C-terminal truncation of the retinoblastoma gene product leads to functional inactivation.
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.
Shew
J Y
JY
Department of Pathology, University of California at San Diego, La Jolla 92093.
Lin
B T
BT
Chen
P L
PL
Tseng
B Y
BY
Yang-Feng
T L
TL
Lee
W H
WH
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D015342:DNA Probes; D005134:Eye Neoplasms / Q000235:genetics*; D005796:Genes; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase; D015183:Restriction Mapping; D012175:Retinoblastoma / Q000235:genetics*; D016160:Retinoblastoma Protein; D013489:Suppression, Genetic
D000906:Antibodies; D015342:DNA Probes; D009363:Neoplasm Proteins; D016160:Retinoblastoma Protein; C062257:retinoblastoma protein p95; D012194:RNA-Directed DNA Polymerase