Kenneth W. Bair

Polyploidy induction as a consequence of topoisomerase inhibition. A flow cytometric assessment.

Following recovery from a 4-hr exposure to clinically achievable concentrations of the topoisomerase II inhibitors Adriamycin, teniposide, or amsacrine or the putative topoisomerase II inhibitor crisnatol, murine erythroleukemic cells remained viable for up to 48 hr, but did not proliferate. Cell cycle analysis after a 24-hr recovery revealed blocks in G2 (4N DNA) or greater than G2 (up to 8N DNA) polyploid stages. The relative percentages of cells in either stage was a function of drug concentration and cell cycle stage at time of exposure: typically, cells exposed during S phase became blocked in G2, whereas those exposed during G2/M progressed into greater than G2 polyploid stages. G2-blocked cells exhibited a 2- to 3-fold increase in nuclear protein content and cellular/nuclear volume (i.e. unbalanced growth) and approximately 5% more DNA stainability (as a consequence of nuclear conformational changes rather than redundant DNA synthesis). In all cases, at the drug concentrations studied, mitotic figures were absent and G2 and greater than G2 blocks were irreversible, indicating that the mechanism of polyploidy induction differs from that of microtubule inhibitors. These findings suggest that although topoisomerase inhibitors interfere with DNA synthesis in the S phase, their induction of greater than G2 polyploid blocks may involve direct or indirect inhibition of chromosome condensation.

An efficient multiple-exposure analysis of the toxicity of crisnatol, a DNA intercalator in phase II clinical trials.

To investigate the toxicity and mechanism of action of crisnatol (CRS), a new DNA intercalator currently in phase II clinical trials, we analyzed cellular and nuclear flow cytometric (FCM) parameters of murine erythroleukemic cells (MELC) exposed to a range of CRS concentrations over three exposure conditions: short-term (4 h), long-term (24 h), and short-term with recovery (4 h+/19 h−). At 0.5–1.0μM CRS, 4 h exposure results in a reversible G2-phase block, while 24 h exposure results in > G2 polyploidy. At 5–10μM CRS concentrations, cells exhibit persistent retardation of S-phase progression or irreversible G2 and/or > G2 blocks, depending on duration of exposure. Cells terminally blocked in G2 exhibit increased nuclear/cellular volumes and increased nuclear fluorescein isothiocyanate (protein) staining, suggestive of unbalanced growth. At 25–50μM CRS concentrations, MELC exhibit severe membrane perturbation (loss of viability) regardless of exposure. In contrast, following similar exposures to an inactive isomer of CRS, MELC exhibit minimal cell cycle effects, suggesting that cell cycle kinetics may be a useful criterion for assessing potential efficacy. Similar analyses with different classes of chemotherapeutic agents reveal that the range of induced cellular/nuclear perturbations varies with the class of compound used. Taken together, these results suggest that drug toxicity can vary with both concentration and duration of exposure and, as such, a selective multiple-exposure FCM analysis may better represent the spectrum of drug action for drug development and pharmacodynamic studies.

Effects of isomeric 2-(arylmethylamino)-1,3-propanediols (AMAPs) and clinically established agents on macromolecular synthesis in P388 and MCF-7 cells.

SummaryThe in vitro effects of the 2-(arylmethylamino)-1,3-propanediols (AMAPs) on macromolecular synthesis have been examined using the murine leukemia, P388, and the human mammary adenocarcinoma, MCF-7, under conditions of short-term drug exposure. AMAPs that were observed to inhibit macromolecular synthesis produced nearly equipotent inhibition of DNA and RNA synthesis. Equivalent inhibition of protein synthesis generally required significantly greater concentrations of AMAP. There is a general correlation between inhibition of polynucleotide synthesis and in vivo antitumor activity. The effects of four clinical candidate AMAPs (crisnatol, 773U82, 502U83, and 7U85) on macromolecular synthesis were further compared with those of actinomycin D, doxorubicin, mitoxantrone, etoposide, amsacrine, and cisplatin in MCF-7 cells. The pattern of AMAP action was most similar to that observed for doxorubicin and mitoxantrone. Finally, the effects of these four AMAPs on the size, specific activity, and rate of incorporation of [3H]-dTTP into DNA of MCF-7 cells synchronized by pretreatment with hydroxyurea was determined. It was found that DNA synthesis was inhibited by AMAPs independent of inhibition of the uptake, phosphorylation, or retention of the metabolic precursors. These results support the theory that antitumor AMAPs interfere with the normal functioning of enzymes, such as topoisomerase II or DNA and RNA polymerases, which interact with DNA.