Data integration from two microarray platforms identifies bi-allelic genetic inactivation of RIC8A in a breast cancer cell line

Background: Using array comparative genomic hybridization (aCGH), a large number of deleted genomic regions have been identified in human cancers. However, subsequent efforts to identify target genes selected for inactivation in these regions have often been challenging. Methods: We integrated here genome-wide copy number data with gene expression data and non-sense mediated mRNA decay rates in breast cancer cell lines to prioritize gene candidates that are likely to be tumour suppressor genes inactivated by bi-allelic genetic events. The candidates were sequenced to identify potential mutations. Results: This integrated genomic approach led to the identification of RIC8A at 11p15 as a putative candidate target gene for the genomic deletion in the ZR-75-1 breast cancer cell line. We identified a truncating mutation in this cell line, leading to loss of expression and rapid decay of the transcript. We screened 127 breast cancers for RIC8A mutations, but did not find any pathogenic mutations. No promoter hypermethylation in these tumours was detected either. However, analysis of gene expression data from breast tumours identified a small group of aggressive tumours that displayed low levels of RIC8A transcripts. qRT-PCR analysis of 38 breast tumours showed a strong association between low RIC8A expression and the presence of TP53 mutations (P = 0.006). Conclusion: We demonstrate a data integration strategy leading to the identification of RIC8A as a gene undergoing a classical double-hit genetic inactivation in a breast cancer cell line, as well as in vivo evidence of loss of RIC8A expression in a subgroup of aggressive TP53 mutant breast cancers.

Zebrafish as a model of Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. Its etiology is unknown and no disease-modifying drugs are available. Thus, more information concerning its pathogenesis is needed. Among other genes, mutated PTEN-induced kinase 1 (PINK1) has been linked to early-onset and sporadic PD, but its mode of action is poorly understood. Most animal models of PD are based on the use of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is metabolized to MPP+ by monoamine oxidase B (MAO B) and causes cell death of dopaminergic neurons in the substantia nigra in mammals. Zebrafish has been a widely used model organism in developmental biology, but is now emerging as a model for human diseases due to its ideal combination of properties. Zebrafish are inexpensive and easy to maintain, develop rapidly, breed in large quantities producing transparent embryos, and are readily manipulated by various methods, particularly genetic ones. In addition, zebrafish are vertebrate animals and results derived from zebrafish may be more applicable to mammals than results from invertebrate genetic models such as Drosophila melanogaster and Caenorhabditis elegans. However, the similarity cannot be taken for granted. The aim of this study was to establish and test a PD model using larval zebrafish. The developing monoaminergic neuronal systems of larval zebrafish were investigated. We identified and classified 17 catecholaminergic and 9 serotonergic neuron populations in the zebrafish brain. A 3-dimensional atlas was created to facilitate future research. Only one gene encoding MAO was found in the zebrafish genome. Zebrafish MAO showed MAO A-type substrate specificity, but non-A-non-B inhibitor specificity. Distribution of MAO in larval and adult zebrafish brains was both diffuse and distinctly cellular. Inhibition of MAO during larval development led to markedly elevated 5-hydroxytryptamine (serotonin, 5-HT) levels, which decreased the locomotion of the fish. MPTP exposure caused a transient loss of cells in specific aminergic cell populations and decreased locomotion. MPTP-induced changes could be rescued by the MAO B inhibitor deprenyl, suggesting a role for MAO in MPTP toxicity. MPP+ affected only one catecholaminergic cell population; thus, the action of MPP+ was more selective than that of MPTP. The zebrafish PINK1 gene was cloned in zebrafish, and morpholino oligonucleotides were used to suppress its expression in larval zebrafish. The functional domains and expression pattern of zebrafish PINK1 resembled those of other vertebrates, suggesting that zebrafish is a feasible model for studying PINK1. Translation inhibition resulted in cell loss of the same catecholaminergic cell populations as MPTP and MPP+. Inactivation of PINK1 sensitized larval zebrafish to subefficacious doses of MPTP, causing a decrease in locomotion and cell loss in one dopaminergic cell population. Zebrafish appears to be a feasible model for studying PD, since its aminergic systems, mode of action of MPTP, and functions of PINK1 resemble those of mammalians. However, the functions of zebrafish MAO differ from the two forms of MAO found in mammals. Future studies using zebrafish PD models should utilize the advantages specific to zebrafish, such as the ability to execute large-scale genetic or drug screens.

