Hans-Herbert Steiner

Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain.

Multidrug resistance proteins (MRPs, symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of organic anions, including cytotoxic and antiviral drugs, from cells. To identify MRP family members possibly involved in the intrinsic resistance of human brain to cytotoxic and antiviral drugs, we analyzed the expression and localization of MRP1-MRP6 in rapidly frozen perilesional samples of several regions of adult human brain obtained during neurosurgery. Quantitative polymerase chain reaction analysis showed expression of MRP1, MRP2, MRP3, MRP4, and MRP5 mRNA, whereas MRP6 mRNA was below detectability. However, immunofluorescence microscopy of cryosections from human brain showed no reactivity for the MRP2 or MRP3 proteins. The proteins MRP1, MRP4, and MRP5 were clearly localized by confocal laser scanning microscopy to the luminal side of brain capillary endothelial cells. The MRP4 and MRP5 proteins were also detected in astrocytes of the subcortical white matter. Notably, MRP5 protein was present in pyramidal neurons. MRP proteins may, thus, contribute to the cellular efflux of endogenous anionic glutathione or glucuronate conjugates (substrates for MRP1), cyclic nucleotides (substrates for MRP4 and MRP5), or glutathione (co-substrate for MRP1 and MRP4); in addition, they may play an important role in the resistance of the brain to several cytotoxic and antiviral drugs.

ABCC Drug Efflux Pumps and Organic Anion Uptake Transporters in Human Gliomas and the Blood-Tumor Barrier

Delivery of therapeutic agents to the brain and its neoplasms depends on the presence of membrane transport proteins in the blood-brain barrier and in the target cells. The cellular and subcellular localization of these membrane transporters determines the drug accessibility to the brain and its tumors. We therefore analyzed the expression and localization of six members of the multidrug resistance protein family of ATP-dependent efflux pumps (ABCC1-ABCC6, formerly MRP1-MRP6) and of six organic anion uptake transporters (OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, and OATP4A1) in 61 human glioma specimens of different histologic subtypes. Real-time PCRs indicated expressions of ABCC1, ABCC3, ABCC4, and ABCC5. In addition, we detected expressions of the OATP uptake transporter genes SLCO1A2, SLCO1C1, SLCO2B1, and SLCO4A1. At the protein level, however, only OATP1A2 and OATP2B1 were detectable by immunofluorescence microscopy in the luminal membrane of endothelial cells forming the blood-brain barrier and the blood-tumor barrier, but not in the glioma cells. ABCC4 and ABCC5 proteins were the major ABCC subfamily members in gliomas, localized both at the luminal side of the endothelial cells and in the glioma cells of astrocytic tumors and in the astrocytic portions of oligoastrocytomas. These results indicate that expression of ABCC4 and ABCC5 is associated with an astrocytic phenotype, in accordance with their expression in astrocytes and with the higher chemoresistance of astrocytic tumors as compared with oligodendrogliomas. Our data provide a basis for the assessment of the role of uptake transporters and efflux pumps in the accessibility of human gliomas for chemotherapeutic agents.

Clinical impact and functional aspects of tenascin-C expression during glioma progression.

The extracellular matrix protein tenascin‐C is expressed in processes like embryogenesis and wound healing and in neoplasia. Tenascin‐C expression in gliomas has been described previously; however, the relation to clinical data remains inconsistent. Generally, analysis of tenascin‐C function is difficult due to different alternatively spliced isoforms. Our studies focus on changes in tenascin‐C expression in human gliomas, correlating these changes with tumor progression and elucidating the functional role of the glioma cell‐specific tenascin‐C isoform pool. Eighty‐six glioma tissues of different World Health Organization (WHO) grades were analyzed immunohistochemically for tenascin‐C expression. The influence of the specific tenascin‐C isoforms produced by glioblastoma cells on proliferation and migration was examined in vitro using blocking antibodies recognizing all isoforms. In general, tenascin‐C expression increased with tumor malignancy. Perivascular staining of tenascin‐C around tumor‐supplying blood vessels was observed in all glioblastoma tissues, whereas in WHO II and III gliomas, perivascular tenascin‐C staining appeared less frequently. The appearance of perivascular tenascin‐C correlated significantly with a shorter disease‐free time. Analysis of proliferation and migration in the presence of blocking antibodies revealed an inhibition of proliferation by around 30% in all 3 glioblastoma cell cultures, as well as a decrease in migration of 30.6–46.7%. Thus we conclude that the endogenous pool of tenascin‐C isoforms in gliomas supports both tumor cell proliferation and tumor cell migration. In addition, our data on the perivascular staining of tenascin‐C in WHO II and III gliomas and its correlation with a shorter disease‐free time suggest that tenascin‐C may be a new and potent prognostic marker for an earlier tumor recurrence.

