RAPID COMMUNICATION
Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

Beate Illek1, Lei Zhang2, Nancy C. Lewis1, Richard B. Moss3, Jian-Yun Dong2, and Horst Fischer1

1 Research Institute and Pulmonary Center, Children's Hospital Oakland, Oakland 94609; 2 Laboratory Medicine, University of California at San Francisco, San Francisco 94143; and 3 Pediatric Pulmonary Medicine, Stanford University Medical Center, Stanford, California 94305


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and Delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was Po = 0.047 ± 0.019. Addition of genistein activated Cl currents ~10-fold, and the Po of G551D-CFTR increased to 0.49 ± 0.12, which is a Po similar to wild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous for the trafficking-impaired Delta F508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These data suggested that genistein activated CFTR mutants that were present in the cell membrane. Therefore, we tested the effects of genistein in CF patients with the G551D mutation in nasal potential difference (PD) measurements in vivo. The perfusion of the nasal mucosa of G551D CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, -2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to beta -adrenergic stimulation) found in healthy subjects.

cystic fibrosis transmembrane conductance regulator mutations; patch clamp; nasal potential difference; flavonoids


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF) is a common genetic disease in the Caucasian population and is caused by mutations in the CF transmembrane conductance regulator (CFTR), the epithelial cAMP-regulated Cl channel. CF is characterized by the absence of a cAMP-stimulated Cl conductance across a variety of epithelia, including the respiratory tree, pancreatic ducts, sweat glands, and the intestine. The commonest mutation is a deletion of phenylalanine at position 508 (Delta F508), which occurs on 66% of CF chromosomes world wide. The glycine-to-aspartic acid missense mutation at codon 551 (G551D) is the third commonest CF mutation, with a worldwide frequency of ~2% on CF chromosomes (25, 27). Higher frequencies of G551D are found in populations of Celtic descent, with incidences of 6.9% and 5.5% in the Irish and Australian CF populations, respectively (3, 12, 25). A low incidence of 0-0.9% was reported in CF populations of central European origin (9, 11, 26).

Delta F508-CFTR is a trafficking-impaired mutation (class II mutation), and the synthesized protein is largely degraded intracellularly. The G551D-CFTR protein traffics to the apical membrane, but Cl channel function is markedly reduced (class III mutation) (27). Compared with CF patients homozygous for the Delta F508 mutation, patients carrying the G551D mutation show a threefold reduction in the incidence of meconium ileus (neonatal intestinal blockage), a trend toward later age at diagnosis of pancreatic insufficiency (12), and milder disease in homozygotes (22). Interestingly, both the Delta F508 mutation and the G551D mutation are in NBD1 of CFTR; however, ATP binding of NBD1 is not affected by the Delta F508 mutation but is significantly impaired in G551D-CFTR (21).

The increasing knowledge about the different cell biological fates of different CFTR mutants revealed selective cellular pharmacological targets. For example, 4-phenylbutyrate (4-PB) was shown to increase expression of Delta F508-CFTR in the plasma membrane (24). We found that the isoflavone genistein and related flavonoids were potent activators of wild-type (wt) CFTR in vitro and in vivo in human subjects (15) and suggested that genistein activates CFTR when it is available in the cell membrane. We therefore tested its effects on G551D-CFTR, which is present in the cell membrane, and on Delta F508-CFTR, once it was translocated in sufficient quantity to the membrane after 4-PB treatment.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of recombinant adenovirus carrying the G551D gene. Recombinant adenovirus carrying the CFTR G551D mutation was generated by a modified ligation procedure. With the use of PCR-mediated point mutation, G551D was generated by converting the codon for glycine (GGT) at position 551 to an aspartic acid codon (GAT). A plasmid containing wtCFTR cDNA, pBQ4.7, was used as the template for the PCR reaction (6). The G551D cDNA was inserted into a shuttle vector, pLadC, which contains the left-hand terminal of adenovirus sequence and a cytomegalovirus promoter. The recombinant adenovirus, AdcG551D, was generated by ligation of the linearized pLadC-G551D to the right-hand portion of the adenovirus sub360, which contains the deletion in the E1a and E3 regions. The ligated products were transfected into 293 cells with Lipofectamine (GIBCO, Life Technologies, Grand Island, NY). Correct recombinants were isolated using a procedure described in Ref. 10. AdcG551D isolated from individual plaques were propagated in 293 cells. The inserted G551D cDNA was confirmed using PCR protocols. This viral suspension was divided into aliquots and stored at -85°C until use.

