Phenothiazine and carbazole-related compounds inhibit mitotic kinesin Eg5 and trigger apoptosis in transformed culture cells.

The effects of phenothiazine and carbazole derivatives on the cell-cycle progression of human transformed culture cells were analyzed. After 2 days incubation, 5 microM 1-phenethylamino-3-phenothiazin-10-yl-propan-2-ol (1) induced strong mitotic arrest followed by cell death, and 20 microM 1-(3,6-dichloro-9H-carbazol-9-yl)-3-phenethylamino-2-propanol (5) and 1-(3,6-dibromo-9H-carbazol-9-yl)-3-phenethylamino-2-propanol (6) also induced cell death. The TUNEL-positive nuclei characteristic of apoptotic cell death were detected in cells treated with the compounds. We observed beta- and gamma-tubulins in the arrested cells after the addition of compound 1, and found that more than 90% of the mitotic cells exhibited the monoastral spindle instead of the normal bipolar spindle. The inhibitory effects of compounds 1, 5, and 6 on the microtubule-activated ATPase activity of mitotic kinesin Eg5, which is essential for bipolar spindle formation, were obtained. The most effective inhibitor, compound 1, had an IC(50) of 1.52 microM. We also examined their toxicities on various cell lines. Compound 1 had less toxicity with the non-transformed cell line WI-38, whereas it exhibited strong toxicity with the transformed cell lines WI38VA13, HL-60 and HeLa. On the other hand, a high dose of compound 6 caused cell death in both types of culture cells. These results suggest that compound 1, an Eg5 inhibitor, selectively kills transformed culture cells.

In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies.

The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells.