Karl-Hermann Schlingensiepen

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.

Antisense Therapeutics for Tumor Treatment: The TGF-beta2 Inhibitor AP 12009 in Clinical Development Against Malignant Tumors

Overexpression of the cytokine transforming growth factor-beta 2 (TGF-beta2) is a hallmark of various malignant tumors including pancreatic carcinoma, malignant glioma, metastasizing melanoma, and metastatic colorectal carcinoma. This is due to the pivotal role of TGF-beta2 as it regulates key mechanisms of tumor development, namely immunosuppression, metastasis, angiogenesis, and proliferation. The antisense technology is an innovative technique offering a targeted approach for the treatment of different highly aggressive tumors and other diseases. Antisense oligonucleotides are being developed to inhibit the production of disease-causing proteins at the molecular level. The immunotherapeutic approach with the phosphorothioate oligodeoxynucleotide AP 12009 for the treatment of malignant tumors is based on the specific inhibition of TGF-beta2. After providing preclinical proof of concept, the safety and efficacy of AP 12009 were assessed in clinical phase I/II open-label dose-escalation studies in recurrent or refractory high-grade glioma patients. Median survival time after recurrence exceeded the current literature data for chemotherapy. Currently, phase I/II study in advanced pancreatic carcinoma, metastatic melanoma, and metastatic colorectal carcinoma and a phase IIb study in recurrent or refractory high-grade glioma are ongoing. The preclinical as well as the clinical results implicate targeted TGF-beta2 suppression as a promising therapeutic approach for malignant tumor therapy.

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.

Transforming growth factor-beta 2 gene silencing with trabedersen (AP 12009) in pancreatic cancer

Pancreatic cancer is one of the most aggressive human cancers with a 5‐year survival rate of <5%. Overexpression of transforming growth factor‐beta 2 (TGF‐β2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF‐β2 mRNA and was successfully tested in a randomized, active‐controlled phase IIb clinical study in patients with high‐grade glioma. Here, we report on the antitumor activity of trabedersen in human pancreatic cancer cells and in an orthotopic xenograft mouse model of human metastatic pancreatic cancer. Trabedersen reduced TGF‐β2 secretion in human pancreatic cell lines with an IC50 in the low μM range without transfection reagent, clearly inhibited cell proliferation, and completely blocked migration of pancreatic cancer cells. Additionally, trabedersen reversed TGF‐β2‐mediated immunosuppression of pancreatic cancer cells targeted by lymphokine activated killer (LAK) cells, resulting in considerably increased LAK cell‐mediated cytotoxicity. Moreover, in an orthotopic mouse model of metastatic pancreatic cancer, intraperitoneal (i.p.) treatment with trabedersen significantly reduced tumor growth, lymph node metastasis and angiogenesis. These promising results warrant further clinical development of trabedersen. Cancer Sci 2011; 102: 1193–1200)

The Antisense Oligonucleotide Trabedersen (AP 12009) for the Targeted Inhibition of TGF-β2

Despite remarkable advances in cancer research, patients with malignant tumors such as high-grade glioma or advanced pancreatic carcinoma still face a poor prognosis. Because of the severe morbidity and mortality of such malignant tumor types, the identification of suitable molecular drug targets for causal treatment approaches is an important area of current research. Transforming growth factor-beta 2 (TGF-β2) is an attractive target because it regulates key mechanisms of carcinogenesis, in particular immunosuppression and metastasis, and is frequently overexpressed in malignant tumors. Here we describe the development of the antisense phosphorothioate oligodeoxynucleotide trabedersen (AP 12009) which was designed for the specific inhibition of TGF-β2 biosynthesis. In vitro and in vivo experiments confirmed the mode of action, efficacy and tolerability of trabedersen and paved the way for clinical studies. In patients with high-grade glioma, intratumoral treatment with trabedersen is currently evaluated in a pivotal, randomized and active-controlled phase III study. Intravenous application of trabedersen for the treatment of patients with advanced pancreatic carcinoma, metastasizing melanoma, or metastatic colorectal carcinoma is assessed in a currently ongoing phase I/II dose escalation study.

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%.