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Recent studies in rat basophilic leukemia cells (RBL-2H3) have shown that two pharmacological agents, ionomycin and thapsigargin, induce leukotriene C4 production and translocation of 5-lipoxygenase from cytosol to membrane, primarily by causing an influx of extracellular calcium. In the present study, we investigate the induction of these events by receptor activation. Cross-linking of high-affinity IgE receptors (Fc epsilon RI) by antigen in RBL-2H3 cells leads to leukotriene C4 production and membrane translocation of 5-lipoxygenase. As in the ionomycin-stimulated cells, leukotriene C4 production in antigen-stimulated cells is calcium-dependent since the amount of leukotriene C4 produced correlates quantitatively with the increase in intracellular free calcium concentration ([Ca2+]i). However, the increase in [Ca2+]i required for equivalent leukotriene C4 production by antigen is not as high as it is using ionomycin. In addition, no threshold [Ca2+]i level is required for leukotriene production by antigen, which is in contrast to the ionomycin stimulation that a [Ca2+]i level of 300-400 nM is required. Furthermore, antigen causes an additive increase in leukotriene C4 production in cells stimulated by the ionomycin. These results suggest that another as yet unidentified intracellular pathway acts in conjunction with Ca2+ for leukotriene synthesis in antigen-stimulated cells. Antigen stimulation causes 20-30% of the total cell 5-lipoxygenase to associate with membranes (compared with 10% in unstimulated cells) as demonstrated by enzyme activity assay and by Western Blot using antibodies to 5-lipoxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)

We have demonstrated that 4'-(9-acridinyl-amino)methanesulfon-m-anisidide (mAMSA), in the presence of Cu(II) ion, causes the breakage of plasmid pDPT275 and pBR322 superhelical form I DNA. In neutral pH, the degradative product was nicked, relaxed form II DNA, resulting from single-stranded DNA breakage. The extent of DNA breakage was both mAMSA concentration and Cu(II) concentration dependent. DNA breakage increased with increasing time of drug treatment. The mAMSA-Cu(II)-induced DNA breakage varied with pH values and also with the nature of the buffer systems. In both Tris-HCl and borate buffers the extent of DNA breakage increased with increasing pH. In Tris-HCl buffer (pH 7-9), only single-strand breaks were obtained, whereas in borate buffer (pH 9-10.5), linear form III DNA was obtained. At equivalent pH, the optimum buffer was borate. No breakage was observed at pH values below 6. The interaction of Cu(II) with mAMSA was examined by using absorption and fluorescence spectroscopies. Interaction of Cu(II) with mAMSA was characterized by a decrease in the absorption at 435 and 420 nm with a simultaneous increase at 330 nm. A highly fluorescent product was obtained upon reacting mAMSA with Cu(II), with an emission spectrum (excitation at 400 nm) showing a doublet at 430 and 450 nm and a shoulder around 480 nm. The spectral changes are also dependent similarly on the pH and the nature of buffer. Other divalent metal ions such as Co(II), Cd(II), Ni(II), and Zn(II) do not induce DNA breakage or spectral changes. The oAMSA isomer, which has no antitumor activity, is less effective in inducing DNA breakage than the mAMSA.

4'-(9-Acridinylamino)methanesulfon-m-anisidide (mAMSA) interacts with Cu(II) ion, as indicated by changes in the mAMSA absorption spectrum induced by Cu(II). The spectral changes are due to the oxidation of mAMSA by Cu(II), resulting in an oxidized mAMSA product and Cu(I). Two lines of evidence for the oxidation of mAMSA are as follows: (1) The spectral changes induced by manganese oxide, an oxidizing agent, were similar to those induced by Cu(II), and (2) the Cu(II)-induced spectral changes were reversed by a reducing agent, NADPH. Thin-layer chromatographic studies showed the oxidized mAMSA product to be N1-methylsulfonyl-N4-(9-acridinyl)-3-methoxy-2,5-cyclohexadiene-1, 4-diimine (mAQDI). The involvement of Cu(I) in the reaction was demonstrated by the use of two Cu(I)-specific chelating agents, neocuproine and bathocuproine. Neocuproine or bathocuproine chelated the Cu(I) ions in the mixture, producing Cu(neocuproine)2+ complex or Cu(bathocuproine)2+ complex. The stoichiometry of mAMSA-Cu(II) interactions was determined by titrating the mAMSA-Cu(II) mixtures with bathocuproine. Job plots of the absorbance at 480 nm showed a clear end point at a Cu(II)/mAMSA ratio of 1.5/1, indicating that 1.5 equiv of Cu(II) reacts with 1 equiv of mAMSA to produce 1.5 equiv of Cu(I). Cu(I) plays an important role in the mAMSA-Cu(II)-induced DNA breakage, since in the presence of neocuproine the DNA breakage is inhibited. Up to 200 microM, Cu(I) by itself is virtually ineffective, in contrast to the mixture of mAMSA and Cu(II).(ABSTRACT TRUNCATED AT 250 WORDS)