First Report of Fruit Rot of Loquat Caused by an Alternaria sp. in Taiwan.

During March 2007, a fruit rot disease was observed in several loquat (Eriobotrya japonica (Thunberg) Lindley) fields located in Taichung, Nantou, and Miaoli counties. Loquat is a valuable fruit crop grown predominantly in central Taiwan, and hence, even a minor yield loss by this new disease is economically significant. Symptoms on fruits initially appeared as small lesions (<1 mm) that later developed into light-to-dark brown, circular, larger (7 mm), sunken lesions, indicating invasion of a pathogen into the fruit. Pieces of rotted fruit tissue (1 × 1 × 1 mm) were immersed for 1 min in 3% commercial bleach, followed by 70% ethanol, cultured on potato dextrose agar (PDA), and incubated under constant fluorescent light (185 ± 35 µE·m-2·s-1) at 24°C for 2 days. Three single conidial isolates (AS1 to AS3) were selected and used in morphological and pathogenicity studies. All three isolates were identified as an Alternaria sp. (1-3) and formed abundant, dark brown mycelium when cultured on PDA with light at 24°C. Conidiophores were 60 to 89 × 3 to 5 µm, densely fasciculate, cylindrical, simple or branched, and had distinct conidial scars. Conidia were 12 to 74 × 6 to 14 µm, golden brown, straight or curved, obclavate with beaks measuring half the length of the conidium, and observed in chains of 10 or more spores with four to seven transverse septa and several longitudinal septa. Pathogenicity tests were conducted twice by inoculating eight surface-sterilized wounded or unwounded fruits with each of the three isolates in each experiment. Two cuts (1 × 1 × 1 mm) were made on each fruit 3 cm apart with a sterile scalpel, and a 300-µl spore suspension (2 × 105 conidia per ml) was placed on each wound. Similarly, a 300-µl spore suspension was placed on unwounded fruits and air dried for 5 min. Control fruits were similarly treated with sterile water. Inoculated fruits were enclosed in a plastic bag and kept at 24 ± 1°C. Symptoms of soft rot were observed on 60% (unwounded) and 100% (wounded) of inoculated fruits 5 days after inoculation, while control fruits did not develop disease symptoms. Reisolation from the symptomatic fruits consistently yielded an Alternaria sp. This fungus previously has been reported as the causal agent of fruit rot or black spot of papaya, mango, kiwifruit, pear, and carambola from Australia, India, Malaysia, South Africa, and the United States (1-3). To our knowledge, this is the first report of fruit rot of loquat caused by an Alternaria sp. in Taiwan. To manage this disease, growers may resort to fungicidal sprays followed by bagging of fruits to reduce pre- and postharvest losses. References: (1) A. L. Jones and H. S. Aldwinckle. Compendium of Apple and Pear Diseases. The American Phytopathological Society. St. Paul, MN, 1990. (2) R. C. Ploetz. Diseases of Tropical Fruit Crops. CABI Publishing. Wallingford, Oxfordshire, UK, 2003. (3) R. C. Ploetz et al. Compendium of Tropical Fruit Diseases. The American Phytopathological Society. St. Paul, MN, 1994.

Characteristics and photocatalytic activity of TiO2 thin film sensitized with a porphyrin dye.

In this study, a novel porphyrin dye, 5, 10, 15, 20-tetraphenyl-21H, 23H-porphine nickel (TPPN) doped TiO2 (TiO2/TPPN) thin film with visible light respondency was prepared using a sol-gel method and characterized with XRD, SEM, UV-Vis instruments. The observation showed that the absorption edge of TPPN dye-doped thin film shifted into the visible light region. The photocatalytic indigo carmine degradation results showed that under visible light irradiation (lambda > 400 nm) for 6 hrs, the photocatalytic activity of TiO2 thin film sensitized with 200 microM of TPPN dye showed the best performance, with an indigo degradation ratio up to 96%. Moreover, the TiO2/TPPN thin film showed a relevant photocatalytic bactericidal effect on Erwinia carotovora subsp. carotovora 7 induced vegetable soft rot disease in the visible spectral region. Evidence for the photocatalytic disinfection technique against a plant pathogen under visible light irradiation will have potential for direct application in future control of plant diseases in irrigation water systems.

First Report of Fruit Rot of Strawberry Caused by an Alternaria sp. in Taiwan.

