Msh2 and Mlh1 proteins from Tetrahymena thermophla are homologues of MutS and MutL from Escherichia coli respectively. MutS and MutL are DNA mismatch repair proteins. In eukaryotes, MutS homologues recognize the replication errors and MutL homologues interact with MutS homologues and other proteins to make the repair occur. Biolistic transformation has been done to make the msh2 and mlh1 single knockouts in the macronuclei of different strains and the knockouts were verified complete. Two strains of WT crossing KO or KO crossing KO, with different mating types, were induced to conjugate. The processes were studied by microscopy using DAPI staining. For the msh2 knockouts, there were no crescent micronuclei formed throughout the conjugation of two knockout cells, and the pairing level was reduced severely. However, a knockout cell and a wild-type cell could conjugate normally at a high level pairing efficiency. Msh2 protein seems to be important to cell pairing and indispensible for the formation of the crescent micronuclei during cell conjugation. For the mlh1 knockouts, the pairing level of a knockout and a wild-type was reduced by half and the pairing level of two knockouts was reduced more than 80%; however, the paired cells in both could complete the conjugation with delay. Pms2 protein may have redundant roles in the MutL heterodimer (Mlh1-Pms2). In addition, chemical mutagens treated knockout was crossed with non-treated wild-type and the conjugation was compared with treated wild-types. Most of the treated knockout cells could not pair after starvation and mixing with non-treated wild-type cells, which means most of the cells could not enter meiotic phase. It is probable that G2/M checkpoint arrested the meiotic cell cycle and the intra-S phase was inactivated. Thus, Msh2 protein may have a role in the meiotic intra-S phase checkpoint system.

The DNA of all organisms is continuously exposed to exogenous and endogenous genotoxic agents. Fortunately, through the concerted actions of several DNA repair and mutation avoidance pathways, DNA damage can be removed and an organism’s genomic stability maintained. DNA base-base mismatches are generated as a result of the inherent replication errors made by the DNA replication machinery, as well as during the meiotic pairing of homologous but non-identical chromosomes. Through the coordinated actions of the highly conserved DNA mismatch repair (MMR) system, these errors are detected, removed and corrected, thus restoring the integrity of the DNA. In the absence of DNA MMR, genetic instability is unavoidable, resulting in the accumulation of mutations, and in mammals, a susceptibility to cancer. To better understand the roles of the MMR system in mutation avoidance during DNA replication, meiosis, and in nuclear apoptosis, we have utilized the nuclear dimorphic, ciliate protozoan Tetrahymena thermophila. We have identified seven putative MMR homologues; two are similar to eukaryotic MLH1 and PMS2, respectively, and five are similar to eukaryotic MutS homologues, one with eukaryotic MSH2 and four with MSH6. Our studies demonstrate that during conjugation, the relative transcript abundance of each MMR homologue is increased compared to vegetatively growing or nutritionally deprived (starved) cells. Also, the expression profile throughout conjugation is bimodal, corresponding to micronuclear (MIC) meiosis and macronuclear (MAC) anlagen development, both periods in which DNA replication occurs. Cells containing macronuclear knockouts of the PMS2, MSH2 and MSH6_1 genes were unable to successfully pair and complete conjugation, but were viable throughout vegetative growth. Cells in which the macronuclear MSH6_2 gene was knocked out had a phenotype that was similar to wild-type cells, during conjugation and vegetative growth. Interestingly, we observed that the MIC of cells containing MAC knockouts of the PMS2 and TML1 genes appear to have decreased copy number of specific “target sequences”, as determined by qPCR using the Random Mutation Capture (RMC) assay. This decrease reflects neither a loss of micronuclei nor a reduction in total micronuclear DNA content. These studies demonstrate that the PMS2, TML1, MSH2, and MSH6_1 homologues are necessary for the maintenance of micronuclear function and stability during conjugal development and vegetative growth, whereas the remaining MSH6 homologues have less pronounced roles in DNA repair and development. Additionally, macronuclear development in Tetrahymena appears less reliant on the DNA mismatch repair system and perhaps uses alternate surveillance mechanisms to maintain genomic stability during asexual and sexual development. / Graduate / 0306 / 0379 / 0307

Dissertation (Ph.D.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 174-180, p. 201-238.

