Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α and death receptor stimulation in resistant variants of the human U937 cell line.

Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

The role of the CpG island methylator phenotype in colorectal cancer prognosis depends on microsatellite instability screening status.

PURPOSE: The aim of this study was to relate the CpG island methylator phenotype (CIMP; characterized by extensive promoter hypermethylation) to cancer-specific survival in colorectal cancer, taking into consideration relevant clinicopathologic factors, such as microsatellite instability (MSI) screening status and the BRAF V600E mutation. EXPERIMENTAL DESIGN: Archival tumor samples from 190 patients from the Northern Sweden Health and Disease Study (NSHDS) and 414 patients from the Colorectal Cancer in Umeå Study (CRUMS), including 574 with cancer-specific survival data, were analyzed for an eight-gene CIMP panel using quantitative real-time PCR (MethyLight). MSI screening status was assessed by immunohistochemistry. RESULTS: CIMP-low patients had a shorter cancer-specific survival compared with CIMP-negative patients (multivariate hazard ratio in NSHDS, 2.01; 95% confidence interval, 1.20-3.37; multivariate hazard ratio in CRUMS, 1.48; 95% confidence interval, 1.00-2.22). This result was similar in subgroups based on MSI screening status and was statistically significant in microsatellite stable (MSS) tumors in NSHDS. For CIMP-high patients, a shorter cancer-specific survival compared with CIMP-negative patients was observed in the MSS subgroup. Statistical significance was lost after adjusting for the BRAF mutation, but the main findings were generally unaffected. CONCLUSIONS: In this study, we found a poor prognosis in CIMP-low patients regardless of MSI screening status, and in CIMP-high patients with MSS. Although not consistently statistically significant, these results were consistent in two separate patient groups and emphasize the potential importance of CIMP and MSI status in colorectal cancer research.

Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosis.

Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-alpha stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-alpha stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation.

Lhx2-/- mice develop liver fibrosis.

Liver fibrosis is a wound-healing response to chronic injury of any type and is characterized by a progressive increase in deposition of extracellular matrix (ECM) proteins, the major source of which are activated hepatic stellate cells (HSCs). Because the LIM homeobox gene Lhx2 is expressed in HSCs and liver development in Lhx2(-/-) mice is disrupted, we analyzed liver development in Lhx2(-/-) embryos in detail. Lhx2(-/-) embryos contain numerous activated HSCs and display a progressively increased deposition of the ECM proteins associated with liver fibrosis, suggesting that Lhx2 inhibits HSC activation. Transfection of Lhx2 cDNA into a human HSC line down-regulates expression of genes characteristic of activated HSCs. Moreover, the Lhx2(-/-) liver display a disrupted cellular organization and an altered gene expression pattern of the intrahepatic endodermal cells, and the increased deposition of ECM proteins precedes these abnormalities. Collectively these results show that Lhx2 negatively regulates HSC activation, and its inactivation in developing HSCs appears therefore to mimic the signals that are triggered by the wound-healing response to chronic liver injury. This study establishes a spontaneous and reproducible animal model for hepatic fibrosis and reveals that Lhx2 expression in HSCs is important for proper cellular organization and differentiation of the liver.