Faculty Bibliography

We have shown that a COOH-terminal peptide of p53 (amino acids 361-382, p53p), linked to the truncated homeobox domain of Antennapedia (Ant) as a carrier for transduction, induced rapid apoptosis in human premalignant and malignant cell lines. Here, we report that human and rat glioma lines containing endogenous mutant p53 or wild-type (WT) p53 were induced into apoptosis by exposure to this peptide called p53p-Ant. The peptide was comparatively nontoxic to proliferating nonmalignant human and rat glial cell lines containing WT p53 and proliferating normal human peripheral marrow blood stem cells. Degree of sensitivity to the peptide correlated directly with the level of endogenous p53 expression and mutant p53 conformation. Apoptosis induction by p53p-Ant was quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and Annexin V staining in human glioma cells in vitro and in a syngeneic orthotopic 9L glioma rat model using convection-enhanced delivery in vivo. The mechanism of cell death by this peptide was solely through the Fas extrinsic apoptotic pathway. p53p-Ant induced a 3-fold increase in extracellular membrane Fas expression in glioma cells but no significant increase in nonmalignant glial cells. These data suggest that p53 function for inducing Fas-mediated apoptosis in gliomas, which express sufficient quantities of endogenous mutant or WT p53, may be restored or activated, respectively, by a cell-permeable peptide derived from the p53 COOH-terminal regulatory domain (p53p-Ant). p53p-Ant may serve as a prototypic model for the development of new anticancer agents with unique selectivity for glioma cancer cells and it can be successfully delivered in vivo into a brain tumor by a convection-enhanced delivery system, which circumvents the blood-brain barrier.

OBJECTIVES: Adjuvant-linked vaccines have been shown to induce anti-tumor immunity in patients with a variety of solid tumors. In this study we describe an in vitro model of active immunotherapy using autologous fibroblasts as immunogen. Correlative results from glioma patients immunized with autologous fibroblasts are also described. METHODS: Peripheral blood lymphocytes (PBLs) from normal subjects were immunized in vitro against autologous skin fibroblasts coupled to the adjuvant muramyl dipeptide. The lymphocytes developed cell-mediated cytotoxicity that was measured with a short-term chromium release assay. Results of in vitro experiments were compared to data derived from glioma patients immunized with subcutaneous injection of an autologous adjuvant-linked fibroblast vaccine. Glioma target cells and fibroblast immunogens were derived from early passage primary tissue culture. RESULTS: A comparison of autologous vs. homologous immunogen indicated that major histocompatibility complex matching was required at the sensitization stage of immunity (17.2 +/- 3.4% specific lysis vs. 0.4 +/- 3.1%, P < 0.01). Pre-treatment of fibroblast immunogen cells with interferon gamma (IFN-gamma) was found to significantly increase immunity (42.2 +/- 10.0%, P < 0.01), as did IFN-gamma pre-treatment of tumor target cells (35.8 +/- 9.0%, P < 0.01). The positive effect of IFN-gamma was diminished by treatment of cells with IFN-alpha. These in vitro results correlated well with in vivo data derived from glioma patients immunized with an autologous adjuvant-linked fibroblast vaccine. PBLs from patients developed direct cell-mediated cytotoxicity against autologous tumor cells. Lysis of tumor targets after in vivo immunization increased over a three-week interval (from 1.2 +/- 3.0% to 21.0 +/- 3.4%, P < 0.01) while lysis of a non-MHC matched control cell line remained essentially unchanged. CONCLUSIONS: Specific lysis of glioma targets in vitro was achieved after in vivo sensitization with autologous adjuvant-linked fibroblasts. Collectively, the data indicate that biochemically modified autologous cells can stimulate anti-glioma immunity in humans. The degree of specific immunity seen in our patients compares favorably with other published series using glioma cells as an antigenic source. Accordingly, fibroblasts may represent a practical alternative to glioma cells for vaccine construction.

OBJECT: The syndrome of normal-pressure hydrocephalus (NPH) refers to the clinical triad of gait disturbance, dementia, and urinary incontinence in association with idiopathic ventriculomegaly and normal intracranial pressure. Ventriculoperitoneal (VP) shunt placement often yields significant clinical improvements, sometimes without apparent reduction of ventricular size. The authors hypothesized that careful volumetric measurements would show a decrease in ventricular volume in these patients. METHODS: Twenty consecutive patients with NPH underwent placement of VP shunts equipped with programmable valves. In 11 patients pre- and postoperative neuroimaging was performed, which allowed volumetric analysis. Volumetric measurements of the lateral ventricles were calculated in triplicate by National Institutes of Health image-processing software to assess standard computerized tomography (CT) scans (eight patients) or magnetic resonance (MR) images (three patients) obtained before and after shunt placement. Ventricular volumes were also assessed by an independent neuroradiologist. Postoperative studies were performed at a time of clinical improvement, between 1 and 9 months postsurgery (mean 5 months). Preoperative and postoperative Unified Parkinson's Disease Rating Scale evaluations were performed in four patients. Significant clinical improvement occurred in all patients after shunt placement (mean follow-up period 17.5 months). Although 10 (91%) of 11 patients demonstrated a calculable decrease in volume in the lateral ventricles (mean decrease 39%), formal interpretation of neuroimages indicated a definite decrease in lateral ventricular volume in only three (27%) of 11 patients. CONCLUSIONS: Volumetric measurements obtained to compare preoperative and postoperative CT or MR studies obtained in patients with NPH in whom clinical improvement was seen after shunt placement surgery show a demonstrable decrease in ventricular size. Volumetric measurements may be helpful in clinical assessment postoperatively and in guiding programmable valve pressure settings