The drought of progress in clinical brain tumor therapy provides an impetus for developing new treatments as well as methods for testing therapeutics in animal models. The inability of traditional assays to simultaneously measure tumor size, location, growth kinetics, and cell kill achieved by a treatment complicates the interpretation of therapy experiments in animal models. To address these issues, tumor volume measurements obtained from serial magnetic resonance images were used to noninvasively estimate cell kill values in individual rats with intracerebral 9L tumors after treatment with 0.5, 1, or 2 ؋ LD 10 doses of 1,3-bis(2-chloroethyl)-1-nitrosourea. The calculated cell kill values were consistently lower than those reported using traditional assays. A dose-dependent increase in 9L tumor doubling time after treatment was observed that significantly contributed to the time required for surviving cells to repopulate the tumor mass. This study reveals that increases in animal survival are not exclusively attributable to the fraction of tumor cells killed but rather are a function of the cell kill and repopulation kinetics, both of which vary with treatment dose.Brain tumors occur frequently in the human population with approximately 35,000 new cases of primary adult central nervous system tumors diagnosed in the United States each year (1). The 1,500-2,000 brain tumors reported annually in children constitute the largest group of solid pediatric neoplasms (2). Despite the use of multimodality therapy, the management of brain tumors in adults and children remains unsatisfactory. In particular, the treatment of glioblastoma multiforme constitutes a major problem caused by the lack of therapeutic responses and a median survival time of only 1 year from the initial diagnosis (3).Promising agents identified through in vitro screening assays with brain tumor cell lines subsequently are evaluated in vivo by using animal brain tumor models (4-6). These studies frequently use rodent brain tumor models (7-10), particularly the rat 9L tumor (11-29). Animal survival, colony-forming efficiency (CFE) assays of cells disaggregated from solid tumors, and measurements of excised tumor weights have been used for two decades to quantitate the efficacy of various treatments on the orthotopic 9L brain tumor (11)(12)(13)(14)(15)(16)(17)(18)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Numerous in vitro and in vivo studies have shown that 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is an effective treatment for the 9L brain tumor (11-15, 17, 18, 20-23, 25-29); in fact, a single bolus of BCNU achieved a 3-4 log cell kill in orthotopic tumors, resulting in a 97% increase in life span (13).BCNU is a mainstay of brain tumor chemotherapy, but the clinical outcome of patients treated solely with BCNU has not proven as efficacious as would be predicted by the responses observed in the 9L tumor model (30-33).In the present study, MRI was used for noninvasive estimation of tumor cell kill in individual animals with orthotopic 9L brain...

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) generated in the stereoselective deamination of D-amino acids catalyzed by D-amino acid oxidase (DAAO). H2O2 readily crosses cellular membranes and damages DNA, proteins, and lipids. The scarcity of DAAO substrates in mammalian organisms and its co-localization with catalase in the peroxisomal matrix suggested that the cytotoxicity of ROS could be harnessed by administration of D-amino acids to tumor cells ectopically expressing DAAO in the cytoplasm. To evaluate this hypothesis, the cDNA encoding the highly active DAAO from the red yeast Rhodotorula gracilis was mutated to remove the carboxy-terminal peroxisomal targeting sequence. A clonal line of 9L glioma cells stably transfected with this construct (9Ldaao17) was found to synthesize active R. gracilis DAAO. Exposure of 9Ldaao17 cells to D-alanine resulted in cytotoxicity at concentrations that were nontoxic to parental 9L cells. Depletion of cellular glutathione further sensitized 9Ldaao17 cells to D-alanine (D-Ala). This result, combined with stimulation of pentose phosphate pathway activity and the production of extracellular H2O2 by 9Ldaao17 cells incubated with D-alanine implicates oxidative stress as the mediator of cytotoxicity. These results demonstrate that expression of R. gracilis DAAO in tumor cells confers chemosensitivity to D-alanine that could be exploited as a novel cancer gene therapy paradigm.