Alterations of cell cycle control genes in human astrocytomas

Abstract

Cytogenetic analyses and molecular genetic studies have revealed a number of chromosomalregions deleted or amplified in astrocytic tumors, presumably harboring tumor suppressorgenes and oncogenes involved in astrocytic oncogenesis. Astrocytomas represent the mostcommon subgroup of primary intracranial tumors. They are subdivided into differentmalignancy grades based on the WHO classification system. Low grade astrocytomas (gradeII) may progress to anaplastic astrocytomas (grade III) and to the most malignant form, theglioblastoma (grade IV). The latter is associated with a very bad prognosis with a survival ofless than one year despite current advanced surgery and irradiation techniques. Withincreasing 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 inhuman astrocytomas. We studied primary human astrocytomas in order to elucidate thegenetic basis underlying astrocytic tumorigenesis and progression.We examined a series of 157 tumors for amplification of the MDM2 gene, a negativeregulator of the p53 tumor suppressor protein. MDM2 was found to be amplified andoverexpressed in 8-10% of anaplastic astrocytomas and glioblastomas and thus appears tofunction as an oncogene in the malignant progression of a subset of these tumors. MDM2amplification occurred exclusively in tumors with wild-type p53 alleles, thereby possiblyrepresenting an alternative mechanism of p53 inactivation. Astrocytomas (grade II) did notshow 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 anaplasticastrocytomas and glioblastomas was defined as lying in between the D9S171 locus and theIFNA gene cluster. Following the cloning of the CDKN2A and CDKN2B genes from thisregion we refined the deletion map. We found these genes homozygously deleted in 41% ofglioblastomas and 19% of anaplastic astrocytomas, suggesting their involvement in astrocyticprogression. These genes code for cyclin-dependent kinase inhibitors (pl6 and pl5) involvedin the regulation of the G1/S progression of the cell cycle. Both CDKN2A and CDKN2B weresequenced, and one missense mutation of the CDKN2A gene was identified in a glioblastomawith hemizygous deletion. Expression of the gene transcripts was variable, and aberrantmethylation of the 5' CpG islands of the CDKN2A gene was excluded as a major mechanismof epigenetic gene inactivation. Amplification with overexpression of CDK4, the target forpl6 and pl5 inhibition, was found to be an alternative mechanism of impairing the regulationof G1/S progression in a significant subset of anaplastic astrocytomas and glioblastomas. Noneof 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 offive, two of which were found in anaplastic astrocytomas. A deletion mapping ofchromosome 13 using microsatellite analysis implicated the RBI gene as the target of thedeletions on this chromosome. Among the cases with hemizygous deletions of RBI, severalmutations were detected by SSCP analysis of the promoter region and all known exons andthese mutations were confirmed by direct sequencing.The cumulative data showed that genes involved in G1/S progression of the cell cycle arefrequently altered in the more malignant astrocytic tumors. Interestingly, these alterationsoccur in a mutually almost exclusive manner with only one of these genes whose products arepart 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 inthe malignant progression of astrocytic tumors.ISBN 91-628-2020-6 Stockholm 1996