Hereditary non-polyposis colon cancer syndrome (HNPCC) is associated with an increased risk of developing several types of cancer and is the most common cause of hereditary ovarian cancer after BRCA1 and BRCA2 mutations. HNPCC results from a germline mutation in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, PMS1, PMS2, MSH6, MSH3 and MLH3. While there has been extensive investigation of MMR deficiency in colorectal cancer, MMR in ovarian cancer is relatively under-investigated. The goal of this project was to study MMR deficiency in ovarian cancer at both the clinical and molecular level. The first aim was to examine the frequency of MMR loss in a large patient cohort and investigate the clinical consequences of MMR deficiency. The second aim was to describe the molecular characteristics of MMR deficiency in ovarian cancer cell lines and establish an in vitro cell line model of MMR deficiency in ovarian cancer. The third aim was to identify synthetic lethal strategies for the treatment of ovarian cancer to maximise cytotoxicity in a MMR-deficient background. In order to characterise the clinical consequences of MMR deficiency, a large patient cohort was studied with regard to MMR status. Three tissue microarrays consisting of 581 ovarian tumours were constructed, and expression of the four most frequently lost MMR proteins: MLH1, MSH2, PMS2 and MSH6 were detected by immunohistochemistry. Afterwards, MMR status and histology subtypes were analysed in combination with the associated clinical data. The overall incidence of MMR deficiency (loss of any MMR protein) was 15.7%, with PMS2 being the most frequently lost protein (9.7%). In addition, MMR deficiency tended to appear in a grouped fashion: MLH1 with PMS2; MSH2 with MSH6. Patients with non-serous subtypes of ovarian cancer, clear cell or mucinous especially, had higher incidence of MMR deficiency compared to patients with serous ovarian cancer. Overall MMR deficient patients were more likely to be diagnosed at early stages compared with MMR proficient patients, and this is probably due to the association between MMR deficiency and non-serous histology. However, platinum-based treatment for patients with MMR deficiency gives no advantage over those without MMR deficiency. Therefore better treatments for this subgroup of patients may be needed. The features of MMR deficiency in ovarian cancer were also characterized at the molecular level. After quantifying mRNA and protein expression of MMR genes in 19 ovarian cell lines, three cell lines (SKOV3, TOV21G and IGROV1) were found to have a defect in MLH1 expression at both the mRNA and protein level. Interestingly, the three cell lines also carried a defect in PMS2 expression at the protein level but not at the mRNA level, which is consistent with our clinical data demonstrating that MLH1 protein and PMS2 protein are paired in loss. In addition, across the 19 cell lines, MLH1 and PMS2 showed positive correlation at both the mRNA level (R=0.53, p=0.02) and protein level (R=0.72, p=0.0006). In order to study co-expression of MLH1 and PMS2, a plasmid encoding the cDNA for MLH1 was transfected into the three MLH1 deficient cell lines; and conversely siRNA targeting MLH1 was transfected into the MMR proficient cell line A2780 and expression of MLH1 protein and PMS2 protein was quantified. The results showed that re-introduction of MLH1 into MLH1 deficient cells resulted in increased expression of PMS2 protein, while knocking down MLH1 in MMR proficient cells leads to decreased PMS2 protein expression. This indicates that MLH1 may play a crucial role in regulating PMS2 protein expression. As the three MLH1 and PMS2 protein deficient cell lines all express PMS2 mRNA, the regulation of PMS2 expression by MLH1 is likely to be at the translational or post-translational level. However, the expression of PMS2 protein was not increased in the absence of MLH1, even when the proteasomal and lysosomal protein degradation pathways were blocked (as seen with SKOV3 cells), suggesting decreased PMS2 protein expression is not due to rapid degradation in the absence of MLH1. Therefore MLH1 may play a role in regulating the synthesis of PMS2 protein at the translational level, rather than preventing the degradation of PMS2. Thus, to investigate the mechanism by which PMS2 protein levels are regulated by MLH1, future work should focus on translational regulation of PMS2. In order to identify synthetic lethal strategies to target MMR deficiency in ovarian cancer, an isogenic cell line model of MMR deficiency was established by stable transfection of a plasmid for MLH1 and its corresponding empty vector into SKOV3 cells. The MLH1+ cell line SAC-1 and MLH1- cell line SN-5 were selected for drug screening based on their phenotype and growth rate. The AlamarBlue assay, with z’ above 0.5, was chosen for drug screening and a kinase inhibitor library containing 362 drugs of known target was screened. Two drugs with similar structures that targeted PLK1 showed greater growth inhibition of SN-5 compared with SAC-1. When the two cell lines were treated with another PLK1 inhibitor, BI2536, with different structure, a 2-fold difference in growth inhibition between SAC-1 and SN-5 was also observed, suggesting PLK1 is a potential synthetic lethal target for MLH1 deficiency in ovarian cancer. Together these data demonstrate that clinically, MMR deficiency is associated with non-serous subtypes of ovarian cancer and specific MMR proteins are paired in loss. While current standard therapy offers no selective benefit to ovarian cancer patients with MMR deficiency, inhibiting PLK1 activity may confer selective benefit.