In March 2005, a fruit rot disease was found in several commercial strawberry (Fragaria × ananassa Duchesne) fields at Fongyuan, 24.25°N, 120.72°E, in Taichung County in central Taiwan. The disease was rare and was negligible in most cultivated areas. However, disease incidence has increased by 4 to 5% over the last 2 years and causes significant postharvest losses. In storage, symptoms on berries include light brown-to-black, sunken, irregularly shaped lesions. The lesions gradually enlarge and become firm with a dark green-to-black, velvety surface composed of mycelia, conidiophores, and conidia. Twelve single conidial isolates (AF-1 to AF-12) of a fungus were isolated by placing portions of symptomatic fruit from four locations onto acidified potato dextrose agar (PDA) and incubating at 24 ± 1°C. One isolate from each of the four locations, AF-2, 6, 9, and 12, was selected for identification and pathogenicity studies. The fungus was identified as an Alternaria sp. according to the morphological descriptions of A. tenuissima (2,3). Conidiophores were simple or branched, straight or flexuous, septate, pale to light brown, 3.0 to 5.0 µm in diameter, and bore two to six conidia in a chain. Conidia were dark brown, obclavate or oval, and multicellular with seven transverse (in most cases) and numerous longitudinal septa. Conidia were 15.5 to 56.5 µm (average 35.0 µm) long × 6.0 to 15.0 µm (average 11.0 µm) wide at the broadest point. The pathogen was consistently isolated from berries in the field or in storage. Pathogenicity tests were conducted by inoculating 12 surface-sterilized berries with each of the four isolates. Approximately 300 µl of a spore suspension (2 × 105 conidia per ml) was placed at two points on the uninjured surface of each fruit and allowed to dry for 5 min. Control fruits were treated with sterile water. The berries were then enclosed in a plastic bag and incubated at 24 ± 1°C for 2 days. Disease symptoms similar to those described above were observed on 95% of inoculated berries 3 days after inoculation, while no symptoms developed in control berries. Reisolation from the inoculated berries consistently yielded the Alternaria sp. described above. Pathogenicity tests were performed three times. Previously, strawberry fruit rot caused by A. tenuissima was reported from Florida (2) and Malaysia (1), however, to our knowledge, this is the first report of fruit rot of strawberry caused by a species of Alternaria in Taiwan. References: (1) W. D. Cho et al. List of Plant Diseases in Korea. Korean Society of Plant Pathology, 2004. (2) C. M. Howard and E. E. Albregts. Phytopathology 63:938, 1973. (3) R. D. Milholland. Phytopathology 63:1395, 1973.

First Report of Gummosis Disease of Plum (Prunus salicina) Caused by a Botryosphaeria sp. in Taiwan.

Plum (Prunus salicina Lindell) is grown on more than 3,870 ha in Taiwan. In 2004, a gummosis disease was observed on plum in the Ming Jian Region of Nantou County (120.675°E, 23.919°N), with 15% of the trees affected. Infections started on the current year's growth, primarily through lenticels, and formed small, sunken, discolored lesions. At later stages, white gum exuded from the lesions. Circular to oval, brown, necrotic areas were seen on the inner bark. Severely infected twigs showed defoliation and dieback. During the winter months, numerous black pycnidia or perithecia formed on infected twigs. Single conidial isolates of the pathogen were obtained from diseased twigs on acidified potato dextrose agar (PDA) and incubated at 25 ± 1°C for 3 days. On the basis of morphological traits, the fungus was identified as a Botryosphaeria sp. according to the CMI descriptions of Botryosphaeria ribis (3). Conidia (14.2 to 26.8 × 4.3 to 7.2 µm) were single celled, hyaline, and spindle shaped. Asci (105 to 135 × 12.5 to 15.5 µm) were hyaline, clavate, and bitunicate. Ascospores (18 to 22 × 7.0 to 8.2 µm) were hyaline and spindle shaped or fusoid. For pathogenicity tests, inoculum was prepared by culturing the fungus on PDA under continuous fluorescent light (128 ± 25 µE·m-2·s-1) at 25°C for 3 days. Two twigs on each of six trees were inoculated. Sharp incisions (3 × 3 × 3 mm) were made on healthy twigs (12 to 15 months old) with a sterilized scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of a conidial suspension (105 conidia/ml) of the fungus. Inoculated areas were covered with moist, sterile cotton and the entire twigs were enclosed in plastic bags. Twigs inoculated with 5-mm PDA discs or sterile water alone served as controls. The symptoms described above were observed on all inoculated twigs 14 days after inoculation, whereas control twigs did not develop any disease symptoms. Reisolation from the inoculated twigs consistently yielded the Botryosphaeria sp., thus fulfilling Koch's postulates. Botryosphaeria spp. have been reported to cause stem blight of many plants in temperate and tropical regions of the world (4). In Taiwan, B. dothidea has been reported as the causal agent of gummosis disease of peach (1) and fruit ring rot of pear (2); however, to our knowledge, this is the first report of a Botryosphaeria sp. causing gummosis of plum. References: (1). Y. Ko et al. Plant Pathol. Bull. 1:70, 1992. (2) Y. Ko et al. Plant Prot. Bull. (Taiwan) 35:211, 1993. (3) E. Punithalingam and P. Holliday. No. 395 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1973. (4) W. A. Sinclair et al. Diseases of Trees and Shrubs. Cornell University Press, Ithaca, NY, 1987.

First Report of Gray Leaf Spot of Mango (Mangifera indica) Caused by Pestalotiopsis mangiferae in Taiwan.

Mango (Mangifera indica L.; family Anacardiaceae) is one of the world's most important fruit crops and is widely grown in tropical and subtropical regions. Since 2001, a leaf spot disease was found in mango orchards of Taiwan. Now, the disease was observed throughout (approximately 21,000 ha) Taiwan in moderate to severe form, thus affecting the general health of mango trees and orchards. Initial symptoms were small, yellow-to-brown spots on leaves. Later, the irregularly shaped spots, ranging from a few millimeters to a few centimeters in diameter, turned white to gray and coalesced to form larger gray patches. Lesions had slightly raised dark margins. On mature lesions, numerous black acervuli, measuring 290 to 328 µm in diameter, developed on the gray necrotic areas. Single conidial isolates of the fungus were identified morphologically as Pestalotiopsis mangiferae (Henn.) Steyaert (2,3) and were consistently isolated from the diseased mango leaves on acidified (0.06% lactic acid) potato dextrose agar (PDA) medium incubated at 25 ± 1°C. Initially, the fungus grew (3 mm per day) on PDA as a white, chalky colony that subsequently turned gray after 2 weeks. Acervuli developed in culture after continuous exposure to light for 9 to 12 days at 20 to 30°C. Abundant conidia oozed from the acervulus as a creamy mass. The conidia (17.6 to 25.4 µm long and 4.8 to 7.1 µm wide) were fusiform and usually straight to slightly curved with four septa. Three median cells were olivaceous and larger than the hyaline apical and basal cells. The apical cells bore three (rarely four) cylindrical appendages. Pathogenicity tests were conducted with either 3-day-old mycelial discs or conidial suspension (105 conidia per ml) obtained from 8- to 10-day-old cultures. Four leaves on each of 10 trees were inoculated. Before inoculation, the leaves were washed with a mild detergent, rinsed with tap water, and then surface sterilized with 70% ethanol. Leaves were wounded with a needle and exposed to either a 5-mm mycelial disc or 0.2 ml of the spore suspension. The inoculated areas were wrapped with cotton pads saturated with sterile water and the leaves were covered with polyethylene bags for 3 days to maintain high relative humidity. Wounded leaves inoculated with PDA discs alone served as controls. The symptoms described above were observed on all inoculated leaves, whereas uninoculated leaves remained completely free from symptoms. Reisolation from the inoculated leaves consistently yielded P. mangiferae, thus fulfilling Koch's postulates. Gray leaf spot is a common disease of mangos in the tropics and is widely distributed in Africa and Asia (1-3); however, to our knowledge, this is the first report of gray leaf spot disease affecting mango in Taiwan. References: (1) T. K. Lim and K. C. Khoo. Diseases and Disorders of Mango in Malaysia. Tropical Press. Malaysia, 1985. (2) J. E. M. Mordue. No. 676 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Surrey, England, 1980. (3) R. C. Ploetz et al. Compendium of Tropical Fruit Diseases. The American Phytopathological Society. St. Paul, MN, 1994.

First Report of Gray Mold Disease of Sponge Gourd (Luffa cylindrica) Caused by Botrytis cinerea in Taiwan.

