Interleukin-21 (IL-21), a recently discovered member of the IL-2 cytokine family, has been shown to have diverse regulatory effects on B cells including the induction of antibody secretion, differentiation, or apoptosis depending on the cell milieu and activation status. However, the effects of IL-21 on B cell neoplasms such as diffuse large B cell lymphoma (DLBCL) are largely unknown. Our research uncovered the widespread expression of the IL-21 receptor (IL-21R) in B cell lymphomas including DLBCL and that IL-21 stimulation resulted in potent phosphorylation of STAT1 and 3 and weak activation of STAT5. However, our findings also showed that treatment of DLBCL cell lines with IL-21 induced cell cycle arrest and apoptosis. The cell death was caspase-dependent and evident in a majority of DLBCL cell lines. To further examine the potential therapeutic applicability of IL-21, we assessed the effects of IL-21 on primary DLBCL tumors and in vivo DLBCL xenografts in mice. In primary tumors, IL-21 induced apoptosis in five of five DLBCLs compared to two of three follicular lymphomas and two of seven chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B lymphocytes. In mice bearing DLBCL xenograft tumors, in situ IL-21 injections induced tumor regression and dramatically extended the overall survival of mice (P<0.001). To elucidate the mechanism of IL-21-induced cell death we analyzed the expression of apoptosis-regulating proteins and observed a strong downregulation of anti-apoptotic Bcl-2 and Bcl-XL and an upregulation of pro-apoptotic Bax post IL-21 stimulation. Subsequent experiments showed that ectopic expression of Bcl-2 or Bcl-XL was able to partially reduce cell death induced by IL-21 while Bax knockdown with siRNA blocked apoptosis completely. To gain insight into the signaling pathways shifting the expression of these proteins toward cell death we performed microarray analysis on sensitive and resistant DLBCL cell lines. The most striking difference in gene expression was observed in C-MYC which was only induced in cell lines exhibiting apoptosis upon IL-21 treatment. Previous reports have shown that c-Myc, which has been studied extensively for its oncogenic properties, can induce apoptosis via downregulation of its transcriptional targets Bcl-2 and Bcl-XL. We then showed that IL-21-induced cell death is dependent on c-Myc by utilizing specific siRNA and shRNA to block the upregulation of this transcription factor and prevent apoptosis. Since c-Myc is a bona-fide target of STAT3 we also showed that siRNA-mediated knockdown of STAT3 abrogated apoptosis by preventing c-Myc upregulation and its subsequent effects on apoptosis-regulating proteins. Our results delineate a novel IL-21 pro-apoptotic signaling pathway and one of the first examples in which the STAT3 - c-Myc pathway, which usually promotes B cell survival and oncogenesis, can be exploited for treatment of cancer. Furthermore, our findings demonstrate that IL-21 is a highly potent anti-DLBCL agent in vitro and in animal models and should be examined in clinical studies of DLBCL.

<p>Cancer is a disease state that arises as a result of multiple alterations in signaling pathways that are critical for making key cell fate decisions in normal cells. Understanding how these pathways operate under normal circumstances, therefore, is crucial for comprehensive understanding of tumorigenic process. With Myc and E2F pathways being central components for controlling cell proliferation, an important property that defines a cancer cell, as well as expanding roles for microRNAs(miRNA) in control of gene expression, we asked if we may better understand the underlying regulatory (transcription factor, microRNA) structure that contribute to Myc and E2F pathway activities. Through integrative analysis of mRNA and miRNA expression profile, we observe a distinct regulatory pattern in which, in the case of Myc pathway, Myc-induced miRNAs were contributing to the repression of negative regulators of cell cycle, including PTEN, while in case of E2F pathway, E2F-induced miRs were forming an incoherent Feed-Forward Loop(iFFL) with a number of E2F-induced genes including cyclin E. We further demonstrate through functional studies, as well as through single cell imaging of gene expression dynamics that miRNAs, depending on the context of either Myc or E2F pathway, play distinct roles in ensuring that cell fate decisions relevant to these pathways are properly executed.</p> / Dissertation

In Drosophila, XX embryos express Sxl from the early promoter, SxlPe, and become females. At the same time, XY embryos with only one X chromosome become males. I investigated the role of repression in the establishment of the strict regulation of SxlPe. I found that the co-repressor Groucho, is responsible for amplification of the two-fold difference in X-encoded activator genes into an all-or-nothing difference in Sxl expression. Three new basic helix-loop-helix repressors of Sxl were identified: Hey, Cwo and the prooncogene Myc, all of which are maternally supplied. I have shown that Myc specific repression is important as early as cycle 10, which is 2 cycles earlier than the onset of normal Sxl expression.

Dissertation (Ph.D.)--University of Toledo, 2009. / Typescript. "Submitted as a partial fulfillment of the requirements for the Doctor of Philosophy degree in Biology." Bibliography: leaves 135-146.

Brain tumors are abnormal tissue masses found, either malignant or benign in nature. Medulloblastoma is a brain tumor subtype found to arise in the hind region of the brain, which is highly malignant and has poor long term prospects in general. On the basis of the driving force behind the tumor, medulloblastoma is further subgrouped into 4 categories: WNT; SHH; Group 3 and Group 4 tumors. Group 3 tumors show a high expression of N-Myc protein which is seen in certain types of cancerous cells. The cell cycle is regulated at several checkpoints by cyclin/cdk inhibitors. The primary cilium is an organelle found on the cellular surface, which has functions in cell growth, differentiation and neurogenesis. Aurora kinase is a protein kinase involved in the regulation and maintainence of the cilium. Often the cilium gets deleted from the cellular surface in tumors coupled with an increase in the kinase level inside the cells. Hence aurora kinase is found to be a viable target for therapy. Aurora kinase is also involved in stabilizing the MYCN gene by protecting it from degradation. In this project, the primary cilum was studied in neural stem cells and followed by study of its presence on tumor cells in culture. The gene involved in cilium development i.e. Kif3a was mutated and its aggressive nature was compared with that of the tumor cells. Aurora kinase was commonly found to be over-expressed in both the tumors and the mutants whereas N-Myc over-expression was seen only in tumors. Experiments suggest that cilia repression in Kif3a mutants takes place via an aurora kinase dependent pathway.

Precancerous lesions from a variety of human tissues display markers of DNA damage suggesting that genetic instability occurs early during the process of carcinogenesis. Consistent with this, several oncogenes can activate ATM and other components of the DNA damage response pathway when expressed in cultured cells. Here we demonstrate that preneoplastic epithelial tissues from four different transgenic mouse models expressing the oncogenes c-myc, SV40 T antigen, human papilloma virus (HPV) E7, or E2F3a display [gamma]-H2AX foci and other markers of DNA damage. Moreover, transgenic expression of these oncogenes leads to increased levels of damaged DNA as measured by the comet assay. In at least the Myc transgenic model, the formation of [gamma]-H2AX foci is dependent on functional ATM. Inactivation of Atm also impairs p53 activation and reduces the level of apoptosis observed in transgenic tissue overexpressing Myc. This correlates with accelerated tumor development in Myc transgenic mice lacking ATM. To understand the mechanism by which oncogenes induce DNA damage, we employed an adenoviral overexpression system. Under conditions in which Myc or E2F3a induced replication is inhibited, we see a reduction in the DNA damage induced by these oncogenes both by comet assay and levels of [gamma]-H2AX. Moreover, Myc and E2F3a induced increased levels of the Cdt1 protein, a replication origin- licensing factor implicated in aberrant DNA replication. Taken together, these findings suggest that deregulated oncogenes induce unscheduled DNA replication leading to DNA damage and activation of the ATM DNA damage response pathway, which is important for the activation of p53, induction of apoptosis and the suppression of tumorigenesis. / text

Although some progress has been made in the treatment of cancer over the last sixty years, the majority of chemotherapeutics has fallen short. Because general chemotherapies that target DNA replication have only a limited efficacy and significant non-target side-effects, a new paradigm for cancer drug development has been adopted. Using a molecular targeted approach, new gene and protein targets have been identified and the development of chemotherapies that are specific to these targets has already begun. In this study, compounds that interact with two key cancer targets, the G-quadruplex of the c-Myc promoter and p-glycoprotein, have been investigated. By developing such compounds, improvements in treatment efficacy is anticipated with an aspiration for decreased mortality attributable to cancer.Formation of DNA secondary structures, such as the G-quadruplex, in the NHE III1 region of the c-Myc promoter has been shown to repress c-Myc transcription. Because c-Myc is an oncogene that is overexpressed in a variety of cancers, stabilization of the G-quadruplex by small molecules would be advantageous in cancer treatment. Using Fluorescence Resonance Energy Transfer, with Taq Polymerase Stop assays for confirmation, a group of compounds were identified that stabilize the c-Myc G-quadruplex structure. Using a colon cancer model, two compounds were shown to decrease c-Myc gene and protein expression. Also, exposure to the compounds for 48 hours results in an induction of caspase-3, indicative of apoptosis. Furthermore, surface plasmon resonance suggests that compound-induced stabilization of the c-Myc G-quadruplex can prevent sustained binding of the regulatory protein NM23-H2 by increasing its dissociation from the G-quadruplex. This may subsequently prevent unraveling of the G-quadruplex.Because resistance to chemotherapy reduces its effectiveness, development of multidrug resistance (MDR) modulators was also studied. Psorospermin is a topoisomerase II-directed DNA alkylating agent active against MDR cell lines. In a study examining the mechanism of psorospermin's P-glycoprotein modulation, Flow Cytometry demonstrated that doxorubicin-resistant multiple myeloma cells pre-treated with psorospermin enhanced intracellular retention of doxorubicin. Because neither transcription of mdr1 nor translation of P-glycoprotein was downregulated by psorospermin, resistance reversal is most likely due to a direct interaction between the side chain of psorospermin and P-glycoprotein, inhibiting drug efflux.

