Elmar Kirches

High frequency of mitochondrial DNA mutations in glioblastoma multiforme identified by direct sequence comparison to blood samples.

In an earlier study, we showed that heteroplasmy in the mitochondrial genome of gliomas sometimes occurs in a D‐loop polycytosine tract. We extended this study by pairwise comparisons between glioma samples and adjacent brain tissue of 55 patients (50 glioblastomas, 1 astrocytoma WHO grade III, 4 astrocytomas WHO grade II). We used a combination of laser microdissection and PCR to detect and quantify variations in the polycytosine tract. New length variants undetectable in the adjacent brain tissue were observed in 5 glioblastomas (9%). In 2 of these cases, samples from a lower tumor stage (WHO grade II) could be analyzed and revealed the early occurrence of these mutations in both cases. Since the mitochondrial D‐loop contains additional repeats and highly polymorphic non‐coding sequences, we compared 17 glioblastomas with the corresponding blood samples of the same patients by direct sequencing of the complete D‐loop. In 6 of these tumors (35%), instability was detected in 1 or 2 of 3 repeat regions; in 1 of these repeats, the instability was linked to a germline T‐to‐C transition. Furthermore, of 2 tumors (12%) 1 carried 1 and the other 9 additional transitions. In the latter patient, 6.7 kb of the protein coding mtDNA sequence were analyzed. Six silent transitions and 2 missense mutations (transitions) were found. All base substitutions appeared to be homoplasmic upon sequencing, and 89% occurred at known polymorphic sites in humans. Our data suggest that the same mechanisms that generate inherited mtDNA polymorphisms are strongly enhanced in gliomas and produce somatic mutations.

Visualization of defective mitochondrial function in skeletal muscle fibers of patients with sporadic amyotrophic lateral sclerosis

The mitochondrial function in skeletal muscle was investigated in skeletal muscle biopsies of 26 patients with sporadic amyotrophic lateral sclerosis (ALS) and compared with investigations of 28 age-matched control muscle samples and biopsies of 6 patients with spinal muscular atrophy (SMA) and two patients with Tay-Sachs disease. In comparison to the control, SMA and Tay-Sachs biopsies, we observed in the ALS samples a significant about two-fold lower activity of complex I of mitochondrial respiratory chain. To visualise the distribution of the mitochondrial defect in skeletal muscle fibers we applied confocal laser-scanning microscopy and video fluorescence microscopy of NAD(P)H and fluorescent flavoproteins. The redox change of mitochondrial NAD(P)H and flavoproteins on addition of mitochondrial substrates, ADP, or cyanide were determined by measurement of fluorescence intensities with dual-photon UV-excitation and single-photon blue excitation. In skeletal muscle fibers of ALS patients with abnormalities of mitochondrial DNA (multiple deletions, n=1, or lower mtDNA levels, n=14) we observed a heterogeneous distribution of the mitochondrial defects among individual fibers and even within single fibers. In some patients (n=3) a mitochondrial defect was also detectable in cultivated skin fibroblasts. These findings support the viewpoint that the observed impairment of mitochondrial function in muscle of certain ALS patients is caused by an intrinsic mitochondrial defect which may be of pathophysiological significance in the etiology of this neurodegenerative disease.

Different Activation of Mitogen-Activated Protein Kinase and Akt Signaling Is Associated with Aggressive Phenotype of Human Meningiomas

Purpose: Activation of intracellular signaling cascades has been implicated in the growth control of benign meningiomas, but their role for meningioma progression and outcome is unknown. Here we determined the expression and function of proteins involved in mitogen-activated protein kinase (MAPK) and phosphinositol-3 kinase (PI3K)/Akt signaling in benign, atypical, and malignant meningiomas and studied their association with clinicopathologic data including meningioma recurrence. Experimental Design: Expression of various MAPK and PI3K signaling proteins was determined in 70 primary meningiomas and, if present, in recurrent tumors by immunohistochemistry and Western blotting. The expression patterns in primary and recurrent tumors were related to clinical data. The effect of MAPK and PI3K pathway inhibition on cell proliferation and apoptosis was determined using a primary malignant meningioma cell culture. Results: Atypical and malignant meningiomas showed higher levels of phospho-Akt compared with benign tumors, and their proliferation could be inhibited by PI3K blocking using wortmannin. PI3K inhibition did not induce apoptosis in malignant meningioma cells. In contrast, expression of phospho-Raf and phospho-MAPK was decreased in aggressive meningiomas compared with benign tumors, but MAPK inhibition by PD98059 resulted in tumor cell apoptosis and decreased proliferation. Reduced MAPK activation was associated with meningioma recurrence, and PI3K activation was associated with poor preclinical condition and brain invasion of malignant meningiomas. Conclusions: Both MAPK and PI3K/Akt pathways are activated at different levels in benign and malignant meningiomas. Activation of PI3K/Akt signaling contributes to the aggressive behavior of malignant meningiomas, whereas MAPK activation is involved in both proliferation and apoptosis of malignant meningiomas.