Genetic and Epigenetic Characterization of Colorectal and Endometrial Cancer

Colorectal cancer is one of the three most common cancers today, for both men and women. Approximately 90% of the cases are sporadic while the remaining 10% is hereditary. Among this 10% is hereditary nonpolyposis colorectal cancer (HNPCC), an autosomal dominant disease, accounting for up to 13% of these cases. HNPCC is associated with germline mutations in four mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2, and is characterized by a familial accumulation of endometrial, gastric, urological, and ovarian tumors, in addition to colorectal cancer. An important etiological characteristic of HNPCC is the presence of microsatellite instability (MSI), caused by mutations of the MMR genes. Approximately 15% of sporadic cases share the MSI+ trait. Colon cancer is believed to be a consequence of an accumulation of mutations in tumor suppressor genes and oncogenes, eventually resulting in tumor development. This phenomena is accelerated in HNPCC due the presence of an inherited mutation in the MMR genes, accounting for one of the two hits proposed to be needed by Knudson (1971) in order for the manifestation of the MSI phenotype. MMR alterations alone, however, do not occur in the majority of sporadic colon cancers, prompting searches for other mechanisms. One such mechanism found to play a role in colon cancer development was DNA methylation, which is known to play a role in MLH1 inactivation. Our objective was clarification of mechanisms associated with tumor development in both HNPCC and sporadic colorectal cancer in relation to tumorigenic mechanisms. Of particular interest were underlying mechanisms of MSI in sporadic colorectal cancers, with attention to DNA methylation changes and their correlation to MSI. Of additional interest were the genetic and epigenetic events leading to the HNPCC tumor spectrum, chiefly colon and endometrial cancers, in regards to what extent the somatic changes in target tissue explained this phenomenon. We made a number of important findings pertaining to these questions. First, MSI tumor development differs epigenetically from stable tumor development, possibly underlying developmental pathway differences. Additionally, while epigenetic modification, principally DNA methylation, is a major mechanism in sporadic MSI colorectal cancer MLH1 inactivation it does not play a significant role in HNPCC tumors with germline MLH1 mutations. This is possibly an explanation for tumorigenic pathways and clinicopathological characteristic differences between sporadic and hereditary MSI colorectal cancers. Finally, despite indistinguishable genetic predisposition for endometrial and colorectal cancers, instability profiles highlighting organ-specific differences, may be important HNPCC tumor spectrum determinants.

Studies on the pro-oxidative properties and neurotoxic potential of Angeli's salt-derived nitroxyl (HNO/NO-)

