Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.

Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss-of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成2年11月22日 竹中徳哉氏の博士論文として提出された

Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss-of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies.

With each heartbeat, major arteries experience circumferential expansion due to internal pressure changes. This pulsatile force is called cyclic strain and has been implicated in playing a pivotal role in the genetic regulation of vascular physiology and pathology. This dissertation investigates the hypothesis that in human umbilical vein endothelial cells (HUVEC), pathological levels of cyclic strain activate the c-Myc promoter, leading to c-Myc transcription and downstream gene induction. To determine expression and time-dependency of c-Myc in HUVEC, mRNA and protein expression of c-Myc under physiological (6-10% cyclic strain) and pathological conditions (20% cyclic strain) were studied. Both c-Myc mRNA and protein expression increased more than three-fold in HUVEC (P4-P5) cyclically-strained at 20%. This expression occurred in a time-dependent manner, peaking in the 1.5-2 hour range and falling to basal levels by 3 hours. Subsequently, the mechanism of c-Myc transcription was investigated by using specific inhibitors to modulate c-Myc transcriptional activation. These compounds, obtained from the University of Arizona Cancer Center, attenuated cyclic-strain-induced c-Myc transcription by about 50%. Having established this reduction in expression, it was investigated how these effects modulate downstream genes that are regulated by c-Myc. The results indicate that direct targeting of the c-Myc promoter may decrease stretch-induced gene expression of vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA) and heat shock protein 60 (HSP60). These findings may help in the development of a novel therapeutic opportunity in vascular diseases.

ABSTRACT TRANSCRIPTION PATTERN COMPARISON OF TWO UBIQUITIN SPECIFIC PROTEASES (USP6 AND USP32) Akhavan Tabasi, Shiva M.Sc., Department of Biology Supervisor: Assist. Prof. Dr. A. Elif Erson August 2007, 93 pages Breast cancer is the most common type of cancer among women worldwide. The incidence of breast cancer is 1 in 8 among women. Usually loss of tumor suppressor genes and overexpression of proto-oncogenes are known to be involved during mammary tumorigenesis. USP32 (Ubiquitin Specific Protease 32) gene is located on chromosomal band 17q23, a region of amplification in breast cancer. Gene amplification is known to be a common mechanism in breast cancer cells, through which proto-oncogenes are overexpressed and contribute to tumor progression. Presence of multiple oncogene candidates on 17q23 requires individual characterization of these genes. USPs (Ubiquitin Specific Protease), have various roles in protein degradation pathways (e.g / by editing the ubiquitin chains, recycling of ubiquitin, v deubiquitinating the target proteins and inhibiting their degradation by the proteasome). Deregulated expression of USPs is likely to interfere with the degradation of many key regulatory proteins in the cell. Therefore, USP32 becomes an interesting oncogene candidate that may have roles in protein degradation pathways based on the fact that it is located on an amplicon region and that it is overexpressed in breast tumors. On the other hand, USP6 (Ubiquitin Specific Protease 6), a known oncogene on 17p13, is also a deubiquitinating enzyme, with conserved histidine and cysteine domains, which are also shared by USP32. Interestingly there is a 97% sequence similarity between bases 3,197 to 7,831 of USP6 and 2,390 to 7,024 of USP32 gene. In this study, we aimed to investigate the expression patterns of USP32 and USP6 (including alternative transcripts) in breast tissue to avoid any possibility of overlapping functions of two enzymes due to their high sequence similarity. In addition, we sub-cloned USP32 gene into TOPO-TA vector, so that further functional studies (e.g / localization and overexpression) can be performed. Further characterizations of Ubiquitin Specific Protease 32, may help us understand its importance in the protein degradation pathway during breast tumorigenesis.

