La régulation de la télomérase s’effectue à de nombreux niveaux dont la transcription de la sous-Unité catalytique (hTERT). Les travaux du laboratoire effectués sur les cellules NB4, modèle de Leucémie Aiguë Promyélocytaire (LAP), ont montré que l'acide rétinoïque tout-Trans (ATRA) réprime la transcription de hTERT. Cette répression peut être associée à la différenciation (cas des cellules NB4) ou en être dissociée conduisant à la mort des cellules (cas des cellules NB4-LR1 résistantes à la maturation induite par l’ATRA). A partir de la lignée NB4-LR1 a été sélectionnée la lignée NB4-LR1SFD résistante à cette mort cellulaire du fait de la ré-Expression de hTERT même en présence d’ATRA. Cependant cette résistance à la répression de hTERT peut être levée par le co-Traitement ATRA et trioxide d’Arsenic (As2O3) qui conduit à la mort des cellules. Il s'agit donc d'une propriété nouvelle de cette lignée dont le mécanisme reste à élucider.Les résultats obtenus par le laboratoire suggèrent l'importance du statut de méthylation de l’ADN du promoteur de hTERT jusque là peu explorée qui pourrait rendre compte de la résistance à la répression de hTERT. Mon projet a pour objectif de valider cette hypothèse en tirant profit de la diversité des réponses biologiques (différenciation, prolifération, mort cellulaire et expression de hTERT) des variants cellulaires du modèle NB4. Une coopération entre le statut épigénétique (méthylation de l’ADN et modification des histones) du promoteur de hTERT et la fixation de facteurs activateurs et/ou répresseurs sera étudiée. Le statut de méthylation du promoteur de hTERT sur une région allant de -2500pb à +1000pb par rapport au site d’initiation de la transcription a été étudié par la technique de séquençage (Illumina) après traitement des cellules NB4-LR1SFD par l’ATRA seul ou en combinaison avec As2O3. Le résultat obtenu à ce jour montre une hypométhylation d’une région limitée du domaine distal (de -1300pb à -800pb) du promoteur de hTERT associée à la répression de hTERT dans les cellules traitées par la combinaison ATRA+ As2O3 par rapport aux traitements seuls par ATRA ou As2O3. Ceci renforce l’importance du statut de méthylation de cette région du promoteur dans la régulation de l’expression de hTERT. Ce co-Traitement induit également une diminution de l’expression protéique de cMyc et WT1, et aussi de l’ADN methyltransférase 1 (DNMT1) suggérant un rôle de cette enzyme dans le maintien de la méthylation de cette région du promoteur de hTERT. Dans le but d’évaluer le rôle de c-Myc dans la régulation de hTERT, nous avons montré qu’un analogue de l’AMPc, le 8-CPT-CAMP, induisait une dégradation (en partie protéasome dépendant) de la protéine c-Myc dès 6h de traitement dans la lignée résistante NB4-LR1SFD et non la lignée parentale NB4. La lignée NB4-LR1SFD est caractérisée par un déficit en sous unité régulatrice PKA RII. Spécifique knock-Down de PKA RII et l’utilisation d’agonistes et d’antagonistes spécifiques de PKAI a montré : 1) PKAI et PKAII ont des rôles différents sur la stabilité de la protéine c-Myc; 2) le rapport PKAI/PKAII déterminait la stabilité de c-Myc suite à l’activation de la signalisation PKA. Ces résultats suggèrent un rôle possible de PKA comme régulatrice de expression de hTERT via son implication dans le maintien de la stabilité de la protéine c-Myc. / The regulation of telomerase occurs at various levels, including the transcriptional regulation of hTERT. Previous results in our laboratory from acute promyolocytic leukemia cell model NB4, have shown that all-Trans retinoid acid (ATRA) repress the transcription of hTERT. This repression can be associated with differentiation (in the case of NB4 cells), or be dissociated with differentiation and triggers cellular death (the case of maturation resistant NB4-LR1 cells). Another variant NB4-LR1SFD cells were isolated from NB4-LR1 cells with continuous presence of ATRA and were resistant to the cellular death induced by ATRA. In fact, this resistance is related to the re-Expression of hTERT in presence of ATRA. However, this resistance can be overcome by combination of ATRA and AS2O3 and triggers cellular death.The results obtained in our laboratory suggested the importance of the DNA methylation status in the promoter region of hTERT and could be the one mechanism of the resistance to