Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

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=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 RARβ 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

Prevalence, Distribution and Functional Significance of the −237C to T Polymorphism in the IL-12Rβ2 Promoter in Indian Tuberculosis Patients

Cytokine/cytokine receptor gene polymorphisms related to structure/expression could impact immune response. Hence, the −237 polymorphic site in the 5′ promoter region of the IL-12Rβ2 (SNP ID: rs11810249) gene associated with the AP-4 transcription motif GAGCTG, was examined. Amplicons encompassing the polymorphism were generated from 46 pulmonary tuberculosis patients, 35 family contacts and 28 miscellaneous volunteers and sequenced. The C allele predominated among patients, (93.4%, 43/46), and in all volunteers and contacts screened, but the T allele was exclusively limited to patients, (6.5%, 3/46). The functional impact of this polymorphism on transcriptional activity was assessed by Luciferase-reporter and electrophoretic mobility shift assays (EMSA). Luciferase-reporter assays showed a significant reduction in transcriptional efficiency with T compared to C allele. The reduction in transcriptional efficiency with the T allele construct (pGIL-12Rb2-T), in U-87MG, THP-1 and Jurkat cell lines, were 53, 37.6, and 49.8% respectively, compared to the C allele construct (pGIL-12Rb2-C). Similarly, densitometric analysis of the EMSA assay showed reduced binding of the AP-4 transcription factor, to T compared to the C nucleotide probe. Reduced mRNA expression in all patients (3/3) harboring the T allele was seen, whereas individuals with the C allele exhibited high mRNA expression (17/25; 68%, p = 0.05). These observations were in agreement with the in vitro assessment of the promoter activity by Luciferase-reporter and EMSA assays. The reduced expression of IL-12Rβ2 transcripts in 8 patients despite having the C allele was attributed to the predominant over expression of the suppressors (IL-4 and GATA-3) and reduced expression of enhancers (IFN-α) of IL-12Rβ2 transcripts. The 17 high IL-12Rβ2 mRNA expressers had significantly elevated IFN-α mRNA levels compared to low expressers and volunteers. Notwithstanding the presence of high levels of IL-12Rβ2 mRNA in these patients elevated IFN-α expression could modulate their immune responses to Mycobacterium tuberculosis

Mycobacterium tuberculosis infection induces il12rb1 splicing to generate a novel IL-12Rβ1 isoform that enhances DC migration

RNA splicing is an increasingly recognized regulator of immunity. Here, we demonstrate that after Mycobacterium tuberculosis infection (mRNA) il12rb1 is spliced by dendritic cells (DCs) to form an alternative (mRNA) il12rb1Deltatm that encodes the protein IL-12Rbeta1DeltaTM. Compared with IL-12Rbeta1, IL-12Rbeta1DeltaTM contains an altered C-terminal sequence and lacks a transmembrane domain. Expression of IL-12Rbeta1DeltaTM occurs in CD11c(+) cells in the lungs during M. tuberculosis infection. Selective reconstitution of il12rb1(-/-) DCs with (mRNA) il12rb1 and/or (mRNA) il12rb1Deltatm demonstrates that IL-12Rbeta1DeltaTM augments IL-12Rbeta1-dependent DC migration and activation of M. tuberculosis-specific T cells. It cannot mediate these activities independently of IL12Rbeta1. We hypothesize that M. tuberculosis-exposed DCs express IL-12Rbeta1DeltaTM to enhance IL-12Rbeta1-dependent migration and promote M. tuberculosis-specific T cell activation. IL-12Rbeta1DeltaTM thus represents a novel positive-regulator of IL12Rbeta1-dependent DC function and of the immune response to M. tuberculosis.This work was supported by the Trudeau Institute and the National Institutes of Health (AI067723 to A. M. Cooper; AI49823 to D. L. Woodland [trainee: R. T. Robinson] and AI084397 to R. T. Robinson)

Analysis of eight genes modulating interferon gamma and human genetic susceptibility to tuberculosis: a case-control association study

<p>Abstract</p> <p>Background</p> <p>Interferon gamma is a major macrophage-activating cytokine during infection with <it>Mycobacterium tuberculosis</it>, the causative pathogen of tuberculosis, and its role has been well established in animal models and in humans. This cytokine is produced by activated T helper 1 cells, which can best deal with intracellular pathogens such as <it>M. tuberculosis</it>. Based on the hypothesis that genes which regulate interferon gamma may influence tuberculosis susceptibility, we investigated polymorphisms in eight candidate genes.</p> <p>Methods</p> <p>Fifty-four polymorphisms in eight candidate genes were genotyped in over 800 tuberculosis cases and healthy controls in a population-based case-control association study in a South African population. Genotyping methods used included the SNPlex Genotyping System™, capillary electrophoresis of fluorescently labelled PCR products, TaqMan^®SNP genotyping assays or the amplification mutation refraction system. Single polymorphisms as well as haplotypes of the variants were tested for association with TB using statistical analyses.</p> <p>Results</p> <p>A haplotype in interleukin 12B was nominally associated with tuberculosis (p = 0.02), but after permutation testing, done to assess the significance for the entire analysis, this was not globally significant. In addition a novel allele was found for the interleukin 12B D5S2941 microsatellite.</p> <p>Conclusions</p> <p>This study highlights the importance of using larger sample sizes when attempting validation of previously reported genetic associations. Initial studies may be false positives or may propose a stronger genetic effect than subsequently found to be the case.</p

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