However, whether PTEN is causally linked to induction of angiogenesis by the tumor cell remains unproven.These and other obser vations led us to hypothesize that PTEN may control tumorinduced angiogenesis and contribute to the high mortality associatedwith malignant brain tumors.Mice, under general anesthesia were placed into the stereotactic dev ice. Membranes were probedwith antisera specific for PTEN, AKT, phosphoSAKT, or TSP.Brief ly, mg of total RNA was precipitated and resuspended in ml of hybridization buf fer contain ing the radioactive probe.The RNA then was heated to C for min and hybridized for hat C.RNA probes were synthesized by using MAX I SCRIPT using PCR templates and T polymerase.Antibody stain ing wasvisualizedwith perox idaseconjugated antimouse and counterstainedwith hematox ylin.Two sections from each tumor were scanned under lowpower magn ification, followed by digitization of three fields from this area.Data were collected from two independent obser verswithout knowledge of which tumors wereviewed.The status of the PTEN gene in each stable cell line was designated as: WT.Comparison of in vitro growth of UMG cells transduced with mutants of PTEN.The UMG cell line is derived from a patient diagnosedwith glioblastoma multiforme, a highly malignant and un iformly fatal brain tumor.This tumor and other human glioblastomas and glioblastoma cell lines contain a mutation in both PTEN alleles, resulting in a null genotype.In light of these obser vations, we Ranitidine Hydrochloride reconstituted the PTEN gene in the parental UMG cells.Stable derivatives of the parental U cells were generated af ter transductionwith retrov iruses encoding cDNA for WT PTEN or specific mutants of this phosphatase.In particular, we used missense mutations in the PT P signature motif to ascertain the importance of the enzymatic activ ity of PTEN to its tumor suppressor function.Tumor cells were characterized biochemically for levels of activated AKT. Therefore, we compared these cell lines further in our in vivo models.Using antiPTEN antisera, we detected the expression of PTEN in all tissues,with the exception of skeletal and heart muscle. These results indicate that the tumor tissue sampled represents predominantly tumor cellderived proteins.The pattern of phosphor ylated AKT was similar when the dif ferent U mutant expressing cell lines were assayed in vitro or in vivo. Despite the similar in vitro growth rate, there was a dramatic difference in the growth of tumors derived from parental U cells compared with cells reconstituted with WT PTEN. Interestingly, the reconstitution of U cells with catalytically impaired PTEN shows an intermediate level of growth suppression, a result that suggests some residual function of these mutants in vivo.These results suggested that something other than the proliferative or apoptotic rate of the UMG versus UMG reconstitutedwith WT PTEN accounted for the dif ference in grow th potential.We obser ved that the loss of inositol Valproic Acid phospholipid phosphatase activ ity results in deregulated tumor grow th comparable to the total ablation of cataly tic activ ity and that the despite dif ferences in overall grow th in vivo, the proliferative rate of these tumors is similar.To examine the ef fect of PTEN on angiogenesis, we compared parental U cells to cells reconstitutedwith WT or mutant PTEN.