Alterations of cell cycle control genes in human astrocytomas

Abstract

Cytogenetic analyses and molecular genetic studies have revealed a number of chromosomal regions deleted or amplified in astrocytic tumors, presumably harboring tumor suppressor genes and oncogenes involved in astrocytic oncogenesis. Astrocytomas represent the most common subgroup of primary intracranial tumors. They are subdivided into different malignancy grades based on the WHO classification system. Low grade astrocytomas (gradeII) may progress to anaplastic astrocytomas (grade III) and to the most malignant form, the glioblastoma (grade IV). The latter is associated with a very bad prognosis with a survival of less than one year despite current advanced surgery and irradiation techniques. With increasing malignancy grade, the tumors show an accumulation of genetic lesions. Only a few genes have been assigned to the targeted regions and found to be altered in human astrocytomas.

We studied primary human astrocytomas in order to elucidate the genetic basis underlying astrocytic tumorigenesis and progression. We examined a series of 157 tumors for amplification of the MDM2 gene, a negative regulator of the p53 tumor suppressor protein. MDM2 was found to be amplified and overexpressed in 8-10% of anaplastic astrocytomas and glioblastomas and thus appears to function as an oncogene in the malignant progression of a subset of these tumors. MDM2 amplification occurred exclusively in tumors with wild-type p53 alleles, thereby possibly representing an alternative mechanism of p53 inactivation. Astrocytomas (grade II) did not show any evidence of amplification. A series of 70 astrocytomas was subjected to a deletion mapping of chromosome 9, concentrating mainly on the short arm.

The region commonly deleted in anaplastic astrocytomas and glioblastomas was defined as lying in between the D9S171 locus and the IFNA gene cluster. Following the cloning of the CDKN2A and CDKN2B genes from this region we refined the deletion map. We found these genes homozygously deleted in 41% of glioblastomas and 19% of anaplastic astrocytomas, suggesting their involvement in astrocytic progression. These genes code for cyclin-dependent kinase inhibitors (pl6 and pl5) involved in the regulation of the G1/S progression of the cell cycle. Both CDKN2A and CDKN2B were sequenced, and one missense mutation of the CDKN2A gene was identified in a glioblastoma with hemizygous deletion. Expression of the gene transcripts was variable, and aberrant methylation of the 5' CpG islands of the CDKN2A gene was excluded as a major mechanism of epigenetic gene inactivation. Amplification with overexpression of CDK4, the target forpl6 and pl5 inhibition, was found to be an alternative mechanism of impairing the regulation of G1/S progression in a significant subset of anaplastic astrocytomas and glioblastomas. None of these abnormalities was detected in any astrocytoma (grade II). An extended series of 195 astrocytomas was characterized as to the status of the CDKN2A, RBI and CDK4 genes. The number of CDKN2A mutations identified increased to a total of five, two of which were found in anaplastic astrocytomas. A deletion mapping of chromosome 13 using microsatellite analysis implicated the RBI gene as the target of the deletions on this chromosome. Among the cases with hemizygous deletions of RBI, several mutations were detected by SSCP analysis of the promoter region and all known exons and these mutations were confirmed by direct sequencing.

The cumulative data showed that genes involved in G1/S progression of the cell cycle are frequently altered in the more malignant astrocytic tumors. Interestingly, these alterations occur in a mutually almost exclusive manner with only one of these genes whose products are part of the same regulatory pathway being affected in any single tumor. In summary, abnormalities of this cell cycle control system appear to represent an almost obligatory step in the malignant progression of astrocytic tumors.