Statement of Problem Telomere dynamics in the three-dimensional (3D) space of the mammalian nucleus plays an important role in the maintenance of genomic stability. However, the telomere distribution in 3D nuclear space of normal and tumor cells was unknown when the study was initiated. Methods Telomere fluorescence in situ hybridization (FISH) and 3D molecular imaging, deconvolution, and analysis were used to investigate telomere organization in normal, immortalized and tumor cells from mouse and human cell lines, and primary tissues. Results Telomeres are organized in a non-overlapping manner and in a cell-cycle dependant fashion in normal cells. In the late G2 phase of cell cycle, telomeres are assembled into a flattened sphere that is termed the telomeric disk In contrast, the telomeric disk is disrupted in the tumor cells. Moreover, telomeric aggregates (TAs) are found in tumor cells. Conditional c-Myc over-expression induces telomeric aggregation leading to the onset of breakage-bridge-fusion cycles and subsequent chromosomal abnormality. Conclusions Telomeres are distributed in a nonrandom and dynamic fashion in the 3D space of a normal cell. Telomeric aggregates are present in cells with genomic instability such as tumor cells and cells with deregulation of c-Myc. Consequently, TA can be a useful biomarker for research in cancer and other disease processes.

Statement of Problem Telomere dynamics in the three-dimensional (3D) space of the mammalian nucleus plays an important role in the maintenance of genomic stability. However, the telomere distribution in 3D nuclear space of normal and tumor cells was unknown when the study was initiated. Methods Telomere fluorescence in situ hybridization (FISH) and 3D molecular imaging, deconvolution, and analysis were used to investigate telomere organization in normal, immortalized and tumor cells from mouse and human cell lines, and primary tissues. Results Telomeres are organized in a non-overlapping manner and in a cell-cycle dependant fashion in normal cells. In the late G2 phase of cell cycle, telomeres are assembled into a flattened sphere that is termed the telomeric disk In contrast, the telomeric disk is disrupted in the tumor cells. Moreover, telomeric aggregates (TAs) are found in tumor cells. Conditional c-Myc over-expression induces telomeric aggregation leading to the onset of breakage-bridge-fusion cycles and subsequent chromosomal abnormality. Conclusions Telomeres are distributed in a nonrandom and dynamic fashion in the 3D space of a normal cell. Telomeric aggregates are present in cells with genomic instability such as tumor cells and cells with deregulation of c-Myc. Consequently, TA can be a useful biomarker for research in cancer and other disease processes.

Regulation of N-myc oncogene expression is an important determinant of the biological behavior of neuroblastoma. The N-myc promoter contains several potential binding sites for transcription factors of the Sp1 family. Mutation of a CT-box motif contained within a 26 base pair region required for N-myc downregulation by retinoic acid decreased basal transcriptional activity and altered DNA-protein interactions of the promoter, while mutations flanking this motif did neither. On gel shift this region generated 3 specific DNA-protein complexes that were reliant on wild type sequence of the core CT element within it. Both Spl and Sp3 bound to the wild type probe as distinct complexes in specifically retarded bands, while neither protein was present on mutated sequences. Lysates from Drosophila S2 cells expressing exogenous Sp1 and Sp3 proteins were able to reproduce the gel shift complexes seen with neuroblastoma nuclear extract. Transient transfections of S2 cells showed that individually or together, Sp1 and Sp3 were able to trans-activate a N-myc CT-box-containing luciferase reporter construct in a dose-dependent manner. Conversely, transfection of CT-box oligonucleotide was able to decrease endogenous N-myc expression in neuroblastoma cells. Together these results suggest that the CT-box element serves a critical functional role, and in the basal state allows for N-myc transactivation by Spl and Sp3.