Exocrine pancreatic cancer is the fifth leading cause of death from malignant disease in Western society and it is one of the most aggressive human tumors. Once diagnosed, the 12-month patient survival is less than 5%. More than 90% of human exocrine tumors are classified as "ductal adenocarcinomas" on the basis of their microscopic appearance. The plasminogen system plays a critical role in intravascular thrombolysis as well as in other biological processes that require cellular migration, such as angiogenesis, inflammatory reactions, tissue remodelling, and tumor progression. There are two types of plasminogen activators that catalyze plasmin generation from plasminogen: tissue-type (tPA) and urokinase-type (uPA). Activation of plasminogen to plasmin results in progressive degradation of fibrin and other extracellular matrix components and may also lead to activation of metalloproteases, latent growth factors, and proteolysis of membrane glycoproteins. All these processes may contribute to tumor development and metastasis. There is extensive evidence supporting the notion that the uPA system, including its receptor and plasminogen activator inhibitor PAI-1, can contribute to tumorigenesis in a variety of tissue types but there is less evidence for such a role regarding tPA and annexin A2 (AnxA2), a putative tPA receptor. Previous studies of our group have shown that tPA is commonly expressed in pancreas cancer tissues and cell lines and appears to be selectively associated with the neoplastic phenotype. Using neutralizing antibodies or chemical inhibitors leads to reduced in vitro tumor invasion. Our results support that - in the pancreas - the tPA system plays an important role in tumor development and/or progression whereas the uPA system may play a more dominant role in pancreatitis. More recent studies have shown that tPA stimulates cell proliferation and angiogenesis in exocrine pancreatic tumors. These results allow new approaches to improve the treatment of this disease, but to do so it is necessary to use of mouse models of disease. In attempt to explore the role of tPA and its receptor, annexin A2, in pancreatic tumorigenesis, we have taken advantage of two well established transgenic mouse models: Ela1-TAg (1-127) and Ela1-myc. In these mice, transgenes are targeted to acinar cells using the Elastase-1 enhancer/promoter. We have also analyzed the pancreas of Ela1-CCK2 and MT-TGFa transgenic mice, as models of acinar-ductal transdifferentiation and ductal complex formation. Our results show that expression of tPA is undetectable in the non-neoplastic pancreatic epithelium and in metaplastic ducts and in acinar tumors. By contrast, tPA is overexpressed in neoplastic pancreatic ducts. This pattern expression is in agreement with the results described in humans, indicating that mouse models of pancreatic cancer may be useful for the study of human pathology. On the other hand, AnxA2 is undetectable in acinar tumors but it is detected in the apical membrane of normal and metaplastic duct epithelium. In addition, AnxA2 is strongly expressed in ductal tumor cells where it shows a non-polarized distribution. These results suggest that different molecular events may participate in the activation of tPA and its receptor, AnxA2, in non-neoplastic ducts. In order to analyze the role of tPA in the progression of pancreatic tumors, we mated Ela1-TAg and Ela1-myc transgenic mice to tPA-deficient mice. The proportion of tumors displaying pure acinar differentiation or mixed acinar/ductal components was similar in both mouse strains, indicating that tPA is not required for in acinar-ductal transdifferentiation. However, it was observed an increased survival in hybrid mice Ela1-myc:tPA-/- supporting a critical role for tPA in the progression of pancreatic ductal tumors. To get insight into the mechanism by wich tPA participates in this process we have analyzed factors related to tumor progression: tumors arising in a tPA-/- genetic background show a lesser vessel density and proliferation rate than those arising in wild type mice. These results indicate that tPA could play a role in angiogenesis stimulation and cell proliferation and suggest that the increase in survival observed in Ela1-myc tPA-/- mice could be a consequence of the inhibition of tumor angiogenesis and cell proliferation. In addition, we have analyzed the differential gene expression between Ela1-myc and Ela1-myc tPA-/- mice by microarrays. This analysis has led to the identification of related genes with tumor progression and invasion that can be a target for the action of tPA, although more work is necessary to determined their role. Finally, we have studied the direct effects of the expression of tPA in the pancreas, by the generation of two transgenic mice which tPA expression is targeted to acinar cells, using the Elastase-1 enhancer/promoter (Ela1-tPA), or to ductal cells using the Citokeratin 19 promoter (CK19-tPA). The results in Ela1-tPA mice show that overexpression of tPA in acinar cells does not affect normal mouse development. The effects on the pancreas analysed are currently being analyzed in greater detail. Altogether, the data described here support the relevant role of tPA in pancreas cancer progression and indicate that mouse models of pancreatic cancer may be useful for the preclinical evaluation of drugs targeting the tPA system.

