912 resultados para Transfection transitoire
Resumo:
La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu frquente des IgG humaines. Pourtant, elle est lobjet de beaucoup dattention depuis que deux articles fondateurs ont t publis, qui montrent lun que la sialylation des IgG diminue leur capacit dclencher la cytotoxicit cellulaire dpendant de lanticorps (ADCC), et lautre que les IgG sialyles en 2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thrapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifre, connaissent depuis la fin des annes 90 un succs et une croissance phnomnaux sur le march pharmaceutique. La matrise de la N-glycosylation du Fc des IgG est une cl de lefficacit des anticorps monoclonaux. Si les IgG sialyles sont des molcules peu frquentes in vivo, elles sont trs rares en culture cellulaire. Dans cette tude, nous avons dvelopp une mthode de production dIgG avec une sialylation de type humain en cellules CHO. Nous avons travaill principalement sur la mise au point dune stratgie de production dIgG sialyles par co-expression transitoire dune IgG1 avec la 1,4-galactosyltransfrase I (4GTI) et la -galactoside-2,6-sialyltransfrase I (ST6GalI). Nous avons montr que cette mthode permettait denrichir lIgG1 en glycane fucosyl di-galactosyl mono-2,6-sialyl G2FS(6)1, qui est le glycane sialyl prsent sur les IgG humaines. Nous avons ensuite adapt cette mthode la production dIgG prsentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. Lanalyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec lIgG1 native ou une version mutante de lIgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilis les IgG1 produites avec diffrents profils de glycosylation afin dvaluer limpact de la sialylation 2,6 sur linteraction de lIgG avec le rcepteur FcRIIIa, principal rcepteur impliqu dans la rponse ADCC. Nous avons montr que la sialylation 2,6 augmentait la stabilit du complexe form par lIgG avec le FcRIIIa, mais que ce bnfice ntait pas directement traduit par une augmentation de lefficacit ADCC de lanticorps. Enfin, nous avons dbut le dveloppement dune plateforme dexpression stable dIgG sialyles compatible avec une production lchelle industrielle. Nous avons obtenu une ligne capable de produire des IgG enrichies en G2FS(6)1 hauteur de 400 mg/L. Cette tude a contribu une meilleure comprhension de limpact de la sialylation sur les fonctions effectrices des IgG, et a permis daugmenter la matrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire.
Resumo:
La possibilit de programmer une cellule dans le but de produire une protine dintrt est apparue au dbut des annes 1970 avec lessor du gnie gntique. Environ dix annes plus tard, linsuline issue de la plateforme de production microbienne Escherichia coli, fut la premire protine recombinante (r-protine) humaine commercialise. Les dfis associs la production de r-protines plus complexes et glycosyles ont amen lindustrie biopharmaceutique dvelopper des systmes dexpression en cellules de mammifres. Ces derniers permettent dobtenir des protines humaines correctement replies et de ce fait, biologiquement actives. Afin de transfrer le gne dintrt dans les cellules de mammifres, le polythylnimine (PEI) est certainement un des vecteurs synthtiques le plus utilis en raison de son efficacit, mais aussi sa simplicit dlaboration, son faible cot et sa stabilit en solution qui facilite son utilisation. Il est donc largement employ dans le contexte de la production de r-protines grande chelle et fait lobjet dintenses recherches dans le domaine de la thrapie gnique non virale. Le PEI est capable de condenser efficacement lADN plasmidique (vecteur dexpression contenant le gne dintrt) pour former des complexes de petites tailles appels polyplexes. Ces derniers doivent contourner plusieurs tapes limitantes afin de dlivrer le gne dintrt au noyau de la cellule hte. Dans les conditions optimales du