Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints.

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.

Sensitizing human colon carcinoma HT-29 cells to cisplatin by cyclopentenylcytosine, in vitro and in vivo.

Cyclopentenylcytosine (CPEC) is cytotoxic to HT-29 cells in vitro and in vivo. Treatment with CPEC resulted in sensitizing HT-29 cells to cisplatin (CDDP), as evidenced by synergistic cytotoxicity. CPEC exhibits potent cytotoxicity to HT-29 cells in vitro, 2 and 24 h exposure providing an LC50 of 2.4 and 0.46 microM, respectively. Exposure of HT-29 cells to CDDP for 2 h resulted in an LC50 of 26 microM. Treatment of HT-29 cells with 1.0 or 1.25 microM CPEC and then incubating with CDDP showed synergistic cytotoxicity. Lesser synergy at very high concentrations of CPEC was demonstrated when HT-29 cells were first exposed to CDDP and then incubated with CPEC. Combination index calculations showed synergistic cytotoxicity in HT-29 cells when CPEC was combined with CDDP. Synergistic antitumor activity was demonstrable in vivo in mice transplanted with HT-29 tumor when treated with a combination of CPEC and CDDP without undue toxicity, since no excessive loss in mouse body weight or overt pathology was observed. CPEC had no influence on the total DNA adduct formation and CDDP did not affect the intracellular levels of CPEC or its metabolites, suggesting that enhanced CDDP cytotoxicity resulted from a step subsequent to excision of platinum-cross-linked DNA. These studies support a new approach for augmenting cytotoxic effect of CPEC with CDDP in treating human colon carcinoma.

RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I.

The acute effect of RNA and DNA synthesis inhibitors on DNA topoisomerase (topo) I localization within cells was examined. Indirect immunofluorescence revealed that topo I was distributed throughout the nuclei but was concentrated in nucleoli of untreated K562 leukemia cells and A549 non-small cell lung cancer cells. Treatment with the DNA polymerase inhibitor aphidicolin did not alter this distribution. In contrast, 30-60 min after addition of the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) at concentrations that inhibited [3H]uridine incorporation into RNA by > or = 50%, topo I was visible throughout the nuclei without nucleolar accentuation. Western blotting and activity assays confirmed that the amount of topo I polypeptide and topo I activity were unaltered by the brief DRB treatment. Within 30 min of DRB removal, topo I relocalized to the nucleoli in the absence or presence of the protein synthesis inhibitor cycloheximide. Collectively, these results suggest a reversible translocation of topo I out of the nucleoli when RNA synthesis is inhibited. Treatment with the topo I poisons topotecan or camptothecin, agents that also inhibit RNA synthesis, likewise caused redistribution of topo I to nonnucleolar regions of the nucleus in a variety of cell types. In DC3F hamster lung fibroblasts, 2.5 microM topotecan or 1.25 microM camptothecin was sufficient to cause this topo I redistribution. In DC3F/C-10 cells that contain a mutant camptothecin-resistant topo I, topo I relocalization required 50-fold higher concentrations of topotecan or camptothecin but not DRB. These observations not only suggest that accumulation of topo I in the nucleolus is related to ongoing RNA synthesis but also raise the possibility of screening for some types of camptothecin resistance at the single-cell level using a rapid immunofluorescence-based assay.

Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3'-processing reaction.

We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions.