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G A Hofmann

Publications and source records attributed to G A Hofmann.

At least 19 recordsLinked to original sources
PubMed1997

Regulation of stress-induced cytokine production by pyridinylimidazoles; inhibition of CSBP kinase.

Members of three classes of pyridinylimidazoles bind with varying affinities to CSBP (p38) kinase which is a member of a stress-induced signal transduction pathway. Based upon SAR and protein homology modeling, the pharmacophore and three potential modes of binding to the enzyme are presented. For a subset of pyridinylimidazoles, binding is shown to correlate with inhibition of CSBP kinase activity, whereas no significant inhibition of PKA, PKC alpha and ERK kinase activity is observed.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Instrumentation.

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Animals↗
PubMed1995

Instrumentation.

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Animals↗
PubMed1995

Instrumentation.

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Animals↗
PubMed1994

Electrochemotherapy--a novel method of cancer treatment.

A critical review is presented on a novel method of treating cancer by a combination of an electric field with chemotherapeutic agents. The work described here summarizes the current state of the technique known as electrical impulse chemotherapy (EIC) or electrochemotherapy (ECT). The review discusses in vitro results with specific cell lines, in vivo work on animals and clinical results on patients with squamous cell carcinomas of the head and neck. In all cases, it has been found that uptake of various drugs by the tumor cells can be increased markedly by EIC/ECT. Partial responses and complete cures have been observed without any damaging side-effects, provided the field strength is kept sufficiently low. ECT followed by injection of a low dose of interleukin-2 (IL-2) or IL-2 secreting cells has shown better results than ECT alone. There appears to be a systemic effect and a strong indication that an immune response may be elicited by this method of treatment. Finally, we discuss the challenges involved in hardware requirements for EIC/ECT and its future prospects for both drug delivery and gene therapy.

Animals↗
PubMed1994

Biological activity of some coumarins from Sri Lankan Rutaceae.

Twelve coumarins isolated from plants of the Rutaceae collected in Sri Lanka have been subjected to a mechanism-based anticancer bioassay employing DNA repair-deficient and repair-proficient yeasts. Of these, seselin [10] and xanthyletin [11] were found to be active. Seselin also exhibited moderate cytotoxicity.

Animals↗
PubMed1993

Topoisomerase II in multiple drug resistance.

Topoisomerase II is a target of alkaloid, anthracycline and related antitumor agents. Two types of multiple drug resistance are associated with these enzymes. In classical (typical) multidrug resistance, inhibitors are actively effluxed from cells by P-glycoprotein. In atypical multidrug resistance, topoisomerase II is either reduced in cellular content or mutated to a form that does not interact with inhibitors. Because cytotoxicity of most antineoplastic topoisomerase II inhibitors is directly related to the number of active topoisomerase II molecules, a reduction in this number leads to resistance. In the topoisomerase II mechanism, through which the DNA linking number is altered, DNA double strands are cleaved, and the termini transiently bound covalently (5') or noncovalently (3') to the enzyme while a second double strand is passed through the break in the first. This transition state complex then decays to enzyme and DNA of altered linking number. Most cytotoxic topoisomerase II inhibitors stabilize these reaction intermediates as ternary complexes, which are converted to lethal lesions when cells attempt to utilize the damaged DNA as templates. Toxicity is related to topoisomerase II content as well as to drug concentration. Thus, multidrug resistance results from either 1) decreasing cellular content of the inhibitor by P-glycoprotein (typical) or 2) decreasing cellular content and/or activity of the target, topoisomerase II, as, for example, when its content or activity is modulated downward by decreased expression, deactivation, or by mutations to the TopII gene, producing an enzyme that reacts poorly with inhibitors (atypical). Mixed types, i.e., both typical and atypical, are known. Attempts to abrogate or prevent both typical and atypical multidrug resistance to topoisomerase II inhibitors have been described.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

In vitro and in vivo effects of clinically important camptothecin analogues on multidrug-resistant cells.

The cytotoxic alkaloid camptothecin (CPT) and several of its analogues, including the clinically relevant topotecan (TPT), irinotecan (CPT-11), and 9-aminocamptothecin, were evaluated for differential cytotoxic effect and DNA damage induction in multidrug-sensitive (AuxB1) and multidrug-resistant (MDR) (CHRC5) Chinese hamster ovary cells. CPT, 10-hydroxycamptothecin, and 10,11-methylenedioxycamptothecin produced equivalent amounts of cell growth inhibition and/or DNA single-strand breakage in the two cell lines. TPT, SN-38 (the active metabolite of CPT-11), and 9-aminocamptothecin were 12-, 9-, and 10-fold, respectively, less toxic to the MDR than to the wild-type cells. These findings are consistent with differences in yields of DNA single-strand breaks produced in AuxB1 and CHRC5 cells by 2-hr incubations with the various compounds. In both assays, the resistance ratios of the topoisomerase I inhibitors were approximately one-tenth those of known MDR drugs such as vinblastine or amsacrine. Thus, cultured cells that overexpress P-glycoprotein have the potential to develop some level of cross-resistance to all three topoisomerase I inhibitors currently in the clinic. The chemical basis for cross-resistance of cultured MDR cell lines to certain CPT analogues is not yet understood, but is likely more complex than positive charge alone. TPT had a reasonable therapeutic effect on B6D2F1 female mice implanted with MDR sublines of P388 leukemia, compared with its effect on mice implanted with wild-type P388 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Camptothecin hyper-resistant P388 cells: drug-dependent reduction in topoisomerase I content.

