Blockade of BFA-mediated apoptosis in macrophages by the HIV-1 Nef protein.

HIV-1 Nef protein has key roles at almost all stages of the viral life cycle. We assessed the role of Nef and of the translation elongation factor eEF1A in primary human macrophages. Nuclear retention experiments and inhibition of the exportin-t (Exp-t) pathway suggested that cytoplasmic relocalization of eEF1A, mediated by Exp-t occurs in Nef-treated monocyte-derived macrophages (MDMs). We observed the presence of tRNA in the Nef/eEF1A complexes. Nucleocytoplasmic relocalization of the Nef/eEF1A complexes prevented stress-induced apoptosis of MDMs treated with brefeldin A. Blockade of stress-induced apoptosis of MDMs treated with HIV-1 Nef resulted from enhanced nucleocytoplasmic transport of eEF1A with decreased release of mitochondrial cytochrome c, and from increased tRNA binding to cytochrome c, ultimately leading to an inhibition of caspase activation. Our results indicate that HIV-1 Nef, through the nucleocytoplasmic relocalization of eEF1A and tRNAs, enhances resistance to stress-induced apoptosis in primary human macrophages.

VDR microRNA expression and epigenetic silencing of vitamin D signaling in melanoma cells.

Malignant melanoma cells express the vitamin D receptor (VDR). However, some melanoma cell lines fail to respond to the antiproliferative effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We reported previously that out of seven melanoma cell lines analyzed, three cell lines (MeWo, SK-Mel28, SM) respond to the antiproliferative effects of 1,25(OH)2D3, while the others (SK-Mel5, SK-Mel25, IGR, Meljuso) are resistant. It was the aim of this study to investigate whether epigenetic mechanisms are of importance for the abrogation of vitamin D signaling in vitamin D resistant melanoma cells. We used the histone deacetylase inhibitor (HDACI) trichostatin A (TSA) and the DNA methyltransferase inhibitor (DNMTI) 5-azacytidine (5-Aza) to elucidate the effects of protein acetylation and of DNA hypermethylation on 1,25(OH)2D3-induced effects on cell proliferation, respectively. Additionally we analyzed the expression of VDR microRNA in 1,25(OH)2D3-responding and resistant melanoma cells. TSA and 5-Aza exerted dose- and time-dependent antiproliferative effects on melanoma cell lines. Interestingly, combination therapy with 1,25(OH)2D3 and TSA exerted synergistic antiproliferative effects in a 1,25(OH)2D3-resistant melanoma cell line (IGR) (p<0.05). Combination therapy with 1,25(OH)2D3 and 5-Aza resulted in synergistic (MeWo after 72 h; p<0.05) or additive (other melanoma cell lines analyzed) antiproliferative effects. Additionally, we could show that VDR mRNA expression is relatively high in two of three 1,25(OH)2D3-responsive melanoma cells as compared to resistant cells, moreover this relatively high VDR expression is associated with low expression of miRNA125b in MeWo and SK-Mel28 cells. Our results suggest that the endogenous VDR mRNA level is inversely associated with expression of miRNA125b in melanoma cell lines analyzed. Moreover, miRNA125b may be involved in the regulation of VDR expression and in the resistance against 1,25(OH)(2)D(3) in melanoma cells. It can be speculated whether miRNA125b may be of prognostic importance and/or may represent a therapeutic target for malignant melanoma. Drugs that influence epigenetic mechanisms might be promising therapeutics for the treatment of metastasized malignant melanoma, alone or in combination with antiproliferative or cytotoxic agents such as 1,25(OH)2D3.

Challenge and promise: the role of miRNA for pathogenesis and progression of malignant melanoma.

microRNAs are endogenous noncoding RNAs that are implicated in gene regulation. More recently, miRNAs have been shown to play a pivotal role in multiple cellular processes that interfere with tumorigenesis. Here we summarize the essential role of microRNAs for human cancer with special focus on malignant melanoma and the promising perspectives for cancer therapies.

Assignment of the NAD-dependent deacetylase sirtuin 5 gene (SIRT5) to human chromosome band 6p23 by in situ hybridization.

