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G J Cote

Publications and source records attributed to G J Cote.

At least 19 recordsLinked to original sources
PubMed2001

Promiscuity of pre-mRNA spliceosome-mediated trans splicing: a problem for gene therapy?

Trans splicing of messenger RNA has been used in experimental settings to replace mutant RNA sequences. We investigated the feasibility of utilizing trans splicing to replace a mutant RET protooncogene sequence known to inappropriately activate this tyrosine kinase receptor. We constructed a pre-trans-splicing molecule (PTM) consisting of a binding domain complementary to the target intron, the 3' splicing signal sequence (3'ss), derived from adenovirus major late transcript intron 1 and a molecular tag sequence. Accurately targeted trans splicing between the human RET exons and the PTM was demonstrated in NIH 3T3 cells cotransfected with the human RET minigene and the PTM. The efficiency of specific trans splicing was estimated to be no more than 15% in the cotransfection experiment. However, in addition to the targeted trans splicing, nontargeted trans splicing to RET exons was observed. Furthermore, the rapid amplification of 5' cDNA ends (5' RACE) analysis demonstrated that nontargeted trans splicing occurred with endogenously expressed pre-mRNAs in TT cells and that specific trans splicing to RET was a rare event. Attempts to reduce nonspecificity by the addition of a stem-loop to the trans-splicing construct designed to suppress nonspecific splicing failed to have the desired effect. These observations suggest that overexpression of a trans-splicing construct containing a 3'ss results in promiscuous trans splicing and raise significant questions about the specificity and usefulness of currently used trans-splicing approaches. In addition, these findings raise the possibility that nonspecific spliced products may be produced by a variety of gene therapy constructs.

3T3 Cells↗
PubMed2001

Arthritic calcitonin/alpha calcitonin gene-related peptide knockout mice have reduced nociceptive hypersensitivity.

Peripheral inflammation induced with a knee joint injection of a mixture of kaolin/carrageenan (k/c) produces primary and secondary hyperalgesia. Inflammatory pain is thought to involve a variety of transmitters released from nerve terminals, including amino acids, substance P (SP) and calcitonin gene-related peptide (CGRP). In the present study, mice deficient in the calcitonin/alpha CGRP gene (CGRP(-/-)) displayed normal responses to noxious stimuli. However, the CGRP knockout mice failed to demonstrate development of secondary hyperalgesia after induction of knee joint inflammation in two tests that assess central sensitization, through testing at sites remote from the primary insult. Nociceptive behavioral responses were assessed using the hot-plate test and paw withdrawal latency (PWL) to radiant heat applied to the hindpaw. The CGRP(-/-) mice showed no signs of secondary hyperalgesia after development of knee joint inflammation, while the expected significant decrease in the PWL was observed in the CGRP(+/+) mice as control. The CGRP(-/-) mice also had a prolonged rather than a shortened response latency in the hot-plate test 4 h after knee joint injection of k/c. Immunohistological study showed that CGRP-like immunoreactivity (CGRP-LI) was absent in the spinal cord and dorsal root ganglia taken from the CGRP(-/-) mice. These results indicate that endogenous CGRP plays an important role in the plastic neurogenic changes occurring in response to peripheral inflammatory events including the development of nociceptive behaviors.

Animals↗
PubMed2001

Structure of the mouse calcitonin/calcitonin gene-related peptide alpha and beta genes.

We report the cloning, genomic organization and sequence of the mouse alpha-CALC and beta-CALC genes. The two genes share extensive sequence homology. The transcription units of both genes contain 6 exons. Transcripts of the alpha-CALC gene were found to alternatively include exon 4 or exons 5 and 6. For the beta-CALC gene exon 4 was not detected in transcripts derived from this gene. The predicted mouse alpha-CGRP was found to be identical to rat alpha-CGRP, however, beta-CGRP predicted amino acid sequences revealed three amino acid differences suggesting these residues are not critical to CGRP function.

