Biomedical subjects

B C Trapnell

Publications and source records attributed to B C Trapnell.

At least 37 records · Page 2Linked to original sources

PubMed1996

Surfactant enhances adenovirus-mediated gene expression in rabbit lungs.

The efficiency and localization of gene transfer for gene therapy in the lung will depend on vector selection and technique used to deliver the vector. We hypothesized that surfactant might facilitate gene transfer because of its biophysical properties that enhance spreading of saline suspensions in the airways. We used a replication defective adenovirus directing the expression of the luciferase reporter gene at different virus doses and surfactant concentrations and instilled 4 ml suspensions into the lungs of adult rabbits. Transgene expression measured as luciferase-dependent light units in tissue homogenates 3 days after instillation had similar lobar distributions for surfactant-viral suspensions and for phosphate-buffered saline viral suspensions. However, total light units were four- to six-fold increased for viral doses of < 10(10) plaque forming units in 10 mg/ml or 25 mg/ml surfactant suspensions. The percentage of total transgene expression in the tracheacarina-bronchus decreased from over 50% to about 10% for virus instilled with surfactant concentrations of 2 mg/ml or greater, indicating preferential expression in airway epithelial cells located more distally in the lung. Surfactant did not enhance persistence of transgene expression 7 or 14 days after instillation. Surfactant facilitates adenovirally mediated gene transfer in the peripheral lung and enhances transgene expression.

Adenoviruses, Human↗

PubMed1995

Pharmacokinetics of adenoviral vector-mediated gene delivery to vascular smooth muscle cells: modulation by poloxamer 407 and implications for cardiovascular gene therapy.

Regional in vivo delivery of therapeutic genes to the cardiovascular system at sites of localized vascular disease is feasible by catheter-mediated delivery of recombinant adenoviral vectors. Vascular smooth muscle cell (SMC) proliferation, which follows angioplasty and contributes to restenosis, is one process that may be amenable to such a gene therapy strategy. The clinical utility of localized delivery strategies such as this critically depends upon successful gene transfer to sufficient numbers of vascular cells, locally, within a clinically acceptable time period. Relatively limited information is available concerning the kinetics of gene transfer by first-generation, replication-deficient, recombinant adenovirus (Av1) vectors. In this context, we evaluated the pharmacokinetics of adenoviral vector-mediated gene delivery to vascular SMC using an Av1 reporter vector (Av1LacZ4) expressing a nuclear-targeted beta-galactosidase (beta-Gal) reporter. Bovine aortic SMC were exposed to Av1LacZ4 for various times at a range of concentrations and multiplicities of infection (MOI). After exposure, cells were washed and evaluated for transduction at 48 hr by X-Gal staining. Transduction occurred with a rate constant typically determined in the range of 10(-10) to 10(-11) events.ml/cell.virion.min. The rate of transduction was directly dependent on virion concentration, but not substantially on the virion-to-cell ratio. Relatively low fractions of the total input vector were found to be consumed, even after prolonged adsorption times. We hypothesized that the cellular transduction rate (and thus overall efficiency) would be improved by agents that could maintain a prolonged, high pericellular vector concentration. To evaluate this, cells were exposed to the vector in the presence of 15 grams/dl poloxamer 407, a viscous biocompatibile polyol, for various times followed by washout and evaluation as described above. Both cells and vector remained viable under these conditions, and poloxamer was found to increase the apparent transduction rate 10-fold or more (1-5 x 10(-9) transduction events.ml/cell.virion.min), with remarkable increases in numbers of cells transduced even after brief exposure periods. These observations demonstrate that the pharmacokinetics of adenoviral-mediated gene delivery to vascular SMC can be modulated by agents such as poloxamer 407, which may improve gene delivery by maintaining high pericellular concentrations of vector. Such modulation may permit achievement of desired levels of gene transfer while requiring lower total viral dosage and exposure time, and in turn may have important implications for in vivo gene delivery to vascular tissues.

Adenoviridae↗

PubMed1995

Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice.

