Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

Leinamycin (1) was isolated from the culture broth of a Streptomyces sp. in 1989, 1-3 and its structure was elucidated by spectroscopic analysis, 1 X-ray crystallography, 4 and chemical synthesis. 5 This antibiotic contains an unusual 1,3-dioxo-1,2dithiolane moiety, which is connected to the 18-membered lactam through a spiro linkage, and appeared to be a new class of natural product. 1 exhibited significant antitumor activity in some murine tumor models. 2 We previously reported that 1 induces single-strand scission of DNA in the presence of thiol cofactors in vitro. We report here the detailed chemistry of thiol-activation and DNA-cleavage induced by 1.Addition of 1.5 equiv of 2-mercaptoethanol (2-ME) to a solution of 1 in MeOH/10 mM phosphate buffer (pH 7) (1/9, v/v) resulted in a rapid conversion to a major product with several minor products. Other thiols including ethanethiol, dithiothreitol, cysteine, and glutathione afforded approximately equal amounts of 2. We isolated 2 from the reaction mixture with reverse-phase HPLC (70%, yield), but the full characterization of 2 failed due to its instability in DMSO. Treatment of 2 with K 2 CO 3 and iodomethane afforded a stable methyl ester 2a (68%, yield). Characterization of 2a by 1D and 2D-NMR experiments established an unexpected structure, in which the 1,3-dioxo-1,2-dithiolane moiety and the 6,7-olefin were missing and a new 3,7-sulfide linkage and 6-hydroxyl group were observed. Treatment of 1 with 2-ME in a MeOH-rich solvent, MeOH/0.5 M phosphate buffer (pH 7) (99/1, v/v), afforded the methanol adduct 2b 6 as the main product.After reaction of 1 with calf thymus DNA in the presence of 2-ME (drug/DNAbp/2ME ) 1/20/1.5), the DNA was purified by ethanol precipitation. The DNA showed a UV spectrum characteristic of the complex with a chromophore. Although it was stable at 4°C, gradual release of the chromophore from the DNA was observed at 37°C. The rate and efficiency of the release increased with further an increase in the temperature. On a preparative scale, we isolated the released chromophore 3 from the leinamycin-treated calf thymus DNA (75% yield from 1). In the 13 C NMR spectrum of 3 all of the resonances were comparable to those of 2, and five additional resonances were found, suggesting the addition of a purine residue to 2. In the 1 H-NMR spectrum, all of the resonances were also comparable to those of 2. One additional nonexchangeable resonance at 7.77 ppm (1H) was found. The only nucleobase that gives one nonexchangeable resonance is guanine. Observation of the NOE between the guanine H-8 and the 6-CH 3 is consistent with alkylation of the C-6 carbon by the N-7 of guanine. These spectroscopic data revealed that 3 is a leinamycin-N7 guanine adduct. This was supported by the molecular formula that was established as the sodium salt C 27 H 31 O 7 N 7 S 2 Na: HRFABMS (M + Na) + m/z 652.1600, calcd 652.1624. 1 did not react with guanosine nucleotide monomer or single stranded DNA, suggesting that the alkylation of DNA could be attributable to the unique...

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