transfert de gne par le PEI, les polyplexes arborent une charge positive nette interagissant de manire lectrostatique avec les protoglycanes hparane sulfate (HSPG) qui dcorent la surface cellulaire. On observe deux familles dHSPG exprims en abondance la surface des cellules de mammifres : les syndcanes (4 membres, SDC1-4) et les glypicanes (6 membres, GPC1-6). Si limplication des HSPG dans lattachement cellulaire des polyplexes est aujourdhui largement accepte, leur rle individuel vis--vis de cet attachement et des tapes subsquentes du transfert de gne reste confirmer. Aprs avoir optimises les conditions de transfection des cellules de mammifres CHO et HEK293 dans le but de produire des r-protines secrtes, nous avons entrepris des cintiques de capture, dinternalisation des polyplexes et aussi dexpression du transgne afin de mieux comprendre le processus de transfert de gne. Nous avons pu observer des diffrences au niveau de ces paramtres de transfection dpendamment du systme dexpression et des caractristiques structurelles du PEI utilis. Ces rsultats prsents sous forme darticles scientifiques constituent une base solide de lenchanement dans le temps des vnements essentiels une transfection efficace des cellules CHO et HEK293 par le PEI. Chaque type cellulaire possde un profil dexpression des HSPG qui lui est propre, ces derniers tant plus ou moins permissifs au transfert de gne. En effet, une tude mene dans notre laboratoire montre que les SDC1 et SDC2 ont des rles opposs vis--vis du transfert de gne. Alors que tous deux sont capables de lier les polyplexes, lexpression de SDC1 permet leur internalisation contrairement lexpression de SDC2 qui linhibe. De plus, lorsque le SDC1 est exprim la surface des cellules HEK293, lefficacit de transfection est augmente de douze pourcents. En utilisant la capacit de SDC1 induire linternalisation des polyplexes, nous avons tudi le trafic intracellulaire des complexes SDC1 / polyplexes dans les cellules HEK293. De plus, nos observations suggrent une nouvelle voie par laquelle les polyplexes pourraient atteindre efficacement le noyau cellulaire. Dans le contexte du transfert de gne, les HSPG sont essentiellement tudis dans leur globalit. Sil est vrai que le rle des syndcanes dans ce contexte est le sujet de quelques tudes, celui des glypicanes est inexplor. Grce une srie de traitements chimiques et enzymatiques visant une approche perte de fonction , limportance de la sulfatation comme modification post-traductionnelle, leffet des chanes dhparanes sulfates mais aussi des glypicanes sur lattachement, linternalisation des polyplexes, et lexpression du transgne ont t tudis dans les cellules CHO et HEK293. Lensemble de nos observations indique clairement que le rle des HSPG dans le transfert de gne devrait tre investigu individuellement plutt que collectivement. En effet, le rle spcifique de chaque membre des HSPG sur la capture des polyplexes et leur permissivit lexpression gnique demeure encore inconnu. En exprimant de manire transitoire chaque membre des syndcanes et glypicanes la surface des cellules CHO, nous avons dtermin leur effet inhibiteur ou activateur sur la capture des polyplexes sans pouvoir conclure quant leffet de cette surexpression sur lefficacit de transfection. Par contre, lorsquils sont prsents dans le milieu de culture, le domaine extracellulaire des HSPG rduit lefficacit de transfection des cellules CHO sans induire la dissociation des polyplexes. Curieusement, lorsque chaque HSPG est exprim de manire stable dans les cellules CHO, seulement une lgre modulation de lexpression du transgne a pu tre observe. Ces travaux ont contribu la comprhension des mcanismes d'action du vecteur polycationique polythylnimine et prciser le rle des protoglycanes hparane sulfate dans le transfert de gne des cellules CHO et HEK293.
Resumo:
Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.
