Jürgen Kraus

SHARED ALLELIC LOSSES ON CHROMOSOMES 1P AND 19Q SUGGEST A COMMON ORIGIN OF OLIGODENDROGLIOMA AND OLIGOASTROCYTOMA

Loss of heterozygosity (LOH) in specific chromosomal regions, which are likely to harbor tumor suppressor genes, has been associated with human gliomas. In this study we have analyzed astrocytic and oligodendroglial tumors for LOH on chromosomes 1 and 19. By microsatellite analysis LOH was found on chromosome arm 1p in 6/15 oligodendrogliomas WHO grade II and III, 12/25 oligoastrocytomas WHO grade II and III, 6/79 glioblastomas WHO grade IV, 5/44 astrocytomas WHO grade II and III and 0/23 pilocytic astrocytomas WHO grade I. The high incidence of LOH on chromosome arm 1p in oligodendrogliomas and oligoastrocytomas indicates that a putative tumor suppressor gene in this region is involved in the formation of gliomas with oligodendroglial features. Furthermore, the frequent involvement of chromosome arm lp in oligodendrogliomas and oligoastrocytomas, but not in astrocytomas, suggests that genetically oligoastrocytoma is more similar to oligodendroglioma than to astrocytoma. In order to support this hypothesis, oligodendroglial and astrocytic areas in three mixed oligoastrocytomas were examined differentially for LOH lp and for LOH 19q, the second genetic region believed to be affected in these tumors. All three tumors had LOH of lp and LOH of 19q in both areas of oligodendroglial and of astrocytic differentiation. These findings show that the astrocytic and oligodendroglial portions of oligoastrocytoma share molecular genetic features and probably are of monoclonal origin.

Induction of apoptosis in human and rat glioma by agonists of the nuclear receptor PPARγ

Malignant astrocytomas are among the most common brain tumours and few therapeutic options exist. It has recently been recognized that the ligand‐activated nuclear receptor PPARγ can regulate cellular proliferation and induce apoptosis in different malignant cells. We report the effect of three structurally different PPARγ agonists inducing apoptosis in human (U87MG and A172) and rat (C6) glioma cells. The PPARγ agonists ciglitazone, LY171 833 and prostaglandin‐J2, but not the PPARα agonist WY14643, inhibited proliferation and induced cell death. PPARγ agonist‐induced cell death was characterized by DNA fragmentation and nuclear condensation, as well as inhibited by the synthetic receptor‐antagonist bisphenol A diglycidyl ether (BADGE). In contrast, primary murine astrocytes were not affected by PPARγ agonist treatment. The apoptotic death in the glioma cell lines treated with PPARγ agonists was correlated with the transient up‐regulation of Bax and Bad protein levels. Furthermore, inhibition of Bax expression by specific antisense oligonucleotides protected glioma cells against PPARγ‐mediated apoptosis, indicating an essential role of Bax in PPARγ‐induced apoptosis. However, PPARγ agonists not only induced apoptosis but also caused redifferentiation as indicated by outgrowth of long processes and expression of the redifferentiation marker N‐cadherin in response to PPARγ agonists. Taken together, treatment of glioma cells with PPARγ agonists may hold therapeutic potential for the treatment of gliomas.

Transcriptional regulation of the cannabinoid receptor type 1 gene in T cells by cannabinoids

Effects of cannabinoids (CBs) are mediated by two types of receptors, CB1 and CB2. In this report, we investigated whether CBs regulate gene expression of their cognate receptors in T cells and studied underlying mechanisms in CD4+ Jurkat T cells. Transcription of the CB1 gene was strongly induced in response to Δ9‐tetrahydroannabinol (THC), whereas the CB2 gene was not regulated. The induction of CB1 gene expression is mediated by CB2 receptors only, as demonstrated by using the CB1 and CB2 agonists R(+)‐methanandamide and JWH 015, respectively, and combinations of THC plus CB1‐ and CB2‐specific antagonists. After activation of CB2 receptors, the transcription factor STAT5 is phosphorylated. STAT5 then transactivates IL‐4. Induction of IL‐4 mRNA as well as IL‐4 protein release from the cells are necessary for the following induction of the CB1 gene. This was demonstrated by using decoy oligonucleotides against STAT5, which blocked IL‐4 and CB1 mRNA induction, and by using the IL‐4 receptor antagonist IL‐4 [R121D, Y124D], which blocked the up‐regulation of CB1 gene transcription. Transactivation of the CB1 gene in response to IL‐4 is then mediated by the transcription factor STAT6, as shown by using decoy oligonucleotides against STAT6. An increase in CB1‐mediated phosphorylation of MAPK in cells prestimulated with CB2‐specific agonists suggests up‐regulation of functional CB1 receptor proteins. In summary, up‐regulation of CB1 in T lymphocytes in response to CBs themselves may facilitate or enhance the various immunomodulatory effects related to CBs.

