²Ä¤@³¡¥÷ Nasopharyngeal carcinoma¡]NPC¡^ is a malignant tumor which occurs at high incidence in southern China. Several risk factors have now been recognized, but the molecular mechanism of this disease is not well understood. To investigate the c-myc¡Bcyclin D1¡Bp16¡Bp27 and Rb gene expression in NPC at protein level, 46 cases of nasopharyngeal carcinoma in southern Taiwan were detected by immunohistochemistry. There was no detectable p16 in 31/45 cases¡]69¢M¡^¡F 34/46 cases¡]73.9¢M¡^had intense staining for the Rb protein¡F 29¡]70.7¢M¡^of 41 cases had c-myc protein expression¡Fcyclin D1 was not overexpression in nasopharyngeal carcinoma¡F 32¡]69.6¢M¡^of 46 cases had high level expression of p27, which was inverse correlation with other tumors. No expression of c-myc protein correlated with higher neck metastasis¡]P¡Õ0.05¡^. No correlation was found between other proteins and any of the clinicopathological parameters. ²Ä¤G³¡¥÷ To better understand nasopharyngeal carcinoma¡]NPC¡^, we have newly established two NPC cell lines. Biopsy specimens from NPC patients were collected, primary culture were set up. Two NPC cell lines were established¡GNPCGK 01 was derived from differentiated carcinoma and NPCGK 02 was derived from undifferentiated carcinoma. Two cell lines have been passaged for more than 25 times. Two cell lines had telomerase activity¡Fstrong expression of hTERT gene and keratin-19 gene were also observed. TGF£] RI protein expression of these NPC cell lines is higher than normal epithelial cell.The oncogenes, c-myc¡Bc-fos and cyclin D1 were overexpressed. The Rb protein was expressed stronger than normal epithelial cells. NPCGK 01 that was derived from differentiated carcinoma had p16 down-regulation and p27 gene not expression, but p21 protein had excess expression. In short, two cell lines had cancer cell characteristics, oncoproteins were overexpression and tumor suppressor proteins were abnormal expression. This result may lead to tumorigenesis of nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) EBNA1 protein mediates the replication and mitotic segregation of the EBV genomes via interactions with the viral oriP sequences. C666-1 is the only known nasopharyngeal carcinoma (NPC) cell line that stably maintains EBV in culture and I investigated whether this is due to differences in oriP-mediated functions in replication and segregation. I found that both C666-1 and EBV-negative NPC cell lines can replicate and maintain oriP plasmids for extended periods but that high EBNA1 levels interfered with plasmid segregation. The segregation element within oriP was recently shown to contain 29 repeated sequences instead of the 20 repeats in initial oriP isolates. I compared the functions of oriP with 20 or 29 repeats and found that the higher number of repeats decreased plasmid replication but increased plasmid maintenance, consistent with a segregation effect. Finally, I identified a potential role for promyelocytic leukemia nuclear bodies in oriP plasmid replication.

The Epstein-Barr virus (EBV) EBNA1 protein mediates the replication and mitotic segregation of the EBV genomes via interactions with the viral oriP sequences. C666-1 is the only known nasopharyngeal carcinoma (NPC) cell line that stably maintains EBV in culture and I investigated whether this is due to differences in oriP-mediated functions in replication and segregation. I found that both C666-1 and EBV-negative NPC cell lines can replicate and maintain oriP plasmids for extended periods but that high EBNA1 levels interfered with plasmid segregation. The segregation element within oriP was recently shown to contain 29 repeated sequences instead of the 20 repeats in initial oriP isolates. I compared the functions of oriP with 20 or 29 repeats and found that the higher number of repeats decreased plasmid replication but increased plasmid maintenance, consistent with a segregation effect. Finally, I identified a potential role for promyelocytic leukemia nuclear bodies in oriP plasmid replication.

