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Reduced expression of NF2 protein in sporadic schwannomas
5134
Basic Sciences- Neuro-oncology
Tuesday, 8 July 1997
strated many small round mononuclear cells . Frozen sections of all meningiomasrecently developed polymerase chain reaction (PCR)-based telomeric repeats showed the COO6positive cells in the tumor parenchyma and interstitial tissue asamplification protocol (TRAP) assay has made it possible to detect telomerase scattered single cells or groups of cells. There were macrophages, monocytes ,activity (N.W. Kim et al. Science, 266, 2011-2015. 1994). We defined a new other small round cells with small nuclei and scanty cytoplasm, and cells thatvaluable indicator the 'relative telomerase activity' (RTA) quantified by TRAP could not be differentiated from tumor cells . Therefore , there may have been assay. The RTA was calculated as a quotient of the total density of ladders of tumor cells that expressed CD68 other than the infiltrated mononuclear cells . the specimen divided by the density of the positive control. Discussion: The CD68 positive cells were supposed to be macroMethods: We analysed RTA in 130 various type of brain tumor samples phagesJmonocyteslmicroglias and possibly to be CD68 expressed tumor cells.(19 of gliomas, 36 of meningiomas, 41 of neurinomas , 14 of metastatic brain Furthermore , the content of CD68 positive cells was higher in meningiomas tumors, 3 of malignant lymphomas, and 13 of miscellaneous tumors). Each than in gliomas and metastatic tumors. There is a possibility that the CD68 sample was treated with ice-cold CHAPS buffer and with extended telomeres positive cell infiltrates have some relation to secretion of various growth factors by telomerase. The mixture was subjected to PCR amplification. The PCR and regulation of tumor growth. Therefore , the CD68 positive cells in this studyproducts were analysed using an NIH image analyzer after electrophoresis . should be differentiated by types and deserve further study in relation to growth Results:The mean RTA was estimated as 16.7 in gliomas , 21.9 in meningiomas, 36.5 in metastatic brain tumors, SO.6 in malignant lymphomas , and 36.2 factors predicting tumor growth and recurrence. in miscellaneous tumors. The RTA was not detected in neurinomas . Discussionsand ConclusIons:Generally, we found that malignant brain P-S-269 I Reducedexpressionof NF2proteinin sporadic ± 22.0) in contrast to benign tutumors presented high levels of RTA ( t 6.6 schwannomas mors (t .t ± 4.6) (p < O.OOt). These results suggest that the quantification of telomerase activity is useful to stratify the risk of malignancy of brain tumors. Joung H. Lee, JyotiA. Harwalkar, Sam E. Kinney, Gordon Hughes, Mladen Golubic.DepartmentofNeurosurgery,ClevelandClinicFoundation,
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Cleveland, Ohio, USA
I P-S-272I Progesteronereceptorsin arachnoidcysts
Introduction:The neurofibromatosis type 2 (NF2) gene is frequently mutated in K.G. Go, M.A. Blankenstein, T.M. Vroom, E.H. Blaauw, F. Dijk, H. Hollema, sporadic meningiomas and vestibular schwannomas (VS). Little is known about A. Verhagen. DepartmentofNeurosurgery,MedicalElectronMicroscopy,and the normal NF2 protein called schwannominlmerlin or about the mutated forms Pathologyof theUniversityofGroningen, theNetherlands, Departmentsof present in these tumors. EndocrinologyandPathologyof theUniversityHospitalofUtrecm, the Methods: Immunoblotting andimmunoprecipitationexperiments with two Netherlands antibodies were used to determine size, subcellulardistribution, and expression level of NF2 protein in 10 sporadic VS, one NF2-related VS, and 6 spinal Introduction:Since arachnoid cysts are closed cystic cavities lacking communication with surrounding CSF spaces, maintenance of cyst fluid volume by schwannomas. Results: The NF2 protein was identified as a single band ot about 66 kDa that secretion of fluid by the cyst lining must be assumed. Previous ultrastructural was predominantly expressed in the Triton