Animals↗
PubMed2001

[Germline mutations of the ret proto-oncogene in Chilean patients with hereditary and sporadic medullary thyroid carcinoma].

BACKGROUND: Medullary thyroid carcinoma (MTC) may occur either as a sporadic or familial disease. Multiple endocrine neoplasia (MEN) type 2, inherited as an autosomal dominant disease, is characterized by MTC only (FMTC) or coexistence of MTC with other endocrine neoplasia (NEM 2A, 2B). Germline mutations of the RET proto-oncogene (cRet) are found in the inherited forms and in some apparently sporadic MTC cases. AIM: To study RET mutations in 8 families with MEN 2. MATERIAL AND METHODS: RET mutations were screened in peripheral blood DNA from 18 patients and 87 high risk carriers belonging to 8 MEN 2 families and 52 sporadic MTC. Exons 10, 11, 13, 14, 15 and 16 of the c-Ret were amplified by polymerase chain reaction (PCR) and examined by direct sequencing of PCR products and/or restriction enzyme analysis. RESULTS: Five MEN 2A and one FMTC families with a germline mutation at codon 634, one MEN 2A and one FMTC family carrying a mutation at codon 620 were identified. Mutations were found in 23 out of 87 high risk carriers. In addition, we detected a S891A (exon 15) germline mutation in a sporadic MTC patient and in one out of her three sons and V804M (exon 14) in another sporadic MTC case and in one out of his six relatives, indicating in both cases the presence of a sporadic misclassified familial disease. CONCLUSIONS: These results underscore the importance of routine application of c-Ret testing in all cases of MTC either familial or sporadic.

Adolescent↗
PubMed2000

Fibroblast growth factor receptor-1 alpha-exon exclusion and polypyrimidine tract-binding protein in glioblastoma multiforme tumors.

Neoplastic transformation of glial cells alters inclusion of the alpha exon in human fibroblast growth factor receptor-1 (FGFR-1) mRNA transcripts. Although normal cells predominantly include the alpha exon, this exon is excluded in most glioblastoma cell transcripts, creating a high-affinity receptor form. In this study, we identified polypyrimidine tract-binding protein (PTB) as a regulator of FGFR-1 splicing. PTB interacted in a sequence-specific manner with the ISS-1 regulatory element in the intron upstream of the a exon. PTB expression was also strongly increased in seven malignant glioblastoma multiforme tumors relative to adjacent normal tissue, but not in a low-grade astrocytoma. These results suggest that increased expression of PTB may contribute to glial cell malignancy.

Cell Transformation, Neoplastic↗
PubMed2000

Multiple endocrine neoplasias.

The multiple endocrine neoplasia syndromes form a distinct group of genetic tumor syndromes. They include multiple endocrine neoplasia types 1 and 2, von Hippel Lindau syndrome, neurofibromatosis, and Carney complex. Research over the past decade has identified a molecular basis for each of these syndromes. This knowledge has revolutionized not only the clinical management but also has illuminated the field of human cancer research by the identification of new and important genes critical for regulation of cell growth, differentiation, and death. This review focuses on the structure, physiologic function, and molecular abnormalities of the genes involved in these syndromes.

Animals↗
PubMed1999

Redundant intronic repressors function to inhibit fibroblast growth factor receptor-1 alpha-exon recognition in glioblastoma cells.

The human fibroblast growth factor receptor-1 primary transcript is alternatively processed to produce receptor forms that vary in their affinity for fibroblast growth factor. The inclusion of a single exon (alpha) in normal brain glial cells produces a low affinity form of the receptor. Recognition of the alpha-exon is dysregulated during neoplastic transformation of glial cells to produce a high affinity receptor form. In this study, we have identified a second intronic repressor of RNA splicing located approximately 250 nucleotides upstream of the alpha-exon. Deletion or mutation of this sequence resulted in a significant increase in exon recognition in glioblastoma cells. This intronic repressor was found to share significant sequence homology with an intronic repressor element located downstream of the alpha-exon. The two repressor elements are functionally redundant in that they are capable of inhibiting alpha-exon recognition when positioned upstream or downstream of the exon. Finally, the elements were found to mediate enhanced exclusion of an unrelated exon, but only the repressors were placed flanking the exon. However, under these conditions, the cell-specific exon exclusion was no longer maintained. These results suggest that although the alpha-exon inclusion is actively repressed in glioblastomas, the absence of trans-activators appears to be key to the production of the high affinity form of fibroblast growth factor receptor-1 in glioblastomas.