Different angiogenic phenotypes in primary and secondary glioblastomas

Primary and secondary glioblastomas (pGBM, sGBM) are supposed to evolve through different genetic pathways, including EGF receptor and PDGF and its receptor and thus genes that are involved in tumor‐induced angiogenesis. However, whether other angiogenic cytokines are also differentially expressed in these glioblastoma subtypes is not known so far, but this knowledge might be important to optimize an antiangiogenic therapy. Therefore, we studied the expression of several angiogenic cytokines, including VEGF‐A, HGF, bFGF, PDGF‐AB, PDGF‐BB, G‐CSF and GM‐CSF in pGBMs and sGBMs as well as in gliomas WHO III, the precursor lesions of sGBMs. In tumor tissues, expression of all cytokines was observed albeit with marked differences concerning intensity and distribution pattern. Quantification of the cytokines in the supernatant of 30 tissue‐corresponding glioma cultures revealed a predominant expression of VEGF‐A in pGBMs and significantly higher expression levels of PDGF‐AB in sGBMs. HGF and bFGF were determined in nearly all tumor cultures but with no GBM subtype or malignancy‐related differences. Interestingly, GM‐CSF and especially G‐CSF were produced less frequently by tumor cells. However, GM‐CSF secretion occurred together with an increased number of simultaneously secreted cytokines and correlated with a worse patient prognosis and may thus represent a more aggressive angiogenic phenotype. Finally, we confirmed an independent contribution of each tumor‐derived cytokine analyzed to tumor‐induced vascularization. Our data indicate that an optimal antiangiogenic therapy may require targeting of multiple angiogenic pathways that seem to differ markedly in pGBMs and sGBMs.

Autocrine Growth Regulation by Granulocyte Colony-Stimulating Factor and Granulocyte Macrophage Colony-Stimulating Factor in Human Gliomas with Tumor Progression

Granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) and/or their receptors are increasingly detected in solid human tumors, although little is known about their function in tumor growth and invasion. We analyzed RNA and protein expression of both factors and their receptors in 22 human gliomas (WHO grade II, III, and IV) and derived cell cultures. G-CSF, GM-CSF, and/or their receptors were expressed in all tumors and derived cell cultures, but coexpression of both factors and receptors was almost exclusively found in grade IV glioblastomas and thus correlated with advanced tumor stage. The functional significance of G-CSF and GM-CSF as regulators for glioma cells was demonstrated by 1) stimulation of proliferation and migration in tumor cells expressing one or both receptors by the corresponding factor; 2) inhibition of growth and migration of glioma cells expressing G-CSF, GM-CSF, and their receptors by neutralizing antibodies to both factors. These results indicate a significant role for both factors in the autocrine regulation of growth and migration in late-stage malignant gliomas and suggest a shift from paracrine to autocrine regulation with tumor progression. The implication of G-CSF and GM-CSF in glioblastoma growth regulation could make these factors further prognostic indicators and raises questions concerning their use in cancer therapy.

Autocrine pathways of the vascular endothelial growth factor (VEGF) in glioblastoma multiforme: clinical relevance of radiation-induced increase of VEGF levels.

In tumour-induced angiogenesis of gliomas, vascular endothelial growth factor (VEGF) and its receptors fms-like tyrosine kinase (Flt-1) and kinase-insert-domain-containing receptor (KDR) play a major role and are promising targets for tumour therapy. Nevertheless, preliminary results of such therapies could not prove clinical efficacy and thus make a profound knowledge of VEGF regulation essential. Based on earlier results, which demonstrated an inhibitory influence of VEGF on Flt-1-expressing glioblastoma cells [1], in the present study we focused on the extent of VEGF and VEGF receptor coexpression and possible therapeutical consequences.Protein expression of VEGF, Flt-1 and KDR was analysed by immunohistochemistry in native tumour tissues of 63 glioblastomas. VEGF could be detected in all glioblastomas. Additionally and independently to the expected Flt-1 and KDR expression in tumour endothelia, we found a coexpression of VEGF with Flt-1 in tumour cells of 46 and with KDR in 45 glioblastomas. After exposure of glioblastoma cells to X-ray radiation we observed a strong dose-dependent increase of VEGF secretion in two glioblastoma cell cultures by up to 46% and 96%, respectively that originated from an increased VEGF mRNA expression. In contrast, under the same conditions secretion of HGF/SF was only slightly elevated and bFGF despite being strongly increased remained at very low overall amounts compared to VEGF. Based on previous data on an autocrine function of VEGF in Flt-1-expressing glioblastoma cells we hypothesise that the X-ray radiation induced upregulation of VEGF might result in a downregulation of tumour cell proliferation and thus lead to a reduced sensitivity to radiation therapy. Therefore our results support the idea that a combination of anti-VEGF and radiation therapy might prove a promising new option in fighting against one of the most fatal tumour types.

Low frequency of chromosomal imbalances in anaplastic ependymomas as detected by comparative genomic hybridization.