X-100-insoluble fraction of humanstudies have demonstrated that the lining of arachnoid cysts consists of the brain tissue and tumors analyzed. The expression levels of NF2 protein were layer of arachnoid mater that borders the dura mater, also designated as the severely reduced in 2110 (20%) sporadic VS, 216 (25%) spinal schwannomas, subdural neurothelium or subdural mesothelium. Moreover, subdural neurotheand in a single NF2-related VS. The intensity of NF2 protein band was reduced Iium is a component of arachnoid granUlations, and Na+K+-ATPase activity has been demonstrated by enzyme cytochemistry in the lining of arachnoid about 50% in 6ItO (60%) sporadic VS, and216 (25%) spinal schwannomas cysts as well as in that of arachnoid granulations , indicating a capacity for fluid compared to human brain tissue and other tumors. Discussionand Conclusions:Barely detectable expression of NF2 protein secretion. Subdural neurothelium is probably also the tissue from which menlnin about 2(}-25 % of tumors indicates the complete inactivation of both copiesgiomas are derived. These tumours have been shown to possess receptors for of NF2 gene. A very weak NF2 band seen in these tumors presumably origi· progesterone rather than for oestrogens. nates from non-tumorous cells. The interpretation of data in other tumors with Material & Methods: In 2 cases of arachnoid cysts, one with a retrocerebellar and the other with a left temporal localization, biopsies of cyst lining have SO"Io reduced NF2 protein expression awaits NF2 gene analysis . itbut might indicate inactivation of only one copy of NF2 gene. Our study of NF2 protein been obtained. On the specimens immunohistochemical studies have been provides a new evidence that inactivat ion of NF2 protein is an important factorconducted for the presence of carcinoembryon ic antigen, glial fibrillary acidic protein, oestrogen and progesterone receptors, and scanning- transmission and in tumorigenesis of sporadic schwannomas. electron microscopy has been performed. Results & Comments:The absence ofcarcinoembryonicantigen and glial fibrillary acidic protein immunostaining demonstrated arachnoid cyst lining (and P-S-270 I TelomeraseactivityIn human braintumors not ependymal or choroid epithelial cyst lining). The scanning-transmission and Kunyu Harada, Kaoru Kurisu, Kazuhiko Sugiyama, Takashi Sadatomo, electron micrographs revealed the cyst lining to resemble subdural neurotneHiroyuki Yoshioka, Seiji Hama. DepartmentofNeurosurgery,Hiroshima Iium. Progesterone receptors were found In the nuclei of cells liningcyst, the UniversitySchoolofMedicine, Hiroshima,Japan which indicates the similarity of the cyst lining to arachnoid granUlations and Telomerase activity was analysed in 69 frozen samples from human brain tu- meningiomas as derivatives of subdural neurothelium , since these also possess mors, 6 of glioma-derived human cell lines, and p16 and p21 transfectants progesterone receptors. protocol method. Seventeen (37.6% of 45 with telomeric repeat amplification gliomas (including low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, gliosarcoma , oligodend roglioma, anaplastic oligodendroglioma) had de- P-S-27S! Braintumorcysts tectable telornerase activity. Only 2 (15.4%) of the 1310w-grade astrocytomas P.N.M. Lohle 1 , HAL Wurzer 2 , P.J. Seelen 3 , LM. Kingma I, K.G. G0 4 . had detectable telomerase activity, while, 14 (48.3%) of 29 high-grade astro1 DepartmentofRadiology,WesteindeHospitalof The Hague, The cytomas (4 of 7 anaplastic astrocytomas and 10 of 22 glioblastomas) showed Netherlands,2Department ofNeurosurgery,WesteindeHospitalof TheHague, telomerase activity. No meningiomas, neurinomas , or pituitary adenomas had TheNetherlands, 3 Departmentof Biochemistry,WasteindeHospitalof The detectable telomerase activity. Among 5 metastatic brain tumors, 3 (60%) had Hague, TheNetherlands,4 DepartmentofNeurosurgery,AcademicHospitalof activity, In five detectable telomerase activity. Normal brain had no telomerase Groningen, TheNetherlands of the six cell lines, telomerase