Chemotherapy is one of the common treatments in cancer therapy. The effectiveness of chemotherapy is limited by several factors one of which is the emergence of multidrug resistance (MDR). MDR is caused by the activity of diverse ATP binding cassette (ABC) transporters that pump drugs out of the cells. There are several drugs which have been used in treatment of cancer. One of them is doxorubicin that intercalates and inhibits DNA replication. However, doxorubicin has been found to cause development of MDR in tumors. It has been reported that there is a correlation between multidrug resistance and invasiveness of cancer cells. Vimentin is a type III intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and also poor prognosis of cancer. There are several studies that have shown the connection between expression level of vimentin and invasiveness. In this study, MCF-7 cell line (MCF-7/S), which is a model cell line for human mammary carcinoma, and doxorubicin resistant MCF-7 cell line (MCF-7/Dox) were used. The resistant cell line was previously obtained by stepwise selection in our laboratory. The main purpose of this study was to investigate changes of metastatic behaviour in MCF-7/Dox cell line, after transient silencing of vimentin gene by siRNA. In conclusion, down-regulation of vimentin gene expression in MCF-7/Dox cell lines was expected to change the characteristics in migration and invasiveness shown by migration and invasion assays.

Η RNase P είναι το ένζυμο που ωριμάζει το 5΄ άκρο των πρόδρομων μορίων tRNA, ενώ έχει βρεθεί και στις τρεις φυλογενετικές περιοχές, καθώς και σε υποκυτταρικά οργανίδια. Είναι ριβονουκλεοπρωτεϊνικής φύσεως στις περισσότερες περιπτώσεις, ενώ έχουν βρεθεί και ένζυμα RNase P αποκλειστικά πρωτεϊνικής φύσεως. Η υπομονάδα RNA των βακτηριακών ολοενζύμων είναι καταλυτικά ενεργή απουσία πρωτεϊνικών παραγόντων in vitro, καθιστώντας την ένα πραγματικό ριβοένζυμο. Η ικανότητα της RNase P να αναγνωρίζει συγκεκριμένες δομές στα μόρια των υποστρωμάτων της και όχι αλληλουχίες, δημιούργησε τη δυνατότητα χρήσης αυτού του ενζύμου ως ενός μοριακού εργαλείου για τη στόχευση πολλών ιικών και παθολογικών μορίων RNA in vitro και in vivo, καταστέλλοντας την έκφραση των μορίων αυτών, μέσω της τεχνολογίας των μορίων EGS (external guide sequence) και των ριβοενζύμων M1GS. Η RNase P, σύμφωνα με πολλές μελέτες, έχει δειχθεί ότι αποτελεί στόχο πολλών φαρμακευτικών παραγόντων, συμπεριλαμβανομένων πολλών γνωστών αντιβιοτικών, οι οποίοι κατά κύριο λόγο αναστέλλουν τη δραστικότητα του ενζύμου. Πρόσφατα δείχτηκε, μέσω αναλυτικής κινητικής μελέτης, ότι ένα μακρολίδιο, η σπιραμυκίνη, ενεργοποιεί σημαντικά τη δραστικότητα της βακτηριακής RNase P και του M1 RNA κατά ένα δοσοεξαρτώμενο τρόπο, λειτουργώντας έτσι ως μη-ειδικός ενεργοποιητής μικτού τύπου. Μέχρι σήμερα, στη διεθνή βιβλιογραφία, δεν έχει αναφερθεί άλλη ουσία η οποία προκαλεί θετική επίδραση στη δραστικότητα της RNase P. Στην παρούσα μελέτη, αρχικά μελετήθηκε η ενεργοποίηση της δραστικότητας της βακτηριακής RNase P και αποκαλύφθηκε ότι η σπιραμυκίνη δεν αλληλεπιδρά με ιοντικούς δεσμούς με το μόριο Μ1 RNA, αλλά προκαλεί αλλαγή διαμόρφωσης στο δομικό στοιχείο P10/11 του ριβοενζύμου. Το δομικό αυτό στοιχείο εμπλέκεται στην αναγνώριση του υποστρώματος, αποτέλεσμα το οποίο έρχεται σε συμφωνία με τις τιμές KD που προσδιορίστηκαν για το σύμπλοκο ριβοενζύμου–υποστρώματος, απουσία και παρουσία σπιραμυκίνης. Με δεδομένο ότι η σπιραμυκίνη δεν επηρεάζει την πρωτεϊνοσύνθεση ή τη δραστικότητα της RNase P των ευκαρυωτικών κυττάρων, κατασκευάστηκε ένα ριβοένζυμο M1GS, ώστε να ελεγχθεί η επίδραση του αντιβιοτικού στη δραστικότητα αυτού του ριβοενζύμου in vivo, σε καλλιεργούμενα ανθρώπινα κύτταρα ΗΕΚ293. Ως στόχος του συγκεκριμένου M1GS, επιλέχτηκε ο μεταγραφικός παράγοντας Ets2 λόγω της μεγάλης κλινικής σημασίας του, εφόσον