Petra Ohneseit

Autophagy contributes to resistance of tumor cells to ionizing radiation

BACKGROUND AND PURPOSE Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used. MATERIALS AND METHODS Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3. RESULTS LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells. CONCLUSION Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance.

Analysis of mdm2 and p53 gene alterations in glioblastomas and its correlation with clinical factors.

Malignant gliomas are the most frequent primary brain tumors. Recent studies defined several genetic markers, which might characterize molecular-biological subsets of glioblastomas with probably prognostic implications. To elucidate the involvement of murine-double-minute (mdm)2 gene amplifications and mutations of the tumor suppressor gene p53 in the tumorigenesis of malignant gliomas we analyzed a series of 75 glioblastomas. The p53 mutations occur in one-third of glioblastomas, mdm2 amplifications were found in 13% of cases. Our analysis revealed a hot spot in the p53 gene locus in codon 156, the same point mutation was detected in 4 tumor samples. None of the mdm2 amplified tumors had p53 mutations, supporting the hypothesis, that mdm2 amplifications are alternative mechanisms for p53 inactivation. Patients with p53 mutated tumors were significantly younger characterized by a mean age of 44 years. Additionally association with longer overall survival could be detected for this subgroup of patients. In our study, survival estimation revealed a significant correlation of mdm2 gene amplification with shorter survival time, and support the hypothesis, that mdm2 oncogene activation appears to occur late in tumor progression and may be characteristic as negative prognostic marker.

Loss of heterozygosity at 11p15 and p53 alterations in malignant gliomas

Purpose: Malignant gliomas are the most frequent primary brain tumors. Recent studies defined several genetic markers, which might characterize molecular-biological subsets of glioblastomas with prognostic implications. In the later steps of tumor-progression, deletions on chromosome 11p15 and mutations of the tumor suppressor gene p53 were determined for different malignancies. To elucidate the involvement of 11p15 deletions in the tumorigenesis of malignant gliomas, we analyzed a series of 50 glioblastomas for loss of heterozygosity (LOH). Methods: Paired tissue and blood samples from 50 patients with glioblastoma multiforme were included. Microsatellite markers located on 11p15.1–11p15.5 were used for LOH analysis. Additionally, mutation analysis of the tumor suppressor gene p53 was performed, which might correlate with favorable survival in glioblastomas. Results: The region 11p15.4–5 was deleted heterozygously in 28% of cases representing 15 cM. Twenty-six glioblastomas did not show allelic loss for any locus. Our data revealed close association of LOH 11p15 with p53 mutations, and survival analysis showed a trend indicating better prognosis in glioblastomas characterized by LOH 11p15. Conclusion: In the tumorigenesis of malignant gliomas, p53 mutations and 11p15 deletions seem to indicate a genetic subset of tumors with favorable prognostic value.

Differential expression of egr1 and activation of microglia following irradiation in the rat brain.

Background:Little is known about the immediate effects of whole-brain γ-irradiation. The authors hypothesize that Egr1 as an immediate early gene and microglia both participate in early reactions.Material and Methods:Both, expression of Egr1 and cellular distribution were studied in a temporal sequence in different brain regions of rats subjected to irradiation with 10 Gy. Brain tissue was examined using immunohistochemistry, real-time RT-PCR (reverse transcription-polymerase chain reaction), and Western blotting.Results:Astroglia and oligodendroglia showed increased Egr1 immunoreactivity within the first hours following irradiation. This was accompanied by a strong peak in CD68 immunoreactivity histologically attributable to activated microglia. A high constitutive expression of Egr1 protein in the nuclei of activated neurons was reduced following irradiation and RT-PCR demonstrated significantly reduced levels of egr1-lv as a neuronal activity-related mRNA variant.Conclusion:The induction of Egr1 in glial cells, as well as the activation of microglia take place earlier than histological changes reported so far. The authors revealed a temporal sequence of reactions that point toward the initiation of an immediate inflammatory response including reduced neuronal activity.Hintergrund:Über die unmittelbaren Effekte der γ-Bestrahlung des Gehirns ist wenig bekannt. Die Autoren untersuchten die Hypothese, dass sowohl Egr1 als “immediate early gene” als auch Mikroglia an den frühen Reaktionen beteiligt sind.Material und Methodik:Beides, Expression von Egr1 und zelluläre Verteilung, wurden in einer zeitlichen Abfolge in unterschiedlichen Regionen des Rattenhirns nach Ganzkörperbestrahlung mit 10 Gy mittels Immunhistochemie, Real-Time-RT-PCR (reverse Transkription-Polymerase-Kettenreaktion) und Western-Blot untersucht.Ergebnisse:Astrozyten wie auch Oligodendrozyten wiesen in den ersten Stunden nach Bestrahlung eine erhöhte Egr1-Immunreaktivität auf. Diese trat gleichzeitig mit einer starken CD68-Induktion auf, die histologisch aktivierter Mikroglia zugeordnet werden konnte. Eine hohe konstitutive Expression des Egr1-Proteins in den Zellkernen von Neuronen wurde durch Bestrahlung reduziert, und die RT-PCR wies signifikant niedrigere Werte für egr1-lv als neuronalen Aktivitätsmarker auf.Schlussfolgerung:Die Induktion von Egr1 in glialen Zellen wie auch die Aktivierung der Mikroglia spielen sich zu einem früheren Zeitpunkt ab als bisher berichtete histologische Veränderungen. Die Autoren zeigen eine zeitliche Sequenz der frühen glialen Reaktionen auf, die auf einen beginnenden inflammatorischen Prozess hinweist und mit einer reduzierten neuronalen Aktivität einhergeht.

