Chromomycin, mithramycin, and olivomycin binding sites on heterogeneous deoxyribonucleic acid. Footprinting with (methidiumpropyl-EDTA)iron(II)

The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA)iron(II) [MPE x Fe(II)] DNA cleavage inhibition pattern technique. Two DNA restriction fragments 117 and 168 base pairs in length containing the lactose operon promoter-operator region were prepared with complementary strands labeled with 32P at the 3' end. MPE x Fe(II) was allowed to partially cleave the restriction fragment preequilibrated with either chromomycin, mithramycin, or olivomycin in the presence of Mg2+. The preferred binding sites for chromomycin, mithramycin, and olivomycin in the presence of Mg2+ appear to be a minimum of 3 base pairs in size containing at least 2 contiguous dG x dC base pairs. Many binding sites are similar for the three antibiotics; chromomycin and olivomycin binding sites are nearly identical. The number of sites protected from MPE x Fe(II) cleavage increases as the concentration of drug is raised. For chromomycin/Mg2+, the preferred sites on the 70 base pairs of DNA examined are (in decreasing affinity) 3'-GGG, CGA greater than CCG, GCC greater than CGA, CCT greater than CTG-5'. The sequence 3'-CGA-5' has different affinities, indicating the importance of either flanking sequences or a nearly bound drug.     

Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation,cytokine production and increased cell proliferation

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.     

The thermodynamics of polyamide-DNA recognition: hairpin polyamide binding in the minor groove of duplex DNA

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.     

Quantitating the concentration of Py-Im polyamide-fluorescein conjugates in live cells

Quantitative fluorescence-based methods have been developed to determine the nuclear concentration of polyamide-fluorescein conjugates in cell culture. Confocal laser scanning microscopy and flow cytometry techniques are utilized to plot calibration curves, from which the nuclear concentration can be interpolated. Upon treatment with polyamide, the concentration in the nucleus of live HeLa cells is calculated to be between 0.1 and 0.5microM, which is significantly lower than the 2microM dosage concentration. In contrast, the observed nuclear concentration in U251 cells is closer to the dosage concentration, indicating a cell line-specific increase in uptake for this class of compounds. Although confocal microscopy and flow cytometry generate disparate values, taken together these experiments suggest that the polyamide concentration inside the cell nucleus is lower than it is outside the cell.     

Inhibition of Moloney murine leukemia virus integration using polyamides targeting the long-terminal repeat sequences

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

Specific activation of open complex formation at an Escherichia coli promoter by oligo(N-methylpyrrolecarboxamide)s: effects of peptide length and identification of DNA target sites

It has previously been shown that open complex formation at a promoter containing a block substitution of nonalternating A-T sequences in the spacer DNA separating the contacted -10 and -35 regions could be accelerated by distamycin. No stimulation was observed at a promoter with a substitution of alternating A-T base pairs in the same region or at the promoter with wild-type spacer. Here we compare the effect of distamycin [tris(N-methylpyrrolecarboxamide), formally a P3] with that of its extended homologues P4, P5, and P6. It is found that the stimulatory potential of these synthetic oligopeptides which bind in the minor groove of DNA ranks in the order P4 greater than (distamycin, P5) greater than P6. The interaction of these peptides with the three promoters was studied by monitoring the positions of the promoter DNA protected from MPE-Fe(II) cleavage in the presence of different concentrations of ligand. The results suggest that a higher affinity of oligopeptide for the spacer DNA than for the -10 and/or -35 region is a necessary, but not sufficient condition for stimulation. Different patterns of protected DNA regions are seen with each of the three promoters; with distamycin, P4, and P5, a unique arrangement of protected regions is observed for the variant containing nonalternating A-T base pairs in its spacer DNA. These data support the hypothesis that differences in the ways the minor-groove binders interact with each of the promoter variants account for the observed differential stimulation. We further postulate that it is a ligand-induced structural change in the nonalternating A-T DNA which is responsible for the activation of open complex formation at the promoter containing this substitution.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Methidiumpropyl-EDTA.Fe(II) and DNase I footprinting report different small molecule binding site sizes on DNA.

DNase I and MPE.Fe (II) footprinting both employ partial cleavage of ligand-protected DNA restriction fragments and Maxam-Gilbert sequencing gel methods of analysis. One method utilizes the enzyme, DNase I, as the DNA cleaving agent while the other employs the synthetic molecule, methidium-propyl-EDTA (MPE). For actinomycin D, chromomycin A3 and distamycin A, DNase I footprinting reports larger binding site sizes than MPE.Fe (II). DNase I footprinting appears more sensitive for weakly bound sites. MPE.Fe (II) footprinting appears more accurate in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA. MPE.Fe (II) and DNase I report the same sequence and binding site size for lac repressor protein on operator DNA.