Adeno-associated satellite virus interference with the replication of its helper adenovirus.

Adeno-associated satellite virus type 4 interferes with the replication of its helper adenovirus. No interferon-like soluble substance could be detected in satellite-infected cultures and other DNA- and RNA-containing viruses were not inhibited by coinfection with satellite virus under conditions which reduced adenovirus yields by more than 90% in monkey cells. Altering the concentration of adenovirus in the presence of constant amounts of satellite resulted in a constant degree of interference over a wide range of adenovirus inocula and suggested that adenovirus concentration was not a significant factor in the observed interference. The interference with adenovirus replication was abolished by pretreating satellite preparations with specific antiserum, ultraviolet light or heating at 80 degrees C for 30 min. This suggested that infectious satellite virus mediated the interference. Satellite virus concentration was found to be a determinant of interference and studies indicated that the amount of interference with adenovirus was directly proportional to the concentration of satellite virus. 8 hr after adenovirus infection, the replication of adenovirus was no longer sensitive to satellite interference. This was true even though the satellite virus was enhanced as effectively as if the cells were infected simultaneously with both viruses. Interference with adenovirus infectivity was accompanied by reduced yields of complement-fixing antigen and of virus particles which suggested that satellite virus interfered with the formation and not the function of adenovirus products. When cells were infected either with adenovirus alone or with adenovirus plus satellite, the same proportion of cells plated as adenovirus infectious centers. However, the number of plaque-forming units of adenovirus formed per cell in the satellite-infected cultures was reduced by approximately 90%, the same magnitude of reduction noted in whole cultures coinfected with satellite and adenovirus. This suggested that all cells infected with the two viruses were producing a reduced quantity of adenovirus.

Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates.

Immunoconjugates (BR96-DOX) were prepared between chimeric monoclonal antibody BR96 and the anticancer drug doxorubicin. The monoclonal antibody binds an antigen related to Lewis Y that is abundantly expressed at the surface of cells from many human carcinomas; it has a high degree of tumor selectivity and is internalized after binding. BR96-DOX induced complete regressions and cures of xenografted human lung, breast, and colon carcinomas growing subcutaneously in athymic mice and cured 70 percent of mice bearing extensive metastases of a human lung carcinoma. Also, BR96-DOX cured 94 percent of athymic rats with subcutaneous human lung carcinoma, even though the rats, like humans and in contrast to mice, expressed the BR96 target antigen in normal tissues.

Regulation of aspartate-derived amino acid homeostasis in potato plants (Solanum tuberosum L.) by expression of E. coli homoserine kinase.

The availability of the carbon backbone O-phosphohomoserine (OPHS) is critical to methionine (met) and threonine (thr) synthesis. OPHS derives from homoserine and is formed by homoserine kinase (HSK). To clarify the function of HSK in cellular metabolism, the E. coli HSK ortholog thrB was expressed in potato plants targeting the EcHSK protein to chloroplasts and to the cytosol. Both approaches resulted in up to 11 times increased total HSK enzyme activity. Transgenic plants exhibited reduced homoserine levels while met and thr did not accumulate significantly. However, the precursor cysteine and upstream intermediates of met such as cystathionine and homocysteine did indicating an accelerated carbon flow towards the end products. Coincidently, plants with elevated cytosolic levels of EcHSK exhibited a reduction in transcript levels of the endogenous HSK, as well as of threonine synthase (TS), cystathionine beta-lyase (CbL), and met synthase (MS). In all plants, cystathionine gamma-synthase (CgS) expression remained relatively unchanged from wild type levels, while S-adenosylmethionine synthetase (SAMS) expression increased. Feeding studies with externally supplied homoserine fostered the synthesis of met and thr but the regulation of synthesis of both amino acids retained the wild type regulation pattern. The results indicate that excess of plastidial localised HSK activity does not influence the de novo synthesis of met and thr. However, expression of HSK in the cytosol resulted in the down-regulation of gene expression of pathway genes probably mediated via OPHS. We integrated these data in a novel working model describing the regulatory mechanism of met and thr homeostasis.

Cell death-mediated antiviral effect of chitosan in tobacco.

