We used a combination of Northern blot analysis, protein truncation assays, and sequence analysis to determine the types and frequency of PTEN mutations in glioma cell lines so that we could define appropriate recipients to assess the growth suppressive function of PTEN by gene transfer.
A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer.
The sequence analysis of PTEN/MMAC1 revealed nucleotide alterations in 1/8 (12.5%) low-grade gliomas in a tumor with LOH at l0q21-qtel and in 5/21 (24%) high-grade gliomas displaying LOH that always included 10q23-26.
Using the mutant PTENglioma cell line, U87MG, as a transfection recipient for a series of PTEN alleles, we provide direct evidence that this capacity requires phosphatase activity.
The authors introduced wild-type PTEN complementary DNA into four human glioma cell lines (T98G, U-251MG, U-87MG, and A172) containing endogenous aberrant PTEN alleles.
In contrast, ectopic expression of wild type PTEN, but not the PTEN(G129R) mutant, in PTEN-mutant gliomas markedly sensitizes these cells to irradiation and to CD95-ligand (CD95L)-induced apoptosis.
Six constitutional missense mutations of the p53 gene were identified (13.6%), but no mutations of the p16 and PTEN genes were found, suggesting that (1) germline p53 mutations contribute to a small portion of astrocytic tumors, (2) inherited mutations of the p16 and PTEN gene do not predispose to the development of gliomas, and (3) other genes are involved in glioma predisposition.
Rsa I digestion may help to identify this putative PTEN pseudogene which, according to RT-PCR analysis on glioma, fibroblast, brain and lung cells, does not appear to be transcribed.
The PTEN gene, located on 10q23.3, has recently been described as a candidate tumour suppressor gene that may be important in the development of advanced cancers, including gliomas.
To characterize the potential cellular functions of MMAC1, we expressed wild-type and several mutant variants of MMAC1 in the human glioma cell line, U373, that lacks endogenous expression.
The tumour suppressor gene PTEN/MMAC1, which is mutated or homozygously deleted in glioma, breast and prostate cancer, is mapped to a region of 10q which shows loss of heterozygosity (LOH) in bladder cancer.
Because PTEN negatively regulates on the downstream effects of phosphatidylinositol-3-kinase (PI3-K), we hypothesized that the loss of PTEN/MMAC1 and the activation of RAS may be largely equivalent because RAS is a known positive upstream regulator of PI3-K. We expanded our previous survey of PTEN/MMAC1 mutations and analyzed the RAS status of 53 cutaneous melanoma cell lines, 18 glioma cell lines, and 17 uncultured cutaneous melanoma metastasis.
These results suggest that mutation of the PTEN gene is a later event than that of the p53 gene in glioma progression and is associated with both the genetic pathways.
Using the C6 glioma cell line, which does not express measurable amounts of PTEN protein and in which in vitro invasiveness is MMP dependent, the authors determined that in vitro glioma cell invasiveness was significantly reduced when cells were preincubated overnight with LY294002 or wortmannin, two specific inhibitors of PI 3-K signaling.
These findings indicate that PTEN gene mutation contributes not only to the neoplastic evolution in gliomas but also to the meningeal dissemination of glioma cells.
The tumour suppressor gene PTEN is also often somatically mutated in glioma, therefore it is attractive as a candidate gene for germline mutations in familial glioma.