<p>A solid-phase sequencing technique was applied to quantify the mitochondrial A3243G mutation in three families with maternally inherited diabetes and deafness. A correlation between the level of heteroplasmy and age at onset was found. The fluctuation of the heteroplasmy levels of the A3243G mutation was monitored from 4 to 18 years, in three female patients. Using the minisequencing method, the level of heteroplasmy was found to decrease over time in endothelial cell samples from all three patients. </p><p> With a similar strategy, the heteroplasmy levels of two neutral polymorphisms in the non-coding region of the mtDNA in healthy individuals were monitored. It has recently been suggested that heteroplasmy occurs frequently at neutral nucleotide positions in the control region of mtDNA and that the heteroplasmy level changes with age. The level of heteroplasmy of the neutral polymorphisms was found to remain unchanged over a time period of up to 25 years in four individuals. </p><p> Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a mitochondrial copper-transporting ATPase. The worldwide prevalence of WD has been estimated to 1 in 30 000. Based on the number of diagnosed patients the estimated prevalence in the Swedish population would be 1 in 300 000. The prevalence of WD in Sweden was estimated indirectly by quantitative minisequencing analysis of two WD-causing mutations in pooled DNA samples. In addition, the population frequencies of eight SNPs in the ATP7B gene were determined. Our results confirmed that WD is truly more rare in Sweden than in other populations.</p><p> A previously nondetectable diversity of alleles at the KIT locus, determining the coat color of pigs, was found by using three quantitative methods, minisequencing, pyrosequencing and the "TaqMan" 5' exonuclease assay. A splice-site mutation and a duplication of the KIT gene, encoding the mast/stem cell growth factor receptor causes the allelic diversity. Despite of a strong selection for white color dating from the medevial era, the desired phenotype has not been fixed. This study provides tools for genotyping the complicated KIT locus in pigs, which may be used for the purpose of breeding true for white color.</p>

<p>Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the <i>PDGFRA</i> gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called <i>Sequence Allocator</i>, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.</p>

Neuroblastoma is a malignant childhood cancer, originating from sympathetic neuroblasts of the peripheral nervous system. Neuroblastoma is a heterogenous group of tumours, while some are highly malignant others can spontaneosly mature into a more benign form or regress. Less than half of the patients survive and this statistics has improved only modestly over the past 20 years. SH-SY5Y is a human neuroblastoma cell line established from a highly malignant tumour. The cells have retained a capacity to differentiate in vitro in response to low concentrations of the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. Differentiated cells are characterised by neurite formation and upregulation of neuronal marker genes. SH-SY5Y are unresponsive to nerve growth factor (NGF), but when transfected to express the NGF-receptor TrkA, they differentiate in response to NGF. Protein kinase C (PKC) is pivotal for the differentiation response to take place. We have investigated the role of signaling through the Ras-MAPK pathway in differentiating SH-SY5Y, with respect to neurite formation, expression of neuronal marker genes and growth control. Our results show that differentiation-promoting treatment induced a sustained activation and nuclear accumulation of the MAPK ERK in SH-SY5Y. The nuclear accumulation of ERK was PKC-dependent. However, nuclear accumulation of ERK was not sufficient for a differentiation response to take place in these cells, but ERK activity was needed for the characteristic upregulation of NPY and GAP-43 induced by TPA. ERK activity did not induce neurite formation, neither was it necessary for TPA-induced neurite formation. Instead, stimulation of a pathway distinct from MEK/ERK, but downstream of Ras, was needed for morphological differentiation. We could also show that differentiated cells still entered S-phase and that there was no correlation between expression of the CKI p21cip1 (an ERK target), BrdU-incorporation or neurite formation.

Glioblastoma multiforme is a malignant brain tumor characterized by heterogeneity.Interactions between heterogeneous tumor cells are supposed to affect the behavior of awhole tumor cell population. In this thesis an in vitro model system of clonal glioma celllines originating from one glioblastoma tumor was used, and the behavior of cells incocultures was studied and compared the behavior of cells grown separately. The resultsindicate the presence of two types of interactions. In one, paracrine signals acted via extra-cellular media. This was associated with increased growth of the whole co-culture followedby a selective force driving one clone to dominance. In the other type, the cell clones grewside by side without signs of paracrine signalling, in a balance resulting in an increasedterminal cell density. Further investigations focused on mechanisms of interactions in thiscombination. Two cell clones were chosen, a GFAP+ and a GFAP-, for further experiments. Withdifferential display PCR it was possible to investigate their specific gene expressionpatterns. Seventeen cDNA fragments were differentially expressed, among them twocorresponded to known transcription factors, ATF3 and prox-1, one to a cytoskeletal protein,α-tropomyosin. The collection also contained eight ESTs (Expressed Sequence Tags) wherethe corresponding genes are unknown at present. Expression of the isolated sequences werealso analyzed in a panel of 12 different glioma cell lines and the results illustrate thecomplexity of gene expression and of tumor heterogeneity. Genes, the expression levels ofwhich were modulated in co-cultures and/or were cell density dependent, were alsoidentified. PDGF B is suggested to play a role in sarcomas. The gene codes for an mRNA transcriptwith long UTRs, parts of which are deleted in the homologous oncogene v-sis. The UTRs ofPDGF B mRNAs in human sarcomas were investigated for deletions similar to v-sis thatmight result in increased protein levels. A new transcript variant was identified, lacking a149 base region in the 3'UTR, but its presence was not associated with increased levels ofprotein. Alterations in the 5'UTR were found more likely to be associated with increasedprotein levels.

Medulloblastoma is a highly malignant cerebellar childhood tumor. As in many other brain tumors, expression of platelet-derived growth factor (PDGF) and its receptors has been shown in medulloblastoma. To reveal the importance of this growth factor in cerebellar development and tumorigenesis, analyses were performed on human medulloblastoma cell lines and on tissue from normal mouse brain at different stages of development. The in vivo effect of a forced expression of PDGF-B in the cerebellar primordium was examined in transgenic mice. In the normal mouse embryo, we found PDGF receptor-α-positive cells in the early neuroepithelium and on neuronal precursors. In the postnatal cerebellum, cells in the external germinal layer and Purkinje cells expressed the receptor. In the medulloblastoma cells, expression of all the three PDGF isoforms and PDGF receptors was seen and correlated to neuronal differentiation. Endogenously activated, i.e. tyrosine phosphorylated, PDGF receptors were identified. To reveal the role of PDGF in normal cerebellar development, we established transgenic mice where a PDGF-B cDNA was introduced via homologous recombination into the engrailed-1 gene. Engrailed-1 is specifically expressed at the mid-/hindbrain boundary of the early neural tube, i.e. in an area from which the cerebellar primordium develops. The ectopic expression of PDGF-B caused a disturbance of cerebellar development. Midline fusion of the cerebellar primordium did not occur properly, which resulted in cerebellar dysplasia in the adult mouse. In a parallel study, the expression pattern of a glial fibrillary acidic protein (GFAP)-lacZ transgene was followed in the embryonic mouse central nervous system. It was shown that the human GFAP promoter was already active by embryonic day 9.5 and as development proceeded, expression occured in different, independent cell populations. Among these cell populations were the radial glial cells in the neocortex.

A solid-phase sequencing technique was applied to quantify the mitochondrial A3243G mutation in three families with maternally inherited diabetes and deafness. A correlation between the level of heteroplasmy and age at onset was found. The fluctuation of the heteroplasmy levels of the A3243G mutation was monitored from 4 to 18 years, in three female patients. Using the minisequencing method, the level of heteroplasmy was found to decrease over time in endothelial cell samples from all three patients. With a similar strategy, the heteroplasmy levels of two neutral polymorphisms in the non-coding region of the mtDNA in healthy individuals were monitored. It has recently been suggested that heteroplasmy occurs frequently at neutral nucleotide positions in the control region of mtDNA and that the heteroplasmy level changes with age. The level of heteroplasmy of the neutral polymorphisms was found to remain unchanged over a time period of up to 25 years in four individuals. Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a mitochondrial copper-transporting ATPase. The worldwide prevalence of WD has been estimated to 1 in 30 000. Based on the number of diagnosed patients the estimated prevalence in the Swedish population would be 1 in 300 000. The prevalence of WD in Sweden was estimated indirectly by quantitative minisequencing analysis of two WD-causing mutations in pooled DNA samples. In addition, the population frequencies of eight SNPs in the ATP7B gene were determined. Our results confirmed that WD is truly more rare in Sweden than in other populations. A previously nondetectable diversity of alleles at the KIT locus, determining the coat color of pigs, was found by using three quantitative methods, minisequencing, pyrosequencing and the "TaqMan" 5' exonuclease assay. A splice-site mutation and a duplication of the KIT gene, encoding the mast/stem cell growth factor receptor causes the allelic diversity. Despite of a strong selection for white color dating from the medevial era, the desired phenotype has not been fixed. This study provides tools for genotyping the complicated KIT locus in pigs, which may be used for the purpose of breeding true for white color.

