Mechanisms leading to hepatitis B virus X-protein mediated hepatocyte transformation
Abstract
Hepatitis B virus X protein (pX) is implicated in the pathogenesis of hepatocellular carcinoma by an unknown mechanism. Utilizing the tetracycline-regulated pX-expressing cell line (4pX-1) derived from the immortalized murine AML 12 hepatocyte cell line, I have discovered two important aspects of pX function. (1) pX induces DNA re-replication, DNA damage and polyploidy and (2) pX activates Polo-like Kinase 1 (Plk1) which deregulates the G2/M DNA damage checkpoint, suppresses DNA repair and leads to transformation of pX-expressing hepatocytes in vitro. Specifically, employing 4pX-1 cells, I demonstrated pX mediates increased expression of replication licensing factors Cdt1 and cdc6. Conversely, geminin, the negative regulator of cdt1 and DNA replication, is repressed by pX. Cdt1 and cdc6, initiators of replication, are induced transcriptionally by pX and their transcript and protein levels remain elevated through G2 phase. Additionally, geminin expression is repressed, leading to continued DNA replication in G2 phase, resulting in DNA damage and polyploidy. Interestingly, pX-expressing cells with DNA damage do not undergo apoptosis but progress through mitosis, propagate DNA damage to daughter cells, and generate partially polyploid hepatocytes. The pX-induced polyploid cell population was isolated by live cells sorting, forming the basis for a cellular model of pX-induced liver cancer progression. Utilizing this cellular model of pX-induced polyploid cells, I have characterized their growth properties for >70 cell doublings. Employing soft agar assays, I demonstrated pX-dependent transformation in vitro occurs only after 30–40 cell doublings. Additionally, expression of Plk1 progressively increases as polyploid cultures undergo pX-mediated transformation. Conversely, expression of the DNA repair protein Mre11 decreases. This led me to ask the question whether Plk1 was deregulating the DNA damage checkpoint at the G2 to M transition, and whether Plk1 regulates Mre11. In pX-expressing 4pX-1 cells I demonstrated that pX-mediated Plk1 activation induces loss of DNA repair protein Mre11 and attenuation of the G2/M DNA damage checkpoint. Specifically, in the presence of pX-induced DNA damage, the protein level of Claspin, required for activation of the DNA damage checkpoint, is stabilized only when Plk1 is inhibited by partial knockdown or Plk1 inhibitors. Likewise, Plk1 inhibition results in Chk1 phosphorylation and activation of the DNA damage checkpoint. Similarly, Plk1 inhibition at the G2/M transition leads to stabilization of both p53 and Mre11 protein, a reduction in polyploid cell number and increase in apoptosis. These studies have identified Plk1 as essential in initiation of pX-mediated transformation.
Degree
Ph.D.
Advisors
Andrisani, Purdue University.
Subject Area
Molecular biology|Genetics|Virology
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