Alternative Splicing↗
PubMed1999

Hammerhead ribozyme-mediated inactivation of mutant RET in medullary thyroid carcinoma.

Activating mutations of the RET proto-oncogene cause hereditary medullary thyroid carcinoma. To examine whether selective inactivation of mutant RET could prevent transformation, a hammerhead ribozyme was designed to cleave RET mRNA containing a transforming mutation of codon 634 TGC --> TAC (Cys634Tyr). In vitro RNA cleavage assay demonstrated that the ribozyme selectively cleaved RET RNA with a Cys634Tyr but not Cys634Arg or the normal sequence. Expression of ribozyme in NIH/3T3 cells prevented RET-mediated colony formation in soft agar. This inhibition required catalytically active ribozyme and was specific for the TAC mutation. Therefore, ribozymes designed to selectively target mutant RET RNA may provide an effective therapeutic in the treatment of this syndrome.

3T3 Cells↗
PubMed1999

Glioblastoma cell-specific expression of fibroblast growth factor receptor-1beta requires an intronic repressor of RNA splicing.

The fibroblast growth factor receptor-1 (FGFR-1) primary transcript is alternatively processed to produce receptors that vary in their ligand affinity and specificity. A high affinity form of this receptor--FGFR-1beta--that lacks the alpha exon is observed on the neoplastic transformation of glial cells. In this study, we have identified a 62-bp sequence located 97 bp downstream from the alpha exon that is required for the exclusion of this exon in a human glioblastoma cell line. Deletion or mutation of this sequence is sufficient to allow enhanced inclusion of the alpha exon or a heterologous exon in glioblastoma cells. Therefore, it would appear that this sequence element plays a key role in the glioblastoma-specific splicing to form FGFR-1beta mRNA.

Base Sequence↗
PubMed1999

A codon 891 exon 15 RET proto-oncogene mutation in familial medullary thyroid carcinoma: a detection strategy.

A ser891ala RET proto-oncogene mutation has been previously discovered in a single kindred with familial medullary thyroid carcinoma (FMTC). An additional two probands with this mutation and with medullary thyroid carcinoma (MTC) without any other manifestations of MEN 2 have been identified. In one of thse families, two other individuals also had the mutant sequence and FMTC. Analysis of both cases showed cosegregation of the mutation and MTC. To facilitate detection of this mutation, a primer was engineered which creates a Hha I recognition site in the presence of the mutant sequence. As a result, this codon 891 exon 15 mutation can be identified with a restriction enzyme digestion.

Amino Acid Substitution↗
PubMed1999

Calcium-induced activation of a mutant G-protein-coupled receptor causes in vitro transformation of NIH/3T3 cells.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is widely expressed, has tissue-specific functions, and regulates cell growth. Activating mutations of this receptor cause autosomal dominant hypocalcemia, a syndrome characterized by hypocalcemia and hypercalciuria. The identification of a family with an activating mutation of the CaR (Thr151Met) in which hypocalcemia cosegregates with several unusual neoplasms led us to examine the transforming effects of this mutant receptor. Transfection of NIH/3T3 cells with the mutant but not the normal receptor supported colony formation in soft agar at subphysiologic calcium concentrations. The mutant CaR causes a calcium-dependent activation of the extracellular signal-regulated protein kinase (ERK) 1/2 and Jun-N-terminal kinase/stress-activated (JNK/ SAPK) pathways, but not P38 MAP kinase. These findings contribute to a growing body of information suggesting that this receptor plays a role in the regulation of cellular proliferation, and that aberrant activation of the mutant receptor in this family may play a role in the unusual neoplastic manifestations.