A disease of sponge gourd (Luffa cylindrica (L.) Roem., family Cucurbitaceae) has become a serious threat to sponge gourd production since 2003 in central Taiwan. Initially, symptoms appear as small, brown spots on the flower petals that spread to the entire flower and cause blossom blight within 2 to 3 days. Subsequently, the pathogen develops abundant mycelium and moves from the petals onto the fruits causing blossom end rot and fruit stem rot. Severely infected fruits become completely rotten and desiccate. Tissues were excised from diseased sponge gourd fruits (sampled from Fongyuan, located at 24.25°N, 120.72°E in Taichung County), immersed in a solution containing 3% sodium hypochlorite and 70% ethanol for 1 min, washed three times with sterile water, and then cultured on potato dextrose agar (PDA) medium. A fungus, identified as Botrytis cinerea, produced abundant mycelium on PDA medium when incubated under constant fluorescent light 185 ± 35 µE·m-2·s-1 at 24°C. The conidia were smooth, hyaline, and globoid or slightly ellipsoid. The conidia measured 9.5 to 19.3 µm (average 13.8 µm) long and 6.0 to 17.8 µm (average 10.1 µm) wide, dimensions that are similar to the descriptions of B. cinerea (11 × 11 to 15 µm) that causes gray mold of strawberry (2). The identity of B. cinerea was also confirmed by the production of numerous black sclerotia on PDA plates incubated either in the dark or under light at 20 to 24°C for 9 to 10 days. Koch's postulates were fulfilled by using 3-day-old mycelial agar discs of the fungus or a spore suspension containing 105 conidia per milliliter of distilled water as inoculum. Shallow (2 × 2 × 2 mm) incisions were made on fresh sponge gourd fruits with a sterile scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of the spore suspension. Inoculated areas were covered with moist sterile cotton, and the fruits were enclosed in a plastic bag and incubated at 20 to 24°C for 3 days. Wounded fruits inoculated with PDA discs or sterile distilled water alone served as controls. Pathogenicity tests were performed three times using five fruits in each trial. Symptoms and signs of the disease similar to those described above were observed in all (100%) the inoculated fruits, while no symptoms developed in the control fruits. Reisolation from the inoculated fruits consistently yielded B. cinerea. Reciprocal inoculations on sponge gourd, guava, and strawberry with mycelial discs or spore suspensions of a B. cinerea isolate obtained from sponge gourd, guava, and strawberry showed cross pathogenicity among isolates and hosts. Important groups of plants that are attacked by B. cinerea are vegetables, small berry fruits, ornamentals, and bulbs (1). Though 80 species of host plants, mostly shrubs and nursery plants, were reported to be the host of B. cinerea in Taiwan (3), to our knowledge, this is the first report of gray mold disease affecting sponge gourd in Taiwan. References: (1) G. N. Agrios. Plant Pathology. Academic Press. San Diego, 2005. (2) J. L. Mass, ed. Page 56 in: Compendium of Strawberry Diseases. The American Phytopathological Society. St. Paul, MN, 1984. (3). Y. Ko et al. Plant Prot. Bull. (Taiwan) 37:439, 1995.

Chronic dosing of oltipraz in people at increased risk for colorectal cancer.

The dithiolethione oltipraz is being developed as a chemopreventive agent for many malignancies, including colorectal cancer, on the basis of its in vivo protective activity against chemically induced tumors in a variety of animal models. This protection has been associated with an enhanced capacity to detoxify reactive carcinogens and, more recently, with increased DNA repair. In a previous single-dose study, elevated detoxification gene expression was observed in the days after oltipraz dosing. Now, in this clinical study, we evaluated the effects of oltipraz when given over a 3-month period. Fourteen individuals with increased risk for colorectal cancer were randomly assigned to one of two oral doses (125 or 250 mg/m2) of oltipraz twice weekly for 12 weeks. Two of seven subjects at the 250 mg/m2 dosage required dose reductions, owing to significant fatigue. The 125 mg/m2 dose level was well tolerated by all patients. Blood or colon tissue (or both) for evaluation of glutathione, glutathione S-transferase, DT-diaphorase activity, and DT-diaphorase mRNA expression were obtained prior to treatment and at weeks 6, 12, and 16. No significant modulation of phase II detoxification enzymes was seen at either dose studied during this period. Phase II trials evaluating a tolerable regimen of oltipraz (as demonstrated in this study) and other possible mechanisms that may be responsible for the protective activity of oltipraz should be pursued.

