Bis-benzimidazole anticancer agents: targeting human tumour helicases.

Certain DNA minor groove binding agents, distamycin, netropsin, and a series of anticancer bis-benzimidazoles can block DNA helicase activity by binding to duplex DNA at specific base sequences. DNA helicases are crucial to cell DNA replication, transcription and repair because these enzymes separate double-stranded DNA, thereby preparing the strands for enzymatic manipulation. From our studies we have developed a hypothesis that focuses on cellular DNA helicase action as a mechanistic site where these minor groove binders can act. A crucial aspect for modulation of DNA activity by drugs is for specificity and selectivity. A series of DNA-interactive bis-benzimidazole analogues of Hoechst 33258 was also prepared to explore the potential for anticancer activity mediated for certain of the drugs via bioreductive activation by endogenous NADH or NADPH. The biological endpoints examined included intracellular distribution in euoxic and hypoxic conditions observed by fluorescence microscopy; relative efficacy as antimetabolites determined by the MTT [tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay in euoxic and hypoxic conditions; and relative inhibitory activities on human DNA helicase, as determined by degree of dissociation of GC B6486 DNA. The intracellular distribution was unique to each of the test compounds. Compounds V-93 and V-153, the respective semiquinone and quinone derivatives, demonstrated the predicted enhanced cytotoxicity and anti-helicase activities, supporting the concept that preferential binding of DNA at 5'-CG and TG sequences provides a novel approach to anticancer drug development.

Enediyne-lexitropsin DNA-targeted anticancer agents. Physicochemical and cytotoxic properties in human neoplastic cells in vitro, and intracellular distribution.

Two series of hybrids of a dynemicin A model and DNA minor groove binding lexitropsins were synthesized and their cytotoxic activities were investigated in a panel of human normal and malignant cell lines using a colorimetric assay. Adriamycin was used as a control. Several of the agents demonstrated cytotoxic activity, the extent of which varied with tumor type. IC50s of the hybrids ranged from approximately 14-48 microM following 96 h incubation in the presence of test compound. Intracellular distribution studies were facilitated through endogenous fluorescence of the compounds. Evidence of nuclear uptake of the hybrid agents was demonstrated by confocal laser scanning microscopy. The results warrant further development of DNA-targeted enediyne-lexitropsin hybrids as potential anticancer agents.

Late outcome and quality of life after complicated heart operations.

BACKGROUND: Patients with severe postoperative complications consume a great deal of the economic resources for intensive care. Our knowledge of the late outcome and quality of life of these patients is scarce. METHODS: One thousand five hundred twenty-two patients undergoing cardiac operations during 1991 and 1992 were studied, and the 100 patients who needed the most expensive treatment were identified. The patients were retrospectively risk scored (Higgins score), and the clinical outcome was studied. The surviving patients were followed up for 2 years after the operation. Their quality of life and remaining symptoms were assessed. RESULTS: No significant age difference between groups was observed. There were significantly more women, emergency cases, high-risk patients, and postoperative complications in the studied group. Mortality rate during the first postoperative year was significantly higher in the studied group. Later the difference in mortality rate between the groups decreased. At the 2-year follow-up all the 72 surviving patients in the study group had returned home with less physical and psychological symptoms related to their heart disease. CONCLUSIONS: The cost of treating severe complications in the intensive care unit is high. However, the results of the present study indicate that even a very complicated postoperative course is not incompatible with a successful outcome in the long run.

Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA.

Stacking between aromatic amino acids and nucleic acid bases may play an important role in the interaction of enzymes with nucleic acid substrates. In such circumstances, disruption of base aromaticity would be expected to decrease enzyme activity on the modified substrates. We have examined the requirement for DNA base aromaticity of five enzymes that act on single-stranded DNA, T4 polynucleotide kinase, nucleases P1 and S1, and snake venom and calf spleen phosphodiesterases, by comparing their kinetics of reaction with a series of dinucleoside monophosphates containing thymidine or a ring-saturated derivative. The modified substrates contained either cis-5R,6S-di-hydro-5,6-dihydroxythymidine (thymidine glycol) or a mixture of the 5R and 5S isomers of 5,6-dihydrothymidine. It was observed that for all the enzymes, except snake venom phosphodiesterase, the parent molecules were better substrates than the dihydrothymidine derivatives, while the thymidine glycol compounds were significantly poorer substrates. Snake venom phosphodiesterase acted on the unmodified and dihydrothymidine molecules at almost the same rate. These results imply that for all the remaining enzymes base aromaticity is a factor in enzyme-substrate interaction, but that additional factors must contribute to the poorer substrate capacity of the thymidine glycol compounds. The influence of the stereochemistry of the dihydrothymidine derivatives was also investigated. We observed that nuclease P1 and S1 hydrolysed the molecules containing 5R-dihydrothymidine approximately 50-times faster than those containing the S-isomer. The other enzymes displayed no measurable stereospecificity.

Influence of intrathecal morphine and naloxone intervention on postoperative ventilatory regulation in elderly patients.

