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The Identification and Characterisation of LRIG Gene Family and Its Expression in Astrocytic Tumours
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Neurosurgery.
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.

Place, publisher, year, edition, pages
2004. , 61 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 932
Keyword [en]
astrocytoma, brain, EGFR, LRIG, leucine-rich repeat, real-time RT-PCR
Research subject
Oncology
Identifiers
URN: urn:nbn:se:umu:diva-370ISBN: 91-7305-771-1 (print)OAI: oai:DiVA.org:umu-370DiVA: diva2:143282
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
List of papers
1. The iCycler iQ detection system for evaluating reference gene expression in normal human tissue
Open this publication in new window or tab >>The iCycler iQ detection system for evaluating reference gene expression in normal human tissue
2002 In: Bio-Rad tech note, Vol. 2804Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-4260 (URN)
Available from: 2004-11-18 Created: 2004-11-18Bibliographically approved
2. Cloning, characterization and expression of human LIG1
Open this publication in new window or tab >>Cloning, characterization and expression of human LIG1
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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.

Keyword
LIG-1, 3p14, EGF, cancer, tumor suppressor, FISH, real-time PCR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-5120 (URN)10.1006/bbrc.2001.5092 (DOI)11414704 (PubMedID)
Available from: 2006-05-03 Created: 2006-05-03 Last updated: 2017-12-14Bibliographically approved
3. Is LRIG1 a tumour suppressor gene at chromosome 3p14.3?
Open this publication in new window or tab >>Is LRIG1 a tumour suppressor gene at chromosome 3p14.3?
2002 (English)In: Acta Oncologica, ISSN 0284-186X, E-ISSN 1651-226X, Vol. 41, no 4, 352-354 p.Article in journal (Refereed) Published
Abstract [en]

The LRIG1 gene (formerly LIG-1), recently cloned by us, displays structural similarities to the Drosophila Kek I gene. Kek I encodes a cell surface protein, Kekkon-1, which inhibits epidermal growth factor receptor-mediated signalling. We localized the LRIG1 gene to chromosome band 3p14.3, a region known to be deleted in various human cancers. In the present study LRIG1 gene expression was examined in different tumour cell lines and corresponding normal tissues by real-time RT-PCR. In many tumour cell lines, LRIG1 expression appeared absent or was down regulated compared to corresponding normal tissues. The results are consistent with LRIG1 being a tumour suppressor gene in humans. However, further studies are justified to elucidate the explicit role of LRIG1 as a negative regulator of oncogenesis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4262 (URN)10.1080/028418602760169398 (DOI)12234026 (PubMedID)
Available from: 2004-11-18 Created: 2004-11-18 Last updated: 2017-12-14Bibliographically approved
4. Characterization and tissue-specific expression of human LRIG2
Open this publication in new window or tab >>Characterization and tissue-specific expression of human LRIG2
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2004 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 332, 35-43 p.Article in journal (Refereed) Published
Abstract [en]

We have recently identified and cloned the human LRIG1 gene (formerly LIG1). LRIG1 is a predicted integral membrane protein with a domain organization reminiscent of the Drosophila epidermal growth factor (EGF)-receptor antagonist Kekkon-1. We have searched for additional members of the human LRIG family and identified LRIG2 (KIAA0806). The LRIG2 gene was localized to chromosome 1p13 and had an open reading frame of 1065 amino acids. The LRIG2 protein was predicted to have the same domain organization as LRIG1 with a signal peptide, an extracellular part containing15 leucine-rich repeats and three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. The LRIG2 amino acid sequence was 47% identical to human LRIG1 and mouse Lrig1 (also known as Lig-1). Northern blotting and RT-PCR revealed LRIG2 transcripts in all tissues analyzed. Quantitative real-time RT-PCR showed the most prominent RNA expression in skin, uterus, ovary, kidney, brain, small intestine, adrenal gland, and stomach. Immunoblotting of COS-7 cell lysates demonstrated that heterologously expressed human LRIG2 had an apparent molecular weight of 132 kDa under reducing gel-running conditions. N-glycosidase F treatment resulted in a reduction of the apparent molecular weight to 107 kDa, showing that LRIG2 was a glycoprotein carrying N-linked oligosaccharides. Cell surface biotinylation experiments and confocal fluorescence laser microscopy demonstrated expression of LRIG2 both at the cell surface and in the cytoplasm. LRIG2 was detected in tissue lysates from stomach, prostate, lung, and fetal brain by immunoblotting. In conclusion, LRIG2 was found to be a glycoprotein which was encoded by a gene on human chromosome 1p13 and its mRNA was present in all tissues analyzed.

