Poly-l-aspartic Acid Enhances and Prolongs Gentamicin-mediated Suppression of the CFTR-G542X Mutation in a Cystic Fibrosis Mouse Model
2009; Elsevier BV; Volume: 284; Issue: 11 Linguagem: Inglês
10.1074/jbc.m806728200
ISSN1083-351X
AutoresMing Du, Kim M. Keeling, Liming Fan, Xiaoli Liu, David M. Bedwell,
Tópico(s)Cystic Fibrosis Research Advances
ResumoAminoglycosides such as gentamicin have the ability to suppress translation termination at premature stop mutations, leading to a partial restoration of protein expression and function. This observation led to studies showing that this approach may provide a viable treatment for patients with genetic diseases such as cystic fibrosis that are caused by premature stop mutations. Although aminoglycoside treatment is sometimes associated with harmful side effects, several studies have shown that the co-administration of polyanions such as poly-l-aspartic acid (PAA) can both reduce toxicity and increase the intracellular aminoglycoside concentration. In the current study we examined how the co-administration of gentamicin with PAA influenced the readthrough of premature stop codons in cultured cells and a cystic fibrosis mouse model. Using a dual luciferase readthrough reporter system in cultured cells, we found that the co-administration of gentamicin with PAA increased readthrough 20–40% relative to cells treated with the same concentration of gentamicin alone. Using a Cftr-/- hCFTR-G542X mouse model, we found that PAA also increased the in vivo nonsense suppression induced by gentamicin. Following the withdrawal of gentamicin, PAA significantly prolonged the time interval during which readthrough could be detected, as shown by short circuit current measurements and immunofluorescence. Because the use of gentamicin to suppress disease-causing nonsense mutations will require their long term administration, the ability of PAA to reduce toxicity and increase both the level and duration of readthrough has important implications for this promising therapeutic approach. Aminoglycosides such as gentamicin have the ability to suppress translation termination at premature stop mutations, leading to a partial restoration of protein expression and function. This observation led to studies showing that this approach may provide a viable treatment for patients with genetic diseases such as cystic fibrosis that are caused by premature stop mutations. Although aminoglycoside treatment is sometimes associated with harmful side effects, several studies have shown that the co-administration of polyanions such as poly-l-aspartic acid (PAA) can both reduce toxicity and increase the intracellular aminoglycoside concentration. In the current study we examined how the co-administration of gentamicin with PAA influenced the readthrough of premature stop codons in cultured cells and a cystic fibrosis mouse model. Using a dual luciferase readthrough reporter system in cultured cells, we found that the co-administration of gentamicin with PAA increased readthrough 20–40% relative to cells treated with the same concentration of gentamicin alone. Using a Cftr-/- hCFTR-G542X mouse model, we found that PAA also increased the in vivo nonsense suppression induced by gentamicin. Following the withdrawal of gentamicin, PAA significantly prolonged the time interval during which readthrough could be detected, as shown by short circuit current measurements and immunofluorescence. Because the use of gentamicin to suppress disease-causing nonsense mutations will require their long term administration, the ability of PAA to reduce toxicity and increase both the level and duration of readthrough has important implications for this promising therapeutic approach. Previous studies have shown that aminoglycosides such as gentamicin and amikacin can suppress translation termination at disease-causing premature stop (nonsense) mutations and partially restore the expression of functional proteins in mammalian cells (for a review, see Ref. 1Keeling K.M. Bedwell D.M. Curr. Pharmacogenomics. 