Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 112-124).

Cyclin D1, a proto-oncogene, is required for progression from the G1 phase into the S phase of the cell cycle. Over-expression of cyclin D1 causes an increase in cell cycle progression and cell proliferation, implicating it in a variety of cancers including renal cell carcinoma (RCC). The rodent RCC cell model, QTRRE, and human RCC cell models, ACHN, 786-O and Caki-2, exhibit elevated levels of cyclin D1. Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor, is an FDA-approved hemorheologic agent used to treat intermittent claudication, stemming from peripheral vascular diseases, as well as other diseases involving defective locoregional blood flow. Treatment of QTRRE, ACHN, 786-O and Caki-2 with PTX caused a time- (0-24 hrs) and dose- (0-1.0 mg/mL) dependent decrease of cyclin D1 protein and p-Rb levels in whole cell lysate as well as cytosolic and nuclear fractions, albeit, to different extents within the models. Concomitant with cyclin D1 and p-Rb decrease, enhanced G1 phase cell cycle arrest was observed in the RCC models. Mechanistic studies in these RCC cell models were carried out to determine PTXs mechanism of action with regard to cyclin D1 protein level decrease. RT-PCR analysis showed no significant changes in cyclin D1 mRNA copy number in time- (0-24 hrs) and dose- (0-1.0 mg/mL) dependent PTX treatments. However, such treatments caused decrease in p-4EBP1 (Ser65), p-4EBP1 (Thr70), and p-4EBP1 (Thr37/46). Because PTX's ability to decrease cyclin D1 protein was prevented in the presence of the proteasome inhibitor, MG-132, studies were performed to determine whether cyclin D1 stability was decreased during PTX treatment. Cyclin D1 degradation is initiated by phosphorylation of residue Thr286 by GSK-3β. Inhibition of GSK-3β with LiCl or knockdown via siRNA in the presence of PTX failed to block cyclin D1 decrease. Moreover, PTX treatment in the presence of MG-132 revealed no significant increase in cyclin D1 p-Thr286 compared to control. Finally, using the protein synthesis inhibitor, CHX, PTX caused no significant decrease in cyclin D1 t₁/₂ (wt-HA and T286A-HA) compared to control. Sorafenib, a broad-spectrum (cRAF, bRAF, KIT, FLT-3, VEGFR-2, VEGFR-3, and PDGFR-β) kinase inhibitor, is FDA-approved for the treatment of RCC. Studies with sorafenib and PTX in the ACHN cell model were carried out to determine PTXs possible adjuvant role in inhibiting cell growth via cyclin D1 decrease and G1 phase arrest. MTS data showed PTX potentiates the anti-proliferative effects of sorafenib. PTX pre-treatment for 24 hrs was also lowered the effective dose of sorafenib from 50 μM to 5 μM. Further, ACHN xenograft tumor volumes from mice treated with PTX and sorafenib displayed significantly higher tumor growth inhibition compared to either drug treatment alone or vehicle. Finally, drug treated ACHN xenograft tissue displayed significantly lower cyclin D1, p-RB and p-4EBP1 levels. These results demonstrate a novel anti-cancer property of PTX and suggest its use as a possible adjuvant therapy in RCC treatment should be further explored.

No description available.

<p>The majority of breast tumors express the estrogen receptor (ER), and more than half of these cancers also express the progesterone receptor (PR). While the actions of ER on breast cancer pathogenesis are well understood, those of PR are still unclear. The Women's Health Initiative trial in 2002 brought into focus the alarming result that women receiving both estrogen and progestins as hormone replacement therapy are at greater risk for breast cancer than women receiving estrogen alone. Thus, there is considerable interest in defining the mechanisms that underlie the pharmacological actions of progestins in the normal and malignant breast. </p><p>Progestins facilitate cell cycle progression through multiple mechanisms, one of which is the induction of phosphorylation of the tumor suppressor retinoblastoma (Rb) protein. Stimulation by growth factors induces the transcription of Cyclin D1 which in turn activates the cyclin dependent kinases (CDKs). The Cyclin D1- Cdk4/6 complex phosphorylates the Rb protein, leading to the release of E2F1, which then binds and activates other target genes, leading to G1-S transition of the cell cycle. Given the reported action of PR to activate MAPK signaling, we initially thought that the progestin-induced Rb phosphorylation was mediated by this pathway. However, we turned to an alternate hypothesis based on our data using MEK inhibitors demonstrating that this was not the case. </p><p>Given the primacy of Cyclin D1 in cell cycle control, we then turned our attention to defining the mechanism by which Cyclin D1 expression is regulated by PR. Interestingly, it was determined that progestin mediated up- regulation of Cyclin D1 is rapid, peaking at 6hrs post hormone addition followed by a decrease in expression reaching a nadir at 18hrs. Unexpectedly, we found that contrary to what has been published before, the induction of Cyclin D1 mRNA expression was a primary transcriptional event and we have demonstrated the specific interaction of PR with PREs (progesterone response elements) located on this gene. We have further determined that the half-life of Cyclin D1 mRNA is decreased significantly by progestin addition explaining how the levels of this mRNA following the addition of hormone are quickly attenuated. Thus, when taken together, our data suggest that progestins exert both positive and negative effects on Cyclin D1 mRNA, the uncoupling of which is likely to impact the pathogenesis of breast cancer</p><p>The observation that PR reduces the Cyclin D1 mRNA stability led us to investigate the effects of PR on RNA binding proteins, especially those which are involved in RNA stability. We discovered that PR induces the expression of several RNA binding proteins. Although the work to determine the effects of these RNA binding proteins on CyclinD1 mRNA stability is still ongoing, we have discovered a role for one of the PR-induced RNA binding proteins tristetraprolin (TTP), in the suppression of the inflammation pathway in breast cancer. We found that while TTP was not required for the PR-mediated decrease in Cyclin D1 mRNA stability, overexpression of this tumor suppressive protein was able to inhibit IL-1&#946;-mediated stimulation of inflammatory genes in our breast cancer model. Since it is established that the upregulation of the inflammatory pathway is oncogenic, we are currently exploring the intersection of PR and TTP-mediated signaling on the inflammation transcriptome in breast cancer. </p><p>Thus, collectively these data provide us with a better picture of the poorly understood actions of PR on breast cancer proliferation and tumorigenesis. We believe that further investigation of the studies developed in this thesis will lead to novel and better-targeted approaches to the use of PR as a therapeutic target in the clinic.</p> / Dissertation

