Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer.

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl(-) ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology.

Cl- channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase.

Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli.

Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.

Repair of psoralen-induced cross-links and monoadducts in normal and repair-deficient human fibroblasts.

SV40-transformed normal, xeroderma pigmentosum (XP) and Fanconi's anemia (FA) fibroblasts have distinct repair capacities for monoadducts and DNA interstrand cross-links produced by exposure to near-UV (320-400 nm) light in the presence of 8-methoxypsoralen or angelicin. Excision repair of monoadducts occurred rapidly in normal and FA cells after exposure but not in XP cells. Cross-links were repaired in normal cells with a t1/2 of about 10 h but not in XP or FA cells. When the total number of adducts induced by 8-methoxypsoralen in normal cells was kept constant, the amount of repair replication decreased as the ratio of cross-links to monoadducts increased. This suggests either that cross-link repair is significantly different from monoadduct repair, involving smaller patches and a much slower rate of patching or that cross-links can inhibit monoadduct repair. Our results show that XP group A and FAH12 cell lines are deficient in cross-link repair. The data also suggest that the mechanism of cross-link repair in human cells involves several enzymes and that different ones may be deficient in XP and FA cells.

Induction of DNA-DNA cross-link formation in human cells by various psoralen derivatives.

Furocoumarin-induced DNA damage, monoadducts, and cross-links were measured in normal human, xeroderma pigmentosum, and Fanconi's anemia cells after exposure to near-UV (356 nm). At similar concentrations and near-UV doses, photoaddition by 8-methoxypsoralen was twice that by angelicin and the substitution of bromodeoxyuridine for thymidine in one strand of DNA did not alter the binding. The rate of cross-linking by 8-methoxypsoralen was twice that of 5-methoxypsoralen. Low frequencies of cross-links were detected from angelicin and 3-carbethoxypsoralen but none were detected from 5-geranoxypsoralen at concentrations up to 25 micrograms/ml and near-UV doses up to 45,000 J/m2.

Ionic selectivity of volume-sensitive currents in human epithelial cells.

The ion selectivity of swelling-activated Cl- currents has been investigated in three different human epithelial cell lines, two derived from the airway epithelium (9HTEo- and CFNPE9o-) and one from a colon carcinoma (T84). The relative permeability of volume-sensitive currents with respect to Cl- is: I- (1.19) greater than NO3- (1.07) approximately Br-(1.05) greater than Cl-(1.0) greater than F-(0.5) approximately HCO3-(0.48) greater than isethionate(0.28) greater than aspartate (0.14) approximately gluconate(0.13) approximately SO4(2-)(0.12). This type of ion selectivity is similar to that described for depolarization-activated outwardly rectifying Cl- channels found in epithelial cells.

Caspases shutdown nonsense-mediated mRNA decay during apoptosis.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that plays integral roles in eliminating mRNAs with premature termination codons to prevent the synthesis of truncated proteins that could be pathogenic. One response to the accumulation of detrimental proteins is apoptosis, which involves the activation of enzymatic pathways leading to protein and nucleic acid cleavage and culminating in cell death. It is not clear whether NMD is required to ensure the accurate expression of apoptosis genes or is no longer necessary since cytotoxic proteins are not an issue during cell death. The present study shows that caspases cleave the two NMD factors UPF1 and UPF2 during apoptosis impairing NMD. Our results demonstrate a new regulatory pathway for NMD that occurs during apoptosis and provide evidence for role of the UPF cleaved fragments in apoptosis and NMD inhibition.

Repair of psoralen adducts in human DNA: differences among xeroderma pigmentosum complementation groups.

Angelicin and 5-methylangelicin formed photoadducts in DNA after illumination with 360-nm radiation that were excised rapidly from normal cells; 80-90% of the initial angelicin adducts and 65% of the initial 5-methylangelicin adducts were excised within 24 h. Xeroderma pigmentosum group A cells excised about 20% of the angelicin adducts, group D cells excised 55-60%, and group E, 80%. This extent of excision resembles that reported for pyrimidine dimers in these complementation groups, except for group D. Repair of psoralen adducts may not, therefore, be identical in every respect to repair of pyrimidine dimers. Group D cells seem exceptionally able to repair angelicin adducts in comparison to their repair of pyrimidine dimers, suggesting that these cells lack a gene product that is required to a greater extent for the repair of pyrimidine dimers than for the repair of angelicin adducts.

Expression sequence tag-specific full-length cDNA cloning: actin cDNAs.

Current strategies for cDNA cloning are based on construction of cDNA libraries and colony screening. The process of obtaining a full-length cDNA clone can be highly time and labor intensive. Using the human actin gene as a model target cDNA, we have developed an RNA-capture method for rapid cloning of full-length cDNAs. The approach involves the capture of mRNA with expressed sequence tag (EST)-derived, biotin labeled antisense "capture" primers and streptavidin-coated magnetic beads. Full-length cDNA is then synthesized from purified EST-specific mRNA and cloned directly into plasmid vectors. The results of using beta-actin-based capture primers on cytoplasmic RNA were the isolation of both beta- and gamma-actin cDNA clones. Of the 16 actin-specific cDNA clones analyzed, 15 (93%) were full-length. This approach for cloning full-length cDNAs from available ESTs or partial cDNA sequences will facilitate a more rapid and efficient characterization of gene structure and function.

In-frame elimination of exon 10 in Cftrtm1Unc CF mice.

Cystic fibrosis (CF) is a severe autosomal-linked inherited disease in humans. Transgenic CF animals play a crucial role in the study of molecular mechanisms underlying disease pathology. In the present study, CFTR mRNA expression was examined in different tissues from one CF mouse model that contains a disruption in exon 10 sequence of the CF transmembrane conductance regulator (CFTR) gene. Multiple tissue samples were collected from new-born normal (+/+), homozygous (-/-), and heterozygous (+/-) mice and compared for their CFTR mRNA expression. Total RNA samples were prepared from eight different tissues (nasal mucosa, trachea, lung, colon, intestine, pancreas, liver, gonads, and brain) and then analyzed by reverse transcription polymerase chain reaction (RT-PCR) amplification. A 329-bp fragment comprising exon 9 through exon 11 of the CFTR gene was amplified from all tissues of the normal mouse. In contrast, a 137-bp fragment was observed in tissue samples from homozygous CF mice. Both the 329-bp and 137-bp fragments were detected in samples from heterozygous mice. Direct sequencing analysis of the amplified fragments showed an exon 9-exon 11 splice junction, indicating that the entire exon 10 sequence was eliminated from homozygous CF mice. RT-PCR analysis of the 3' end of CFTR mRNA showed the presence of a 682-bp, exon 20-24 fragment in -/- and +/- mice. These results demonstrate that an alternately spliced CFTR mRNA is produced in this CF 'knock-out' mouse. A semi-quantitative comparison of the wild-type and exon 10 minus CFTR (CFTR-E10) mRNA in heterozygote animals indicated that less (but a detectable amount) mutant CFTR mRNA was present in all organs tested. There was, however, a significant reduction of CFTR-E10 mRNA in the liver and the pancreas. Since the deletion of exon 10 is in-frame, the significance of the CFTR-E10 mRNA in terms of CFTR protein and function requires further analysis.

Extracellular 2-chloroadenosine and ATP stimulate volume-sensitive Cl- current and calcium mobilization in human tracheal 9HTEo- cells.

The perforated-patch whole-cell technique was used to record membrane currents in epithelial cells (9HTEo-) obtained from the human tracheal epithelium. Extracellular application of 2-chloroadenosine and ATP (0.01-100 microM) caused activation of Cl- currents similar to those regulated by cell volume in airway and intestinal cells. This response was inhibited by increasing extracellular osmolality, by omission of extracellular Ca2+, or by the addition of the A2 adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Fluorimetric measurements with fura-2 reveal that 2-chloroadenosine and ATP elicited both a Ca2+ influx through the plasma membrane and a release from intracellular stores.

Transfer and expression of foreign genes in mammalian cells.

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

Differential sensitivity of normal and cystic fibrosis airway epithelial cells to epinephrine.

