Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the ß-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Perspectives of protein kinase C (PKC) inhibitors as anti-cancer agents.

Although the role of serine/threonine protein kinase C (PKC) in malignant transformation is known from decades, an anti-PKC based approach in cancer therapy was hampered for the difficulties in developing pharmacological compounds able to selectively inhibit specific PKC isoforms. In this review, the role of PKC-epsilon and PKC-delta in promoting and counteracting tumor progression in different types of cancer, respectively, will be discussed in relationship with promising therapeutic perspectives based either on small molecule inhibitors or on natural compounds. Among a myriad of molecules able to modulate PKC activity, we will focus on the role of the enzastaurin and briostatin-1, which already entered clinical trials for several human cancers.

Effects of a hydroxyapatite-based biomaterial on gene expression in osteoblast-like cells.

Biostite is a hydroxyapatite-derived biomaterial that is used in periodontal and bone reconstructive procedures due to its osteoconductive properties. Since the molecular effects of this biomaterial on osteoblasts are still unknown, we decided to assess whether it may specifically modulate osteoblast functions in vitro. We found that a brief exposure to Biostite significantly reduced the proliferation of MG-63 and SaOS-2 osteoblast-like cells to approximately 50% of the plateau value. Furthermore, gene array analysis of MG-63 cells showed that Biostite caused a differential expression of 37 genes which are involved in cell proliferation and interaction, and related to osteoblast differentiation and tissue regeneration. Results were confirmed by RT-PCR, Western blot, and by an increase in alkaline phosphatase (ALP) specific activity. Biostite also increased levels of polycystin-2, a mechano-sensitive Ca(2+) channel, a promising new marker of bone cell differentiation. Biostite, therefore, may directly affect osteoblasts by enhancing chondro/osteogenic gene expression and cytoskeleton-related signaling pathways, which may contribute to its clinical efficacy.

Phosphoinositide 3-kinase activity is essential for all-trans-retinoic acid-induced granulocytic differentiation of HL-60 cells.

Phosphoinositide 3-kinase (PI 3-K) activity increases in HL-60 cells that are induced to granulocytic differentiation by all-trans-retinoic acid. Immunochemical and immunocytochemical analyses by confocal microscopy also reveal an increase in the amount of the enzyme, which is particularly evident at the nuclear level. Inhibition of PI 3-K activity by nanomolar concentrations of wortmannin and of its expression by transfection with an antisense fragment of p85alpha prevented the differentiative process. The data obtained indicate that PI 3-K activity plays an essential role in promoting granulocytic differentiation.

HIV-1 Tat induces tyrosine phosphorylation of p125FAK and its association with phosphoinositide 3-kinase in PC12 cells.

OBJECTIVE: To evaluate the signal transduction potential of HIV-1 Tat in a neuronal cell model. METHODS: The tyrosine phosphorylation levels of the focal adhesion kinase p125FAK and its association with phosphoinositide 3-kinase (PI 3-K) were evaluated in serum-starved rat pheochromocytoma PC12 cells, either treated with low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein or stably transfected with Tat cDNA. RESULTS: Extracellular Tat induced a rapid increase of p125FAK tyrosine phosphorylation and p125FAK-associated PI 3-K activity. By using recombinant mutated Tat proteins, it was found that deletion of amino acids 73-86 encoded by the second exon of the tat gene resulted in a significant decrease of the ability of Tat to induce p125FAK tyrosine phosphorylation. Paradoxically, mutations in the basic region encoded by the first exon of tat, which is essential for nuclear localization and HIV-1 LTR transactivation, increased the ability of Tat to stimulate p125FAK tyrosine phosphorylation. Moreover, in comparison with cells transfected with a control vector, PC12 cells stably transfected with tat cDNA showed greater amounts of p125FAK protein, an increase in p125FAK tyrosine phosphorylation and higher levels of p125FAK-associated PI 3-K activity. The addition of anti-Tat neutralizing antibody to tat-transfected PC12 cells in culture blocked both the p125FAK tyrosine phosphorylation and its association with PI 3-K but did not affect the total amount of p125FAK. CONCLUSION: HIV-1 Tat protein enhanced both the expression and the functionality of p1 25FAK in PC12 neuronal cells. Whereas the first event required intracellular Tat, the increased p125FAK phosphorylation was strictly dependent upon extracellular Tat.

Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.

OBJECTIVE: To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells. METHODS: CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity. RESULTS: Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin. CONCLUSIONS: Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.

Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR.

In the present paper, biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect the Trp1282Ter mutation (W1282X) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We first immobilized on a SA5 sensor chip a single-stranded biotinylated oligonucleotide containing the sequence involved in this mutation, and the efficiency of hybridization of oligonucleotide probes differing in length was determined. Second, we immobilized on different SA5 sensor chips biotinylated polymerase-chain reaction (PCR) products from a normal subject as well as from heterozygous and homozygous W1282X samples. The results obtained show that both allele-specific 10- and 12-mer oligonucleotides are suitable probes to detect W1282X mutations of the cystic fibrosis gene under standard BIA experimental conditions. During the association phase performed at 25 degrees C, discrimination between mismatched and full matched hybrids was readily and reproducibly observed by using the 10-mer W1282X probes. By contrast, when the 12-mer DNA probes were employed, discrimination between mismatched and full matched hybrids was observed during the dissociation phase. Taken together, the results presented suggest that BIA is an easy, speedy, and automatable approach to detect point mutations leading to cystic fibrosis. By this procedure, it is possible to perform real-time monitoring of hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal or heterozygous subjects, and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one-step, non-radioactive protocol to perform diagnosis.

Analysis of the human HLA-DRA gene upstream region: evidence for a stem-loop array directed by nuclear factors.

Sequence analysis of the far-upstream region of the human HLA-DRA gene has revealed the presence of Y' and X' boxes, highly homologous to the well characterized Y and X boxes present within the proximal-promoter region. Comparison of Y, Y', X, and X' box sequences present within different class II MHC genes of different species demonstrates that these boxes are conserved during evolution, suggesting an important role in regulation of gene expression. The far-upstream region and the proximal promoter region of the class II MHC genes could be organized in secondary structures, as suggested for the EA gene, the murine counterpart of the human HLA-DRA gene. The essential feature of this model is a dimerization of the proteins binding to X and X' and/or Y and Y' boxes resulting in a loop-out of the intervening DNA and a rapprochement of the far-upstream and proximal-promoter regions, and consequently of any proteins binding to them. We set up an in vitro approach in order to determine whether proteins bound to sequences present within far-upstream and proximal-promoter regions of the human HLA-DRA gene could direct a secondary structure assembly of regulative regions. Moreover, by gel retardation and DNase I footprinting assays, we demonstrate that similar proteins bind to Y and Y' boxes and, among these proteins, NF-Y was unambiguously identified by antibody-super shift experiments. Taken together, the data presented in this paper provide evidence supporting the hypothesis that a stem-loop array of the 5'-upstream region of the human HLA-DRA gene could be directed by nuclear factors. In this manner, additional nuclear factors bound to the far region could be driven in close proximity of the transcription initiation site.

Peptide nucleic acids (PNA)-DNA chimeras targeting transcription factors as a tool to modify gene expression.

Peptide nucleic acids (PNAs)-DNA chimeras have been recently described as DNA mimics constituted of a part of PNA and of a part of DNA. We have demonstrated that double stranded molecules based on PNA-DNA chimeras bind to transcription factors in a sequence-dependent manner. Accordingly, these molecules can be used for transcription factor decoy (TFD) pharmacotherapy. Effects of double stranded PNA-DNA chimeras targeting NF-kappaB and Sp1 were determined on in vitro cultured human cells and were found to be comparable to those observed using double-stranded DNA decoys. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments for non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

Synthesis and antitumor activity of new benzoheterocyclic derivatives of distamycin A.