2005; 3: 259-269Crossref Scopus (21) Google Scholar). In particular, gentamicin has been shown to suppress nonsense mutations and partially restore protein expression in mouse models of Duchenne muscular dystrophy (2Barton-Davis E.R. Cordier L. Shoturma D.I. Leland S.E. Sweeney H.L. J. Clin. Investig. 1999; 104: 375-381Crossref PubMed Scopus (497) Google Scholar) and cystic fibrosis (CF) 2The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; PAA, poly-l-aspartic acid; PBS, phosphate-buffered saline. (3Du M. Jones J.R. Lanier J. Keeling K.M. Lindsey J.R. Tousson A. Bebok Z. Whitsett J.A. Dey C.R. Colledge W.H. Evans M.J. Sorscher E.J. Bedwell D.M. J. Mol. Med. 2002; 80: 595-604Crossref PubMed Scopus (149) Google Scholar, 4Du M. Keeling K.M. Fan L. Liu X. Kovacs T. Sorscher E. Bedwell D.M. J. Mol. Med. 2006; 84: 573-582Crossref PubMed Scopus (58) Google Scholar). However, the use of aminoglycosides is commonly associated with serious side effects, including nephrotoxicity and ototoxicity (5Hutchin T. Cortopassi G. Antimicrob. Agents Chemother. 1994; 38: 2517-2520Crossref PubMed Scopus (107) Google Scholar, 6Mingeot-Leclercq M.P. Tulkens P.M. Antimicrob. Agents Chemother. 1999; 43: 1003-1012Crossref PubMed Google Scholar). The high dose of gentamicin (34 mg/kg) initially used to demonstrate readthrough in mouse models also resulted in serum concentrations that were far in excess of their maximum clinically recommended levels (2Barton-Davis E.R. Cordier L. Shoturma D.I. Leland S.E. Sweeney H.L. J. Clin. Investig. 1999; 104: 375-381Crossref PubMed Scopus (497) Google Scholar, 3Du M. Jones J.R. Lanier J. Keeling K.M. Lindsey J.R. Tousson A. Bebok Z. Whitsett J.A. Dey C.R. Colledge W.H. Evans M.J. Sorscher E.J. Bedwell D.M. J. Mol. Med. 2002; 80: 595-604Crossref PubMed Scopus (149) Google Scholar, 4Du M. Keeling K.M. Fan L. Liu X. Kovacs T. Sorscher E. Bedwell D.M. J. Mol. Med. 2006; 84: 573-582Crossref PubMed Scopus (58) Google Scholar). More recently, we demonstrated that a lower dose of gentamicin (5 mg/kg) produced peak serum concentrations in a CF mouse model that were within the accepted clinical range for these compounds (4Du M. Keeling K.M. Fan L. Liu X. Kovacs T. Sorscher E. Bedwell D.M. J. Mol. Med. 2006; 84: 573-582Crossref PubMed Scopus (58) Google Scholar). Functional CFTR protein was produced under those conditions, as shown by immunofluorescence and short circuit current measurements. However, the level of suppression obtained was significantly less than was observed with higher doses. Consistent with the efficacy of these clinically relevant doses in mice, small clinical trials have suggested that gentamicin can suppress premature stop mutations in patients with Duchenne muscular dystrophy (7Politano L. Nigro G. Nigro V. Piluso G. Papparella S. Paciello O. Comi L.I. Acta Myol. 2003; 22: 15-21PubMed Google Scholar) and CF (8Clancy J.P. Bebok Z. Ruiz F. King C. Jones J. Walker L. Greer H. Hong J. Wing L. Macaluso M. Lyrene R. Sorscher E.J. Bedwell D.M. Am. J. Respir. Crit. Care Med. 2001; 163: 1683-1692Crossref PubMed Scopus (221) Google Scholar, 9Wilschanski M. Famini C. Blau H. Rivlin J. Augarten A. Avital A. Kerem B. Kerem E. Am. J. Respir. Crit. Care Med. 2000; 161: 860-865Crossref PubMed Scopus (170) Google Scholar, 10Wilschanski M. Yahav Y. Yaacov Y. Blau H. Bentur L. Rivlin J. Aviram M. Bdolah-Abram T. Bebok Z. Shushi L. Kerem B. Kerem E. N. Engl. J. Med. 2003; 349: 1433-1441Crossref PubMed Scopus (435) Google Scholar). However, negative results have also been obtained in other clinical trials for both CF (11Clancy J.P. Rowe S.M. Bebok Z. Aitken M.L. Gibson R. Zeitlin P. Berclaz P. Moss R. Knowles M.R. Oster R.A. Mayer-Hamblett N. Ramsey B. Am. J. Respir. Cell Mol. Biol. 2007; 37: 57-66Crossref PubMed Scopus (72) Google Scholar) and Duchenne muscular dystrophy (12Wagner K.R. Hamed S. Hadley D.W. Gropman A.L. Burstein A.H. Escolar D.M. Hoffman E.P. Fischbeck K.H. Ann. Neurol. 