1. Despite extensive research on oral squamous cell carcinoma (OSCC), its five-year survival rate has not improved for the last two decades. Effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, DDRT-PCR analysis was used to identify molecular markers, which could be used as therapeutic targets. 2. DDRT-PCR in combination with reverse Northern analysis identified 25 differentially expressed genes in oral tumors. Fourteen genes did not show homology to any known gene in the database and therefore may represent non-specific genomic DNA sequences or novel genes that have not yet been identified. The remaining 11 genes showed homology to known genes such as DIAPH1, NJMU-R1, RBM28, PCNA, GLTP, MTATP6, ZKSCAN1, TNKS2, PAM, TUBB2C and C14orf154. TNKS2, PAM, TUBB2C and C14orf154 showed downregulation and the remaining seven genes were upregulated in oral tumor samples. 3. To reconfirm the results of DDRT-PCR and reverse Northern blot analyses, Northern blot analysis was carried out on matched normal and tumor samples for a few genes. As expected, PCNA, NJMU-R1 and ZKSCAN1 showed upregulation, whereas TUBB2C showed downregulation in the tumor sample. PCNA was also found to be upregulated in tumor samples at the protein level. 4. The expression of eight differentially expressed genes (viz., DIAPH1, NJMU-R1, RBM28, PCNA, GLTP, TNKS2, PAM and TUBB2C) was also validated in a panel of 16 matched normal and tumor samples. The mean mRNA expression levels of GLTP, PCNA, RBM28, NJMU-R1 and DIAPH1 were significantly greater in tumor samples than in normal samples. The mean expression levels of TNKS2, PAM and TUBB2C were significantly lower in tumor samples than in normal samples. 5. As some of the genes like NJMU-R1, RBM28, GLTP and PAM are found to differentially regulated in a majority of the tumors, they could be used as potential markers in oral cancer. 6. Tuberin and hamartin have been placed as a complex in the insulin signaling pathway and are known to negatively regulate this pathway. Since overexpression of TSC2 has been previously shown to exert antitumor effect on two oral cancer cell lines, and some components of the insulin signaling pathway have already been implicated in head and neck cancers, we reasoned that both TSC genes and other key players of this pathway might be differentially regulated in oral tumors. Northern blot analysis showed downregulation of the TSC2 gene in an oral tumor sample. In order to further validate the expression pattern of the TSC2 gene, a semiquantative RT-PCR analysis was carried out in a panel of 16 matched normal and tumor samples. The mean expression level of TSC2 was significantly lower in tumor samples than in normal tissue samples. The mean expression level of its interacting partner TSC1 was also significantly lower in tumor samples than in normal tissue samples, suggesting the involvement of these genes in the etiology of oral cancer. TSC1 and TSC2 were also downregulated in eight matched normal and tumor samples at the protein level. We wanted further to determine the expression of both TSC genes in cell lines. Interestingly, TSC2 did not show a detectable level of expression in an oral cancer cell line SCC 131, whereas it was expressed in two other oral cancer cell lines KB and SCC 104 as well as in four non-oral cell lines: A549, HEK-293T, HeLa and HepG2 at the protein level. The TSC2 expression in KB was, however, lower than in other cell lines. TSC1 was expressed in all the cell lines, albeit at different levels. The TSC1 expression was lower in SCC 131 as compared to two other cell lines KB and SCC 104. 7. Given the fact that both are tumor suppressors, it was hypothesized that LOH, inactivating somatic mutations and/or promoter methylation might be playing a role for their downregulation in oral tumors. Mutation analysis of all the coding regions of both the TSC genes failed to detect any mutation in a panel of 25 tumor samples. However, seven normal population variants were identified in different patients. Our analysis of the matched peripheral blood and tumor DNA samples from 52 patients showed LOH at both the TSC loci. At the TSC1 locus, 17/48 (35.42%) tumors showed an allelic loss for one or more markers. At the TSC2 locus, LOH was found in 18/48 (37.5%) informative cases. Nine patients (9/48, 18.75%) had LOH at both the TSC loci. Since PTEN is another tumor suppressor in the insulin signaling pathway, we then sought to determine if LOH is also present in the PTEN candidate region in a panel of 50 matched samples. Microsatellite analysis using three markers showed a low LOH rate of 13% in tumor samples. 