Restoration of p53 function for selective Fas-mediated apoptosis in human and rat glioma cells in vitro and in vivo by a p53 COOH-terminal peptide.

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.

Lack of B7 expression, not human leukocyte antigen expression, facilitates immune evasion by human malignant gliomas.

OBJECTIVE: Lack of human leukocyte antigens and costimulatory molecules have been suggested as mechanisms by which human malignant gliomas avoid immune recognition and elimination. METHODS: Using quantitative multiparameter flow cytometric analysis, tumor cells from patients with glioblastoma multiforme (n = 18) were examined ex vivo for the expression of human leukocyte antigen Class I and II molecules and the costimulatory molecules B7-1 and B7-2. They were compared with reactive astrocytes from peritumoral brain metastases (n = 7), and astrocytes removed during nontumor surgery (n = 5). RESULTS: In contrast to the vast majority of solid peripheral human tumors, these results demonstrate that glioblastoma multiforme frequently express both human leukocyte antigen Class I and II molecules. Like most solid peripheral tumors, glioblastoma multiforme tumor cells express few or no B7 costimulatory molecules. Functional assays using heterogeneous ex vivo tumor preparations or pure populations of ex vivo tumor cells and microglia obtained using fluorescence-activated cell sorting indicate that CD4+ T-cells are activated by tumor cells only in the presence of exogenous B7 costimulation (provided by addition of soluble agonist anti-CD28 monoclonal antibody). CONCLUSION: Thus, in contrast to many solid peripheral tumors, failure to present tumor antigens is not a likely impediment to immunotherapeutic strategies against malignant gliomas. Rather, immunotherapeutic strategies need to overcome low levels of B7 costimulation.

Changes in the immunologic phenotype of human malignant glioma cells after passaging in vitro.

Although immunotherapeutic strategies against glioblastomas have been promising both in vitro and in animal models, similar successes have not been realized in human clinical trials. One reason may be that immunotherapeutic strategies are based on prior studies that primarily have used human glioblastoma cell lines passaged in vitro, which may not accurately reflect the in vivo properties of glioblastoma cells. In this report, we used flow cytometry to quantify the expression of immunological cell surface molecules on human glioblastomas directly ex vivo (prior to any in vitro culturing) and after varying passages in vitro. Furthermore, we used ELISA to quantitate cytokine secretion after various passages in vitro. We demonstrate that in vitro culturing of established cell lines led to increases in the cell surface expression of MHC class I and ICAM-1 and secretion of IL-6 and TGF-beta(2). Furthermore, there were significant changes in the expression of MHC class I, MHC class II, B7-2, ICAM-1, and FasL when comparing ex vivo tumor cells to those after a single passage in vitro. After passaging once in vitro, there were also significant changes in the secretion of TGF-beta(2) and IL-10. This report indicates that in vitro culturing leads to significant changes in both cell surface molecules and secreted cytokines, which are known to affect the ability of immune cells to initiate an anti-tumor immune response. These changes in the immunological phenotype of glioblastomas after in vitro culturing may in part explain the limited success of immunotherapeutic strategies against glioblastomas in human clinical trials.