Resumo:
La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu frquente des IgG humaines. Pourtant, elle est lobjet de beaucoup dattention depuis que deux articles fondateurs ont t publis, qui montrent lun que la sialylation des IgG diminue leur capacit dclencher la cytotoxicit cellulaire dpendant de lanticorps (ADCC), et lautre que les IgG sialyles en 2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thrapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifre, connaissent depuis la fin des annes 90 un succs et une croissance phnomnaux sur le march pharmaceutique. La matrise de la N-glycosylation du Fc des IgG est une cl de lefficacit des anticorps monoclonaux. Si les IgG sialyles sont des molcules peu frquentes in vivo, elles sont trs rares en culture cellulaire. Dans cette tude, nous avons dvelopp une mthode de production dIgG avec une sialylation de type humain en cellules CHO. Nous avons travaill principalement sur la mise au point dune stratgie de production dIgG sialyles par co-expression transitoire dune IgG1 avec la 1,4-galactosyltransfrase I (4GTI) et la -galactoside-2,6-sialyltransfrase I (ST6GalI). Nous avons montr que cette mthode permettait denrichir lIgG1 en glycane fucosyl di-galactosyl mono-2,6-sialyl G2FS(6)1, qui est le glycane sialyl prsent sur les IgG humaines. Nous avons ensuite adapt cette mthode la production dIgG prsentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. Lanalyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec lIgG1 native ou une version mutante de lIgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilis les IgG1 produites avec diffrents profils de glycosylation afin dvaluer limpact de la sialylation 2,6 sur linteraction de lIgG avec le rcepteur FcRIIIa, principal rcepteur impliqu dans la rponse ADCC. Nous avons montr que la sialylation 2,6 augmentait la stabilit du complexe form par lIgG avec le FcRIIIa, mais que ce bnfice ntait pas directement traduit par une augmentation de lefficacit ADCC de lanticorps. Enfin, nous avons dbut le dveloppement dune plateforme dexpression stable dIgG sialyles compatible avec une production lchelle industrielle. Nous avons obtenu une ligne capable de produire des IgG enrichies en G2FS(6)1 hauteur de 400 mg/L. Cette tude a contribu une meilleure comprhension de limpact de la sialylation sur les fonctions effectrices des IgG, et a permis daugmenter la matrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire.
Resumo:
La possibilit de programmer une cellule dans le but de produire une protine dintrt est apparue au dbut des annes 1970 avec lessor du gnie gntique. Environ dix annes plus tard, linsuline issue de la plateforme de production microbienne Escherichia coli, fut la premire protine recombinante (r-protine) humaine commercialise. Les dfis associs la production de r-protines plus complexes et glycosyles ont amen lindustrie biopharmaceutique dvelopper des systmes dexpression en cellules de mammifres. Ces derniers permettent dobtenir des protines humaines correctement replies et de ce fait, biologiquement actives. Afin de transfrer le gne dintrt dans les cellules de mammifres, le polythylnimine (PEI) est certainement un des vecteurs synthtiques le plus utilis en raison de son efficacit, mais aussi sa simplicit dlaboration, son faible cot et sa stabilit en solution qui facilite son utilisation. Il est donc largement employ dans le contexte de la production de r-protines grande chelle et fait lobjet dintenses recherches dans le domaine de la thrapie gnique non virale. Le PEI est capable de condenser efficacement lADN plasmidique (vecteur dexpression contenant le gne dintrt) pour former des complexes de petites tailles appels polyplexes. Ces derniers doivent contourner plusieurs tapes limitantes afin de dlivrer le gne dintrt au noyau de la cellule hte. Dans les conditions optimales du transfert de gne par le PEI, les polyplexes arborent une charge positive nette interagissant de manire lectrostatique avec les protoglycanes hparane sulfate (HSPG) qui dcorent la surface cellulaire. On observe deux familles dHSPG exprims en abondance la surface des cellules de mammifres : les syndcanes (4 membres, SDC1-4) et les glypicanes (6 membres, GPC1-6). Si limplication des HSPG dans lattachement cellulaire des polyplexes est aujourdhui largement accepte, leur rle individuel vis--vis de cet attachement et des tapes subsquentes du transfert de gne reste confirmer. Aprs avoir optimises les conditions de transfection des cellules de mammifres CHO et HEK293 dans le but de produire des r-protines secrtes, nous avons entrepris des cintiques de capture, dinternalisation des polyplexes et aussi dexpression du transgne afin de mieux comprendre le processus de transfert de gne. Nous avons pu observer des diffrences au niveau de ces paramtres de transfection dpendamment du systme dexpression et des caractristiques structurelles du PEI utilis. Ces rsultats prsents sous forme darticles scientifiques