Phase I clinical/pharmacokinetic and pharmacodynamic trial of the c-raf-1 antisense oligonucleotide ISIS 5132 (CGP 69846A).

PURPOSE: Raf-1 is a protein kinase that plays a broad role in oncogenic signaling and acts as a downstream effector of Ras in the mitogen-activated protein kinase pathway. The present study was designed to determine the maximum-tolerated dose (MTD), toxicity profile, pharmacokinetics, and antitumor activity of the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A; ISIS Pharmaceuticals Inc, Carlsbad, CA). The effect of ISIS 5132 on c-raf-1 gene expression in peripheral-blood mononuclear cells (PBMCs) of treated patients was studied using a reverse transcriptase polymerase chain reaction assay. PATIENTS AND METHODS: Patients with refractory malignancies received ISIS 5132 as a 2-hour intravenous infusion three times weekly for 3 consecutive weeks. Pharmacokinetic sampling was performed during the first cycle in all patients; PBMCs for c-raf-1 mRNA analysis were collected at baseline and on days 3, 5, 8, and 15 of cycle 1 and on day 1 of each cycle thereafter. RESULTS: Thirty-one patients received ISIS 5132 at one of nine dose levels ranging from 0.5 mg/kg to 6.0 mg/kg. Clinical toxicities included fever and fatigue, but these were not dose limiting. A clinically defined MTD was not reached. The harmonic mean half-life of ISIS 5132 was 59.8 minutes (range, 35.5 to 107.3 minutes). The area under the concentration-time curve increased linearly with dose, and mean plasma clearance was 1.86 mL/kg/min (range, 1.21 to 2.41 mL/kg/min). Two patients experienced prolonged stable disease lasting more than 7 months, which was associated with persistent reduction in c-raf-1 expression in PBMCs. Significant decreases in c-raf-1 expression were identified at time points after the baseline value (P <.05) at doses >/= 2.5 mg/kg. CONCLUSION: ISIS 5132 is well tolerated at doses up to 6.0 mg/kg when administered as a thrice weekly 2-hour infusion for 3 consecutive weeks. The pharmacokinetic behavior of the drug is reproducible, and suppression of target gene expression is observed in circulating PBMCs.

c-raf-1 depletion and tumor responses in patients treated with the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A).

Abnormally regulated signaling through proliferative signal transduction pathways characterizes many of the common solid tumors. The best described of these involves potentially oncogenic proteins of the Ras family, which activate Raf proteins in the early steps of the mitogen-activated protein kinase cascade. ISIS 5132, a phosphorothioate antisense oligodexoynucleotide directed to the 3' untranslated region of the c-raf-1 mRNA, inhibits the growth of human tumor cell lines in vitro and in vivo in association with specific down-regulation of target message expression. Using a semiquantitative reverse transcription-PCR assay, we analyzed changes in c-raf-1 mRNA expression in peripheral blood mononuclear cells collected from patients with advanced cancers treated with ISIS 5132 as part of a clinical trial. Specimens were collected for analysis pretreatment and on days 3, 5, 8, and 15 of the first cycle and on day 1 of each subsequent cycle. We observed significant reductions of c-raf-1 expression from baseline by day 3 in 13 of 14 patients (P = 0.002). The time course and depletion of c-raf-1 message in peripheral blood mononuclear cells paralleled the clinical benefit in two patients. These findings demonstrate that ISIS 5132 specifically reduces target gene expression in treated patients and that peripheral blood mononuclear cells are suitable tissues for biomarker studies in future trials.

Phase II trial of topotecan as a 21-day continuous infusion in patients with advanced or metastatic adenocarcinoma of the pancreas.

The aim of this study was to determine the efficacy and toxicity of topotecan administered as a 21-day continuous intravenous infusion in patients with advanced or metastatic adenocarcinoma of the pancreas. 26 previously untreated patients with advanced or metastatic pancreatic adenocarcinoma received topotecan at a dose of 0.5 mg/m2/day or 0.6 mg/m2/day as a continuous intravenous infusion for 21 days. Courses were repeated every 28 days. 26 patients were assessable for response and toxicity on an intent-to-treat basis. The initial 8 patients at a starting dose of 0.6 mg/m2/day experienced unacceptable myelosuppression and dose delays. The subsequent 18 patients, therefore began therapy at a dose of 0.5 mg/m2/day. The major toxicity of topotecan at this dose and schedule was myelosuppression, which was reversible and non-cumulative. There were no complete responses and two partial responses for a total response rate of 8% (95% confidence interval, 1-25%). Response durations were 17 and 45 weeks. Stable disease was seen in 3 patients. The median time to progression for all patients was 8 weeks and the median survival was 20 weeks. Topotecan given as a 21-day continuous intravenous infusion has a similar response rate and median survival to our previously reported study of the 5-day short infusion regimen in pancreatic carcinoma.

Clinical studies of reversal of drug resistance based on glutathione.

The greater affinity of electrophiles for thiol groups than for hydroxyl or amine groups provides a teleological basis for the evolution of this mechanism to assist in the maintenance of cellular homeostasis. As the most abundant cellular non-protein thiol, glutathione (GSH) is pivotal in the protection of cells from electrophiles created during normal respiration and protection after exposure to environmental mutagens. Mutagens and many anti-cancer drugs, e.g. cisplatin and alkylating agents, have the same target, i.e. DNA. This suggested that one mechanism by which cancer cells might circumvent the action of cancer chemotherapeutic agents would be by increasing their cellular GSH and/or enhanced conjugation of these drugs to this abundant tripeptide. This chapter describes the abundant preclinical data that support this mechanism of resistance to platinum drugs and alkylating agents. This data was the rationale for the development of pharmacologic strategies to lower GSH and inactivate the gluathione-S-transferases to make anti-cancer drugs more effective. The positive outcome of preclinical studies to lower GSH and enhance the activity of melaphalan are described as is the status of on going clinical trials built around this data.

Cellular kinetics of induction by oltipraz and its keto derivative of detoxication enzymes in human colon adenocarcinoma cells.

Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] is a synthetic dithiolethione with chemopreventive activity against carcinogen-induced neoplasia of liver, lung, and colon in several animal model systems. Protection from tumor formation is associated with elevation of Phase II enzymes, including glutathione (GSH) transferase and NAD(P)H:quinone oxidoreductase (DT-diaphorase) in experimental carcinogenesis models in vivo. To investigate the time and dose relationships of the pharmacological action of oltipraz and to develop a model for its investigation, a human colon adenocarcinoma HT29 cell line was primarily used. In this cell line, oltipraz resulted in increased activity of both GSH transferase and DT-diaphorase. At the maximum effective concentration (100 microM), the elevation of GSH transferase was 3-fold and that of DT-diaphorase was 2-fold. The optimal duration of oltipraz exposure to HT29 cells was 24 h, following which the peak in enzyme activity was observed at 24 h after removal of the drug, and activity had almost returned to control levels after 72 h in drug-free media. Steady-state mRNA levels for DT-diaphorase were observed to increase during the period of drug exposure and remained elevated, even as catalytic activities declined to control levels, suggesting additional mechanisms for control of the activity of this enzyme. More prolonged drug exposure was associated with less induction of the detoxication enzymes, prompting an investigation of the possible toxicity of oltipraz to these cells. Although the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed inhibition of proliferation (IC50, 100 microM oltipraz), a clonogenic assay demonstrated no loss of clonogenicity. Oltipraz is known to be extensively metabolized in many species; two major metabolites include a 3-ketone (metabolite 2, M2) and a molecular rearrangement to a pyrrolopyrazine derivative (metabolite 3, M3), numerous conjugates of which are formed in vivo. To investigate the potential cause of the lag in response, we synthesized two major oltipraz metabolites (M2 and M3) and tested their efficacy in enzyme induction. The activity of DT-diaphorase was induced similarly by both oltipraz and M2 (2.6- versus 2.8-fold baseline) at 100 microM, whereas M3 was inactive at all concentrations. M2 also resulted in a 5.8-fold elevation of steady-state DT-diaphorase mRNA levels. Both enzyme activity and steady-state mRNA peaked at 24 h as with the parent compound. Thus, the oxidative desulfuration of oltipraz results in the formation of an active metabolite, but this process is not rate limiting for the induction of detoxicating enzymes. These data support the use of intermittent schedules in oltipraz in clinical trials of chemoprevention because of evidence of attenuation of response. The metabolite M2, but not M3, is as active as the parent compound and may be considered for clinical development in its own right.

