Wolfgang Brysch

Block of c-Fos and JunB Expression by Antisense Oligonucleotides Inhibits Light-induced-Phase Shifts of the Mammalian Circadian Clock

Light‐induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c‐Jun, JunB, c‐Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c‐Fos and JunB, the predominantly expressed AP‐1 proteins for the phase‐shifting effects of light, we blocked the expression of c‐Fos and JunB in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 μ1 of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c‐fos and JunB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induced a significant phase shift (by 125 ± 15 min) of the circadian locomotor activity rhythm, whereas application of AS0 6 h before the light pulse completely prevented this phase shift. Application of control nonsense oligodeoxynucleotides had no effect. ASO strongly reduced the light‐induced expression of c‐Fos and JunB proteins. In contrast, light pulses with or without the control nonsense oligodeoxynucleotides evoked strong nuclear c‐Fos and JunB immunoreactivity in the rat suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription factors such as c‐Fos and JunB are an essential part of fundamental biological processes in the adult mammalian nervous system, e.g. of light‐induced phase shifts of the circadian pacemaker.

Transforming growth factor-beta-mediated autocrine growth regulation of gliomas as detected with phosphorothioate antisense oligonucleotides

Transforming growth factors‐β1 and ‐β2 (TGF‐β1 and ‐β2) are important growth‐regulatory proteins for astroglial neoplasms. We analyzed their role in tumor‐cell proliferation in 12 glioma cell lines, employing phosphorothioate antisense oligodeoxynucleotides (S‐ODNs, 14 mer), specifically targeted against the coding sequences of TGF‐β1‐mRNA and TGF‐β2‐mRNA. TGF‐β1‐S‐ODNs inhibited cell proliferation in 5 of 12 gliomas, whereas TGF‐β2‐S‐ODNs reduced the cell proliferation in all glioma cell lines, compared to nonsense‐S‐ODN‐treated and S‐ODN‐untreated cells as controls. The efficacy and specificity of antisense effects was validated by Northern‐blot analysis and determination of protein concentrations in culture supernatants (ELISA). Exogenous hrTGF‐β1 either stimulated or inhibited the cell lines, whereas pnTGF‐β2 stimulated the proliferation of most glioma cells. Blocking the extracellular pathway of TGF‐β by neutralizing antibodies only slightly inhibited those cell lines, which were markedly stimulated by TGF‐βs. As the effects of TGF‐β2‐S‐ODNs were much stronger than those of TGF‐β neutralizing antibodies, we postulate that the endogenously produced TGF‐β2 control glioma‐cell proliferation, in part by an intracellular loop.

High basal expression of the Full-size image (<1 K) immediate early gene in cortical layers IV and VI, in CA1 and in the corpus striatum - an in situ hybridization study.

The localization of zif/268 gene expression in adult rat and mouse brain was studied with in the situ hybridization, using 32P-labeled 30 mer oligonucleotide probes. Basal expression without intentional neuronal stimulation was examined. Distribution of zif/268 mRNA was highly differential. In the neocortex a layer specific expression was seen with high levels in layers IV and VI, most prominent in the visual and somatosensory cortices. In the hippocampus labeling was strongest in CA1 but very low in the dentate gyrus. Strong expression was also seen in the primary olfactory cortex, the corpus striatum, the nuclei amygdaloidei, the nucleus accumbens and the cerebellar cortex.

Regional and temporal expression of sodium channel messenger RNAs in the rat brain during development

SummaryThe distribution of mRNA expression for three types of voltage gated neuronal sodium-channels was studied in the rat brain at different developmental stages (embryonal day E18, postnatal day P5 and adult). With the in-situ hybridization technique, using synthetic DNA-oligomer probes, pronounced regional and temporal variations in the expression levels of the different channel subtypes could be detected. In comparison with types I and III, sodium channel II mRNA was the most abundant subtype at all developmental stages. Maximal expression of sodium channel II mRNA was seen at P5 in virtually all parts of the grey matter, except for the cerebellum. In adult rat brain in contrast, sodium channel II mRNA levels were maximal in the granular layer of the cerebellum, whereas in all other regions expression had decreased to roughly 50% of postnatal levels. Na channel I expression was virtually absent at E18 and showed highest levels at P5, with maxima in the caudate nucleus and hippocampus. In the adult brain, expression of Na-channel I was nearly absent in the neocortex, but well detectable in the cerebellum and, at lower levels in the striatum and thalamus. Sodium channel III was mainly expressed at the embryonal stage and showed a decrease to very low levels with little regional preferences in the adult.

Autocrine growth regulation in neuroectodermal tumors as detected with oligodeoxynucleotide antisense molecules.

The cell lines of three neuroectodermal tumors, two glioblastomas (HTZ-146, HTZ-17) and one melanoma (HTZ-19) were established and screened for the expression of growth factors by northern blotting and immunochemical methods. All three tumors were positive for platelet-derived growth factor- (PDGF-) A-, -B-chain, and basic fibroblast growth factor (bFGF) messenger ribonucleic acids. Cultured cells as well as original tumor material were also positive for PDGF-AA-, PDGF-BB, and bFGF protein, as shown by immunochemistry. To investigate the possible pathophysiological role of PDGF and bFGF, antisense technology was employed with chemically modified nuclease-stable 14-mer phosphorothioate oligodeoxynucleotides. Proliferation of all three tumors was reduced to a different extent with antisense phosphorothioate oligodeoxynucleotides in vitro, targeted against PDGF-A-chain-, -B-chain-, and -bFGF-messenger ribonucleic acid. These data indicate autocrine stimulatory loops for PDGF and bFGF, which may be blocked, may have different relevance in neuroectodermal tumors in vitro, and may have conceivable future therapeutic implications.