D Cyclins play an essential role connecting exogenous stimulation to the intrinsic cell cycle machinery. This family of proteins is composed of three members sharing a highly homologous domain, the cyclin box (coded by exons 1-3), which is responsible for their redundant role in the phosphorylation of the retinoblastoma protein upon association with cydin-dependent kinases Cdk4/6. Both mature T and B-cells have a remarkable division capability after antigen stimulation, essential to the generation of efficient immune responses, raising the interest of D Cyclins in lymphopoiesis. Cyclin Dl, although weakly expressed by lymphocytes, is the D Cyclin most commonly implicated in lymphoid cancers and as having a Cdk-independent transcriptional role. To study the role of Cyclin Dl, we used mice deficient for the Dl cyclin box but sparing exons 4-5. Surprisingly, individual mice have very different phenotypes that we subdivided into four arbitrary groups. Group I mice show the most precocious block in lymphoid lineage differentiation, illustrated by a low cellularity of common lymphoid progenitor cells (CLP). The thymi showed very few CD4*CD8*, double positive (DP) cells, while the CD4 CD8TCR, triple negative (TN) populations were found to be mostly constituted by the early CD44*CD25' (TNI) and few CD44*CD25* (TN2). TNl's early thymocyte progenitors (ETP) were virtually absent. At the B-cell lineage level in the bone marrow (BM) there was a major block in pre-proB differentiation. The number of peripheral T-cells was severely reduced, mainly in LN, since group I T-cells lack CCR7 expression. Group II mice presented moderate thymus atrophy. The block on TN differentiation occurs at a later stage, i.e., in the TN3 to TN4 transition, and the TNI population was characterized by a less severe depletion of the ETP. Group II mice showed a partial pre-proB block and a reduction in pre-B-cells. CLPs were also reduced but to a lesser extent than in group I mice. Group ill and group IV mice appear to have a normal thymocyte population distribution but showed an increase on ETP compartment. Group IV mice displayed thymic hyperplasia while group III mice possessed normal thymus cellularity. B-cell differentiation on both groups appeared to be normal but BM precursors had an increase in both CLP and early haematopoietic progenitor's (LSK) levels as compared with wild type mice. Cyclin Dl involvement in G1 to S transition led us to analyse in vivo division rates. Strikingly, group I mice were virtually devoid of cycling cellsin all lymphoid compartments, explaining why lymphoid lineage cells do not differentiate in these mice. In contrast, in all other groups we observed an increased BrdU incorporation. These contradicting phenotypes correlated with the expression or absence of a truncated Dl protein coded by exons 4-5. The presence of the cyclin Dl truncated mRNA was not found in group I mice but high levels of expression are consistently observed in the remaining groups. In the absence of the Dl truncated protein only trace values of Cyclins D2 and D3 were found, highlighting the role of this protein as a master D cyclin regulator, which further supports the profound aplasia and arrest in lymphoid lineage division on cells that predominantly express Cyclin D2. These results suggest that, while the function of the Dl cyclin box is redundant, the regulatory domain coded by exons 4-5 is fundamental for lymphopoiesis. Full Dl protein was also eliminated by RNA interference both in vitro and in vivo. These experiments reproduced the phenotype of group I mice. We have developed a lentiviral vector with a truncated Dl (exons 4-5) and conditional knockout (KO) mice by floxing exons 4-5 of cyclin Dl. These tools will allow us to show Cyclin Dl Cdk-independent role as a transcription regulator in lymphopoiesis and to attribute this function to exons 4-5. Understanding how exons 4-5 regulate different transcription factors might be a key in...

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.

Cdk7 (Cyclin-dependent kinase 7) is conserved from yeast to human and involved in multiple functions. Cdk7 acts as a CAK (Cdk activating kinase) in a trimeric complex with Cyclin H and Mat1. The CAK activity is required for the full activation of the Cdks that directly regulate the cell cycle transitions. In addition, Cdk7 is the kinase subunit of TFIIH, the general transcription/DNA repair factor IIH. TFIIH is required for the general transcription of messenger RNAs by RNA polymerase II and for the transcription-coupled nucleotide excision repair functions. As in other systems, Drosophila Cdk7 has multiple functions. In order to understand how different functions of Cdk7 are regulated, I performed genetic screens to identify the regulators or downstream factors of multiple functions of Cdk7. Several candidate dominant suppressors and enhancers were identified in these screens. One strong suppressor of cdk7, xpd, encodes another subunit of TFIIH. The genetic suppression by xpd attracted me to further characterize the biological significance of this interaction. I showed that Xpd does have a novel function in regulating CAK activity of cdk7 , it down-regulates mitotic CAK activity. Furthermore, I found that Xpd protein levels are cell cycle dependent, being down-regulated at the beginning of the mitosis. Based on these data, I propose a model that mitotic down-regulation of Xpd results in increased CAK activity, positively regulating mitotic progression. Simultaneously, this down-regulation can be expected to contribute to the mechanisms of mitotic silencing of basal transcription.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 140-172).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 3 uppsatser.