Functional Enhancement of CFTR Expression by Mitomycin C

Maitra, Rangan; Shaw, Collin M.; Hamilton, Joshua W.

doi:10.1159/000047796

Cited by 6 publications

(4 citation statements)

References 13 publications

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“…Noncytotoxic doses of mitomycin C, a DNA cross‐linking reagent have been shown to preferentially alter the expression of inducible genes [19]. Mitomycin C was also shown to induce CFTR mRNA and protein levels in colon carcinoma cells lines [10]. We have shown that activation of the Capan1 pancreatic adenocarcinoma cells by a low dose of mitomycin C reproducibly enhanced the intensity of the DHS in intron 18 in comparison to nonactivated cells.…”

Section: Discussionmentioning

confidence: 91%

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Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene

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Nuthall

et al. 2002

European Journal of Biochemistry

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The cystic ®brosis transmembrane conductance regulator (CFTR) gene shows a complex pattern of expression, with temporal and spatial regulation that is not accounted for by elements in the promoter. One approach to identifying the regulatory elements for CFTR is the mapping of DNase I hypersensitive sites (DHS) within the locus. We previously identi®ed at least 12 clusters of DHS across the CFTR gene and here further evaluate DHS in introns 2, 3, 10, 16, 17a, 18, 20 and 21 to assess their functional importance in regulation of CFTR gene expression. Transient transfections of enhancer/reporter constructs containing the DHS regions showed that those in introns 20 and 21 augmented the activity of the CFTR promoter. Structural analysis of the DNA sequence at the DHS suggested that only the one intron 21 might be caused by inherent DNA structures. Cell speci®city of the DHS suggested a role for the DHS in introns 2 and 18 in CFTR expression in some pancreatic duct cells. Finally, regulatory elements at the DHS in introns 10 and 18 may contribute to upregulation of CFTR gene transcription by forskolin and mitomycin C, respectively. These data support a model of regulation of expression of the CFTR gene in which multiple elements contribute to tightly co-ordinated expression in vivo.Keywords: CFTR; regulation; DNase I hypersensitive sites.The cystic ®brosis transmembrane conductance regulator (CFTR) gene shows a tightly regulated pattern of temporal and spatial expression though the elements responsible for this remain poorly characterized. We previously identi®ed DNase I hypersensitive sites (DHS) across 400 kb¯anking the CFTR gene in order to locate potential regulatory elements [1±5]. These DHS lie 5¢ to the gene at )79.5 and )20.9 kb with respect to the translational start site; in introns 1, 2, 3, 10, 16, 17a, 18, 20 and 21; and 3¢ to the gene at +5.4 to +7.4 and +15.6 kb (Fig. 1). DHS are often, but not always, associated with regulatory elements in chromatin. As we have identi®ed multiple clusters of DHS, it is possible that not all of these represent important regulatory elements for the CFTR gene. The aim of this work was to evaluate the regions of the CFTR gene containing the DHS to identify those containing important regulatory elements. In vitro analyses of the DHS regions have included evaluation in enhancer/reporter gene constructs where luciferase activity is driven by the CFTR basal promoter and DNA¯anking the DHS is inserted into the enhancer site of the vector. The results suggest that in addition to the DHS in intron 1 (at 185 + 10 kb) which was previously shown to increase CFTR promoter activity [2], the DHS in intron 20 (at 4005 + 4 kb) also augments promoter activity and the DHS in intron 21 (at 4095 +7.2 kb) has modest enhancer activity.The majority of the DHS were initially identi®ed in the Caco2 colon carcinoma cell line [5]. We have now looked for tissue-speci®c regulatory elements by analysing chromatin structure at these DHS in two pancreatic adenocarcinoma cell lines Capan1 [6] and NP31 [7...

