The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.

Multidrug resistance (MDR) is characterized by a decrease inof anthracyclines (N-acetyl-daunorubicin) or vinca-alkaloids the efficiency of chemotherapeutic agents correlated with the (vindoline), steroids (progesterone), and other lipophilic catexpression and activity of a membrane protein: the ionic molecules (amiodarone, dipyridamole, S9788 5 ) (for permeability-glycoprotein (Pgp 170). Clinically, detection of reviews see Refs 6 and 7). Some compounds including verapa-

Ellipticine (E) seems to be included in two steps by two y-cyclodextrin molecules. The first inclusion is detected by changes in the absorbance spectrum of ellipticine with added cyclodextrin. It occurs from the pyridine side of the molecule to form E-CD, the first inclusion complex, with a dissociation constant K, = 9.80 x low5 rnol dm-3 and a T-jump-measured second-order rate constant k, = 3.6 x lo6 dm3 mo1-l s-l. E-CD is then included by another y-cyclodextrin molecule to form E-CD,, the second inclusion complex. As evidenced b y changes in the absorbance and fluorescence spectra of E, this second inclusion occurs from the indole side of the molecule with a dissociation constant K2 = 1.85 x 1 0-3 rnol dm-3 and with a second-order rate constant k, > 10' dm-3 mol-' s-'. These results may be of interest in chemotherapy based o n the use of planar DNA-intercalating agents.Ellipticine-a natural plant alkaloid '-and its synthetic derivatives are DNA-intercalating planar pyridocarbazole systems 1,2 which are known to inhibit the replication of DNA and the transcription of RNA both in vitro and in u i ~o . ~ Some of these derivatives display antitumour properties and exhibit high cytotoxic activity against various tumour cells including breast cancers, renal-cell carcinoma, small and non-small lung cancer cells, and osteolitic breast cancer meta~tasis.~