the repression of hTERT induced by ATRA. My project is by taking the diversity of biological response of the NB4 cells variants to validate the hypothesis. And the cooperation between epigenetic modifications and the binding of transcriptional factors will be equally studied.The DNA methylation status in the promoter region of hTERT from -2500bp to +1000bp has been analyzed with the sequencing technique (illumina) in NB4-LR1SFD treated by ATRA alone or in combination with AS2O3. The results showed a distal hypomethylated region from -1300bp to -800bp associated with the repression of hTERT by the co-Treatment of ATRA and AS2O3 compared with the treatment by ATRA or AS2O3 alone. This result strengthens the importance of methylation status in this region in the regulation of hTERT. The co-Treatment induces also a diminution in protein expression of cMyc, WT1 and DNA methyltransferase 1 (DNMT 1), suggesting this enzyme may play a role in the maintenance of methylation level in this region.In order to evaluate the role of cMyc in the regulation of hTERT, we have shown that an analog de cAMP, 8-CPT-CAMP, induces degradation (partly proteasome-Dependent) of c-Myc protein since 6h in NB4-LR1SFD cells but not in NB4 cells. NB4-LR1SFD cells are characterized by a defect of the PKA regulatory subunit II. Specific knockdown of PKA RII and utilizations of agonists and antagonists of PKA I have shown that: 1) PKA I and PKA II have distinct functional roles on the steady-State of c-Myc protein. 2) The ratio of PKA I/PKA II determines the stability of c-Myc protein with the activation of PKA signalization. These results suggest a possible role of PKA in the regulation of hTERT expression through its modulation on the stability of c-Myc.

Pour assurer la production de milliards de globules rouges, l’érythropoièse doit parfaitement contrôler les processus de prolifération et de différenciation. Ces deux processus sont régulés par l’expression de gènes spécifiques dépendant d’une coordination entre l’activité des facteurs de transcription (FT) et les fonctions épigénétiques portées par exemple par les protéines à bromodomaine. Cette étude se concentre sur les conséquences de l’association ou la dissociation du FT clef de l’érythropoièse GATA-1 avec les FT déterminant pour le cycle cellulaire, pRb et E2F. Dans la première partie de ma thèse, j’ai participé à l’étude du rôle de l’association/dissociation de GATA-1 et FOG-2 avec pRb/E2F dans le contrôle la balance prolifération/différenciation cellulaire. Nos résultats montrent que les souris exprimant une mutation de GATA-1 sur la sérine 310 (GATA-1S310A), qui a la capacité accrue à séquestrer E2F-2, présentent une anémie létale lorsqu’un mécanisme de compensation de production de E2F-2 induit par l’IGF-1 est inhibé. Puis, nous avons trouvé que les propriétés décrites pour GATA-1 sont partagées par le FT FOG-2 et montré que l’abrogation de sa fixation avec pRb induit une perturbation de l’adiposité dans des souris FOG-2pRb-. Dans la deuxième partie, l’expression de c-Myc étant régulé différentiellement par GATA-1 et E2F, j’ai testé si la drogue « JQ1 », premier inhibiteur épigenétique chimique de l’expression de c-Myc, pouvait contrôler l’érythropoièse. Pour cela, j’ai utilisé la ligné érythroleucémique UT7 qui prolifère sans se différencier en présence d’érythropoiétine (stade proérythroblaste). Les résultats montrent que le traitement par JQ1 bloque la prolifération des cellules UT7 et permet de réinitier le programme de différentiation érythroide terminale. J’ai alors recherché les mécanismes moléculaires impliqués dans cette régulation et trouvé que l’inhibition transcriptionnelle de c-Myc par JQ1 est associée à l’inhibition de l’activité transcriptionnelle de STAT5 sans modification de son état de phosphorylation. Enfin, j’ai montré que JQ1 pouvait avoir une activité comparable à celle du TGF-b mais sans implication les voies Smad. Des études in vivo montre que JQ1 augmente la viabilité cellulaire et accélère la maturation des cellules érythroides à la fois chez les souris sauvages et thalassémiques. Cette différence d’action de JQ1 sur l’érythropoièse normale et pathologique implique des modifications épigénétiques différentielles entre ces deux types cellulaires et sont à la base de nouvelles stratégies du traitement du cancer. Le rôle clef de