Cells. The cervical adenocarcinoma cell line HeLa was cultured in H21 DMEM supplemented with 10% FCS. For patch clamping, cells were seeded at low density on cover glasses. Twenty-four hours before use, cells were infected with viral vectors containing G551D-CFTR, wtCFTR, or LacZ as control at a multiplicity of infection of ~100. A virus-based infection of cells was used to achieve the transduction of all cells for patch clamping. The JME cell line is a continuous cell line derived from the nasal epithelium of a homozygous Delta F508 CF patient and was cultured as described in Ref. 16.

Patch-clamp recordings. Cells were patch clamped on the stage of an inverted microscope in a constantly perfused chamber at 37°C as described in Refs. 8 and 15. In the whole cell mode, a 29:150 mM Cl gradient from pipette to bath was used as a driving force while the cell membrane potential (Vm) was clamped to zero. Because there were no other significant gradients or driving forces, the resulting membrane current (Im) is a Cl current. The bath solution had the following composition (in mM) 145 N-methyl-D-glucamine chloride (NMDG-Cl), 1.7 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 10 sucrose, pH = 7.4. The pipette solution contained (in mM) 27 NMDG-Cl, 2 EGTA, 1 MgCl2, 10 HEPES, 5 glucose, 110 NMDG-gluconate, 5 MgATP, and 0.1 NaGTP, pH = 7.4. Current-voltage (I-V) relations were fitted with the Goldman equation (15), which resulted in fit estimates for the apparent intracellular Cl concentration (Cli) and the reversal potential (Erev). Membrane conductance (Gm) was calculated at 0 mV with Ohm's law of the form Gm = Im/Erev.

The average single channel open probability (Po) of the population of channels in whole cell recordings was calculated from current variance (sigma 2) to mean current plots (7). Data were fitted to Sigworth's parabola of the form sigma 2 = Im · i - Im2/N, which estimated the single channel current (i) and the number of channels in the recording (N). Po was then calculated from Po = Im/(N · i).

Recordings in the cell-attached patch-clamp mode were performed with the 145 mM NMDG-Cl solution in both bath and pipette. Po and i were determined with pCLAMP version 7 (Axon Instruments, Foster City, CA) using standard methods, and N was defined as the maximum number of open levels observed in the total length of the recording.

Nasal potential difference measurements. Patients with at least one G551D-CFTR allele were recruited from Children's Hospital Oakland and from the Stanford CF Center at Lucile Salter Packard Children's Hospital. Measurements were approved by the Internal Review Board of Children's Hospital Oakland and done exactly as described in Ref. 15. The NaCl solution contained (in mM) 145 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH = 7.4. In Cl-free solutions all Cl salts were replaced by the respective gluconate salts.

Chemicals. Unless otherwise mentioned, chemicals were from Sigma (St. Louis, MO). Forskolin (Calbiochem, La Jolla CA), genistein (4',5,7-trihydroxy-isoflavone), and quercetin (3',3,4',5,7-pentahydroxy-flavone) were made as 100 mM stock solutions in DMSO. 4-PB was made as a 5 M stock in DMSO and used at 5 mM. Amiloride was made as a 10 mM stock and isoproterenol as a 100 mM stock in water.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of genistein on G551D-CFTR in patch-clamp recordings. HeLa cells infected with the G551D-CFTR-containing adenovirus were patch clamped in the whole cell and the cell-attached mode. Figure 1 shows a whole cell recording from a G551D-CFTR-expressing cell. Stimulation with forskolin (10 µM) caused only a very small increase in current (Fig. 1A). On average forskolin-stimulated conductance was 0.19 ± 0.10 nS (n = 4). Addition of genistein (30 µM) stimulated large Cl currents, which were time and voltage independent (Fig. 1, B and C). Data were well fitted with the Goldman equation (Fig. 1D), which are typical biophysical characteristics of CFTR-mediated currents. Average forskolin-plus-genistein-activated conductance was 2.1 ± 0.61 nS (n = 4). Current variance to mean current plots was used to calculate the average Po of G551D-CFTR-mediated whole cell current. Figure 1E shows an example of this analysis applied to the current trace shown in Fig. 1A. Note that current noise significantly increased during stimulation of current. Po remained low in the presence of forskolin, reaching a maximum of 0.04 (Fig. 1E, open circles). Stimulation with genistein increased Po to a maximum of 0.46 (Fig. 1E, closed circles). Figure 1F shows average Po values of G551D-CFTR compared with wtCFTR. Note the low Po of G551D-CFTR during forskolin stimulation (0.047 ± 0.19, n = 4), which was increased by genistein to 0.49 ± 0.12, a value not different from wtCFTR. LacZ-infected control HeLa cells (n = 4) or uninfected control HeLa cells (n = 4) showed no responses to treatment with forskolin and genistein (data not shown).


