Downregulation of p21^waf/cip-1 mediates apoptosis of human hepatocellular carcinoma cells in response to interferon-γ

Abstract

There is no effective treatment for advanced hepatocellular carcinoma (HCC). We therefore explored the molecular mechanisms of interferon-γ (IFN-γ)-mediated growth regulation in human HCC cell lines. IFN-γ receptor expression, signal transduction, and regulation of effectors were examined by RT-PCR, immunoprecipitation, immunoblotting, and reporter gene assays. Growth and apoptosis were determined based on cell numbers, cell cycle analyses, kinase assays, DNA fragmentation, and PARP cleavage. HCC cell lines express functionally intact IFN-γ receptors and downstream effectors. IFN-γ profoundly inhibited growth of HCC cells via two different mechanisms: inhibition of G1 cell cycle progression and induction of apoptosis. Analyses in SK-Hep-1 cells revealed a deficient cyclin D induction in IFN-γ-treated cells, resulting in reduced activity of CDK4 and CDK2 kinases and pRB hypophosphorylation. In contrast, apoptosis prevailed in IFN-γ-treated HepG2 cultures. A survey of apoptosis relevant IFN-γ effectors including IRF-1, caspase-1, caspase-3, and p21^waf/cip-1 documented a dramatic transcriptional downregulation of p21^waf/cip-1 exclusively in apoptosis-susceptible HepG2 cells. Reconstitution of p21^waf/cip-1 rescued HepG2 cells from IFN-γ-induced apoptosis, indicating that p21^waf/cip-1 reduction was required for apoptosis execution. Inversely, downregulation of p21^waf/cip-1 sensitized SK-Hep-1 cells to IFN-γ-induced apoptosis. Thus, downregulation of p21^waf/cip-1 in HCC cells functions as a novel, critical determinant of alternative growth inhibitory pathways in response to IFN-γ.

Introduction

Hepatocellular carcinoma (HCC)² is one of the most frequent malignancies worldwide [1]. HCC often arises in a multifocal manner [2], suggesting specific and coordinate transformation events. A wide spectrum of alterations appears to be involved in the hepatocyte transformation process including growth factor signaling pathways, oncogenes and tumor suppressors, matrix interaction, and angiogenesis [3]. Currently, the prognosis of patients diagnosed with HCC remains poor unless curative surgery or transplantation can be performed [1], emphasizing the need for systemic treatment alternatives.

The cytokine interferon-γ (IFN-γ) is capable of eliciting potent antiproliferative actions in epithelial tumors [4]. This growth inhibitory action has been attributed to direct effects on the transformed cells as well as indirect mechanisms involving immunomodulation [5] and inhibition of angiogenesis [6].

All mechanisms involved in IFN-γ action critically depend on the activation of the downstream effector and putative tumor suppressor STAT-1 [7], [8]. IFN-γ-induced activation of STAT-1 is mediated by the IFN-γ receptor-associated tyrosine kinases of the Janus-kinases family JAK-1 and JAK-2 and requires ligand-induced sequential tyrosine phosphorylation of JAK kinases, the IFN-γ receptor chain, and STAT-1 [9]. Upon phosphorylation, Stat-1 proteins convert from the latent to the DNA-binding state, dimerize, and translocate to the nucleus where they activate transcription from IFN-γ response elements [10].

Despite these well-conserved signaling events, direct antiproliferative effects on tumor cells appear to be highly tissue and cell type specific [11], likely due to differences in the repertoire and activity of STAT-1 interaction partners and/or in the tumor-specific expression pattern of oncogenic alterations. Furthermore, a specific loss of biological responsiveness to IFN-γ may occur in the context of malignant transformation [12], [13], [14].

Depending on the cell model investigated, IFN-γ is capable of differentially affecting central cellular growth-control pathways, resulting in profound changes of cell cycle progression [15] and cell survival [16], [17]. Under physiological conditions, apoptotic cell death is induced predominantly by stimulation of death domain containing receptors like Fas (CD 95) or TNFα, which have been demonstrated to be upregulated by IFN-γ [18]. Direct effects of IFN-γ on tumor cell survival also occur, ranging from potent apoptosis induction [19] to antiapoptotic protection [20], [21], [22] or complete apoptosis resistance [12], [23].

The molecular determinants of either apoptosis induction or cell cycle arrest have not been identified yet. Nonetheless, the clinical availability of IFN-γ would make the cytokine an attractive candidate drug for the treatment of HCC, if (i) consistent antiproliferative effects were observed, (ii) a subgroup of responsive tumors could be identified prior to treatment, or (iii) a specific and reliable response parameter could be identified. In view of these requirements, an understanding of the molecular mechanisms underlying putative antiproliferative actions of IFN-γ in HCC cells is required.

Here, we analyzed direct effects of IFN-γ on growth and survival of human HCC cells and present two alternative effector pathways of growth inhibition: induction of apoptosis versus G1 cell cycle inhibition. We further provide evidence that these alternative pathways are determined by a novel mechanism: downregulation of the cell cycle inhibitor p21^waf/cip-1.