The antiviral activity induced by chitosan (CHT), and the mechanisms underlying it, were studied in a tobacco-tobacco necrosis necrovirus (TNV) pathosystem. Treatments with 0.1% CHT enhanced tobacco inducible defenses against TNV, reducing significantly the virus-induced necrotic lesions (in a range from 32% to 83%). In planta, this resistance was associated with a network of callose deposits, micro-oxidative bursts and micro-hypersensitive responses (micro-HRs), as assessed, respectively, by aniline blue, 3,3'-diaminobenzidine (DAB) and Evans blue staining. In order to verify if CHT-elicited cell death could be regarded as an apoptotic process, tobacco bright yellow 2 (BY2) cell cultures were treated with different CHT concentrations, ranging from 0.01% to 0.1%. After 6 h about half of the cultured cells incubated in 0.05% CHT were Evans blue positive, showing some typical morphological features of apoptosis, such as cytoplasm shrinkage and nuclear chromatin condensation. The latter was checked by 4',6-diamino-2-phenylindole (DAPI) and ethidium bromide nuclear staining and was visible already at 2 h after treatment. Moreover, the cell death kinetic induced by CHT was delayed by Verapamil(R), a calcium channel blocker. Finally, electrophoresis of genomic DNA extracted from cultured cell after 48 h treatment showed internucleosomal fragmentation, visualized as a distinct ladder of DNA bands corresponding to oligonucleosomal units.

Doxorubicin disaccharide analogue: apoptosis-related improvement of efficacy in vivo.

BACKGROUND: Although doxorubicin remains one of the most effective agents for the treatment of solid tumors, there is an intensive effort to synthesize doxorubicin analogues (compounds with similar chemical structures) that may have improved antitumor properties. We have synthesized a novel doxorubicin disaccharide analogue (MEN 10755) and have characterized some of its relevant biochemical, biologic, and pharmacologic properties. METHODS: The antitumor activity of this compound (MEN 10755) was studied in a panel of human tumor xenografts, including xenografts of A2780 ovarian tumor cells, MX-1 breast carcinoma cells, and POVD small-cell lung cancer cells. MEN 10755 was compared with doxorubicin according to the optimal dose and schedule for each drug. The drug's cytotoxic effects, induction of DNA damage, and intracellular accumulation were studied in A2780 cells. DNA cleavage mediated by the enzyme topoisomerase II was investigated in vitro by incubating fragments of simian virus 40 DNA with the purified enzyme at various drug concentrations and analyzing the DNA cleavage-intensity patterns. Drug-induced apoptosis (programmed cell death) in tumors was determined with the use of MX-1 and POVD tumor-bearing athymic Swiss nude mice. RESULTS: MEN 10755 was more effective than doxorubicin as a topoisomerase II poison and stimulated DNA fragmentation at lower intracellular concentrations. In addition, MEN 10755 exhibited striking antitumor activity in the treatment of human tumor xenografts, including those of the doxorubicin-resistant breast carcinoma cell line MX-1. CONCLUSIONS: The high antitumor activity of MEN 10755 in human tumor xenografts, including doxorubicin-resistant xenografts, and its unique pharmacologic and biologic properties make this disaccharide analogue a promising candidate for clinical evaluation.

Relationship between activity and amino sugar stereochemistry of daunorubicin and adriamycin derivatives.

The effects of 4'-epi-daunorubicin, 4'-epi-adriamycin, and the corresponding beta anomers on the in vitro activity of Escherichia coli DNA polymerase I and RNA polymerase were determined and compared with the effects of the parent compounds. The observed effects parallel the cytotoxic activities, assayed by inhibition of mouse embryo fibroblast proliferation, and the inhibitory activities on DNA synthesis in cultured cells. The data indicate that the inverted configuration at position 1 of the amino sugar results in a markedly reduced biological activity. This conclusion is also substantiated by the data obtained with the beta anomer of adriamycin. A preliminary investigation on the binding properties of these derivatives suggests that the inverted configuration at C-1' produces a significant decrease in the binding to DNA. In contrast, epimerization at position 4' did not produce any significant change in activity. The relationship between biological and biochemical activity and DNA binding properties of the tested compounds are discussed with particularly reference to antitumor activity.

Relationship between effects on nucleic acid synthesis in cell cultures and cytotoxicity of 4-demethoxy derivatives of daunorubicin and adriamycin.