2001; 49: 706-711Crossref PubMed Scopus (226) Google Scholar). These discrepancies suggest that further refinement of aminoglycoside-based treatment strategies is needed. Several approaches have been investigated to reduce aminoglycoside toxicity (6Mingeot-Leclercq M.P. Tulkens P.M. Antimicrob. Agents Chemother. 1999; 43: 1003-1012Crossref PubMed Google Scholar). Among these, one of the most potent protectants against the renal toxicity associated with these compounds is poly-l-aspartic acid (PAA). The co-administration of PAA with gentamicin has been shown to provide a significant level of protection against aminoglycoside-induced nephrotoxicity in rats, as measured by the absence of functional and pathological changes caused by lysosomal phospholipidosis in proximal tubular cells. Because phospholipidosis results from the intralysosomal accumulation of aminoglycosides and their binding to the acidic head groups of phospholipids in the lysosomal membrane, it was proposed that PAA exerts its protective effect by forming complexes with gentamicin following their protonation within lysosomes, thus preventing their membrane association (13Beauchamp D. Laurent G. Maldague P. Abid S. Kishore B.K. Tulkens P.M. J. Pharmacol. Exp. Ther. 1990; 255: 858-866PubMed Google Scholar, 14Gilbert D.N. Wood C.A. Kohlhepp S.J. Kohnen P.W. Houghton D.C. Finkbeiner H.C. Lindsley J. Bennett W.M. J. Infect. Dis. 1989; 159: 945-953Crossref PubMed Scopus (100) Google Scholar, 15Kishore B.K. Ibrahim S. Lambricht P. Laurent G. Maldague P. Tulkens P.M. J. Pharmacol. Exp. Ther. 1992; 262: 424-432PubMed Google Scholar, 16Williams P.D. Hottendorf G.H. Bennett D.B. J. Pharmacol. Exp. Ther. 1986; 237: 919-925PubMed Google Scholar). In this report, we show that the co-administration of PAA with gentamicin induced a higher level of suppression of a readthrough reporter in cultured cells than gentamicin alone. The co-administration of PAA with gentamicin also resulted in an increased and prolonged level of suppression of the CFTR-G542X nonsense mutation in a CF mouse model. Because it is well documented that the co-administration of PAA with gentamicin reduces the toxicity of aminoglycosides (13Beauchamp D. Laurent G. Maldague P. Abid S. Kishore B.K. Tulkens P.M. J. Pharmacol. Exp. Ther. 1990; 255: 858-866PubMed Google Scholar, 14Gilbert D.N. Wood C.A. Kohlhepp S.J. Kohnen P.W. Houghton D.C. Finkbeiner H.C. Lindsley J. Bennett W.M. J. Infect. Dis. 1989; 159: 945-953Crossref PubMed Scopus (100) Google Scholar, 15Kishore B.K. Ibrahim S. Lambricht P. Laurent G. Maldague P. Tulkens P.M. J. Pharmacol. Exp. Ther. 1992; 262: 424-432PubMed Google Scholar, 16Williams P.D. Hottendorf G.H. Bennett D.B. J. Pharmacol. Exp. Ther. 1986; 237: 919-925PubMed Google Scholar), these results suggest that the co-administration of these compounds may alleviate the major limitation of this therapeutic approach while enhancing its efficacy. Cell Culture and Dual Luciferase Readthrough Assays-HEK293T cells maintained as monolayer cultures were grown in Dulbecco's modified Eagle's medium with 4.5 gm/liter d-glucose, 584 mg/liter l-glutamine, and 110 mg/liter sodium pyruvate supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Gentamicin was from Abbott Laboratories (North Chicago, IL). Poly-l-aspartic acid (sodium