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Antisense-mediated specific inhibition of P120 protein expression prevents G1- to S-phase transition
A Fonagy1, C Swiderski, M Dunn
1Department of Surgery, Lucille Markey Cancer Center, University of Kentucky Medical Center, Lexington 40536.
Abstract:
A pentadecadeoxyribonucleotide (5'-AAAGCCCCCCACCAC), complementary to a splice junction site of mRNA for human proliferation-associated nucleolar protein P120, inhibited expression of the P120 gene and the mitogen-induced proliferation of human lymphocytes. The inhibition of P120 gene expression and proliferation was concentration dependent and reached 90% at 200 microM, as measured by [3H]thymidine uptake and by densitometric scanning of Northern (mRNA) and Western (protein) blots of P120. Inhibition was not observed in cells treated with the correspondent nonsense oligomer. P120 antisense oligomer treatment prevented S-phase entry of mitogen-stimulated lymphocytes, as determined by flow cytometric analysis, but did not block G0-G1 transition assessed by morphological blast transformation and induction of [3H]uridine incorporation. Results of this study suggest that P120 expression may be required for the upregulation of nucleolar function necessary for cell proliferation.
Insights
An antisense oligomer targeting the proliferation-associated nucleolar protein P120 gene inhibited human lymphocyte proliferation. This suggests P120 is crucial for cell division and nucleolar function.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- The proliferation-associated nucleolar protein P120 plays a role in cell growth.
- Understanding the regulatory mechanisms of P120 is essential for controlling lymphocyte proliferation.
Purpose of the Study:
- To investigate the effect of inhibiting P120 gene expression on human lymphocyte proliferation.
- To determine the role of P120 in the cell cycle progression of mitogen-stimulated lymphocytes.
Main Methods:
- A pentadecadeoxyribonucleotide antisense oligomer complementary to P120 mRNA splice junction was synthesized.
- Oligomer effects on P120 gene expression and lymphocyte proliferation were assessed using [3H]thymidine uptake, Northern/Western blotting, and flow cytometry.
Main Results:
- The P120 antisense oligomer significantly inhibited P120 gene expression and mitogen-induced lymphocyte proliferation in a concentration-dependent manner.
- Inhibition reached 90% at 200 microM, with no effect observed from a nonsense oligomer.
- Antisense treatment blocked S-phase entry but not G0-G1 transition in stimulated lymphocytes.
Conclusions:
- P120 gene expression is necessary for the upregulation of nucleolar function required for cell proliferation.
- Targeting P120 with antisense oligomers offers a potential strategy to control lymphocyte proliferation.