Four new derivatives of daunorubicin and two new derivatives of Adriamycin characterized by the absence of the methoxyl groups at the C-4 position have been studied in cell cultures in vitro to establish structure-activity relationships. 4-Demethoxydaunorubicin was 27 to 100 times more active than was daunorubicin when inhibiting the cloning efficiency of exponential-phase HeLa cells and, like daunorubicin, was slightly active on early plateau-phase cells. DNA synthesis in mouse embryo fibroblasts stimulated by fetal calf serum was inhibited equally by the two compounds, although 4-Demethoxydaunorubicin was slightly more active than was daunorubicin when inhibiting RNA synthesis. The beta anomer of 4-demethoxydaunorubicin showed a reduced activity on HeLa cells compared to its alpha anomer, but it was equally active on DNA synthesis. The stereoisomers of 4-demethoxydaunorubicin bearing the inverted configuration in positions 7 and 9 were devoid of significant cytotoxic activity and were only slightly active on DNA synthesis at the doses tested. 4-demethoxyadriamycin and 4-demethoxy-4'-epi-adriamycin were 65 to 500 times more active than was Adriamycin on HeLa cell cloning efficiency and about 10 times more active on DNA synthesis in mouse embryo fibroblasts. Cell uptake in mouse embryo fibroblasts was also investigated for all the new derivatives tested.

Mechanisms of resistance to etoposide and teniposide in acquired resistant human colon and lung carcinoma cell lines.

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.

Chemical and biological characterization of 4'-iodo-4'-deoxydoxorubicin.

4'-Iodo-4'-deoxydoxorubicin is a doxorubicin (DXR) analogue with greater lipophilicity and reduced basicity of the amino group. In vitro 4'-iodo-4'-deoxydoxorubicin is more cytotoxic than DXR against a panel of human and murine cell lines and is characterized by a higher and faster uptake. In vivo, the spectrum of activity of 4'-iodo-4'-deoxydoxorubicin is comparable to that of DXR, but the new compound has higher activity against murine P388 leukemia resistant to DXR and against pulmonary metastases from Lewis lung carcinoma. Moreover, the new analogue exhibits antitumor activity also after p.o. administration and shows no cardiotoxicity in experimental systems.

Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol).

Eleutherobin is a novel natural product isolated from a marine soft coral that is extremely potent for inducing tubulin polymerization in vitro and is cytotoxic for cancer cells with an IC50 similar to that of paclitaxel. This compound is cross-resistant along with other multidrug-resistant agents against P-glycoprotein-expressing cells and is cross-resistant with paclitaxel against a cell line that has altered tubulin. In mechanistic studies, eleutherobin shares with paclitaxel the ability to induce tubulin polymerization in vitro and is most likely cytotoxic by virtue of this mechanism. Human colon carcinoma cells exposed to eleutherobin contain multiple micronuclei and microtubule bundles, and they arrest in mitosis, depending on concentration, cell line, and length of exposure. These morphological abnormalities appearing in cultured cells are indistinguishable from those induced by paclitaxel. Electron microscopy reveals that eleutherobin induces homogeneous populations of long, rigid microtubules similar to those formed by paclitaxel. Thus, eleutherobin is a new chemotype with a mechanism of action similar to that of paclitaxel and, as such, has promising potential as a new anticancer agent.

Effect of linker variation on the stability, potency, and efficacy of carcinoma-reactive BR64-doxorubicin immunoconjugates.

The internalizing anti-Le(y) monoclonal antibody (MAb) BR64 was conjugated to the anticancer drug doxorubicin (DOX) using an acid-labile hydrazone bond to the DOX and either a disulfide or thioether bond to the MAb. The resulting disulfide (BR64-SS-DOX) and thioether (BR64-S-DOX) conjugates were evaluated for stability, potency, and antigen-specific activity in both in vitro and in vivo model systems. The BR64-SS-DOX conjugates demonstrated antigen-specific activity both in vitro and when evaluated against antigen-expressing, DOX-sensitive human carcinoma xenografts. However, the stability and potency of disulfide conjugates were poor, and in vivo activity superior to unconjugated DOX was seen only at doses approaching the maximum tolerated dose. Furthermore, BR64-SS-DOX conjugates were not active against antigen-expressing, DOX-insensitive colon tumor xenografts. In contrast, the BR64-S-DOX conjugates demonstrated good stability both in vitro and in vivo. The increased stability of the BR64-S-DOX conjugates resulted in the delivery of more biologically active DOX to tumors with a concomitant increase in potency and efficacy over that which could be achieved with either unconjugated DOX or BR64-SS-DOX conjugates. Delivery of DOX by BR64-SS-DOX conjugates resulted in complete regressions and cures of both DOX-sensitive lung xenografts and DOX-intensitive colon tumor xenografts. These results demonstrate the importance of linker stability when delivering drugs such as DOX to carcinomas via internalizing antibodies and are likely to have direct relevance to the clinical utility of MAb-directed delivery.

