Faculty Bibliography

Cell cycle progression requires precise expression and activation of several cyclins and cyclin-dependent kinases. Geldanamycin (GA) affects cell cycle progression in various kinds of cells. We analyzed GA-induced cell cycle regulation in glioblastoma cells. GA-induced G2 or M arrest in glioblastoma cells in a cell line-dependent manner. GA decreased the expression of Cdc2 and cyclin B1 in U87MG cells. And phosphorylated Cdc2 decreased along with Cdc2 in the GA-treated cells. This cell line showed G2 arrest after GA treatment. In contrast, GA failed to down-regulate these cell cycle regulators in U251MG cells. In U251MG cells, the cell cycle was arrested at M phase in addition to G2 by GA. Next, we analyzed the mechanism of the GA-induced regulation of Cdc2 and cyclin B1 in U87MG cells. Cdc2 and cyclin B1 were ubiquitinated by GA. MG132 abrogated the GA-induced decrease of Cdc2 and cyclin B1 indicating that these proteins were degraded by proteasomes. In conclusion, GA controls the stability of Cdc2 and cyclin B1 in glioblastomas cell species-dependently. Cdc2 and cyclin B1 might be responsible for the different responses of glioblastoma cell lines to GA

We describe a lesion with the magnetic resonance imaging (MRI) characteristics of a glioblastoma mutiforme and demonstrate how perfusion MRI and proton MR spectroscopic imaging can be used to differentiate necrotizing cerebritis from what appeared to be a high-grade glioma. A 43-year-old woman presented to her physician complaining of progressive visual disturbance and headache for several weeks. Conventional MRI demonstrated a parietal peripherally enhancing mass with central necrosis and moderate to severe surrounding T2 hyperintensity, suggesting an infiltrating high-grade glioma. However, advanced imaging, including dynamic susceptibility contrast MRI (DSC MRI) and magnetic resonance spectroscopic imaging (MRSI), suggested a nonneoplastic lesion. The DSC MRI data demonstrated no hyperperfusion within the lesion and surrounding T2 signal abnormality, and the MRSI data showed overall decrease in metabolites in this region, except for lactate. Because of the aggressive appearance to the lesion and the patients' worsening symptoms, a biopsy was performed. The pathologic diagnosis was necrotizing cerebritis. After the commencement of steroid therapy, imaging findings and patient symptoms improved. This report will review the utility of advanced imaging for differentiating inflammatory from neoplastic appearing lesions on conventional imaging

PURPOSE: To determine if increased perfusion using dynamic susceptibility contrast perfusion MRI (DSC MRI) in gliomas may be predictive of 1p19q deletions. Loss of heterozygosity of chromosomes 1p and 19q confers responsiveness to chemotherapy improving survival in gliomas. MATERIALS AND METHODS: We retrospectively reviewed 16 patients who had DSC MRI and molecular studies of their excised gliomas for 1p19q deletions. Allelic status was assessed by loss of heterozygosity using polymerase chain reaction (PCR). DNA was extracted from paraffin curls of brain tumor sections and nail clippings. Relative cerebral blood volume (rCBV) measurements were then statistically compared with the presence of 1p and 19q deletions. RESULTS: Patients with 1p19q deletions (N = 7) demonstrated rCBV values of 10.54 +/- 2.93. Patients without 1p deletions (N = 9) had rCBV values of 4.84 +/- 2.4 (P = 0.012). Logistic regression demonstrated that rCBV was able to predict the presence of a 1p deletion to significance levels of 0.038 and 0.044, adjusted and not adjusted for age and sex, respectively. The kappa coefficient for the agreement between predicted deletion status using rCBV and the truedeletion status was 0.746 (P = 0.0028). Deletions of 19q alone, or together with 1p deletions, were not associated with high rCBV. CONCLUSION: Histopathologic, molecular, and imaging evidence supports increased neovascularity in gliomas with 1p deletions in this preliminary study. We propose a diagnostic algorithm to obtain molecular studies in gliomas demonstrating high rCBV.

Geldanamycin is a naturally occurring benzoquinone ansamycin product of Streptomyces geldanus that binds the protein chaperone heat shock protein 90. As geldanamycin binds to heat shock protein 90 interfering with its function and heat shock protein 90 is overexpressed in many cancers, heat shock protein 90 has become a target for cancer therapy. As the geldanamycin analogue 17-allylamino-17-demethoxygeldanamycin has a favorable toxicity profile, it is being tested extensively in clinical trials in patients with advanced cancer. In this study, GL261 glioma cells from C57BL/6 mice were used to investigate the anti-tumor effect of 17-allylamino-17-demethoxygeldanamycin both in vitro and in vivo. Heat shock protein 90 inhibitors possess potent anti-proliferative activity, usually at low nanomolar ranges, owing to their pharmacological characteristics of binding tightly to heat shock protein 90, coupled with a slow dissociation rate. We found that 17-allylamino-17-demethoxygeldanamycin at doses as low as 200 nmol/l showed anti-tumor activity within 24 h of treatment. Treatment with 17-allylamino-17-demethoxygeldanamycin arrested GL261 cells in the G2 phase of the cell cycle associated with the downregulation of cyclin B1. Low doses of 17-allylamino-17-demethoxygeldanamycin significantly inhibited migration of GL261 cells within 16 h of treatment, concomitant with the downregulation of phosphorylated focal adhesion kinase and matrix metalloproteinase 2 secretion. Using an orthotopic glioma model with well-established intracranial tumors, 3 weekly cycles of 17-allylamino-17-demethoxygeldanamycin significantly reduced tumor volumes of treated animals compared with untreated controls (P=0.002). Given these promising results, clinical testing of 17-allylamino-17-demethoxygeldanamycin or other novel heat shock protein 90 inhibitors being developed should be considered for glioma patients whose tumors remain refractory to most current treatment regimens

