Functional interaction between human topoisomerase IIα and retinoblastoma protein

DNA topoisomerase II—an essential nuclear enzyme in DNA replication and transcription, chromatin segregation, and cell cycle progression—is also a target of clinically useful anticancer drugs. Preliminary observations of a positive correlation between the expression of topoisomerase (topo) IIα and the retinoblastoma protein (Rb) in a series of rhabdomyosarcoma cells prompted us to ask whether these two proteins interact in vivo. Using human rhabdomyosarcoma and leukemic cell lines, we found a physical association between topo IIα and Rb protein by reciprocal immunoprecipitation and immunoblotting, in which topo IIα appeared to interact primarily with the underphosphorylated form of Rb. Experiments with truncated glutathione S-transferase-Rb fusion proteins and nuclear extracts of Rh1 rhabdomyosarcoma cells indicated that topo IIα binds avidly to the A/B pocket domain of Rb, which contains the intact spacer amino acid sequence. To determine whether this interaction has functional consequences in vivo, we expressed wild-type and mutant Rb in human cervical carcinoma cells lacking functional Rb. Wild-type, but not mutant, Rb inhibited topo II activity in nuclear extracts of these transfected cells. Moreover, purified wild-type Rb inhibited the activity of purified human topo IIα, indicating a direct interaction between these two proteins. We conclude that topo IIα associates physically with Rb in interactions that appear to have functional significance.