Cell cycle regulatory genes in human astrocytic tumors

Abstract

Cancer is a genetic disorder of somatic cells. Neoplastic transformation and malignant progression may be a consequence of accumulated multiple genetic changes in a cell. Astrocytic gliomas are the commonest brain tumors in humans. They are subclassified into different malignancy grades, and malignant progression of astrocytomas (malignancy grade II) to anaplastic astrocytomas (malignancy grade III) and glioblastomas (malignancy grade IV) is a well-documented phenomenon. The aim of the study described in this thesis was to improve and extend the characterization of the genetic changes associated with the oncogenesis and progression of astrocytic gliomas and to identify some genes involved in these processes. The areas of the genome studied primarily included 9p and 12q. The findings in these regions led to the identification and detailed study of cell cycle regulatory gene alterations in gliomas.

Homozygous deletion of the interferon a (IFNA) locus, located at 9p22, was known to occur frequently in glioblastomas. In order to investigate whether IFNA is the real target of such deletions, precise deletion mapping around the IFNA locus was carried out combining RFLP analysis, densitometry and microsatellite analysis in astrocyticgliomas of different malignancy grades. The commonly deleted region was defined asbeing between and not including the IFNA and D9S171 loci, suggesting the loss of an asyet unidentified tumor suppressor gene. The CDKN2A (p161MTSl) and CDKN2B (p151MTS2) genes, coding for inhibitors of the Cyclin D/CDK4 complex, were identified and localized within this region by others. A more detailed allelic assessment of the region in our tumors showed that the frequency of homozygous deletion was the highest at the CDKN2A locus. Approximately 40% of all glioblastomas had homozygous deletions and an additional 30% had hemizygous deletions of the CDKN2A gene with anaplastic astrocytomas showing a similar pattern but at lower frequencies. The homozygous and hemizygous deletions involved both CDKN2A and CDKN2B genes in the vast majority of tumors. Hemizygous deletions of the CDKN2A were infrequently associated with mutations, a total of five being identified among 14 anaplastic astrocytomas and 39 glioblastomas. No mutations of CDKN2B were identified. Variable expression of CDKN2A was documented in cases retaining at least one allele. Hypermethylation of the 5'CpG islands of CDKN2A did not correlate with a lack of gene expression.

In parallel with this study, a mapping of the MDM2 amplicon on 12ql3-14 in gliomas was carried out. CDK4 and SAS, not MDM2, were identified as being most frequently amplified. Amplification of all these three genes, but not other co-amplified genes, was invariably accompanied by overexpression of transcripts. When the data from 9p and 12q were compiled, amplification of CDK4 was preferentially found among glioblastomas and anaplastic astrocytomas without CDKN2A deletions. Normally CDK4 forms a kinase complex with Cyclin Dl and phosphorylates pRB in the G, phase of the cell cycle. Phosphorylated pRB releases the E2F transcription factors which transcriptionally activate the genes required for S phase entry and DNA replication. The protein product of CDKN2A, p 16, is a specific inhibitor of Cyclin D/CDK4 kinase complex. Thus alteration of one of these genes, involved in the same cell-cycle regulatory pathway was shown to be a common abnormality in anaplastic astrocytomas and glioblastomas. In order to obtain further evidence to support the hypothesis that alteration of cell cycle regulatory genes plays an important role in astrocytic tumor progression, the RBI gene was examined in the same tumors. A total lack of wild-type RBI was observed preferentially among tumors with normal CDKN2A and CDK4. In total, 64% of glioblastomas contained either no wild-type type CDKN2A or RBI, or had amplification of CDK4. Only 6% retained two, apparently wild-type, copies of these genes. Astrocytoma grade II had no amplification of CDK4 and always retained one wild-type allele of CDKN2A and RBI. Our results implicate CDKN2A, CDK4 and RBI in their progression and malignant progression. Alterations of G,/S phase cell cycle regulation appeared to be a cardinal mechanism in the development and progression of human anaplastic astrocytomas and glioblastomas.