Mutation or inactivation of RB occurs in most human tumors and results in the deregulation of several E2F family transcription factors. Among the E2F family, E2F3 has been implicated as a key regulator of cell proliferation and E2f3 gene amplification and overexpression is detected in some human tumors. To study the role of E2F3a in tumor development, we established a transgenic mouse model expressing E2F3a in a number of epithelial tissues via a keratin 5 (K5) promoter. Transgenic expression of E2F3a leads to hyperproliferation, hyperplasia and increased levels of p53-independent apoptosis in transgenic epidermis. Consistent with data from human cancers, the E2f3a transgene is found to have a weak oncogenic activity on its own and to enhance the response to a skin carcinogenesis protocol. While E2F3a induces apoptosis in the absence of p53, the inactivation of both p53 and p73, but not p73 alone, significantly impairs apoptosis induced by E2F3a. This suggests that both p53 and p73 contribute to E2F3a induced apoptosis but that their function is compensatory. Even though data suggest that E2F3a carries out its unique apoptotic activity in part through another E2F family member E2F1, unlike E2F1, the ARF tumor suppressor is required for E2F3a-induced apoptosis. While both E2F3a and E2F1 require ATM for apoptosis, E2F3a activates ATM through a distinct mechanism from E2F1. The overexpression of E2F3a results in the accumulation of DNA damage in K5 transgenic keratinocytes and normal human fibroblasts (NHFs). In response to this, the DNA damage checkpoint kinase ATM is activated, and phosphorylation of the downstream targets p53 and the histone variant H2AX are significantly increased. Additional studies show that increased Cdk activity and aberrant DNA replication contributes to DNA damage, ATM activation and apoptosis in response to deregulated E2F3a, which suggest that aberrant replication imposed by deregulated E2F3a plays an important role in the activation of the ATM DNA damage response pathway. Activation of ATM by E2F3a is not affected by loss of ARF or E2F1. Meanwhile, E2F3a-induced ARF upregulation is not affected by E2F1 loss. The above results indicate that E2F3a engages several parallel pathways involving E2F1, ARF and the ATM kinase, and these pathways cooperate to promote apoptosis.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Με σκοπό να διερευνήσουμε τους μηχανισμούς επαγωγής απόπτωσης από την TRAIL και την επιλεκτικότητα την οποία δείχνει απέναντι στα καρκινικά κύτταρα, χρησιμοποιήσαμε κυτταρικές σειρές, προερχόμενες από το παχύ έντερο ανθρώπου, οι οποίες αντιπροσωπεύουν τα διάφορα στάδια της καρκινογένεσης, από το πρώιμο αδένωμα στο καρκίνωμα. Παρατηρήθηκε μια ανθεκτικότητα των πρώιμων αδενωμάτων απέναντι στην TRAIL σε αντίθεση με τα κύτταρα που προέρχονται από καρκίνους προχωρημένου σταδίου, ενώ σημαντικό ρόλο φαίνεται να παίζει, εκτός από το στάδιο καρκινογένεσης και η ύπαρξη ορισμένων ογκογονιδίων στον καθορισμό της ευαισθησίας των κυττάρων απέναντι στην TRAIL. Συγκεκριμένα, οι διάφορες ισομορφές του ογκογονιδίου RAS παίζουν ιδιαίτερο ρόλο τόσο όσο αφορά την ανταπόκριση του κύτταρου στην επαγωγή με TRAIL, όσο και στη