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Interruption of activin A autocrine regulation by antisense oligodeoxynucleotides accelerates liver tumor cell
K Takabe1, J J Lebrun, Y Nagashima
1The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037, USA.
Abstract:
Administration of activin A, a member of the transforming growth factor-beta superfamily inhibits hepatocyte proliferation in vitro and reduces liver mass in vivo. However, a role of endogenous activin A in local growth modulation has not been established in any system. The aim of this study was to examine the production of activin A in the human hepatoma cell line HLF and to explore a possible autocrine role of activin as a cell growth inhibitor by blocking production of endogenous activin using antisense oligodeoxynucleotides. Administration of exogenous activin A suppressed HLF cell growth, and immunoreactive activin A was shown to be produced in the cells at confluency by Western blotting analysis. Cells were exposed to phosphorothioate-modified oligodeoxynucleotides, synthesized with antisense or randomly shuffled base sequences of activin betaA subunit messenger RNA, under serum-free conditions. Uptake of the oligodeoxynucleotides into the cells was confirmed by use of fluorescein isothiocyanate-labeled oligodeoxynucleotides. Administration of antisense oligodeoxynucleotides reduced activin A production as confirmed by both competitive PCR and Western blotting. Activin betaA antisense oligodeoxynucleotides significantly increased cell proliferation compared with controls. These findings are consistent with the existence of an autocrine role of activin A as an inhibitor of hepatocyte proliferation.
Insights
Activin A, a growth factor, inhibits liver cell proliferation. Blocking its production with antisense oligodeoxynucleotides in hepatoma cells increased proliferation, suggesting an autocrine inhibitory role for activin A in liver growth.
Area of Science:
- Hepatology
- Molecular Biology
- Cell Signaling
Background:
- Activin A, a TGF-β superfamily member, inhibits hepatocyte proliferation in vitro and reduces liver mass in vivo.
- The role of endogenous activin A in local growth modulation remains unestablished.
- Understanding activin A's autocrine function is crucial for liver growth regulation.
Purpose of the Study:
- To investigate activin A production in the human hepatoma cell line HLF.
- To explore the autocrine role of activin A as a cell growth inhibitor.
- To block endogenous activin A production using antisense oligodeoxynucleotides.
Main Methods:
- HLF cells were treated with exogenous activin A to assess its effect on proliferation.
- Western blotting was used to detect immunoreactive activin A production at confluency.
- Antisense oligodeoxynucleotides targeting activin betaA subunit mRNA were administered under serum-free conditions.
- Competitive PCR and Western blotting confirmed reduced activin A production.
- Cell proliferation was measured following antisense treatment.
Main Results:
- Exogenous activin A suppressed HLF cell growth.
- Immunoreactive activin A was produced by HLF cells at confluency.
- Antisense oligodeoxynucleotides successfully reduced activin A production.
- Activin betaA antisense treatment significantly increased HLF cell proliferation compared to controls.
Conclusions:
- Endogenous activin A is produced by HLF cells.
- Blocking activin A production with antisense oligodeoxynucleotides enhances hepatocyte proliferation.
- These findings support an autocrine role for activin A as a hepatocyte proliferation inhibitor.