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Transcriptional Profiling Identifies Gene Expression Changes Associated with IFN- Tolerance in Hepatitis C–Related Hepatocellular Carcinoma Cells

Departments of ¹ Anatomical and Cellular Pathology and ² Surgery, Chinese University of Hong Kong; ³ Oncology Centre, Hong Kong Sanatorium & Hospital, Hong Kong, China; ⁴ Microarray Centre, Clinical Genomics Centre, University Health Network; and ⁵ Departments of Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Requests for reprints: Nathalie Wong, Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong, China. Phone: 852-2632-1128; Fax: 852-2648-8842; E-mail: natwong{at}cuhk.edu.hk.

Purpose: Treatment with IFN- therapy has been shown to exhibit antitumor effects on patients with hepatocellular carcinoma (HCC). However, individual responses remained unpredictable because of the frequent presence of intrinsic or acquired IFN- resistance. Hence, delineation of molecular targets implicated in the resistant pathway holds value in refining the therapeutic benefits of IFN-.

Experimental Design: The current study analyzed the effect of IFN- in human HCC cells. Three hepatitis C virus (HCV)–related, five hepatitis B virus (HBV)–related and two non-B non-C–related cell lines were subjected to IFN- treatment and the cytotoxic effect on cell viability was measured. Further analysis by cDNA microarray and quantitative reverse transcription-PCR were conducted to examine the gene expression changes that mediated the IFN- resistance observed.

Results: According to the IC₅₀ values determined, HCV-related cell lines indicated distinct resistance (IC₅₀, 389-1468 units/mL) compared with the HBV-related (IC₅₀, 11-77 units/mL) and non-B non-C–related cell lines (IC₅₀, 24-108 units/mL). Unsupervised hierarchical clustering on array data indicated three HCV-related cell lines to cluster independently from the sensitive cell lines, suggesting discrete features in association with IFN- tolerance. Moreover, Significance Analysis of Microarrays analysis indicated the differential expression of 149 expressed sequence tags that represented 51 up-regulated and 98 down-regulated genes in the resistant cell lines. Comparing the temporal pattern of gene expression between 6- and 24-hour treatments, candidate genes that were considerably induced with time were further highlighted in the tolerant HCV-related cell lines. These candidates were verified by quantitative reverse transcription-PCR, which confirmed the down-regulation of UBA2, ZNF185, and FOXF1 and up-regulation of UBE4B in the drug-tolerant cells.

Conclusions: Our present study showed that the insensitivity to IFN- therapy in HCC cells is associated with drug-inducible transcriptional alterations. Furthermore, our investigation highlighted potential candidate genes in conferring an anti-apoptotic effect toward IFN- treatment.

Key Words: HCV-related hepatocellular carcinoma cells • IFN- • cDNA microarray • UBA2 • ZNF185 • FOXF1 • UBE4B

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