A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.

Animals↗
PubMed1991

Synergistic cell killing by ionizing radiation and topoisomerase I inhibitor topotecan (SK&F 104864).

Topotecan (SK&F 104864), a water-soluble analogue of the topoisomerase I inhibitor camptothecin, is currently in Phase II clinical trial for solid tumors. We have characterized topotecan in terms of its effect upon gamma-radiation-induced cell killing. In colony formation experiments, subtoxic concentrations of topotecan (2 microM) potentiated radiation-induced killing of exponentially growing Chinese hamster ovary or P388 murine leukemia cultured cells. Survival curve shoulders were reduced; the slopes of the exponential portions of the curves were decreased to a small extent. D37 and D10 (radiation dose resulting in 37 and 10% survival of colony-forming ability) values were reduced by approximately 60 and 50%, respectively, in the case of Chinese hamster ovary cells. In P388 cells, topotecan reduced D37 by 35 to 40% and D10 by 20 to 25%. Potentiation of radiation-induced cell killing by topotecan was absolutely dependent upon the presence of the topoisomerase I inhibitor during the first few (less than 30) min after irradiation. Association of topoisomerase I with this effect was confirmed in studies of Chinese hamster ovary cells previously made resistant to camptothecin (and cross-resistant to topotecan), resulting in decreased cellular content of topoisomerase I. These cells were found to be 2- to 3-fold hypersensitive to gamma-radiation-induced killing. P388 camptothecin-resistant cells were further sensitized to the lethal effects of ionizing radiation by nontoxic treatment with the topoisomerase II inhibitor novobiocin, consistent with increased dependence of topoisomerase I-deficient cells upon topoisomerase II.

Animals↗
PubMed1990

Differences between normal and ras-transformed NIH-3T3 cells in expression of the 170kD and 180kD forms of topoisomerase II.

The activity of topoisomerase II and the cellular content of the 170kD and 180kD forms of the enzyme were studied as functions of transformation and growth state by using normal and ras-transformed NIH-3T3 cells. Total topoisomerase II activity, as measured by the unknotting of P4 DNA, was higher in ras-transformed than in normal cells in similar growth states, and was higher in exponentially growing than in plateau cells for both cell lines. Total topoisomerase II levels, as measured by immunoblotting, showed a similar dependence on transformation and growth state. The relative amounts of the 170kD and 180kD forms of the enzyme varied as a function of transformation and growth state. The proportion of 170kD topoisomerase II was higher in ras-transformed than in untransformed cells and depended much less on growth state in the ras-transformed cells. The topoisomerase II activity in extracts of ras-transformed cells was more sensitive to inhibition by teniposide and merbarone, drugs which selectively inhibit the 170kD form of topoisomerase II. The ras-transformed cells were also more sensitive to the cytotoxic effects of these drugs. An increase in the relative cellular content of 170kD topoisomerase II is characteristic of ras-transformed 3T3 cells, and the levels of this form of the enzyme appear to be less dependent on proliferation state than in untransformed cells. The susceptibility of certain tumors to killing by topoisomerase II-directed drugs may be due to a higher proportion of 170kD enzyme as well as a higher level of total topoisomerase II activity.

Animals↗
PubMed1990

Quantitative adaptation of the bacteriophage P4 DNA unknotting assay for use in the biochemical and pharmacological characterization of topoisomerase II.

The ATP-dependent unknotting of phage P4 DNA is a highly specific assay for type II topoisomerases. Despite the unique specificity of the assay, however, its semiquantitative design has limited its use in studying the biochemical properties of these enzymes. To overcome this problem, we have modified the P4 DNA unknotting assay to provide a sensitive and reproducible method for quantifying topoisomerase II activity. Methods are described for accurate measurement of 10-100 ng of unknotted P4 DNA. Under the assay conditions employed, the initial rate of topoisomerase II activity was linear through 30 min. The quantitative assay has been used to determine biochemical and pharmacological parameters of purified topoisomerase II (p170). No topoisomerase II activity was observed in the absence of ATP; enzymatic activity was optimal between 0.5 and 1.0 mM ATP, but substrate inhibition occurred at concentrations above 1 mM. Eadie-Hofstee analysis with varying ATP concentrations gave an apparent Km for ATP of 0.24 mM and a maximal velocity under these conditions of 7.4 ng P4 DNA unknotted/min/ng topoisomerase II. IC50 values were determined for several topoisomerase inhibitors, including amsacrine, teniposide, and novobiocin. Inhibition by teniposide was found to be uncompetitive versus ATP, with a Ki of 3.7 microM. In contrast, inhibition by novobiocin was competitive versus ATP, indicating that teniposide and novobiocin inhibit topoisomerase II by different mechanisms.