Magnetic resonance imaging (MRI) is one of medical images used by doctors for diagnosing diseases. MRI shows higher quality in displaying soft tissues and tumors. Pharmacokinetic dynamic gadolinium-enhanced MR imaging and functional MR imaging (fMRI) were used in this dissertation. Dynamic MR images are obtained using fast spin-echo sequences at consecutive time after the injection of gadolinium-diethylene-triamine penta-acetic (Gd-DTPA) acid. A pharmacokinetic model analyzes time-signal intensity curves of suspected lesions. Functional MR imaging produces images of activated brain regions by detecting the indirect effects of neuronal activity on local blood volume, flow, and oxygen saturation. Thus it is a promising tool for further understanding the relationships between brain structure, function, and pathology. Because of patients' movement during imaging, serially acquired MR images do not correspond in the same pixel position. Therefore, matching corresponding points from MR images is one of fundamental tasks in this dissertation. Least-squares estimation is a standard method for parameter estimation. However, outliers (due to non-Gaussian noise, lesion evolution, motion-related artifacts, etc.) may exist and thus may cause the motion parameter estimation result to deteriorate. In this dissertation, we describe two robust estimation algorithms for the registration of serially acquired MR images. The first estimation algorithm is based on the Newton method and uses the Tukey's biweight objective function. The second estimation algorithm is based on the Levenberg-Marquardt technique and uses a skipped mean objective function. The robust M-estimators can suppress the effects of the outliers by scaling down their error magnitudes or completely rejecting outliers using a weighting function. Experimental results show the accuracy of the proposed robust estimation algorithms is within subpixel. MR imaging has been used to evaluate nasal papilloma. However, postoperative MR imaging of nasal papilloma becomes more complicated because repair with granulation and fibrosis occurs after surgery. Therefore, it is possible to misclassify recurrences as postoperative changes or to misclassify postoperative changes as recurrences. Recently, dynamic gadolinium-enhanced MR imaging with pharmacokinetic analysis has been successfully used to identify the post-treatment recurrence or postoperative changes in rectal and cervical carcinoma. Nasopharyngeal carcinoma (NPC) comprising malignant tumors is a disease more common in Asia than in other parts of the world. Hence, in this dissertation, we evaluate the feasibility of dynamic gadolinium-enhanced MR imaging with pharmacokinetic analysis in detecting NPC and distinguishing recurrent nasal papilloma from postoperative changes (fibrosis or granulation tissue). In this dissertation, a new approach to differentiate recurrent nasal papilloma from postoperative changes using pharmacokinetic dynamic gadolinium-enhanced MR imaging and robust motion correction is presented. First, a robust estimation technique is incorporated into nonlinear minimization method to robustly register dynamic gadolinium-enhanced MR images. Next, user roughly selects the region of interest (ROI) and an active contour technique is used to extract a more precise ROI. Then, the relative signal increase (RSI) is calculated. We use a three-parameter mathematical model for pharmacokinetic analysis. The pharmacokinetic parameters A (enhancement amplitude) and Tc (tissue distribution time) are calculated by a nonlinear least-squares fitting technique. The calculated A and Tc are used to characterize tissue. Pharmacokinetic analysis shows that recurrent nasal papilloma has faster tissue distribution time (Tc, 41 versus 88 seconds) and higher enhancement amplitude (A, 2.4 versus 1.2 arbitrary units) than do postoperative changes. A cut-off of 65 seconds for tissue distribution time and 1.6 units for enhancement amplitude yields an accuracy of 100% for differentiating recurrent nasal papilloma from postoperative changes. Though the above methods obtained good results, finding the region of interest (ROI) was done in a semi-automatic manner. For diagnosing NPC and improve the drawback, a system that automatically detects and labels NPC with dynamic gadolinium-enhanced MR imaging is presented. This system is a multistage process, involving motion correction, gadolinium-enhanced MR data quantitative evaluation, rough segmentation, and rough segmentation refinement. Three approaches, a relative signal increase method, a slope method and a relative signal change method, are proposed for the quantitative evaluation of gadolinium-enhanced MR data. After the quantitative evaluation, a rough NPC outline is determined. Morphological operations are applied to refine the rough segmentation into a final mask. The NPC detection results obtained using the proposed methods had a rating of 85% in match percent compared with these lesions identified by an experienced radiologist. However, the proposed methods can identify the NPC regions quickly and effectively. In this dissertation, the proposed methods provide significant improvement in correcting the motion-related artifacts and can enhance the detection of real brain activation and provide a fast, valuable diagnostic tool for detecting NPC and differentiating recurrent nasal papilloma from postoperative changes.

