Reduction in p48-ISGFgamma levels confers resistance to interferon-alpha2a in MHCC97 cells.
OBJECTIVE
Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in China and, due to the limited efficacy of currently available therapies, is responsible for a large number of deaths. IFN-alpha therapy has shown promise in the treatment of various forms of human cancer and is considered in the treatment of HCC. Previous results from our group showed that high doses of IFN-alpha exert a significant antiproliferative effect on MHCC97 human xenografts in nude mice, but not on MHCC97 cells when tested in vitro. Here we present experiments designed to characterize the molecular mechanism underlying the defective response of MHCC97 cells to IFN-alpha. Elucidation of the mechanism underlying the defective response of MHCC97 to IFN-alpha may help to explain and possibly to overcome clinical failures of this form of tumor therapy.


METHODS
IFN-alpha(2a) was administered between 3,000 and 10,000 IU/ml, a range strongly inhibiting proliferation in other cell lines. Gene expression profiles of MHCC97 cells were obtained before and after treatment with IFN-alpha(2a) using cDNA microarray analysis. The transcriptional activity of relevant genes responding to IFN-alpha(2a) in the cDNA microarray experiments was confirmed by RT-PCR and Northern blot analysis. Transient transfection with an expression vector was used to restore p48-ISGFgamma (IRF9) protein levels. Cell proliferation was evaluated using the MTT assay.


RESULTS
Although IFN-alpha treatment caused the activation of several signal transduction pathways in MHCC97 cells, the lack of an antiproliferative effect was found to mainly derive from a defect in the activation of the transcription factor ISGF3 required for Jak/STATS signaling. We show that the defect in ISGF3 activation is mainly caused by the absence of one of its essential components, the protein p48-ISGFgamma from MHCC97 cells. Indeed, transient expression of p48-ISGFgamma restores sensitivity to IFN-alpha(2a). Although the mRNA levels of p48-ISGFgamma were normal in MHCC97 cells, mutations could be detected in the gene coding for the protein. We hypothesize, therefore, that these mutations alter the message or protein stability, leading to the reduced protein levels observed.


CONCLUSIONS
Our results confirm the important role of Jak/STATS signaling in the antiproliferative effects of IFN-alpha in tumor cells and indicate that defects in ISGF3 can cause resistance to IFN-alpha(2a) treatment.
Wu
Wei-Zhong
WZ
Liver Cancer Institute and Zhongshan Hospital, Shanghai, P.R. China.
Sun
Hui-Chuan
HC
Gao
Yan-Qin
YQ
Li
Yan
Y
Wang
Lu
L
Zhou
Kang
K
Liu
Kang-Da
KD
Iliakis
George
G
Tang
Zhao-You
ZY
D000970:Antineoplastic Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000737:chemistry* / Q000188:drug therapy; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D000077190:Interferon alpha-2; D050832:Interferon-Stimulated Gene Factor 3; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D016898:Interferon-alpha / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000737:chemistry* / Q000188:drug therapy; D020411:Oligonucleotide Array Sequence Analysis; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor; D050795:STAT2 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism*
D000970:Antineoplastic Agents; D017931:DNA Primers; D004268:DNA-Binding Proteins; C494227:IRF9 protein, human; D000077190:Interferon alpha-2; D050832:Interferon-Stimulated Gene Factor 3; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D016898:Interferon-alpha; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050795:STAT2 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors