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- Rolén, Ulrika
(författare)
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Modulation of the deubiquitinating system in viral infection, lymphoid cell activation and malignant transformation
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Most cellular proteins are affected by the conjugation of Ubiquitin (Ub), which may regulate their stability, activity or localization. The family of deubiquitinating enzymes (DUBs) removes Ub from conjugates and regulates the production and recycling of Ub. DUBs are thereby critically involved in the control of important functions such as cell growth, differentiation and apoptosis. Increasing evidence implicates deregulation of DUBs in malignant transformation. The human genome contains many putative DUB-encoding genes but little is known about their tissue distribution, pattern of expression, activity and substrate specificity. This thesis describes the use of a chemistry-based functional proteomics approach to identify active DUBs in human tumor cells of different tissue origin, in primary resting and mitogen stimulated cells, in Epstein-Barr virus (EBV) infected B lymphocytes, as well as in human papilloma virus (HPV) E6/E7 immortalized keratinocytes. The role of the ubiquitin C-terminal hydrolase (UCH)-L1 in the EBV-related B cell tumor Burkitt´s lymphoma (BL) is further investigated. Both tumor specific and tissue specific patterns of DUB activity were identified in a panel of tumor cell lines of different tissue origin. HPV carrying cervical carcinomas showed a diverse activity pattern when compared to the adjacent normal tissue, suggesting a role for some of the DUBs in the development of this tumor. The activity of specific enzymes, including USP5, -7, -9, -13, -15 and -22, was upregulated by mitogen activation or virus infection in normal T- and B-lymphocytes. UCH-L1 was highly expressed in tumor cell lines of epithelial and hematopoietic cell origin but was not detected in freshly isolated and mitogen activated cells. Upregulation of this DUB was a late event in the establishment of EBV immortalized lymphoblastoid cell lines (LCLs) and correlated with enhanced proliferation suggesting a possible role in growth transformation. UCH-L1 was shown to regulate lymphocyte function-associated antigen (LFA)-1 dependent homotypic adhesion in B-lymphocytes. Integrin-mediated cell adhesion is essential in the development and activation of B-cells, and tight LFA-1 mediated adhesion may hinder malignant cell growth and invasion. LFA-1 dependent homotypic adhesion was activated following UCH-L1 knockdown in BL cells and this correlated with slow-down of BL cell proliferation in suspension and in semisolid agar and an accumulation in the G1 phase of the cell cycle. This suggests that UCH-L1 is required for maintaining LFA-1 in a low-avidity, non-adhesive state that favors the proliferation of malignant B-cells.
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| 2. |
- Rolén, Ulrika
(författare)
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Modulation of the deubiquitnating system during lymphoid cell activation and viral infection
- 2005
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The family of ubiquitin-specific proteases (USPs) removes ubiquitin from its conjugates and regulates the production and recycling of ubiquitin. USPs are thereby critically involved in the control of important functions such as cell growth, differentiation and apoptosis. Increasing evidence implicates USPs deregulation in malignant transformation. The human genome contains many putative USP-encoding genes but little is known about USP tissue distribution, pattern of expression, activity and substrate specificity. We have used a chemistry-based functional proteomics approach to identify active USPs in human tumor cells of different tissue origin, in primary resting and mitogen stimulated cells, in Epstein-Barr virus (EBV) infected B-lymphocytes, as well as HPV E6/E7 immortalized keratinocytes. Both tumor specific and tissue specific patterns could be identified when profiling USP activity in a panel of tumor cell lines of different tissue origin. Only few USPs were active in primary cells of hematopoietic origin. The activity of specific USPs, including USP5, -7, -9, -13, -15 and -22, was upregulated by mitogen activation or virus infection in normal T- and B-lymphocytes. UCH-L1 was highly expressed in several tumor cell lines of epithelial and hematopoietic cell origin but was not detected in freshly isolated and mitogen activated cells. Upregulation of this LISP was a late event in the establishment of EBV immortalized lymphoblastoid cell lines and correlated with enhanced proliferation suggesting a possible role in growth transformation. To further investigate the role of USPs in tumor development, the activity profiles of human papillomavirus (HPV) carrying cervical carcinomas and the adjacent normal tissue were compared. The activity of UCH-L3 and UCH37 was upregulated in the majority of tumor tissues as compared to the adjacent normal tissue. UCH-L1 activity appeared to be lower in a significant proportion of the tumors, to a less extent in more advanced stages of the tumor. In order to assess the contribution of HPV proteins, LISP activity was also monitored in a panel of HPV positive and negative cervical carcinoma cell lines and in HPV E6/E7 immortalized human keratinocytes. In accordance with the relatively low UCH-L1 activity in tumor biopsies, UCH-L1 was detected only in one out of eight cervical carcinoma lines while seven out of eight cell lines expressed significant levels of USP7 that was only occasionally detected in tumor biopsies. UCHL1, UCH-L3, USP7 and USP9X were upregulated in HPV E6/E7 immortalized keratinocytes suggesting that changes in the pattern of activity of these enzymes may play a role in growth transformation.
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| 3. |
- Rolén, Ulrika, et al.
(författare)
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The ubiquitin C-terminal hydrolase UCH-L1 regulates B-cell proliferation and integrin activation
- 2009
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Ingår i: Journal of Cellular and Molecular Medicine. - : John Wiley & Sons. - 1582-1838 .- 1582-4934. ; 13:8b, s. 1666-1678
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Tidskriftsartikel (refereegranskat)abstract
- The ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme that catalyses the hydrolysis of polyubiquitin precursors and small ubiquitin adducts. UCH-L1 has been detected in a variety of malignant and metastatic tumours but its biological function in these cells is unknown. We have previously shown that UCH-L1 is highly expressed in Burkitt's lymphoma (BL) and is up-regulated upon infection of B lymphocytes with Epstein-Barr virus (EBV). Here we show that knockdown of UCH-L1 by RNAi inhibits the proliferation of BL cells in suspension and semisolid agar and activates strong LFA-1-dependent homotypic adhesion. Induction of cell adhesion correlated with cation-induced binding to ICAM-1, clustering of LFA-1 into lipid rafts and constitutive activation of the Rap1 and Rac1 GTPases. Expression of a catalytically active UCH-L1 promoted the proliferation of a UCH-L1-negative EBV transformed lymphoblastoid cell line (LCL) and inhibited cell adhesion, whereas a catalytic mutant had no effect, confirming the requirement of UCH-L1 enzymatic activity for the regulation of these phenotypes. Our results identify UCH-L1 as a new player in the signalling pathways that promote the proliferation and invasive capacity of malignant B cells.
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