<p>Breast cancer mortality in Sweden is now in decline, thanks to early detection and the wide use of adjuvant endocrine therapy and chemotherapy. </p><p>While hormone receptor status is predictive of response to endocrine treatment, there is no clinically useful predictive marker of a patient’s response to chemotherapy. Consequently, patients receive chemotherapy with considerable toxicity but minimal benefit. The aim of this thesis was to investigate a number of methods with the potential to predict response to chemotherapy and thus enhance treatment efficacy in breast cancer patients.</p><p>We found that topo IIα, the key target enzyme of topo II inhibitors, is significantly expressed in nonproliferating breast cancer cells. This finding may explain why topo II inhibitors are effective in patients with slow growing tumors and a low proliferation rate.</p><p>Topo IIα gene amplification was suggestive of increased response to anthracyclines in advanced breast cancer, whereas the oncogene HER2 had no predictive value by itself. These findings are in accordance with current knowledge.</p><p>Cyclin A, a marker of cell proliferation, showed good prognostic value but did not predict response to chemotherapy in advanced breast cancer.</p><p>In vitro chemosensitivity testing with FMCA predicted tumor response in patients with advanced breast cancer with a sensitivity of 89% and a specificity of 53%. Our results are consistent with the results from similar assays, which predict drug resistance with good accuracy while clinical drug sensitivity is less reliably predicted. The use of FMCA and similar assays is not yet recommended outside clinical trials; their main utility is in preclinical testing of new anti-cancer drugs, including targeted therapies.</p><p>The combination of epirubicin, capecitabine, and cisplatin (EXC) demonstrated high clinical response rate (74%) and pathological complete response rate (22%) in locally advanced breast cancer, but with cumbersome toxicity. The fluoropyrimidine biomarkers TS, TP, and DPD did not predict response to the EXC regimen.</p>

Transcription and DNA replication of herpes simplex virus type 1 (HSV-1) occur in nuclear domains adjacent to structures named ND10. The HSV-1 single-stranded DNA binding protein ICP8 localizes to these nuclear domains to direct the assembly of the pre- and replication compartments. Inhibition of cyclin dependent kinases with roscovitine inhibits HSV-1 DNA replication, even in the presence of all required HSV-1 proteins, at an unidentified step. Here I show that roscovitine inhibits the localization of pre-expressed ICP8 to new replication sites. Therefore, the inhibition of HSV-1 DNA replication occurs at a step prior to initiation. I next evaluated the mechanisms of inhibition of proper ICP8 localization. ICP8 was extracted at lower salt concentrations from roscovitine-treated than untreated cells, but the affinity of ICP8 for ssDNA in vitro was not affected. I propose that roscovitine inhibits HSV-1 DNA replication by inhibiting DNA accessibility. I also discuss alternative mechanisms.

Breast cancer mortality in Sweden is now in decline, thanks to early detection and the wide use of adjuvant endocrine therapy and chemotherapy. While hormone receptor status is predictive of response to endocrine treatment, there is no clinically useful predictive marker of a patient’s response to chemotherapy. Consequently, patients receive chemotherapy with considerable toxicity but minimal benefit. The aim of this thesis was to investigate a number of methods with the potential to predict response to chemotherapy and thus enhance treatment efficacy in breast cancer patients. We found that topo IIα, the key target enzyme of topo II inhibitors, is significantly expressed in nonproliferating breast cancer cells. This finding may explain why topo II inhibitors are effective in patients with slow growing tumors and a low proliferation rate. Topo IIα gene amplification was suggestive of increased response to anthracyclines in advanced breast cancer, whereas the oncogene HER2 had no predictive value by itself. These findings are in accordance with current knowledge. Cyclin A, a marker of cell proliferation, showed good prognostic value but did not predict response to chemotherapy in advanced breast cancer. In vitro chemosensitivity testing with FMCA predicted tumor response in patients with advanced breast cancer with a sensitivity of 89% and a specificity of 53%. Our results are consistent with the results from similar assays, which predict drug resistance with good accuracy while clinical drug sensitivity is less reliably predicted. The use of FMCA and similar assays is not yet recommended outside clinical trials; their main utility is in preclinical testing of new anti-cancer drugs, including targeted therapies. The combination of epirubicin, capecitabine, and cisplatin (EXC) demonstrated high clinical response rate (74%) and pathological complete response rate (22%) in locally advanced breast cancer, but with cumbersome toxicity. The fluoropyrimidine biomarkers TS, TP, and DPD did not predict response to the EXC regimen.

Cardiovascular diseases are the leading cause of death in humans throughout the world and “congenital heart defects” (CHDs) are the major cause of infant mortality and morbidity. GATA4 is one of the most critical and intensely studied cardiac transcription factor. It is important for proliferation of cardiomyocytes as well as their survival and adaptive response. The focus of the following thesis was to identify GATA4 mediators and cofactors in cardiac growth. The first part focused on cyclin D2 (CycD2), a growth inducible cell cycle protein. We identified Ccnd2 (gene encoding CycD2) as a direct transcriptional target of GATA4 in postnatal cardiomyocytes and Ccnd2 cardiomyocyte specific overexpression in Gata4 heterozygote mice was able to rescue their heart size and function. We further uncovered a novel regulatory loop between GATA4 and CycD2. CycD2 enhanced GATA4 activation of its target promoters. GATA4 was able to physically interact with CycD2 and its cyclin dependent kinase CDK4 suggesting that GATA4 recruits CycD2/CDK4 to its target promoters. Together, our data uncover a role of CycD2 in the developing and postnatal heart and provide novel insight for the potential of targeting the cell cycle in cardiac therapy. The second part of the project focused on KLF13, a cell specific cofactor of GATA4. KLF13 is a member of the Krϋppel-like transcription factors that are important regulators of cell proliferation and differentiation. Klf13 is highly enriched in the developing heart where it is found in both myocardial and endocardial cells. To determine its role in the mammalian heart, we deleted the Klf13 gene in transgenic mice. Klf13-/- mice were born at 50% reduced frequency and presented with variable cardiac phenotypes. Epithelial-mesenchymal transformation (EMT) was affected in these mice and reduced cell proliferation was evident in the AV cushion. These data uncover a role for a new class of transcription factors in heart formation and point to KLF13 as a regulator of endocardial cell proliferation and a potential CHD causing gene. Future discovery of more cardiac regulators and understanding the molecular basis of CHDs is essential for preventions of these defects and possible development of therapeutic approaches for myocardial repair.

