Role of Tripeptidyl peptidase II in cell cycle regulation and tumor progression

Protein degradation is essential for many cellular functions and the ubiquitin-proteasome system controls the turnover of most cellular proteins and plays a pivotal role in the regulation of basic physiological processes and in the pathogenesis of many diseases. Alterations in the metabolism of proteins involved in the regulation of cell cycle and apoptosis may result in uncontrolled cell division, which em lead to malignant transformation and tumor development One melt example is the Burkitt's lymphoma (BL), a highly malignant B-cell tumor characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Proteolysis in BLs is characterized by impaired proteasome function and the upregulation of the cytosolic protease tripeptidyl peptidase II (TPPII) and dcubiquifinating enzymes. Treatment with an inhibitor of TPPII induced apoptosis in BL cells suggesting that this enzyme may regulate processes associated with survival in these cells. The general aim of this thesis was to elucidate the mechanisms behind the alterations in proteolysis in BL cells and the implications of these changes for malignant transformation. TPPII is m evolutionary conserved serine peptidase composed of 138 kDa submits that form large complexes localized in the cytoplasm and the plasma membrane of most cell types. TPPII is m exopeptidase that removes tripeptides from the free N-terminus of oligopeptides but was also suggested to possess endopeptidase activity towards intact proteins or long polypeptides. TPPII was upregulated in cells adapted to grow in the presence of toxic doses of proteasome inhibitors suggesting that it may assist the proteasome activity in the production of free amino acids from longer precursors. A more specific role in the regulation of apoptosis was suggested by the finding that increased TPPII activity correlated with upregulation of members of the inhibitors of apoptosis (IAPs) family in mouse lymphoma cells and induced rapid tumor formation in vivo. We have analyzed the effects of TPPII on cell growth and apoptosis and showed that overexpression of TPPII resulted in faster cell proliferation and higher mitotic index in 293 human embryonic kidney (HEK) cells transfected with a TPPII construct. TPPII overexpressing cells exhibited aberrant chromosome numbers and centrosome abnormalities in much higher percentages than the control cells. BL cells, that spontaneously express high levels of TPPII, displayed similar abnormalities in centrosome structure and formation of the mitotic spindle. Functional knockdown of TPPII by infection of BL cells with a TPPII specific shRNA expressing lentivirus vector resulted in growth retardation and the accumulation of polynucleated cells. These findings suggest that TPPII may regulate cellular functions that am critical to, the induction and/or maintenance of genetic instability in malignant cells. We have also explored the possible involvement of TPPII in the regulation of mitotic checkpoints and apoptosis. We found that the length of mitosis and of the entire cell cycle were significantly reduced in TPPII overexpressing HEK293 cells as compared to the controls. TPPII overexpressing B-cells evade mitotic arrest induced by spindle poisons and display high levels of polypIoidy despite the constitutively high expression of major components of the spindle checkpoint. Overexpression of TPPIl correlated with upregulation of certain IAPs and with resistance to mitochondria-dependent apoptosis induced by p53 stabilization, a pathway that is readily triggered by mitotic checkpoint activation. Our data imply that the spindle checkpoint that ensures the equal distribution of chromosomes to the daughter cells during mitosis is compromised in TPPII overexpressing B-cells. Therefore, TPPII appears to promote malignant cell growth by allowing exit from mitosis and the survival of cells with severe mitotic spindle damage. Analysis of cellular localization by immunofluorescence showed that TPPII accumulates in a ring structure around the centrosome at the onset of S phase till prometaphase. Thew ring structures co-localize with the pericentriolar Golgi apparatus and disperse in parallel with Golgi fragmentation at the onset of mitosis. Chemical inhibition of TPPII activity resulted in Golgi enlargement and cell death in pm-metaphase suggesting a role for TPPII in the Golgi fragmentation. Microarray data analysis in TPPII knockdown BL cells showed that TPPII downregulation modulated the expression of regulatory proteins involved in cell cycle and centrosome function such as Polo-like kinase 1. Thus, TPPII may exert its role in cell division by participating in processes ensuring the normal function of Golgi and centrosome apparatuses