Abstract
Mutational inactivation of the retinoblastoma (RB) gene has been implicated in the genesis of retinoblastoma, osteosarcoma, and other human tumors. Our strategy has been to characterize naturally occurring mutants from tumor cells to pinpoint potential domains of RB protein crucial for tumor suppression. We show here that osteosarcoma cell line Saos-2 contains an abnormal endogenous RB protein of 95 kDa (p95) that is located mainly in the cytoplasm. This protein was identified by antibodies recognizing several different RB epitopes, but not by one directed solely against the C terminus, suggesting C-terminal truncation. This conclusion was supported by analysis of mRNA and genomic DNA, which revealed that a transcriptionally active RB allele had a deletion of exons 21-27. In contrast to normal RB protein, this truncated protein was not phosphorylated and did not bind to the large tumor (T) antigen encoded by simian virus 40. We previously reported that introduction of normal RB protein into Saos-2 cells suppressed their neoplastic phenotype, indicating functional inactivation of their endogenous RB genes. These results provide an initial step to elucidate domains crucial to the cancer-suppression function of RB protein; its C-terminal portion is evidently important for this activity.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bookstein R., Lee E. Y., Peccei A., Lee W. H. Human retinoblastoma gene: long-range mapping and analysis of its deletion in a breast cancer cell line. Mol Cell Biol. 1989 Apr;9(4):1628–1634. doi: 10.1128/mcb.9.4.1628. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bookstein R., Lee E. Y., To H., Young L. J., Sery T. W., Hayes R. C., Friedmann T., Lee W. H. Human retinoblastoma susceptibility gene: genomic organization and analysis of heterozygous intragenic deletion mutants. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2210–2214. doi: 10.1073/pnas.85.7.2210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen P. L., Scully P., Shew J. Y., Wang J. Y., Lee W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. doi: 10.1016/0092-8674(89)90517-5. [DOI] [PubMed] [Google Scholar]
- DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [PubMed] [Google Scholar]
- Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. doi: 10.1126/science.2537532. [DOI] [PubMed] [Google Scholar]
- Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Friend S. H., Bernards R., Rogelj S., Weinberg R. A., Rapaport J. M., Albert D. M., Dryja T. P. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986 Oct 16;323(6089):643–646. doi: 10.1038/323643a0. [DOI] [PubMed] [Google Scholar]
- Fung Y. K., Murphree A. L., T'Ang A., Qian J., Hinrichs S. H., Benedict W. F. Structural evidence for the authenticity of the human retinoblastoma gene. Science. 1987 Jun 26;236(4809):1657–1661. doi: 10.1126/science.2885916. [DOI] [PubMed] [Google Scholar]
- Harbour J. W., Lai S. L., Whang-Peng J., Gazdar A. F., Minna J. D., Kaye F. J. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science. 1988 Jul 15;241(4863):353–357. doi: 10.1126/science.2838909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hong F. D., Huang H. J., To H., Young L. J., Oro A., Bookstein R., Lee E. Y., Lee W. H. Structure of the human retinoblastoma gene. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5502–5506. doi: 10.1073/pnas.86.14.5502. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horowitz J. M., Yandell D. W., Park S. H., Canning S., Whyte P., Buchkovich K., Harlow E., Weinberg R. A., Dryja T. P. Point mutational inactivation of the retinoblastoma antioncogene. Science. 1989 Feb 17;243(4893):937–940. doi: 10.1126/science.2521957. [DOI] [PubMed] [Google Scholar]
- Huang H. J., Yee J. K., Shew J. Y., Chen P. L., Bookstein R., Friedmann T., Lee E. Y., Lee W. H. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science. 1988 Dec 16;242(4885):1563–1566. doi: 10.1126/science.3201247. [DOI] [PubMed] [Google Scholar]
- Lee E. Y., Bookstein R., Young L. J., Lin C. J., Rosenfeld M. G., Lee W. H. Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell line Y79. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6017–6021. doi: 10.1073/pnas.85.16.6017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee E. Y., To H., Shew J. Y., Bookstein R., Scully P., Lee W. H. Inactivation of the retinoblastoma susceptibility gene in human breast cancers. Science. 1988 Jul 8;241(4862):218–221. doi: 10.1126/science.3388033. [DOI] [PubMed] [Google Scholar]
- Lee W. H., Bookstein R., Hong F., Young L. J., Shew J. Y., Lee E. Y. Human retinoblastoma susceptibility gene: cloning, identification, and sequence. Science. 1987 Mar 13;235(4794):1394–1399. doi: 10.1126/science.3823889. [DOI] [PubMed] [Google Scholar]
- Lee W. H., Bookstein R., Lee E. Y. Studies on the human retinoblastoma susceptibility gene. J Cell Biochem. 1988 Nov;38(3):213–227. doi: 10.1002/jcb.240380309. [DOI] [PubMed] [Google Scholar]
- Lee W. H., Shew J. Y., Hong F. D., Sery T. W., Donoso L. A., Young L. J., Bookstein R., Lee E. Y. The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature. 1987 Oct 15;329(6140):642–645. doi: 10.1038/329642a0. [DOI] [PubMed] [Google Scholar]
- Ludlow J. W., DeCaprio J. A., Huang C. M., Lee W. H., Paucha E., Livingston D. M. SV40 large T antigen binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell. 1989 Jan 13;56(1):57–65. doi: 10.1016/0092-8674(89)90983-5. [DOI] [PubMed] [Google Scholar]
- Radke K., Carter V. C., Moss P., Dehazya P., Schliwa M., Martin G. S. Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses. J Cell Biol. 1983 Nov;97(5 Pt 1):1601–1611. doi: 10.1083/jcb.97.5.1601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rappolee D. A., Mark D., Banda M. J., Werb Z. Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping. Science. 1988 Aug 5;241(4866):708–712. doi: 10.1126/science.3041594. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shew J. Y., Ling N., Yang X. M., Fodstad O., Lee W. H. Antibodies detecting abnormalities of the retinoblastoma susceptibility gene product (pp110RB) in osteosarcomas and synovial sarcomas. Oncogene Res. 1989;4(3):205–214. [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whyte P., Buchkovich K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. doi: 10.1038/334124a0. [DOI] [PubMed] [Google Scholar]