salt; molecular weight, 8,900–10,300; catalogue number P-5387) was from Sigma. For dual luciferase assays, HEK293T cells were split into 96-well plates and treated with different concentrations of gentamicin with or without PAA for the indicated time period. The cells were transfected with the dual luciferase UGA readthrough reporter (pDB691) or CGA sense control reporter (pDB690) using FuGENE 6 (Roche Applied Science). Each well was transfected with 0.75 μl of FuGENE 6 and 0.25 μg of dual luciferase constructs, and cells were grown for an additional 24 or 48 h before harvesting for assays. Dual luciferase assays were performed using a dual luciferase assay kit (Promega Corp.), and light emission was quantitated using a Berthold Lumat LB9507 luminometer. The dual luciferase reporters used to monitor readthrough of stop codons in mammalian cells have been described previously (17Grentzmann G. Ingram J.A. Kelly P.J. Gesteland R.F. Atkins J.F. RNA. 1998; 4: 479-486Crossref PubMed Scopus (44) Google Scholar, 18Howard M.T. Anderson C.B. Fass U. Khatri S. Gesteland R.F. Atkins J.F. Flanigan K.M. Ann. Neurol. 2004; 55: 422-426Crossref PubMed Scopus (95) Google Scholar). The percentage of readthrough was defined as the firefly/Renilla luciferase activity (nonsense) divided by firefly/Renilla luciferase activity (sense) × 100. All of the results are expressed as the means ± S.D. The Student's t test was used for statistic analysis. Measurement of Intracellular Gentamicin Concentrations-Gentamicin was extracted from HEK293T cells treated with gentamicin ± PAA by a sodium hydroxide extraction method (19Brown S.A. Sugimoto K. Smith G.G. Garry F.B. Antimicrob. Agents Chemother. 1988; 32: 595-597Crossref PubMed Scopus (16) Google Scholar). Briefly, the cells were grown in 10-cm culture dishes in the presence of gentamicin (± PAA) at different concentrations for 24 h. The cells were washed three times with PBS to remove any remaining extracellular gentamicin. 300 μl of PBS was then added to the dish, and the cells were homogenized. The homogenizer was rinsed with another 300 μl of PBS, which were pooled with the original homogenate. An equal volume of 2 m sodium hydroxide was added to the homogenate, and the mixture was incubated at 70°C for 20 min. The samples were centrifuged at 13,000 × g for 20 min, and the supernatant was collected and neutralized to pH 7.0 prior to measuring the gentamicin and protein concentrations. Gentamicin concentrations were determined using a fluorescence polarization immunoassay, whereas protein concentrations were measured using a dye binding assay (Bio-Rad). Mice and Treatment Protocols-The CFTR-G542X mice used in this study contained the Cftrtm1Cam knock-out (20Ratcliff R. Evans M.J. Cuthbert A.W. MacVinish L.J. Foster D. Anderson J.R. Colledge W.H. Nat. Genet. 1993; 4: 35-41Crossref PubMed Scopus (213) Google Scholar) and expressed a human CFTR transgene with the G542X premature stop mutation (3Du M. Jones J.R. Lanier J. Keeling K.M. Lindsey J.R. Tousson A. Bebok Z. Whitsett J.A. Dey C.R. Colledge W.H. Evans M.J. Sorscher E.J. Bedwell D.M. J. Mol. Med. 2002; 80: 595-604Crossref PubMed Scopus (149) Google Scholar, 4Du M. Keeling K.M. Fan L. Liu X. Kovacs T. Sorscher E. Bedwell D.M. J. Mol. Med. 2006; 84: 573-582Crossref PubMed Scopus (58) Google Scholar, 21Du M. Liu X. Welch E.M. Hirawat S. Peltz S.W. Bedwell D.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2064-2069Crossref PubMed Scopus (210) Google Scholar) (referred to as Cftr-/- hCFTR-G542X mice). Control mice were C57BL/6J (referred to as wild type or Cftr+/+ mice) or Cftrtm1Unc knock-out mice (22Snouwaert J.N. Brigman K.K. Latour A.M. Malouf N.N. Boucher R.C. Smithies O. Koller B.H. Science. 