We screened 26 ependymomas in 22 patients (7 WHO grade I, myxopapillary, myE; 6 WHO grade II, E; 13 WHO grade III, anaplastic, aE) using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). 25 out of 26 tumors showed chromosomal imbalances on CGH analysis. The chromosomal region most frequently affected by losses of genomic material clustered on 13q (9/26). 6/7 myE showed a loss on 13q14‐q31. Other chromosomes affected by genomic losses were 6q (5/26), 4q (5/26), 10 (5/26), and 2q (4/26). The most consistent chromosomal abnormality in ependymomas so far reported, is monosomy 22 or structural abnormality 22q, identified in approximately one third of Giemsabanded cases with abnormal karyotypes. Using FISH, loss or monosomy 22q was detected in small subpopulations of tumor cells in 36% of cases. The most frequent gains involved chromosome arms 17 (8/26), 9q (7/26), 20q (7/26), and 22q (6/26). Gains on 1q were found exclusively in pediatric ependymomas (5/10). Using FISH, MYCN proto‐oncogene DNA amplifications mapped to 2p23‐p24 were found in 2 spinal ependymomas of adults. On average, myE demonstrated 9.14, E 5.33, and aE 1.77 gains and/or losses on different chromosomes per tumor using CGH. Thus, and quite paradoxically, in ependymomas, a high frequency of imbalanced chromosomal regions as revealed by CGH does not indicate a high WHO grade of the tumor but is more frequent in grade I tumors.

Cell death induction by betulinic acid, ceramide and TRAIL in primary glioblastoma multiforme cells

SummaryBackground. Glioblastoma multiforme (WHO Grade IV, GBM) is the most malignant brain tumour with a mean survival time of less than one year. Betulinic acid, ceramide and TRAIL (TNF-related apoptosis inducing ligand) represent novel therapeutic agents for potential use in GBM. Method. Primary GBM cells of 21 patients with macroscopically complete tumour resection were tested in vitro for cell death induction by betulinic acid, ceramide, TRAIL and established therapeutics (BCNU, cisplatin, doxorubicin, vincristin and γ-irradiation). Findings. At peak plasma concentrations (PPC), Betulinic acid, ceramide and TRAIL induced cell death in primary GBM cells at higher rates than established cytotoxic drugs. Specific cell death ≥75% was observed in 43% (9/21), 38% (8/21), and 19% (4/21) for betulinic acid, ceramide, and TRAIL respectively, while this was only found in 5% (1/21) of γ-irradiated and cisplatin-treated cells, and in none of the GBM cultures, where BCNU or vincristin were applied in PPC. Conclusion. Due to a markedly improved cell death of GBM cells as compared with established therapeutics, Betulinic acid, ceramide and TRAIL might represent potent substances for future treatment of GBM.

Entrapment neuropathy at the cubital tunnel: simple decompression is the method of choice.

SummaryWe present a retrospective study of 41 patients treated by simple decompression for ulnar neuropathy.Pre- and postoperatively, patients were evaluated clinically and electrophysiologically. The median follow-up was 2 years (minimum: 0.5 years, maximum: 5.1 years). The leading pre-operative sign was motor loss in the ulnar distribution (36 patients = 89%) with consecutive atrophy of ulnar innervated muscles (30 patients = 75%). The secondary complaint was sensory impairment in 59% of all cases, less frequently patients presented with pain or paraesthesia. In the majority of cases the aetiology remained unknown (27 patients = 65%). When aetiology was known, previous trauma to the elbow was reported most frequently (9 patients = 22%).Motor nerve conduction velocity (mNCV), compared to the contralateral, non-involved arm, was lower at least for 10 m/s. In cases with atrophy of the ulnar innervated muscles the difference was greater than 15 m/s.In 89%, postoperative results were good or even very good. In 8% (3 patients) no improvement was observed. Worsening due to surgery did not occur. We could demonstrate a significant increase in postoperative mNCV of 7.95 m/s in all patients (p < 0.05).There is still disagreement as to the correct surgical treatment of this disorder. We favour simple decompression (SD) as the appropriate operative technique for cubital tunnel syndrome.

Lower limb compartment syndrome following lumbar discectomy in the knee-chest position

We report on two cases of compartment syndrome following lumbar discectomy in the knee-chest position. This complication has only been described once since 1953. Seven cases of compartment syndrome following other surgical procedures were found in the literature. Any increases in tissue pressure of a muscle compartment exceeding 35–40 mmHg over a longer period of time can result in this complication for example, too tight cast, space-occupying intrafascial bleeding, or postischemic swelling. The diagnosis is purely clinical and is based on the typical combination of extremely painful edema with rapid onset of sensory loss and subsequent motor deficits. The muscle necrosis leads to myoglobinaemia and myoglobinuria, recognizable by brown urine. The therapy consists of urgent fasciotomy of swollen compartements without skin suture. The prognosis is highly dependent on the time of the surgical decompression: within 6 hours serious deficits are avoidable; after 24 hours irreversible necrosis of muscle occurs. It seems that the possibility of a compartment syndrome is a specific, but a rare risk of a prolonged knee-chest position.