The hepatitis B virus HBx protein modulates cell cycle regulatory proteins in cultured primary human hepatocytes

Abstract

There are over 350 million people chronically infected with the Hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). While the precise mechanism of HBV-associated HCC remains undefined, it is believed to involve a combination of the host immune response to infection and activities of HBV proteins including the nonstructural X protein (HBx). HBx is a multifunctional protein that can modulate various cellular processes including cell proliferation. The exact effect of HBx on cell proliferation has varied depending on the cell line and exact conditions used in the study. Our previously published reports have demonstrated that HBx modulates the levels of cell cycle regulatory proteins in primary rat hepatocytes; however, the effect of HBx on cell cycle regulatory proteins in primary human hepatocytes, the natural host for HBV infection, has not been studied. Here we have examined the effect of HBx on cell cycle regulatory proteins in cultured, primary human hepatocytes. We demonstrate that HBx decreases the levels of cell cycle proteins that prevent progression into G1 phase and increases the levels of cell cycle proteins active in G1 phase. We have also shown that HBx modulation of cell cycle regulatory proteins requires cytosolic calcium, similar to the results we previously obtained in primary rat hepatocytes. Cumulatively, our results are the first demonstration that HBx modulates the levels of cell cycle regulatory proteins in a calcium-dependent manner in primary human hepatocytes.

Figure 1. Confirmation of differentiated primary human hepatocytes

(A-B) RNA was extracted from hepatocytes upon arrival or hepatocytes cultured for 72 hours. RNA was then reverse-transcribed and PCR-amplified using primers specific for transferrin (TFN), albumin (ALB), connexin 26 (CX26), or connexin 43 (CX43). Expected band sizes are 574 base pairs (bp) for TFN, 761 bp for ALB, 618 bp for CX26, and 717 bp for CX43. A human total liver cDNA library (BioChain Institute, Inc.) was used as the positive control for CX43. (-) signifies samples where isolated RNA was directly PCR amplified, without undergoing the reverse transcription step, to confirm the absence of DNA contamination. Results shown are representative samples from experiments performed in duplicate from three human donors.

Figure 2. HBx modulates cell cycle regulatory proteins in primary human hepatocytes

Primary human hepatocytes were infected with AdGFP and AdGFP-HBx recombinant adenoviruses and collected 48 hours post-infection. Lysates were resolved via SDS-PAGE and subjected to Western blot analysis for (A-C) β-actin, HBx, p15, p16, p21, p27, cyclin D1, cyclin E, cyclin A, and PCNA. Results shown are representative samples from independent experiments performed in duplicate in hepatocytes from six human donors. *fold difference plus/minus standard error between AdGFP-HBx and AdGFP was statistically significant as determined using a Student’s t-test (p≤0.05).

Figure 3. HBx requires cytosolic calcium signaling to modulate cell cycle regulatory proteins in primary hepatocytes

Primary human hepatocytes were infected with AdGFP or AdGFP-HBx recombinant adenoviruses, treated with 50μM BAPTA-AM 24 hours post-infection, and collected 24 hours post-treatment. Lysates were resolved via SDS-PAGE and subjected to Western blot analysis for (A-B) β-actin, HBx, p15, p16, p21, p27, cyclin D1, and cyclin E. Results shown are representative samples from independent experiments performed in duplicate in hepatocytes from 3 human donors. *fold difference plus/minus standard error between AdGFP-HBx and AdGFP was statistically significant as determined using a Student’s t-test (p≤0.05).