Plexin signaling hampers integrin-based adhesion, leading to Rho-kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility.

Plexins encode receptors for semaphorins, molecular signals guiding cell migration, and axon pathfinding. The mechanisms mediating plexin function are poorly understood. Plexin activation in adhering cells rapidly leads to retraction of cellular processes and cell rounding "cell collapse"). Here we show that, unexpectedly, this response does not require the activity of Rho-dependent kinase (ROCK) nor the contraction of F-actin cables. Interestingly, integrin-based focal adhesive structures are disassembled within minutes upon plexin activation; this is followed by actin depolymerization and, eventually, by cellular collapse. We also show that plexin activation hinders cell attachment to adhesive substrates, blocks the extension of lamellipodia, and thereby inhibits cell migration. We conclude that plexin signaling uncouples cell substrate-adhesion from cytoskeletal dynamics required for cell migration and axon extension.

Paclitaxel (Taxol(R)) inhibits motility of paclitaxel-resistant human ovarian carcinoma cells.

The effect of paclitaxel on the adhesive and motility properties of human ovarian carcinoma cell lines was investigated. Paclitaxel significantly inhibited the motility of OVCAR 5, SK-OV-3, and HOC-1OTC ovarian carcinoma cell lines (IC50 = 2.1 x 10(-8), 2 x 10(-9), and 1.9 x 10(-8) m, respectively) but did not affect the adhesion of these cells to the subendothelial matrix. The association between inhibition of motility and cytotoxic activity was investigated using an A2780 subclone (1A9) and three paclitaxel-resistant variants (designated 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18). Although paclitaxel did not significantly affect the adhesion to subendothelial matrix of the sublines, it completely inhibited their migration. Inhibition of migration was similar in 1A9 cells and the resistant sublines, with an IC50 of 1 x 10(-8) for 1A9 cells and 5.4 x 10(-9), 1.1 x 10(-8), and 5.2 x 10(-9) m for 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively. Paclitaxel inhibited motility induced by soluble attractant (chemotaxis) and immobilized attractant (haptotaxis). Inhibition of cell motility occurred in the absence of an antiproliferative effect, because higher concentrations of paclitaxel were required to inhibit tumor cell proliferation (IC50 = 1.9 x 10(-7) and 4.6 x 10(-6), 1 x 10(-5), and 3.1 x 10(-6) m for 1A9 and 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively). These data show that paclitaxel is a potent inhibitor of ovarian carcinoma cell motility and that this activity is independent of its cytotoxic activity.

BAY 12-9566, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity.

Matrix metalloproteinases (MMPs) have been implicated in tumor cell invasion, metastasis, and angiogenesis. BAY 12-9566, a novel, non-peptidic biphenyl MMP inhibitor, has shown preclinical activity on a broad range of tumor models and is currently in clinical development. The purpose of this study was to investigate the antiangiogenic activity of BAY 12-9566. In vitro, BAY 12-9566 prevented matrix invasion by endothelial cells in a concentration-dependent manner (IC50 = 8.4x10(-7) M), without affecting cell proliferation. In vivo, oral daily administration of BAY 12-9566 (50-200 mg/kg) inhibited angiogenesis induced by basic fibroblast growth factor in the Matrigel plug assay, reducing the hemoglobin content of the pellets. Histological analysis showed a reduction in the amount of functional vessels within the Matrigel. We conclude that the MMP inhibitor BAY 12-9566 inhibits angiogenesis, a property that further supports its clinical development as an antimetastatic agent.

Improved efficacy and enlarged spectrum of activity of a novel anthracycline disaccharide analogue of doxorubicin against human tumor xenografts.

On the basis of a structure-activity study of a new series of anthracycline disaccharides, we recently identified a doxorubicin analogue (MEN 10755) with a promising antitumor activity. In the present study, to better support the pharmacological interest of MEN 10755, we extended the preclinical evaluation of antitumor efficacy to a large panel of 16 human tumor xenografts, which originated from different clinicopathological types. Tumors with typical multidrug-resistant phenotype were excluded because MEN 10755 was found unable to overcome resistance mediated by transport systems. In the doxorubicin-responsive series, MEN 10755 exhibited a higher activity in three of five tumors, as documented by a more marked tumor growth inhibition and an increased value of log-cell kill. In the series of doxorubicin-resistant tumors, MEN 10755 was found effective in 6 of 11 tumors (1 breast, 3 lung, and 2 prostate carcinomas). The overall response rates were 31% and 69% for doxorubicin and MEN 10755, respectively. The improvement in drug efficacy was also supported by a substantial increase in the long-term survivor rate of animals implanted with responsive tumors. Most of the tumors refractory to doxorubicin and responsive to MEN 10755 were characterized by overexpression of the antiapoptotic protein Bcl-2. In one of these tumors (MX-1 breast carcinoma), we examined the ability of MEN 10755 to induce phosphorylation of Bcl-2 after a single treatment with therapeutic doses. The results indicated that, unlike doxorubicin, MEN 10755 induced protein phosphorylation. A similar modification was produced by Taxol, which is known to be very effective against the tumor. The correlation between drug efficacy and Bcl-2 phosphorylation may underly a peculiar feature related to improvement of efficacy of the disaccharide analogue. In conclusion, the present study supports some favorable features of the novel doxorubicin analogue in terms of both efficacy and tolerability with comparison to doxorubicin, although the improvement is somewhat tumor- and schedule-dependent.

Naamidine A is an antagonist of the epidermal growth factor receptor and an in vivo active antitumor agent.

The known 2-aminoimidazole alkaloid naamidine A (1) was isolated from a Fijian Leucetta sp. sponge as an inhibitor of the epidermal growth factor (EGF) receptor. The compound exhibited potent ability to inhibit the EGF signaling pathway and is more specific for the EGF-mediated mitogenic response than for the insulin-mediated mitogenic response. Evaluation in an A431 xenograft tumor model in athymic mice indicated that naamidine A exhibited at least 85% growth inhibition at the maximal tolerated dose of 25 mg/kg. Preliminary mechanism of action studies indicate that the alkaloid fails to inhibit the binding of EGF to the receptor and has no effect on the catalytic activity of purified c-src tyrosine kinase.

Synthesis and antitumor properties of new glycosides of daunomycinone and adriamycinone.

The synthesis of 4'-epi-daunorubicin and of 4'-epi-adriamycin was performed by condensation of 2,3,6-trideoxy-3-trifluoroacetamido-4-O-trifluoroacetyl-alpha-L-arabino-hexopyranosyl chloride with daunomycinone or the protected adriamycinone derivative 17, respectively. Both the alpha and beta anomers were obtained and characterized. All new compounds are biologically active in cultured cells and the alpha anomers display noticeable activity in experimental tumors in mice. Interestingly, 4'-epi-adriamycin (4) appears nontoxic to cultured heart cells up to a concentration of 5 mug/ml.

Preparation and functional characterization of thylakoids from Arabidopsis thaliana.

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O(2) and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate.

In vivo evaluation of epidermal growth factor and transforming growth factor beta 1 in mouse tumor models.

The influence of modulating circulating levels of epidermal growth factor (EGF) and transforming growth factor beta 1 (TGF-beta 1) on tumor growth was examined in a variety of mouse models. Removal of the EGF-rich submandibular gland from host mice failed to alter the growth of a variety of human tumor xenografts or a C3H mouse tumor. Infusion of EGF from Alzet minipumps raised circulating EGF levels. However, only the A549 human tumor xenograft showed any significant increase in growth in the presence of EGF infusion and this response was marginal. The growth of Wehi 3BD+ and A549 tumor lines in culture was inhibited by TGF-beta 1. The growth of these lines in vivo, however, was not significantly altered by the administration of TGF-beta 1 via a variety of routes.

Structure-activity relationships of VP-16 analogues.

A total of 27 selected analogues of VP-16 and VM-26 were compared with VP-16 and VM-26 for their relative abilities to stabilize the enzyme-substrate intermediate normally formed between eukaryote topoisomerase II and DNA. This activity was compared with cytotoxicity results obtained using the human colon HCT116 cell line and antitumor results obtained after intraperitoneal injection of mice with murine leukemia P388. The most potent analogues were those containing OH groups (demethyl) in either the 3' and 4' or the 3', 4', and 5' positions, the latter being twice as potent as VP-16. VM-26 was only 40% more potent than VP-16 in this assay. It was generally found that the 4'-esters had little activity in vitro, yet were cytotoxic and had antitumor activities. All other analogues with little in vitro activity were not very cytotoxic and had little if any antitumor activity. A very good correlation exists between stabilization of topoisomerase II-DNA intermediates, cytotoxicity, and antitumor activity.