έχει συσχετιστεί με αρκετούς τύπους καρκίνου και παθολογικές καταστάσεις, καθώς και με διαδικασίες διαφοροποίησης. Ο σπουδαίος ρόλος του Ets2, σε συνδυασμό με τα ελλιπή δεδομένα σχετικά με την έκφρασή του, είχαν αποτρέψει μέχρι σήμερα την αποτελεσματική στόχευσή του με τη χρήση των υπαρχουσών μεθοδολογιών που βασίζονται στο RNA, όπως το RNAi. Μετά από ανάλυση της δευτεροταγούς δομής του Ets2 mRNA, σχεδιάστηκαν δύο οδηγοί αλληλουχίες. Οι αλληλουχίες αυτές, αρχικά, δοκιμάστηκαν ως εξωτερικές οδηγοί αλληλουχίες (EGS) σε συνδυασμό με το βακτηριακό ολοένζυμο της RNase P. Η EGS303 (το νούμερο υποδεικνύει το νουκλεοτιδικό κατάλοιπο του στόχου που δρα η RNase P), εμφάνισε τη μεγαλύτερη ικανότητα να επάγει τη δράση της RNase P in vitro. Η οδηγός αυτή αλληλουχία, στη συνέχεια κλωνοποιήθηκε στο 3΄ άκρο του M1 RNA, παράγοντας το ριβοένζυμο M1GS303, το οποίο είναι δραστικό έναντι του μορίου–στόχου του in vitro. Η δραστικότητα του συγκεκριμένου ριβοενζύμου ενεργοποιείται εντυπωσιακά κατά 160% παρουσία σπιραμυκίνης. Προκειμένου να ελεγχθεί η δραστικότητα αυτού του ριβοενζύμου in vivo, το μόριο–στόχος και το ριβοένζυμο εκφράστηκαν ελεγχόμενα σε κύτταρα E. coli, προκαλώντας μείωση της έκφρασης του μορίου–στόχου από το M1GS303 κατά 95% μετά από 12 ώρες έκφρασης των μορίων. Μείωση στα ίδια επίπεδα ανιχνεύτηκε μόλις μετά από 4 ώρες έκφρασης εφόσον στα κύτταρα είχε προστεθεί σπιραμυκίνη, γεγονός που υποστηρίζει την εντυπωσιακά θετική επίδραση της σπιραμυκίνης επί της δραστικότητας του ριβοενζύμου. Η ίδια σειρά πειραμάτων επαναλήφθηκε σε ευκαρυωτικά κύτταρα, με έκφραση του ριβοενζύμου σε HEK293 κύτταρα. Η δραστικότητα του ριβοενζύμου προσδιορίστηκε ποιοτικά και ποσοτικά, από την έκφραση της χιμαιρικής φθορίζουσας πρωτεΐνης Ets2–EGFP (μόριο–στόχος), σε διαφορετικούς χρόνους έκφρασης. Παρατηρήθηκε ότι το M1GS δρα αποτελεσματικά έναντι του μορίου–στόχου του και σε ευκαρυωτικά κύτταρα in vivo, προκαλώντας μείωση στην έκφραση του Ets2, η οποία αυξάνεται επιπλέον παρουσία σπιραμυκίνης. Τα παραπάνω αποτελέσματα δείχνουν τη σημαντική ενεργοποίηση της δραστικότητας του M1GS σε ανθρώπινα κύτταρα και καθιστούν τη σπιραμυκίνη ένα σημαντικό ενεργοποιητή στη χρήση των ριβοενζύμων M1GS ως εργαλεία γονιδιακής αποσιώπησης. Ο συνδυασμός βελτιωμένων ριβοενζύμων M1GS με την παρουσία σπιραμυκίνης αυξάνει ακόμα περισσότερο την πρακτική χρήση της συγκεκριμένης τεχνολογίας τόσο in vitro όσο και in vivo, επιτυγχάνοντας ακόμα πιο αποτελεσματική αποσιώπηση της γονιδιακής έκφρασης. / RNase P is the enzyme that endonucleolytically cleaves the precursor tRNA transcripts to produce their mature 5΄ ends. It has been found in all three phylogenetic domains of life, as well as in subcellular organelles. In most cases, it has been described as a ribonucleoprotein complex. However, few RNase P enzymes that are exclusively proteinaceous have been also reported recentrly. The RNA subunit of bacterial holoenzyme is catalytically active in the absence of protein factors in vitro, making it a true ribozyme. The ability of RNase P to recognize specific structures in its substrate molecules instead of specific sequences, allowed the use of this enzyme as a molecular tool for targeting pathological and viral RNA molecules in vitro and in vivo, by suppressing gene expression through the technology of EGS (external guide sequence) and M1GS ribozymes. RNase P, according to numerous studies, has been the target of several pharmaceutical agents, including most of the mainstream antibiotics. It has been shown recently, through analytical kinetic studies that the macrolide spiramycin significantly enhances the activity of bacterial RNase P and M1 in a dose dependent manner, acting as a non-specific mixed-type activator. Until now, no other compound has been reported to induce a positive effect on RNase P activity. In the present