Sirtuin 5 (SIRT5) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase and belongs to the Silent information regulator 2 (Sir2) family of sirtuin histone deacetylases (HDACs), which play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. We have isolated and characterized the human SIRT5 genomic sequence, which spans a region of 28,182 bp and which has one single genomic locus. Human SIRT5 consists of eight exons and is found in two isoforms, which encode a 310 aa and a 299 aa protein, respectively. Human SIRT5 is most predominantly expressed in heart muscle cells and in lymphoblasts. Fluorescence in situ hybridization analysis localized the human SIRT5 gene to chromosome 6p23.

First experiences with DIN ISO 14835-1 in the context of vibration-induced white finger disease.

OBJECTIVES: In order to establish an international standard of cold provocation test in the assessment of vibration-induced white finger (VWF) disease, an ISO-working group tentatively created the DIN ISO 14835-1. Based on this new standard, previously existing testing conditions had to be modified. Since a comparison of current and previous evaluation procedures is necessary for both the individual assessment and the performance of metaanalyses, the revision and validation of criteria for the examination of the cold provocation tests are appropriate and necessary. METHODS: Twenty-one individuals suffering from VWF disease whose disorder was accepted as an occupational disease underwent the cold provocation test on two successive days following a 2- and a 5-min-long exposure to the cold. As a benchmark for classification as 'normal' or 'pathological', the 15-min mark after a 2-min-long exposure was chosen. A skin temperature of 28 degrees C was selected for discrimination between 'non-pathological' (at least 28 degrees C) and 'pathological' test results. RESULTS: It could be shown, that exposures to cold water (12 degrees C) over 2 and 5 min lead to similar rewarming profiles, who differ in median systematically by 1 degrees C. A modification of the former classification rule should be considered. After a 5 min exposure, the classification criterion can be based on the temperature assessments measured after 20 min; alternatively the cut point can be reduced from 28 to 27 degrees C while maintaining the previous assessment time of t = 15 min. CONCLUSIONS: The shown results represent the first attempt of modifying the previous classification criteria of the cold provocation test within the scope of the VWF disease. In view of the described problems of the study design there is no doubt that continuing modifications and their validation on the base of larger collectives groups are necessary.

Far-Western based protein-protein interaction screening of high-density protein filter arrays.

Even though a rough sketch of the human genome is now available and the number of newly discovered genes, which carry the potential of being biologically and medically relevant is currently greater than ever, only a small proportion has been assigned a biological function. Therefore, enormous attention is now increasingly being drawn towards functional genomics, i.e. the functional characterization of these newly identified sequences. In order to elucidate the role of a particular gene product within its cellular context, we have screened high-density protein filter arrays for protein-protein interactions on the basis of a 'Far-Western' based approach. The methodology described herein easily allows the identification and isolation of cDNAs of proteins, which interact with specific ligands (interacting proteins, antibodies and DNA/RNA sequences), and represents an alternative to tedious conventional protein interaction analyses. Far-Western screening in the context of a whole-genome expression analysis not only facilitates the assignment of biological functions to specific, newly identified protein and DNA sequences, but also is useful in studies that assess the binding capacity of mutant proteins to their interaction partner and in the identification of domains and amino acids involved in known protein-protein interactions. Taken together, we describe an approach that allows the easy and reproducible identification of protein ligands on the basis of a whole-genome expression analysis.

Macrophages and T-cell apoptosis in HIV infection: a leading role for accessory cells?

Recent studies indicate that macrophages modulate T-cell apoptosis in HIV infection. Macrophages have been shown to trigger apoptosis of uninfected bystander T cells and to protect HIV-infected T cells from apoptosis. This article raises the possibility that macrophages, via modulation of T-cell apoptosis, play a crucial role in both immune suppression and the formation of viral reservoirs during HIV infection.

Resistance to apoptosis in HIV-infected CD4+ T lymphocytes is mediated by macrophages: role for Nef and immune activation in viral persistence.

Apoptosis or programmed cell death may play a critical role in AIDS pathogenesis through depletion of both CD4(+) and CD8(+) T lymphocytes. Using a reporter virus, a recombinant HIV infectious clone expressing the green fluorescent protein (GFP), apoptosis was measured in productively infected CD4(+) T lymphocytes, in the presence and absence of autologous macrophages. The presence of macrophages in the culture increased the frequency of nonapoptotic GFP-positive productively infected CD4(+) T lymphocytes. The appearance of nonapoptotic productively infected CD4(+) T lymphocytes in the culture required intercellular contacts between macrophages and PBLs and the expression of the HIV Nef protein. The presence of macrophages did not reduce apoptosis when CD4(+) T lymphocytes were infected with a GFP-tagged virus deleted for the nef gene. TNF-alpha (TNF) expressed on the surface of macrophages prevented apoptosis in nef-expressing, productively infected CD4(+) T lymphocytes. Similarly, following TNF stimulation, apoptosis was diminished in Jurkat T cells transfected with a nef-expressing plasmid. TNF stimulation of nef-expressing Jurkat T cells resulted in NF-kappaB hyperactivation, which has been shown to deliver anti-apoptotic signals. Our results indicate that intercellular contacts with macrophages increase the rate of productively infected nonapoptotic CD4(+) T lymphocytes. The survival of productively infected CD4(+) T lymphocytes requires Nef expression as well as activation by TNF expressed on the surface of macrophages and might participate in the formation and maintenance of viral reservoirs in HIV-infected persons.

Chromosomal organization and localization of the human histone deacetylase 5 gene (HDAC5).

Histone deacetylases (HDACs) are important participants in the remodeling of chromatin structure and in the regulation of eukaryotic proliferation and differentiation. We have isolated and characterized the human HDAC5 genomic sequence, which spans a region of 39,138 bp and which has one single chromosomal locus. Determination of the exon-intron splice junctions established that HDAC5 is encoded by 26 exons ranging in size from 22 bp (exon 1) to 285 bp (exon 12). Characterization of the 5' flanking genomic region revealed that the human HDAC5 promoter lacks both the canonical TATA and CCAAT boxes. The human HDAC5 mRNA encodes a 1122 aa protein with a predictive molecular mass of 121.9 kDa and an isoelectric point of 5.84. Fluorescence in situ hybridization analysis localized the human HDAC5 gene to chromosome 17q21, a region which is characterized by frequent gains and losses of chromosomal material in several types of cancer.

Histone acetylation modifiers in the pathogenesis of malignant disease.

Chromatin structure is gaining increasing attention as a potential target in the treatment of cancer. Relaxation of the chromatin fiber facilitates transcription and is regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which modify the acetylation state of histone proteins and other promoter-bound transcription factors. While HATs, which are frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, HDACs tend to associate with multisubunit core-pressor complexes, which result in chromatin condensation and transcriptional repression of specific target genes. HATs and HDACs are known to be involved both in the pathogenesis as well as in the suppression of cancer. Some of the genes encoding these enzymes have been shown to be rearranged in the context of chromosomal translocations in human acute leukemias and solid tumors, where fusions of regulatory and coding regions of a variety of transcription factor genes result in completely new gene products that may interfere with regulatory cascades controlling cell growth and differentiation. On the other hand, some histone acetylation-modifying enzymes have been located within chromosomal regions that are particularly prone to chromosomal breaks. In these cases gains and losses of chromosomal material may affect the availability of functionally active HATs and HDACs, which in turn disturbs the tightly controlled equilibrium of histone acetylation. We review herein the recent achievements, which further help to elucidate the biological role of histone acetylation modifying enzymes and their potential impact on our current understanding of the molecular changes involved in the development of solid tumors and leukemias.

When the band begins to play: histone acetylation caught in the crossfire of gene control.

Increasing evidence from recent research suggests a connection between cancer and a deranged equilibrium of histone acetylation, which is maintained by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs). It is our hypothesis that a significant proportion of leukemias and possibly also solid tumors have abnormalities involving HATs or HDACs at the genomic level through genetic mutations or chromosomal alterations. In these cases, altered levels of HATs or HDACs may derange the tightly regulated equilibrium of histone acetylation, which may affect the expression of a broad spectrum of cellular genes. On the other hand, HATs and HDACs may be carried to defined target promoters as cofactors of transcription factor-bound repressor or enhancer complexes and thereby carry out unwanted enzymatic activities in the wrong place at the wrong time. We therefore propose a model for disease being associated with a deranged equilibrium of acetylation that affects histone proteins and promoter-bound transcription factors.

High resolution physical mapping of human HDAC3, a potential tumor suppressor gene in the 5q31 region.

Histone deacetylases have been described as crucial cofactors of mammalian transcriptional complexes. We have recently identified human histone deacetylase HDAC3 on chromosome 5q31 by fluorescence in situ hybridization (FISH) in a region commonly deleted in malignant myeloid disease. Since HDAC3 carries strong potential to be a tumor suppressor gene, we report herein its exact position between the CD14 and GRIA1 genes within the 5q31.1 subband.