To evaluate the role of cell-mediated immunity during gene transfer to the respiratory epithelium, the time course of luciferase activity was assessed after intratracheal administration of Av1Luc1, an E1a-E3-deleted adenoviral (Ad5) vector expressing firefly luciferase, to FVB/N, BALB/c and BALB/c-nu/nu adult mice. Adenovirus-mediated luciferase activity was rapidly lost from the respiratory tract between 2 and 14 days after treatment of both FVB/N and BALB/c wild-type mice. In the wild-type mice, loss of luciferase activity was associated with an early inflammatory response consisting of infiltration with macrophages and polymorphonuclear leukocytes and a more prolonged response characterized by lymphocytic infiltration. In the immune-deficient nu/nu mice, luciferase activity was maintained at higher levels than in immune-competent mice after exposure to virus and was associated with a distinct pattern of inflammation, consisting primarily of macrophages and polymorphonuclear cells but lacking the lymphocytic infiltrates typical of the inflammation in wild-type mice. Adenoviral DNA was rapidly cleared from the lungs of both nu/nu and wild-type mice. Markedly increased expression of proliferating cell nuclear antigen (PCNA) was observed in bronchiolar and alveolar epithelial cells and in inflammatory cells after exposure to Av1LUc1. The proliferative response of the respiratory epithelium was more extensive and persistent in wild-type than in nu/nu mice. To assess further the impact of the immune system on adenovirus-mediated gene expression, cotton rats treated with cyclosporin A or dexamethasone were exposed to Av1Luc1. Both agents decreased lung inflammation and significantly increased lung luciferase activity. The loss of lung luciferase activity is dependent, in part, on the immune-mediated clearance of respiratory epithelial cells, which may limit the extent and duration of gene expression with recombinant adenoviral vectors.

Adenoviridae Infections↗

PubMed1995

Adenovirus-mediated gene transfer to the respiratory tract of fetal sheep in utero.

Many human genetic diseases, such as congenital surfactant protein B deficiency, manifest in the perinatal period. Prenatal gene therapy may be necessary to minimize morbidity in these diseases. We hypothesized that bacterial beta-galactosidase (beta-Gal) gene could be transferred to and expressed in the pulmonary epithelium of fetal sheep in utero using a replication-deficient adenovirus (Av1LacZ4). We instilled Av1LacZ4 (1.5 x 10(11) plaque-forming units, n = 10) or saline (n = 2) intratracheally to chronically instrumented fetal sheep at 112-134 days gestation (term = 145 days). Lung fluid was collected before and after Av1LacZ4 administration for cytological analysis. Lung tissue was examined for transgenic beta-Gal activity and evidence of toxicity. Transgenic beta-Gal activity was visualized as blue nuclear staining of tissue treated with X-Gal and was detected in the lungs of 5 animals for up to 14 days after administration. Transgenic beta-Gal activity was not detected in the lungs of animals analyzed beyond 14 days after treatment. Pulmonary histopathology was detected in most Av1LacZ4-treated animals and manifested as a mixed cellular infiltrate consisting of neutrophils, macrophages, and lymphocytes. Fetal lung fluid analysis revealed a predominantly lymphocytic response in most Av1LacZ4-treated animals within 3 days (2.88 x 10(6) vs. 4 x 10(3) total cells/ml in control animals). We have demonstrated that adenovirus vectors can direct gene transfer to the lungs of fetal sheep in utero. The transferred gene expression was transient and possibly limited by the induced inflammatory response.

Adenoviridae↗

PubMed1995

Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues.