Preeclampsia and molar pregnancy are two devastating placental pathologies characterized by an immature proliferative trophoblast phenotype accompanied by excessive cell death. It is therefore of paramount importance to study the regulation of cell fate in the placenta, to gain a further understanding of the mechanisms that contribute to these diseases. In this dissertation we report that during normal placental development and in preeclampsia, Matador (Mtd), a pro-apoptotic member of the Bcl-2 family, has a dual function in regulating trophoblast cell proliferation and death. Importantly, we reveal a novel role of Mtd-L in promoting cyclin E1 expression and cell cycle progression. Of clinical importance, we also identify that both cyclin E1 and the CDK inhibitor p27, are increased in severe early onset preeclampsia. However, the inhibitory function of p27 in this pathology may be hampered due to its increased phosphorylation at Ser10, resulting in its nuclear export. Of equal importance, data presented demonstrate that placentae from severe early onset preeclampsia display a molecular profile distinct from late onset preeclampsia or intrauterine growth restricted pregnancies. In the final data chapter we demonstrate that Mtd is highly expressed in molar tissue, where it localizes to both apoptotic and proliferative cells. Our data suggests that an abundance of Mtd and cyclin E1 in conjunction with the low level of p27 may contribute to the hyperproliferative nature of the disorder. The body of work in this dissertation uncovers novel insights into the regulation of trophoblast cell fate. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel mechanism found to regulate trophoblast cell proliferation in normal and pathological placentation. Equally important is our identification of molecular differences between placental pathologies that may help to differentiate early and late onset preeclampsia, IUGR and molar pregnancy.

Preeclampsia and molar pregnancy are two devastating placental pathologies characterized by an immature proliferative trophoblast phenotype accompanied by excessive cell death. It is therefore of paramount importance to study the regulation of cell fate in the placenta, to gain a further understanding of the mechanisms that contribute to these diseases. In this dissertation we report that during normal placental development and in preeclampsia, Matador (Mtd), a pro-apoptotic member of the Bcl-2 family, has a dual function in regulating trophoblast cell proliferation and death. Importantly, we reveal a novel role of Mtd-L in promoting cyclin E1 expression and cell cycle progression. Of clinical importance, we also identify that both cyclin E1 and the CDK inhibitor p27, are increased in severe early onset preeclampsia. However, the inhibitory function of p27 in this pathology may be hampered due to its increased phosphorylation at Ser10, resulting in its nuclear export. Of equal importance, data presented demonstrate that placentae from severe early onset preeclampsia display a molecular profile distinct from late onset preeclampsia or intrauterine growth restricted pregnancies. In the final data chapter we demonstrate that Mtd is highly expressed in molar tissue, where it localizes to both apoptotic and proliferative cells. Our data suggests that an abundance of Mtd and cyclin E1 in conjunction with the low level of p27 may contribute to the hyperproliferative nature of the disorder. The body of work in this dissertation uncovers novel insights into the regulation of trophoblast cell fate. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel mechanism found to regulate trophoblast cell proliferation in normal and pathological placentation. Equally important is our identification of molecular differences between placental pathologies that may help to differentiate early and late onset preeclampsia, IUGR and molar pregnancy.

<p>Post-transcriptional regulation plays an important role in governing various processes in all organisms. The development of the early embryo of <italic>Drosophila melanogaster</italic> is governed solely by post-transcriptional mechanisms; therefore, further insights into post-transcriptional regulation can be gained by studying the <italic>Drosophila </italic> embryo. This thesis addresses the actions of the translational repressor, Pumilio, in regulating two mRNAs during early embryogenesis. First, we examined the ability of Pumilio to regulate the mRNA stability of <italic>bicoid</italic>, a gene required for <italic>Drosophila </italic> head development. <italic>bicoid</italic> mRNA contains the canonical Pumilio recognition site, termed the Nanos response element (NRE), within the 3'UTR. Interestingly, we show that Pumilio binds to the NRE both in vitro and in vivo; however, no physiological significance is associated with this interaction. Furthermore, in <italic> pumilio</italic> mutant embryos <italic>bicoid</italic> mRNA stability and translation are unaltered, demonstrating that Pumilio does not regulate <italic>bicoid</italic> mRNA. Second, Pumilio has been shown to negatively regulate <italic>Cyclin B</italic>, the cyclin necessary for mitotic entry, in the somatic cytoplasm of the embryo and this repression is alleviated by the PNG Kinase complex through currently unidentified mechanisms. We further investigated the actions of Pumilio in regulating <italic>Cyclin B</italic> and discovered that the canonical partner of Pumilio, Nanos, is not involved in repressing somatic <italic>Cyclin B</italic>. Furthermore, we show that the 3'UTR of <italic>Cyclin B</italic> is not required for the regulation by Pumilio and the PNG Kinase complex. Lastly, through genetic analyses, we conclude that Pumilio may actually act upstream of the PNG Kinase complex to regulate <italic>Cyclin B</italic>.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-144).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 69-77).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 103-119).