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Cloning, characterization and expression of human LIG1
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
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2001 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 284, no 5, 1155-1161 p.Article in journal (Refereed) Published
Abstract [en]

Growth factor receptors are frequently amplified and over-expressed in various human cancers. Recently, a Drosophila cell surface protein, Kekkon-1, was found to participate in an epidermal growth factor (EGF) driven negative feedback loop. Kekkon-1 is induced by EGF, binds to the EGF-receptor, and inhibits receptor-mediated signaling. Here, we have searched for human genes with homologies to Kekkon-1 and identified human LIG1. The gene is the human homologue of mouse Lig-1 and is located on chromosome band 3p14, a region frequently deleted in various human cancers. It is predicted to encode a transmembrane cell-surface protein with extracellular leucine-rich repeats and immunoglobulin-like domains. LIG1 mRNA was detected in all tissues analyzed. The highest and lowest relative expression levels were found in brain and spleen, respectively, and differed by more than 200-fold. Taken together, our data are compatible with a role for LIG1 as a growth and tumor suppressor in human tissues.

Place, publisher, year, edition, pages
2001. Vol. 284, no 5, 1155-1161 p.
Keyword [en]
LIG-1, 3p14, EGF, cancer, tumor suppressor, FISH, real-time PCR
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:umu:diva-5120DOI: 10.1006/bbrc.2001.5092PubMedID: 11414704OAI: oai:DiVA.org:umu-5120DiVA: diva2:144511
Available from: 2006-05-03 Created: 2006-05-03 Last updated: 2017-12-14Bibliographically approved
In thesis
1. The LRIG-family: identification of novel regulators of ErbB signaling with clinical implications in astrocytoma
Open this publication in new window or tab >>The LRIG-family: identification of novel regulators of ErbB signaling with clinical implications in astrocytoma
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Astrocytic tumors are the most common malignancies of the central nervous system, accounting for more than 60% of all primary brain tumors. The prognosis for high grade astrocytoma patients is dismal and there is no curative treatment, today. A molecular hallmark of astrocytic tumors is dysregulated receptor tyrosine kinase signaling, especially of the epidermal growth factor receptor (EGFR, ErbB1). The aim of the present thesis was to identify endogenous human proteins that downregulate the function of the ErbB1 receptor. We identified a human gene family, the leucine-rich repeats and immunoglobulin-like domains family (LRIG), consisting of LRIG1, LRIG2 and LRIG3, which might fulfill this criterion.

Two candidates were identified, LRIG1 and LRIG2, which genes were localized to regions frequently deleted in human cancers, chromosome bands 3p14 and 1p13, respectively. LRIG1 and LRIG2 mRNA were expressed in all tissues analyzed, with high expression in brain and other organs. The LRIG mRNA were predicted to encode integral membrane proteins. Antibodies against LRIG1 and LRIG2 were developed and the protein expression was analyzed. LRIG1 was found to have an apparent molecular weight of 143 kDa and was expressed in most tissues with high expression in glandular tissues of the breast and prostate, and the peptic cells of the stomach. LRIG2 was slightly smaller and had an apparent molecular weight of 132 kDa. The LRIG proteins were localized to the cell surface and to perinuclear structures, where LRIG1 co-localized with the trans-Golgi network and early endosomes.

LRIG1 was found to restrict growth factor signaling by enhancing receptor ubiquitylation and degradation. We showed that LRIG1 interacted with all four members of the ErbB family and induced their downregulation. The interaction with ErbB1 involved both the LRR-domains and the Ig-like domains of LRIG1. LRIG1 enhanced receptor degradation by recruiting the E3 ubiquitin ligase c-Cbl to the LRIG1-ErbB1 complex.

LRIG1, LRIG2, and LRIG3 were expressed in glioma cell lines and tumor tissues. The LRIG expression was analyzed in 404 astrocytic tumor samples. We found that perinuclear LRIG protein expression correlated with increased survival of patients with astrocytic tumors. Especially perinuclear LRIG3 showed strong correlations with patient survival and tumor cell proliferation index.