Adenovirus infected cells provide a good model system for studying nuclear organization during RNA production and transport. This thesis is focused on the dynamic organization of splicing factors during the late phase of Adenovirus infection in HeLa cells, the nuclear localization of viral RNA, and the pathway used for viral RNA transport to the cytoplasm. Splicing factors are relocalized from interchromatin granule clusters to sites of transcription in Adenovirus infected cells at intermediate times of infection. Later, splicing factors and viral RNA accumulate posttranscriptionally in interchromatin granule clusters. The release of the splicing factors from transcription sites was energy dependent or preceded by energy requiring mechanisms. Our data indicated that phosphorylation events inhibited by staurosporine, and 3' cleavage of the transcript are two possible mechanisms involved prior to the release of the RNP complex from transcription sites. A viral protein derived from orf6 of early region 4, 34K, is important for the nuclear stability and transport of late viral mRNA derived from the major late transcription unit. A viral mutant lacking this region is defective for posttranscriptional accumulation of viral mRNA in interchromatin granule clusters, and for the accumulation of viral RNA in the cytoplasm. These results suggest that posttranscriptional accumulation of viral RNA in interchromatin granule clusters may contribute to the maturation of the RNP complex or sorting of RNAs and proteins, to prepare the final RNP complex for transport to the cytoplasm. A previous model suggested that adenoviral late mRNA is transported to the cytoplasm by utilizing the CRM-1 pathway. This pathway can be blocked by the drug leptomycin B. The data presented in paper IV suggests that this model might not be applicable, since leptomycin B did not inhibit adenoviral late gene expression.

Platelet-derived growth factor receptor α (PDGFRα) plays an important role during embryogenesis. After implantation, the patterns of expression of Pdgfrα and its ligand Pdgf-A undergo an "autocrine-paracrine transition", in that Pdgf-A becomes expressed in the ectoderm and epithelia, while Pdgfrα is expressed in the adjacent mesenchymal tissue. In human tumors, such as malignant glioma, both PDGF and PDGFRα are overexpressed within the same tissue, indicating that an autocrine PDGF loop is generated in the tumors. This thesis is focused on the in vivo functionality of the PDGFRα gene (PDGFRA) promoter, arid on the effect of autocrine PDGF-A stimulation in transgenic n-iice during embryogenesis. To test the in vivo promoter function of a human PDGFRA 2.2 kb 5' flanking fragment, we generated transgenic mouse lines and found that the 2.2 kb fragment was able to promote lacZ reporter gene expression in most of the endogenous Pdgfra expressing tissues. Absence of expression and "ectopic" expression of the transgenic lacZ were also observed. To investigate the autocrine PDGF effect, we produced autocrine PDGF-As (A short-chain) transient transgenic embryos. These transgenic embryos carried a 6 kb mouse Pdgfra 5' flanking sequence linked to a human PDGF-As cDNA. The pattern of expression of the PDGF-As transgene mRNA was similar to that of lacZ. Some of the transgenic embryos exhibited severe abnormal phenotypes, such as midline fusion defects in the cephalic and craniofacial region and small body size, and these embryos die at mid-gestation stage. These findings indicate that a paracrine pattern of expression and the dosage of PDGF are important for sustaining normal embryo development, especially with regard to the middline fusion in craniofacial regions. The possible signaling pathways that may be involved in regulating Pdgfra activity were also studied by comparison of patterns of mRNA expression of Gli, Ptc, and Paxl with that of Pdgfra. The results pointed to the possibility that the Shh signaling pathway may be involved in the regulation of Pdgfra expression for example during early bone and foregut development. The specific regulatory mechanisms may vary for different tissues.

