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Jie Lu, Zhengping Zhuang, Debbie K. Song, Gautam U. Mehta, Barbara Ikejiri, Harry Mushlin, Deric M. Park and Russell R. Lonser

Object

Nuclear receptor corepressor (N-CoR) forms a complex that maintains neural stem cells in an undifferentiated state through transcriptional repression. Recently, it has been shown that N-CoR is overexpressed in glioblastoma multiforme (GBM) tumor stem cells and has a putative role in maintaining these cells in an undifferentiated immortal state. To determine the effects of disruption of N-CoR complex function by serine/threonine protein phosphatase 2A (PP2A) inhibition on GBM tumor cell differentiation and proliferation, the authors developed and investigated a competitive small molecule inhibitor (LB1) of PP2A in GBM.

Methods

The authors investigated the effects of LB1 on GBM proliferation and molecular differentiation pathways using in vitro and in vivo studies.

Results

The LB1 inhibited PP2A, leading to increased levels of phosphorylated Akt kinase and decreased NCoR expression, as well as dose-dependent antiproliferative activity in cultured U87 and U251 malignant glioma cells (dose range 1–10 μM). Systemic LB1 treatment (1.5 mg/kg/day for 21 days) had significant tumor antiproliferative effects in mice harboring U87 glioma xenografts (73% mean reduction in tumor volume compared with controls; p < 0.001). Moreover, a reduction in PP2A expression and activity after LB1 treatment in vivo correlated with increased Akt phosphorylation, reduced nuclear N-CoR expression and N-CoR cytoplasmic translocation, and increased accumulation of acetylated core histones, which coincided with the appearance of glial fibrillary acidic protein–expressing tumor cells.

Conclusions

These findings indicate that PP2A inhibition effectively disrupts N-CoR complex function/expression and leads to cytoplasmic translocation of N-CoR with subsequent tumor cell differentiation and/or death. Therapeutic paradigms that target N-CoR function in the cancer stem cell component of malignant gliomas may have treatment utility.

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Timothy W. A. Vogel, Alexander O. Vortmeyer, Irina A. Lubensky, Youn-Soo Lee, Makoto Furuta, Barbara Ikejiri, H. Jeffrey Kim, Russell R. Lonser, Edward H. Oldfield and Zhengping Zhuang

Object. Von Hippel—Lindau (VHL) disease is characterized by multiple tumors in specific organs. The cell of origin and the reason for the particular organ distribution of the tumors remains unknown. Endolymphatic sac tumor (ELST) is one of the lesions associated with VHL disease. Data from previous studies of VHL disease—associated hemangioblastomas (HBs) and renal cell carcinomas (RCCs) have indicated that VHL gene deficiency causes coexpression of erythropoietin (Epo) and its receptor (Epo-R), which facilitates tumor growth.

Methods. The authors studied ELSTs from five patients with VHL germline mutations. Analysis of the five ELST samples revealed loss of the wild-type allele, consistent with Knudson's two-hit hypothesis for tumorigenesis. All five ELST specimens were characterized microscopically and by immunohistochemical analysis. Coexpression of Epo and Epo-R was found in all five tumors on immunohistochemical studies and confirmed through reverse transcription—polymerase chain reaction and Western blot analysis.

Conclusions. Expression of Epo appears to be a result of VHL gene deficiency, whereas the simultaneous coexpression of Epo-R may reflect a developmental mechanism of tumorigenesis. Coexpression of Epo and Epo-R in ELSTs together with the morphological and genetic similarities of these lesions with other VHL disease—associated tumors indicates that VHL disease—associated tumors in different organs share common pathogenetic pathways.

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Martin A. Proescholdt, Marsha J. Merrill, Barbara Ikejiri, Stuart Walbridge, Aytac Akbasak, Steven Jacobson and Edward H. Oldfield

Object. Immunotherapy for glioblastoma has been uniformly ineffective. The immunological environment of the brain, with its low expression of major histocompatibility complex (MHC) molecules and limited access for inflammatory cells and humoral immune effectors due to the blood—brain barrier (BBB), may contribute to the failure of immunotherapy. The authors hypothesize that brain tumors are protected from immune surveillance by an intact BBB at early stages of development. To investigate the immunological characteristics of early tumor growth, the authors compared the host response to a glioma implanted into the brain and into subcutaneous tissue.

Methods. Samples of tumors growing in the brain or subcutaneously in rats were obtained for 7 consecutive days and were examined immunohistochemically for MHC Class I & II molecules, and for CD4 and CD8 lymphocyte markers. Additionally, B7-1 costimulatory molecule expression and lymphocyte-specific apoptosis were examined.

Conclusions. On Days 3 and 4 after implantation, brain tumors displayed significantly lower MHC Class II expression and lymphocytic infiltration (p < 0.05). After Day 5, however, no differences were detected. The MHC Class II expressing cells within the brain tumors appeared to be infiltrating microglia. Minimal B7-1 expression combined with lymphocyte-specific apoptosis were detected in both brain and subcutaneous tumors. Low MHC Class II expression and low lymphocytic infiltration at early time points indicate the importance of the immunologically privileged status of the brain during early tumor growth. These characteristics disappeared at later time points, possibly because the increasing perturbation of the BBB alters the specific immunological environment of the brain. The lack of B7-1 expression combined with lymphocyte apoptosis indicates clonal anergy of glioma-infiltrating lymphocytes regardless of implantation site.

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Robert J. Plunkett, Stephen C. Saris, Krzysztof S. Bankiewicz, Barbara Ikejiri and Richard J. Weber

✓ Although several experimental therapies such as dopaminergic cell implantation in parkinsonian models and intratumoral placement of lymphokine-activated killer cells require intracerebral deposition of dispersed cell suspensions, a successful technique of needle implantation of cells into primate brain has not been demonstrated. The authors have sought to establish a stereotaxic technique to predictably deposit dispersed cells in primate brain. Human lymphocytes were cultured in recombinant interleukin-2, labeled with sodium 51 chromate (51Cr), and stereotaxically injected into the frontal white matter of six anesthetized rhesus monkeys. A 10-µl aliquot of cell suspension (2 × 107 cells/ml) was deposited 16 mm deep to the dura at 5 µl/min via Hamilton No. 22s or 26s needles. Five control aliquots were counted for each injection. Reflux out of the needle track was absorbed on gauze, and the recovered cells were counted. The animals were sacrificed 1 hour after implantation and the brain was removed and sectioned such that the cortex and white matter along the needle track were separate. The tissue sections were then counted. Recovery was expressed as the percentage of total injected radioactivity (cpm) that was present in each brain section. Two additional injected hemispheres were processed for autoradiography and histological study.

Cell recovery in the brain (mean ± standard deviation) was 87.2% ± 13.9% (3.3% ± 4.9% in cortex and 83.9% ± 15.9% in white matter). The autoradiograms and histological examination showed a dense accumulation of radioactivity (cells) at the target site and minimal radioactivity (cells) in the needle track. Accurate intracerebral deposition of dispersed cells in primates was achieved with the technique described. This knowledge permits reliable stereotaxic implantation of cells into the brains of nonhuman primates and humans for investigation and therapy.