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Fig. 1.   G551D-CFTR-mediated current is stimulated by genistein. A: whole cell recording from a HeLa cell expressing G551D-CFTR. Forskolin (10 µM) resulted in a very small stimulation, and perfusion with genistein (30 µM) activated large Cl current. During breaks in current trace current-voltage (I-V) relations were recorded. B and C: I-V step protocols. Plotted currents are difference currents (Delta I) calculated by subtracting control currents from stimulated currents. Membrane voltage (Vm) was stepped from -80 to +80 mV in 20-mV increments. Initial transients are uncompensated capacitative transients. B was recorded with forskolin in the bath, C with forskolin and genistein in the bath. Note time independence of currents. Delta Im are baseline subtracted Im. D: I-V plots. Line is best fit of data to Goldman equation. During forskolin (open circle ), intracellular Cl concentration (Cli) = 35.9 mM and Cl conductance (GCl) = 0.36 nS; and during forskolin + genistein (), Cli = 51.8 mM, GCl =3.09 nS. E: current variance to mean current plot. Variance (sigma 2), mean current (Im), and corresponding open probabilities (Po, top axis) were calculated from 40-s intervals of current trace filtered at 10 Hz. Line is best fit of data to Sigworth's parabola, which yielded single channel current (i) = 0.39 pA and number of channels (N) = 482. Data points are during treatment with forskolin (open circle ), forskolin + genistein (), and washout (triangle ). Maximal Po after treatment with forskolin was 0.04, after forskolin + genistein Po = 0.46. F: average calculated Po values during forskolin treatment (fsk, open bars) and forskolin + genistein treatment (geni, solid bars) in HeLa cells expressing G551D or wild-type (wt) CFTR. Genistein significantly increased Po of G551D-CFTR (P = 0.01, n = 4) and wtCFTR (P = 0.02, n = 4, paired t-tests).

Figure 2 shows the effects of genistein on the G551D-CFTR channel in a cell-attached patch-clamp recording. In this recording Po values were calculated, assuming that two channels were present. Under unstimulated conditions little channel activity was observed (Fig. 2A, control, Po = 0.017). Perfusion of the bath with forskolin stimulated few channel openings (Fig. 2A, forskolin, Po = 0.036). Addition of genistein increased Po to 0.385 (Fig. 2A, forskolin + genistein). Figure 2B shows the average single channel I-V relation for G551D-CFTR recorded in the cell-attached mode. Single channel I-V relations were typically outwardly rectifying in the cell-attached mode (7), with a conductance in the positive voltage range of g = 8.7 ± 1.0 pS (n = 4, forskolin) and 9.6 ± 0.8 pS (forskolin + genistein), which were similar to wtCFTR (g = 8.6 ± 0.91 pS, n = 3) recorded under the same conditions.


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Fig. 2.   Cell-attached recording of G551D-CFTR. A: each trace shows 20 s of a continuous recording from a cell that was first stimulated with forskolin and then with forskolin + genistein. Patch contained 2 channels. Under control conditions (top trace) Po = 0.017, after forskolin (10 µM, middle trace) Po = 0.036, and treatment with forskolin + genistein (30 µM) increased Po to 0.385. Holding potential was 72 mV. B: single channel I-V plot in cell-attached mode. Single channel conductance between 50 and 100 mV was g = 8.7 ± 1.0 pS (n = 4, open circle ) during forskolin stimulation and g = 9.6 ± 0.8 pS (, not significantly different) during forskolin + genistein stimulation.