activity was detected . In our previous report, Introduction: There are no exact figures for the frequency of cystic brain tumors , by the transfecwe confirmed that the growth of malignant glioma was inhibited tion with p16 gene and p2t gene. In p21 transfectants (U 251-MG and T9BG), but it is estimated that about 10% of all brain tumors are cystic. These cysts telomerase activities were detectable . There was no difference between non- may contribute to the space occupying character of the tumor, and occasionally transfectant and neo-transfectant in telomerase activity. In p16 transfectant,may even be the main space occupying component of the tumor. It has often been assumed that the cyst is the result of necrosis the of tumor, but as results were Ihe same as for the p21 transfectanl. we have demonstrated previously (Lohle et al. Neurosurgery1993) from the prevalence of plasma proteins among the total protein content of brain tumor blood-branbarrier impairment , probably cysts, cyst fluid indicates an origin from [P-S-271 Rela~ive telomeraseactivityin 130 braintumor by liquefaction of peritumoral edematous tissue, while the small component of specimens endogenous proteins in brain tumor cyst fluid argues against necrosis as the Tetsuro Sano, Akio Asai, Kazuhiko Mishima, Takamitsu Fujimaki, origin of tumor cysts. Proton-Magnetic Resonance Spectroscopicstudies have Takaaki Kirino.Departmentof Neurosurgery,FacultyofMedicine, Universityof shown the presence of lactate within brain tumor cysts, which may originate Tokyo, Tokyo, Japan from aerobic glycolysis in the tumor, or from ischemia by elevated regional tissue Introduction:Telomerase is a ribonucleoprotein that synthesizes telomeric pressure, and the presence of lactate might also suggest acidosis. However, repeats; TTAGGG onto the chromosome end. Unlimited proliferation in immor- Phosphorus Magnetic Resonance Spectroscopic data have indicated pH values talized cells is believed to be highly dependent on telomerase activity. The within tumors which are not consistent with acidosis.
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I
Basic Sciences- Neuro-oncology
Tuesday, 8 July 1997
strated many small round mononuclear cells . Frozen sections of all meningiomasrecently developed polymerase chain reaction (PCR)-based telomeric repeats showed the COO6positive cells in the tumor parenchyma and interstitial tissue asamplification protocol (TRAP) assay has made it possible to detect telomerase scattered single cells or groups of cells. There were macrophages, monocytes ,activity (N.W. Kim et al. Science, 266, 2011-2015. 1994). We defined a new other small round cells with small nuclei and scanty cytoplasm, and cells thatvaluable indicator the 'relative telomerase activity' (RTA) quantified by TRAP could not be differentiated from tumor cells . Therefore , there may have been assay. The RTA was calculated as a quotient of the total density of ladders of tumor cells that expressed CD68 other than the infiltrated mononuclear cells . the specimen divided by the density of the positive control. Discussion: The CD68 positive cells were supposed to be macroMethods: We analysed RTA in 130 various type of brain tumor samples phagesJmonocyteslmicroglias and possibly to be CD68 expressed tumor cells.(19 of gliomas, 36 of meningiomas, 41 of neurinomas , 14 of metastatic brain Furthermore , the content of CD68 positive cells was higher in meningiomas tumors, 3 of malignant lymphomas, and 13 of miscellaneous tumors). Each than in gliomas and metastatic tumors. There is a possibility that the CD68 sample was treated with ice-cold CHAPS buffer and with extended telomeres positive cell infiltrates have some relation to secretion of various growth factors by telomerase. The mixture was subjected to PCR amplification. The PCR and regulation of tumor growth. Therefore , the CD68 positive cells in this studyproducts were analysed using an NIH image analyzer after electrophoresis . should be differentiated by types and deserve further study in relation to growth Results:The