Design and application of antisense oligonucleotides in cell culture,in vivo, and as therapeutic agents

Summary1. Synthetic oligonucleotides can inhibit the expression of a gene in a sequence specific manner on the transcriptional and translational level. These molecules are usually referred to as antisense oligonucleotides.2. Antisense mediated inhibition of gene expression is a valuable tool to analyze the function of a genein vivo and can also be used for therapeutic gene suppression.3. A number of factors such as the mode of action, specificity, chemistry, and pharmacology must be carefully considered for the design and successful application of antisense oligonucleotides.4. Assay systems and controls must be chosen as to assure that the observed biological effects of antisense oligonucleotides do in fact reflect the result of a specific gene inhibition.5. This article critically discusses these factors in view of the literature and our own experience with a wide range of cell types and animal models, targeting different genes. The emphasis is on the use of phosphorothioate oligodeoxynucleotides in cell cultures,in vivo, and as potential drugs.

Sequence specific impairment of learning by C-jun antisense oligonucleotides

Hybridization studies revealed a differential accumulation of c-jun and jun B mRNA in the hippocampus and cerebral cortex of rats trained on a foot-shock-motivated brightness discrimination. Supposing that early gene expression is functionally significant for plastic changes in the brain, we used antisense phosphorothioate oligo- deoxynucleotides (S-ODN) in vivo to study effects of specific inhibition of c-jun and jun B gene expression on learning and memory formation in rat brain. Discrimination performance of rats was impaired after intrahippo- campal injection of anti-c-jun S-ODN but not of anti-jun B S-ODN. These results imply that topically injected anti- sense oligonucleotides affect processes involved in learning and memory in a sequence-specific manner.

Developmental expression of the transcription factor zif268 in rat brain

Changes in the distribution pattern of mRNA encoding the zif268 transcription factor (also referred to as NGFI-A, Krox-24 or EGR-1) were investigated by in situ hybridization histochemistry during postnatal rat brain development. Marked changes in zif268 expression patterns were seen in particular in the cerebral cortex and the hippocampal formation during the first 3 wk. In the 1st postnatal week, zif268 mRNA levels were highest in the corpus striatum and the piriform cortex. In the neocortex, expression rose sharply in the sensorymotor area between postnatal days (PNDs) 10 and 12. In the frontal and occipital cortex, in contrast, an increase in zif268 mRNA levels was first seen on PND 14. After PND 17, levels decreased in the sensorymotor and the frontal cortex but remained high in the occipital and the piriform cortex. In the hippocampus, an initially uniform increase in expression during the 2nd week was followed by a marked dissociation in expression levels between CA1, with continuously high expression levels on the one hand, and CA3, CA4 and the dentate gyrus, with a strong decline of expression during the 3rd week, on the other hand. Our results indicate that zif268 expression displays a highly dynamic expression pattern during plastic adaptations of different cerebral subregions during postnatal development, suggesting a possible involvement in gene regulatory processes during these phases.

bFGF induces its own gene expression in astrocytic and hippocampal cell cultures

Basic FGF mRNA induction by bFGF was investigated in cell cultures from rat brain, i.e. postnatal day 2 cortex and embryonic day 18 hippocampus. In situ hybridization shows that after bFGF treatment (10(-10) M) for 14 h neurones and glial cells show a remarkable increase in bFGF mRNA production. Incubation of astrocytes with antisense bFGF phosphorothioate oligodeoxynucleotides (bFGF-PTOs) resulted in an inhibition of both bFGF induced and serum induced proliferation. The results indicate that bFGF is capable of inducing its own mRNA production. This induction, i.e. new synthesis of bFGF mRNA, seems to be essential for the mitogenic effect of both bFGF and serum components.

Reversal of multiple drug resistance in vitro by phosphorathioate oligonucleotides and ribozymes

In the present study we investigated the effectiveness of 14, 15 and 18 nucleotide antisense phosphorothioate oligonucleotides (S-ODNs) directed to four different regions of the published mdr-1 gene sequence to reduce the level of mdr-1 gene product (p170, P-glycoprotein) and its function in the over-expressing cell lines LoVoDxR, S180DxR and KBChR8–5. The highest efficiency in reduction of multiple drug resistance was obtained at a concentration of 2 μM. In proliferation assays a growth reduction of 50% was observed after exposure of doxorubicin-resistant cells to S-ODN3. p170 protein expression of the resistant cell line LoVoDxR was reduced to the level of the sensitive cell line LoVo as shown by Western blot analysis. S-ODN3 reduced the ID50 of the two human cell lines up to 60% (LoVoDxR) and 21% (KBChR8–5), respectively, but showed no effect in the murine cell line S180DxR. In contrast, S-ODN1 was most effective in the murine system (67% reduction of the ID50), less effective in LoVoDxR (34%) and exhibited no effect in cell line KBChR8–5. Based on the results with the antisense oligonucleotides, a ribozyme directed against the mRNA target region of S-ODN3 was designed. This ribozyme was able to reduce the mdr-1 mRNA in total RNA preparations from cell line LoVoDxR up to 80% after an incubation time of 6 h in the presence of 10 mM MgCI2 at pH 7.5. A modified ribozyme was investigated in cell culture and reduced chemo-resistance of the resistant cell line LoVoDxR and ex vivo cultured blasts of acute myelold leukemia patients up to 50%.