Keyword
Leucine-rich repeats and immunoglobulin-like domains, LRIG1, Lig-1
National Category
Medical and Health Sciences
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-15347 (URN)10.1016/j.gene.2004.02.002 (DOI)15145052 (PubMedID)
Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2017-12-14Bibliographically approved
5. The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues and a homolog in ascidiacea
Open this publication in new window or tab >>The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues and a homolog in ascidiacea
2004 (English)In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 84, no 1, 157-165 p.Article in journal (Refereed) Published
Abstract [en]

Human LRIG1 (formerly LIG1), human LRIG2, and mouse Lrig1 (also known as Lig-1) encode integral membrane proteins. The human genes are located at chromosomes 3p14 and 1p13, which are regions frequently deleted in human cancers. We have searched for additional members of the LRIG family and by molecular cloning identified human LRIG3 and its mouse ortholog Lrig3. Human LRIG3 is located at chromosome 12q13. In silico analysis of public databases revealed a mouse Lrig2 mRNA, three LRIG homologs in the puffer fish Fugu rubripes, and one LRIG homolog in the ascidian tunicate Ciona intestinalis. The human and mouse LRIG polypeptides have the same predicted domain organization: a signal peptide, 15 tandem leucine-rich repeats with cysteine-rich N- and C-flanking domains, three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. The extracellular part—especially the IgC2.2 domain, the transmembrane domain, and the membrane-proximal part of the cytoplasmic tail—are the most conserved regions. Northern blot analysis and real-time RT-PCR revealed that the three LRIG paralogs are widely expressed in human and mouse tissues. In conclusion, the LRIG gene family was found to have three widely expressed mammalian paralogs, corresponding orthologs in fish, and a homolog in Ascidiacea.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4264 (URN)10.1016/j.ygeno.2004.01.013 (DOI)15203213 (PubMedID)
Available from: 2004-11-18 Created: 2004-11-18 Last updated: 2017-12-14Bibliographically approved
6. Perinuclear leucine-rich repeats and immunoglobulin-like domain proteins (LRIG1-3) as prognostic indicators in astrocytic tumors
Open this publication in new window or tab >>Perinuclear leucine-rich repeats and immunoglobulin-like domain proteins (LRIG1-3) as prognostic indicators in astrocytic tumors
Show others...
2006 (English)In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 111, no 3, 238-346 p.Article in journal (Refereed) Published
Abstract [en]

We have previously characterized three human leucine-rich repeats and immunoglobulin-like domains (LRIG) genes and proteins, named LRIG1-3 and proposed that they may act as suppressors of tumor growth. The LRIG1 transmembrane protein antagonizes the activity of epidermal growth factor receptor family receptor tyrosine kinases. In this study, we evaluated the mRNA expression level of LRIG1-3 in human glioma cell lines and control-matched glioma tissues, characterized the sub-cellular localization of an LRIG3–GFP fusion protein, and analyzed the relationship between sub-cellular localization of LRIG1-3 and clinical parameters in 404 astrocytic tumors by immunohistochemistry. LRIG1-3 mRNA was detected in all human glioma cell lines and matched glioma samples, with large differences in the expression levels. Ectopically expressed LRIG3–GFP localized to perinuclear and cytoplasmic compartments, and to the cell surface of transfected glioma cells. Perinuclear staining of LRIG1-3 was associated with low WHO grade and better survival of the patients. Perinuclear staining of LRIG3 was associated with a lower proliferation index and was in addition to tumor grade, an independent prognostic factor. Furthermore, within the groups of grade III and grade IV tumors, perinuclear staining of LRIG3 significantly correlated with better survival. These results indicate that expression and sub-cellular localization of LRIG1-3 might be of importance in the pathogenesis and prognosis of astrocytic tumors.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-13620 (URN)10.1007/s00401-006-0032-5 (DOI)16532360 (PubMedID)
Available from: 2007-12-06 Created: 2007-12-06 Last updated: 2017-12-14Bibliographically approved

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