No abstract

We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n ¼ 11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARb and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas.

IL‐12 is a potent inducer of IFN‐γ production and drives the development of Th1 cells. Human polarized Th2 cells do not express the signaling β2‐subunit of the IL‐12R and, therefore, do not signal in response to IL‐12. The question was raised as to what extent the loss of the IL‐12Rβ2 chain in Th2 cells has bearing on the stability of the human Th2 phenotype. In the present report, we show that restimulation of human fully polarized Th2 cells in the presence of IL‐12 primes for a shift towards Th0/Th1 phenotypes, accompanied by suppression of GATA‐3 expression and induction of T‐bet expression. These reversed cells are further characterized by a marked IL‐12Rβ2 chain expression and fully restored IL‐12‐inducible STAT4 activation. The IL‐12‐induced phenotypic shift proved to be stable as a subsequent restimulation in the presence of IL‐4 and in the absence of IL‐12 could not undo the accomplished changes. Identical results were obtained with cells from atopic patients, both with polyclonal Th2 cell lines and allergen‐specific Th2 cell clones. These findings suggest the possibility of restoring IL‐12 responsiveness in established Th2 cells of atopic patients by stimulation in the presence of IL‐12, and that IL‐12‐promoting immunotherapy can be beneficial for Th2‐mediated immune disorders, targeting both naive and memory effector T cells.

Interleukin 12 (IL-12) is a potent enhancer of interferon ␥ production by activated T cells. The high-affinity IL-12 receptor (IL-12R) is a heterodimer of a ␤1 and a ␤2 subunit. Expression of the signaling IL-12R␤2 chain is usually low, as compared with the more abundant ␤1 chain, and may be rate-limiting for IL-12 sensitivity. Little is known about the mechanisms controlling IL-12R␤2 gene expression. Reporter gene assays in IL-12R␤2-expressing Jurkat cells showed that truncation of the region from ؊151 to ؊61 abrogated promoter activity. The proximal promoter region does not contain a typical TATA box, suggesting a role for SP-1. Indeed, mutagenesis of the ؊63 SP-1 consensus site decreased transcription by 50%. Electrophoretic mobility shift experiments confirmed the binding of SP-1 and SP-3 at this site. In contrast, truncation of ؊252 to ؊192 increased promoter activity. Likewise, mutagenesis of the consensus nuclear factor of activated T cells site at ؊206 increased promoter activity by 70%, suggesting silencer activity of this element. Electrophoretic mobility shift experiments with primary Th (T helper) cells showed the formation of a specific, T-cell receptor-inducible complex at this site that is sensitive to cyclosporin A and supershifted with anti-NFATc2 in both Th1 and Th2 cells. Accordingly, cyclosporin A dose-dependently increased IL-12R␤2 mRNA expression. These first data on IL-12R␤2 gene regulation indicate a TATA-less promoter, depending on SP-1/SP-3 transcription factors, and a negative regulatory NFAT element at ؊206. This element may contribute to the overall low level of IL-12R␤2 expression on Th cells.

Interleukin-12 (IL-12) is a potent inducer of interferon-gamma production by T cells and is a major factor for the development of T-helper 1 (Th1) cells. It exerts its biological effects through binding to the IL-12 receptor (IL-12R), a heterodimer composed of a 1 and a beta2 subunits. The signaling beta2 chain is expressed on Th1 cells and to a lesser extent on Th0 cells, but not on Th2 cells, rendering these latter cells unresponsive to IL-12. Polymorphisms in the coding region of the IL-12Rbeta2 gene were shown to be associated with atopic disease. Here, we analyzed the 5'-regulatory region of the human IL-12Rbeta2 gene by denaturing high-performance liquid chromatography (Transgenomic WAVE system, San Jose, CA). We found five novel single-nucleotide polymorphisms (SNPs) in the proximal 1.2 kb IL-12Rbeta2 promoter region, i.e. -237C/T, -465A/G, -1023A/G, -1033T/C, and -1035A/G. SNP -465A/G is of particular interest as it determines the integrity of a GATA consensus site. By functional comparison of both -465 alleles in transient transfection assays, we show that promoter activity is increased in case of the -465G allele, disrupting the intact GATA site. Comparison of the prevalence of -465A/G SNP alleles in small cohorts of allergic asthmatic and healthy control individuals provided no evidence for an altered distribution in the asthmatic population. In conclusion, we have identified a novel polymorphic GATA site that may affect transciptional activity of the human IL-12Rbeta2 gene under GATA3-mediated, Th2-polarizing conditions.