The promoter of the human cystic fibrosis transmembrane conductance regulator gene directing SV40 T antigen expression induces malignant proliferation of ependymal cells in transgenic mice.

Transgenic mice bearing a human cystic fibrosis transmembrane conductance regulator (CFTR) promoter-SV40 T antigen fusion transgene were generated in order to localize in vivo the potential oncogenesis linked to the tissue-specific activity of the promoter for the CFTR gene. Surprisingly, the only site of tumors resulting from expression of the reporter onc gene was ependymal cells lining the brain ventricles. SV40 T antigen expression in these cells led to a consistent pathology in the first weeks of age: ependymoma and consequent hydrocephaly. Tumor-derived cell lines were established, characterized and shown to originate from SV40 T antigen-induced ependymoma. No pathological alterations were found in other organs, such as lungs and pancreas, in which cystic fibrosis is pathologically manifest in humans. Such transgenic mice and derived cell lines may represent valid models for analysing (1) the role of SV40 T antigen in ependymoma formation and (2) CFTR function in ependymal cells.

Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery.

A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature.

Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial.

Ad CFTR, a replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator (CFTR), was administered by aerosolization in a single escalating dose to three pairs (cohorts) of cystic fibrosis (CF) patients. Buffer only was administered to the nose and lungs 9-14 days before nasal instillation of virus followed the day after by aerosolization of Ad CFTR to the lung. Nasal doses (defined in terms of viral plaque forming units, pfu) were 10(5), 10(7), and 4 x 10(8), whereas aerosolized doses were 10(7), 10(8), 5.4 x 10(8) for each cohort, respectively. No acute toxic effects were observed in the first 4 weeks after virus treatment. Shedding of infectious Ad CFTR was never detected, whereas detection of vector DNA sequences and CFTR expression demonstrated DNA transfer to the nose and airways of patients. No significant deviations in immunological and inflammatory parameters were observed in serum and in bronchoalveolar lavage (BAL). Importantly, for all patients, the serum anti-adenovirus antibody levels did not change significantly from baseline and no antibodies against adenovirus were found in BAL.

A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk.

We have investigated whether DNA regions present in the rabbit whey acidic protein (WAP) promoter/5' flanking sequence could potentially confer, in vivo, high level expression of reporter genes. Transgenic mice were generated expressing a variant of human alpha 1-antitrypsin, which has inhibitory activity against plasma kallikrein under the control of a 17.6 kbp DNA fragment located upstream of the rabbit WAP gene. Up to 10 mg/ml of active and correctly processed recombinant protein were detected in mouse milk, thus suggesting that the far upstream DNA sequences from the rabbit WAP gene might be useful for engineering efficient protein production in the mammary glands of transgenic animals.

In vitro and in vivo effects of glucocorticoids on gene transfer to skeletal muscle.

As a pharmacological approach to potentially improve gene transfer efficiency into skeletal muscle cells, glucocorticoids were shown here to allow efficient transfection of cultured and mouse human myoblasts, human pulmonary A549 cells, but not dog myoblasts, independently of the transfection protocol, the reporter gene and the transcription promoter employed. Transduction with adenovirus was also increased by dexamethasone. Pretreatment of cells 48 h prior to transfection was most effective and was shown to be concentration-dependent. This effect is mediated by binding to the glucocorticoid receptor, but not by glucocorticoid responsive elements present in the vectors. The acute dexamethasone effect could be due to increased plasmid entry into the cells as suggested by Southern blot, whereas the sustained increase of luciferase activity in dexamethasone-treated cultures may be related to intracellular mechanisms following cell entry. In mice in vivo, a similar increase of luciferase activity upon glucocorticoid treatment was found.

Proliferation of rat astrocytes, but not of oligodendrocytes, is stimulated in vitro by protease inhibitors.

Various natural protease inhibitors stimulate the proliferation of rat astrocytes grown in primary culture in the absence of serum. They are inactive on the proliferation of oligodendrocytes. The mean level of stimulation of the astrocyte proliferation elicited by the protease inhibitors is higher when the cells are in the growth phase, at low cell density than when they are quiescent, at high cell density. Among the protease inhibitors tested three serum proteins, alpha 1-antitrypsin, alpha 2-macroglobulin and anti-thrombin III were the most active. The present results, taken together with our previous finding that thrombin and some other proteases also stimulate the proliferation of astroglial cells but not of oligodendroglial cells, suggest that proteases and protease inhibitors participate, through still unclear mechanisms, in the control of the proliferation of astrocytes, but not in that of oligodendrocytes, during brain ontogenesis.

Thrombin is a potent mitogen for rat astroblasts but not for oligodendroblasts and neuroblasts in primary culture.