constituent une base solide de lenchanement dans le temps des vnements essentiels une transfection efficace des cellules CHO et HEK293 par le PEI. Chaque type cellulaire possde un profil dexpression des HSPG qui lui est propre, ces derniers tant plus ou moins permissifs au transfert de gne. En effet, une tude mene dans notre laboratoire montre que les SDC1 et SDC2 ont des rles opposs vis--vis du transfert de gne. Alors que tous deux sont capables de lier les polyplexes, lexpression de SDC1 permet leur internalisation contrairement lexpression de SDC2 qui linhibe. De plus, lorsque le SDC1 est exprim la surface des cellules HEK293, lefficacit de transfection est augmente de douze pourcents. En utilisant la capacit de SDC1 induire linternalisation des polyplexes, nous avons tudi le trafic intracellulaire des complexes SDC1 / polyplexes dans les cellules HEK293. De plus, nos observations suggrent une nouvelle voie par laquelle les polyplexes pourraient atteindre efficacement le noyau cellulaire. Dans le contexte du transfert de gne, les HSPG sont essentiellement tudis dans leur globalit. Sil est vrai que le rle des syndcanes dans ce contexte est le sujet de quelques tudes, celui des glypicanes est inexplor. Grce une srie de traitements chimiques et enzymatiques visant une approche perte de fonction , limportance de la sulfatation comme modification post-traductionnelle, leffet des chanes dhparanes sulfates mais aussi des glypicanes sur lattachement, linternalisation des polyplexes, et lexpression du transgne ont t tudis dans les cellules CHO et HEK293. Lensemble de nos observations indique clairement que le rle des HSPG dans le transfert de gne devrait tre investigu individuellement plutt que collectivement. En effet, le rle spcifique de chaque membre des HSPG sur la capture des polyplexes et leur permissivit lexpression gnique demeure encore inconnu. En exprimant de manire transitoire chaque membre des syndcanes et glypicanes la surface des cellules CHO, nous avons dtermin leur effet inhibiteur ou activateur sur la capture des polyplexes sans pouvoir conclure quant leffet de cette surexpression sur lefficacit de transfection. Par contre, lorsquils sont prsents dans le milieu de culture, le domaine extracellulaire des HSPG rduit lefficacit de transfection des cellules CHO sans induire la dissociation des polyplexes. Curieusement, lorsque chaque HSPG est exprim de manire stable dans les cellules CHO, seulement une lgre modulation de lexpression du transgne a pu tre observe. Ces travaux ont contribu la comprhension des mcanismes d'action du vecteur polycationique polythylnimine et prciser le rle des protoglycanes hparane sulfate dans le transfert de gne des cellules CHO et HEK293.
Resumo:
Dissertao de Mestrado, Cincias Biomdicas, Departamento de Cincias Biomdicas e Medicina, Universidade do Algarve, 2016
Resumo:
Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.
Resumo:
Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB
Resumo:
Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an artificial niche required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.
Resumo:
To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.
Resumo:
To date, attempts to regenerate a complete tooth, including the critical periodontal tissues associated with the tooth root, have not been successful. Controversy still exists regarding the origin of the cell source for cellular cementum (epithelial or mesenchymal). This disagreement may be partially due to a lack of understanding of the events leading to the initiation and development of the tooth roots and supportive tissues, such as the cementum. Osterix (OSX) is a transcriptional factor essential for osteogenesis, but its role in cementogenesis has not been addressed. In the present study, we first documented a close relationship between the temporal- and spatial-expression pattern of OSX and the formation of cellular cementum. We then generated 3.6 Col 1-OSX transgenic mice, which displayed accelerated cementum formation vs. WT controls. Importantly, the conditional deletion of OSX in the mesenchymal cells with two different Cre systems (the 2.3kb Col 1 and an inducible CAG-CreER) led to a sharp reduction in cellular cementum formation (including the cementum mass and mineral deposition rate) and gene expression of dentin matrix protein 1 (DMP1) by cementocytes. However, the deletion of the OSX gene after cellular cementum formed did not alter the properties of the mature cementum as evaluated by backscattered SEM and resin-cast SEM. Transient transfection of Osx in the cementoblasts in vitro significantly inhibited cell proliferation and increased cell differentiation and mineralization. Taken together, these data support 1) the mesenchymal origin of cellular cementum (from PDL progenitor cells); 2) the vital role of OSX in controlling the formation of cellular cementum; and 3) the limited remodeling of cellular cementum in adult mice.
