Yasuo Tohma

PTEN (MMAC1) mutations are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas.

Loss of heterozygosity (LOH) on chromosome 10 is the most frequent genetic alteration associated with the evolution of malignant astrocytic tumors and it may involve several loci. The tumor suppressor gene PTEN (MMAC1) on chromosome 10q23 is mutated in approximately 30% of glioblastomas (WHO Grade IV). In this study, we assessed the frequency of PTEN mutations in primary glioblastomas, which developed clinically de novo, and in secondary glioblastomas, which evolved from low-grade (WHO Grade II) or anaplastic astrocytomas (WHO Grade III). Nine of 28 (32%) primary glioblastomas contained a PTEN mutation and an additional case showed a homozygous PTEN deletion. This indicates that after overexpression/ amplification of the EGF receptor, loss of PTEN function is the most common alteration in primary glioblastomas. In this series, 5 of 28 (18%) primary glioblastomas showed both a PTEN mutation and EGFR amplification. In contrast, only 1 of 25 (4%) secondary glioblastomas contained a PTEN mutation, and none of them showed a homozygous PTEN deletion. The secondary glioblastoma with a PTEN mutation developed from an anaplastic astrocytoma that already carried the mutation. The observation that secondary glioblastomas have a p53 mutation as a genetic hallmark but rarely contain a PTEN mutation supports the concept that primary and secondary glioblastomas develop differently on a genetic level.

Alterations of cell cycle regulatory genes in primary (de novo) and secondary glioblastomas.

Abstract Primary glioblastomas develop rapidly de novo through a genetic pathway characterized by amplification/overexpression of EGFR and of MDM2 genes. Secondary glioblastomas develop more slowly through progression from low grade or anaplastic astrocytoma and show a high incidence of a p53 mutation. In the present study, primary and secondary glioblastomas were analyzed for p16 deletions and CDK4 amplification by differential PCR and for loss of expression of the retinoblastoma (RB) gene by immunohistochemistry. Except for one case, alterations in the structure or expression of p16, CDK4 and RB were mutually exclusive. The overall incidence of aberrant expression of these genes coding for components of the cell-cycling-regulatory system was similar in primary (14/28; 50%) and secondary glioblastomas (9/23; 39%). However, p16 deletions were significantly more frequent in the former (10/28; 36%) than in the latter (1/23, 4%; P = 0.0075), suggesting that this alteration constitutes an additional genetic hallmark of the primary (de novo) glioblastoma.

Fas Ligand Expression in Glioblastoma Cell Lines and Primary Astrocytic Brain Tumors

Fas/APO‐1 (CD95) is a cell surface receptor that mediates apoptosis when it reacts with Fas ligand (FasL) or Fas antibody. We previously reported that Fas expression is predominantly induced in perinecrotic glioma cells, suggesting that Fas induction is associated with apoptosis and necrosis formation, a histological hallmark of glioblastomas. In this study, we assessed the expression of FasL in 10 glioblastoma cell lines and in 14 astrocytic brain tumors (three low‐grade astrocytomas and 11 glioblastomas). Reverse transcriptase (RT)‐PCR revealed that all glioblastoma cell lines and primary astrocytic brain tumors express FasL. Immunohistochemically, FasL was predominantly expressed on the plasma membrane of glioma cells. These results suggest that FasL expression is common in human astrocytic brain tumors and may cause apoptosis of glioma cells if Fas expression is induced.

Necrogenesis and Fas/APO-1 (CD95) expression in primary (de novo) and secondary glioblastomas.

Glioblastomas may develop rapidly without clinical and histopathological evidence of a less malignant precursor lesion (de novo or primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). Primary glioblastomas typically show overexpression of EGFR, but rarely p53 mutations, while secondary glioblastomas frequently carry a p53 mutation, but usually lack overexpression of EGFR, suggesting that these glioblastoma subtypes develop through distinct genetic pathways. In the present study, we assessed the expression of Fas/APO-1 (CD95), an apoptosis-mediating cell membrane protein, and its relation to necrosis phenotype in primary and secondary glioblastomas. Large areas of ischemic necroses were observed in all 18 primary glioblastomas, but were significantly less frequent in secondary glioblastomas (10 of 19, 53%; p=0.0004). Fas expression was predominantly observed in glioma cells surrounding large areas of necrosis and was thus significantly more frequent in primary glioblastomas (18 of 18, 100%) than in secondary glioblastomas (4 of 19, 21%; p< 0.0001), suggesting that these clinically and genetically defined subtypes of glioblastoma differ in the extent and mechanism of necrogenesis. Necrosis and microvascular proliferation are histologic hallmarks of the glioblastoma. Following incubation of glioblastoma cell lines under hypoxic/anoxic conditions for 24-48 hours, Fas mRNA levels remained unchanged, whereas VEGF expression was markedly upregulated. This suggests that in contrast to VEGF, Fas expression is not induced by ischemia/hypoxia. Analysis of Fas mRNA levels in a glioblastoma cell line containing a p53 mutation and an inducible wild-type p53 gene showed little difference under induced and noninduced conditions, suggesting that in glioblastomas, Fas expression is not directly linked to the p53 status.

Effect of Intragastric Application of N-Methylnitrosourea in p53 Knockout Mice

Nullizygous p53 knockout (p53−/−) mice are highly susceptible to spontaneous tumorigenesis, in particular malignant lymphomas at an early age. Heterozygous p53 knockout (p53+/−) mice develop spontaneous tumors less frequently but may show increased susceptibility to chemical carcinogens. In this study, p53−/−, p53+/−, and p53 wild‐type (p53+/+) mice were treated with N‐methylnitrosourea (MNU) by gastric intubation (5 μg/g body weight) three times per week for 5 wk, starting at 5–6 wk of age. The surviving mice were killed when they were 56–57 wk old. All eight p53−/− mice treated with MNU developed malignant lymphomas with a shorter latent period (mean age  =  16.4±0.5 wk) than their spontaneous tumors (61%, at age 23.3±1.4 wk). In p53+/− mice treated with MNU, malignant lymphomas developed at a higher frequency (eight of 27, 30%) than did spontaneous lymphomas (5%). Development of sarcomas in p53−/− and p53+/− mice was also significantly enhanced by treatment with MNU. All eight thymic lymphomas and three sarcomas in the p53+/− mice showed a loss of the remaining wild‐type p53 allele. These results indicate that intragastric MNU treatment significantly enhanced spontaneous development of malignant lymphomas and sarcomas in both p53−/− and p53+/− mice. In the stomachs of 12 p53+/− mice, that were killed at the end of the experiment, two adenomas, one carcinoma in situ, and four adenocarcinomas were observed. In the stomachs of 31 p53+/+ mice, eight adenomas and one carcinoma in situ were detected. The overall incidence of tumorous changes in the stomachs of p53+/− (seven of 12, 58%) and p53+/+ (nine of 31, 29%) mice were not significantly different (P = 0.090). However, adenocarcinomas invading the submucosa were observed in p53+/− mice (four of 12, 33%) but not in p53+/+ mice (zero of 31; P = 0.004), suggesting a slightly higher susceptibility to gastric carcinogenesis induced by MNU in p53+/− mice. Mol. Carcinog. 28:97–101, 2000.