Increased human polo-like kinase-1 expression in gliomas

PLK-1 (polo-like kinase) belongs to the family of serine/threonine kinases and is involved in spindle formation, centrosome cycles and chromosome segregation. Hence, the kinase is tightly linked to cell proliferation. We could detect immunohistochemically highly expressed PLK protein in astrocytic tumours depending on the grade of anaplasia, in commercially available human glioma cell lines (U87MG, U118MG, U138MG), in one immortalized cell culture derived from a glioblastoma patient and in a primary culture derived from a glioblastoma patient. The highest labelling of PLK-1 was demonstrated in glioblastomas. There was a significant correlation between the PLK expression and the nuclear immunoreactivity of MIB-1. PLK-mRNA, found in all tumour specimens investigated emphasizes the close correlation to proliferation and growth. Furthermore, the relation of the PLK-1 expression to the Mitogen-activated Protein Kinase Cascades was studied by applying various highly specific inhibitors. While all inhibitors minimized the cell density, only the PLCγ inhibitor clearly lead to a reduced PLK-1 expression in the three cell lines U87MG, U118MG, U138MG.

Preliminary results of active specific immunization with modified tumor cell vaccine in glioblastoma multiforme.

AbstractObject Treatment for glioblastoma multiforme has failed to show any progress for decades. While specific immunization with tumor cells modified with Newcastle-Disease-Virus (NDV) has been reported successful in some extracerebral tumors, its effect on glioblastoma is unknown. We report on 11 patients, in whom this approach was analyzed. Methods A vaccine was produced from autologous tumor cell cultures of 11 patients with glioblastoma. After completed surgery and radiotherapy an intracutaneous vaccination was performed 4 times with a 2 week interval and finally after 3 months. Local reactions, general side effects and survival were monitored closely. Results The local reaction of the skin after injection of vaccine increased from 1.67 to 4.05 cm2 in 8 weeks. The skin reaction after parallel injection of inactivated, untreated tumor cells increased from 0.11 to 1.09 cm2. The median survival was 46 weeks (mean 60 weeks). No side effects were noted. Conclusion Active specific immunization with NDV-modified glioblastoma cells produced a noticeable peripheral immune response. In this preliminary series survival of patients was not significantly longer after active specific immunization than after combined treatment of surgery, radiotherapy and chemotherapy. As there were no side effects, however, active specific immunization may be nobreak considered an alternative in the management of glioblastoma.

Immunohistochemical and molecular analysis of p53, RB, and PTEN in malignant peripheral nerve sheath tumors

Abstract. The molecular basis of both sporadic and neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs) is yet largely undetermined. Therefore, we analyzed a series of 12 MPNSTs – including two cases which arose in the setting of NF1 – for molecular alterations in the p53, retinoblastoma (Rb), and PTEN tumor suppressor genes. Furthermore, the immunohistochemical expression of p53, RB, and PTEN protein was examined in these tumors. One mutation (8%), an A to T transversion leading to an amino acid exchange, was found in exon 5 of the p53 gene in a sporadic MPNST. In two other sporadic tumors (20%), loss of heterozygosity (LOH) of the p53 gene occurred. Nuclear overexpression of p53 protein was observed in ten tumors (83%). Loss of RB protein expression was seen in two MPNSTs (17%), and LOH of the Rb gene was detected in four tumors (44%), including the two NF1-associated MPNSTs, one of them showing concomitant loss of RB protein expression. No mutation in the PTEN gene was detected, and cytoplasmic immunoreactivity for the PTEN protein was maintained in eight MPNSTs (67%). We suggest that alterations in the p53 and RB pathway, both are essential in controlling the cell-cycle progression, are critical points in the tumorigenesis of sporadic and NF1-associated MPNSTs, whereas the PTEN gene seems to play no significant role in this process.

Application of inhibitor titrations for the detection of oxidative phosphorylation defects in saponin-skinned muscle fibers of patients with mitochondrial diseases.

Inhibitor titrations were applied to characterize functional changes in mitochondrial energy metabolism in the skeletal muscle of patients with mitochondrial diseases. For this we titrated the maximal mitochondrial respiration rate of saponin-skinned muscle fibers isolated from the skeletal muscle biopsy with the specific inhibitors of mitochondrial oxidative phosphorylation complexes I, IV and V-rotenone, azide and oligomycin. For three patients with deletions of mitochondrial DNA and one patient with a complex I deficiency the titrations revealed at rather normal respiration activities of saponin-skinned fibers significant differences to healthy controls: (i) The inhibitor titration curves of the affected enzyme were much steeper and (ii) for almost complete inhibition of respiration a smaller amount of the inhibitor is necessary. The detailed analysis of the titration curves within the framework of metabolic control theory indicated elevated flux control coefficients of the respective complex of respiratory chain. On the other hand, for one patient with a mitochondrial DNA depletion syndrome, decreased respiration activities of skinned fibers but no redistribution of flux control was observed. We conclude, therefore, that application of inhibitor titrations and the quantitative description of the titration curve can be a valuable approach to elucidate functional defects of mitochondrial oxidative phosphorylation.