Resumo:
The obligate endosymbiont Wolbachia pipientis is found in a wide range of invertebrates where they are best known for manipulating host reproduction. Recent studies have shown that Wolbachia also can modulate the lifespan of host insects and interfere with the development of human pathogens in mosquito vectors. Despite considerable study, very little is known about the molecular interactions between Wolbachia and its hosts that might mediate these effects. Using microarrays, we show that the microRNA (miRNA) profile of the mosquito, Aedes aegypti, is significantly altered by the wMelPop-CLA strain of W. pipientis. We found that a host miRNA (aae-miR-2940) is induced after Wolbachia infection in both mosquitoes and cell lines. One target of aae-miR-2940 is the Ae. aegypti metalloprotease gene. Interestingly, expression of the target gene was induced after Wolbachia infection, ectopic expression of the miRNA independent of Wolbachia, or transfection of an artificial mimic of the miRNA into mosquito cells. We also confirmed the interaction of aae-miR-2940 with the target sequences using GFP as a reporter gene. Silencing of the metalloprotease gene in both Wolbachia-infected cells and adult mosquitoes led to a significant reduction in Wolbachia density, as did inhibition of the miRNA in cells. These results indicate that manipulation of the mosquito metalloprotease gene via aae-miR-2940 is crucial for efficient maintenance of the endosymbiont. This report shows how Wolbachia alters the host miRNA profile and provides insight into the mechanisms of host manipulation used by this widespread endosymbiont.
Resumo:
We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR) - linked survival pathways (phosphoinositol-3-kinase/AKT/ mammalian target of rapamycin and extracellular signal - regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death - related gene expression. Our results suggest that EGFR phosphorylation is causally linkedto pERK and pAKT activation by asbestos in normal and SV40 Tag - immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.
Resumo:
We previously showed that integrin alphavbeta3 overexpression and engagement by its ligand vitronectin increased adhesion, motility, and proliferation of human ovarian cancer cells. In search of differentially regulated genes involved in these tumor biological events, we previously identified the integrin-linked kinase (ILK) to be under control of alphavbeta3. In the present investigation we demonstrated significantly upregulated ILK protein as a function of alphavbeta3 in two ovarian cancer cell lines, OV-MZ-6 and OVCAR-3, and proved co-localization at the surface of alphavbeta3-overexpressing cells adherent to vitronectin. Increase of ILK protein was reflected by enhanced ILK promoter activity, an effect, which we further characterized with regard to transcriptional response elements involved. Abrogation of NF-kappaB/c-rel or p53 binding augmented ILK promoter activity and preserved induction by alphavbeta3. The AP1-mutant exhibited decreased promoter activity but was also still inducible by alphavbeta3. Disruption of the two DNA consensus motifs for Ets proteins led to divergent observations: mutation of the Ets motif at promoter position -462 bp did not significantly alter promoter activity but still allowed response to alphavbeta3. In contrast, disruption of the second Ets motif at position -85 bp did not only lead to slightly diminished promoter activity but also, in that case, abrogated ILK promoter induction by alphavbeta3. Subsequent co-transfection studies with ets-1 in the presence of the second Ets motif led to additional induction of ILK promoter activity. Taken together, these data suggest that ets-1 binding to the second Ets DNA motif strongly contributes to alphavbeta3-mediated ILK upregulation. By increasing ILK as an important integrin-proximal kinase, alphavbeta3 may promote its intracellular signaling and tumor biological processes arising thereof in favor of ovarian cancer metastasis.
Resumo:
Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell-extracellular matrix (ECM) and growth factor interactions based upon their sulfation patterns. When compared with non-osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3-E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant fibroblast growth factor receptor (FGFR) from FGFR1 (0.5-fold decrease) to FGFR3 (1.5-fold increase). The change in FGFR expression profile of the osteogenic-committed cultures was reflected by their inability to sustain an FGF-2 stimulus, but respond to BMP-2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican-3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N- and O-sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (21 kDa) HS chain. With the HSPG glypican-3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican-3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2-null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican-3.