1992; 257: 1083-1088Crossref PubMed Scopus (769) Google Scholar) (referred to as Cftr-/- mice). Transcription of the hCFTR-G542X transgene was driven by the rat intestinal fatty acid-binding protein promoter. Gentamicin and PAA were dissolved separately in PBS. Treatments consisted of subcutaneous injections of 5 mg/kg gentamicin alone or 5 mg/kg gentamicin plus 70 mg/kg PAA delivered in the hind limb once daily for the time periods indicated. The treatment of all mice was initiated 16 days after birth; the mice were weaned 23 days after birth. Because of the high incidence of intestinal blockage in CF mice (22Snouwaert J.N. Brigman K.K. Latour A.M. Malouf N.N. Boucher R.C. Smithies O. Koller B.H. Science. 1992; 257: 1083-1088Crossref PubMed Scopus (769) Google Scholar), all of the mice were maintained on a liquid diet (Peptamen® complete elemental diet; Nestlé) after weaning. The animal protocols used in this work were reviewed and approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Short Circuit Current Measurements-Transepithelial short circuit current measurements were carried out using an Ussing chamber under conditions previously described (3Du M. Jones J.R. Lanier J. Keeling K.M. Lindsey J.R. Tousson A. Bebok Z. Whitsett J.A. Dey C.R. Colledge W.H. Evans M.J. Sorscher E.J. Bedwell D.M. J. Mol. Med. 2002; 80: 595-604Crossref PubMed Scopus (149) Google Scholar, 4Du M. Keeling K.M. Fan L. Liu X. Kovacs T. Sorscher E. Bedwell D.M. J. Mol. Med. 2006; 84: 573-582Crossref PubMed Scopus (58) Google Scholar, 21Du M. Liu X. Welch E.M. Hirawat S. Peltz S.W. Bedwell D.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2064-2069Crossref PubMed Scopus (210) Google Scholar). Forskolin (10 μm) was added to both the mucosal and serosal solutions, and the short circuit current was continuously monitored for ≥10 min to ensure that a sustained response was obtained. Glybenclamide (200 μm) was then added to both the mucosal and serosal solutions to block the forskolin-activated CFTR short circuit current. In all experiments, the current measurements obtained immediately before and 10 min after forskolin addition were used to calculate the current change in each sample. Immunofluorescence-Immunofluorescence experiments were carried out as previously described (3Du M. Jones J.R. Lanier J. Keeling K.M. Lindsey J.R. Tousson A. Bebok Z. Whitsett J.A. Dey C.R. Colledge W.H. Evans M.J. Sorscher E.J. Bedwell D.M. J. Mol. Med. 2002; 80: 595-604Crossref PubMed Scopus (149) Google Scholar, 4Du M. Keeling K.M. Fan L. Liu X. Kovacs T. Sorscher E. Bedwell D.M. J. Mol. Med. 2006; 84: 573-582Crossref PubMed Scopus (58) Google Scholar, 21Du M. Liu X. Welch E.M. Hirawat S. Peltz S.W. Bedwell D.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2064-2069Crossref PubMed Scopus (210) Google Scholar). CFTR-specific antiserum 4562 was raised against an antigen that included hCFTR NBD1 and a portion of the R domain (hCFTR amino acids 521–828). Treatment of Cultured Cells with PAA and Gentamicin Induces More Readthrough than Gentamicin Alone-Previous studies have shown that PAA protects against the renal toxicity associated with aminoglycosides. Surprisingly, this protection was accomplished even though PAA increases the intracellular aminoglycoside concentration (13Beauchamp D. Laurent G. Maldague P. Abid S. Kishore B.K. Tulkens P.M. J. Pharmacol. Exp. Ther. 1990; 255: 858-866PubMed Google Scholar, 14Gilbert D.N. Wood C.A. Kohlhepp S.J. Kohnen P.W. Houghton D.C. Finkbeiner H.C. Lindsley J. Bennett W.M. J. Infect. Dis. 1989; 159: 945-953Crossref PubMed Scopus (100) Google Scholar, 15Kishore B.K. Ibrahim S. Lambricht P. Laurent G. Maldague P. Tulkens P.M. J. Pharmacol. Exp. Ther. 1992; 262: 424-432PubMed Google Scholar, 16Williams P.D. Hottendorf G.H. Bennett D.B. J. Pharmacol. Exp. Ther. 