The design, synthesis, and in vivo and in vitro antileukemic activity of a novel series of compounds (13-22 and 34), in which different benzoheterocyclic rings, bearing a nitrogen mustard or a benzoyl nitrogen mustard or an alpha-bromoacryloyl group as alkylating moieties, are tethered to a distamycin frame, are reported, and structure-activity relationships are discussed. The new derivatives were prepared by coupling nitrogen mustard-substituted, benzoyl nitrogen mustard-substituted, or alpha-bromoacryloyl-substituted benzoheterocyclic carboxylic acids 23-32 with desformyldistamycin (33) or in one case with its two-pyrrole analogue 35. With very few exceptions, the activities of compounds bearing the same alkylating moiety are slightly affected by the kind of the heteroatom present on the benzoheterocyclic ring. All novel compounds, with one exception, showed in vitro activity against L1210 murine leukemia cell line comparable to or better than that of tallimustine. The compounds in which the nitrogen mustard and the alpha-bromoacryloyl moieties are directly linked to benzoheterocyclic ring showed potent cytotoxic activities (IC(50) ranging from 2 to 14 nM), while benzoyl nitrogen mustard derivatives of benzoheterocycles showed reduced cytotoxic activities, and one compound (16) of this cluster was the sole derivative devoid of significant activity. Compound 18, a 5-nitrogen mustard N-methylindole derivative of distamycin, showed the best antileukemic activity in vivo, with a very long survival time (%T/C = 457), significantly increased in comparison to tallimustine (%T/C = 133), and was selected for further extensive evaluation. Arrested polymerase chain reaction and direct DNA fragmentation assays were performed for compound 18 and the structurally related compounds 13-17 and 19. The results obtained have shown that both alkylating groups and oligopeptide frames play a crucial role in the sequence selectivity of these compounds.

Synthesis, in vitro antiproliferative activity, and DNA-binding properties of hybrid molecules containing pyrrolo[2,1-c][1, 4]benzodiazepine and minor-groove-binding oligopyrrole carriers.

The synthesis, biological activity, and DNA-binding properties of a series of four hybrids prepared by combining polypyrrole minor groove binders and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) 13, related to the naturally occurring anthramycin (3) and DC-81 (4), have been described, and structure-activity relationships have been discussed. These hybrids 22-25 contain from one to four pyrrole units, respectively. To investigate sequence selectivity and stability of drug/DNA complexes, DNase I footprinting and arrested polymerase chain reaction (PCR) were performed on human c-myc oncogene, estrogen receptor gene, and human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) gene sequences. The antiproliferative activity of the hybrids has been tested in vitro on human myeloid leukemia K562 and T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1, its tetrapyrrole homologue 17, DC 81 (4), and the PBD methyl ester 12. The results obtained demonstrate that the hybrids 22-25 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 12. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids 22-25 and stability of drug/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 25 containing the four pyrroles is more active than 22, 23, and 24 both against K562 and Jurkat cell lines.

Use of an automated laboratory workstation for isolation of genomic DNA suitable for PCR and allele-specific hybridization.

In this paper we describe the isolation of genomic DNA by using anion exchange chromatography performed on a Biomek 1000 Automated Laboratory Workstation. This procedure allows the automated isolation of DNA suitable for most molecular analyses employed in diagnosis of genetic pathologies and infectious diseases. The genomic DNA isolated by using the Biomek 1000 was indeed found to be suitable for polymerase chain reaction and allele-specific hybridization.

Targeting of the Sp1 binding sites of HIV-1 long terminal repeat with chromomycin. Disruption of nuclear factor.DNA complexes and inhibition of in vitro transcription.

Sequence selectivity of DNA-binding drugs has recently been reported in a number of studies employing footprinting and gel retardation approaches. In this paper, we studied the biochemical effects of the sequence-selective binding of chromomycin to the long terminal repeat of the human immunodeficiency type I virus. Deoxyribonuclease I (E.C.3.1.21.1) footprinting, arrested polymerase chain reaction, gel retardation and in vitro transcription experiments have demonstrated that chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1. Accordingly, interactions between nuclear proteins and Sp1 binding sites are inhibited by chromomycin, and this effect leads to a sharp inhibition of in vitro transcription.