la régulation de l’association/dissociation de GATA-1 ou FOG-2 avec pRb/E2F dans l’érythropoièse et l’adipogénèse, nous a conduit, dans une troisième partie, à déterminer in vivo, les conséquences physiologiques de la séquestration de E2F par pRb. Pour cela nous avons crée une souris transgénique exprimant de façon conditionnelle un peptide contenant la partie N terminale de GATA-1 qui se fixe à pRb (GATA-1Nter). In vitro, ce peptide séquestre E2F dans le complexe GATA-1Nter/pRb et inhibe la prolifération cellulaire de façon irréversible. In vivo, aucune souris transgéniques exprimant le peptide GATA-1Nter n’a pu être sélectionnée et une mortalité au stade embryonnaire est observée. Une expression induite de ce peptide au stade adulte ne produit que des souris chimériques avec une fréquence de recombinaison du transgène GATA-1Nter importante. L’établissement de lignées stables de souris exprimant le peptide GATA-1Nter permettra de déterminer les conséquences physiologiques de la séquestration de E2F dans le complexe GATA-1Nter/pRb. / To ensure the generation of billions of erythrocytes daily, erythropoiesis must be well controlled by proliferation and differentiation processes. These two processes are regulated by expressions of specific genes, coordinated by transcription factors (TFs) and epigenetic factors, such as bromodomain proteins. This study focused on the effects of the binding and dissociation of a key erythroid TF, GATA-1, to the crucial cell cycle TFs, pRb and E2F. In the first part of this thesis, the role of GATA-1 and FOG-2 binding to pRb/E2F in a control balances between cell proliferation and differentiation was studied. Mice bearing a GATA-1 mutation (GATA-1S310A) displayed higher levels of E2F2 sequestration and suffered from fatal anemia when the compensatory pathway of E2F2 production via IGF-1 signaling was also inhibited. The properties described for GATA-1 were found to be common to FOG-2, and the abolition of FOG-2 binding to pRb led to obesity resistance in FOG-2pRb- mice. In the second part of this work, as c-Myc is regulated by GATA-1 and E2F, the first chemical epigenetic inhibitor repressing c-Myc expression to be described, JQ1, was investigated to see if it could control erythropoiesis. The UT7 erythroleukemia cell line, which proliferates without differentiating was used. This cell line stops differentiation at the proerythroblast stage, in response to erythropoietin. JQ1 treatment inhibited UT7 proliferation and restored terminal erythroid differentiation. The molecular mechanism underlying this regulation by JQ1 was shown that the inhibition of c-Myc expression was associated with the inhibition of STAT5 transcription, with no change in the phosphorylation of this protein. It was found that JQ1 had a putative TGF--like activity, which did not involve the Smad pathway. It was shown in the ex vivo studies that JQ1 increased the viability of erythroid cells and accelerated the maturation of these cells in both WT and thalassemic mice. The observed differences between leukemic and normal erythropoiesis involved differential epigenetic modifications that could be at the basis of new strategies regarding cancer treatment.The key role of the association of GATA-1 or FOG-2 had with pRb/E2F, and the dissociation of these factors, in erythropoiesis and adipogenesis, respectively, led us to investigate, in vivo, the physiological consequences of E2F sequestration by pRb. As a result, transgenic mice displaying conditional expression of a peptide containing the N-terminal part of GATA-1 that binds to pRb (GATA-1Nter) were developed. In vitro, this peptide traps E2F in a GATA-1Nter/pRb complex, resulting in the irreversible inhibition of cell proliferation. The yield of transgenic mice expressing the GATA-1Nter peptide in vivo was unsuccessful, as this expression lead to lethality at the embryonic stage. Using an alternative approach, based on the inducible expression of the peptide in adults, chimeric mice with a high frequency of recombination of the GATA-1Nter transgene were obtained for this study. The establishment of a stable mouse line expressing the GATA-1Nter peptide should make it possible to determine the pathophysiological consequences of E2F sequestration in the GATA-1Nter/pRb complex.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 92-109).

