även på vikten av p53 vad gäller tumörens respons av strålbehandling. Genom att identifiera viktiga proteiner för mitotisk katastrof, senescence, och apoptos, kan man utveckla inhibitorer mot dessa och använda de i kobination med RT och RIT för att förbättra behandlingseffekten.

<p>In diploid yeast cells, mitotic recombination is very important for repairing double-strand breaks (DSB). When repair of a DSB results in crossovers, it may cause loss of heterozygosity (LOH) of markers centromere-distal to the DSB in both daughter cells. Gene conversion events unassociated with crossovers cause LOH for an interstitial section of a chromosome. Alternatively, DSBs can initiate break-induced replication (BIR), causing LOH in only one of the daughter cells. Mapping mitotic LOH contributes to understanding of mechanisms for repairing DSBs and distribution of these recombinogenic lesions. Methods for selecting mitotic crossovers and mapping the positions of crossovers have recently been developed in our lab. Our current approach uses a diploid yeast strain that is heterozygous for about 55,000 SNPs, and employs SNP-Microarrays to map LOH events throughout the genome. These methods allow us to examine selected crossovers on chromosome V and unselected mitotic recombination events (crossovers, gene conversion events unassociated with crossovers, and BIR events) at about 1 kb resolution across the genome.</p><p>Mitotic recombination can be greatly induced by UV radiation. However, prior to my research, the nature of the recombinogenic lesions and the distribution of UV-induced recombination events were relatively uncharacterized. Using SNP microarrays, we constructed maps of UV-induced LOH events in G1-synchronized cells. Mitotic crossovers were stimulated 1500-fold and 8500-fold by UV doses of 1 J/m2 and 15 J/m2, respectively, compared to spontaneous events. Additionally, cells treated with 15 J/m2 have about eight unselected LOH events per pair of sectors, including gene conversions associated and unassociated with crossovers as well as BIR events. These unselected LOH events are distributed randomly throughout the genome with no particular hotspots; however, the rDNA cluster was under-represented for the initiation of crossover and BIR events. Interestingly, we found that a high fraction of recombination events in cells treated with 15 J/m2 reflected repair of two sister chromatids broken at roughly the same position. In cells treated with 1 J/m2, most events reflect repair of a single broken sister chromatid (Chapter 2). </p><p>The primary pathway to remove pyrimidine dimers introduced by UV is the nucleotide excision repair (NER) pathway. In NER, the dimer is excised to generate a 30-nucleotide gap that can be replicated to form DSBs if not filled in before DNA replication. The NER gap can also be expanded by Exo1p to form single stranded gaps greater than one kilobase. Alternatively, in the absence of NER, unexcised dimers could result in blocks of DNA replication forks. Resolving the stalled replication fork could lead to recombinogenic breaks. In Chapter 3 and Chapter 4, we analyzed recombination events in strains defective in various steps of processing of UV-induced DNA damage, including exo1 and rad14 mutants. </p><p>In Chapter 3, I show that Exo1p-expanded NER gaps contribute to UV-induced recombination events. Interestingly, I also found that Exo1p is also required for the hotspot activity of a spontaneous crossover hotspot involving a pair of inverted Ty repeats. In addition to its role of expanding a nick to a long single-stranded gap, Exo1p is also a major player in DSB end resection. Therefore, I examined the gene conversion tract lengths in strains deleted for EXO1. I found that, although crossover-associated gene conversion tracts become shorter in the exo1 mutant as expected, noncrossover tract lengths remained unaffected. As a result, noncrossover tracts are longer than crossover tracts in the exo1 mutant while the opposite result was observed in the wild-type strains. I proposed models to rationalize this observation.</p><p>In Chapter 4, to investigate whether the substantial recombinogenic effect in UV in G1-synchronized cells requires NER, we mapped UV-induced LOH events in NER-deficient rad14 diploids treated with 1 J/m2. Mitotic recombination between homologs was greatly stimulated, which suggests that dimers themselves can also cause recombination without processing by NER. We further show that UV-induced inter-homolog recombination events (noncrossover, crossover and BIR) depend on the resolvase Mus81p, and are suppressed by Mms2p-mediated error-free post-replication repair pathway. </p><p>The research described in Chapters, 2, 3, and 4 are in the publications Yin and Petes (2013), Yin and Petes (2014), and Yin and Petes (2015), respectively.</p> / Dissertation

Cells must faithfully segregate their chromosomes at division; errors in this process causes cells to inherit an incorrect number of chromosomes, a hallmark of birth defects and cancer. The machinery required to segregate chromosomes is called the spindle, a bipolar array of microtubules that attach to chromosomes through the kinetochore. Replicated chromosomes contain two sister chromatids whose kinetochores must attach to microtubules from opposite poles to ensure correct inheritance of chromosomes. The spindle checkpoint monitors the attachment to the spindle and prevents cell division until all chromatids are attached to opposite poles. Both the spindle and the checkpoint are critical for correct segregation, and we sought to understand the regulation of these two components. The spindle is assembled to a characteristic metaphase length, but it is unknown what determines this length. It has been proposed that spindle length could be regulated a balance of two forces: one generated by interaction between microtubules that elongates the spindle and a second due to interactions between kinetochores and microtubules that shortens the spindle. We tested this force-balance model which predicts that altering the number of kinetochores will alter spindle length. We manipulated the number of kinetochores and found that spindle length scales with the number of kinetochores; introducing extra kinetochores produces shorter spindles and inhibiting kinetochores produces longer spindles. Our results suggest that attachment of chromosomes to the spindle via kinetochores produces an inward force that opposes outward force. We also found that the number of microtubules in the spindle varied with the number of kinetochores. In addition to establishing a spindle, cells must also guarantee that chromosomes are correctly attached to it. Correct attachment generates tension as the chromatids are pulled toward opposite poles but held together by cohesin until anaphase. The spindle checkpoint monitors this tension which causes stretching of chromatin and kinetochores. Lack of tension on activates the checkpoint, but is unknown if the checkpoint measures stretch between kinetochores (inter-kinetochore stretch) or within kinetochores (intra-kinetochore). We tethered sister chromatids together to inhibit inter-kinetochore stretch and found that the checkpoint was not activated. Our results negate inter-kinetochore models and support intra-kinetochore models.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb 10, 2009). Includes bibliographical references.