Transforming growth factor-beta2 (TGF-beta2) is known to suppress the immune response to cancer cells and plays a pivotal role in tumor progression by regulating key mechanisms including proliferation, metastasis, and angiogenesis. For targeted protein suppression the TGF-beta2-specific antisense oligodeoxynucleotide AP 12009 was developed. In vitro experiments have been performed to prove specificity and efficacy of the TGF-beta2 inhibitor AP 12009 employing patient-derived malignant glioma cells as well as peripheral blood mononuclear cells (PBMCs) from patients. Clinically, the antisense compound AP 12009 was assessed in three Phase I/II-studies for the treatment of patients with recurrent or refractory malignant (high-grade) glioma WHO grade III or IV. Although the study was not primarily designed as an efficacy evaluation, prolonged survival compared to literature data and response data were observed, which are very rarely seen in this tumor indication. Two patients experienced long-lasting complete tumor remissions. These results implicate targeted TGF-beta2-suppression using AP 12009 as a promising novel approach for malignant gliomas and other highly aggressive, TGF-beta-2-overexpressing tumors.

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The 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.

Here, we provide first evidence that long-term continuous infusion of highly purified antisense phosphorothioate oligodeoxynucleotides (S-ODN) into brain parenchyma is well tolerated and thus highly suitable for in vivo application. AP 12009 is an S-ODN for the therapy of malignant glioma. It is directed against human transforming growth factor-beta (TGF-beta2) mRNA. In the clinical setting, AP 12009 is administered intratumorally by continuous infusion directly into the brain tumor. In view of this clinical application, the focus of our data is on local toxicology studies in rabbits and monkeys to evaluate the safety of AP 12009. AP 12009 was administered either by intrathecal bolus injection into the subarachnoidal space of the lumbar region of both cynomolgus monkeys and rabbits or by continuous intraparenchymatous infusion directly into the brain tissue of rabbits. Intrathecal bolus administration of 0.1 ml of 500 microM AP 12009 showed neither clinical signs of toxicity nor macroscopically visible or histomorphologic changes. After a 7-day intraparenchymatous continuous infusion of 500 microM AP 12009 at 1 microl/h in rabbits, there was no evidence of toxicity except for local mild to moderate lymphocytic leptomeningoencephalitis. Additionally, AP 12009 showed good tolerability in safety pharmacology as well as in acute toxicity studies and 4-week subchronic toxicity studies in mice, rats, and monkeys. This favorable safety profile proves the suitability of AP 12009 for local administration in brain tumor patients from the point of view of toxicology.

1. To investigate the role of the Jun transcription factors in neuronal differentiation, programmed neuronal cell death, and neuronal plasticity, we used phosphorothioate oligodeoxynucleotides (S-ODN) to inhibit selectively the expression of c-Jun, JunB, and JunD. 2. We have shown previously that in contrast to c-Jun, the JunB and JunD transcription factors are negative regulators of cell growth in various cell lines. Here we confirm this finding in primary human fibroblasts. 3. c-Jun and JunB are counterplayers not only with respect to proliferation, but also in cell differentiation. Since JunB expression is essential for neuronal differentiation, we analyzed possible posttranslational modifications of JunB after induction of PC-12 cell differentiation by nerve growth factor (NGF). 4. JunB was strongly phosphorylated after induction of PC-12 cell differentiation with NGF but not after stimulation of cell proliferation with serum. Thus, while cell proliferation is associated with c-Jun phosphorylation, cell differentiation is correlated with JunB phosphorylation. This supports the finding that c-Jun and JunB play antagonistic roles in both proliferation and differentiation. 5. The JunB transcription factor together with the c-Fos transcription factor is also induced in vivo in the suprachiasmatic nucleus (SCN) of rat brain after a light stimulus that induces resetting of the circadian clock. 6. Using antisense oligonucleotides injected into the third ventricle, we selectively cosuppressed the two transcription factors in vivo as shown by immunohistochemistry. Expression of c-Jun, JunD, and FosB was not affected. Inhibition of JunB and c-Fos expression prevented the light-induced phase shift of the circadian rhythm. In contrast, rats injected with a randomized control oligonucleotide showed the same phase shift as untreated animals. 7. In primary rat hippocampal cultures, anti-c-jun S-ODN selectively inhibited neuronal cell death and promoted neuronal survival. This indicates a causal role of c-Jun in programmed neuronal cell death. 8. These findings demonstrate the essential role of inducible transcription factors in the reprogramming of cells to a different functional state. Jun transcription factors play an essential role not only in fundamental processes such as cell proliferation, differentiation, and programmed neuronal cell death, but also in such complex processes as plastic adaptations in the mature brain. The inhibition of neuronal cell death by anti-c-jun S-ODN shows the great therapeutic potential of selective antisense oligonucleotides.

Induction of the jun-B and/or c-jun transcription factors is part of the immediate early response to diverse stimuli that induce alterations in cellular programs. While c-jun is a protooncogene whose expression is required for induction of cell proliferation, jun-B has recently been found to be induced by stimuli inducing differentiation in various cell lines. Furthermore, its expression is largely restricted to differentiating cells during embryogenesis. To determine the functional significance of these findings, we used antisense phosphorothioate oligodeoxynucleotides to inhibit expression of the two genes in proliferating and neuronally differentiating cells. While selective inhibition of c-jun expression reduced proliferation rates, inhibition of jun-B protein synthesis markedly increased proliferation in 3T3 fibroblasts, human mammary carcinoma cells and PC-12 pheochromocytoma cells, suggesting jun-B involvement in negative growth control. Neuronal differentiation of PC-12 cells induced by nerve growth factor (NGF) was prevented by inhibition of jun-B protein synthesis. PC-12 cells not only failed to grow neurites but also remained in the proliferative state. Furthermore, in cultured primary neurons from rat hippocampus, inhibition of jun-B expression, again, markedly reduced morphological differentiation. Conversely, inhibition of c-jun protein synthesis enhanced morphological differentiation of both primary neurons and PC-12 tumor cells. Thus, jun-B expression is required for neuronal differentiation and its balance with c-jun activity is involved in regulating key steps in proliferation and differentiation processes.

This paper addresses the question of a general topological principle of thalamo-cortical projections. In the lissencephalic primate brain of the common marmoset (Callithrix jacchus), large injections of horseradish peroxidase were made in various parts of the neocortex. These injections were placed in different animals and hemispheres along various caudo-rostral and medio-lateral gradients. Labelled cells in the thalamus were plotted and the labelling-zones resulting from several injections along a medio-lateral and two caudo-rostral cortical vectors were drawn into semi-schematic thalamic maps. These composite maps reveal a topological organization of the whole thalamo-cortical projection. The thalamic representation of the caudo-rostral and medio-lateral gradients indicate a rotation of the posterior relative to the anterior thalamus. An attempt is made to relate the organization of the thalamo-cortical projection to the development of the thalamus and the cortex. The cortex is divided into concentric zones around the sensory-motor and insular cortex. The thalamus is divided into corresponding projection zones. The topology of thalamo-cortical connections can then be regarded as a consequence of corresponding thalamic and cortical growth gradients. This is not only consistent with the general thalamo-cortical topology and the inversion of maps from thalamus to cortex, but also explains the continuity and overlap of thalamic projection zones in the pulvinar to widely separated cortical areas as the parietal, temporal and frontal association cortex.

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