Functional interaction between human topoisomerase IIalpha and retinoblastoma protein

Abstract

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) IIalpha 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 IIalpha and Rb protein by reciprocal immunoprecipitation and immunoblotting, in which topo IIalpha 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 IIalpha 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 IIalpha, indicating a direct interaction between these two proteins. We conclude that topo IIalpha associates physically with Rb in interactions that appear to have functional significance.

Figure 1

Constitutive expression of topo IIα and Rb proteins in RMS cell lines. (A) Representative immunoblots based on analysis of total cell extracts with specific antibodies (Ab-284, topo IIα; G3–245, Rb). Equal loading of the proteins was confirmed by Ponceau-S staining (Materials and Methods). Values beneath the lanes are mean levels of topo IIα or Rb expression (relative to the levels in Rh1 cells) from five separate experiments. SDs ranged from 0.08 to 0.47. (B) Correlation of topo IIα expression with Rb expression in RMS cell lines. r = Pearson’s correlation coefficient; the P value was determined by Student’s t test.

Figure 2

Coimmunoprecipitation of Rb by topo IIα-specific antibody in total extracts of Rh1 cells. Immunoprecipitations (IP) were performed with antibodies to the C-terminal region of topo IIα (Ab-1), Rb (G3–245), and E2F-1 (KH-95) or rabbit IgG. The immunoblots represent three separate experiments. (Top) Blotted with Ab-284. (Middle) Blotted with G3–245. (Bottom) Blotted with KH-95. Lanes 1–4, IP with the indicated antibodies; lane 5, direct immunoblots of the cell lysate used in IP.

Figure 3

Reciprocal coimmunoprecipitations of topo IIα and Rb in Rh1 cell lysates treated with alkaline phosphatase (+) or untreated controls (−). Shown is a representative immunoblot of three separate experiments. (A) Coimmunoprecipitation of topo IIα by Rb-specific antibody (XZ-77) before (lane 1) and after (lanes 2–4) treatment with alkaline phosphatase. Total cell extracts were treated with alkaline phosphatase, immunoprecipitated with XZ-77, p53 (Ab-6, irrelevant isotype-matched antibody), or mouse IgG2a, and immunoblotted with Ab-284. Lanes 5 and 6, direct immunoblot of the lysates. (B) Coimmunoprecipitation of Rb by topo IIα-specific antibody Ab-284 before (lane 1) and after (lanes 2–4) treatment with alkaline phosphatase in Rh1 cells. Total cellular extracts were treated with alkaline phosphatase and immunoprecipitated with Ab-284, anti-Mcl-1 (an irrelevant rabbit antibody), or rabbit IgG, as indicated, and immunoblotted with G3–245 (Rb-specific). Lanes 5 and 6, direct immunoblot of lysates used in IP.

Figure 4

Topo IIα-binding domains in Rb protein determined by in vitro binding with GST-Rb fusion proteins. (A) Structures of the truncated GST-Rb fusion proteins used for in vitro binding with nuclear extracts from Rh1 cells. (Upper) Protein-binding domains illustrating the A/B and C pockets, insert (I) region, and the large pocket (LP) in human retinoblastoma protein. The numbers in parentheses refer to the amino acids from the N terminus; dl, deletion. The extent of binding of the proteins to topo IIα is indicated at the right; ++++, strong; ++, moderate; +, weak; −, none. (B) A representative immunoblot (of three separate experiments) illustrating the comparative binding of topo IIα from Rh1 nuclear extracts to the GST-Rb fusion proteins. The extracts were incubated with GST-Rb fusion proteins (equivalent amounts) bound to GST-Sepharose. The protein bound beads were separated by SDS/PAGE and immunoblotted with Ab-284. Lanes: 1–7, topo IIα binding to the respective GST-Rb fusion proteins (as numbered); GST, binding with GST alone; a, nuclear extract of Rh1 used for binding assay (no beads); b, total protein extract from HeLa cells (no beads; used as topo IIα marker).

Figure 5

(A) Topo II activity in C33A cells transfected with wild-type or mutant Rb. The cytomegalovirus expression vectors were transfected in C33A cells, nuclear proteins were extracted, and topo II activity was measured by decatenation of k-DNA. Displayed here is a representative (negative) image of a 1% agarose gel (ethidium bromide-stained) showing the decatenation of k-DNA by the indicated amounts of nuclear extracts. Lanes: 1 and 2, extracts from wild-type Rb transfected cells; 4 and 5, extracts from mutant Rb-transfected cells; 7 and 8, extracts from mock-transfected cells; 9, no extract control; 10, catenated k-DNA marker; 11, decatenated k-DNA marker; 12, linear k-DNA marker. Lanes 3 and 6 were left blank. (B) Immunoblots of nuclear extracts from C33A cells transfected with wild-type Rb (379–928) (lane 1) or mutant Rb (379–928) (lane 2) or from mock-transfected (lane 3) cells showing topo IIα (Upper) and Rb (Lower) expression. wt, wild type; mock-tran, mock-transfected; cat, catenated; decat, decatenated.

Figure 6

Effect of purified wild-type and mutant Rb on activity of purified human topo IIα. Indicated amounts of purified human topo IIα were incubated with 12 ng of purified wild-type [GST-Rb (379–928)] or mutant [GST-Rb (379–928), amino acids 706 C → F] Rb. Topo II activity was measured by decatenation of k-DNA. A representative (of three separate experiments) negative image of 1% agarose gel (ethidium bromide-stained) is shown. Lanes: 1–4, untreated control (no Rb); 5–8, treated with wild-type Rb; 10–13, treated with mutant Rb; 14, control (no topo IIα); 15, decatenated k-DNA marker; and 16, linear k-DNA marker. Lane 9 was left blank.

Figure 7

Suggested model for the regulatory role of topo IIα-Rb interactions in vivo.