Amsacrine↗
PubMed1990

Development of a stable camptothecin-resistant subline of P388 leukemia with reduced topoisomerase I content.

A camptothecin-resistant subline of P388 leukemia (P388/CPT) was developed by repeated transplantation of P388 cells in mice treated with therapeutic doses of camptothecin. In mice bearing the resistant tumor, a maximally tolerated dose of camptothecin produced no net reduction in tumor cell burden, in contrast to a 5-log cell kill in the parental P388 (P388/S). The IC50 of camptothecin, as determined by colony formation assays of cultured cells, was 8 times greater for the cloned P388/CPT cell line than for P388/S. P388/CPT cells were not cross-resistant to other antineoplastic agents, including topoisomerase II inhibitors. The type I topoisomerases purified from P388/CPT and P388/S cells were identical with respect to molecular weight, specific activity, in vitro camptothecin sensitivity, and DNA cleavage specificity. Camptothecin induced fewer protein-associated DNA single-strand breaks in the resistant cells than in the wild-type P388 cells. Topoisomerase I mRNA, immunoreactivity, and extractable enzymatic activity were 2-4 times lower for P388/CPT cells than for P388/S cells. As resistance to camptothecin developed, topoisomerase I extractable activity decreased, concomitant with an increase in topoisomerase II extractable activity. Furthermore, the appearance of camptothecin resistance was associated with specific rearrangements of the topoisomerase I gene. These results suggest that development of resistance to inhibitors of topoisomerase I can occur by down-regulation of the target enzyme, thus reducing the production of lethal enzyme-mediated DNA damage. The enhanced topoisomerase II activity in these cells suggests that resistance to camptothecin may be overcome by co-treatment with topoisomerase II inhibitors.

Animals↗
PubMed1989

Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II.

The p170 and p180 forms of topoisomerase II have been compared. The concentration dependence of ATP for catalytic activity of the two forms of the enzyme was identical, and each was equally sensitive to novobiocin. Orthovanadate was found to be a potent inhibitor of catalytic activity of both p170 and p180, with an IC50 value of about 2 microM for each. Under standard reaction conditions, relaxation of supercoiled pBR322 by p180 was highly processive, while p170 performed the same reaction in a distributive manner. The optimal concentration of KCl for catalytic activity of p180 was 20-30 mM higher than that for p170. Comparison of their thermal stability showed that p180 was inactivated at twice the rate of p170. Teniposide and merbarone selectively inhibited catalytic activity of p170, requiring concentrations 3-fold and 8-fold lower, respectively, than those required for equivalent inhibition of p180. Similar selectivity for p170 was seen for teniposide-stimulated DNA cleavage or its inhibition by merbarone. Analysis of sites of DNA cleavage indicated a subset of sites that were either preferred or unique for each of the enzymes. A synthetic oligonucleotide representative of p170 sites selectively inhibited the p170 enzyme. Immunoblotting of p170 and p180 from U937 cells at different stages of proliferation showed that p170 levels declined as the cells reached the plateau phase of growth, while p180 levels were low during rapid proliferation and increased as the growth rate slowed. The data indicate that the p170 and p180 forms of topoisomerase II can be distinguished biochemically, pharmacologically, and by differential cellular regulation.

Adenosine Triphosphate↗
PubMed1989

In vitro and intracellular inhibition of topoisomerase II by the antitumor agent merbarone.

Merbarone has previously been shown to have antitumor activity of unknown mechanism in P388 and L1210 tumor models (A. D. Brewer et al., Biochem. Pharmacol., 34:2047-2050, 1985) and is currently undergoing Phase I clinical trials. Here we report that merbarone is an inhibitor of topoisomerase II. Merbarone inhibited purified mammalian topoisomerase II with a 50% inhibitory concentration of 20 microM, as assessed by ATP-dependent unknotting of P4 phage DNA or relaxation of supercoiled pBR322 plasmid. In contrast to the type II enzyme, inhibition of catalytic activity of topoisomerase I required about 10-fold higher concentrations of merbarone, with a 50% inhibitory concentration of approximately 200 microM. Unlike epipodophyllotoxin analogues and certain DNA intercalative agents which stabilize the topoisomerase II-DNA "cleavable complex," merbarone did not cause detectable topoisomerase II-induced DNA cleavage. Furthermore, merbarone inhibited the production by amsacrine or teniposide of topoisomerase II-associated DNA strand breaks; under identical conditions novobiocin did not decrease these breaks, setting merbarone apart from a novobiocin-like class of topoisomerase II inhibitor. In L1210 cells, merbarone produced only small numbers of protein-associated DNA strand breaks, and only at very high concentrations. Merbarone reduced in a concentration-dependent manner the number of amsacrine- or teniposide-stimulated protein-associated DNA strand breaks in L1210 cells or their isolated nuclei. The data suggest that merbarone represents a novel type of topoisomerase II inhibitor.

Amsacrine↗