ρύθμιση των μορίων που εμπλέκονται άμεσα στο μονοπάτι σηματοδότησης του TRAIL. Τα ογκογονίδια RAS παίζουν σημαντικό ρόλο στην καρκινική εξαλλαγή ενεργοποιώντας μια σειρά από μονοπάτια σηματοδότησης που οδηγούν στην ανεξέλεγκτη κυτταρική διαίρεση και στην προστασία από τα αποπτωτικά σήματα. Εντούτοις, η TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing Ligand) έχει τη δυνατότητα να προκαλεί απόπτωση κυρίως στα καρκινικά κύτταρα, ενεργοποιώντας τους υποδοχείς DR4 και DR5 (Death Receptors). Σε αυτή τη μελέτη δείξαμε ότι σε αδενοκαρκινώματα του παχέος εντέρου ανθρώπου η έκφραση των υποδοχέων της TRAIL ρυθμίζεται από την ενεργότητα της κινάσης MEK (MAPK/ERK Kinase). Η ευαισθησία στην TRAIL των κυτταρικών σειρών που χρησιμοποιήσαμε φάνηκε να συσχετίζεται με το βαθμό κακοήθους εξαλλαγής. Συγκεκριμένα, οι κυτταρικές σειρές AAC1 και RGC2, που προέρχονται απο πρώιμα αδενώματα και η κυτταρική σειρά Caco2, που προέρχεται απο μέσο αδένωμα παρουσίασαν μεγάλη ανθεκτικότητα στην TRAIL. Αντίθετα, οι κυτταρικές σειρές DLD-1 και HT-29, που προέρχονται απο αδενοκαρκινώματα προχωρημένου στάδιου ήταν ευαίσθητες στην TRAIL. Επιλέχθηκε η κυτταρική σειρά Caco2 η οποία είναι ανθεκτική στην TRAIL και οι δύο κυτταρικές σειρές που προέρχονται από προχωρημένα αδενοκαρκινώματα για να εξεταστεί η έκφραση σημαντικών μορίων για την αποπτωτική σηματοδότηση από την TRAIL. Φάνηκε μια αντιστοιχία της έκφρασης των υποδοχέων DR4 και DR5 με την ευαισθησία των κυττάρων στην TRAIL, ενώ τα επίπεδα έκφρασης των FADD, κασπάση 8, κασπάση 3 και FLIP δεν παρουσίασαν παρόμοια αντιστοιχία. Επιπλέον, παρατηρήθηκε παρατεταμένη ενεργοποίηση των κινασών MEK, ERK, JNK1/2 και p38 μετά από επαγωγή με TRAIL, σε αντίθεση με την αντίστοιχη κινητική ενεργοποίησης των ίδιων κινασών μετά από επαγωγή με ορό. Αντίστοιχη κινητική με αυτή των ΜΕΚ και ERK παρουσίασε η έκφραση του γονιδίου c- FOS μετά από επαγωγή με TRAIL ή ορό. Τα κύτταρα Caco2, DLD-1 και HT- 29 επωάστηκαν με χημικούς αναστολείς των κινασών ΜΕΚ για 16 h και ελέγχθηκε η έκφραση των DR4 και DR5. Τα επίπεδα των DR4 και DR5 μειώθηκαν και στις τρεις κυτταρικές σειρές ως συνέπεια της αναστολής της ΜΕΚ, ενώ η αναστολή των ΜΕΚ δεν επιρρέασε τα επίπεδα των FADD, FLIP, κασπάση 8, κασπάση 3 και τον υποδοχέα FAS που ανήκει στην ίδια οικογένεια με τους DR4 και DR5. Επιπλέον, ελέγχθηκε με ανάλυση FACS η έκφραση των υποδοχέων DR4, DR5 και FAS in vivo σε ζωντανά κύτταρα που προεπωάστηκαν με τον αναστολέα της ΜΕΚ και στους αντίστοιχους μάρτυρες και τα αποτελέσματα ήταν αντίστοιχα με αυτά των in vitro πειραμάτων. Οι παραπάνω παρατηρήσεις αποτελούν ισχυρές ενδείξεις για το συσχετισμό της ενεργοποίησης της οδού MEK/ERK/FOS με με την έκφραση των υποδοχέων DR4 και DR5 και με την ευαισθησία των κυττάρων στην TRAIL, καθώς κύτταρα HT-29 που προεπωάστηκαν με τον αναστολέα των ΜΕΚ παρουσίσαν μειωμένη ευαισθησία στην TRAIL. Για τον προσδιορισμό των διακριτών επιδράσεων των ογκογονιδίων RAS στον καρκίνο του παχέος εντέρου, διαμολύνθηκαν κύτταρα που προέρχονται από μέσο αδένωμα του παχέος εντέρου (Caco2) με τα ογκογονίδια Ki- και Ha-RAS. Μετά από εξέταση πολλών διαφορετικών κλώνων επιλέχθηκαν για λεπτομερέστερη ανάλυση κλώνοι οι οποίοι δεν υπερεκφράζουν την RAS σε