Cell cycle regulatory genes in human astrocytic tumors

Abstract: Cancer is a genetic disorder of somatic cells. Neoplastic transformation andmalignant progression may be a consequence of accumulated multiple genetic changes in acell. Astrocytic gliomas are the commonest brain tumors in humans. They aresubclassified into different malignancy grades, and malignant progression ofastrocytomas (malignancy grade II) to anaplastic astrocytomas (malignancy grade III) andglioblastomas (malignancy grade IV) is a well documented phenomenon. The aim of thestudy described in this thesis was to improve and extend the characterization of the geneticchanges associated with the oncogenesis and progression of astrocytic gliomas and toidentify some genes involved in these processes. The areas of the genome studiedprimarily included 9p and 12q. The findings in these regions led to the identification anddetailed study of cell cycle regulatory gene alterations in gliomas. Homozygous deletion of the interferon a (IFNA) locus, located at 9p22, wasknown to occur frequently in glioblastomas. In order to investigate whether IFNA is thereal target of such deletions, precise deletion mapping around the IFNA locus was carriedout 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 identifiedand localized within this region by others. A more detailed allelic assessment of the regionin our tumors showed that the frequency of homozygous deletion was the highest at theCDKN2A locus. Approximately 40% of all glioblastomas had homozygous deletions andan additional 30% had hemizygous deletions of the CDKN2A gene with anaplasticastrocytomas showing a similar pattern but at lower frequencies. The homozygous andhemizygous deletions involved both CDKN2A and CDKN2B genes in the vast majorityof tumors. Hemizygous deletions of the CDKN2A were infrequently associated withmutations, a total of five being identified among 14 anaplastic astrocytomas and 39glioblastomas. No mutations of CDKN2B were identified. Variable expression ofCDKN2A was documented in cases retaining at least one allele. Hypermethylation of the5'CpG islands of CDKN2A did not correlate with a lack of gene expression. In parallelwith this study, a mapping of the MDM2 amplicon on 12ql3-14 in gliomas was carriedout. 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 invariablyaccompanied by overexpression of transcripts. When the data from 9p and 12q werecompiled, amplification of CDK4 was preferentially found among glioblastomas andanaplastic astrocytomas without CDKN2A deletions. Normally CDK4 forms a kinasecomplex with Cyclin Dl and phosphorylates pRB in the G, phase of the cell cycle.Phosphorylated pRB releases the E2F transcription factors which transcriptionally activatethe genes required for S phase entry and DNA replication. The protein product ofCDKN2A, p 16, is a specific inhibitor of Cyclin D/CDK4 kinase complex. Thusalteration of one of these genes, involved in the same cell-cycle regulatory pathway wasshown to be a common abnormality in anaplastic astrocytomas and glioblastomas. Inorder to obtain further evidence to support the hypothesis that alteration of cell cycleregulatory genes plays an important role in astrocytic tumor progression, the RBI genewas examined in the same tumors. A total lack of wild-type RBI was observedpreferentially among tumors with normal CDKN2A and CDK4. In total, 64% ofglioblastomas contained either no wild-type type CDKN2A or RBI, or had amplificahonof 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-typeallele of CDKN2A and RBI. Our results implicate CDKN2A, CDK4 and RBI in theirprogression and malignant progression. Alterations of G,/S phase cell cycle regulationappeared to be a cardinal mechanism in the development and progression of humananaplastic astrocytomas and glioblastomas.ISBN 91-628-2021-4 Stockholm 1996