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.