C-terminal truncation of the retinoblastoma gene product leads to functional inactivation
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1Department of Pathology, University of California at San Diego, La Jolla 92093.
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.
Insights
Mutational inactivation of the retinoblastoma (RB) gene is linked to cancer. Researchers identified a truncated RB protein in osteosarcoma cells, indicating the C-terminal portion is crucial for tumor suppression.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Mutational inactivation of the retinoblastoma (RB) gene is a key factor in the development of various human tumors, including retinoblastoma and osteosarcoma.
- Understanding the functional domains of the RB protein is critical for elucidating its tumor suppressor mechanisms.
Purpose of the Study:
- To characterize naturally occurring RB mutants in tumor cells to identify domains essential for tumor suppression.
- To investigate the structural and functional properties of an abnormal RB protein found in the Saos-2 osteosarcoma cell line.
Main Methods:
- Immunological identification of RB protein variants using antibodies against different epitopes.
- Analysis of RB gene expression at the mRNA and genomic DNA levels.
- Assessment of RB protein phosphorylation and its interaction with simian virus 40 T antigen.
Main Results:
- An abnormal, truncated RB protein (p95) was identified in Saos-2 cells, primarily localized in the cytoplasm.
- The truncated RB protein resulted from a deletion of exons 21-27 in the RB gene.
- This abnormal RB protein was not phosphorylated and failed to bind to the SV40 T antigen, unlike the normal RB protein.
Conclusions:
- The C-terminal portion of the RB protein is essential for its tumor suppressor activity.
- Functional inactivation of RB protein, due to C-terminal truncation, contributes to the neoplastic phenotype of osteosarcoma cells.