PURPOSE: Rhabdoid tumors are rare but aggressive pediatric malignancies characterized by biallelic loss of INI1/hSNF5. Reintroduction of INI1 causes cell arrest and senescence in rhabdoid cells. Our purpose was to identify INI1-downstream genes and to determine their functional and therapeutic significance for rhabdoid tumors. EXPERIMENTAL DESIGN: INI1 downstream targets in rhabdoid cells were identified using a cDNA microarray analysis and the expression of selected INI1 targets was confirmed by quantitative reverse transcription-PCR, Western analysis, and/or immunohistochemical analysis of rhabdoid cells and primary rhabdoid tumors. To determine the functional significance of downstream targets, activated targets of INI1 were induced and repressed targets of INI1 were knocked down (by using RNA interference) in rhabdoid cells, in the absence of INI1. Consequence of altered expression of INI1 downstream targets for rhabdoid cell survival, cell cycle, and apoptosis was assessed. RESULTS: Microarray studies indicated that INI1 activated IFN-stimulated genes at early time points and senescence markers at late time points and repressed mitotic genes such as Polo like kinase 1 (PLK1), selectively in rhabdoid cells. Treatment of rhabdoid cells with recombinant IFNs resulted in induction of IFN-stimulated genes, G1 arrest, and flat cell formation. PLK1 was overexpressed in primary human and mouse rhabdoid tumors. RNA interference-mediated knock down of PLK1 in rhabdoid cells resulted in mitotic arrest, aberrant nuclear division, decreased survival, and induction of apoptosis. CONCLUSIONS: Targeting downstream effectors of INI1 such as IFN pathway and mitotic genes leads to antiproliferative effects in rhabdoid cells. IFN treatment and down-modulation of PLK1 constitute potential novel therapeutic strategies for rhabdoid tumors

OBJECTIVE:To determine whether relative cerebral blood volume (rCBV) can predict patient outcome, specifically tumor progression, in low-grade gliomas (LGGs) and thus provide a second reference standard in the surgical and postsurgical management of LGGs. METHODS:Thirty-five patients with histologically diagnosed LGGs (21 low-grade astrocytomas and 14 low-grade oligodendrogliomas and low-grade mixed oligoastrocytomas) were studied with dynamic susceptibility contrast-enhanced perfusion magnetic resonance imaging. Wilcoxon tests were used to compare patients in different response categories (complete response, stable, progressive, death) with respect to baseline rCBV. Log-rank tests were used to evaluate the association of rCBV with survival and time to progression. Kaplan-Meier time-to-progression curves were generated. Tumor volumes and CBV measurements were obtained at the initial examination and again at follow-up to determine the association of rCBV with tumor volume progression. RESULTS:Wilcoxon tests showed patients manifesting an adverse event (either death or progression) had significantly higher rCBV (P = 0.003) than did patients without adverse events (complete response or stable disease). Log-rank tests showed that rCBV exhibited a significant negative association with disease-free survival (P = 0.0015), such that low rCBV values were associated with longer time to progression. Kaplan-Meier curves demonstrated that lesions with rCBV less than 1.75 (n = 16) had a median time to progression of 4620 +/- 433 days, and lesions with rCBV more than 1.75 (n = 19) had a median time to progression of 245 +/- 62 days (P < 0.005). Lesions with low baseline rCBV (< 1.75) demonstrated stable tumor volumes when followed up over time, and lesions with high baseline rCBV (> 1.75) demonstrated progressively increasing tumor volumes over time. CONCLUSION/CONCLUSIONS:Dynamic susceptibility contrast-enhanced perfusion magnetic resonance imaging may be used to identify LGGs that are either high-grade gliomas, misdiagnosed because of sampling error at pathological examination or that have undergone angiogenesis in the progression toward malignant transformation. This suggests that rCBV measurements may be used as a second reference standard to determine the surgical management/risk-benefit equation and postsurgical adjuvant therapy for LGGs.