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Section: Discussionmentioning

confidence: 91%

“…The pancreatic lines show a different predominance of DHS than the Caco2 cell line, while the intensity of DHS in Calu3 chromatin is very weak. Finally we evaluated the effect of known activators of CFTR transcription, including forskolin [9] and mitomycin C [10] on the DHS.…”

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confidence: 99%

Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene

Phylactides

Rowntree

Nuthall

et al. 2002

European Journal of Biochemistry

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“…In any case, the mild CF phenotype of patients with the P67S or the P67L mutations suggests that the residual function of these mutant channels is sufficient to prevent severe symptoms. In addition, mutant CFTR channels with residual function may respond particularly well to novel pharmacological approaches designed to rescue channel function by using chloride channel openers [39] or chaperones [40,41].…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of a Novel CFTR Mutation P67S Identified in a Patient with Atypical Cystic Fibrosis

Kraus

Reis²,

Naehrlich³

et al. 2007

Cell Physiol Biochem

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Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR serves as a cAMP-stimulated chloride channel in a wide range of epithelial tissues and its dysfunction is a hallmark of CF. Over 1400 mutations in the CFTR gene are known, but functional data exist only for a minority of the mutant channels. The aim of the present study was to functionally characterize a novel CFTR mutation identified in a patient with atypical CF. Full length sequencing of the patient’s CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution. Mutant and wild-type CFTR were heterologously expressed in Xenopus laevis oocytes. CFTR whole-cell currents were studied using the two-electrode voltage-clamp technique. Channel surface expression was assessed by a chemiluminescence assay. Expression of P67S-CFTR resulted in functional CFTR chloride channels. However, the CFTR chloride conductance observed in oocytes expressing the mutant channel averaged only 24% of that in oocytes expressing wild-type CFTR. Similarly, surface expression of the mutant channel was reduced. In contrast, the mutation did not alter the anion selectivity of the channel, and Western blot analysis indicated a similar protein expression level of mutant and wild-type CFTR. Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression. The mild disease phenotype of the patient indicates that the residual function of the mutant channel is sufficient to prevent the development of severe CF symptoms.

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“…There is evidence of co-regulation of CFTR and Pgp expression in vitro and in vivo [11][12][13][14]. Accordingly, our previous work demonstrated that certain Pgp modulators could affect CFTR expression and function [14][15][16]. In particular, the anthracycline compound doxorubicin (Dox), increased CFTR-associated chloride secretion over two-fold in T-84 cells and partially corrected ΔF508-CFTR localization, which ultimately resulted in enhanced chloride secretion in an engineered MDCK-C7 cell line [15].…”

Section: Introductionmentioning

confidence: 98%

Altered Biogenesis of ΔF508-CFTR Following Treatment with Doxorubicin

Maitra¹,

Hamilton²

2007

Cell Physiol Biochem

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Cystic fibrosis (CF) is caused by mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common of these mutations is deletion of a phenylalanine residue at position 508 (ΔF508), which accounts for ñ70% of all CF alleles. This mutation interferes with the biogenesis and maturation of ΔF508-CFTR to the plasma membrane. However, ΔF508-CFTR can partially function upon proper localization. Thus, pharmacological correction of ΔF508-CFTR maturation holds promise in CF therapy. Our previous studies indicate that a single non-cytotoxic dose of the anthracycline doxorubicin (Dox) significantly increase ΔF508-CFTR-associated chloride secretion in MDCK cells by increasing the expression of this protein at the apical plasma membrane. We report here that Dox alters the biogenesis of ΔF508-CFTR. Treatment with Dox increases the resistance of ΔF508-CFTR to trypsin digestion, possibly by expediting protein folding. Further, treatment with Dox reduces the amount of polyubiquitinated ΔF508-CFTR in cells and prolongs the half-life of this protein. Concomitantly, treatment with Dox decreases the association of ΔF508-CFTR with HSP70 but does not alter the expression of major HSP70 family members. Based on these results, we propose that Dox expedites the folding and maturation of ΔF508-CFTR by acting as a pharmacological chaperone, which consequently promotes the functional expression of this protein in MDCK cells.

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Functional Enhancement of CFTR Expression by Mitomycin C

Cited by 6 publications

References 13 publications

Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene

Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene

Functional Characterization of a Novel CFTR Mutation P67S Identified in a Patient with Atypical Cystic Fibrosis

Altered Biogenesis of ΔF508-CFTR Following Treatment with Doxorubicin

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