1986; 237: 919-925PubMed Google Scholar). However, it is unknown how the co-administration of PAA with aminoglycosides affects the suppression of nonsense mutations. To examine this question, we used a dual luciferase readthrough reporter system in cultured HEK293T cells. In this system, the Renilla and firefly luciferase reporter genes are located upstream and downstream of an in-frame UGA stop codon, respectively (17Grentzmann G. Ingram J.A. Kelly P.J. Gesteland R.F. Atkins J.F. RNA. 1998; 4: 479-486Crossref PubMed Scopus (44) Google Scholar, 18Howard M.T. Anderson C.B. Fass U. Khatri S. Gesteland R.F. Atkins J.F. Flanigan K.M. Ann. Neurol. 2004; 55: 422-426Crossref PubMed Scopus (95) Google Scholar) (Fig. 1A). The cells were transfected with the relevant dual luciferase plasmids, cultured for 24 h, and treated with gentamicin ± PAA for another 24 h. They were then harvested and lysed for readthrough assays. Treatment of HEK293T cells with gentamicin induced a dose-dependent increase in readthrough (Fig. 1B), as previously reported using various cell-based systems (23Bedwell D.M. Kaenjak A. Benos D.J. Bebok Z. Bubien J.K. Hong J. Tousson A. Clancy J.P. Sorscher E.J. Nat. Med. 1997; 3: 1280-1284Crossref PubMed Scopus (280) Google Scholar, 24Howard M. Frizzell R.A. Bedwell D.M. Nat. Med. 1996; 2: 467-469Crossref PubMed Scopus (408) Google Scholar, 25Keeling K.M. Bedwell D.M. J. Mol. Med. 2002; 80: 367-376Crossref PubMed Scopus (114) Google Scholar, 26Keeling K.M. Brooks D.A. Hopwood J.J. Li P. Thompson J.N. Bedwell D.M. Hum. Mol. Genet. 2001; 10: 291-299Crossref PubMed Google Scholar). In preliminary experiments, the optimal gentamicin to PAA ratio was determined (data not shown). We found that treatment of cells for 24 h with 0.5 mm gentamicin plus 6.5 μm PAA (a gentamicin to PAA molar ratio of ∼77:1) stimulated readthrough of the UGA nonsense codon 1.4-fold more than the same concentration of gentamicin alone, a significant increase (p value < 0.01). Treatment of cells with 1 mm gentamicin plus 13 μm PAA resulted in a 1.2-fold increase in readthrough compared with gentamicin alone, again a significant increase (p value < 0.01). We next asked whether the increased readthrough observed when cells are treated with gentamicin plus PAA correlated with an increased intracellular gentamicin concentration. HEK293T cells were incubated in the presence of 0.5 or 1.0 mm gentamicin ± PAA for 24 h. The cells were then washed, and the amount of intracellular gentamicin was quantitated using fluorescence polarization immunoassay. We found that cells treated with gentamicin plus PAA contained 1.2–1.3-fold more of the aminoglycoside than cells treated with gentamicin alone (Fig. 2A), which represented a significant increase (p value < 0.05). This suggested that the intracellular gentamicin concentration increased in proportion to the amount of gentamicin in the culture medium. Further analysis of these data revealed a very strong correlation between the readthrough level and the intracellular gentamicin concentration (correlation coefficient, 0.998; Fig. 2B). These results indicate that the co-administration of gentamicin with PAA leads to a higher level of intracellular gentamicin than treatment with gentamicin alone, leading to increased readthrough of the UGA nonsense codon. We next asked whether the enhanced suppression of nonsense mutations was maintained following extended treatments with gentamicin plus PAA. The cells were grown in continuous culture in medium containing 1 mm gentamicin ± PAA for 1, 8, or 16 days, and dual luciferase readthrough reporters were transfected into the cells 24 h before harvesting. At the end of the treatment period the cells were harvested, and dual luciferase readthrough assays were