Molecular genetic analysis of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 tumour-associated genes in supratentorial primitive neuroectodermal tumours and glioblastomas of childhood

Supratentorial primitive neuroectodermal tumours (sPNETs) are malignant central nervous system tumours of childhood which are histologically characterized by poorly differentiated neuroepithelial cells with the capacity for divergent differentiation into glial, neuronal, myogenic or melanotic lines. The histological differential diagnosis between sPNET and glioblastoma multiforme (GBM) may be difficult, particularly as GBMs can sometimes demonstrate a poorly differentiated PNET‐like phenotype. To identify molecular genetic markers that may distinguish sPNET and GBM, we investigated 12 cerebral sPNETs and six GBMs from paediatric patients for genetic alterations of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 genes, as well as for allelic loss on chromosome arms 10q and 17p. Mutations of the TP53 tumour suppressor gene were found in one of 12 sPNETs (8%) and two of six GBMs (33%). None of the sPNETs but two of six GBMs (33%, including one GBM with a TP53 mutation) showed allelic losses on chromosome arm 17p. PTEN mutations were detected in one of 12 sPNET (8%) and one of six GBMs (17%). None of the sPNETs and GBMs carried a homozygous deletion involving the CDKN2A tumour suppressor gene. No amplification of the EGFR, CDK4 or MDM2 proto‐oncogenes was detected. Taken together, our results indicate that paediatric GBMs differ from sPNETs by a higher incidence of allelic losses on 17p and TP53 mutations. In addition, the patterns of genetic alterations in sPNETs and paediatric GBMs appear to be distinct from those in cerebellar medulloblastomas and adult GBMs, respectively.

Long-term survival of glioblastoma multiforme: Importance of histopathological reevaluation

Abstract The overall prognosis for patients with glioblastoma multiforme is extremely poor. However, a small proportion of patients enjoy prolonged survival. This study investigated retrospectively the extent to which erroneous histopathological classification may contribute to long-term survival of patients initially diagnosed with “glioblastoma multiforme”. We compared two age- and gender-matched patient groups with different postoperative time to tumor progression (TTP), defined as “short-term” for TTP of less than 6 months (n=54) and “long-term” for TTP of more than 12 months (n=52). Histological specimens of the corresponding tumors, all primarily diagnosed as glioblastome multiforme, were reevaluated according to the current World Health Organization (WHO) classification of central nervous system tumors, with the investigators being blinded to clinical outcome. Among the tumors from short-term TTP patients, one tumor (2%) was reclassified as anaplastic oligoastrocytoma (WHO grade III) while the remaining 53 were confirmed as glioblastoma multiforme. In contrast, 13 tumors (25%) from the long-term TTP patients were reclassified, mostly as anaplastic oligodendroglioma (WHO grade III; n=7) or anaplastic oligoastrocytoma (WHO grade III, n=2), respectively. In addition, three were reclassified as anaplastic astrocytoma (WHO grade III), and one was identified as anaplastic pilocytic astrocytoma (WHO grade III). Our data indicate that a sizable proportion of glioblastoma patients with long-term survival actually carry malignant gliomas with oligodendroglial features. The correct histopathological recognition of these tumors has not only progrostic but also therapeutic implications, since oligodendroglial tumors are more likely to respond favorably to chemotherapy.

Comparative analysis of mu-opioid receptor expression in immune and neuronal cells

Morphine modulates neuronal and immune cell functions via mu-opioid receptors. In primary and Jurkat T cells, and Raji B cells mu-opioid receptor transcripts were detected only after stimulation of the cells with IL-4 or TNF-alpha. Moreover, the amount of the induced mu-opioid receptor mRNA in the immune cells was 15 to 200 times less than those in primary cortical and SH SY5Y neuronal cells. Nevertheless, mu-opioid receptor mRNA in immune cells is processed to functional receptors, as demonstrated by morphine-mediated phosphorylation of mitogen activated protein kinase, morphine-mediated up-regulation of IL-4 mRNA and coupling to adenylyl cyclase in Jurkat cells.

Loss of heterozygosity on chromosome 1 in human hepatoblastoma

In previous studies we have found loss of heterozygosity (LOH) on chromosome arm 11p in 33% of hepatoblastomas (HBs). In addition, cytogenetic studies have revealed aberrations of chromosome arm 1p in single cases. Therefore, we have used the PCR to amplify 10 microsatellites on the short arm and 7 microsatellites on the long arm of chromosome 1 to assess allelic loss in 32 cases of HB. LOH on chromosome 1 was found in 11 cases. Seven HBs showed LOH on chromosome 1p, 7 cases had LOH on 1q, and 3 tumors had LOH on both 1p and 1q. Six HBs with LOH on 1p had LOH at DIS243 (1p36.3), and one tumor had a loss at DIS80 maintaining heterozygosity at DIS243. A common region of overlap was present at the telomeric chromosomal portion of 1p between DIS80 and DIS243. Of the HBs with LOH on 1q, 4 showed a common region of overlap at 1q31‐q32.1, and the other 3 at DIS1609 located more telomerically. The parental origin of the lost allele was of random distribution for chromosome arm 1p and of paternal origin for chromosome arm 1q. Our data suggest that tumor suppressor genes located at the telomeric region of chromosome arm 1p and different regions of chromosome arm 1q may be involved in the pathogenesis of HB.

