P53 gene expression in medulloblastoma by quantitative polymerase chain reaction

Massimo Loda, Felice Giangaspero, Paola Capodieci

Research output: Contribution to journalArticle

Abstract

The frequent cytogenetic abnormality—isochromosome 17q [i(17)q]—observed in medulloblastomas (MB) may result in altered expression of the oncosuppressor gene p53 that is located on 17p. p53 expression was therefore evaluated in five MBs and in one MB cell line derived from one of these tumors. Expression levels of p53 utilizing serially diluted unfractionated RNA from tumors and the cell line were assessed both by dot-blot and by reverse transcription (RT) followed by the polymerase chain reaction (PCR). The quality of RNA, efficiency of reaction, and transcript quantitation were determined by simultaneous transcription and amplification of a similarly sized fragment of the α-tubulin gene. All MBs showed low levels of expression of p53 compared to those found in normal tissues. p53 messenger RNA (mRNA) was significantly increased (two- to threefold) in the MB cell line compared to its tumor of origin and to the other MBs. Immunoperoxidase studies performed with monoclonal antibodies to the p53 protein product showed focal nuclear expression in one of five of the original tumors while most cells grown in vitro and in the xenograft were positive. The results indicate that (a) p53 mRNA was present in all MBs tested, thereby excluding the possibility of homozygous deletions of 17p, even though one tumor had i(17)q and another had a rearranged 17p; (b) p53 mRNA was not present in MBs in quantities significant enough to suspect inactivation by a combination of loss of heterozygosity and allelic point mutation; (c) p53 mRNA was overexpressed only in the MB cell line and xenograft suggesting an acquired p53 mutation conferring a growth advantage to selected clones of MB cells; and (d) RT-PCR is a rapid and simple nonisotopic technique that utilizes minimal quantities of total RNA for the assessment of gene expression.

Original languageEnglish
Pages (from-to)36-44
Number of pages9
JournalDiagnostic Molecular Pathology
Volume1
Issue number1
Publication statusPublished - 1992

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Keywords

  • Medulloblastoma
  • P53
  • Quantitative PCR

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pathology and Forensic Medicine

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