πρωτεϊνικό επίπεδο πάνω από 3 φορές σε σχέση με την ενδογενή RAS των μητρικών κυττάρων. Επιπλέον, ελέγχθηκε αν οι κλώνοι που υπερεκφράζουν τις ογκογόνες RAS παρουσιάζουν αυξημένη σηματοδότηση προς μονοπάτια κυτταρικής σηματοδότησης που είναι γνωστό οτι ενεργοποιούνται από τις RAS. Τόσο οι κλώνοι Ki-RAS, όσο και οι Ha-RAS, έδειξαν να ενεργοποιούν τα μονοπάτια σηματοδότησης RAF/MEK/ERK και PI3K/AKT με τους Ha-RAS να προκαλούν ισχυρότερη ενεργοποίηση. Σχεδιάστηκαν πειράματα για τον καθορισμό του βαθμού της in vitro και in vivo κακοήθους εξαλλαγής. Τα πειράματα σε μαλακό άγαρ δείχνουν την αποτελεσματικότητα των καρκινικών κυττάρων να αναπτύσσονται δημιουργώντας αποικίες χωρίς να προσκολλώνται σε επιφάνεια. Οι δυο ισομορφές της RAS αύξησαν σημαντικά την ιδιότητα των Caco2 να σχηματίζουν αποικίες σε μαλακό άγαρ, ενώ η Ha-RAS παρουσίασε τη μεγαλύτερη ικανότητα. Για τον προσδιορισμό του βαθμού κακοήθους εξαλλαγής των κλώνων in vivo έγινε υποδόριος εμβολιασμός με περίπου 106 κύτταρα από τον κάθε κλώνο σε ποντίκια SCID. Τόσο η Κi-RASV12 όσο και η Ha-RASV12 αύξησαν την ικανότητα των Caco2 να σχηματίζουν όγκους σε ποντίκια SCID με την Ha-RAS να προκαλεί τον σχηματισμό περισσότερων και μεγαλύτερων όγκων. Για την περαιτέρω κατανόηση των μηχανισμών εξαλλαγής των κυττάρων από τα ογκογονίδια RAS αξιολογήθηκαν αποτελέσματα από ανάλυση γονιδιακών μικροσυστοιχιών. Μετά από ανάλυση του γονιδιακού προφίλ των CACO-RAS κλώνων και τα δυο ογκογονίδια βρέθηκαν να επάγουν την έκφραση γονιδίων που εμπλέκονται στην αγγειογένεση και στην προώθηση του καρκίνου, όπως τα γονίδια που κωδικοποιούν τους υποδοχείς VEGF και TGFβ. Σημειώνεται ότι μεταστατικοί δείκτες, όπως η Vimentin, που είναι και δείκτης της μετάβασης από επιθηλιακό σε μεσεγχυματικό φαινότυπο, βρέθηκαν να υπερεκφράζονται μόνο στους κλώνους Ha-RASV12. Ο in vitro και in vivo χαρακτηρισμός εξαλλαγμένων κυττάρων από τα ογκογονίδια RAS, καθώς και η ανάλυση μικροσυστοιχειών γονιδίων έδειξε ότι στο κυτταρικό μοντέλο που χρησιμοποιήθηκε το ογκογονίδιο Ha-RAS έχει αυξημένες εξαλλακτικές ιδιότητες. Τα ογκογονίδια RAS αύξησαν το βαθμό κακοήθους εξαλλαγής των κυττάρων Caco2 και ελέγχθηκε αν αυτό συνοδεύεται από αυξημένη ευαισθησία των κλώνων CACO2-RAS στην TRAIL. Μετρήθηκε η βιωσημότητα των κλώνων CACO2-RAS μετά από επαγώγη με TRAIL και φάνηκε ότι και τα δύο ογκογονίδια αύξησαν την ανταποκρισιμότητα των κυττάρων Caco2 στην TRAIL. Τα Caco2 που εξαλλάχθηκαν με την Ki- RASV12 ήταν λιγότερο ευαίσθητα στην TRAIL από ότι αυτά με τη Ha- RASV12 σε όλους τους κλώνους που ελέγχθηκαν. Ο έλεγχος της έκφρασης σημαντικών παραγόντων για την απόπτωση από την TRAIL έδειξε ότι οι πρωτεΐνες που παρουσίασαν τις μεγαλύτερες διαφοροποιήσεις στους κλώνους που υπερεκφράζουν τις RAS, σε σχέση με τα κύτταρα μάρτυρα, ήταν οι υποδοχείς DR4 και DR5. Παρόλο που οι κλώνοι Ha-RAS παρουσίασαν μεγαλύτερη ευαισθησία στην TRAIL, τα επίπεδα έκφρασης των DR4 και DR5 ήταν παρόμοια μεταξύ των κλώνων Ki-RAS και Ha-RAS. Για να ελεγχθεί κατά πόσο το μονοπάτι σηματοδότησης MEK/ERK παίζει ρόλο στην υπερέκφραση των υποδοχέων από τα ογκογονίδια RAS χρησιμοποιήθηκαν χημικοί αναστολείς της ΜΕΚ και ελέγθηκε η επίδρασή τους στα επίπεδα των DR4 και DR5. H χημική αναστολή της