dc-88-a and stallimycin

Duocarmycin A (Duo), which is one of well-known antitumor antibiotics, efficiently alkylates adenine N3 at the 3' end of AT-rich sequences in the DNA. The addition of a minor groove binder, distamycin A (Dist), not only accerelates the reactivity of Duo with oligonucleotide duplex but also switches the DNA-alkylation site to guanine in GC-rich sequences. Here we examined cytotoxic effect of Duo in the coexistence of Dist using human lung carcinoma (HLC-2) cells. The cytotoxicity of Duo to HLC-2 cells was enhanced 10 times by the addition of 0.5microg/ml Dist, which was much lower than the IC(50) value of 16microg/ml. Addition of Duo alone to HLC-2 cells resulted in typically apoptotic changes, including chromatin condensation, sub-G1 accumulation in DNA histogram pattern, and decrease in procaspase-3 and 9 levels. Interestingly, these apoptotic characteristics in Duo-treated cells were suppressed by the addition of 0.5microg/ml Dist, and the G2/M population in the cell cycle progression of HLC-2 cells was largely unchanged in the coexistence of Dist along with the extremely low accumulation of p53 and higher induction of p21. In contrast, the treatment of HLC-2 cells with Dist (16microg/ml) alone was observed to induce the accumulation of p53 and cell cycle arrest at the G1 phase. These results indicate that Dist suppresses apoptosis induced by Duo as well as enhances Duo-induced cytotoxicity in living cells, and may contribute to chemotherapy for tumors resistant to inducing apoptotic cell death.

Topics: Alkylating Agents; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Survival; Distamycins; Duocarmycins; Flow Cytometry; Humans; Indoles; Inhibitory Concentration 50; Lung Neoplasms; Pyrrolidinones; Time Factors; Tumor Suppressor Protein p53

Duo A (Duo) normally alkylates adenine N3 at the 3' end of A + T-rich sequence in DNA. The addition of another minor groove binder, dist A (Dist), dramatically modulate the site of DNA alkylation by Duo with great acceleration of the reaction rate. In order to examine the mode of alkylation, the kinetics of the reaction under various conditions were examined. Based on the simulation of experimental data, a new reaction pathway was proposed.

Topics: Alkylating Agents; Alkylation; Antiviral Agents; Base Sequence; Dimerization; Distamycins; DNA; Duocarmycins; Indoles; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Pyrrolidinones

Duocarmycin A (Duo) normally alkylates adenine N3 at the 3' end of A + T-rich sequences in DNA. The efficient adenine alkylation by Duo is achieved by its monomeric binding to the DNA minor groove. The addition of another minor groove binder, distamycin A (Dist), dramatically modulates the site of DNA alkylation by Duo, and the alkylation switches preferentially to G residues in G + C-rich sequences. HPLC product analysis using oligonucleotides revealed a highly efficient G-N3 alkylation via the cooperative binding of a heterodimer between Duo and Dist to the minor groove. The three-dimensional structure of the ternary alkylated complex of Duo/Dist/d(CAGGTGGT).d(ACCACCTG) has been determined by nuclear Overhauser effect (NOE)-restrained refinement using 750 MHz two-dimensional NOE spectroscopy data. The refined NMR structure fully explains the sequence requirement of such modulated alkylations. This is the first demonstration of Duo DNA alkylation through cooperative binding with another structurally different natural product, and it suggests a promising new way to alter or modify the DNA alkylation selectivity in a predictable manner.

Topics: Alkylation; Distamycins; DNA; Duocarmycins; Indoles; Models, Molecular; Protein Conformation; Pyrrolidinones; Sequence Analysis, DNA

Antitumor antibiotic Duocarmycin A alkylates adenines at the 3 end of sequences of three or more consecutive A or T base pairs through binding to the minor groove of DNA. In the presence of distamycin A, duocarmycin A was found to alkylate guanine residue in G-C rich sequences, which are not alkylated by duocarmycin A alone. Efficient guanine alkylation through cooperatively binding of a heterodimer in the minor groove of DNA will be discussed.

Topics: Alkylation; Antibiotics, Antineoplastic; Antineoplastic Agents, Alkylating; Binding Sites; Dimerization; Distamycins; DNA; Duocarmycins; Guanine; In Vitro Techniques; Indoles; Models, Molecular; Molecular Structure; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Pyrrolidinones

Duocarmycin A, a novel antitumor antibiotic, has a reactive cyclopropane ring, which has been reported to alkylate adenine at the 3' end of sequences of three or more consecutive A or T in DNA [Boger, D. L., et al. (1990) J. Am. Chem. Soc. 112, 8961-8971]. In order to study the DNA recognition, the reaction of DNA with duocarmycin A was performed in the presence of DNA ligands. Distamycin A, berenil, Hoechst 33258, and 4',6-diamidino-2-phenylindole (DAPI), which are minor-groove binders with affinity to A.T-rich sequences, were used. DNA-sequencing experiments showed that treatment of DNA with duocarmycin A plus distamycin A caused alkylation of guanine residues in G.C-rich sequences, which are not alkylated by duocarmycin A alone. Guanine alkylation by duocarmycin A was not observed with berenil, Hoechst 33258, or DAPI. HPLC product analysis showed that duocarmycin A reacted with a double-helical DNA octamer d(CCCCGGGG)2 in the presence of distamycin A to produce duocarmycin A-guanine adduct, while duocarmycin A alone did not react with the octamer. Chromomycin A3, which binds as a Mg(II)-coordinated dimer to G.C-rich sequences in the minor groove, inhibited the guanine alkylation by duocarmycin A in the presence of distamycin A. A footprinting experiment showed that there is a distamycin A-binding site close to the alkylated guanine residue. These results suggest that two different molecules, duocarmycin A and distamycin A, cooperatively recognize DNA sequences including consecutive G.C base pairs resulting in alkylation at the novel guanine sites. The cooperative drug recognition can be designated as "concerted DNA recognition".

Topics: Alkylation; Animals; Antibiotics, Antineoplastic; Base Sequence; Cattle; Chromatography, High Pressure Liquid; Distamycins; DNA; DNA Damage; Duocarmycins; Electrophoresis, Polyacrylamide Gel; Indoles; Ligands; Molecular Sequence Data; Molecular Structure; Pyrrolidinones