Mutation of the p53 gene appears to be an early event in skin cancer development. The present study is based on morphology and represents a cellular and genetic investigation of p53 alterations in normal human skin and basal cell cancer. Using double immunofluorescent labelling, we have demonstrated an increase in thymine dimers and p53 protein expression in the same keratinocytes following ultraviolet radiation. Large inter-individual differences in the kinetics of thymine dimer repair and subsequent epidermal p53 response were evident in both sunscreen-protected and non-protected skin. The formation of thymine dimers and the epidermal p53 response were partially blocked by topical sunscreen. We have optimized a method to analyze the p53 gene in single cells from frozen tissue sections. In chronically sun-exposed skin there exist clusters of p53 immunoreactive keratinocytes (p53 clones) in addition to scattered p53 immunoreactive cells. Laser assisted microdissection was used to retrieve single keratinocytes from immunostained tissue sections, single cells were amplified and the p53 gene was sequenced. We have shown that p53 mutations are prevalent in normal skin. Furthermore, we detected an epidermal p53 clone which had prevailed despite two months of total protection from ultraviolet light. Loss of heterozygosity in the PTCH and p53 loci as well as in the sequenced p53 gene was determined in basal cell cancer from sporadic cases and in patients with Gorlin syndrome. Allelic loss in the PTCH region was prominent in both sporadic and hereditary tumors, while loss of heterozygosity in the p53 locus was rare in both groups. p53 mutations found in the hereditary tumors differed from the typical mutations found in sporadic cases. In addition, we found genetically linked subclones with partially different p53 and/or PTCH genotypes in individual tumors. Our data show that both genes are important in the development of basal cell cancer.

All-trans retinoic acid (ATRA), a biologically active metabolite of vitamin A, is a powerful inducer of terminal differentiation and growth arrest of several myeloid cell lines in vitro. Although the efficacy of ATRA as an anti-cancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), knowledge concerning the molecular mechanisms directing ATRA-induced differentiation and cell cycle arrest of myeloid cells is lacking. Our results show, for the first time, that the complex regulation of cell cycle proteins and myeloid-specific transcription factors induced by ATRA relies on functional Stat1. We found that Stat1 is activated by both tyrosine-701 and serine-727 phosphorylation upon ATRA-induced differentiation of the human monoblastic cell line U-937. Expression of phosphorylation deficient mutants of Stat1 (Stat1Y701F or Stat1S727A) inhibited both ATRA-induced differentiation and cell cycle arrest of U-937 cells, pointing to a requirement of active Stat1 in these processes. Detailed analysis of the molecular mechanism of ATRA-induced cell cycle arrest and differentiation showed that the onset of cell cycle arrest was associated with a decrease in c-Myc and cyclin E levels and upregulation of p27Kip1 and p21WAF1/CIP1. This was followed by a rapid fall in cyclin A and B and a coordinate dephosphorylation of the retinoblastoma protein (pRb). The inhibition of ATRA-induced cell-cycle arrest by constitutive expression of Stat1Y701F or Stat1S727A was associated with impaired regulation of these cyclins and p27Kip1, positioning Stat1 activation upstream of these events. To further understand the process of ATRA-induced differentiation, the regulation of myeloid-specific transcription factors was investigated during ATRA-treatment. Notably, ATRA-induced upregulation of Stat2, ICSBP and C/EBP-ε was selectively impaired in sublines expressing Stat1Y701F or Stat1S727A, suggesting an important function of these factors downstream Stat1. Taken together, the work in this thesis clearly demonstrates that Stat1 plays a key role in ATRA-induced terminal differentiation of myeloid cells, through regulation of cell cycle proteins and myeloid-specific transcription factors.