Involvement of activator protein-1 and nuclear factor-kappaB transcription factors in the control of the DT-diaphorase expression induced by mitomycin C treatment.

The antitumor antibiotic mitomycin C is activated by several bioreductive enzymes, including DT-diaphorase. In HT29 cells, mitomycin C treatment results in the induction of DT-diaphorase as reflected in elevated steady state DT-diaphorase mRNA levels. An increase in the transcriptional rate was demonstrated by nuclear run-on assay. To investigate the molecular basis of the change in transcriptional activity caused by mitomycin C treatment, electrophoretic mobility shift assays were used to demonstrate the induction of nuclear factor binding to elements in the 5' flanking region of the DT-diaphorase gene. Treatment of HT29 cells with mitomycin C resulted in the dose-dependent induction of binding activity directed to the activator protein-1 (AP-1) binding element with a time course similar to that of mRNA elevation. Supershift assays using specific antibodies to Jun and Fos demonstrated the participation of both proteins in the binding activities generated. A binding activity for the nuclear factor-kappaB (NF-kappaB) site was induced with a similar time course. Both competitor and supershift experiments indicated that a heterodimer of the NF-kappaB proteins p50 and p65 was contained in the bound complex. To further investigate the functional consequences of such binding, we transfected HT29 cells with a plasmid containing 3 kb of the DT-diaphorase 5' region upstream of a reporter gene, chloramphenicol acetyltransferase. Treatment with mitomycin C resulted in a 5.5-fold increase in the expression of a chloramphenicol acetyltransferase construct containing 3 kb of DT-diaphorase promoter sequence. Using a series of deletion mutations based on this full-length construct, we found that two regions of the DT-diaphorase promoter region, positions -346 to -588 (containing the AP-1 element) and positions -785 to -890 (containing the NF-kappaB element) are required for the full expression of the mitomycin C response. The specific involvement of these binding elements was confirmed using mutational analysis. The results demonstrate that mutation of either element alone or of both diminishes the response, indicating an additive interaction between the elements at a minimum. However, inducibility characterizes a promoter fragment as small as 78 base-pairs from the transcription start site. Treatment of cells with mitomycin C induced binding to a 38-base-pair region (-40 to -78) devoid of known transcription factor binding elements. These data suggest that mitomycin C induces the overexpression of DT-diaphorase through a mechanism involving both the AP-1 and NF-kappaB response elements and that inducibility depends on a novel factor binding element.

The chemopreventive agent oltipraz stimulates repair of damaged DNA.

Carcinogens may damage DNA either through the production of radicals that cause base modification in situ or through the formation of bulky adducts at relatively nucleophilic sites. Preclinical studies have demonstrated that administration of the dithiolethione oltipraz protects laboratory animals from the development of tumors following subsequent exposure to a variety of carcinogens. This may occur through a mechanism involving the induction of detoxicating gene expression. In some models, oltipraz treatment following carcinogen exposure may also confer protection. To investigate a possible mechanism for this observation, we studied the effects of oltipraz on base excision repair and platinum-DNA damage formation and removal. No effect of oltipraz was observed on base excision repair as determined by an in vitro assay measuring the repair of apurinic/apyrimidinic sites by untreated and oltipraz-treated HT-29 whole-cell extracts. Treatment of HT-29 cells with cisplatin in the absence or presence of 30 and 100 microM oltipraz decreased the accumulation of platinum in DNA. A dose-dependent reduction in DNA platination was also observed in purified DNA treated concurrently with cisplatin and increasing concentrations of oltipraz. When DNA was first platinated and subsequently incubated with oltipraz, no decrease in platinum content in DNA was found. Preincubation of HT-29 cells with oltipraz enhanced the rate of removal of total platinum-DNA adducts and interstrand cross-links. These data support a novel mechanism through which dithiolethiones may protect carcinogen-exposed animals from tumor formation and may expand their potential role in the clinic.

Modulation of gene expression in subjects at risk for colorectal cancer by the chemopreventive dithiolethione oltipraz.

