ABSTRACT
<p><b>OBJECTIVE</b>Major histocompatibility complex class I C-related molecules A and B (MICA and MICB) are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. Our previous study showed that 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, can induce the expression of MICB and sensitized cells to NKL-cell-mediated cytolysis. The aim of this study was to determine the expression level of MICA in HepG2 cells (an HCC cell line) and L02 cells ( a normal liver cell), and to investigate the effect of 5-aza-dC on MICA expression in HepG2 cells.</p><p><b>METHODS</b>Cells were treated with 5-aza-dC, caffeine and ATM-specific siRNA. The cell surface MICA protein on HepG2 cells and L02 cells was determined using flow cytometry. The mRNA level was detected using real time RT-PCR.</p><p><b>RESULTS</b>MICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression was upregulated in response to 5-aza-dC treatment (P less than 0.05), and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase (P less than 0.05).</p><p><b>CONCLUSION</b>Our data suggest that 5-aza-dC induces the expression of MICA by a DNA damage-dependent mechanism.</p>
Subject(s)
Humans , Ataxia Telangiectasia Mutated Proteins , Azacitidine , Pharmacology , Caffeine , Pharmacology , Carcinoma, Hepatocellular , Metabolism , Cell Cycle Proteins , Metabolism , Cell Line , Cell Membrane , Metabolism , DNA Damage , DNA-Binding Proteins , Metabolism , Flow Cytometry , Hep G2 Cells , Hepatocytes , Metabolism , Histocompatibility Antigens Class I , Genetics , Metabolism , Liver Neoplasms , Metabolism , Protein Serine-Threonine Kinases , Metabolism , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Proteins , Metabolism , Up-RegulationABSTRACT
<p><b>OBJECTIVE</b>To investigate whether there is intercellular transfer of functional P-glycoprotein(P-gp) from P-gp-positive cells to P-gp-negative cells in vitro.</p><p><b>METHODS</b>HepG2/GFP cells, a HepG2 cell line stably expressing GFP, were co-cultured with HepG2/ADM cells, an adriamycin-resistant cell line derived from HepG2 cells. The distribution of P-gp in hepatocellular carcinoma cell was observed under laser scanning confocal microscope (LSCM). Immunomagnetic beads were used to separate HepG2/GFP cells from the mixed culture. The abundance of P-gp was analyzed by western blot, and the expression of mdr1 mRNA was detected by qRT-PCR.</p><p><b>RESULTS</b>Yellow fluorescence was detected in HepG2/aqMDR cells, green fluorescence was detected in HepG2/GFP cells, red fluorescence was detected in HepG2/ADM cells by LSCM. The level of P-gp protein in HepG2/aqMDR cells was lower than that in HepG2/ADM cells, but higher than that in HepG2/GFP cells (q = 35.07, P < 0.05) and HepG2 cells (q = 36.87, P < 0.05). The expression of mdr1 mRNA in HepG2/ADM cells was higher than that in HepG2/aqMDR, HepG2 and HepG2/GFP cells, but there was no significant difference in mdr1 mRNA among HepG2/aqMDR, HepG2 and HepG2/GFP cells (F = 2.30, P > 0.05).</p><p><b>CONCLUSIONS</b>P-gp can transfer from drug resistant hepatocellular cells to sensitive hepatocellular carcinoma cells. This study suggests a novel mechanism of multidrug resistance in hepatocellular carcinoma.</p>