Next we tested the effects of genistein in unstimulated cells using the whole cell mode (Fig. 3). Genistein showed no effects on Cl current, indicating that 1) HeLa cells had no endogenous genistein-activated Cl current, and 2) unstimulated G551D-CFTR could not be activated by genistein alone. Subsequent addition of forskolin in the presence of genistein stimulated Cl currents. This indicated that genistein was not a direct channel opener of G551D-CFTR but relied on regulation by the cAMP/protein kinase A (PKA) pathway and that G551D-CFTR was only active when both genistein was present and PKA was active. In summary, these patch-clamp data show that G551D-CFTR is a channel with markedly reduced open probability. Treatment of G551D-CFTR with genistein effectively restored regulation of Po by the cAMP pathway similar to wtCFTR.


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Fig. 3.   Genistein has no effect on G551D-CFTR-mediated Cl currents in unstimulated cells. Whole cell recording of a HeLa cell expressing G551D-CFTR. In absence of forskolin, genistein (30 µM) did not significantly affect Cl conductance (53 ± 211 pS, n = 4, not different from 0). Addition of forskolin (10 µM) in presence of genistein showed a significant activation of a Cl conductance (by 2.2 ± 0.51 nS, P = 0.02, one sample t-test).

Effects of genistein on Delta F508-CFTR in patch-clamp recordings. Delta F508-CFTR was investigated in a nasal epithelial cell line derived from a CF patient homozygous for the Delta F508 mutation. We selected JME cells because these cells have been shown to express several phenotypical features of CF cells, including very low Delta F508-CFTR expression in the membrane (16). In whole cell experiments, no currents were activated by forskolin or genistein (Fig. 4A, control), indicating that no measurable CFTR activity was present in the plasma membrane of these CF cells. After pretreatment of JME cells with 4-PB (5 mM for 3 days), Cl currents were stimulated by forskolin and further increased by genistein (Fig. 4A, 4-PB). Both forskolin- and genistein-activated currents showed rectification according to the Goldman equation (Fig. 4B). Figure 4C shows the average responses of JME cells to forskolin and genistein treatment. On average, genistein increased the forskolin-activated Cl conductance in 4-PB-treated cells by an additional 177% (Fig. 4C). These data indicate that genistein stimulates Delta F508-CFTR when it is available in the cell membrane, such as after treatment with 4-PB.


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Fig. 4.   Effects of genistein on Delta F508-CFTR-mediated Cl currents. A: whole cell recordings of JME cells homozygous for Delta F508-CFTR. Bottom trace (control) was recorded from an untreated control JME cell; top trace was recorded from a cell that had been incubated with 5 mM 4-phenylbutyrate (4-PB) for 3 days. Forskolin (fsk, 10 µM) and forskolin + genistein (geni, 30 µM) stimulated Cl currents in 4-PB-treated cells. Drug additions were additive. B: I-V plots from 4-PB-treated cell. Line is Goldman fit to data. Estimated parameters were: forskolin (open circle ), Cli = 77.6 mM, GCl = 3.69 nS; and forskolin + genistein (), Cli = 74.5 mM, GCl = 6.45 nS. C: summary of activated conductances. In control JME cells, treatment with forskolin or forskolin + genistein (geni) resulted in no significant Cl conductance (Gm, one sample t-test). In 4-PB-treated JME cells treatment with forskolin + genistein significantly increased Gm compared with forskolin treatment (by 177%, P < 0.05, paired t-test).

