Methylation of CIITA promoter IV causes loss of HLA-II inducibility by IFN-gamma in promyelocytic cells.

The human promyelocytic cell line THP-1 expresses high level of HLA class II (HLA-II) molecules after IFN-gamma treatment. Here, we report a variant of THP-1 that does not express HLA-II after IFN-gamma. The variant's HLA-II phenotype is constant over time in culture and it is not related to a defective IFN-gamma-signalling pathway. Transfection of CIITA, the HLA-II transcriptional activator, under the control of a cytomegalovirus promoter rescues high level of HLA-DR surface expression in the variant indicating that the biosynthetic block resides in the expression of CIITA and not in the CIITA-dependent transactivation of the HLA-II promoters. Treatment of the variant with 5-azacytidine (5-aza), which inhibits CpG methylation, restores inducibility of HLA-II by IFN-gamma both at transcriptional and phenotypic level and antigen presenting and processing function of the variant. DNA studies demonstrate that the molecular defect of the THP-1 variant originates from the methylation of the CIITA promoter IV. Furthermore, treatment with 5-aza produces a substantial demethylation of CIITA promoter IV and a significant increase of IFN-gamma-dependent HLA-II expression in another myelomonocytic cell line, U937. Therefore hyper-methylation of CIITA promoter IV may be a relevant mechanism of epigenetic control preventing HLA-II IFN-gamma inducibility in the myelomonocytic cell lineage.

A novel isoform of pro-interleukin-18 expressed in ovarian tumors is resistant to caspase-1 and -4 processing.

Interleukin-18 (IL-18) is a proinflammatory cytokine synthesized as a 24 kDa inactive precursor (pro-IL-18) by several cell types, and is processed to a bioactive molecule of 18 kDa by the proteinases caspase-1 or caspase-4. All ovarian carcinoma cell lines express pro-IL-18, only in some instances coexpress caspase-1, and always express caspase-4; in any case, they display a defective processing of IL-18. We analysed whether pro-IL-18, present in two ovarian carcinoma cell lysates, could be processed 'in vitro' by recombinant active caspase-1. While most of pro-IL-18 could be cleaved by caspase-1, a residual of pro-IL-18 appeared to be resistant. Cloning and sequence analysis of the whole pro-IL-18 open reading frame demonstrated the existence of an alternatively spliced mRNA variant, which lacked exon-3 (Delta3pro-IL-18). The 12 bp exon-3 encodes for the AEDD amino-acid sequence, which is N-terminal with respect to the cleavage site of caspase-1. Both pro-IL-18 and Delta3pro-IL-18 mRNA isoforms were detected in all ovarian cancer cell lines analysed, while Delta3pro-IL-18 mRNA was undetectable in normal ovarian epithelial cells. The Delta3pro-IL-18 cDNA induced synthesis of an alternative Delta3pro-IL-18 protein upon transfection into a murine cell line. The Delta3pro-IL-18 protein was resistant to proteolytic activation by caspase-1 and -4, although it was capable to bind caspase-1. Aternative splicing of pro-IL-18 exon-3 may represent a novel mechanism of regulation of bioactive IL-18 production in human ovarian tumors.

Different levels of control prevent interferon-gamma-inducible HLA-class II expression in human neuroblastoma cells.

The HLA class II expression is controlled by the transcriptional activator CIITA. The transcription of CIITA is controlled by different promoters, among which promoter-IV is inducible by IFN-gamma. We analysed the regulation of HLA class II molecules by IFN-gamma in a large series of human neuroblastoma cell lines. No induction of surface or intracellular HLA class II molecules and of specific mRNA was observed, in all neuroblastomas, with the exception of a nonprototypic cell line, ACN. In a large subset of neuroblastomas IFN-gamma induced expression of CIITA mRNA, derived from promoter-IV, which was not methylated. In contrast, in another subset of neuroblastomas, CIITA was not inducible by IFN-gamma and CIITA promoter-IV was either completely or partially methylated. Interestingly, the use of DNA demethylating agents restored CIITA gene transcriptional activation by IFN-gamma, but not HLA class II expression. The defect of HLA class II was not related to alterations in RFX or NF-Y transcription factors, as suggested by EMSA or RFX gene transfection experiments. In addition, the transfection of a functional CIITA cDNA failed to induce HLA class II expression in typical neuroblastoma cells. Confocal microscopy and Western blot analysis suggested a defective nuclear translocation and/or reduced protein synthesis in CIITA-transfected NB cells. Altogether, these data point to multiple mechanisms preventing HLA class II expression in the neuroblastoma, either involving CIITA promoter-IV silencing, or acting at the CIITA post-transcriptional level.