Thirty elderly patients undergoing major hip surgery under spinal analgesia were randomly allocated in a double-blind manner into three groups. The aim was to evaluate the influence of intrathecal morphine and postoperative naloxone infusion on the regulation of ventilation. The Bupivacaine Group received spinal analgesia with 20 mg bupivacaine intrathecally. The Morphine Group received spinal analgesia with 20 mg bupivacaine + 0.3 mg morphine intrathecally. The Naloxone Group received spinal analgesia with 20 mg bupivacaine + 0.3 mg morphine intrathecally + postoperative naloxone infusion intravenously (1 microgram/kg/h over 12 h, 0.25 micrograms/kg/h over the next 12 h). Evaluation of resting ventilation and the ventilatory responses to hypercarbia and hypoxaemia was made on three occasions: before surgery, and 8, and 24 h after the intrathecal injection. Intrathecal morphine had no significant effect on ventilatory regulation in elderly patients undergoing major hip surgery performed under bupivacaine spinal analgesia. Postoperative administration of opioids or sedatives after intrathecal morphine as well as postoperative blood loss associated with a fall in blood pressure appeared to increase the risk of developing respiratory depression. Naloxone infusion seemed to reduce the risk of developing respiratory depression. Furthermore, one third of the elderly had a poor response to hypoxaemia before surgery.

Nuclear magnetic resonance spectroscopy and sensitizer-adduct measurements of photodynamic therapy-induced ischemia in solid tumors.

Dunning R3327-AT prostate carcinomas growing in Fischer X Copenhagen rats were treated with interstitial photodynamic therapy (PDT--15 mg/kg Photofrin II 4 hours before illumination with 630-nm light via four parallelly implanted optical fibers) at different light intensities. Forty to 60 minutes after treatment, 31P-nuclear magnetic resonance spectra of tumors in anesthetized animals were obtained at 2.35 Tesla using surface coil localization. Areas under resonance peaks were normalized to the area under the peak of a phosphorus standard positioned at a fixed distance on the opposite side of the surface coil. Tumor concentrations of phosphomonoesters and phosphodiesters showed no change after tumor light doses up to 3000 J. Phosphocreatine, alpha-adenosine triphosphate (ATP), beta-ATP, and gamma-ATP signals decreased and inorganic phosphate signals increased with increasing light doses. The intratumor pH did not change significantly at these short times after PDT. In other R3327-AT and R3327-H tumor-bearing animals, [3H]misonidazole was administered 30 minutes prior to PDT treatments of both tumors. Twenty-four hours later, the tumors were resected in toto, and levels of retained [3H]misonidazole were determined in lased tumor specimens by liquid scintillation procedures. The amount of [3H]misonidazole activity in tumor tissue (covalently bound after hypoxic reduction) increased with light doses up to 3000 J. Sensitizer-adduct formation was found to correlate with the ratio of the concentration of inorganic phosphate to that of beta-ATP, both of which are presumed measures of tumor oxygenation status. These measurements have high-lighted the heterogenous nature of the oxygenation status of these experimental tumors. The precision of each assay for estimating tumor oxygenation is discussed.

32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini.

A 32-P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level. The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5' to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing "dinucleoside" monophosphates. Because these fragments contain an unmodified nucleoside at the 5'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and [gamma-32P]ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC. We observed a linear induction of total damage in DNA irradiated with 5-50 Gy. Virtually no damage was detected when the DNA was irradiated in solution containing 1 M DMSO, implicating hydroxyl radicals in the formation of these lesions. Evidence for the presence of thymine glycols and phosphoglycolate groups came from (i) a comparison of the radiation-induced products with those produced by OsO4 and KMnO4 and (ii) incubation of irradiated DNA with Escherichia coli endonuclease III and exonuclease III before analysis by the postlabeling procedure. This was confirmed by comigration of the radiogenic products with chemically synthesized markers. G values of 0.0022 and 0.0105 mumol J-1 were obtained for thymine glycol and phosphoglycolate production, respectively. The identity of the 5'-nucleotide of each isolated compound was obtained by nuclease P1 digestion. This analysis of nearest-neighbor bases to thymine glycols and phosphoglycolates indicated a nonrandom interaction between radiation-induced hydroxyl radicals and DNA.

Metabolic binding of misonidazole to mouse tissues. Comparison between labels on the ring and side chain, and the production of tritiated water.

The 2-nitroimidazole, misonidazole, is of current interest as an imaging agent for hypoxic regions in tumors and in vascular disease such as stroke. The basis of this technique is the reductive activation and binding of nitroheterocycles which is much more efficient in the absence of oxygen. The appropriate molecular location for an active isotope on the nitroheterocyclic probe depends on the nature of the metabolites retained in tissues after the parent drug has been cleared. Previous studies with tumor cells in vitro indicated that a ring label (2-14C) and a side-chain label (3H) were retained equally efficiently in the acid-insoluble fraction, whereas 1.5 to 3 times more side-chain label was retained in the total pool (acid soluble plus acid insoluble) of metabolites in several normal murine tissues. We show here that the excess side-chain label in six normal tissues, plasma and EMT6 tumors was found entirely in the acid-soluble fraction as a volatile component. This volatile component was tentatively identified as tritiated water. It appeared that, in general, molecular products of misonidazole metabolism were retained in mouse tissues, with the exceptions that a small excess of ring label was found in liver and heart and that tritiated water appeared in the acid-soluble fraction of all tissues. Tritiated water would not be important in imaging studies but could be a factor in studies in which scintillation counting of tritiated nitroheterocyles is used.