Stimulation of Cl transport by genistein in CF patients. We performed nasal potential difference (PD) measurements in CF patients bearing the G551D mutation on at least one allele and in healthy human subjects. To isolate and amplify the Cl-selective PD, the nasal mucosa was perfused with amiloride (to block Na transport) and with Cl-free solution (to establish a driving force for Cl) according to the protocol of Knowles et al. (20). The nasal epithelium was then stimulated with isoproterenol and with genistein. Figure 5 shows measurements of nasal PD from a healthy volunteer (Fig. 5A) and a G551D CF patient (Fig. 5B). Average responses of nasal PD are summarized in Fig. 5C. Perfusion with Cl-free solution and subsequent addition of isoproterenol hyperpolarized nasal PD in healthy subjects, whereas nasal PD was depolarized in G551D CF patients, indicating a lack of a beta -adrenergic-stimulated Cl conductance in CF patients. However, perfusion of the nasal mucosa with genistein significantly hyperpolarized nasal PD both in G551D CF patients and in healthy subjects, indicative of a genistein-induced Cl conductance in both groups. In normal subjects and CF patients genistein caused a slow activation of nasal PD (as in Fig. 5), typical for the activation of CFTR. In G551D CF patients, genistein caused a hyperpolarization of, on average, -2.4 ± 0.6 mV (n = 5), which corresponds to 16.9% of the average response of normal subjects to treatment with Cl-free solution and isoproterenol (-14.0 ± 2.1 mV, n = 8). Another tested flavonoid, quercetin, was also active but less potent than genistein in stimulating nasal PD in G551D CF patients (by -0.9 ± 0.3 mV, n = 4, significantly different from 0, one sample t-test).


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Fig. 5.   Genistein activates Cl-selective potentials in CF patients with G551D mutation. A: measurement of nasal potential difference (PD) in a healthy subject. Negative intranasal potentials are plotted downward in all graphs. All drug treatments were additive. Note that perfusion with Cl-free solution, isoproterenol (isoprot), and genistein resulted in significant hyperpolarizations, respectively. B: measurement of nasal PD in a G551D CF patient. Patient was a 14-year-old Caucasian female with genotype G551D/Delta F508. Note y-axis scaling. Perfusion with Cl-free solution consistently caused a depolarization likely caused by a tip potential at voltage-sensing electrode. Isoproterenol (10 µM) in Cl-free solution showed no effect, which is typical for recordings in CF patients (20). Genistein (30 µM) significantly hyperpolarized nasal PD. C: average responses of G551D CF patients (n = 5) to treatment with amiloride (amil, 50 µM), Cl-free solution (exchanged for gluconate) and 10 µM isoproterenol (Cl free/iso), and 30 µM genistein (geni), in comparison to measurements in healthy subjects (n = 8). CF patients were 2 males and 3 females, ages 10, 14, 15, 22, and 37. Genotypes (and responses of PD to genistein) of patients were: three G551D/Delta F508 (-0.8, -2.4, and -2.3 mV), one G551D/G551D (-2.0 mV), and one G551D/unknown (-4.3 mV). Responses of nasal PD to treatment with amiloride and Cl free/iso were significantly different between CF and normal subjects (P < 0.01), whereas response to treatment with genistein was not (P > 0.05, t-tests). Average nasal PD during initial NaCl perfusion was -16.1 ± 1.4 mV in normals. In G551D CF patients average nasal PD values were: NaCl, -33.8 ± 3.2 mV; amiloride, -11.7 ± 0.9 mV; isoproterenol/Cl free, -6.4 ± 1.7 mV; and genistein, -8.7 ± 1.6 mV. Readings were taken after >2 min after change of conditions when PD reached stable values and represent the numerical average of a 20-s period.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Genistein restores cAMP-dependent G551D-CFTR activity. For this study we selected the G551D mutation because the G551D-CFTR protein was described as a trafficking-competent mutant (23). We found that acute treatment of cells with genistein in the presence of forskolin resulted in G551D-CFTR activity that was functionally similar to wtCFTR during stimulation with forskolin. The genistein-stimulated G551D-CFTR had a single channel conductance of 9.6 ± 0.8 pS, showed time- and voltage-independent currents, and Po was 0.49 ± 0.12, which are typical biophysical characteristics of wtCFTR. In addition, stimulation of G551D-CFTR by genistein required intrinsic regulation of the channel by the cAMP/PKA system. The recovery of a cAMP-regulated Cl channel appears fundamental for a drug-based treatment of the Cl channel defect in CF, since CF epithelial cells express significant differently regulated Cl conductances [e.g., by intracellular Ca (28)], which do not functionally substitute for the cAMP-regulated CFTR in human CF patients.