In summary, this thesis contains the discovery and characterization of human LRIG1 and LRIG2. LRIG1 was found to interact with ErbB receptors and downregulate their function. In a clinical material, expression of LRIG proteins correlated with survival in patients with astrocytic tumors.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2006. 41 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1027
Keyword
astrocytic tumors, EGFR, ErbB, glioblastoma multiforme, LRIG, negative regulation.
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-783 (URN)91-7264-074-X (ISBN)
Public defence
2006-05-24, 244 Lionssalen, By 7, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2006-05-03 Created: 2006-05-03 Last updated: 2012-04-03Bibliographically approved
2. The Identification and Characterisation of LRIG Gene Family and Its Expression in Astrocytic Tumours
Open this publication in new window or tab >>The Identification and Characterisation of LRIG Gene Family and Its Expression in Astrocytic Tumours
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gliomas are the most common primary brain tumours, and their capacity to invade surrounding normal brain prevents complete removal of the tumour. Malignant glioma has still a poor prognosis. However, with the rapid development of molecular biology our understanding about glioma has increased dramatically. Among known growth factors, EGF and its receptor are frequently amplified and over expressed in malignant glioma. Therefore, it is of interest to find approaches to hamper the activity of EGF/EGFR. The aim of this thesis was to identify and characterize human analogues to a recently identified gene in Drosophilia, kekkon-1, which negatively regulates the activity of Drosophilia EGF receptor.

In the first part, we set up a quantitative real-time RT-PCR assay, which showed good linearity, reproducibility and uniformity. We analyzed the expression of the most commonly used reference genes, and showed that 18S was the most reliable endogenous reference gene in this study.

In the second part, we cloned, identified, and sequenced a gene family, which we named leucine-rich repeats and immunoglobulin–like domains family (LRIG). The LRIG gene family had three vertebrate paralogs and one homolog in ascidiacea. The proteins encoded by human LRIG genes shared an overall structure with a signal peptide, 15 tandems leucine-rich repeats with N- and C- terminal flanking regions followed by 3 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. Northern blot showed the mRNA sizes to be 5.5 kb for LRIG1, 4.8 kb for LRIG2, and 5.1 kb for LRIG3. LRIG1-3 mRNAs were detected in all human and mouse tissues analyzed, however, at various levels. FISH and BLAST analysis showed that LRIG1 was located at 3p14, LRIG2 at 1q13, and LRIG3 at 12q13. LRIG1 was shown to be down-regulated in several cancer cell lines and proposed to be a tumour suppressor gene.

In the third part, we analysed the expression of LRIG gene family in human astrocytic tumours. LRIG1-3 mRNAs were detected in all human glioma cell lines, in primary tumour tissues and control-matched normal brain tissues, at various levels. Subcellular localizations of LRIG1-GFP fusion proteins were visualized in nuclear, perinuclear, and cytoplasmic compartment. According to the predicted protein sequences, short peptides were synthesized and used to raise antibodies in rabbits. The antibodies were used for immunohistochemical analysis of LRIG1-3 in 404 human astrocytic tumours in a tissue micro array. The pattern of immunoreactivity of LRIG1-3 was heterogeneous with staining in nuclear, perinuclear and cytoplasmic compartment of positive tumour cells. Perinuclear staining of LRIG1-3 displayed a significant inverse correlation with WHO grade and especially positive LRIG3 perinuclear and cytoplasmic staining correlated with a low proliferation index. The LRIGs correlated with survival, and LRIG3 perinuclear staining was in addition to tumour grade an independent prognostic factor. The results suggest that LRIGs may play a role in normal tissue, and may be of importance in the pathogenesis and prognosis of tumours. The exact function of LRIG1-3 remains to be established.

Publisher
61 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 932
Keyword
astrocytoma, brain, EGFR, LRIG, leucine-rich repeat, real-time RT-PCR
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-370 (URN)91-7305-771-1 (ISBN)
Public defence
2004-12-11, Lionsalen, Onkologi, Norrlands universitetssjukhus, Umeå, 11:00 (English)
Opponent
Available from: 2004-11-18 Created: 2004-11-18 Last updated: 2010-04-19Bibliographically approved

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