c-MYC est un facteur de transcription oncogénique dont l’expression est dérégulée dans 78% des gliomes. On observe d’ailleurs une corrélation positive entre sa surexpression et le grade des gliomes. De plus, cette surexpression serait essentielle à la survie des cellules souches tumorales, cellules qui seraient davantage résistantes à la chimiothérapie et à la radiothérapie en plus d’avoir un caractère plus invasif. Il a aussi été démontré que l’inhibition de c-MYC par ARN interférents peut sensibiliser les cellules cancéreuses à l’apoptose et réduire leur prolifération. Sa surexpression relative dans les glioblastomes (GBM) est signe de la malignité et l’espérance de vie des patients atteints par ces tumeurs est réduite chez les patients plus âgés. c-MYC doit s’hétérodimériser avec MAX, son partenaire obligatoire afin de se lier aux promoteurs de ses gènes cibles contenant des EBox (CANNTG) et ainsi activer leur transcription. Cependant, il a été proposé que MAX pourrait homodimériser et agir comme antagoniste en compétitionnant pour les mêmes sites de reconnaissance que l’hétérodimère c-MYC/MAX sur l’ADN. Notre étude vise donc à évaluer l’effet dose-dépendant d’un traitement exogène de MAX*WT, correspondant à une version tronquée du facteur de transcription MAX, sur différentes lignées cellulaires de GBM. Nous avons d’abord étudié les capacités de la protéine à transloquer dans les cellules par microscopie. Ceci a permis de déterminer que le peptide s’internalise rapidement (15 minutes) pour ensuite s’accumuler au niveau nucléaire (24 h, 48 h). Par la suite, des analyses de FACScan ont démonté qu’un traitement de 72 heures provoque une inhibition de la prolifération cellulaire. À l’aide de chambres de Boyden et d’essais de croissance en sphéroïdes dans une matrice de Matrigel(indice supérieur TM]), nous avons observé une diminution importante du caractère invasif des lignées de gliomes malins suite au traitement avec MAX*WT. Ces résultats démontrent que la protéine MAX*WT semble avoir un effet antinéoplasique sur plusieurs lignées de gliomes malins et que la voie de signalisation de c-MYC pourrait constituer une cible thérapeutique intéressante.

In this dissertation, we explore the transcriptional regulatory roles of Gquadruplex- forming motifs and the involvement of specific transcriptional factors, which interact with the same elements, in the control of human c-myc and bcl-2 gene expression. The G-quadruplex structures within the NHE III1 region of the c-myc promoter and their ability to repress transcription has been well established. However, a longstanding unanswered question is how these stable DNA secondary structures are transformed to activate c-myc transcription. NDPK-B has been recognized as an activator of c-myc transcription via interactions with NHE III1 region of the c-myc gene promoter. Through the use of RNAi, we confirmed the transcriptional regulatory role of NDPK-B. We demonstrate that NDPK-B has DNA binding activity and the nuclease activity results from a contaminating protein. NDPK-B preferentially binds to the singlestranded guanine-rich strand of the c-myc NHE III₁. Potassium ions and G-quadruplexinteractive agents, which stabilize G-quadruplex structures, had an inhibitory effect on NDPK-B DNA binding activity. Based on our studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing singlestranded transcription factors to bind and activate c-myc transcription. This model provides a rationale for how the stabilization of G-quadruplexes within the c-myc gene promoter region can inhibit NDPK-B from activating c-myc transcription. Similarly, the human bcl-2 gene contains a GC-rich region within its promoter region, which is critical in the regulation of bcl-2 expression. We demonstrate that the guanine rich strand within this region can form three intramolecular G-quadruplex structures. Based on NMR studies, the central G-quadruplex forms a mixed parallel/antiparallel structure with three tetrads connected by loops of one, seven, and three bases. The Gquadruplex structures in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. This suggests that targeting these individual structures could lead to different biological outcomes. We also found that Telomestatin upregulates bcl-2 gene expression, which we propose is a result of inhibiting the binding of the WT1 repressor protein by the formation of a drug-stabilized G-quadruplex structure.