A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1alpha mutant, or a short hairpin RNA directed against HIF-1alpha. In human RCC biopsies, expression of E-cadherin and HIF-1alpha was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1-dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin

Purpose: To determine retrospectively whether relative cerebral blood volume (CBV) measurements can be used to predict clinical response in patients with low-grade gliomas. Materials and Methods: Approval for this retrospective HIPAA-compliant study was obtained from the Institutional Board of Research Associates, with waiver of informed consent. Thirty-five patients (23 male and 12 female patients; median age, 39 years; range, 4-80 years) with histologically diagnosed low-grade gliomas (21 low-grade astrocytomas and 14 low-grade oligodendrogliomas and low-grade mixed oligoastrocytomas) were examined with dynamic susceptibility-weighted contrast material-enhanced perfusion magnetic resonance (MR) imaging. Wilcoxon tests were used to compare patients in different response categories (complete response, stable, progressive, death) with respect to baseline relative CBV. Kaplan-Meier survival curves, log-rank tests, and Weibull survival models were used to characterize and evaluate the association of baseline relative CBV with time to progression. Tumor volumes and relative CBV measurements were obtained at initial examination and follow-up. Results: Lesions with relative CBV less than 1.75 had a median time to progression of 4620 days +/- 433 (standard deviation), and lesions with relative CBV more than 1.75 had a median time to progression of 245 days +/- 62. Patients who had an adverse event (either death or progression) had significantly higher (P = .003) relative CBV than did patients without adverse events (either complete response or stable disease). Lesions with low baseline relative CBV had stable tumor volumes at follow-up over time, whereas those with high baseline relative CBV (>1.75) had progressively increasing tumor volumes over time. Conclusion: Dynamic susceptibility-weighted contrast-enhanced perfusion MR imaging can help to identify low-grade gliomas that will progress rapidly and a subset of low-grade gliomas that have a propensity for malignant transformation. (c) RSNA, 2006

Response of a solid tumor to radiation treatment depends, in part, on the intrinsic radiosensitivity of tumor cells, the proliferation rate of tumor cells between radiation treatments and the hypoxic state of the tumor cells. A successful radiosensitizing agent would target S-phase cells and hypoxia. Recently, we demonstrated the anti-tumor effects of flavopiridol in the GL261 murine glioma model might involve 1) recruitment of tumor cells to S-phase (Newcomb et al Cell Cycle 2004; 3:230-234) and 2) an anti-angiogenic effect on the tumor vasculature by downregulation of hypoxia-inducible factor -1alpha (HIF-1alpha) (Newcomb et al Neuro-Oncology 2005; 7:225-235). Given that flavopiridol has demonstrated radiosensitizing activity in several murine tumor models, we tested whether it would enhance the response of GL261 tumors to radiation. In the present study, we evaluated the intrinsic radiation sensitivity of the GL261 glioma model using the tumor control/cure dose of radiation assay (TCD(50)). We found that a single dose of 65 Gy (CI 57.1-73.1) was required to cure 50% of the tumors locally. Using the tumor growth delay assay, fractionated radiation (5 fractions of 5 Gy over 10 days) combined with flavopiridol (5 mg/kg) given three times weekly for 3 cycles produced a significant growth delay. Our results indicate that the GL261 murine glioma model mimics the radioresistance encountered in human gliomas, and thus should prove useful in identifying promising new investigational radiosensitizers for use in the treatment of glioma patients

Overexpression of hypoxia-inducible factor-1 (HIF-1) is a common feature in solid malignancies related to oxygen deficiency. Since increased HIF-1 expression correlates with advanced disease stage, increased angiogenesis and poor prognosis, HIF-1 and its signaling pathway have become targets for cancer chemotherapy. In this study, we identified noscapine to be a novel small molecule inhibitor of the HIF-1 pathway based on its structure-function relation-ships with HIF-1 pathway inhibitors belonging to the benzylisoquinoline class of plant metabolites and/or to microtubule binding agents. We demonstrate that noscapine treatment of human glioma U87MG and T98G cell lines exposed to the hypoxic mimetic agent, CoCl2, inhibits hypoxia-mediated HIF-1alpha expression and transcriptional activity as measured by decreased secretion of VEGF, a HIF-1 target gene. Inhibition of hypoxia-mediated HIF-1alpha expression was due, in part, to its ability to inhibit accumulation of HIF-1alpha in the nucleus and target it for degradation via the proteasome. One mechanism of action of microtubule binding agents is their antiangiogenic activity associated with disruption of endothelial tubule formation. We show that noscapine has similar properties in vitro. Thus, noscapine may possess novel antiangiogenic activity associated with two broad mechanisms of action: first, by decreasing HIF-1alpha expression in hypoxic tumor cells, upregulation of target genes, such as VEGF, would be decreased concomitant with its associated angiogenic activity; second, by inhibiting endothelial cells from forming blood vessels in response to VEGF stimulation, it may limit the process of neo-vascularization, correlating with antitumor activity in vivo. For more than 75 years, noscapine has traditionally been used as an oral cough suppressant with no known toxic side effects in man. Thus, the studies reported here have found a novel function for an old drug. Given its low toxicity profile, its demonstrated antitumor activity in several animal models of cancer and its potential to inhibit the HIF-1 pathway, noscapine should be considered as an antiangiogenic chemotherapy for glioma