Subject(s)
Algorithms , Gene Expression Profiling/methods , In Situ Hybridization, Fluorescence/methods , Microscopy, Fluorescence/methods , Models, Genetic , Oligonucleotide Array Sequence Analysis/methods , Computer Simulation , Fluorescent Dyes , Models, Statistical , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The NF-kappaB/Rel family of transcription factors regulates a wide variety of genes whose products play a fundamental role in inflammatory and immune responses. The implication of NF-kappaB/Rel proteins and their IkappaB regulatory subunits in the control of cellular growth and oncogenesis, was suggested by the induction of fatal lymphomas in birds by the v-rel oncoprotein, and the rearrangement and amplification of several genes encoding the NF-kappaB/Rel/IkappaB signal transduction factors in human malignancies, primarily of lymphoid origin. Hodgkin's disease (HD) is a lymphoma characterized by a low frequency of malignant Hodgkin and Reed-Sternberg (H/RS) cells in a reactive background of non-neoplastic cells. The peculiar activated phenotype of Hodgkin and Reed-Sternberg cells and their pattern of cytokine secretion are believed to be a consequence of constitutive activation of the NF-kappaB transcription factor. Here, we report the detection of mutations of the IkBa gene, in two HD-derived cell lines and in two out of eight biopsy samples from patients with relapsed Hodgkin's disease. The presence of defective IkappaBalpha is thus likely to explain the constitutive activation of NF-kappaB in these cells and suggests that IkappaBalpha is a tumour suppressor controlling the oncogenic activation of NF-kappaB in Hodgkin and Reed-Sternberg cells.
Subject(s)
DNA-Binding Proteins/genetics , Genes, Tumor Suppressor , Hodgkin Disease/genetics , I-kappa B Proteins , Mutation , Alleles , Base Sequence , Biopsy , DNA Primers , Hodgkin Disease/pathology , Humans , NF-KappaB Inhibitor alpha , RNA, Messenger/genetics , Sequence DeletionABSTRACT
The CEF-4/9E3 gene is expressed aberrantly in chicken embryo fibroblasts transformed by the Rous sarcoma virus. This aberrant expression is dependent on transcriptional activation and on the stabilization of the CEF-4 mRNA. The characterization of the CEF-4 promoter indicated that three distinct regulatory elements corresponding to an AP-1 binding site, a PRDII/ kappaB domain and a CAAT box are involved in the activation by pp60v-src. Several v-src responsive genes are controlled by AP-1 and members of the Ets family but few appear to be dependent on NF-kappaB. In this study we characterize the expression of genes regulated by NF-kappaB in normal and RSV-transformed CEF. Run-on transcription analysis indicated that pp60v-src induces the transcription of several genes controlled by NF-kappaB but at different levels. While the transcription of CEF-4 was strongly stimulated, that of NF-kappaB1, c-rel, p53 or IkappaB-alpha was activated more modestly by pp60v-src. In addition the CEF-4 mRNA was the only mRNA species to accumulate significantly in transformed CEF. The ectopic expression of RelA or Rel resulted in the stimulation of the transcription of several known targets of NF-kappaB. However, the mRNA for IkappaB-alpha was the only mRNA species to accumulate considerably in the RelA- or Rel-expressing cells. Hence for most kappaB-controlled genes, transcriptional activation was not sufficient to obtain a significant increase in mRNA expression. Likewise, RelA or Rel enhanced the transcription of the CEF-4 gene without a significant accumulation of the CEF-4 mRNA. However, transformation by v-src caused a massive accumulation of the CEF-4 mRNA but not of other mRNA species in the RelA- and Rel-expressing cells. Transient expression assays, run-on transcription and Northern blotting analyses indicated that the effect of pp60v-src on CEF-4 expression was mediated predominantly at the post-transcriptional level in these cells. Therefore transcriptional and post-transcriptional mechanisms determine the restricted pattern of activation of kappaB-controlled genes in RSV-transformed CEF.
Subject(s)
Avian Proteins , Cytokines/genetics , Gene Expression Regulation , NF-kappa B/pharmacology , Oncogene Protein pp60(v-src)/pharmacology , Protein Processing, Post-Translational , Animals , Cell Transformation, Viral , ChickensABSTRACT
Protein H16, which we have identified previously in mammalian cell lines, binds in vitro to two single stranded DNA sites on the late strand of the early promoter of SV40. It has no other single strand binding site in the SV40 genome and does not bind to double stranded DNA. In vitro, H16 can be shown to stimulate strongly the activity of purified RNA polymerase II. Here we have purified this 70 kDa protein from cultured monkey cells and have sequenced three of its tryptic peptides. The analysis indicates that H16 is the simian homolog of human protein K, a nuclear RNA-binding protein found in heterogeneous nuclear ribonucleoprotein (hnRNP) particles, which contains a KH domain present in several proteins including the fragile X mental retardation gene product (FMR1). The binding affinities of protein K/H16 for RNA and DNA were subsequently compared in detail. They showed that under conditions where K/H16 binds strongly to its single stranded DNA site, it binds very weakly to the corresponding RNA sequence. This result suggests a possible shuttling of the protein from RNA to DNA during processes which involve opening of the DNA double helix.