1. Exposure to epinephrine has been shown to have a range of effects on cells and tissues. A recent study suggested that the proliferative ability of CF epithelial cells, exposed to high concentrations of epinephrine (200 - 300 microM), was reduced when compared to that of normal cells. This approach could potentially provide a means to effectively separate cells with functional cyclic AMP-dependent Cl-ion transport from those defective in this pathway. 2. The sensitivity to killing by epinephrine is reported here for four different CF cell lines, three normal cell lines, and two CF epithelial cell lines complemented with wild-type (wt) CF transmembrane conductance regulator (CFTR) cDNA. 3. While each cell line exhibited varying sensitivity to 200 microM epinephrine, no predictable pattern was observed between the expression of wt-CFTR and cell survival following epinephrine exposure. Overall, normal cell lines did exhibit a greater resistance to epinephrine-induced cell death although, the most resistant cell line was derived from CF tracheal epithelium (SigmaCFTE29o-). 4. The expression of exogenous wt-CFTR increased the survival of one cell line (CFDEo-) when compared to the parent line, but in another complemented line, survival was reduced. 5. These findings suggest that while epinephrine induces cell killing, it is not consistently effective for preferential selection of normal over CF cells. Although CFTR may play a role in the mechanism(s) of epinephrine killing, other factors such as cell density, proliferative ability, cell type origin and phenotype are involved.

Stimulation of chloride secretion by P1 purinoceptor agonists in cystic fibrosis phenotype airway epithelial cell line CFPEo-.

1. P1 purinoceptor agonists like adenosine have been shown to stimulate Cl- transport in secretory epithelia. In the present study, we investigated whether P1 agonist-induced Cl- secretion is preserved in cystic fibrosis airway epithelium and which signalling mechanism is involved. The effects of purinoceptor agonists on Cl- secretion were examined in a transformed cystic fibrosis airway phenotype epithelial cell line, CFPEo-. 2. Addition of adenosine (ADO; 0.1-1 mM) markedly increased 125I efflux rate. The rank order of potency of purinoceptor agonists in stimulating 125I efflux was ADO > AMP > ADP approximately equal to ATP. A similar order of potency was seen in transformed cystic fibrosis nasal polyp cells, CFNPEo- (ADO > ATP > AMP > ADP). These results are consistent with the activation of Cl- secretion via a P1 purinoceptor. 3. The P1 agonists tested (at 0.01 and 0.1 mM) revealed a rank order of potency of 5'-N-ethylcarboxamine adenosine (NECA) > 2-chloro-adenosine (2-Cl-ADO) > R-phenylisopropyl adenosine (R-PIA). 4. The known potent A2 adenosine receptor (A2AR) agonist, 5'-(N-cyclopropyl) carboxamidoadenosine (CPCA, 2 microM) but not the A1 adenosine receptor agonist, N6-phenyl adenosine (N6-phenyl ADO, 10 microM) markedly increased 125I efflux rate (baseline, 5.9 +/- 2.0% min-1, + CPCA, 10.9 +/- 0.6% min-1; P < 0.01). The stimulant effect of CPCA (10 microM) was abolished by addition of the A2AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (100 microM; reported K(i) = 11 +/- 3 microM). These results favour the involvement of A2AR. 5. ADO (0.1-mM) and CPCA (2 microM) both induced a marked increase in intracellular [Ca2+] ([Ca2+]i); the effect of the latter was again abolished by pretreatment of the cells with DMPX. By contrast, N6-phenyl ADO did not affect [Ca2+]i. 6. In patch-clamp experiments, ADO (1 mM) induced an outwardly-rectified whole-cell Cl- current (baseline, 2.5 +/- 0.8 pA pF-1, + ADO, 78.4 +/- 23.8 pA pF-1; P < 0.02), which was largely inhibited in cells internally perfused with a selective inhibitory peptide of the multifunctional Ca2+/calmodulin-dependent protein kinase, CaMK [273-302] (20 microM), as compared to a control peptide, CaMK [284-302]. Addition of BAPTA (10 mM), a Ca2+ chelator, to the perfusion pipette also abolished the ADO-elicited Cl- current. 7. In conclusion, our results suggest that A2AR participates in regulation of airway C1 secretion via aCa2+-dependent signalling pathway, which involves CaMK and appears to be at least partially conserved in cystic fibrosis airway epithelial cells.

Formation and repair of psoralen-DNA adducts and pyrimidine dimers in human DNA and chromatin.

DNA damage and repair in human cells exposed to ultraviolet light (254 nm) or to psoralen derivatives plus 360 nm light were compared by means of a variety of analytic techniques. The two kinds of damage show considerable structural similarity; both involve cyclobutyl bonds to 5,6 positions of pyrimidines as major products and have various minor products. In purified DNA, pyrimidine dimers, but not psoralen adducts, cause structural distortions that are substances for digestion with single-strand-specific nucleases. Whereas pyrimidine dimers are randomly produced in chromatin, psoralen adducts, are concentrated approximately 2- to 4-fold in linker regions of chromatin at doses that are not highly lethal. Chromatin shows considerable mobility; assignment of DNA to linker or core regions is not permanent, and psoralen adducts initially concentrated in linker regions become randomized after 10 hr. Pyrimidine dimers and psoralen adducts are excised by normal cells but not by repair-deficient xeroderma pigmentosum cells. This repair process requires DNA polymerase alpha, but its rate in ultraviolet-damaged cells is twice that in psoralen-damaged cells. Conversion of monoadducts to DNA-DNA crosslinks reduces the rate of repair because of the increased complexity of the damaged site.

Transformation and establishment of fragile X cell lines from amniocytes.

Fragile X [fra(X)] cell lines have been established from an expressing 46,XX amniocyte culture. The amniocyte cells were transformed by the addition of an origin defective pSV40 vector. Fra(X) expression was observed at a frequency of 3% in the parental amniocyte cell line, and the transformed clones expressed the fra(X) site at a frequency of 9-13%. These cell lines maintain the cytogenetic phenotype and can serve as positive controls for testing the efficacy of the inducing systems during prenatal diagnostic studies.

Established cell lines used in cystic fibrosis research.

The development of immortalized cell lines has been a significant benefit to the study of human disease, due to limitations in using primary cells and the availability of tissue. The immortalization of cells from cystic fibrosis (CF) patients as well as cells from non-CF individuals from tissues relevant to CF has been critical to enhancing our understanding of the physiological, biochemical and genetic mechanisms underlying CF and for the development of therapeutic strategies designed to manage CF pathology. A comprehensive list of immortalized cells from various tissue and species, with an emphasis on epithelial cells, is presented and discussed here.

Infection of cultured human airway epithelial cells by influenza A virus.

The lack of an adequate in vitro model has hampered study of the cellular basis by which influenza A virus causes disease in the human airway. We report in vitro infection of human airway epithelial cells by influenza A virus. Fetal and adult human tracheal and bronchial epithelial cells cultured from explants and SV40 transformed adult human tracheal epithelial cells were exposed to a recently isolated strain of influenza A virus (H1N1) and a laboratory passaged strain (WSN) of influenza A virus at similar multiplicity of infection. All cultures derived from explants showed hemadsorption (approximately 30% of the cells) with the H1N1 virus. No hemadsorption was detected with the WSN virus. One of two transformed cell lines showed a 5-10% hemadsorption to cells after H1N1 exposure and none following exposure to WSN. Immunofluorescent staining for influenza A-specific antigens in virus-exposed, explant-derived cells indicated viral infection and replication in these cells. Hemagglutinating material in the growth medium of infected, explant-derived cell lines, increased as a function of time, indicating the production of virus proteins. Exposure of rhesus monkey kidney cells and new human tracheal epithelial cultures to supernatant from these cells resulted in hemadsorption, indicating the presence of infectious virus in the supernatant. Light microscopic examination of virally infected bronchial epithelial cells demonstrated that the common types of cytopathic changes were rarely seen while cell proliferation continued over time. The data indicate that influenza A virus can infect, replicate, and produce infectious virus in cultured human tracheal and bronchial epithelial cells.

Response of cultured human airway epithelial cells to X-rays and energetic alpha-particles.

Radon and its progeny, which emit alpha-particles during decay, may play an important role in inducing human lung cancer. To gain a better understanding of the biological effects of alpha-particles in human lung we studied the response of cultured human airway epithelial cells to X-rays and monoenergetic helium ions. Our experimental results indicated that the radiation response of primary cultures was similar to that for airway epithelial cells that were transformed with a plasmid containing an origin-defective SV40 virus. The RBE for cell inactivation determined by the ratio of D0 for X-rays to that for 8 MeV helium ions was 1.8-2.2. The cross-section for helium ions, calculated from the D0 value, was about 24 microm 2 for cells of the primary culture. This cross-section is significantly smaller than the average geometric nuclear area (approximately 180 microms 2), suggesting that an average of 7.5 alpha-particles (8 MeV helium ions) per cell nucleus are needed to induce a lethal lesion.