Targeting oncogene promoters: a novel heterocyclic cations as G-quadruplex stabilizing ligands Lenka Pohlová Abstract: The diploma thesis studies an effect of newly synthesized group of compounds - helquats - on the expression of c-myc as a major player in malignant transformation and tumorigenesis via the stabilization of G-quadruplex in c-myc promotor. The G-quadruplex c-myc stabilization ability was tested for 101 helquats using dual luciferase reporter assay. The G-quadruplex c-myc stabilization ability was found for 13 helquats by this method. 8 successful helquats was selected by a comparison of the results from dual luciferase reporter assay and FRET melting assay. Effect on cell viability of tumor (HeLa S3) and non-tumor (HUVEC) cell lines was evaluated for these 8 helquats. Three of them exhibited cytotoxic effect on tumor cells but no effect was observed on viability of non-tumor cells. Moreover, an effect of these 3 helquats on c-myc expression on both mRNA and protein level, where significant effect on c-myc mRNA expression was not found for most of incubation periods. The 30% decrease in mRNA level was observed only for 24 hours incubation period for two helquats (LS702 and MJ656). The decrease in the expression on protein level was observed for all tested helquats, and helquat LS702 had the...

This PhD aimed to elucidate the mechanisms by which polymorphisms may alter androgen-induced transactivation of androgen receptor (AR) target genes which may be important in prostate cancer aetiology. The second aspect of this PhD focused on identifying and characterising functional polymorphisms that may have utility as predictive risk indicators for prostate cancer and which may aid in earlier therapeutic intervention and better disease management. Analyses were carried out on the kallikrein-related peptidase 3 (KLK3), also known as the prostate specific antigen (PSA), gene and the kallikrein-related peptidase 4 (KLK4) gene. The PSA and KLK4 genes are part of the serine protease family that have trypsin or chymotrypsin like activity and are thought to play a role in the development of hormone-dependent cancers in tissues such as those in the prostate, breast, endometrium and ovaries. In the prostate, PSA is regulated by androgens and three androgen response elements (AREs) have been described in the promoter and upstream enhancer region. The PSA ARE I harbours a polymorphism at -158 bp from the transcription initiation site (TIS) that results in a G to A transition (G-158A). This PhD investigated the functional significance of the PSA G-158A polymorphism which has been reported to be associated with prostate cancer risk. Electromobility shift assays (EMSAs) investigating the interaction of ARE I variants with the AR DNA binding domain (AR-DBD) demonstrated that the A allele had a two-fold increased binding affinity for the AR-DBD when compared with the G allele. This was confirmed with endogenous AR in limited proteolysis-EMSA experiments. The limited proteolysis-EMSA experiments also demonstrated differential sensitivities of PSA ARE I alleles to trypsin digestion, which suggests that the G-158A polymorphism has an allosteric effect on the AR that alters AR/ARE I complex stability. Furthermore, Chromatin Immunoprecipitation (ChIP) assays suggest that the A allele more readily recruited the AR in vivo when compared with the G allele and is consistent with the in vitro binding data. Luciferase reporter assays carried out in both LNCaP and 22Rv1 prostate cancer cells, and using the natural (dihydrotestosterone; DHT) ligand demonstrated that the A allele was more responsive to androgens in LNCaP cells. Hence, this study has elucidated the potential mechanisms by which the G-158A polymorphism may differentially regulate PSA expression (of which up-regulation of PSA is thought to be important in prostate cancer development and progression). KLK4 has similar tissue-restricted expression as PSA and is up-regulated by steroid hormones in many endocrine cells including those in the prostate. A putative ARE (KLK4-pARE) located at -1,005 to -1019 relative to the more predominantly used transcription initiation site, TIS3, was initially found in supershift assays using AR antibodies to interact with endogenous AR. However, subsequent EMSA analysis using purified AR-DBD suggest that KLK4-pARE may be interacting with the AR indirectly. To investigate this hypothesis, a tandem construct of KLK4-pARE was cloned into the pGL3-Promoter vector for hormone-induced reporter assays. However, reporter assays did not demonstrate any responsiveness of KLK4-pARE to androgens, estradiol or progestins. Consequently, Real-Time PCR was carried out to reassess the hormonal regulation of KLK4 at the mRNA level. Consistent with the literature, data from this study suggests that KLK4 may be up-regulated by androgens, progestins and estradiol in a cyclical manner. Hormone-induced luciferase reporter assays were then carried out on seven promoter constructs that span 2.8 kb of the KLK4 promoter from TIS3. However, none of the seven promoter constructs demonstrated any significant responsiveness to androgens, estradiol or progestins. This study suggests that hormone response elements (HREs) that may drive the hormonal regulation of KLK4 in prostate cancer may be located further upstream from the promoter region investigated in this PhD, or alternatively, may lie 3' of TIS3. The characterisation of KLK4 promoter polymorphisms and their flanking sequences were also carried out in parallel to the functional work with the intent to assess the functional significance of any polymorphisms that may be located within HREs. In total 19 polymorphisms were identified from the public databases and from direct sequencing within 2.8 kb of the KLK4 promoter from TIS3. However, the functional and clinical significance of these 19 polymorphisms were not further pursued given the negative findings from the functional work. The PSA AR enhancer region was also assessed for potential polymorphisms that may be associated with prostate cancer risk. A total of 12 polymorphisms were identified in the PSA enhancer of which two (A-4643G and T-5412C) have been reported to alter functionality of the enhancer region and thus, prioritised for further analysis. Association analysis for prostate cancer risk was then carried out on these PSA enhancer polymorphisms as none of the KLK4 promoter polymorphisms were found in functional HREs. No significant association for either the A-4643G or T-5412C polymorphism with prostate cancer risk was found at the P = 0.05 level. However, under an age-adjusted dominant model a 1.22- (95% CI = 1.16-1.26) and 1.23-fold (95% CI = 1.17-1.29) increased risk for prostate cancer was found for the A-4643G or T-5412C polymorphisms, respectively. Both polymorphisms were also assessed for association with tumour grade and stage and PSA levels. Genotypes were significantly different for the A-4643G and T-5412C polymorphisms with tumour stage and PSA levels, respectively. However, these results are likely to be biased by the case population which consist primarily of men who presented with incidental (pT1) and organ-confined (pT2) tumours. To summarise, the A-4643G and T-5412C polymorphisms are unlikely to be associated with prostate cancer risk, PSA levels or stage/grade of disease. However, further analyses in a larger cohort is warranted given that these polymorphisms alter androgen responsiveness of the PSA enhancer and that elevated PSA levels are indicative of men with prostate cancer. To summarise, this PhD has elucidated the functional significance of the PSA G-158A polymorphism in prostate cancer and which may be important in prostate cancer patho-physiology. This PhD has also furthered the understanding of the hormonal regulation of KLK4 in prostate cancer cells. Finally, this PhD has carried out a pilot study on two functional PSA enhancer polymorphisms (A-4643G and T-5412C) with prostate cancer risk.

Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.