3T3 Cells↗
PubMed1999

Screening for MEN1 mutations in patients with atypical endocrine neoplasia.

BACKGROUND: Most patients from typical multiple endocrine neoplasia type 1 (MEN1) kindreds harbor mutations in the MEN-1 gene, MEN1. We hypothesized that some patients with atypical endocrine neoplasia would also have mutations in MEN1. METHODS: DNA sequencing analysis of mutations in the coding region of MEN1 was performed with genomic DNA obtained from peripheral blood lymphocytes in a total of 21 patients who had: typical MEN1 (n = 8), clinical features suggestive of MEN1 but without a family history of endocrinopathy (n = 7), and atypical endocrine neoplasia and a family history of endocrinopathy suggestive of MEN1 (n = 6). RESULTS: All 8 patients with typical MEN1 had mutations in MEN1. None of the 7 patients with features of MEN1, but without a family history of endocrinopathy, had a MEN1 mutation. In contrast, 4 of 6 patients with atypical endocrine neoplasia that included components of MEN1 and a family history of endocrinopathy had mutations in MEN1, including 2 patients with pheochromocytoma. CONCLUSIONS: Genomic mutations in MEN1 may frequently be identified in patients with atypical endocrine neoplasia, especially in the setting of a family history of endocrinopathy. Atypical presentations of MEN1 may include pheochromocytoma.

Adrenal Gland Neoplasms↗
PubMed1999

Surgical strategy for the treatment of medullary thyroid carcinoma.

OBJECTIVE: To evaluate surgical complications, patterns of lymph node metastases, and calcitonin response to compartment-oriented lymphadenectomy in patients with primary or recurrent medullary thyroid carcinoma (MTC). SUMMARY BACKGROUND DATA: The majority of patients with invasive MTC have metastasis to regional lymph nodes at the time of diagnosis, as evidenced by the frequent finding of persistently elevated calcitonin levels after thyroidectomy and the high rates of recurrence in the cervical lymph nodes reported in retrospective studies. These data have provided the rationale for surgeons to perform a more extensive lymphadenectomy at the time of initial thyroidectomy and to consider reoperative cervical lymphadenectomy in patients with persistently elevated calcitonin levels after thyroidectomy. METHODS: Forty patients underwent surgery for MTC from 1991 to 1997 (23 sporadic cases, 17 familial cases). Patients were divided into three groups based on whether they had undergone previous thyroidectomy and on the results of standardized staging studies performed after referral to the authors' institution. Group 1 (11 patients) had received no previous surgery; group 2 (13) underwent thyroidectomy before referral and had an elevated calcitonin level without radiologic evidence of local regional or distant metastases; and group 3 (16) underwent thyroidectomy before referral and had an elevated calcitonin level with radiologic evidence of local-regional recurrence. The central neck compartment was dissected in all patients; preoperative staging and the extent of previous surgery dictated the need for lateral (modified radical) neck dissection. After primary or reoperative surgery, calcitonin levels were assessed. RESULTS: All patients had major reductions in postoperative calcitonin levels. Seven (29%) of 24 patients in groups 1 and 2 achieved normal calcitonin values compared with only 1 (6%) of 16 in group 3. Postoperative complications included seven cases (17%) of permanent hypoparathyroidism; five (71%) of these occurred in group 3. There were no iatrogenic recurrent laryngeal nerve injuries; one patient required recurrent nerve resection to achieve complete tumor extirpation. At a median follow up of 35 months, local recurrence was documented in 5 (13%) of 40 patients. CONCLUSIONS: Compartment-oriented lymphadenectomy performed early in the course of MTC is safe and may return calcitonin levels to normal in up to 25% of carefully selected patients. However, reoperation for bulky cervical disease (group 3) rarely results in normal calcitonin levels and is associated with a high incidence of permanent hypoparathyroidism.

Adolescent↗
PubMed1999

A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland.