mean RTA was estimated as 16.7 in gliomas , 21.9 in meningiomas, 36.5 in metastatic brain tumors, SO.6 in malignant lymphomas , and 36.2 factors predicting tumor growth and recurrence. in miscellaneous tumors. The RTA was not detected in neurinomas . Discussionsand ConclusIons:Generally, we found that malignant brain P-S-269 I Reducedexpressionof NF2proteinin sporadic ± 22.0) in contrast to benign tutumors presented high levels of RTA ( t 6.6 schwannomas mors (t .t ± 4.6) (p < O.OOt). These results suggest that the quantification of telomerase activity is useful to stratify the risk of malignancy of brain tumors. Joung H. Lee, JyotiA. Harwalkar, Sam E. Kinney, Gordon Hughes, Mladen Golubic.DepartmentofNeurosurgery,ClevelandClinicFoundation,
I
Cleveland, Ohio, USA
I P-S-272I Progesteronereceptorsin arachnoidcysts
Introduction:The neurofibromatosis type 2 (NF2) gene is frequently mutated in K.G. Go, M.A. Blankenstein, T.M. Vroom, E.H. Blaauw, F. Dijk, H. Hollema, sporadic meningiomas and vestibular schwannomas (VS). Little is known about A. Verhagen. DepartmentofNeurosurgery,MedicalElectronMicroscopy,and the normal NF2 protein called schwannominlmerlin or about the mutated forms Pathologyof theUniversityofGroningen, theNetherlands, Departmentsof present in these tumors. EndocrinologyandPathologyof theUniversityHospitalofUtrecm, the Methods: Immunoblotting andimmunoprecipitationexperiments with two Netherlands antibodies were used to determine size, subcellulardistribution, and expression level of NF2 protein in 10 sporadic VS, one NF2-related VS, and 6 spinal Introduction:Since arachnoid cysts are closed cystic cavities lacking communication with surrounding CSF spaces, maintenance of cyst fluid volume by schwannomas. Results: The NF2 protein was identified as a single band ot about 66 kDa that secretion of fluid by the cyst lining must be assumed. Previous ultrastructural was predominantly expressed in the Triton X-100-insoluble fraction of humanstudies have demonstrated that the lining of arachnoid cysts consists of the brain tissue and tumors analyzed. The expression levels of NF2 protein were layer of arachnoid mater that borders the dura mater, also designated as the severely reduced in 2110 (20%) sporadic VS, 216 (25%) spinal schwannomas, subdural neurothelium or subdural mesothelium. Moreover, subdural neurotheand in a single NF2-related VS. The intensity of NF2 protein band was reduced Iium is a component of arachnoid granUlations, and Na+K+-ATPase activity has been demonstrated by enzyme cytochemistry in the lining of arachnoid about 50% in 6ItO (60%) sporadic VS, and216 (25%) spinal schwannomas cysts as well as in that of arachnoid granulations , indicating a capacity for fluid compared to human brain tissue and other tumors. Discussionand Conclusions:Barely detectable expression of NF2 protein secretion. Subdural neurothelium is probably also the tissue from which menlnin about 2(}-25 % of tumors indicates the complete inactivation of both copiesgiomas are derived. These tumours have been shown to possess receptors for of NF2 gene. A very weak NF2 band seen in these tumors presumably origi· progesterone rather than for oestrogens. nates from non-tumorous cells. The interpretation of data in other tumors with Material & Methods: In 2 cases of arachnoid cysts, one with a retrocerebellar and the other with a left temporal localization, biopsies of cyst lining have SO"Io reduced NF2 protein expression awaits NF2 gene analysis . itbut might indicate inactivation of only one copy of NF2 gene. Our study of NF2 protein been obtained. On the specimens immunohistochemical studies have been provides a new evidence that inactivat ion of NF2 protein is an important factorconducted for the presence of carcinoembryon ic antigen, glial fibrillary acidic protein, oestrogen and progesterone receptors, and scanning- transmission and in tumorigenesis of sporadic schwannomas. electron microscopy has been performed. Results & Comments:The absence ofcarcinoembryonicantigen and glial fibrillary acidic protein