8. As the OSCC cell line SCC 131 did not show a detectable level of TSC2 expression, we treated this cell line with methylation inhibition drug 5-azacytidine. The treatment restored the expression of TSC2 and increased the expression of TSC1, suggesting that the promoter methylation and LOH are the important mechanisms for their downregulation. In order to see if the downregulation of the TSC genes is due to their promoters being methylated in tumors from the patients, we examined the methylation status of their promoters in 16 oral tumors, three normal oral tissues, two peripheral blood DNA samples from normal individuals and two cell lines HeLa and SCC 131 by COBRA. Our repeated efforts to amplify the TSC1 promoter using different DNA polymerases failed. However, we were able to successfully amplify the 571 bp long TSC2 promoter. Our analysis showed methylation of the TSC2 promoter in all tumors and two cell lines. As expected, the TSC2 promoter was not methylated in normal oral tissues and control blood DNA samples. Our bisulfite sequencing data suggested a low level and a considerable heterogeneity of methylation. 9. Using Fisher’s exact test, no correlation was found between LOH at the TSC loci and different clinical parameters such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis. 10. Using Fisher’s exact test, no correlation was found between the TSC2 promoter methylation and its downregulation in 16 tumor samples. We believe that this could be due to small sample size. 11. Since TSC1 and TSC2 are important regulators of the insulin pathway, it was hypothesized that other key players of this pathway might also be dysregulated in oral cancer. To this end, the expression pattern of some of the major regulators of the insulin pathway (viz., PI3K, AKT, PDK1, RHEB, mTOR, S6K1, S6, eIF4E, 4E-BP1, PTEN, 14-3-3ﾟ and IRS1) was investigated using semiquantative RT-PCR in a panel of 16 matched normal and tumor samples. The mean expression levels of the following genes showed significant upregulation in tumor samples: AKT, PI3K, PDK1, RHEB, mTOR, S6K1, S6 and eIF4E. On the other hand, 4E-BP1 and PTEN showed significant downregulation in tumor tissues. No significant difference in the expression was found for 14-3-3ﾟ and IRS1 between tumor and normal tissues. The expression pattern of some of these genes was also analyzed at the protein level using Western blot analysis and eight matched normal and tumor tissues. The level of total AKT was upregulated in 2/8 tumor samples only. However, phosphorylated-AKT (Thr308) showed upregulation in 6/8 samples. p70S6K1 and phosphorylated-p70S6K1 (Thr389) were upregulated in 8/8 and 6/8 tumor samples, respectively. Increase in the phosphorylated forms of both AKT and its downstream effector p70S6K1 suggested an increase in their kinase activity, indicating a constitutive activation of this pathway in oral cancer. 12. Based on our findings of mutation analysis, LOH study, 5-azacytidine treatment of an oral cancer cell line and COBRA analysis, we suggest that LOH at the TSC gene loci and promoter methylation are important mechanisms for the downregulation of the TSC genes. Loss of function of these genes may thus contribute to the constitutive activation of the insulin signaling pathway in oral cancer, leading to overall cell growth and proliferation. Our studies have shown that several key members of this pathway show aberrant expression in a subset of cancers of the oral cavity and can provide useful therapeutic targets. Several inhibitors of the insulin signaling pathway, such as rapamycin and its derivatives which inhibit mTOR and the PI3K inhibitor wortmannin, are now being actively evaluated for clinical trials for other cancers. We suggest that these inhibitors could also be evaluated for the treatment of oral cancer in future. Our differential display analysis has served to identify several genes that may be important for the onset and progression of oral cancer. Further analysis of these genes is warranted.

Microarray expression analysis was carried out to identify genes with a role in epithelial ovarian cancer (EOC). The U133A Affymetrix GeneChipRTM was used to determine the expression patterns of the 3p25.3-ptel genes represented on the microarray in 14 primary cultures of normal ovarian surface epithelial (NOSE) samples, 25 frozen malignant ovarian tumor samples and four EOC cell lines. Seven genes with differential expression patterns in the tumor samples compared to the NOSE samples were identified as candidates for further analysis, starting with ARPC4, SRGAP3 and ATP2B2. Although none of the candidates had been previously studied in ovarian cancer, several had either family or pathway members that had. Expression patterns seemed unaffected by either tumor histopathological subtype or the allelic imbalances observed with loss of heterozygosity (LOH) analysis. The absence of association with genomic context suggested that differential expression was the result of transcriptional regulation rather than direct targeting.

DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes. / QC 20100819

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Colorectal cancer (CRC) is a common malignancy which develops due to old age and lifestyle factors, low percent of patients afflicted by a genetic disorders. Half of all colorectal cancer patients are diagnosed after metastasis. The high rate of the late detection, emphasizes on the requirement of convenient and inexpensive diagnostic methods for comprehensive screening programs. The aim of this study was to discover proto-oncogenes mutation and assessment of tumor suppressor genes expression. Formalin fixed paraffin embedded (FFPE) histologically verified colorectal cancer samples were used. APC, KRAS and BRAF mutations were investigated using polymerase chain reaction (PCR) fragments and direct sequencing. Gene expression assessment of MLH1, MGMT and CDKN2A were achieved via quantitative polymerase chain reaction (qPCR). In the present study we could detect a novel transversion heterozygous mutation in APC gene codon 1365 in three patients. BRAF codon 600 mutation were detected in one patient. KRAS codon 12 mutation was discovered in one sample and also a novel transition mutation in codon 15 was detected in 6 patients. In 80% of cases, MLH1 and MGMT expression were undetectable, in remaining 20%, MLH1 expression were reduced, but MGMT showed both reduced and increased expression compared to control. In 100% of patients CDKN2A expression was undetectable. The rate of mutations in predetermined hotspot codons and amount of uncommon mutations into APC, BRAF and KRAS in Nepalese patients indicates the requirement of further investigation in CRC patients from that part of the world. Also, the expression rate of MLH1, MGMT, CDKN2A and deficiency of an information source emphasizes the necessity of whole genome CRC expression profiling data to comparison and conclusion. / <p>-</p> / -

One important objective of prostate cancer (PCa) research is to understand the molecular basis underlying the progression of these cancers from an androgen dependent to an androgen independent state. Hypermethylation of the promoter CpG islands is associated with the transcriptional silencing of specific gene sets in each tumor type and subtype. Transcriptional silencing of antitumor genes via CpG island hypermethylation could be a mechanism mediating PCa progression from an androgen-dependent to an androgen-independent state. Hypermethylation associated gene silencing has been reported for a great number of genes in PCa with the exception of the genes that undergo methylation associated silencing specifically during cancer development to androgen independence. The first aim of this thesis is to identify novel glenes which undergo DNA hypermethylation associated gene silencing during the cancer progression. The androgen-dependent (AD, as defined as the inability of celill to proliferate in the absence of androgen) PCa cell line LNCaP gives rise to the androgen-independent (AI) subline LNCaPcs generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. This LNCaP cell model was used to identify differentially methylated sequences between the two genomes using the Methylation-Sensitive Restriction Fingerprinting (MSRF) technique. One sequence identified is located in a 5' CpG island, which encompasses part of the promoter, exon 1, and part of intron 1, of the Peroxisomal Membrane Protein 24 KD (PMP24) gene. PMP24 is silenced in concert with the hypermethylation of its CpG island in AI LNCaPcsand PC-3 cell lines. The silencing is reactivated by the treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AZAdC). PMP24 is specifically silenced in PCa cancer cell lines and shows potential antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence. The next study investigated the molecular mechanism for DNA methylation associated gene silencing of PMP24 in AI LNCaPcs and PC-3 cell lines. We demonstrated that PMP24 transcription is repressed by the disruption of transcription factor binding to a critical cis-element by hypermethylation of its promoter CpG island. We found a CpG containing activator protein 2 (AP-2) cis-element in the intron 1 of PMP24 whose first CpG dinucleotidle is essential for the sequence-specific protein binding and the promoter activity of the gene. We presented first in cellulo evidence that the methylation of AP-2 cis-element alone but not the whole CpG island, using a newly developed methylated oligonucleotides treatment, is sufficient for the downregulation of PMP24. Our study is the first to report that the silencing mechanism for PMP24 in AI LNCaPcs and PC-3 is mediated by the complete methylation of a single GpG site of AP-2 cis-element in the intron 1 part of the CpG island, which interferes with transcription factor binding. Most interestingly, the promoter CpG island of PMP24 is hypermethylated in AD LNCaP cells with the incomplete methylation specifically at the AP-2 cis-element. The silencing of PMP24 in AD LNCaP cells was reactivated not by the 5AZAdC treatment but by the treatment with Trichostatin A (TSA), a histone deacetylase inhibitor. An alternative silencing mechanism for PMP24 other than the interference with transcription factor binding by methylation is therefore likely involved at this androgen-dependent stage. During the androgen ablation process, this mechanism is either evolved by the spread of methylation in the promoter CpG island or selected against, leading to the methylation-dominant silencing mechanism in the AI cells as seen in LNCaPcsand PC-3 cells. Taken together, this thesis emphasized the important role of DNA methylation in the progression of PCa into androgen independence. Particular respect should be paid to the specific CpG dinucleotides in cis-elements critical for the promoter activity, whose complete methylation could dominate the silencing mechanism which is independent of androgen. This thesis also pointed to the importance of monitoring the effects of cell culture on the methylation status of genes. Most importantly, this thesis raised the possibility that the silencing mechanisms for PMP24 could be different in AD LNCaP cells as compared to AI LNCaPcs and PC-3 cells. Either the evolution of such mechanism or the selectivity against it during the androgen ablation process would result in a methylation-dominant silencing mechanism of the genes such as PMP24 in AI cells and may contribute to the overall androgen independence of the cells.