The biological function of nitric oxide and its oxidized forms has received a great deal of attention over the past two decades. However much less attention has been focused on the reduced nitric oxide, nitroxyl (HNO). Unlike NO, HNO is highly reactive species and thus it needs to be generated by using donor compounds under experimental conditions. Currently there is only one donor available, Angeli s salt, which releases HNO in a controlled fashion under pysiological conditions. Prior studies have shown the pro-oxidative and cytotoxic potential of Angeli s salt compared to NO donors. The high reactivity of HNO with cysteine thiols is considered to form the biochemical basis for its unique properties compared to other nitrogen oxides. Such thiol modification cold result in disturbances of vital cellular functions and subsequently to death of disturbance sensitive cells, such as neurons. Therefore modification of proteins and lipids was studied in vitro and the potential neurotoxicity was studied in vivo by local infusion of Angeli s salt into the rat central nervous system. The results show that under aerobic in vitro conditions, HNO can, subsequent to autoxidation, cause irreversible oxidative modification of proteins and lipids. These effects are not however seen in cell culture or following infusion of Angeli s salt directly into the rat central nervous tissue likely due to presence of lower oxygen and higher thiol concentration. However, due to high reactivity with thiols, HNO can cause irreversible inactivation of cysteine modification sensitive enzymes such as cysteine proteases papain in vitro and cathepsin B in cell culture. Furthermore it was shown that infusion of HNO releasing Angeli s salt into the rat central nervous system causes necrotic cell death and motor dysfunction following infusion into the lumbal intrathecal space. In conclusion, the acute neurotoxic potential of Angeli s salt was shown to be relatively low, but still higher compared to NO donors. HNO was shown to affect numerous cellular processes which could result in neurotoxicity if HNO was produced in vivo.

The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

The Role Of Fumarase (FH) In Tumorigenesis

In this study, a predisposing gene for a recently characterized cancer syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC), was identified and the role of the gene was investigated in other familial cancers and in nonsyndromic tumorigenesis. HLRCC is a dominantly inherited disorder predisposing predominantly to uterine and skin leiomyomas, and also to renal cell cancer and uterine leiomyosarcoma. The disease gene was recently localized in Finnish families to 1q42-q43 by a genome-wide linkage search. Independently in the UK, a clinically similar condition, multiple cutaneous and uterine leiomyomata (MCUL), was linked to the same chromosomal region, strongly suggesting that HLRCC and MCUL are actually a single syndrome. Linkage results were confirmed by detecting loss of heterozygosity (LOH) at the disease locus in most of the patients' tumors, suggesting that this predisposing gene acts as a tumor suppressor. Through detailed investigation by genotyping of microsatellite markers and haplotype construction in Finnish and UK HLRCC/MCUL families we were able to narrow the disease locus down to 1.6 Mb. Extensive mutation screening of known and predicted transcripts in the target region resulted in identification of the HLRCC predisposing gene, fumarase (fumarate hydratase, FH). FH is a key enzyme in energy metabolism, catalyzing fumarate to malate in the tricarboxylic acid cycle (TCAC) in mitochondria. Germline alterations in FH segregating with the disease were detected in 25 of 42 HLRCC/MCUL families including whole-gene deletions, truncating small deletions/insertions and nonsense mutations, as well as substitutions or deletions of highly conserved amino acids. Biallelic inactivation was detected in almost all studied tumors of HLRCC patients. Furthermore, FH enzyme activity was reduced in the patients' normal tissues and was completely or virtually absent from tumors. Based on these findings, we extensively demonstrated that mutations in FH underlie the HLRCC/MCUL syndrome. In our studies of other familial cancers, evidence for involvement of FH defects was not found in familial prostate and breast cancers. To investigate the role of FH in sporadic tumorigenesis, we analyzed 652 lesions, including a series of 353 nonsyndromic counterparts of tumor types associated with HLRCC. Mutations in nonsyndromic tumors were rare and appeared to be limited to tumor types observed in the hereditary form of the disease. Biallelic inactivation of FH was detected in a uterine leiomyosarcoma, a cutaneous leiomyoma, a soft-tissue sarcoma, and in two uterine leiomyomas. In the uterine leiomyosarcoma and the cutaneous lesion FH mutations originated from the germline whereas the soft-tissue sarcoma harbored purely somatic changes. In uterine leiomyomas somatic mutations were detected in the two out of five tumors with LOH at the FH locus. Our findings demonstrate that FH inactivation is also involved in nonhereditary tumor development, and further support the hypothesis that FH acts as a tumor suppressor. The role of FH in predisposition to malignancies, renal cell carcinoma and leiomyosarcoma is important in the diagnosis and prevention of cancer among HLRCC patients. This study is of general clinical interest, because prior to our findings, little was known about the molecular genetics of uterine leiomyomas, the most common tumors of women.