carried out. We found that a similar level of gentamicin-induced readthrough was sustained after 1, 8, or 16 days (Table 1). A significant 1.3–1.4-fold increase in readthrough was also observed in cells grown in the presence of gentamicin plus PAA after each time period (p value < 0.01). These results indicate that readthrough promoted by gentamicin (or gentamicin plus PAA) can be sustained in cultured cells over an extended growth period.TABLE 1Persistence of readthrough stimulation by PAA in HEK293 cellsTreatment timePercentage of readthroughaThe percentage of readthrough is expressed as the means ± standard deviationp valueGentamicinb1 mm gentamicin, 13 μm PAA (as indicated)Gentamicin + PAAb1 mm gentamicin, 13 μm PAA (as indicated)Fold stimulation1 day4.30 ± 0.225.45 ± 0.241.27<0.018 days4.10 ± 0.355.21 ± 0.491.27<0.0116 days4.09 ± 0.345.57 ± 0.231.36<0.01a The percentage of readthrough is expressed as the means ± standard deviationb 1 mm gentamicin, 13 μm PAA (as indicated) Open table in a new tab We next asked whether the co-administration of gentamicin plus PAA extended the time period during which readthrough occurred after gentamicin was removed from the culture medium. HEK293T cells were treated with the four protocols shown in Fig. 3A. Protocol 1 was a control without any treatment. In protocol 2, the cells were initially grown in culture medium with 1 mm gentamicin for 24 h and then changed to fresh medium without gentamicin and grown for an additional 24 or 48 h. In protocol 3, the cells were treated with 1 mm gentamicin plus 13 μm PAA for 24 h, then changed to fresh medium without either compound, and grown for an additional 24 or 48 h. In protocol 4, the cells were grown in culture medium with 1 mm gentamicin plus 13 μm PAA for 24 h, then changed to fresh medium without gentamicin (but with 13 μm PAA), and grown for an additional 24 or 48 h. In all four protocols, the dual luciferase plasmid constructs were transfected into the cells when the medium was changed to initiate the chase period. After the 24- or 48-h chase period, the cells were harvested, and dual luciferase assays were carried out to monitor the level of readthrough. Fig. 3B shows readthrough after the 24-h chase period, whereas Fig. 3C shows readthrough following the 48-h chase. In both cases, treatment with gentamicin plus PAA (protocol 3) increased readthrough by 20% more than gentamicin alone (protocol 2), a significant increase (p value < 0.05). A further 10% increase in readthrough was observed when PAA was present during the chase period (protocol 4). This latter result represented a significant increase when compared with not only to gentamicin alone (protocol 2; p value < 0.05) but also when compared with gentamicin plus PAA (protocol 3; p value < 0.05). When taken together, these results indicate that the elevated readthrough resulting from the co-administration of gentamicin plus PAA persisted following the removal of gentamicin from the culture medium. PAA Enhances Gentamicin-induced Readthrough in Cftr-/- hCFTR-G542X Mice-We previously reported that once daily subcutaneous injections of 5 mg/kg gentamicin or 15 mg/kg amikacin resulted in suppression of the hCFTR-G542X mutation and a partial restoration of CFTR protein and function in Cftr-/- hCFTR-G542X mice (4Du M. Keeling K.M. Fan L. Liu X. Kovacs T. Sorscher E. Bedwell D.M. J. Mol. Med. 2006; 84: 573-582Crossref PubMed Scopus (58) Google Scholar). Notably, those doses also resulted in peak serum aminoglycoside levels within the accepted clinical range. However, the readthrough afforded by either aminoglycoside at those low dosages was weaker than the readthrough observed with higher doses. Because our in vitro results indicated that the co-administration of gentamicin