Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations.

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

The nucleotide sequence of leuB from Salmonella typhimurium.

The nucleotide sequence and deduced polypeptide sequence of the Salmonella typhimurium leuB are reported, as well as a conserved region that might bind the enzyme substrate.

Reconstitution, identification, and purification of the Torpedo californica electroplax chloride channel complex.

A voltage-gated chloride channel was identified in the electric organ of the marine ray Torpedo californica by White and Miller (J. Biol. Chem. 254, 10161-10166 (1979)). The experiments reported here concern the purification and identification of this channel which was accomplished by solubilization of electric organ plasma membranes and reconstitution of the channel into vesicles made of phosphatidylethanolamine, phosphatidylserine, and cholesterol. Channel activity was measured in these vesicles by assaying 36Cl- uptake against an outwardly directed chloride chemical gradient as described by Garty et al. (J. Biol. Chem. 258, 13094-13099 (1983)). Maximal uptake occurred by 15 s. Addition of valinomycin after 10 min released intravesicular 36Cl- suggesting that chloride is moving through a channel. Channel activity was inhibited by DIDS (K0.5 of 56 mM) and NBD chloride (K0.5 of 176 mM). In a 40 lipid/1 protein (w/w) reconstitution, approx. 30% of the vesicles contained a functional chloride channel, based upon uptake done in the presence of chlorotriphenyltin (an anion ionophore), indicating that the Torpedo electric organ is an enriched source as shown by White and Miller (Biophys. J. 35, 455-462 (1981)). The chloride channel was purified approx. 40-fold by sedimentation velocity. In this purified preparation, four polypeptides (210, 95, 55, and 40 kDa) were visible by silver-staining after nonreducing SDS-PAGE. Of the four polypeptides, the largest (210 kDa) is not sufficient for Cl- channel activity by itself, but it is labeled by DIDS, an inhibitor of channel activity. Channel activity was approx. 20-fold greater in material that bound to concanavalin A compared to the concanavalin A flow-through; all four polypeptides were present in the bound materia. It is possible that some of these polypeptides are subunits of the chloride channel.

Transcription termination sites at the distal end of the leu operon of Salmonella typhimurium.

Transcription terminates at two different sites at the distal end of the leucine operon of Salmonella typhimurium. The first of these sites (leut), located 140 base-pairs past the end of leuD, contains a G + C-rich palindrome followed by a run of T residues in the non-coding strand. Termination at leut, both in vitro and in vivo, is independent of rho protein, but is stimulated by the NusA protein. The second termination site (leut'), located 145 base-pairs beyond the first, is rho-dependent both in vitro and in vivo, and is not influenced by NusA protein. The organization of transcription termination sites at the distal end of the leu operon (a rho-independent site followed by a rho-dependent site) is similar to that for the trp operon of Escherichia coli.

Effect of DNA superhelicity on transcription termination.

Restriction fragments containing either leut (a rho-independent transcription termination site) and/or leut' (a rho-dependent transcription termination site) were cloned into plasmid pOL4. Treatment of plasmid-containing Escherichia coli strains with coumermycin resulted in loss of in vivo plasmid superhelicity 10 min after antibiotic addition. Galactokinase levels specified by these plasmid-containing strains were the same regardless of whether functional DNA gyrase was present. These results suggest that transcription termination is unaffected by the superhelical state of DNA.

Characterization of the 3' end of the leucine operon of Salmonella typhimurium.

The nucleotide sequence of the leuD gene of Salmonella typhimurium and of the downstream flanking region are presented. S1 mapping experiments identified 3' endpoints of leu mRNA 140 and 285 nucleotides downstream of the UAA stop codon of leuD mRNA. Experiments employing pulse-labeled RNA suggest that these endpoints result from transcription termination rather than RNA processing. Our results indicate that the organization of the 3' non-translated region of the leu operon from S. typhimurium resembles that of the trp operon of Escherichia coli. Further, our results suggest that the leu operon of S. typhimurium does not contain structural genes other than those identified by genetic experiments, i.e. leu, A,B,C and D.

Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.

Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.

Transfer of a constitutive viral promoter-cystic fibrosis transmembrane conductance regulator cDNA to human epithelial cells conveys resistance to down-regulation of cAMP-regulated Cl- secretion in the presence of inflammatory stimuli.

The expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be down-regulated by inflammatory stimuli such as phorbol myristate acetate (PMA). Since the respiratory manifestations of cystic fibrosis (CF) are characterized by intense chronic airway inflammation very early in life, successful gene therapy for CF will require that expression of the transferred normal CFTR gene be resistant to down-regulation by inflammatory mediators. To evaluate the concept that a viral promoter--human CFTR cDNA unit would be resistant to this form of down-regulation, a retrovirus promoter (5' long terminal repeat of the Moloney murine leukemia virus)--human CFTR cDNA unit was transferred to T84 human colon carcinoma cell line using a retrovirus vector. Exposure of the retrovirus-modified T84 cells to PMA resulted in down-regulation of the endogenous CFTR mRNA transcripts (6.5 kb), but did not affect the level of exogenous CFTR transcripts (8.0 kb). Importantly, in parallel with the persistence of the exogenous CFTR transcripts, the modified cells still maintained cAMP-regulated CI- secretion in the presence of PMA. These in vitro data suggest that a constitutive viral promoter--CFTR cDNA unit should be resistant to modulation by inflammatory stimuli, a likely requirement for successful gene therapy for CF.

Recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate expression of cyclin proteins.

First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (DeltaE1) or lacking the Ad E3 region in addition to E1 sequences (DeltaE1DeltaE3) induced G2 cell cycle arrest and inhibited traverse across G1/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34(cdc2) protein levels. In some instances, infection with DeltaE1 or DeltaE1 DeltaE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation DeltaE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in DeltaE1 or DeltaE1 DeltaE3 Ad vectors induce G2 growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium.

Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl- permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11-14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.