The activated product of the c-MYC proto-oncogene is one of the strongest known activators of carcinogenesis. It has been estimated that as many as one-seventh of all cancer deaths are associated with alterations in the c-MYC gene or its expression [1]. Therefore, understanding the regulation of c-MYC expression is a key factor in understanding carcinogenesis in many histologic classes of malignancy. The nuclease hypersensitive element (NHE) III₁ region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. In this dissertation, we investigate the role that nucleolin, a critical player in ribosome biogenesis and cell stress sensing, plays on the transcriptional regulation of the c-MYC promoter through its interaction with the c-MYC G-quadruplex structure. Our studies initiated with the design of a c-MYC G-quadruplex affinity column intended to trap potential c-MYC G-quadruplex-binding proteins that were then identified by LC-MS/MS. After careful examination of the literature of the list of potential c-MYC G-quadruplexbinding proteins, we realized that several of the proteins identified had been previously reported to interact directly with nucleolin. Consequently, we chose to focus our studies on nucleolin, as it could be a central regulator of the (NHE) III region. By performing chromatin immunoprecipitation in HeLa cells, we found that nucleolin indeed interacts with the c-MYC promoter region containing the NHE III₁ element. This binding activity was confirmed by both electromobility shift assay and polymerase stop assay. We provide evidence that nucleolin can induce the formation of the c-MYC G-quadruplex structure from single-stranded DNA, both in linear and circular DNA forms. We show that upon binding, nucleolin increases the stability of the c-MYC G-quadruplex structure leading to repression of c-MYC promoter activity. We also show that nucleolin binds with much higher affinity to G-quadruplex structures with topology similar to that of the parallel c-MYC G-quadruplex, such as those found in the VEGF and PDGF-A promoters; in comparison to G-quadruplexes found in telomeres or the c-MYB promoter, whose have significantly different topology. Interestingly, we also demonstrate that nucleolin binds with higher affinity to the c-MYC G-quadruplex than to its consensus RNA substrate, the nucleolin recognition element (NRE). Furthermore, we show that the C-terminal domain of nucleolin is critical for its interaction and stabilization of the c-MYC G-quadruplex structure. Lastly, we show that the binding of nucleolin to the (NHE) III region causes repression of c-MYC transcription. On the basis of these results, we propose that nucleolin may play an important role in the transcriptional regulation of c-MYC in vivo by inducing the formation of the c-MYC G-quadruplex structure.

The transcription factor c-Myc is an early downstream target of oestrogen action in breast cancer cells in culture and it has been speculated that aberrant c-Myc expression may mediate antioestrogen resistance. However, studies of c-Myc protein expression as either a prognostic or predictive marker in human breast cancer have been limited and contradictory, as have been studies of c-Myc expression during breast cancer evolution. In order to assess the relationship between c-Myc protein expression and outcome from breast cancer, a representative cohort of 292 women with invasive ductal carcinoma (IDC) and linked clinicopathological data was assembled and tissue microarrays (TMA) generated from the archived breast cancer specimens. Detailed assessments of the expression of cyclin D1, cyclin E, p21WAF1/Cip1 and p27Kip1 were also conducted and analysed in relation to c-Myc expression using immunohistochemistry. Changes in c-Myc protein expression in a TMA model of breast cancer evolution were also conducted. Finally the cell-cycle effects of low-level constitutive c-Myc expression and high-level inducible c-Myc expression were evaluated in MCF-7 cells in vitro. Key novel results obtained were that c-Myc protein expression changed from preferentially nuclear to preferentially cytoplasmic during the evolution of breast cancer. In women with early invasive breast carcinoma, a "high-risk" cytoplasmic predominant c-Myc expression pattern was defined (~13% of cases) that independently predicted for poor outcome generally, among ER positive cases and in ER postive cases treated with endocrine therapy. In vitro studies confirmed that c-Myc overexpression was associated with resistance to the anti-proliferative effects of anti-oestrogens with persistence of both cyclin D1-cdk4 and cyclin E-cdk2 activities in the face of anti-oestrogen treatment. Further novel findings were that high cyclin D1 expression (upper 10% of expressors) was an independent predictor of poor outcome among ER positive breast cancer cases. Amongst ER + PR positive cases, both "high-risk" c-Myc expression and high level cyclin D1 expression were independent predictors of poor outcome. In summary, these data indicate that aberrant expression of the cell cycle proteins c-Myc and cyclin D1 may result in poor breast cancer outcomes in hormone receptor positive breast cancer and reinforces the importance of the cell cycle as a potential site of therapeutic manipulation in endocrine-resistant breast cancer.

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.

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.