Astroblasts from brain of newborn rat can survive and even proliferate to some extent in a chemically defined medium containing no other growth factor than insulin, providing they are grown first in the presence of fetal calf serum for at least 4 days (Weibel et al., 1984, Int. J. devl Neurosci. 2, 355-366). We found that thrombin is a potent mitogen for these cells, in vitro. The mitogenic activity of thrombin for astroblasts can be compared to that of the astroglial growth factor on astroblasts. However, in contrast to the bFGF, thrombin does not modify significantly the morphology of the cells and their synthesis of glutamine synthetase, an astroglial marker in rat brain. Some other proteases are also able to stimulate the proliferation of astroblasts, but to a lesser extent than thrombin. Thrombin does not stimulate the proliferation of oligodendroblasts from newborn rat and of neuroblasts from 13-day-old rat embryo. These results suggest that in the central nervous system thrombin might play a role in the induction of astrocyte proliferation after brain injury.

Effects of acidic and basic fibroblast growth factors on proliferation and maturation of cultured rat oligodendrocytes.

A pure culture of oligodendrocytes has been developed starting from brain hemispheres of newborn rats. Various effects of acidic and basic fibroblast growth factors (FGFs) on the development of oligodendrocytes have been examined and compared. Both factors elicited similar effects, i.e. stimulation of the proliferation, inhibition of the specific activity of the marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase and decrease of the ratio of myelin basic protein positive cells. These results indicate that FGFs are very potent mitogens for oligodendrocytes, even in the absence of other cell types, but that they elicit a negative effect on the cell maturation, possibly related to their strong effect on proliferation.

Platelet-derived growth factor is a mitogen for glial but not for neuronal rat brain cells in vitro.

The effects of platelet-derived growth factor (PDGF) on the proliferation of isolated rat neural cells grown in serum-free chemically defined media have been investigated. It was found that PDGF drastically stimulates the proliferation of astroblasts and oligodendroblasts, but has no effect on the proliferation of neuroblasts in primary culture. A role of PDGF in the reactive gliosis, occurring after brain injury, can be suggested.

Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.

Primary cultures of astrocytes from different brain areas of newborn rats and effects of basic fibroblast growth factor.

The effects of basic fibroblast growth factor (bFGF) on the morphology and the expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) in cultured astrocytes prepared from various areas of newborn rat brain was studied. The brain was dissected in two ways, either the telencephalon (area A) and the diencephalon (area B) were dissected out of the brain (without olfactory bulbs, mesencephalon and cerebellum) or the brain was cut transversely into 3 parts (areas 1, 2 and 3). Area 1 (the anterior part) included the frontal cortex, the olfactory nuclei, the neostriatum, the accumbens nucleus and the septum; area 2 (the medial part) included the cortex, hippocampus, amygdale, thalamus and hypothalamus, and area 3 (the posterior part) included the occipital cortex, the posterior part of hippocampus and thalamus and the mamillary bodies. Essentially two different morphological aspects were observed. Most cells from areas A, 1 and 3, were flat, large, presented an irregular shape and were loosely arranged; cells from areas B and 2 were essentially polygonal in shape and closely apposed to each other. The various control cultures showed nearly the same immunostaining pattern for GFAP, but different patterns for GS. Most astroglial cells responded to bFGF and became fibrous. The GFAP immunoreaction was intense and localized in the cell bodies and processes of most cells from area A, but essentially in the processes for cells from areas 1 and 2. The immunoreactivity was weaker in cells from areas B and 3. GS-positive cells, heavily and weakly stained, were found in all treated cultures, and very strongly stained cells were located in certain zones of cultures from area A. But GS-negative cells were also seen in these treated cultures as well as in control cultures. Measurements of GS activities revealed no differences. These results indicate that astrocytes from different regions of the brain in primary culture show differences in their responsiveness to bFGF. The astroglial cells from the cerebral cortex and from the thalamus seem to present the highest and the lowest response to bFGF, respectively.

Neuronal-derived factors regulating glial cell proliferation and maturation.

We purified to homogeneity two active factors, named astroglial growth factors (AGFs: AGF2 and AGF1), from bovine brain after two and three chromatographic steps, respectively. We found that AGFs have a strong affinity for heparin. Therefore, heparin affinity chromatography was used to purify rapidly and efficiently these growth factors. The purified AGF1 is an acidic protein (pI: 5.5) with an apparent molecular weight of about 17,500 daltons; the AGF2 is a basic protein (pI: 9.5) of 18,500 daltons. The comparison of the physico-chemical properties, the aminoacid composition and the amino-terminal sequence of the AGFs with that of other growth factors isolated from the brain and affecting the proliferation of other cell types has indicated that AGF1 and AGF2 are identical to the acidic and basic fibroblast growth factor (FGF), respectively. Both factors stimulate the proliferation as well as the morphological and biochemical maturation of the astroglial cells. Both factors enhance also the multiplication of oligodendroglial cells. Polyclonal and monoclonal antibodies against AGFs have been prepared and used for immunocytochemical localization of these molecules in the rat brain and cerebellum. AGFs are found exclusively in neuronal cells.