study, the enhancement of bacterial RNase P activity by spiramycin was tested initially, and it was revealed that spiramycin does not interact with the M1 RNA molecule through ionic bonds. On the contrary, it induces a conformational change of the P10/11 structural element of M1 RNA, which is mainly responsible for substrate recognition. The above results are in agreement with the KD values determined for the ribozyme-substrate complex, in the absence or in the presence of spiramycin. Since spiramycin does not affect eucaryotic protein synthesis or eucaryotic RNase P activity, an M1GS ribozyme was constructed, in order to examine the effect of spiramycin on the ribozyme activity in vivo, using human HEK293 cells. The target of this M1GS was the transcription factor Ets2, a factor with great clinical importance, since it has been associated with several types of cancer and disease, as well as essential processes during differentiation. The important role of Ets2 in combination with the lack of data on Ets2 expression, had hitherto prevented its effective targeting by using the existing methodologies based on RNA, such as RNAi. After analysis of the secondary structure of Ets2 mRNA, two guide sequences were designed. These sequences were originally tested in trans as external guide sequences (EGS), in combination with the bacterial RNase P. The EGS303 (the number indicates the nucleotide residue cleaved by RNase P), showed an ability to induce RNase P activity in vitro. The guide sequence was then cloned and fused into the 3' end of M1 RNA ribozyme, thus producing the M1GS303 ribozyme, which was found to be effective against the target molecule. The activity of this specific ribozyme is impressively enhanced by 160% in the presence of spiramycin. In order to examine the activity of this ribozyme in vivo, the expression of the target molecule and the ribozyme were induced in E. coli cells. After 12 hours of expression, a reduced level of the target molecule was detected, because of the M1GS303 activity (about 95%). Reduction to similar levels was observed after only 4 hours from the induction of both molecules expression, in the presence of spiramycin. This observation strongly supports spiramycin’s striking positive effect on the ribozyme activity. The same set of experiments was repeated in human HEK293 cells. The activity of the ribozyme was determined qualitatively and quantitatively, by the determination of the expression of the chimeric fluorescent protein Ets2-EGFP (target molecule) at different times of expression. The M1GS ribozyme cleaves efficiently the target molecule in human cells as well in vivo, resulting in a reduction in the expression of Ets2, which is further increased in the presence of spiramycin. This result indicates the significant activation of M1GS activity in human cells, making spiramycin an important activator in using M1GS ribozymes as tools in gene silencing. The combination of improved M1GS ribozymes in the presence of spiramycin, further increases the practical utilization of this technology both in vitro and in vivo, thus achieving an even more effective suppression in gene expression.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

Genome sequencing has yielded unparalleled insights into fundamental biological processes and the genetics that guide them. In contrast to expectations that protein-coding genes would be the primary output of eukaryotic genomes, however, it is now clear that the vast majority of transcription is devoted to noncoding RNAs (ncRNAs). Although originally regarded as 'transcriptional noise', it is now clear that these transcripts are essential regulators of genetic activity. In this thesis I build upon the hypothesis that the genomes of eukaryotes encode a regulatory 'RNA machine' dominated by ncRNAs. In