ΜΕΚ προκάλεσε μείωση των επιπέδων των φωσφορυλιωμένων ERK1/2 και των επιπέδων έκφρασης των DR4 και DR5 τόσο στους CACO-Κ15, όσο και στους CACO-Η2. Τέλος, ελέγχθηκε κατά πόσο η μείωση των DR4 και DR5 από την χημική αναστολή των ΜΕΚ μπορέι να έχει επίδραση στη δράση της TRAIL απέναντι στους κλώνους CACO2-RAS και βρέθηκε ότι προεπώση των Ha-RASV12 κλώνων για 16 h με χημικό αναστολέα των ΜΕΚ μέιωσε σημαντικά την ευαισθησία τους στην TRAIL. / In order to study the mechanism by which TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing Ligand) induces apoptosis almost exclusively to cancer cells we used human colon cancer cell lines that represent different stages of carcinogenesis, from early adenoma to carcinoma. It was observed that early adenoma cells were resistant to TRAIL-induced apoptosis. On the contrary, cells that were derived from carcinomas were sensitive; while a correlation of the sensitivity of the cells to TRAIL-induced apoptosis to the presence of certain activated oncogenes was observed. In particular, the various isoforms of RAS oncogenes appear to play an important role in the responsiveness of the cancer cells to TRAIL as well as in regulating specific components which are essential for TRAIL signaling. RAS oncogenes play an important role in oncogenic transformation by activating various signaling pathways that favor tumor growth also by controlling cell division and resistance to apoptotic stimuli. TRAIL, however, has the unique ability to cause apoptosis preferentially to cancer cells by activating DR4 and DR5 receptors. In this study we show that in human colorectal adenocarcinomas cells the expression of DR4 and DR5 is partially regulated by the activity of MEK (MAPK/ERK Kinase). The sensitivity of the cell lines to TRAIL seemed to be correlated with the level of oncogenic transformation of the cells. In particular, the AAC1 and RGC2 cell lines that were derived from early adenomas and the Caco2 cell line that was derived from an intermediate adenoma were very resistant to TRAIL induced apoptosis. On the contrtary, the DLD-1 and HT-29 cell lines, which came from advanced stage carcinomas were sensitive to TRAIL. The Caco2 cell line, which is resistant to TRAIL and the cell lines that derived from advanced stage adenocarcinomas were chosen for expression analysis of important molecules in TRAIL-induced apoptosis. There was a correlation of DR4 and DR5 expression with the sensitivity of the cell lines to TRAIL, while the expression levels of FADD, Caspase 3, Caspase 8 and FLIP did not seem to correlate with TRAIL sensitivity in these cell lines. In addition, it was observed a prolonged activation of MEK, ERK1/2, JNK1/2 and p38 after induction with TRAIL, which did not follow the kinetics of serum-induced activation of ERK1/2. The activation of MEK and ERK1/2 was correlated to the kinetics of c-FOS proto-oncogene expression induced by TRAIL or serum. The expression levels of DR4 and DR5 were analysed after incubation for 16 h of the Caco2, DLD-1 and HT-29 cell lines with MEK inhibitors. There was a decrease of DR4 and DR5 expression levels in response to MEK inhibition, while the expression levels of FADD, Caspase 3, Caspase 