Nasopharyngeal carcinoma is a commonly occuring tumor in Southern China. However, the genetic basis underlying its tumorigenicity is still unclear. In eukaryotic cells, progression of the cell cycle is regulated by interactions of cyclins, cyclin dependent kinase (CDKs) and CDK inhibitors (CDKIs). These cell cycle-regulator proteins play important roles in growth of both normal and tumor cells. Many human tumors exhibit deregulation of one or more genes which involved in regulation of cell cycle progression. p27Kip1, a member of the Cip/Kip family, inhibits both cyclin D-CDK4, and cyclin E-CDK2 complexes and regulates progression of the cell cycle from G1 to S phase. Although p27Kip1 gene mutations are rare in human tumors, low expression of p27Kip1 are observed in several cancers, such as colon, breast and esophagus. In our previous study, p27Kip1 shown lower expression in two NPC cell lines compared with NNE and 293 (HEK). A doxycycline inducible construct, pBIG2r/p27Kip1, included a full length of human p27Kip1 cDNA was transfected into two NPC cell lines. Expression of several cell cycle-related genes were analyzed. By increasing p27Kip1 in NPC cell lines, we found that the G1 phase and the doubling time were lengthened. Protein expression of cyclin E and CDK2 were up-regulated. These data suggest that the overexpression of p27Kip1 might be cause NPC cells to arrest at G1 phase and might lead to apoptosis.

Cette thèse de doctorat est une étude de la biologie et de la dynamique des microARN circulants, démontrant leur potentiel comme biomarqueurs pour l’amélioration de la surveillance et de l’évaluation pronostique dans le cancer. Il s’agit des ARN non codants simple-brin, d'environ 19-25 nt de longueur. Ils jouent un rôle clé dans la régulation de l'expression génomique au niveau post-transcriptionnel, en ciblant et réprimant la traduction des ARNm par complémentarité partielle avec leur 3'-UTR. Ils sont également libérés dans le milieu extracellulaire, la circulation et les liquides biologiques. Leur stabilité remarquable et leur diversité dans la circulation, ainsi que leur provenance maligne ou normale, en font des candidats biomarqueurs intéressants, reflétant potentiellement l'état et la dynamique d’une tumeur. Nous nous sommes focalisés sur les carcinomes ovariens (OvCa) et nasopharyngés (NPC), essayant d'élucider la relation entre les miARN plasmatiques et le pronostique des OvCa après une première ligne de traitement, ainsi que d’évaluer leur utilité dans la détection d’une réponse précoce des NPC au traitement. Le carcinome ovarien séreux est la malignité gynécologique la plus agressive. L'absence de symptômes précoces et l'insuffisance des moyens modernes pour détecter la maladie résiduelle et évaluer les résultats du traitement signalent le besoin de nouveaux biomarqueurs diagnostiques et pronostiques. En utilisant des prélèvements plasmatiques séquentiels OvCa avant et après traitement, nous avons étudié un groupe de miARN, en comparaison à des lésions pelviennes bénignes et des femmes en bonne santé. MiR-200b avait une concentration nettement plus élevée dans les échantillons malins avant traitement par rapport aux deux groupes non cancéreux. L'analyse pré- et post-traitement de miR-200b en parallèle avec le biomarqueur standard CA125 a révélé des variations distinctes et une corrélation significative de la variation de miR-200b avec le temps de rémission (PFS). Nous concluons que miR-200b pourrait éventuellement être utilisé comme biomarqueur supplémentaire pour l'estimation de la rémission à la fin du traitement. Le carcinome nasopharyngé (NPC) d'autre part est une tumeur constamment associée à une infection latente des cellules malignes par le virus d’Epstein-Barr (EBV), présentant une distribution géographique particulière. La position de la tumeur rend difficile l'approche chirurgicale, les biomarqueurs évaluant différentes approches thérapeutiques étant de grande importance. Etudiant une nouvelle forme orale de l'agent démethylant 5-azacytidine, démontrée prometteuse pour un tiers des patients NPC qui l’ont reçue, nous avons essayé d'évaluer son impact sur l'expression des miARN et des protéines virales. Malgré l'infection virale latente, les miARN viraux sont abondamment exprimés. En traitant pendant deux semaines quatre modèles de tumeur NPC développés in vivo, nous avons observé une réponse nette dose-dépendante dans les deux. L'analyse protéique a montré une induction de la protéine activatrice du cycle lytique BZLF1, renforçant les preuves antérieures d'induction partielle du cycle lytique viral par la 5-azacytidine. L'analyse des miARN a confirmé l'expression robuste des miARN BART