Replication-deficient adenoviruses have been used to transfer various genes of interest to mammalian tissues in vivo. Effective gene therapy for inborn genetic defects presenting with significant morbidity and mortality at birth will require correction of the defect prenatally. To test the hypothesis that intra-amniotically administered adenovirus transfers gene expression to fetal tissues, replication-deficient human type 5 adenovirus carrying the lacZ gene which encodes nuclear-targeted bacterial beta-galactosidase (Av1LacZ4) was instilled into the amniotic cavity of fetal sheep (10(10) to 1.5 x 10(11) pfu) and fetal mice (10(9) pfu) at 0.8 term gestation. Amniotic membranes and gastrointestinal and respiratory tract tissues were harvested after 3 d, bacterial beta-galactosidase activity was determined by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-gal) enzyme-histochemistry, and tissue integrity was assessed in sections stained with hematoxylin and eosin. Bacterial beta-galactosidase activity was abundant in amniotic membranes and present in lower levels in esophagus, stomach, and small intestine as well as in conducting airways and pulmonary alveoli. To determine whether gene transfer by intraamniotic injection of adenovirus was dose-dependent, Av1Luc1, an adenoviral vector carrying the gene for luciferase (10(5)-10(9) pfu), was injected intraamniotically into fetal mice at 0.8 term gestation. Luciferase activity measured after 3 d in tissue homogenates of Av1Luc1-treated fetal mice revealed a linear dose response in amniotic membranes and gastrointestinal and respiratory tract organs. Intraamniotic administration of an adenoviral gene vector leads to expression of the transferred gene in amniotic membranes as well as in fetal gastrointestinal and respiratory tract tissues in a dose-dependent manner.

Adenoviruses, Human↗

PubMed1995

Transduction of normal and malignant oral epithelium by an adenovirus vector: the effect of dose and treatment time on transduction efficiency and tissue penetration.

We tested an adenovirus vector that carries a beta-D-galactosidase marker gene for its ability to transduce normal oral mucosa and oral carcinoma cells. Topical application of adenovirus to normal oral mucosa of mice at 1 x 10(10) plaque-forming units (pfu)/mL for 1 minute did not result in transduction of epithelial cells. Similarly, topical application to human oral mucosa ex vivo was not successful. However, systemic administration by intracardiac injection of hamsters did transduce normal oral mucosa effectively. To evaluate transduction of carcinomas, the Tu138 human oral cancer cell line was used. A single application of virus at 1 x 10(8) pfu/mL in vitro resulted in 30% of oral carcinoma cells expressing the marker gene, and 2 x 10(8) pfu/mL transduced 60% of cells. After two applications of virus at 2 x 10(8) pfu/mL with an interval of 18 hours, 100% of oral carcinoma cells expressed the marker gene. Topical application to a raft culture of Tu138 cells for 1 hour produced gene expression that penetrated up to four layers of cells. To emulate the effect of treating a carcinoma, Tu138 cells were implanted subcutaneously in nude mice, allowed to grow to a tumor 1 cm in diameter, and then injected directly with virus. This produced diffuse transduction of around 30% of cells in the tumor, and expression was seen in cells at a significant distance from the injection site. Adenovirus vectors are therefore capable of transferring novel genetic information to both normal and malignant oral mucosa. They may have potential for use in gene therapy in the prevention or treatment of oral squamous carcinomas.

Adenoviruses, Human↗

PubMed1994

Gene therapy using adenoviral vectors.

Growing interest in adenoviral gene-transfer vectors, stimulated by efforts to develop in vivo gene therapy for cystic fibrosis, has led to an evaluation of their use in many other applications of in vivo gene therapy. Studies are beginning to define strategies for the efficient, albeit transient, expression of the transferred gene and have further identified and partially characterized important host responses to in vivo gene transfer that modulate the duration of expression of the transgene. Ongoing work is actively exploring these issues, with a view to the design of the next generation of adenoviral vectors.

Adenoviridae↗

PubMed1994

Evaluation of the efficacy and safety of in vitro, adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA.

Cystic fibrosis (CF) is a common, fatal recessive disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene manifested by abnormalities in the regulation of chloride ion (Cl-) secretion across the apical membrane of epithelial cells throughout the body. Adenovirus-mediated delivery of the normal CFTR cDNA and correction of the CF epithelial cell Cl- secretory phenotype suggests the feasibility of gene therapy for CF lung disease. Few studies, however, have focused on the evaluation of the safety of the adenovirus-mediated gene transfer approach. This study presents in vitro data on the efficacy and safety of adenovirus-mediated transfer of the human CFTR cDNA using Av1Cf2. Av1Cf2-mediated transfer of the human CFTR cDNA complemented the abnormal cAMP-regulated Cl- permeability of cells with the CF epithelial phenotype. Av1 vectors did not replicate infectious virus in HeLa cells infected in vitro, although trace vector DNA synthesis was observed at very high multiplicity of infection. Expression of the adenoviral late gene for the hexon capsid protein was observed at trace levels in Av1 vector-infected HeLa cells, but not in freshly isolated human bronchial epithelial cells, consistent with the pattern of DNA synthesis observed in these different target cells. Although, these observations support the efficacy and safety of use of Av1Cf2 for treatment of the fatal pulmonary component of CF.

