OVERPRODUCTION OF RB PROTEIN AFTER THE G(1) S BOUNDARY CAUSES G(2) ARREST/

The Rb protein is known to exert its activity at decision points in th e G1 phase of the cell cycle. To investigate whether it may also play some role(s) at later points in the cell cycle, we used a system of ra pid inducible gene amplification to conditionally overexpress Rb prote in during G2 phase. A cell line expressing a temperature-sensitive sim ian virus 40 large T antigen (T-Ag) was stably transfected with plasmi ds containing the Rb cDNA linked to the simian virus 40 origin of repl ication: pRB-wt, pRB-fs, and pRB-Dra, carrying wild-type murine Rb cDN A, a frameshift mutation close to the beginning of the Rb coding regio n, and a single-amino-acid deletion in the EIA/T-Ag binding pocket, re spectively. Numerous independent cell lines were isolated at the nonpe rmissive temperature; cell lines displaying a high level of episomal a mplification of an intact Rb expression cassette following shiftdown t o the permissive temperature were chosen for further analysis. Plasmid pRB-fs did not express detectable Rb antigen, while pRB-Dra expressed full-length Rb protein. The Dra mutation has previously been shown to abrogate phosphorylation as well as T-Ag binding. Fluorescence-activa ted cell sorting (FACS) analysis revealed that cultures induced to ove rexpress either wild-type or Dra mutant Rb proteins were significantly enriched for cells with a G2 DNA content. Cultures that amplified pRB -fs or rearranged pRB-wt and did not express Rb protein had normal cel l cycle profiles. Double-label FACS analysis showed that cells overexp ressing Rb or Rb-Dra proteins were uniformly accumulating in G2, where as cells expressing endogenous levels of Rb were found throughout the cell cycle. These results indicate that Rb protein is interacting with some component(s) of the cell cycle-regulatory machinery during G2 ph ase.