Expression of the plasminogen activator system and the inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and brain injuries following dramatic circulatory arrests: an immunohistochemical study.

Plasminogen activators as inducible extracellular serine proteases are involved in a variety of processes, such as the degradation of brain structures. In regions of brain degradation, an increase in the expression of genes encoding cytokines and proteinases has recently been demonstrated. We tested the hypothesis, whether the plasminogen activator system as well as the plasminogen activator inhibitors are expressed and possibly involved in a proteolytic cascade that breaks down the extracellular matrix as a result of ischemic or posttraumatic brain destructions. To study this supposition, we investigated immunohistochemically the expression of tPA, uPA and its receptor, the plasminogen activator inhibitors PAI-1 and PAI-2, tetranectin as well as the laminin breakdown as an event of secondary brain injury. Brain tissue from 21 autopsy cases with severe brain injuries, material from 14 ischemic infarcts and 11 controls with acute hypoxia were used. All components of the plasminogen activator system studied were over-expressed immunohistochemically in reactive astrocytes, microglia and endothelial cells around the lesion zone. Tetranectin showed an analogous distribution to the plasminogen activator system. A reduced immunoreactivity of laminin within the identical region of destruction was detected concomitant with laminin remnants in perivascular macrophages, so that a remarkable role of the plasmin cascade in the degradation of extracellular matrix proteins in the brain is taken into consideration.

Immunohistochemical expression of P-glycoprotein and glutathione S-transferases in cerebral gliomas and response to chemotherapy.

Abstract The expression of the drug resistance-related proteins glutathione S-transferases (GST) and P-glycoprotein (Pgp) was analyzed quantitatively in samples of 53 astrocytic gliomas (eight WHO grade 1, 11 WHO grade 2, 9 WHO grade 3 and 25 glioblastomas, WHO grade 4). Sections of these tumors were immunohistochemically stained with antibodies to Pgp (MDR1-gene product) and to GST subclasses alpha, mu and pi. Pgp expression was not detected in tumor cells of the majority of low-grade astrocytomas (69%) and the percentage of Pgp stained cells generally increased with tumor grade. However, 4 of the 34 malignant gliomas were negative. Many neoplastic cells of most tumors were dominantly stained for GST-pi. The other two subclasses were expressed in a less consistent fashion with no linear correlation to grading. Grade 2 astrocytomas exhibited the highest percentage of cells with GST expression. GST-alpha was absent in 9 tumors, GST-mu in 8 and GST-pi in 4. Four tumors showed no expression of any GST subclass or Pgp in neoplastic cells. Of 13 patients 5 with a more favorable clinical course after radiation and chemotherapy had a lower percentage of neoplastic cells immunostained for Pgp and the three GST subclasses than 8 patients with a worse clinical course. These results suggest a relationship between expression of drug resistance-related proteins in gliomas and response to chemotherapy with ACNU/VM26.

Review: iron metabolism and the role of iron in neurodegenerative disorders.

Iron plays a role for the biogenesis of two important redox‐reactive prosthetic groups of enzymes, iron sulphur clusters (ISC) and heme. A part of these biosynthetic pathways takes plays in the mitochondria. While several important proteins of cellular iron uptake and storage and of mitochondrial iron metabolism are well‐characterized, limited knowledge exists regarding the mitochondrial iron importers (mitoferrins). A disturbed distribution of iron, hampered Fe‐dependent biosynthetic pathways and eventually oxidative stress resulting from an increased labile iron pool are suggested to play a role in several neurodegenerative diseases. Friedreichs ataxia is associated with mitochondrial iron accumulation and hampered ISC/heme biogenesis due to reduced frataxin expression, thus representing a monogenic mitochondrial disorder, which is clearly elicited solely by a disturbed iron metabolism. Less clear are the controversially discussed impacts of iron dysregulation and iron‐dependent oxidative stress in the most common neurodegenerative disorders, i.e. Alzheimers disease (AD) and Parkinsons disease (PD). Amyotrophic lateral sclerosis (ALS) may be viewed as a disease offering a better support for a direct link between iron, oxidative stress and regional neurodegeneration. Altogether, despite significant progress in molecular knowledge, the true impact of iron on the sporadic forms of AD, PD and ALS is still uncertain. Here we summarize the current knowledge of iron metabolism disturbances in neurodegenerative disorders.