Molecular basis of colorectal cancer predisposition

Colorectal cancer (CRC) is one of the most frequent malignancies in Western countries. Inherited factors have been suggested to be involved in 35% of CRCs. The hereditary CRC syndromes explain only ~6% of all CRCs, indicating that a large proportion of the inherited susceptibility is still unexplained. Much of the remaining genetic predisposition for CRC is probably due to undiscovered low-penetrance variations. This study was conducted to identify germline and somatic changes that contribute to CRC predisposition and tumorigenesis. MLH1 and MSH2, that underlie Hereditary non-polyposis colorectal cancer (HNPCC) are considered to be tumor suppressor genes; the first hit is inherited in the germline and somatic inactivation of the wild type allele is required for tumor initiation. In a recent study, frequent loss of the mutant allele in HNPCC tumors was detected and a new model, arguing against the two-hit hypothesis, was proposed for somatic HNPCC tumorigenesis. We tested this hypothesis by conducting LOH analysis on 25 colorectal HNPCC tumors with a known germline mutation in the MLH1 or MSH2 genes. LOH was detected in 56% of the tumors. All the losses targeted the wild type allele supporting the classical two-hit model for HNPCC tumorigenesis. The variants 3020insC, R702W and G908R in NOD2 predispose to Crohn s disease. Contribution of NOD2 to CRC predisposition has been examined in several case-control series, with conflicting results. We have previously shown that 3020insC does not predispose to CRC in Finnish CRC patients. To expand our previous study the variants R702W and G908R were genotyped in a population-based series of 1042 Finnish CRC patients and 508 healthy controls. Association analyses did not show significant evidence for association of the variants with CRC. Single nucleotide polymorphism (SNP) rs6983267 at chromosome 8q24 was the first CRC susceptibility variant identified through genome-wide association studies. To characterize the role of rs6983267 in CRC predisposition in the Finnish population, we genotyped the SNP in the case-control material of 1042 cases and 1012 controls and showed that G allele of rs6983267 is associated with the increased risk of CRC (OR 1.22; P=0.0018). Examination of allelic imbalance in the tumors heterozygous for rs6983267 revealed that copy number increase affected 22% of the tumors and interestingly, it favored the G allele. By utilizing a computer algorithm, Enhancer Element Locator (EEL), an evolutionary conserved regulatory motif containing rs6983267 was identified. The SNP affected the binding site of TCF4, a transcription factor that mediates Wnt signaling in cells, and has proven to be crucial in colorectal neoplasia. The preferential binding of TCF4 to the risk allele G was showed in vitro and in vivo. The element drove lacZ marker gene expression in mouse embryos in a pattern that is consistent with genes regulated by the Wnt signaling pathway. These results suggest that rs6983267 at 8q24 exerts its effect in CRC predisposition by regulating gene expression. The most obvious target gene for the enhancer element is MYC, residing ~335 kb downstream, however further studies are required to establish the transcriptional target(s) of the predicted enhancer element.