Adenoviruses, Human↗

PubMed1994

In vivo evaluation of the safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lung.

Cystic fibrosis (CF) is a common, fatal hereditary disease resulting from mutations of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene in which epithelial cells throughout the body manifest altered regulation of apical membrane chloride secretion. Although the disease affects multiple organs throughout the body, over 90% of patients die of complications of the lung involvement. The feasibility of adenovirus-derived vectors for in vivo delivery of the human CFTR cDNA to treat the pulmonary component of CF is currently being evaluated using in vitro and in vivo approaches. Defining the therapeutic window between biological efficacy and toxicity is an important part of this work. Here we present data regarding the preclinical evaluation of the safety of in vivo delivery of the human CFTR cDNA to the cotton rat airway epithelium using the replication-deficient adenoviral vector Av1Cf2 or a similar vector, Av1LacZ4, expressing the Escherichia coli LacZ gene as a histologic marker. Gene transfer to the respiratory epithelium was efficient, as demonstrated by in situ hybridization and histochemical staining. Administration of these vectors resulted in a mild, transient, dose-dependent cellular inflammatory response similar in character to that seen with adenovirus 5 (Ad5), but far less in intensity, which was not associated with structural lung damage or mortality. Av1Cf2 DNA sequences were easily detected in the lung after pulmonary administration, but could not be demonstrated in organs other than the lung. These preclinical observations suggest that adenovirus-mediated gene transfer to the airway epithelium can be achieved efficiently, but is accompanied by a dose- and time-dependent inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗

PubMed1994

Adenoviral-mediated gene transfer of human surfactant protein B to respiratory epithelial cells.

Human surfactant protein B (SP-B) is a 79-amino acid, phospholipid-associated polypeptide expressed by respiratory epithelial cells of the lung. SP-B is essential for lung function, enhancing the spreading and stability of surfactant phospholipids that serve to reduce surface tension at the alveolar air-liquid interface. Congenital absence of SP-B results in neonatal respiratory failure and death. In the present work, we constructed a replication-deficient adenoviral vector, Av1SP-B1, in which the human SP-B cDNA is expressed under control of the Rous sarcoma virus (RSV) promoter in an E1-E3-deleted adenovirus type 5 (Ad5)-based vector system. Av1SP-B1 was produced in 293 kidney cells, directing the synthesis of the SP-B protein and SP-B peptides. Av1SP-B1 directed the synthesis of SP-B mRNA, precursor and active 8-9 kD polypeptide in immortalized mouse lung epithelial cells (MLE-12 cells), demonstrating complete processing to the human SP-B protein by these cells. Synthesis of human SP-B mRNA was detected as early as 12 h after infection and was maximal 48 h after infection in vitro. Northern blot analysis demonstrated that human SP-B mRNA was expressed in the lungs of cotton rats infected with Av1SP-B1 but not in those of uninfected animals or in animals infected with a reporter adenoviral vector, Av1LacZ4. In situ hybridization demonstrated the abundance and localization of the transferred human SP-B mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗

PubMed1994

Adenovirus-mediated gene transfer for cystic fibrosis: quantitative evaluation of repeated in vivo vector administration to the lung.

Adenoviral vectors have an important role as in vivo gene delivery vehicles in developing human gene therapy for the fatal pulmonary component of cystic fibrosis. In this study we evaluated the immune responses to wild-type adenovirus and replication-deficient, first generation adenoviral (Av1) vectors in the cotton rat (Sigmodon hispidus) and then quantitatively evaluated the efficiency of gene delivery and expression of single and repeated in vivo administration of Av1 vectors to the respiratory tract. Av1 vector reporter gene expression was quantitatively evaluated by employing a luciferase expression vector (Av1Luc1) and measuring luciferase activity in whole lung tissue homogenates by routine luminometry. Gene transfer and expression in naive animals (e.g. first Av1 vector dose) was efficient. A repeat dose also resulted in successful gene transfer and expression, although at a significantly reduced level (p < 0.01) compared with naive animals. This reduction inversely correlated with serum human adenovirus neutralizing antibody (HANA) titers. Importantly, increasing doses of Av1Cf2, an Av1 vector expressing the human CFTR cDNA, resulted in a graded HANA response consistent with a lack of in vivo replication. These observations have significant implications for repeated administration of adenoviral vectors to the lungs of individuals with cystic fibrosis.