Caspase-8 gene expression in neuroblastoma.

Neuroblastoma (NB) is a solid tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic/differentiation program. Indeed, the level of expression of molecules involved in the regulation of apoptosis, such as p73 or survivin, is a prognostic factor in NB patients. The caspase-8 gene (CASP8) encodes a key enzyme at the top of the apoptotic cascade. Although methylation of a putative regulatory region of the CASP8 gene reportedly inhibits its transcription in some MYCN-amplified NB, our results indicate that the transcriptional inactivation of caspase-8 occurs in a subset of primary NB independently of MYCN amplification or CpG methylation. In addition, the apoptotic agent fenretinide (4HPR) and interferon-gamma (IFN-gamma) induce caspase-8 expression without modifying the methylation status of this gene. Nevertheless, the methylation level of CASP8 intragenic and promoter regions is higher in MYCN-amplified tumors as compared to nonamplified samples. This phenomenon might reflect the existence of distinct DNA methylation errors in MYCN-amplified and MYCN-single copy tumors. To gain information on the mechanisms that regulate the expression of this crucial apoptotic gene, we searched for potential CASP8 regulatory regions and cloned a DNA element at the 5' terminus of this gene that functionally acts as a promoter only in NB cell lines that express caspase-8. The retinoic acid analogue 4HPR, IFN-gamma, and the demethylating agent 5-aza-cytidine activate this promoter in NB cells that lack endogenous caspase-8, indicating that this element may regulate both constitutive and inducible CASP8 expression. These results indicate also that demethylation of the cellular genome may upregulate CASP8 through the action of trans-acting factors. Our results provide new insights to the regulation of CASP8, a gene with an essential role in a variety of physiologic and pathologic conditions.

An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cells.

We previously identified a 1.2 Kb DNA element (P-1161/+16), 5' to caspase-8 exon-1, that acts as promoter in caspase-8-positive, but not in caspase-8-negative neuroblastoma (NB) cells. The P-1161/+16 DNA element regulates both constitutive and interferon IFN-gamma-inducible caspase-8 expression. Two GAS (IFN-activated sequence, STAT-1 binding site) and two ISRE (interferon sensitive response element, IRF binding site) were present in P-1161/+16. Deletion studies indicated that elements essential for promoter activity in NB cells were present in a 167 bp region 5' flanking exon-1 (P-151/+16), which contains an ISRE at position -32. The transcription initiation site was mapped by 5' rapid amplification of cDNA end (RACE) at position -20 from caspase-8 cDNA reference sequence. Disruption of the ISRE-32 indicated that it is required for both constitutive and IFN-gamma-inducible caspase-8 expression. IRF-1 and IRF-2 transcription factors bind to the (-151/+16) DNA fragment in vitro. Chromatin immunoprecipitation (ChIP) assays showed that IRF-1 and IRF-2 bind to the DNA region at the 5' of caspase-8 gene in NB cells, which show constitutive expression but not in caspase-8 negative cells. In these last cells, up-regulation of caspase-8 by IFN-gamma was associated to induction of IRF-1 and IRF-2 binding to caspase-8 promoter and increased histone acetylation. Moreover, RNA interference experiments also supported the involvement of IRF-1 and IRF-2 in constitutive caspase-8 expression in NB cells.