8, FLIP and FAS receptor were not affected. Moreover, FACS analysis of living cells showed that MEK inhibition reduces the levels of DR4 and DR5 but not of other receptors of the same family, in vivo. It appears that the activation of the MEK/ERK/FOS axis plays a role in the positive feedback loop of TRAIL its receptors DR4 and DR5. To determine the distinct effects of different RAS oncogenes in cancer cells colon intermediate adenoma cells (Caco2) were chosen for transfection with the Ki- and Ha-RAS oncogenes. Clones that did not express more than 3 times the endogenous levels of RAS proteins were selected for further analysis. In addition it was examined whether the CACO2-RAS clones are able to activate the RAF/MEK/ERK and PI3K/AKT pathways, which are known effectors of RAS proteins. Both RAS isoforms activated these two pathways with the Ha-RASV12 clones presenting better potential in activating both RAF/MEK/ERK and PI3K/AKT pathways. In order to characterize the in vitro and in vivo oncogenic potential the ability of the CACO2-RAS clones to grow in soft agar and to grow tumors in nude mice was examined. The in vitro and in vivo characterization of the Caco2 RASV12-transformed cells showed that the Ha-RAS oncogene has a higher in vitro and in vivo transforming ability relative to the Ki-RAS oncogene in these cells. Results from cDNA microarray analysis were evaluated in order to further understand the mechanisms by which the RAS oncogenes cause oncogenic transformation. Gene expression profile analysis of the CACO2-RAS clones showed that both oncogenes induced expression of genes involved in angiogenesis and tumor promotion such as VEGF and TGFβ. Notably, metastatic markers, such as Vimentin, which is also an epithelial to mesenchymal transition marker, were overexpressed only in the Caco2 cells transformed with the Ha-RAS oncogene. The RAS oncogenes increased the oncogenic potential of the Caco2 cells and it was examined if the increased oncogenic potential correlated with increased sensitivity to TRAIL. Moreover, the examination of the expression levels of molecules important for TRAIL singnaling showed that Ki-RAS as well as Ha-RAS oncogenes can induce DR4 and DR5 expression with similar efficiency. However, in spite of similar induction of DR4 and DR5 by the two RAS oncogenes, the Ki-RAS-transformed cells were less susceptible to TRAIL induced apoptosis. Finally, in order to see whether the increased MEK/ERK activation observed in the CACO-RAS clones played a role in increasing DR4 and DR5 levels the CACO-RAS clones were incubated for 16h in the presence of MEK inhibitors. MEK inhibition resulted in decreased DR4 and DR5 expression levels, as well as decreased sensitivity of the clones to TRAIL induced apoptosis.

Errata is tipped in behind bibliography. / Bibliography: leaves 191-215. / xiv, 216, [29] leaves, [81] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reports the partial cloning and characterisation of the human uroplakin 1B gene which has allowed analysis and characterisation of the gene with regard to its structure, chromosomal localisation and integrity. / Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 1999?