Adenoviruses, Human↗

PubMed1993

Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA.

Variable in-frame skipping of exon 9 in cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts (exon 9-) occurs in the respiratory epithelium. To explore the genetic basis of this event, we evaluated respiratory epithelial cells and blood leukocytes from 124 individuals (38 with cystic fibrosis (CF), 86 without CF). We found an inverse relationship between the length of the polythymidine tract at the exon 9 splice branch/acceptor site and the proportion of exon 9- CFTR mRNA transcripts. These results strongly indicate a genetic basis in vivo modulating post-transcriptional processing of CFTR mRNA transcripts.

Adult↗

PubMed1993

Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice.

Gene therapy strategies designed to combat haemophilia B, caused by defects in clotting factor IX, have so far concentrated on ex vivo approaches. We have now evaluated adenoviral vector-mediated expression of human factor IX in vivo. Injection of the vector Av1H9B, which encodes human factor IX cDNA, into the tail veins of mice resulted in efficient liver transduction and plasma levels of human factor IX that would be therapeutic for haemophilia B patients. However, levels slowly declined to baseline by nine weeks and were not re-established by a second vector injection. These results address both the advantages and obstacles to the use of adenoviral vectors for treatment of haemophilia B.

Adenoviruses, Human↗

PubMed1993

Quantitative evaluation of gene expression in freshly isolated human respiratory epithelial cells.

Recent advances in molecular biology have led to development of methods to evaluate the in vivo expression of specific genes in epithelial cells of the human respiratory tract. These methods involve collection of small samples of respiratory epithelium from the human airway with a cytology brush, together with sensitive techniques to enumerate cells and to detect and quantify RNA using the polymerase chain reaction (PCR). Thus, using the typical kinds of tissue samples obtainable in a clinical setting, one can now evaluate in vivo expression of specific genes in human airway epithelium. Specifically, mRNA transcript levels can be determined on a copy number per cell basis even when the level of expression is in the range of 1-10 mRNA transcripts/cell. The currently available techniques for measuring mRNA transcript levels in small tissue samples are reviewed with emphasis on the use of quantitative PCR assays.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed1993

In vivo antioxidant gene expression in human airway epithelium of normal individuals exposed to 100% O2.

Human bronchial epithelium is exquisitely sensitive to high O2 levels, with tracheobronchitis usually developing after 12 h of exposure to 100% O2. To evaluate whether this vulnerability results from inability of the bronchial epithelium to provide adequate antioxidant protection, we quantified antioxidant gene expression in bronchial epithelium of normal volunteers at baseline and after exposure to 100% O2 in vivo. After 14.8 +/- 0.2 h of 100% O2, 24 of 33 individuals had evidence of tracheobronchitis. Baseline gene expression of CuZn superoxide dismutase (SOD), MnSOD, and catalase in bronchial epithelium was very low (CuZnSOD 4.1 +/- 0.8 transcripts/cell, MnSOD 5.1 +/- 0.9, catalase 1.3 +/- 0.2), with control gamma-actin expression relatively abundant (50 +/- 6 transcripts/cell). Importantly, despite 100% O2 exposure sufficient to cause tracheobronchitis in most individuals, antioxidant mRNA transcripts/cell in bronchial epithelium did not increase (P > 0.5). Catalase activity in bronchial epithelium did not change after exposure to hyperoxia (P > 0.05). Total SOD activity increased mildly (P < 0.01) but not sufficiently to protect the epithelium. Together, the very low levels of expression of intracellular antioxidant enzymes and the inability to upregulate expression at the mRNA level with oxidant stress likely have a role in human airway epithelium susceptibility to hyperoxia.

Actins↗

PubMed1993

In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries.