MR imaging of experimental meningeal melanomatosis in nude rats.

MR imaging of the rat brain has become an increasingly frequently used method in experimental neuroradiology. On a generally available 1.5 T whole body tomograph, supplemented with an individually made small coil and a special SE sequence we obtained fairly fine images of the structures of the rat brain. With gadolinium-DTPA, we were able to visualize posterior fossa and cervical leptomeningeal growth of intrathecally injected B16 melanoma in nude rats. Using MRI to follow experimental leptomeningeal metastasis, may provide a new means for diagnostic evaluation and preclinical testing of treatment modalities.

Intrathecal ACNU treatment of B16 melanoma leptomeningeal metastasis in a new athymic rat model

SummaryTo evaluate new cytotoxic drugs for intrathecal treatment we developed an experimental model of leptomeningeal metastasis by intracisternal injection of 104B16-F10 melanoma cells in nude rats. One hour in vitro incubation with 20 μg/ml ACNU (area under the drug concentration-time curve = 1200 μgxmin/ml) induced a 4-log kill of B16 melanoma cells. A single or repeated non-toxic dose of 1 mg/kg was injected into the cisterna magna of rats inoculated with tumor (area under the drug concentration-time curve assuming an even cerebrospinal fluid distribution > 7000 μgxmin/ml). Median survival free of symptoms was 16 days (range 14–27) for controls (n = 9) and 18 days (range 17–23) for rats treated with ACNU on day 4 (n = 9). Animals treated both on day 2 and 8 (n = 8) developed symptoms on day 21 (range 13–35). Neurological symptoms and neuropathological examination in animals with increased survival indicated local suppression of tumor growth in the cisterna magna but increased spinal seeding and mass growth. From these results and the available pharmacokinetic data on ACNU it is concluded that bolus injection of ACNU — although locally effective — is not a sufficient treatment of widespread leptomeningeal metastasis. An increased therapeutic efficacy might be achieved by ventriculolumbar perfusion.

Differential response of tumor cells and normal fibroblasts to fractionated combined treatment with topotecan and ionizing radiation

Purpose : The impact of the topoisomerase-I inhibitor topotecan (Hycamtin ®) as a single agent or in combination with ionizing radiation on clonogenic cell survival has been evaluated in three tumor cell lines and in normal fibroblasts. Both agents have been applied in a fractionated scheme in order to assess clinically relevant conditions. Materials and methods : Cell inactivation was investigated in human glioblastoma cell lines U118 and U138, lung carcinoma cell line A549 and normal fibroblasts of the skin (HSF6) and lung (CCD32) using the colony formation assay. Results : The glioblastoma cell lines were highly sensitive to the drug, whereas normal fibroblasts were much less sensitive. A significant antagonistic effect of the drug combined with irradiation was found for normal fibroblasts, while glioblastoma cells showed no evidence of interaction, indicating additivity. A549 cells showed a biphasic response to topotecan alone and in combination with irradiation, suggesting induction of resistence to the drug. Conclusion : Differential effects of combined topotecan/radiation treatment were detected between cells of normal tissue and tumor cells, being antagonistic in fibroblasts of the skin and lung, but not in tumor tissue, especially in glioblastoma cells.