Memory performance in multiple sclerosis patients correlates with central brain atrophy

Objective To assess whole brain and central brain atrophy as well as their differential relation to memory, cognitive performance, fatigue, depression and quality of life in patients with relapsingremitting multiple sclerosis (RRMS). Methods A 3D flow compensated gradient recalled T1-weighted MRI was acquired in 45 RRMS patients. An automated analysis tool was used to calculate brain parenchymal fraction (BPF) and ventricular brain fraction (VF). All patients were assessed with neuropsychological tests focusing on memory and self-rating scales for depression, fatigue and quality of life. Age corrected partial correlations between brain atrophy, motor performance, psychological scales and test scores were calculated. Results BPF correlated moderately (0.35≤r<0.5) with duration of symptoms and disease, the Expanded Disability Status Scale (EDSS), the upper extremity motor performance, and with mental aspects of quality of life. VF correlated moderately with EDSS, upper and lower extremity motor performance and memory functions. Neither BPF nor VF correlated with fatigue and depression. Results of several cognitive tests correlated moderately with depression and fatigue, the Paced Auditory Serial Addition Test (PASAT) showing the largest correlation. Conclusions Memory performance shows a correlation with relative ventricular size in RRMS patients, indicating the strategic location of the ventricle system along the structures of the limbic system and its vulnerability in MS. The PASAT and several other cognitive tests show moderate correlations with depression and fatigue, arguing for an inter relation between the cognitive functioning and the emotional state of patients. However, this relation is independent of measurable brain atrophy.

Distinct palindromic extensions of the 5′-TTC…GAA-3′ motif allow STAT6 binding in vivo

STATs (signal transducers and activators of transcription) are transcription factors downstream of cytokine and growth factor signals. All of the seven different STATs bind to regulatory promoter elements with the common core motif 5’‐TTC(N)2‐4GAA‐3’. A key question is how the different STAT factors recognize “their” response elements, that is, what distinguishes for example STAT1 from STAT6 binding sites. In vivo, binding of the different STATs to DNA elements is highly specific and disruption of the genes for the different STAT factors is accompanied with distinct, non‐overlaping phenotypical effects. As a first step towards discrimination of target sequences for the various STATs, we determined requirements for binding sites for STAT6. In functional assays, six sequences were identified. These have palindromic extensions of the core motif in common (underlined): 5’‐TTTCNNNGAAA‐3’, 5’‐CTTCNNNGAAG‐3’, 5’‐TTTCNNNNGAAA‐3’, 5’‐CTTCNNNNGAAG‐3’, 5’‐TTCCNNGGAA‐3’ and 5’‐TTCANNTGAA‐3’. Different approaches and mutational analysis demonstrated the functionality of these sequences and high specific binding to STAT6. (I) These elements mediate transcriptional induction by interleukin‐(IL)‐4, IL‐13, IL‐15, and platelet‐derived growth factor. (II) When used as “decoy” oligonucleotides, they bind STAT6 and disrupt its function in vivo, attenuating (a) STAT6/IL‐4‐mediated reporter gene transcription and (b) STAT6/IL‐4‐mediated induction of μ‐opioid receptor mRNA of Raji cells.

TP53 gene mutations, nuclear p53 accumulation, expression of Waf/p21, Bcl-2, and CD95 (APO-1/Fas) proteins are not prognostic factors in de novo glioblastoma multiforme

Glioblastoma multiforme (WHO grade IV; GBM) is the most common primary brain tumor with a median survival of less than one year despite multimodal treatment regimens. However, a small subgroup of GBM patients has a better clinical outcome, with a small number of patients surviving several years. Apoptosis, a genetically determined program of cell suicide, may be induced as a consequence of critical DNA damage. However, due to defects in the signaling pathways, cancer cells may escape apoptosis, despite carrying irreversible DNA damage. In the present study, we have analyzed tumors of two age-matched, equally treated groups of GBM patients with different postoperative time to tumor progression (TTP), defined as ‘short-term’ for TTP of less than 6 months (n = 54), and ‘long-term’ for TTP of more than 12 months (n = 39) for alterations in apoptosis regulatory pathways: Mutations of the TP53 tumor suppressor gene and/or nuclear accumulation of its gene product p53, expression of Waf/p21, CD95 (Apo1/Fas), and Bcl-2. TP53 mutations were found in 12 out of 54 (22%) GBMs of short-term survivors and 8 out of 35 (23%) tumors of long-term survivors; the respective numbers for nuclear p53 protein accumulation were 12/53 (23%) and 10/37 (27%). Waf1/p21 expression was found in 13/53 (25%) tumors of short-term survivors and 9/35 (26%) GBMs of long-term survivors. The respective numbers for Bcl-2 expression were 25/42 (60%) and 22/36 (61%) and for CD95 (Apo1/Fas) expression 20/49 (41%) and 14/36 (39%) GBMs. The percentage of alterations in genes/proteins involved in the apoptotic pathway investigated here was virtually identical in the two groups of clinically different GBM patients. Thus, our data imply that none of these alterations investigated per se has a strong impact on the overall survival of GBM patients.