plus PAA enhanced suppression, we next investigated whether PAA could enhance the readthrough of a nonsense mutation by a low dose of gentamicin in Cftr-/- hCFTR-G542X mice. Several studies have previously shown that the co-administration of gentamicin with PAA at molar ratios ranging from 1.5:1 to 8:1 significantly reduced gentamicin-induced nephrotoxicity while increasing the intracellular gentamicin concentration in the renal cortex (13Beauchamp D. Laurent G. Maldague P. Abid S. Kishore B.K. Tulkens P.M. J. Pharmacol. Exp. Ther. 1990; 255: 858-866PubMed Google Scholar, 14Gilbert D.N. Wood C.A. Kohlhepp S.J. Kohnen P.W. Houghton D.C. Finkbeiner H.C. Lindsley J. Bennett W.M. J. Infect. Dis. 1989; 159: 945-953Crossref PubMed Scopus (100) Google Scholar, 15Kishore B.K. Ibrahim S. Lambricht P. Laurent G. Maldague P. Tulkens P.M. J. Pharmacol. Exp. Ther. 1992; 262: 424-432PubMed Google Scholar). Based on these results, we chose to examine the effect of PAA on gentamicin-induced readthrough in our CF mouse model using a gentamicin:PAA molar ratio of 1.5 to 1. The CFTR protein is a cAMP-activated chloride channel, and transepithelial chloride conductance attributable to CFTR can be monitored in intestinal tissues mounted in an Ussing chamber following the addition of the cAMP agonist forskolin. To systematically examine the effect of PAA on readthrough of the hCFTR-G542X nonsense mutation, Cftr-/- hCFTR-G542X mice were included in six different treatment groups as shown in Fig. 4A. Treatments consisted of subcutaneous injections of 5 mg/kg gentamicin alone or 5 mg/kg gentamicin plus 70 mg/kg PAA delivered once daily in a hind limb. Groups 1–3 contained Cftr-/- hCFTR-G542X mice that were treated as indicated for 14 days. Group 1 contained control mice that were left untreated for 14 days. Group 2 contained mice treated with gentamicin alone for 14 days, whereas group 3 contained Cftr-/- hCFTR-G542X mice that were administered gentamicin plus PAA for 14 days. Following the 14-day treatment period, the mice were sacrificed, and intestinal short circuit current measurements were carried out. Groups 4–6 contained Cftr-/- hCFTR-G542X mice that were again treated for 14 days as described above, followed by a 4-day chase period before assays were performed. Group 4 mice were treated with gentamicin for 14 days and then maintained without treatment for another 4 days. Group 5 mice were given gentamicin plus PAA for 14 days and then maintained without treatment for 4 days. Group 6 mice were given gentamicin plus PAA for 14 days and then administered PAA alone for the next 4 days. As controls, both wild type mice (Cftr+/+, group 8) and Cftr knock-out mice without the hCFTR-G542X transgene (Cftr-/-, group 10) were treated with gentamicin plus PAA for 14 days with continuing administration of PAA for another 4 days. These mice were then sacrificed for cAMP-stimulated short circuit current measurements, and the results were compared with untreated mice of the same genotype (groups 7 and 9, respectively). The results of short circuit current measurements from the 10 different treatment groups of Cftr-/- hCFTR-G542X, wild type mice, and Cftr-/- mice are shown in Fig. 4B. A summary and statistical analysis of short circuit currents from the 10 different treatment groups are shown in Table 2. The results from group 1 (untreated Cftr-/- hCFTR-G542X mice) revealed that 11% of samples (3/28) exhibited cAMP-stimulated short circuit currents, resulting in an average current of only 0.2 μA/cm2. The infrequent response observed in untreated Cftr-/- hCFTR-G542X mice may be attributable to a low baseline level of endogenous readthrough of the hCFTR-G542X transgene, because these currents ar
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