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Original Article
Cancer Gene Therapy (2010) 17, 814–825; doi:10.1038/cgt.2010.39; published online 13 August 2010
Ribozyme-mediated compensatory induction of menin-oncosuppressor function in primary fibroblasts from MEN1 patients
E Luzi1, F Marini1, I Tognarini1, S Carbonell Sala1, G Galli1, A Falchetti1 and M L Brandi1
1Department of Internal Medicine, Metabolic BoneUnit, Regional Center for Hereditary Endocrine Tumors, Azienda Ospedaliera Universitaria Careggi, Florence, Italy
Correspondence: Dr ML Brandi, Department of Internal Medicine, University of Florence, Viale Pieraccini 6, Florence 50139, Italy. E-mail: m.brandi@dmi.unifi.it
Received 22 October 2009; Revised 9 January 2010; Accepted 7 April 2010; Published online 13 August 2010.
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AbstractMultiple endocrine neoplasia type 1 (MEN1) syndrome is characterized by the occurrence of tumors of parathyroids, neuroendocrine cells of the gastro-enteropancreatic tract and anterior pituitary. MEN1 gene encodes menin-oncosuppressor protein. Loss of heterozygosity at 11q13 is typical of MEN1 tumors. We have analyzed the MEN1 mRNA and menin expression in fibroblasts from normal skin biopsies andfrom MEN1 patients (two with a frameshift 738del4 (exon 3) mutation, introducing a premature stop codon, and an individual with an R460X (exon 10) nonsense mutation). The expression of full-length menin protein did not differ between MEN1 and normal fibroblasts. Wild-type alleles mRNAs were expressed in MEN1 patients, whereas mutant alleles were partially degraded by nonsense-mediated mRNA decaypathway, suggesting a mechanism of compensation for allelic loss by the up-regulation of wild-type menin expression at a post-transcriptional level. Small-interfering RNA silencing of the wild-type mRNA allele abolished menin compensation, whereas the ribozyme silencing of the MEN1-mutated mRNA allele resulted in strongly enhanced wild-type menin expression. Gel-retardation analysis showed that invitro-specific RNA–protein complexes bound to MEN1 mRNA. These findings contribute to the understanding of tumorigenesis in MEN1, offering the basis for the development of RNA-based therapies in MEN1 gene mutation carriers.
ribozymes; siRNAs; multiple endocrine neoplasia type 1 (MEN1) syndrome; loss of heterozygosity; post-transcriptional regulation
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Thehallmark of multiple endocrine neoplasia type 1 (MEN1) is the development of tumors in the parathyroids, in the enteropancreatic endocrine tissue and in the anterior pituitary. Other associations found in MEN1 patients include foregut carcinoids, facial angiofibromas, lipomas, collagenomas, meningiomas and smooth muscle cell tumors.1, 2, 3, 4, 5 MEN1 patients typically inherit germline loss offunction mutations in the MEN1 gene, and tumors arise when the somatic loss of the retained wild-type allele occurs. Thus, the MEN1 gene follows the classic ‘two-hit’ tumor-suppressor model, first proposed by Knudson6 for retinoblastoma. The ubiquitously expressed product of MEN1, menin, is a 67-kDa protein found predominantly in the nucleus.7 Studies in Men1 knockout mice support a tumor-suppressorfunction for menin.8, 9, 10, 11, 12, 13, 14
The development of tumors in an endocrine-specific pattern is puzzling as menin appears to be expressed in all tissues. Menin has been reported to interact with a multitude of proteins including JunD, SMAD family members, Pem, NFκB, FANCD2, RPA2, NMMHC II-A, GFAP, vimentin and Hsp70.15 The diverse functions of the menin partners suggest functions for meninin transcriptional regulation, DNA processing, repair and recombination, cytoskeletal organization and protein degradation, although to date none of the interacting partners have been directly proved important in MEN1 pathophysiology.15, 16, 17, 18, 19, 20, 21
Conversely, little is known about how MEN1 gene itself is regulated. Studies on cell cycle-dependent expression of menin have led to...
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