et l'absence des miARN BHRF1 dans les NPC sans traitement. Lors du traitement, nous avons détecté les miR-BHRF1 à la fois dans la tumeur et le plasma des souris traitées. Cette induction a été validée par un traitement ultérieur d'une semaine qui a aussi mis en évidence l'induction de l’expression de l'ARNm de BHRF1, transcrit par le locus situé parmi les miARN BHRF1. Une induction de ces miARN a été observée après traitement par des agents de chimiothérapie, suggérant une utilité clinique potentielle des miR-BHRF1 comme biomarqueurs pour l’évaluation précoce de l’efficacité du traitement. Notre objectif actuel est de valider cette induction par chimiothérapie et étendre nos études au plasma humain. / This doctorate thesis provides an insight into the biology and dynamics of circulating microRNAs, demonstrating their potential to become crucial biomarkers for a better surveillance and prognosis of cancer. MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs, 19-25 nt long, with a key role in the post-transcriptional regulation of gene expression, repressing the translation of target mRNAs through partial base-pair complementarity with their 3’-UTR. They can be released in the extracellular medium, being protected from RNases by association with various transporters and reach body fluids and circulation, participating in intercellular communication. Their remarkable stability and manageable diversity in circulation, as well as the fact that they derive from both malignant and normal cells make them very attractive biomarker candidates, potentially reflecting tumor state and dynamics. We have been focusing on ovarian (OvCa) and nasopharyngeal (NPC) carcinomas, attempting to elucidate the relation between plasma miRNAs and the prognosis of OvCa after first-line treatment, as well as to evaluate their use in the detection of early response of NPC tumors to treatment. Serous epithelial ovarian carcinoma is the most frequent ovarian and the most aggressive gynecologic malignancy. The absence of early symptoms and the insufficiency of modern means to accurately map residual disease and assess treatment outcome highlight the need for new diagnostic and prognostic biomarkers. Using sequential plasma samples from OvCa patients before and after first-line treatment, we studied a pre-selection of miRNAs, comparing them to samples from benign pelvic lesions and healthy women. MiR-200b exhibited a distinct higher concentration in malignant samples before treatment compared to both non-cancerous groups. Pre- and post-treatment assessment of miR-200b in parallel with the standard biomarker CA125 revealed distinct variations and a significant correlation of miR-200b variation with the progression-free survival (PFS) of the patient. We suggest that miR-200b could eventually be used as a supplementary biomarker for estimation of the remission upon treatment completion. Nasopharyngeal carcinoma (NPC) on the other hand is a tumor consistently associated to latent Epstein-Barr virus (EBV) infection of the malignant cells, presenting a unique geographical incidence pattern. The deep position of the tumor makes it tough to access surgically, with biomarkers assessing different therapeutic approaches being greatly needed. Studying a new oral form of the demethylating agent 5-azacytidine, proven to be promising for one third of NPC patients receiving it as a monodrug, we attempted to identify impact of the drug on the expression of viral miRNAs and proteins. Despite the latent viral infection, viral miRNAs are abundantly expressed, attracting interest in EBV-associated malignancies. Treating four in vivo developed NPC tumor models for two weeks, we observed clear response in two of them, in a dose-dependent manner. Protein analysis showed an induction of the immediate-early lytic protein BZLF1, solidifying previous evidence of partial activation of the viral lytic cycle by 5-azacytidine. MiRNA analysis confirmed robust expression of BART and absence of BHRF1 miRNAs at baseline status of NPC. Upon treatment, we observed an induction of BHRF1 miRNAs in both tumor and plasma of treated mice. This induction was successfully validated in a following one-week treatment and completed by a recorder de novo expression of the BHRF1 mRNA, transcribed within the BHRF1 miRNA loci. A weaker induction of BHRF1 miRNAs was also recorded after treatment with standard chemotherapeutic agents, suggesting a potential clinical utility of these miRNAs as circulating biomarkers for detection of early response to treatment. We are further working to confirm this induction by chemotherapy and extend our study to plasma samples derived from treated patients.