Induction of erythroid differentiation of human K562 cells by cisplatin analogs.

Human leukemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including hemin, butyric acid, 5-azacytidine, and cytosine arabinoside. Differentiation of K562 cells is associated with an increase in the expression of embryo-fetal globin genes, such as the zeta-, epsilon-, and gamma-globin genes. Therefore, the K562 cell line has been proposed as a very useful in vitro model system for determining the therapeutic potential of new differentiating compounds as well as for studying the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation that stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper, we analyzed the effects of a series of cisplatin analogs on both cell growth and differentiation of K562 cells. Among seven cisplatin analogs studied, three were found to be potent inducers of erythroid differentiation. Erythroid differentiation was associated with an increase in the accumulation of (a) hemoglobins Gower 1 and Portland and (b) gamma-globin mRNA.

Distamycin analogues with improved sequence-specific DNA binding activities.

In the present study we have investigated the effect of unprecedented chemical modifications introduced in the distamycin molecule, with the aim of assessing their ability to interfere with sequence-specific DNA-protein interactions in vitro. By using an electrophoretic mobility shift assay, we have been able to identify novel distamycin analogues with improved displacing abilities on the binding of octamer nuclear factors to their target DNA sequence. While variations in the number of pyrrole rings and/or reversion of an internal amide bond result in distamycin-like compounds with identical or very similar properties, the reversion of the formamido into a carboxyamido group or its replacement with the charged formimidoyl moiety significantly improves the ability of the resulting novel distamycin derivatives to compete with OCT-1 (octamer 1 nuclear factor) for its target DNA sequence. Tissue-specific octamer-dependent in vitro transcription is similarly affected by these chemical modifications, suggesting that the ability of distamycins to bind octamer sequences has a direct influence on the functional state of octamer-containing promoters. These data represent an initial, successful attempt to rationalize the design of DNA binding drugs, using distamycins as a model.

N1-substituted tetra-benzamidines - inhibition of DNA-protein interactions and invitro tumor-cell growth.

The pharmacological-mediated inhibition of the interaction between regulatory proteins and target DNA sequences could represent a potential experimental strategy to control growth of neoplastic cells, viral DNA replication and biological life cycle of infectious microorganisms. Aromatic polyamidines are powerful inhibitors of DNA-protein interactions, in vitro proliferation of tumor cell lines and in vivo growth of tumorigenic cells xenografted into nude mice. In order to obtain more detailed information on structure-activity relationships, we have analysed the effects of different aromatic polyamidines on the binding of a recombinant protein, the Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA-1) to the DNA target sequence of EBV, containing the 12 bp palindromic consensus TAGCATATGCTA sequence. The results obtained suggest that aromatic polyamidines differentially inhibit the interactions between DNA-binding proteins and target DNA sequences, leading to differential effects on tumor cell growth.

Detection of hepatitis-C virus by unbalanced polymerase-chain reaction, hybridization to synthetic oligonucleotides and capillary electrophoresis.

Polymerase chain reaction (PCR) has been reported as one of the most efficient techniques to detect hepatitis C virus (HCV) RNA. The determination of the specificity of PCR products is usually based on 'nested' PCR, Southern blotting and hybridization of the amplified DNA to radioactive oligonucleotide probes recognizing sequences comprised between the PCR primers. The recent introduction of capillary electrophoresis (CE) to analyse DNA fragments and PCR products appears to be very interesting because this technology is rapid, reproducible, sensitive and could be suitable to detect DNA/DNA and DNA/RNA hybrids. We demonstrate that specific hybridization of an HCV oligonucleotide probe to single stranded HCV-DNA obtained by unbalanced PCR is detectable by capillary electrophoresis, therefore enabling a one-step, non-radioactive protocol to demonstrate the specificity of amplification of HCV sequences by PCR.

A nonradioactive automated protocol to study protein-DNA interactions by dnase-I footprinting.

Regulation of gene expression is operated, at transcriptional level, by the interactions between proteins (transcription factors) and elements present within eukaryotic and viral promoters (transcription signals) exhibiting specific nucleotide sequences. In this study we performed DNase I cleavage and analysis of the cleavage products using the Pharmacia ALF(TM) DNA sequencing system. As model system we employed the long terminal repeat (LTR) of the human immunodeficiency type I (HIV-1) virus, containing the DNA sequences recognized by a number of transcription factors, including NF-kB, Sp1 and TFIID. The main conclusion of our experiments is that automated analysis of DNAse I footprinting employing the ALF DNA sequencing system is a fast and reliable technique to study protein-DNA interactions.

In vivo and in vitro modulatory effect of human immunodeficiency virus type-1 (HIV-1) Tat protein on protein kinase C activity.

Extracellular HIV-1 Tat protein shows a pleiotropic activity on the survival/proliferation of different cell types, which may be relevant to the pathogenesis of the immune suppression as well as of the frequent neoplastic disorders observed during the course of HIV-1 disease. Therefore, we investigated the effect of recombinant Tat on the protein kinase C (PKC) activity in Jurkat CD4(+) T lymphoma cells by using a serine substituted specific PKC peptide substrate, which allowed the evaluation of the whole catalytic activity of both Ca++-dependent and Ca++-independent PKC isoforms. High concentrations of recombinant Tat (1 mu g/ml) induced an early (5 min) stimulation followed by a secondary (30-60 min) inhibition of PKC in whole Jurkat cell homogenates. Immuno-localization experiments showed that recombinant Tat protein was rapidly taken up by Jurkat cells within the first 5 min from the addition in culture, thus suggesting the possibility that the secondary inhibitory phase of Tat on PKC activity in Jurkat cells could be due to a direct interaction between the two proteins. Consistently, PKC immunoprecipitated from Jurkat cells or purified from rat brain was significantly inhibited by the addition of high (0.1-1 mu g) but not low (1-10 ng) doses of Tat in a cell-free in vitro assay. The inhibition of PKC catalytic activity mediated by 1 mu g of Tat was at least partially due to competition among substrates. The present data may help in understanding the opposite effects on the survival/proliferation of different cell types observed in the presence of picomolar (stimulation) vs nanomolar (inhibition) concentrations of recombinant Tat.

Surface plasmon resonance for real-time monitoring of molecular interactions between a triple helix forming oligonucleotide and the Sp1 binding sites of human Ha-ras promoter: effects of the DNA-binding drug chromomycin.

DNA-binding molecules have been recently proposed as potential inhibitors of molecular interactions between transcription factors and target DNA sequences. Among DNA-binding drugs, chromomycin binds to GC-rich sequences of the Sp1 binding sites of the Ha-ras oncogene. These sites are also molecular targets of a triple-helix forming oligonucleotide [Sp1(Ha-ras)TFO] which is able to inhibit Ha-ras oncogene transcription. We studied molecular interactions between triple-helix forming oligonucleotides and target Sp1 binding sites of the human Ha-ras promoter in the presence of the DNA-binding drug chromomycin. This study was performed by (a) surface plasmon resonance and biosensor technology, (b) gel retardation assay and (c) magnetic capturing of molecular complexes between TFO, chromomycin and target DNA. The main conclusion of our study is that low concentrations of chromomycin allow binding of the triplex-forming oligonucleotide to Sp1 target DNA sequences of the Ha-ras oncogene promoter. Higher concentrations of this DNA-binding drug fully suppress molecular interactions between the Sp1(Ha-ras)TFO and target DNA. Additionally, low concentrations of chromomycin potentiate the effects of the Sp1(Ha-ras)TFO in inhibiting the molecular interactions between purified Sp1 transcription factor and target DNA sequences.