Gene profiling studies of patients with chronic lymphocytic leukemia (CLL) has revealed increased expression of Ror1, a cell surface receptor tyrosine kinase. The aim of present study was to analyze gene and protein expression of Ror1 in CLL cells and normal blood leukocytes. Gene expression analysis reverse transcriptionpolymerase chain reaction of ROR1 revealed that all patients with CLL (n 5 100) spontaneously expressed ROR1 mRNA whereas enriched blood B and T cells as well as granulocytes from healthy donors (n 5 10) were negative. A strong nonphysiological activation signal (PMA/ionomycin) was required to induce expression in vitro in normal lymphocytes. Major genomic aberrations (mutations or truncation) of ROR1 were not observed. Protein expression was analyzed by Western blot using a panel of polyclonal anti-Ror antibodies as well as flow cytometry. Blood lymphocytes from 18/18 CLL patients, but none of the 10 healthy donors, expressed surface Ror1. The majority of CLL cells exhibited Ror1 surface expression (71% mean; range 36-92%) with a mean fluorescence intensity (MFI) of 20 (range 10-45). The corresponding MFI of CD19 on CLL cells was 26 (range 9-48). There was no difference in the Ror1 protein expression comparing IgVH mutated and unmutated cases as well as progressive and nonprogressive CLL patients. Two different variants of the Ror1 protein, 105 and 130 kDa, were identified. The Ror1 protein expression in patients with CLL but not in normal leukocytes merits further studies of its role in the pathobiology of CLL, which may provide a basis for development of Ror1 directed targeted therapy. ' 2008 Wiley-Liss, Inc. Key words: B-CLL; tyrosine kinase receptors; Ror1Chronic lymphocytic leukemia (CLL) originates from B lymphocytes, which differ in activation and maturation stage and are derived from antigen experienced B cells with different immunoglobulin heavy chain variable (IgVH) gene mutations. 1 Patients with mutated IgVH genes have a better prognosis compared to patients with unmutated genes. 2,3 Global gene expression profiling studies have revealed partly distinguishing but in general overlapping expression profiles in mutated and unmutated leukemic B cells, suggesting a common phenotype. 4,5 Gene expression profiling studies showed a 43.8-fold increase of the orphan receptor tyrosine kinase (RTK) ROR1 in CLL cells. 4 Ror1 is a member of the RTK family of orphan receptors related to muscle specific kinase and Trk neurotrophin receptors. [6][7][8] Ror receptors are cell surface receptors participating in signal transduction, cell-cell interaction, regulation of cell proliferation, differentiation, cell metabolism and survival. 7,9 They are evolutionarily highly conserved between different species e.g., human, mouse, Drosophila and C. elegans, suggesting important biological functions.The human ROR1 gene has a coding region of 2814 bp with a predicted 937 amino acids sequence and 105 kDa protein size including an Ig-like domain, cysteine-rich domain, kringle domain, tyrosine kinase domain and p...
SummaryWe have previously demonstrated that ROR1 and FMOD (fibromodulin) are two genes upregulated in chronic lymphocytic leukaemia (CLL) cells compared to normal blood B cells. In this study, siRNAs were used to specifically silence ROR1 and FMOD expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75-95%) in FMOD and ROR1 mRNA. Western blot analysis with specific antibodies for FMOD and ROR1 demonstrated that the proteins were significantly downregulated 48 h after siRNA treatment. Silencing of FMOD and ROR1 resulted in statistically significant (P £ 0AE05-0AE001) apoptosis of CLL cells but not of B cells from normal donors. Human fibroblast cell lines treated with FMOD and ROR1 siRNA did not undergo apoptosis. This is the first report demonstrating that ROR1 and FMOD may be involved in the survival of CLL cells. ROR1 in particular is further explored as potential target for therapy in CLL.
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