Endogenous lectin CSL is present on the membrane of cilia of rat brain ependymal cells.

An endogenous brain lectin, with a great affinity for oligomannosidic glycans, called CSL (for 'cerebellar soluble lectin'), was detected on the surface of the cilia of ependymal cells both in cultures and in vivo. The lectin is not synthesized by the ependymal cells themselves. In vivo it is neither found in cerebrospinal fluid nor in cells of the choroid plexus. Probably, lectin CSL is produced by subependymal astrocytic cells. The membranes of ependymal cells seem to possess glycoprotein ligands for the lectin which explain the specific adhesion of CSL on the surface of these cells, particularly on the cilia. The localization of this adhesive molecule on cilia of ependymal cells suggests that it may play a role in trapping foreign cells, micro-organisms or debris.

An endogenous lectin found in rat astrocyte cultures has a role in cell adhesion but not in cell proliferation.

The presence of an endogenous cerebellar soluble lectin (CSL) has been demonstrated in cultured rat astrocytes by using immunocytochemical techniques. In these cells, the location of lectin CSL was found intracellularly as well as on the external surface of the plasma membrane of the cell bodies and processes, especially in the zones of contact between cells. This suggested that CSL could have a role in adhesion of astrocytes to sister cells. Kinetics of adhesion of astrocytes to culture dishes precoated with CSL showed a rapid binding of these cells. In confluent astrocyte cultures, anti-CSL Fab fragments affected the shape and organization of astrocytes (retraction of the cytoplasm), but they did not detach cells from the substratum. These results indicated that CSL has adhesive properties for astroglial cells and is probably involved 1) in adhesion of astrocytes to sister cells; 2) in binding of protoplasmic regions of astrocyte membrane to the substratum. Further support for these roles came from demonstration of the presence in cultures of glycoprotein ligands recognized by this lectin. The problem of the mitogenic properties of the lectin was also questioned. The addition of CSL to confluent astroglial cultures was able to stimulate only by 40% the proliferation of these cells at an optimal concentration of 5 micrograms CSL lectin/ml of culture medium. This indicated that CSL is not a powerful growth factor for astrocytes.

Comparison of the morphological effects of acidic and basic fibroblast growth factors on rat astroblasts in culture.

The effects of acidic and basic fibroblast growth factors (aFGF and bFGF) on the morphology of cultured rat astroblasts and on the expression of glial fibrillary acidic protein (GFAP) were compared. The addition of either aFGF or bFGF affected the morphology of the flat, irregular, polygonal-shaped astroblasts, which formed processes and acquire a fibrous appearance. Appreciable different morphological aspects were observed between aFGF- and bFGF-induced cells, essentially between 11 and 14 days in culture. In the presence of bFGF the astroglial cells were more fibrous with a more compact perikaryon as compared to aFGF treated cells. At the ultrastructural level abundant intermediate filaments were observed in astroglial cells as an effect of aFGF and rare filaments but numerous microtubules were seen in bFGF-treated cells. The immunoreactivity for GFAP increased with time in culture and was much stronger in aFGF-treated cells compared to bFGF-treated cells at day 14. An intense positive staining was observed in the somata of the astroglial cells and their processes in the presence of aFGF, while essentially the processes were stained in the presence of bFGF. After 21 days in culture GFAP immunoreaction was also found in the perikarya of cells treated with bFGF. These results show that rat astroglial cells respond somewhat differently to aFGF and bFGF.

Effects of acidic and basic fibroblast growth factors (aFGF and bFGF) on the proliferation and the glutamine synthetase expression of rat astroblasts in culture.

The two fibroblast growth factors called acidic and basic FGF (aFGF and bFGF) show a strong homology (55%) of their amino acid sequence (Esch et al.: Proc. Nat. Acad. Sci. USA 85:6507-6511, 1985). The effects of these factors on the rate of proliferation of rat astroblasts and on the expression of glutamine synthetase activity in cells grown in primary culture were investigated and compared under various culture conditions. In all the experimental conditions used, both growth factors triggered the proliferation of the cells to the same extent and with similar dose dependence. The mitogenic activities of aFGF and bFGF were potentiated similarly by heparan sulfate and by heparin, with a maximum stimulation of about 100% at 100 micrograms/ml heparin. Treatment of the cells with either of the two factors resulted in identical enhancement of the activity of glutamine synthetase relative to total proteins. These results suggest that both factors act either through the same membrane receptors or through different receptors that mediate nearly identical effects.

