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Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression
E S Knudsen1, C Buckmaster, T T Chen
1Department of Biology, University of California, San Diego (UCSD), La Jolla, California 92093-0322 USA.
Abstract:
The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.
Insights
Constitutively active retinoblastoma tumor suppressor protein (RB) inhibits S-phase entry and DNA replication. This novel RB function, distinct from G1/S control, requires continued RB phosphorylation for DNA replication completion.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- The retinoblastoma tumor suppressor protein (RB) is a key negative regulator of cell proliferation.
- RB's growth-inhibitory function is modulated by phosphorylation.
- Mutations creating constitutively active RB (PSM-RB) enhance its growth-suppressive properties.
Purpose of the Study:
- To investigate the role of RB in cell cycle progression beyond the G1/S transition.
- To determine if constitutively active RB affects DNA replication.
- To elucidate the mechanisms by which RB regulates S-phase entry and progression.
Main Methods:
- Utilized Rat-1 cells expressing wild-type RB and phosphorylation-site-mutated RB (PSM-RB).
- Assessed cell cycle progression using techniques like S-phase entry inhibition and DNA synthesis assays.
- Employed ectopic expression of cyclins (E, A) and viral oncoproteins (SV40 T-antigen, adenovirus E1A) and E2F-1 to modulate RB activity and cell cycle.
Main Results:
- PSM-RB inhibited S-phase entry in Rat-1 cells, unlike wild-type RB.
- PSM-RB-arrested cells showed normal cyclin E levels but inhibited cyclin A expression.
- Ectopic cyclin E restored cyclin A expression and S-phase entry, but DNA replication completion was impaired.
- Microinjection of PSM-RB inhibited DNA synthesis in cells past the G1 restriction point.
- SV40 T-antigen, E1A, and high E2F-1 expression attenuated PSM-RB's S-phase inhibitory effects, but cyclins E and A did not.
Conclusions:
- RB plays a novel role in inhibiting S-phase progression, separate from its G1/S checkpoint function.
- Continued RB phosphorylation after G1/S is necessary for the complete execution of DNA replication.
- Dysregulation of RB phosphorylation may contribute to uncontrolled cell proliferation and cancer development.