Genetic studies on recurrent miscarriage

Recurrent miscarriage (RM) is defined as three consecutive pregnancy failures and is estimated to affect ~1% of couples trying to conceive. The cause of RM remains unknown in approximately 50% of cases. In this study, it was hypothesized that some of the underlying factors yet to be discovered are genetic. The aim was to search for mutations in genes AMN, EPCR, TM, and p53 known to cause miscarriage in mouse models and thereby find new genetic causes for unexplained miscarriages in humans. In addition, the mitochondrial genome was studied because mitochondria are involved in processes important in early development. Furthermore, sex chromosome characteristics suggested to underlie miscarriage were also studied. A total of 40 couples and 8 women with unexplained RM were collected for this study and screened for mutations in the candidate genes. Six interesting exonic or potential splice site disrupting variations were detected. However, their phenotypic effects cannot be determined without further investigations. Additionally, an association between the C11992A polymorphism of the p53 gene and RM was detected. The results indicate that women carrying the C/A or A/A genotype have a two-fold higher risk for RM than women with a C/C genotype. This strengthens the results of previous studies reporting that p53 sequence variations may cause miscarriage. The role of variation C11992A in embryonic development is, however, difficult to predict without further studies When screening the mitochondrial genome a heteroplasmic mtDNA variation was found in an unexpected high number of women, as heteroplasmic variations are reported to be rare. One novel variation and 18 previously reported polymorphisms were detected in the mitochondrial genome. Although the detected variations are likely to be neutral polymorphisms, a role in the aetiology of miscarriage cannot be excluded as some mtDNA variations may be pathogenic only when a threshold is reached. Recent publications have reported skewed X chromosome inactivation and Y chromosome microdeletions to be associated with RM. Therefore, these sex chromosome abnormalities in the context of RM were investigated. No associations between skewed X chromosome inactivation or Y chromosome microdeletions and RM in the Finnish patients were detected. Data on ancestral birthplaces of the patients were collected to study any possible geographic clustering, which would indicate a common predisposing factor. The results showed clustering of the birthplaces in eastern Finland in a subset of patients. This suggests a possibility of an enriched susceptibility gene which may contribute to RM.

Microarrays in Molecular Profiling of Ewing Sarcoma Family of Tumors and CDKN2A Aberrations

Background: The Ewing sarcoma family of tumors (ESFT) are rare but highly malignant neoplasms that occur mainly in bone or but also in soft tissue. ESFT affects patients typically in their second decade of life, whereby children and adolescents bear the heaviest incidence burden. Despite recent advances in the clinical management of ESFT patients, their prognosis and survival are still disappointingly poor, especially in cases with metastasis. No targeted therapy for ESFT patients is currently available. Moreover, based merely on current clinical and biological characteristics, accurate classification of ESFT patients often fails at the time of diagnosis. Therefore, there is a constant need for novel molecular biomarkers to be applied in tandem with conventional parameters to further intensify ESFT risk-stratification and treatment selection, and ultimately to develop novel targeted therapies. In this context, a greater understanding of the genetics and immune characteristics of ESFT is needed. Aims: This study sought to open novel insights into gene copy number changes and gene expression in ESFT and, further, to enlighten the role of inflammation in ESFT. For this purpose, microarrays were used to provide gene-level information on a genomewide scale. In addition, this study focused on screening of 9p21.3 deletion sizes and frequencies in ESFT and, in another pediatric cancer, acute lymphocytic leukemia (ALL), in order to define more exact criteria for highrisk patient selection and to provide data for developing a more reliable diagnostic method to detect CDKN2A deletions. Results: In study I, 20 novel ESFT-associated suppressor genes and oncogenes were pinpointed using combined array CGH and expression analysis. In addition, interesting chromosomal rearrangements were identified: (1) Duplication of derivative chromosome der(22)(11;22) was detected in three ESFT patients. This duplication included the EWSR1-FLI1 fusion gene leading to increase in its copy number; (2) Cryptic amplifications on chromosomes 20 and 22 were detected, suggesting a novel translocation between chromosomes 20 and 22, which most probably produces a fusion between EWSR1 and NFATC2. In study II, bioinformatic analysis of ESFT expression profiles showed that inflammatory gene activation is detectable in ESFT patient samples and that the activation is characterized by macrophage gene expression. Most interestingly, ESFT patient samples were shown to express certain inflammatory genes that were prognostically significant. High local expression of C5 and JAK1 at the tumor site was shown to associate with favorable clinical outcome, whereas high local expression of IL8 was shown to be detrimental. Studies III and IV showed that the smallest overlapping region of deletion in 9p21.3 includes CDKN2A in all cases and that the length of this region is 12.2 kb in both Ewing sarcoma and ALL. Furthermore, our results showed that the most widely used commercial CDKN2A FISH probe creates false negative results in the narrowest microdeletion cases (<190 kb). Therefore, more accurate methods should be developed for the detection of deletions in the CDKN2A locus. Conclusions: This study provides novel insights into the genetic changes involved in the biology of ESFT, in the interaction between ESFT cells and immune system, and in the inactivation of CDKN2A. Novel ESFT biomarker genes identified in this study serve as a useful resource for future studies and in developing novel therapeutic strategies to improve the survival of patients with ESFT.

