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Cyclin A is a functional target of retinoblastoma tumor suppressor protein-mediated cell cycle arrest
K E Knudsen1, A F Fribourg, M W Strobeck
1Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521, USA. Erik.Knudsen@UC.Edu
Abstract:
Although RB inhibits the G(1)-S transition, the mechanism through which RB prevents cell cycle advancement remains unidentified. To delineate the mechanism(s) utilized by RB to exert its anti-proliferative activity, constitutively active RB proteins (which cannot be inactivated by phosphorylation) or p16ink4a (which prevents RB inactivation) were utilized. Both proteins inhibited the G(1)-S transition, whereas wild-type RB did not. We show that active RB acts to attenuate cyclin A promoter activity, and that overexpression of cyclin E reverses RB-mediated repression of the cyclin A promoter. Although cyclin A is an E2F-regulated gene, and it has been long hypothesized that RB mediates cell cycle advancement through binding to E2F and attenuating its transactivation potential, cyclin E does not reverse dominant negative E2F-mediated repression of the cyclin A promoter. Although active RB repressed both cyclin A and two other paradigm E2F-regulated promoters, only cyclin A transcription was restored upon co-expression of cyclin E. Additionally, we show that RB but not dominant negative E2F regulates the cyclin A promoter through the CCRE element. These data identify cyclin A as a downstream target of RB-mediated arrest. Consistent with this idea, ectopic expression of cyclin A reversed RB-mediated G(1) arrest. The findings presented suggest a pathway wherein cyclin A is a downstream target of RB, and cyclin E functions to antagonize this aspect of RB-mediated G(1)-S inhibition.
Insights
Active Retinoblastoma (RB) protein inhibits cell cycle progression by repressing cyclin A promoter activity. Cyclin E counteracts this RB-mediated G1-S phase inhibition, identifying cyclin A as a key downstream target.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- The Retinoblastoma (RB) protein is a crucial tumor suppressor that inhibits cell cycle progression.
- The precise molecular mechanisms by which RB enforces the G1-S phase transition blockade are not fully understood.
Purpose of the Study:
- To elucidate the specific mechanisms employed by RB to inhibit cell proliferation.
- To identify downstream targets and pathways regulated by RB during cell cycle arrest.
Main Methods:
- Utilized constitutively active RB proteins and p16ink4a to inhibit RB inactivation.
- Assessed effects on cyclin A promoter activity and E2F-regulated promoters.
- Investigated the role of cyclin E and dominant-negative E2F in modulating RB's effects.
Main Results:
- Active RB proteins and p16ink4a inhibited the G1-S transition.
- Active RB attenuated cyclin A promoter activity, an effect reversed by cyclin E overexpression.
- RB, but not dominant-negative E2F, regulated the cyclin A promoter via the CCRE element.
- Ectopic cyclin A expression reversed RB-mediated G1 arrest.
Conclusions:
- Cyclin A is identified as a direct downstream target of RB-mediated G1-S phase arrest.
- Cyclin E antagonizes RB's inhibitory effect on cyclin A transcription, thus promoting cell cycle progression.
- These findings reveal a novel pathway where RB controls cell cycle entry through regulation of cyclin A, with cyclin E acting as a key modulator.