We studied the ability of adenoviral vectors to achieve gene transfer into injured arteries. A recombinant adenoviral vector expressing a nuclear-targeted beta-galactosidase gene was constructed and infused into balloon-injured rat carotid arteries. Three days after gene transfer, recombinant gene expression was assessed quantitatively by (1) measuring beta-galactosidase antigen and activity in tissue extracts and (2) histochemical staining and counting of cells expressing beta-galactosidase. Exposure of injured carotid arteries to increasing concentrations of the vector (10(8) to 10(10) plaque-forming units per milliliter) resulted in a dose-responsive increase in beta-galactosidase expression, with peak expression of approximately 43 mU or 25 ng beta-galactosidase per vessel. Microscopic examination of histochemically stained arteries demonstrated gene transfer limited to the vascular media; transduced cells were identified immunohistochemically as smooth muscle cells. Counting of both histochemically stained and total nuclei in the media revealed that approximately 30% of the cells in the media of the injured vessels were transduced. Calculations based on both counting cells and on the level of beta-galactosidase expression in tissue extracts suggested the presence of 5000 to 10,000 transduced cells per 10 mm of vessel. Arteries infused with either vehicle only, a control adenoviral vector, or liposomes combined with the vector plasmid contained little or no evidence of beta-galactosidase expression. High levels of in vivo beta-galactosidase expression persisted for at least 7 days after gene transfer but declined significantly by day 14. We conclude that adenoviral vector-mediated gene transfer into the injured rat carotid artery results in efficient gene transfer into the vascular media, with levels of recombinant protein production significantly higher than any previously reported in arterial gene transfer studies. Adenoviral vectors appear to be particularly useful agents for in vivo arterial gene transfer.

Adenoviridae↗

PubMed1992

Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene.

Based on the knowledge that expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be modulated at the transcriptional level, and that the CFTR gene promoter contains sequences homologous to elements in other promoters that respond to tumor necrosis factor-alpha (TNF), we evaluated the hypothesis that TNF might modulate CFTR gene expression in epithelial cells. Studies with HT-29 cells, a colon epithelium-derived tumor cell line known to express the CFTR gene, demonstrated that TNF downregulated CFTR mRNA transcript levels in a dose- and time-dependent fashion. Interestingly, nuclear run-on analyses demonstrated that TNF did not affect the rate of transcription of CFTR gene, but exposure of the cells to TNF did modify the stability of CFTR mRNA transcripts, resulting in a mRNA half-life that was reduced to 65% of the resting level. These observations suggest that CFTR gene expression can be modulated by TNF, at least in part, at the posttranscriptional level.

Blotting, Northern↗

PubMed1992

Expression of the cystic fibrosis transmembrane conductance regulator gene can be regulated by protein kinase C.

Epithelial cells utilize at least two types of apical Cl- channels, the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) and the Ca2+/calmodulin-dependent Cl- channel. While phorbal ester (PMA) activates only CFTR-dependent Cl- secretion and the Ca2+ ionophore A23187 only the Ca2+/calmodulin-dependent Cl- secretion, PMA and A23187 share the ability to down-regulate expression of the CFTR gene at the transcriptional level. Since both PMA and A23187 can activate protein kinases, we hypothesized that protein kinase pathways may be involved in the regulation of CFTR gene expression. Exposure of HT-29 human colon carcinoma cells to the protein kinase C activator SC9 down-regulated CFTR mRNA levels in a dose-dependent fashion, similar to that seen with PMA. The reduction in CFTR transcript levels by SC9 and PMA was blocked by H7, an inhibitor of protein kinases. In a similar fashion, the down-regulation of CFTR transcript levels by A23187 was blocked by H7 as well as staurosporine, another protein kinase inhibitor. Interestingly, both H7 and staurosporine themselves increased CFTR mRNA levels. Quantification of CFTR gene transcription rate showed a reduction by SC9 (similar to that with PMA and A23187) that was prevented by H7 and that H7 by itself increased CFTR transcription. Together, these observations suggest that protein kinase pathways, likely including protein kinase C, are involved in the regulation of CFTR gene expression, with activation or inhibition of protein kinase activity down-regulating or up-regulating CFTR gene expression, respectively.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