Defective human retinoblastoma protein identified by lack of interaction with the E1A oncoprotein

• Published in: Cancer research (Chicago, Ill.) Vol. 54; no. 4; pp. 1098 - 1104
• Main Authors: PAGGI, M. G, MARTELLI, F, FANCIULLI, M, FELSANI, A, SCIACCHITANO, S, VARMI, M, BRUNO, T, CARAPELLA, C. M, FLORIDI, A
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 15.02.1994
• Subjects: Adenovirus E1A Proteins - metabolism; Biological and medical sciences; Blotting, Northern; Blotting, Southern; Genes, Retinoblastoma; Glioma - genetics; Glioma - metabolism; Humans; Medical sciences; Neurology; Precipitin Tests; Retinoblastoma Protein - metabolism; RNA, Messenger - analysis; Tumor Cells, Cultured; Tumors of the nervous system. Phacomatoses; Adenoviridae; Methodology; Gene product; V-Onc gene; Inactivation; Adenovirus 5; Malignant glioma; Rapid technique; Investigation method; Human adenovirus; Established cell line; Tumor cell; Tumor suppressor gene; Human; Nervous system diseases; Interaction; Exploration; Malignant tumor; Medical screening; In vitro; Virus; Biological fixation; Central nervous system disease; Mastadenovirus; Mutation
• ISSN: 0008-5472; 1538-7445

Inactivating mutations of the retinoblastoma susceptibility gene (Rb) are involved in the pathogenesis of hereditary and sporadic retinoblastoma. Alterations in the Rb gene have also been found in several other human tumors occurring with epidemiological incidence higher than that of retinoblastoma. Four human malignant glioma cell lines were examined for abnormalities in the retinoblastoma gene product (pRb), using a procedure based on the interaction of pRb with an in vitro-translated adenovirus E1A oncoprotein. In the CRS-A2 cell line, derived from a glioblastoma multiforme, pRb did not bind with the in vitro-translated E1A protein. Restriction analysis of the CRS-A2 Rb gene and Rb mRNA expression provided patterns that could not be distinguished from the other glioma cell lines. Further investigation revealed the presence of a truncated pRb in the CRS-A2 cell line, due to a nucleotide insertion in the coding sequence at position 2550. In addition, this truncated Rb protein was undetectable in phosphorylated form. The binding assay with the in vitro-translated E1A was also used to study other cell lines with known mutations in the Rb gene. This method, which evaluates the interaction between in vitro-translated E1A and the pRb, is proposed as a rapid screening for detecting functional alterations in the retinoblastoma protein.