Mismatch repair corrects errors made during DNA replication and inactive mismatch repair is associated with Lynch Syndrome and sporadic cancer. Genome replication in eukaryotes is accompanied by chromatin formation. The first step in chromatin establishment is nucleosome assembly, that starts with histone tetramer deposition. It is not clear how three important cellular processes: genome replication, mismatch repair and nucleosome assembly are coordinated. Here we analyzed human mismatch repair in the presence of histone deposition in a reconstituted system. We showed that mismatch repair factor inhibits nucleosome assembly on the DNA region with the replicative error. Such a mechanism is important, since in this way DNA with errors remains accessible for mismatch repair system to perform the repair. The DNA synthesis step in mismatch repair is performed by DNA polymerase. Eukaryotes possess two major replicative DNA Polymerases: DNA Polymerase delta and DNA Polymerase epsilon. DNA polymerase delta is involved in mismatch repair. However, it was unknown whether DNA polymerase epsilon can also work in mismatch repair. Here we analyzed human mismatch repair with DNA Polymerase delta and DNA Polymerase epsilon in the environment of histone deposition. Our results indicated that repair activity with both polymerases was activated by histone deposition. Here it was first shown that human DNA Polymerase epsilon performs DNA synthesis during mismatch repair in vitro. Importantly, recent studies have revealed association of Polymerase epsilon mutations with cancer. Since our data showed activity of DNA Polymerase epsilon in mismatch repair, a possible tumor development mechanism may involve inactivation of mismatch repair due to Polymerase epsilon mutations. Overall, our study expanded the understanding of the mechanism of human mismatch repair in the chromatin environment.

Abstract Breast cancer is the leading cause of death from cancer among Finnish women, but the ultimate causation of carcinogenesis still remains unclear. Reactive oxygen species (ROS) is a collective term for several types of reactive oxygen metabolites that are continuously generated in human cells mainly as by-products of aerobic respiration. ROS, including nitric oxide and its derivatives, play highly important roles in cell physiology. If ROS production exceeds the capacity of detoxification systems, principally antioxidant enzymes, oxidative stress is said to occur. This state is known to contribute to all stages of carcinogenesis. To explore the widely unstudied role of ROS and cell redox state modulating enzymes in breast carcinomas, the extent of ROS-derived macromolecule damage and the expression of the vast majority of known antioxidant enzymes were assessed in a large series of breast carcinomas, and the results were compared to the patients' clinicopathological parameters. The results were also compared to angiogenesis, DNA repair enzymes, cell proliferation, NF-κB, p53 expression, and survival. Immunohistochemistry was the main method applied, but western blotting and immunoelectron microscopy were also used. There is extensive oxidative damage in breast carcinomas, which seems to associate with tumor development. Oxidative macromolecule damage is notable even in stage I tumors. Cell redox state regulating enzymes, such as peroxiredoxin V, thioredoxin, thioredoxin reductase, and glutamate-cysteine ligase, associate with more aggressive phenotypes of tumors, including larger primary tumors, growth of metastases, increased cell proliferation, and poor differentiation. This indirectly suggests that cell redox state modulating enzymes may be inductive of tumor promotion in an oxidated environment. The results of this thesis support the importance of ROS in all stages of carcinogenesis. These observations are largely in line with the previous studies on different carcinomas, but there seem to be certain carcinoma type specific differences in the expression of these enzymes. Since the expression of given cell redox state modulating enzymes distinctly associates with clinicopathological parameters, these enzymes may be useful as prognostic indicators and facilitate the choice of appropriate treatment in the future.