Genetic Mechanisms of Tumourigenesis in von Hippel-Lindau-Associated Tumours with the Emphasis on Capillary Hemangioblastoma

The von Hippel-lindau (VHL) disease is a dominantly inherited neoplastic disorder which predisposes patients to multiple tumours including capillary haemangioblastomas (CHBs), pheochromocytomas (PCCs), renal cell carcinomas (RCCs). CHBs are the most common manifestations of VHL disease, occurring sporadically or as a manifestation of VHL disease. Inactivation of the VHL gene at 3p25-26 is believed to cause both familial and sporadic VHL-associated tumours and germ-line mutation of the VHL gene have been detected in 100% of the CHBs studied. However, a limited number of sporadic CHBs, PCCs display VHL inactivation. Other molecular alterations involved in tumourigenesis of sporadic CHBs, PCCs remain largely unknown. The purpose of the present work was to search for genetic alterations, or other mechanisms of inactivation, in addition to the VHL gene, that may be important in the development of VHL-associated tumours. Though less satisfactory than cure, prevention and early detection are the most promising and feasible means reducing cancer morbidity and mortality. This work is based on the view that increasing knowledge about the molecular events underlying tumour development will eventually aid in early detection and lead to improved treatment. We evaluated a large set of VHL-associated patients, searched for a clinical and radiologic signs of the disease. We succesfully performed a germ-line mutation analysis and characterised three patient groups, VHL, suspect VHL and sporadic, a germ-line mutation analysis revealed a 50% mutation rate only in the VHL groups, no sporadic or suspect cases displayed any mutation. We also utilized comparative genomic hybridization (CGH) to screen for DNA copy number changes in both sporadic and VHL-associated CHB. Our analysis revealed (27%) DNA copy number losses. The most common finding was loss of chromosomal arm 6q, seen in (23%) cases, No differences were noted between VHL-associated and sporadic tumours. Furthermore a loss of heterozygosity (LOH) study on chromosome 3p and 6q was done with the purpose to determine allele losses not observable by CGH, and to uncover the location of putative tumour suppressor genes important in CHB and PCC tumourigenesis. We identified loss of chromosome 6q and a minimal deleted area at 6q23-24 in CHBs. We also showed LOH at 6q23-24 in PCCs and identified the ZAC1 (6q24-25) as a candidate gene, ZAC1 is a maternally imprinted tumour suppressor gene with anti proliferative properties. To study further the role of ZAC inactivation in CHBs, we investigated LOH, promoter hypermethylation and expression status of the ZAC1 gene in mainly sporadic CHBs. Our LOH analysis revealed that the majority of the tumours with allele loss. The gene promoter methylation analysis similarly detected predominance of the methylated ZAC sequence in almost all tumours. Immunohistochemistry exhibited a strongly reduced expression of ZAC in stromal cells of all CHBs studied. Our current results indicate that the absence of the unmethylated, ZAC1 promoter sequence was highly concurrent with LOH for the ZAC1 region or 6q loss. This observation together with lack of ZAC expression, points to preferential loss of the non imprinted, expressed ZAC allele in CHB, in summary, our series of studies reveal a new chromosomal region 6q, emphasizes the importance of ZAC1 gene in the development of CHB and PCC, particularly in non-VHL associated cases.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

HPLC analysis of plant sterol oxidation products

Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.