the Introduction (Chapter 1) I discuss how prior gene models may have inadvertently prevented a full understanding of ncRNAs, review the transcriptional landscape of eukaryotes, and examine the biogenesis and function of small regulatory RNAs. In support of a role for ncRNAs in complex metazoa, Chapter 2 presents data showing a positive correlation between the proportion of non-protein-coding DNA and biological complexity, suggesting that the evolutionary trajectory of intricate developmental phenotypes may have been facilitated by ncRNAs. In the following chapters two more 'gears' are added to the RNA machine. Chapter 3 details the discovery of snoRNA-derived RNAs - an evolutionarily ancient class of Argonaute-assocaited RNA whose biogenesis overlaps with microRNAs (miRNAs) and silencing RNAs (siRNAs). Likewise, Chapter 4 reports a new class of ~18 nt transcription initiation RNAs (tiRNAs) derived from regions proximal to transcription start sites. tiRNAs are enriched at GC-rich promoters and regions of active transcription, implicating them in transcriptional regulation. Chapter 5 presents evidence that tiRNAs are restricted to metazoa, and describes a model of RNA Polymerase II dependent tiRNA biogenesis. This thesis concludes with a general discussion of the implications of these findings, and the potential development of RNA therapeutics. Gathering evidence suggests that eukaryotic genomes are driven by a complex and interwoven network of RNA regulatory feedback loops. This thesis takes a small step towards developing a complete picture of this system.

Genome sequencing has yielded unparalleled insights into fundamental biological processes and the genetics that guide them. In contrast to expectations that protein-coding genes would be the primary output of eukaryotic genomes, however, it is now clear that the vast majority of transcription is devoted to noncoding RNAs (ncRNAs). Although originally regarded as 'transcriptional noise', it is now clear that these transcripts are essential regulators of genetic activity. In this thesis I build upon the hypothesis that the genomes of eukaryotes encode a regulatory 'RNA machine' dominated by ncRNAs. In the Introduction (Chapter 1) I discuss how prior gene models may have inadvertently prevented a full understanding of ncRNAs, review the transcriptional landscape of eukaryotes, and examine the biogenesis and function of small regulatory RNAs. In support of a role for ncRNAs in complex metazoa, Chapter 2 presents data showing a positive correlation between the proportion of non-protein-coding DNA and biological complexity, suggesting that the evolutionary trajectory of intricate developmental phenotypes may have been facilitated by ncRNAs. In the following chapters two more 'gears' are added to the RNA machine. Chapter 3 details the discovery of snoRNA-derived RNAs - an evolutionarily ancient class of Argonaute-assocaited RNA whose biogenesis overlaps with microRNAs (miRNAs) and silencing RNAs (siRNAs). Likewise, Chapter 4 reports a new class of ~18 nt transcription initiation RNAs (tiRNAs) derived from regions proximal to transcription start sites. tiRNAs are enriched at GC-rich promoters and regions of active transcription, implicating them in transcriptional regulation. Chapter 5 presents evidence that tiRNAs are restricted to metazoa, and describes a model of RNA Polymerase II dependent tiRNA biogenesis. This thesis concludes with a general discussion of the implications of these findings, and the potential development of RNA therapeutics. Gathering evidence suggests that eukaryotic genomes are driven by a complex and interwoven network of RNA regulatory feedback loops. This thesis takes a small step towards developing a complete picture of this system.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.