immunostaining demonstrated arachnoid cyst lining (and P-S-270 I TelomeraseactivityIn human braintumors not ependymal or choroid epithelial cyst lining). The scanning-transmission and Kunyu Harada, Kaoru Kurisu, Kazuhiko Sugiyama, Takashi Sadatomo, electron micrographs revealed the cyst lining to resemble subdural neurotneHiroyuki Yoshioka, Seiji Hama. DepartmentofNeurosurgery,Hiroshima Iium. Progesterone receptors were found In the nuclei of cells liningcyst, the UniversitySchoolofMedicine, Hiroshima,Japan which indicates the similarity of the cyst lining to arachnoid granUlations and Telomerase activity was analysed in 69 frozen samples from human brain tu- meningiomas as derivatives of subdural neurothelium , since these also possess mors, 6 of glioma-derived human cell lines, and p16 and p21 transfectants progesterone receptors. protocol method. Seventeen (37.6% of 45 with telomeric repeat amplification gliomas (including low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, gliosarcoma , oligodend roglioma, anaplastic oligodendroglioma) had de- P-S-27S! Braintumorcysts tectable telornerase activity. Only 2 (15.4%) of the 1310w-grade astrocytomas P.N.M. Lohle 1 , HAL Wurzer 2 , P.J. Seelen 3 , LM. Kingma I, K.G. G0 4 . had detectable telomerase activity, while, 14 (48.3%) of 29 high-grade astro1 DepartmentofRadiology,WesteindeHospitalof The Hague, The cytomas (4 of 7 anaplastic astrocytomas and 10 of 22 glioblastomas) showed Netherlands,2Department ofNeurosurgery,WesteindeHospitalof TheHague, telomerase activity. No meningiomas, neurinomas , or pituitary adenomas had TheNetherlands, 3 Departmentof Biochemistry,WasteindeHospitalof The detectable telomerase activity. Among 5 metastatic brain tumors, 3 (60%) had Hague, TheNetherlands,4 DepartmentofNeurosurgery,AcademicHospitalof activity, In five detectable telomerase activity. Normal brain had no telomerase Groningen, TheNetherlands of the six cell lines, telomerase activity was detected . In our previous report, Introduction: There are no exact figures for the frequency of cystic brain tumors , by the transfecwe confirmed that the growth of malignant glioma was inhibited tion with p16 gene and p2t gene. In p21 transfectants (U 251-MG and T9BG), but it is estimated that about 10% of all brain tumors are cystic. These cysts telomerase activities were detectable . There was no difference between non- may contribute to the space occupying character of the tumor, and occasionally transfectant and neo-transfectant in telomerase activity. In p16 transfectant,may even be the main space occupying component of the tumor. It has often been assumed that the cyst is the result of necrosis the of tumor, but as results were Ihe same as for the p21 transfectanl. we have demonstrated previously (Lohle et al. Neurosurgery1993) from the prevalence of plasma proteins among the total protein content of brain tumor blood-branbarrier impairment , probably cysts, cyst fluid indicates an origin from [P-S-271 Rela~ive telomeraseactivityin 130 braintumor by liquefaction of peritumoral edematous tissue, while the small component of specimens endogenous proteins in brain tumor cyst fluid argues against necrosis as the Tetsuro Sano, Akio Asai, Kazuhiko Mishima, Takamitsu Fujimaki, origin of tumor cysts. Proton-Magnetic Resonance Spectroscopicstudies have Takaaki Kirino.Departmentof Neurosurgery,FacultyofMedicine, Universityof shown the presence of lactate within brain tumor cysts, which may originate Tokyo, Tokyo, Japan from aerobic glycolysis in the tumor, or from ischemia by elevated regional tissue Introduction:Telomerase is a ribonucleoprotein that synthesizes telomeric pressure, and the presence of lactate might also suggest acidosis. However, repeats; TTAGGG onto the chromosome end. Unlimited proliferation in immor- Phosphorus Magnetic Resonance Spectroscopic data have indicated pH values talized cells is believed to be highly dependent on telomerase activity. The within tumors which are not consistent with acidosis.
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