L'inactivation du système MMR (mismatch repair) favorise un processus oncogénique d'instabilité des microsatellites du génome (MSI). Au cours de ma thèse, j'ai étudié d'une part le rôle de l'azathioprine (Aza) dans l'induction de tumeurs MSI chez la souris. Des études épidémiologiques avaient rapporté une corrélation entre l'émergence de cancers MSI tardifs chez l'homme, et la prise au long cours de cet immunosuppresseur dont la cytotoxicité in vitro est médiée par l'activité MMR. Dans une étude dose-réponse, j'ai observé l'émergence de rares lymphomes MSI de survenue tardive chez la souris de génotype sauvage traitée par l'Aza, mais pas par la ciclosporine (autre immunosuppresseur utilisé en comparaison). Ces résultats permettent d'établir in vivo que l'Aza est un facteur de risque pour l'émergence de tumeurs MSI lors d'une exposition prolongée. D'autre part, je me suis intéressée au syndrome CMMRD (constitutional MMR deficiency), une prédisposition majeure et rare, aux cancers MSI. Les patients atteints étant porteurs de mutations germinales bialléliques d'un gène MMR, le diagnostic repose sur le génotypage constitutionnel, une méthode non-contributive quand un variant de signification inconnue est détecté (30% patients). Dans ce contexte, j'ai développé une méthode d'aide au dépistage de ce syndrome chez les sujets à risque, l'hypothèse étant que 2 caractéristiques fonctionnelles des cellules tumorales MMR-déficientes, le phénotype MSI et la tolérance aux agents génotoxiques tels que l'Aza, pouvaient être objectivées dans les tissus sains des patients CMMRD. Mes travaux proposent un test diagnostique sensible et spécifique qui répond aux limites de l'analyse génétique. / Inactivation of the MMR (mismatch repair) system promotes the oncogenic process of microsatellite instability (MSI). During my PhD, I firstly investigated the role of azathioprine (Aza) in the induction of MSI tumors in mice. Epidemiological studies reported a correlation between the occurrence of late MSI cancers in humans and long-term treatment with this immunosuppressant whose cytotoxicity was shown in vitro to be mediated by MMR activity. Using a dose-response study, I observed the occurrence of rare late-onset MSI lymphomas in wild-type mice treated with Aza, but not with ciclosporin (another immunosuppressant used for comparison). These results established in vivo that long-term Aza exposure is a risk factor for the emergence of MSI tumors. Secondly, I was interested in the CMMRD syndrome (constitutional MMR deficiency), a major and rare predisposition to MSI cancers. Since CMMRD patients are carriers of biallelic germline mutations of a MMR gene, diagnosis is based on constitutional genotyping, a method that was found non-contributory when a variant of unknown significance is detected (30% patients). In this context, I developed a complementary approach for the detection of this syndrome in at-risk patients, based on the hypothesis that two functional features of MMR-deficient tumor cells, i.e. the MSI phenotype and the tolerance to genotoxic agents such as Aza, can be demonstrated in non-neoplastic tissues of CMMRD patients. We provided a sensitive and specific method that may constitute a valuable tool when diagnosis of CMMRD could not be confirmed by genetic testing.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23114号 / 医科博第125号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 篠原 隆司, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

To study hybrid sterility our laboratory uses mouse strains PWD/Ph (PWD), derived from Mus musculus musculus wild mice and the common laboratory strain C57BL/6J (B6) mostly of Mus musculus domesticus origin as a model. Crossing between PWD female and B6 male results in sterile male progeny. F1 hybrid males carry defects in the repair mechanisms of asymmetric double-strand DNA breaks (DSBs). Functional interplay of SPO11 and PRDM9 proteins in the meiotic prophase I is necessary for repairs. Its defect leads to incorrect synapse formation between homologous chromosomes, leading to halt in spermatogenesis and thus male sterility. The formation of DSBs and their subsequent repair is essential for first meiotic division. The working hypothesis stems from the findings in yeast model, where supposed antirecombinatorial mechanism of mismatch repair genes Msh6 and Msh2 prevents DSBs repairs during meiosis. Despite the functional mechanism of these two genes is not explicitly known, existence of similar repair system in mice is presumed. Variety of methods was implemented in this thesis. The effects of Msh6 deletion on meiotic prophase I and sperm maturation were performed by designing guide RNAs for CRISPR/Cas9 for creation of three knock-outs in B6 mice. The PCR was used to amplify regions adjacent to the...

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18110号 / 理博第3988号 / 新制||理||1575(附属図書館) / 30968 / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 秋山 秋梅, 教授 沼田 英治, 教授 疋田 努 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM