Copper-nuclease efficiency correlates with cytotoxicity for the 4-methoxypyrrolic natural products.

The DNA-targeting activities of the 4-methoxypyrrolic natural products, that include prodigiosin (1), tambjamine E (2), and the blue pigment (3), have been compared using fluorescence spectroscopy to study DNA binding and agarose gel electrophoresis to assess their ability to facilitate oxidative copper-promoted DNA cleavage. Fluorescence emission titration of 3 with calf-thymus DNA (CT-DNA) shows that the natural product occupies a site size (n) of ca. two base pairs and possesses an affinity constant (K) of approximately 6x10(5) x M(-1). Similar to prodigiosin (1), the blue pigment 3 was found to facilitate oxidative double-strand DNA (dsDNA) cleavage without the aid of an external reducing agent. Quantitation of ds- (n2) and ss- (n1) breaks provided n1:n2 ratios of approximately 8-12, which were significantly greater than the number expected from the accumulation of ss-breaks (approximately 120). This was contrasted by the nicking activity of tambjamine E (2), which only generates ss-breaks in the presence of copper. The superior copper-nuclease activity of 1 and 3 also correlated with their superior anticancer properties against leukemia (HL-60) cells. These results are discussed with respect to the mode of cytotoxicity by the 4-methoxypyrrolic natural products.

Design, synthesis, and biological activity of a novel non-cisplatin-type platinum-acridine pharmacophore.

Platinum-acridine conjugates were prepared from [PtCl2(ethane-1,2-diamine)] and the novel acridinylthioureas MeHNC(S)NMeAcr (6) and MeHNC(S)NMe(CH2CH2)NHAcr (15) by replacing one chloro leaving group in the cisplatin analogue with thiourea sulfur. In HL-60 leukemia cells, IC(50) values for 7 (Pt-tethered 6) and 16 (Pt-tethered 15) were 75 and 0.13 microM, respectively. In the ovarian cell lines 2008 and C13, 16 was active at micromolar concentrations and showed only partial cross-resistance with clinical cisplatin. Possible structure-activity relationships are discussed.

Role of c-Jun N-terminal kinase/p38 stress signaling in 1-beta-D-arabinofuranosylcytosine-induced apoptosis.

1-beta-D-Arabinofuranosylcytosine (ara-C) induced apoptosis in HL-60 cells, which was preceded by the activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK). 2'-Amino-3'-methoxyflavone (PD098059) and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) were used to inhibit the activity of ERK and p38, respectively. SEK-AL, a dominant-negative mutant of SEK1, was transfected into HL-60 cells (HL-60/SEK-AL) to assess the role of JNK/SAPK activity in apoptosis. PD098059 (25 microM) inhibited ara-C-induced caspase-3-like activity but was ineffective in altering ara-C-mediated apoptotic DNA fragmentation and clonogenicity. On the other hand, SB203580 (20 microM) inhibited ara-C-induced caspase-3-like activity, apoptotic DNA fragmentation, and clonogenicity. The inhibition of JNK1 activation in HL-60/SEK-AL cells did not block ara-C-induced apoptotic DNA fragmentation. These results suggest that ara-C-induced apoptotic DNA fragmentation and loss of clonogenicity occur through a p38-dependent pathway.

Antineutrophil cytoplasmic antibodies: clinical characteristics of 10 patients with the perinuclear pattern of immunofluorescence on ANCA testing.

Antineutrophil cytoplasmic antibodies (ANCA) have recently been described in association with necrotizing glomerulonephritis, systemic vasculitis, and other autoimmune-mediated connective tissue diseases, including systemic lupus erythematosus (SLE) and polychondritis. At least two distinct classes of ANCA have been described, differentiated by characteristic immunofluorescence patterns using neutrophils as substrate for indirect immunofluorescence assay (IFA). A focal, centrally accentuated, finely granular cytoplasmic staining pattern (c-ANCA) is both sensitive and specific for Wegener's granulomatosis (WG) and is thus a useful clinical adjunct in the diagnosis and monitoring of disease activity in WG. The second class of ANCA, defined by a perinuclear immunofluorescent staining pattern (p-ANCA) on standardized IFA, has not been studied as extensively. It appears to occur in a variety of connective tissue diseases, most often necrotizing glomerulonephritis other than WG, systemic vasculitis with renal involvement, and SLE. In screening more than 2000 serum samples received in our rheumatology laboratory for ANA testing, we found p-ANCA in 10 patients. All 10 had evidence of systemic autoimmune disease, though with wide variation in extent and severity of disease. All 10 had other autoantibodies, most frequently ANA (60%). Our studies suggest that p-ANCA define a heterogeneous patient population with a spectrum of autoimmune disease, most frequently necrotizing glomerulonephritis and systemic vasculitis. Future studies will establish the role of p-ANCA in clinical medicine and broaden our understanding of the origin and possible pathogenesis of ANCA and autoantibodies in general.

Detection of anti-neutrophil cytoplasmic autoantibodies using the promyelocytic HL-60 cell-line.

IgG autoantibodies against antigen in the cytoplasm of cells of the neutrophil-monocyte cell lineage have been found in the sera of patients with Wegener's granulomatosis (WG). The indirect immunofluorescence test (IFT) is proving to be a valuable screening test for these antibodies, but obtaining neutrophils for substrate is time-consuming, and interpretation of the fluorescence patterns in ethanol-fixed cells requires considerable experience. We report an improved IFT using HL-60 cells as substrate. The myeloid reactivity of HL-60 cells was characterized and compared to that of neutrophils, with and without prior ethanol fixation. In contrast to neutrophils, myeloperoxidase (MPO) was more completely extracted from HL-60 cells by prior ethanol fixation, eliminating the confusion inherent in trying to distinguish anti-MPO antibodies from Wegener's granulomatosis associated anti-neutrophil cytoplasmic autoantibodies (WG-ANCA) in the IFT. The WG-ANCA reactivity remained intact with ethanol fixation, producing a distinct crescent and half-moon pattern of specific immunofluorescence. This WG-ANCA positive pattern was found in 25 sera from 11 WG patients and was absent in over 1200 control sera from patients referred for autoantibody testing.

Tissue factor and factor VII messenger RNAs in human alveolar macrophages: effects of breathing ozone.

This study was performed to determine if genes for tissue factor and factor VII proteins are expressed and regulated in vivo in lung macrophages during inflammation. Human alveolar macrophages and alveolar fluids were obtained 18 hours after healthy male adults were exposed, for 2 hours during intermittent exercise, to either air or air with 0.4 ppm ozone, added as a model toxic respiratory agent. Messenger RNA (mRNA) for both tissue factor and factor VII were demonstrated in macrophages isolated after subjects were exposed to unpolluted control air. With the same subjects examined after breathing ozone, the following changes were observed: tissue factor mRNA concentration in macrophages increased 2.6 +/- 0.47-fold. Factor VII mRNA concentration was reduced 0.64 +/- 0.24-fold. Total numbers of macrophages recovered did not change significantly. Ratios of nuclear:cytoplasmic areas of cytocentrifuged macrophages were augmented by 24.8% +/- 3%, giving morphometric evidence that immature cell forms increased in the population. In the lavage, tissue factor activity was increased 2.1 +/- 0.3-fold, while amounts of lipid phosphorous, which estimate total membrane lipids, and estimated volumes of alveolar fluid were not significantly changed. Factor VII activity and fibrinopeptide A levels in lavage were increased approximately twofold. These results using rapidly isolated, noncultured cells indicate that tissue factor and factor VII mRNA are synthesized in the alveolar macrophage population in vivo. In addition, evidence was found that as a result of breathing ozone, a shift in alveolar macrophage maturity occurred in association with tissue factor mRNA, tissue factor activity, and factor VII activity increases, and with formation of fibrinopeptide A in alveolar fluids.

Anticardiolipin antibody: a marker of immune reactivity.

An enzyme-linked immunosorbent assay for anticardiolipin (ACL) antibody was performed on 250 consecutive antinuclear antibody (ANA) or anticytoplasmic antibody (ACA) positive sera and 50 consecutive ANA/ACA negative sera submitted to a rheumatology reference laboratory for ANA testing. Of the 250 ANA/ACA positive sera, 33 (13%) were found to be ACL antibody positive. This compared with only 2 (4%) ACL antibody positive samples among the 50 ANA/ACA negative controls. Chart review revealed only one documented case of thrombosis in ACL antibody positive patients. We conclude that among ANA/ACA positive patients, ACL antibody is a frequent finding. ACL antibody in the population studied is not associated with thrombosis. ACL antibody in this group appears to more accurately reflect immune reactivity than a thrombotic state.