Effects of genistein in G551D CF patients. Our nasal PD measurements in G551D CF patients showed the typical abnormalities present in other CF patients, which are: a hyperpolarized basal nasal PD, an increased amiloride-sensitive PD, a depolarization of nasal PD with Cl-free solutions, and a lack of response to the beta -adrenergic agonist isoproterenol (20). In contrast, perfusion of the nasal epithelium of G551D CF patients with genistein resulted in a hyperpolarization of nasal PD in all patients, which under the experimental conditions corresponds to an activation of a Cl conductance. To our knowledge, no other CFTR-activating drug has been shown before to hyperpolarize nasal PDs in CF patients in vivo.

Several studies suggested that only a fraction of the normal function of CFTR is required to ameliorate CF symptoms. Highsmith et al. (13) have shown that the expression of only ~4% of normal CFTR mRNA leads to an unusually mild CF phenotype. Johnson et al. (18) have shown that 6-10% of CFTR-expressing cells in an epithelial monolayer are sufficient to correct the electrophysiological parameters in CF epithelia. A study by Dorin et al. (5) suggested that 5% of CFTR function per cell may be sufficient to prevent intestinal pathology in CF mice. To relate the measured nasal PD responses to stimulation of Cl current, the transepithelial resistance (Rt) of the nasal epithelium is required. Rt cannot be determined from nasal PD measurements directly; however, Rt was previously estimated in human primary airway cultures. Willumsen et al. (29) found Rt values of 340 Omega  · cm2 in normal subjects and slightly higher values (430 Omega  · cm2) in CF patients. Assuming that 1) Rt in vivo is similar to Rt in cultures, and 2) there are no significant differences in unstirred layers (and thus driving forces for Cl) between normal and CF, we can estimate the equivalent Cl current from the measured nasal PD responses (normal, -14 mV; CF, -2.4 mV) with Ohm's law (-41.2 and -5.6 µA/cm2 for normal and CF, respectively). Thus the responses to genistein of nasal PD in G551D CF patients correspond to 13.6% of normal in terms of Cl current, which may have therapeutic significance.

CF drug development. Currently, treatment of CF is directed toward minimizing the effects of secondary complications caused by the defective CFTR. No treatment is in use that targets the defect of the channel. We suggest that pharmacological treatment strategies for CF, which are aimed at correcting the defect in CFTR-dependent Cl secretion, will depend on the kind of mutation present in CFTR. Welsh and Smith (27) distinguished four classes of CFTR mutations causing defective 1) protein production, 2) processing, 3) regulation, and 4) conduction. Because these defects create vastly different pharmacological targets in the cell, it appears likely that the different classes of mutations will need selective drug treatment. In several studies small molecules were tested that target the defect in protein production (14), protein processing (2, 17, 24), and CFTR regulation (1, 4, 19, and the present study). Some of these drugs may complement one another. For example, we show that Delta F508-CFTR can be used in combination with 4-PB to maximize Cl currents.

It has been shown that genistein has a variety of biological targets (see Ref. 15) and it will be important to study its toxicological properties. We suggest that genistein can serve as a lead compound for mutant-specific drug development projects that are aimed at 1) improving its selectivity as an activator of CFTR in the membrane and 2) exploiting synergistic pharmacological effects in combination with drugs that correct protein production or processing mutations.


    ACKNOWLEDGEMENTS

We thank all the CF patients that participated in this study.


    FOOTNOTES

This study was funded by the Commercial Endowment of Children's Hospital Oakland, by National Institutes of Health (NIH) Grant SCOR IP50HL60288-01, by a pilot project of the NIH Gene Therapy Core at the University of California San Francisco, and by NIH Grant M01RR01271-16 to the Pediatric Clinical Research Center at Children's Hospital Oakland.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. Fischer, Children's Hospital Oakland, Research Institute, 747 52nd St., Oakland, CA 94609 (E-mail: hfischer{at}mail.cho.org).

Received 30 April 1999; accepted in final form 15 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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