Introdução: Os micro RNA (miRNA) são formados a partir de RNA precursores de fita dupla que contém entre 60 a 110 nucleotídeos e formam estruturas do tipo hairpin. Imediatamente após sua transcrição pela RNA polimerase II a enzima Dicer promove a clivagem do RNA precursor em seqüências menores contendo 19 a 22 nucleotídeos. Após a clivagem, o miRNA integra-se ao complexo silenciador induzido pelo RNA (RISC) que o conduz ao seu RNA mensageiro (mRNA) homólogo recém transcrito. Esta associação promove a degradação do mRNA, ou interfere na tradução da proteína caracterizando um grande mecanismo de controle da expressão dos genes. Este mecanismo está relacionado ao desenvolvimento de órgãos e tecidos, e está envolvido no processo de carcinogênese. Nosso objetivo é identificar um perfil de expressão de miRNA que defina o adenocarcinoma de próstata de prognóstico favorável e desfavorável considerando os níveis de PSA e dados anatomopatológicos. Materiais e métodos: Foram selecionados 53 pacientes com tumores desfavoráveis (mediana do escore de Gleason igual a 8, 79,2% estadiados pT3, mediana de PSA 10,1 ng/mL e mediana do volume tumoral de 23%) e 45 considerados favoráveis (mediana do escore de Gleason igual a 5, 80% estadiados pT2, mediana de PSA de 7,8 ng/mL e mediana do volume tumoral de 11,5%). O controle foi representado por 7 pacientes com hiperplasia prostática benigna (HPB). Todos os pacientes foram submetidos a prostatectomia radical pelo mesmo cirurgião. Os espécimes cirúrgicos foram examinados na sua totalidade pelo mesmo patologista. A análise dos miRNA foi feita a partir de tecido congelado e tecido incluído em parafina usando a técnica da reação em cadeia da polimerase em tempo real quantitativa (qRT-PCR) utilizando primers e sondas Taqman® específicas. O RNU43 foi usado como controle interno. Resultados: Com exceção dos miRNA 199a, 21, 15a, 16 e 25 que se mostraram subexpressos tanto nos casos desfavoráveis como nos favoráveis, houve uma diminuição global na expressão dos miRNA com redução estatisticamente significativa na expressão dos miRNA 143, 145 e 146a, 191, 218 e Let7c em tumores desfavoráveis em relação aos tumores favoráveis. Conclusão: Demonstramos que no processo de transição entre os carcinomas favoráveis e desfavoráveis de próstata existe uma perda global na expressão de miRNA que podem ser importantes controladores de expressão de uma série de genes relacionados a progressão desta neoplasia. Dados experimentais avaliando o papel desses miRNA devem ser conduzidos para que possamos definir seu papel na evolução do câncer de próstata / Introduction: micro RNA (miRNA) are formed from double-stranded RNA precursors that contain between 60-110 nucleotides and form structures such as hairpin. Immediately after their transcription by RNA polymerase II, the enzyme Dicer promotes the cleavage of precursor RNA sequences containing minor 19-22 nucleotides. After cleavage, the miRNA is part of the RNA-induced silencing complex (RISC) that leads to its messenger RNA (mRNA) newly transcribed counterpart. This association promotes the degradation of mRNA, or interferes with the protein translation characterizing a great mechanism for controlling gene expression. This mechanism is related to the development of organs and tissues, and may be involved in the process of carcinogenesis. Our goal is to identify a miRNA expression profile that distinguishes prostate adenocarcinoma of favorable and unfavorable prognosis considering the PSA and pathological findings. Material and Methods: We studied 53 patients with tumors considered unfavorable (Median of Gleason score 8, 79.2% staged pT3, median of PSA 10.1 ng/mL and median of tumor volume of 23%) and 45 considered favorable (Median of Gleason score 5, 80% staged pT2, median of PSA 7.8 ng/mL and median of tumor volume of 11.5%). The control group was represented by seven patients with benign prostatic hyperplasia (BPH). All patients underwent radical prostatectomy by the same surgeon. The surgical specimen was examined entirely by the same pathologist. The analysis of miRNA was made from frozen and paraffin embedded tissue by quantitative real-time polymerase chain reaction (qRT-PCR) using the Taqman® specific primers and probes. The RNU43 was used as a internal control. Results: Except for miRNAs 199a, 21, 15a, 16 e 25 that were underexpressed by both favorable and unfavorable prostate cancer, there was a global decrease of all miRNAs studied, and some differences were statistically significant as miRNAs 143, 145 e 146a, 191, 218 e Let7c that were underexpressed in unfavorable carcinomas compared favorable tumor. Conclusion: We have demonstrated that in the process of transition between favorable and unfavorable prostate cancer there is a global loss of expression of miRNAs. These molecules can be important controllers of a series of genes related to cancer progression. Experimental studies are needed in order to comprehend the role of these genes in prostate carcinogenesis