Functional interaction between human topoisomerase II alpha and retinoblastoma protein

DNA topoisomerase II-an essential nuclear enzyme in DNA replication and tra nscription, chromatin segregation, and cell cycle progression-is also a tar get of clinically useful anticancer drugs. Preliminary observations of a po sitive correlation between the expression of topoisomerase (topo) II alpha and the retinoblastoma protein (Rb) in a series of rhahdomyosarcoma cells p rompted us to ask whether these two proteins interact in vivo. Using human rhabdomyosarcoma and leukemic cell lines, we found a physical association b etween topo II alpha and Rb protein by reciprocal immunoprecipitation and i mmunoblotting, in which topo II alpha appeared to interact primarily with t he underphosphorylated form of Rb. Experiments with truncated glutathione S -transferase-Rb fusion proteins and nuclear extracts of Rh1 rhabdomyosarcom a cells indicated that topo II alpha binds avidly to the A/B pocket domain of Rb, which contains the intact spacer amino acid sequence. To determine w hether this interaction has functional consequences in vivo, we expressed w ild-type and mutant Rb in human cervical carcinoma cells lacking functional Rb. wild-type, but not mutant, Rb inhibited topo II activity in nuclear ex tracts of these transfected cells. Moreover, purified wild-type Rb inhibite d the activity of purified human topo II alpha, indicating a direct interac tion between these two proteins. We conclude that topo II alpha associates physically with Rb in interactions,that appear to have functional significa nce.