Background& Aims: ¿1-Antichymotrypsin (¿1-ACT), a member of the serpin family (SERPINA3), is an acute-phase protein secreted by hepatocytes in response to cytokines such as oncostatin M. ¿1-ACT is a protease inhibitor thought to limit tissue damage produced by excessive inflammation-associated proteolysis. However, ¿1-ACT also is detected in the nuclei of cells, where its activities are unknown. Expression of ¿1-ACT is down-regulated in human hepatocellular carcinoma (HCC) tissues and cells; we examined its roles in liver regeneration and HCC proliferation.
Methods: We measured levels of ¿1-ACT messenger RNA in human HCC samples and healthy liver tissue. We reduced levels of ¿1-ACT using targeted RNA interference in human HCC (HepG2) and mouse hepatocyte (AML12) cell lines, and overexpressed ¿1-ACT from lentiviral vectors in Huh7 (HCC) cells and adeno-associated viral vectors in livers of mice. We assessed proliferation, differentiation, and chromatin compaction in cultured cells, and liver regeneration and tumor formation in mice.
Results: Reducing levels of ¿1-ACT promoted proliferation of HCC cells in vitro. Oncostatin M up-regulated ¿1-ACT expression and nuclear translocation, which inhibited HCC cell proliferation and activated differentiation of mouse hepatocytes. We identified amino acids required for ¿1-ACT nuclear localization, and found that ¿1-ACT inhibits cell-cycle progression and anchorage-independent proliferation of HCC cells. HCC cells that overexpressed ¿1-ACT formed smaller tumors in mice than HCC cells that did not express the protein. ¿1-ACT was observed to self-associate and polymerize in the nuclei of cells; nuclear ¿1-ACT strongly bound chromatin to promote a condensed state that could prevent cell proliferation.
Conclusions: ¿1-ACT localizes to the nuclei of hepatic cells to control chromatin condensation and proliferation. Overexpression of ¿1-ACT slows the growth of HCC xenograft tumors in nude mice.