L'adenocarcinoma pancreàtic és una neoplàsia amb mal pronòstic per la que no existeixen marcadors específics. En aquesta tesi s'han estudiat possibles alteracions de les estructures glucídiques de la ribonucleasa pancreàtica humana (RNasa 1) de sèrum amb l'objectiu de determinar el seu ús diagnòstic. S'han descrit les estructures glucídiques de la RNasa 1 sèrica i de línies cel·lulars endotelials, i s'ha observat un increment en la proporció d'estructures biantenàries amb Fc en la RNasa 1 sèrica de pacients amb càncer de pàncreas, fet que podria ser d'utilitat diagnòstica. També, donada la gran similitud entre les estructures glucídiques descrites per la RNasa 1 sèrica i per l'endotelial, l'origen de la RNasa 1 sèrica sembla ser principalment endotelial. La RNasa 1 també s'ha avaluat per electroforesi bidimensional i s'ha establert una correlació entre el contingut d'àcid siàlic i el seu pI, fet que pot ajudar a la interpretació dels mapes bidimensionals d'altres glicoproteïnes. / Pancreatic adenocarcinoma has one of the highest mortality rates of all neoplasias and it has no specific markers. In this work, possible alterations in the glycan structures of human pancreatic ribonuclease (RNase 1) present in serum are investigated with the aim of determining its diagnostic utility. Serum and endothelial RNase 1 glycan structures have been described and an increase of biantennary structures with Fc in serum RNasen 1 from pancreatic cancer patients could be observed, what can be of diagnosis utility. Also, due to the high similarity between serum RNase 1 and endothelial RNase 1 glycan structures, it was concluded that endothelium can be the main producer of serum RNase 1. RNase 1 was also evaluated by two-dimensional electrophoresis and a correlation between the sialic acid content and the observed pI decrease could be establish, what may be very significant for understanding and interpreting two-dimensional maps from other glycoproteins.

El sistema TK/GCV es, problamente, la estrategia suicida mejor caracterizada hasta el momento. No obstante, se desconocen muchos aspectos relacionados con su mecanismo de acción. Con el objetivo de indentificar condicionantes de la respuesta TK/GCV, realizamos un estudio comparativo de la expresión de genes y de las vías de señalización que se activan en células sensibles y en células resistentes al tratamiento. Así, pudimos asociar la actividad de la quinasa Chk1, y la expresión de genes involucrados en el control del ciclo celular, con una mayor respuesta al sistema suicida. Así mismo, determinamos que la combinación de TK/GCV con el inhibidor de Chk1 UCN-01 produce un efecto antagónico en las células sensibles a TK/GCV. Por otro lado, la terapia combinada capaz de lisar las células e inducir muerte celular por fosforilación de GCV, en un único agente (ICOVIR11), resultó en una potenciación de sus efectos citotóxicos, permitiendo la compensación de la pérdida de potencia secundaria al uso de un promotor selectivo de tumor. Más aún, la expresión de TK como gen tardío de ICOVIR11,permitió la monitorización in vivo y de manera no invasiva, de la actividad TK y la replicación viral. / Although extensively characterized, the paradigmatic suicide system TK/GCV conceals the details of its ultimate mechanism of action. In order to shed some light on this issue, we conducted a series of experiments with resistant and sensitive cell lines, allowing us to identify cell cyclerelated genes that are deregulated in cells with induced resistance to TK/GCV. In addition, the association of Chk1 activation with a greater sensitivity to TK/GCV, pointed out the relevance of the cell cycle status at the moment of receiving the treatment, and its control in response to genotoxic insults. Treatment with a Chk1 inhibitor induced, in sensitive cells, an antagonistic effect on TK/GCV cytotoxicity. On the other hand, single-agent combination therapy of TK/GCV with adenoviral lysis resulted in enhanced cytotoxicity. In this setting the expression of TK as a late gene in an oncolytic adenovirus minimized the loss of potency associated to the conditioning of viral replication. On top of that, TK expression allowed for in vivo, real time, non-invasive monitoring of viral replication in mice, and was used to analyze the effects of treatment schedule on treatment outcome.