Transfection of myoblasts in primary culture with isomeric cationic cholesterol derivatives.

Transfection of satellite cells from dog muscle (myoblasts) in primary culture has been optimized with respect to the position of the cholesteryl moiety along the polyamine chain of spermidine or spermine. Spermidine or spermine were derivatized with cholesterylchloroformate giving rise to three isomers in the case of spermidine and two isomers for spermine that were separated by reversed-phase high-performance liquid chromatography (rp-HPLC). The position of the cholesteryl moiety was assigned by 13C-NMR and coelution with synthetic isomers of defined structure. The isomeric cationic lipids were evaluated for their transfection activity in myoblasts from dog muscle and a human lung epithelial cell line (A549) using plasmid DNA expressing the luciferase reporter gene. The results showed that the position of the cholesteryl moiety is of critical importance for efficient transfection of myoblasts in primary culture with isomers having a derivatized secondary amine being significantly more effective than those with a derivatized primary amine. On the contrary, differences in the A549 cell line were less pronounced and did not follow the same pattern. The results show that slight structural differences between cationic lipids lead to significantly different transfection efficiencies for myoblasts in primary culture. This may also represent an advantage in view of cell or organ targeting.

Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse).

A number of therapeutic plasma proteins are synthesized by human hepatocytes. Since many of these proteins undergo liver-specific post-translational modifications which are required for full biological activity, it may therefore be necessary to develop hepatocyte-based expression systems for their production. Using transgenic mice we have developed a transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human alpha 1 antitrypsin (alpha 1 AT), a plasma protein which is produced mainly in liver cells. This was achieved by co-expression of the mouse c-myc proto-oncogene and a genomic copy of the human alpha 1 AT gene, both under the control of the human alpha 1 AT promoter. Transgenic mice carrying this construct developed hepatomas producing human alpha 1 AT. Under defined culture conditions, cell lines secreting active alpha 1 AT were derived from these tumours. These cells maintain a differentiated hepatic phenotype and continue to secrete human alpha 1 AT for at least 40 generations.

Transforming growth factor type beta 1 modulates the effects of basic fibroblast growth factor on growth and phenotypic expression of rat astroblasts in vitro.

In a search of the growth factors possibly involved in brain ontogenesis we have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth and phenotypic expression of rat astroblasts in primary culture. Along TGF-beta 1 elicited only a slight negative effect on the growth of these cells. However, this factor was found to modulate the mitogenic effects of other growth factors. On quiescent cells it potentiates the mitogenic effect of basic fibroblast growth factor (bFGF) but not that of other growth factors, namely, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and thrombin. TGF-beta 1 did not modulate significantly the stimulatory effect of these growth factors on the activity of the enzyme glutamine synthetase (GS); but kinetic studies showed that TGF-beta 1 delays the stimulation of GS activity. DNA synthesis monitored by the incorporation of [125I]iododeoxyuridine (125I-dUrd) was maximum after 24-30 h of treatment with bFGF. With bFGF plus TGF-beta 1 the maximum was shifted to 30-36 h. This shift is compatible with the idea that TGF-beta 1 induces responsiveness in some cells which are otherwise unresponsive to the mitogenic action of bFGF, and that this induction requires some time. This hypothesis is sustained by the observation that in cells treated for only 12 h with bFGF, the treatment with TGF-beta 1 for the same 12 h or for longer time did not stimulate significantly the cell growth. Stimulation occurred only when the bFGF treatment was continued after 12 h. Potentiation of the mitogenic effect of bFGF and shift of the maximum 125I-dUrd incorporation towards 24 h was seen with cells pretreated with TGF-beta 1. This potentiation effect decreased with increasing time between the two treatments. The potentiation effect of TGF-beta 1 is not mediated by an induction of new bFGF membrane receptors as seen by binding studies.

Reactive astrogliosis after basic fibroblast growth factor (bFGF) injection in injured neonatal rat brain.

Reactive gliosis was revealed by immunocytochemistry using antibodies against the glial fibrillary acidic protein (GFAP) after a stab or an electrolytic lesion administered to the cerebral cortex, corpus callosum, striatum, or hippocampus of a 6-day-old rat. The intensity of the gliosis was about the same in the various structures injured and did not change with the delay of 3, 7, or 20 days between the injury and the sacrifice of the animals. When basic fibroblast growth factor (bFGF) was injected in the lesion locus just after the lesion was performed, it resulted (as soon as 3 days after injury) in a strong astrogliosis that was enhanced after a delay of 7 days, the astrocytes in the lesion area exhibiting enlarged cell processes and intense GFAP-positive immunoreactivity. After a delay of 20 days, the astrocytes were not dispersed any more but packed in three or four layers along the borders of the lesion, thus reducing its extension. This suggests a possible role for bFGF in promoting scar formation following brain injury.