Mutations in genes encoding the ATP-regulated potassium (K(ATP)) channels of the pancreatic beta-cell (SUR1 and Kir6.2) are the major known cause of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We collected all cases of PHHI diagnosed in Finland between 1983 and 1997 (n = 24). The overall incidence was 1:40,400, but in one area of Central Finland it was as high as 1:3,200. Haplotype analysis using polymorphic markers spanning the SUR1/Kir6.2 gene cluster confirmed linkage to the 11p region. Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No K(ATP) channel activity was observed in beta-cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Thus, the mutation produces a nonfunctional channel and, thereby, continuous insulin secretion. This unique SUR1 mutation explains the majority of PHHI cases in Finland and is strongly associated with a severe form of the disease. These findings provide diagnostic and prognostic utility for suspected PHHI patients.

ATP-Binding Cassette Transporters↗
PubMed1998

Exon sequence is required for regulated RNA splicing of the human fibroblast growth factor receptor-1 alpha-exon.

Alternative RNA processing of the human fibroblast growth factor receptor-1 transcript results in receptor forms that vary in their affinity for fibroblast growth factor. An alternative RNA processing event involving recognition of the alpha-exon is deregulated during neoplastic transformation of glial cells. We have previously established a splicing reporter/transfection cell culture model system to identify sequences involved in recognition of this exon. In this study, the system was used to identify two sequence elements that differentially function to regulate splicing of this exon. Exclusion of the alpha-exon in glioblastoma cells specifically required the downstream intron sequence comprising the 5'-splice site. Replacement or mutation of this sequence increasing complementarity to U1 RNA resulted in enhanced exon recognition in SNB-19 glioblastoma cells. Sequences within the exon were found to be required for alpha-exon inclusion. Deletion and gain-of-function experiments identified a 69-nucleotide exon sequence that was specifically required for alpha-exon inclusion. These studies indicate that multiple sequences are required for the regulated recognition of the alpha-exon.

Alternative Splicing↗
PubMed1998

A human succinate-ubiquinone oxidoreductase CII-3 subunit gene ending in a polymorphic dinucleotide repeat is located within the sulfonylurea receptor (SUR) gene.

We report the cloning of two variant genes encoding the CII-3 subunit of succinate-ubiquinone oxidoreductase complex II. One gene is located within intron 10 of the human sulfonylurea receptor gene. The 3' boundary of this gene ends in a polymorphic dinucleotide repeat. The second gene CII-3b is expressed at a low level and contains a 102-bp internal deletion compared to CII-3 cDNA. These genes should prove valuable in the characterization of Complex II disorders.

ATP-Binding Cassette Transporters↗
PubMed1998

Mutational analysis of the GDNF/RET-GDNFR alpha signaling complex in a kindred with vesicoureteral reflux.

Glial cell line-derived neurotrophic factor (GDNF) mediates signaling across the cell membrane by interaction with the RET-GDNFR alpha receptor complex. We identified a family in which one member had medullary thyroid carcinoma (MTC) and four members had vesicoureteral reflux (VUR). Knowledge that mutations in the RET proto-oncogene cause MTC and studies documenting genitourinary abnormalities in RET or GDNF knockout mice led us to examine the GDNF/RET-GDNFR alpha signaling complex in this family. RET and GDNF were excluded as the causative VUR gene by haplotype and sequence analysis. The GDNFR alpha gene was mapped to chromosome 10q25-26 by radiation hybrid techniques and was eliminated as the causative gene by haplotype analysis and sequencing of cDNA from an obligate carrier. Sequencing identified a 15-nucleotide deletion in GDNFR alpha mRNA, which was found to code for a single exon; analysis of several cell types revealed an identical mRNA form, indicating that this variant is a product of alternative RNA processing. We conclude that GDNFR alpha maps to 10q25-26 and that its RNA transcript is alternatively processed. Mutation abnormalities in the GDNF/RET-GDNFR alpha signaling system do not cause VUR in this family.

Adult↗