Prolonged exposure to mutagenic substances is strongly associated with an individual's risk of developing colorectal cancer. Clinical investigation of oltipraz as a chemopreventive agent is supported by its induction of the expression of detoxication enzymes in various tissues, and its protective activity against the formation of chemically induced colorectal tumors in animals. The goals of the present study were: to determine if oltipraz could induce detoxicating gene expression in human tissues; to identify effective non-toxic doses for more extensive clinical testing; and to establish a relationship between effects in the colon mucosa and those in a more readily available tissue, the peripheral mononuclear cell. 24 evaluable patients at high risk for colorectal cancer were treated in a dose-finding study with oltipraz 125, 250, 500, or 1,000 mg/m2 as a single oral dose. Biochemical analysis of sequential blood samples and colon mucosal biopsies revealed increases in glutathione transferase activity at the lower dose levels. These effects were not observed at the higher doses. More pronounced changes were observed in detoxicating enzyme gene expression in both tissues at all doses. Peripheral mononuclear cell and colon mRNA content for gamma-glutamylcysteine synthetase (gamma-GCS) and DT-diaphorase increased after dosing to reach a peak on day 2-4 after treatment, and declined to baseline in the subsequent 7-10 d. The extent of induction of gene expression in colon mucosa reached a peak of 5.75-fold for gamma-GCS, and a peak of 4.14-fold for DT-diaphorase at 250 mg/m2 ; higher doses were not more effective. Levels of gamma-GCS and DT-diaphorase correlated closely (P < or = 0.001) between peripheral mononuclear cells and colon mucosa both at baseline and at peak. These findings demonstrate that the administration of minimally toxic agents at low doses may modulate the expression of detoxicating genes in the tissues of individuals at high risk for cancer. Furthermore, peripheral mononuclear cells may be used as a noninvasive surrogate endpoint biomarker for the transcriptional response of normal colon mucosa to drug administration.

Increased DT-diaphorase expression and cross-resistance to mitomycin C in a series of cisplatin-resistant human ovarian cancer cell lines.

In a series of ovarian carcinoma cell lines selected in vitro for resistance to cisplatin by continuous exposure to increasing drug concentrations, the level of resistance is proportional to the expression of gamma-glutamylcysteine synthetase (gamma-GCS). To determine if other detoxicating genes are coordinately expressed, we measured the activity of DT-diaphorase and cytochrome P450 reductase. The specific activity of DT-diaphorase, but not that of cytochrome P450 reductase, increased with increasing resistance to cisplatin. Steady-state mRNA levels for DT-diaphorase correlated with enzyme activity and hence with cisplatin resistance. Since the activity of DT-diaphorase has been associated with sensitivity to quinones, we studied the cytotoxicity of mitomycin C under oxic conditions. Unexpectedly, resistance to mitomycin C increased proportionally with that to cisplatin (r = 0.997). Pretreatment with buthionine sulfoximine, which inhibits glutathione (GSH) synthesis, failed to sensitize either the sensitive or the resistant lines to mitomycin C. Thus, the basis for collateral resistance to mitomycin C in the cisplatin-resistant lines under oxic conditions is unrelated to overproduction of GSH. Under hypoxia, the toxicity of mitomycin C to the most sensitive (A2780) cell line was unchanged. However, the most resistant (C200) line was 2-fold more resistant to mitomycin C under hypoxic conditions. The coordinate overexpression of DT-diaphorase and gamma-GCS in the resistant cell lines is thus associated with hypoxic cell resistance, and supports the involvement of shared mechanisms of gene regulation in the observed resistant phenotype.

Alternative splicing and differential expression of DT-diaphorase transcripts in human colon tumors and in peripheral mononuclear cells in response to mitomycin C treatment.

The two-electron bioreductive enzyme DT-diaphorase catalyzes the metabolism of quinones. The existence of several distinct sizes of DT-diaphorase mRNA transcripts has been observed in human tissues. One of these, an alternatively spliced mRNA that lacks exon 4, has been recently found to be expressed at levels comparable to those of the full-length mRNA. The protein encoded by the mRNA lacking exon 4 has minimal catalytic activity, consistent with the elimination of the quinone-binding site coded for by this exon. We have pursued a number of approaches to examine the significance of this splice variant. We identified a similar truncated transcript in a human HepG2 cDNA library. To determine the frequency of expression of this form of DT-diaphorase in the general population, we examined mRNA obtained from the peripheral mononuclear cells of 16 patients and found substantial interindividual variability in the patterns of transcript expression. Following treatment of these 16 patients with 20 mg/m2 mitomycin C (MMC), the induction of DT-diaphorase transcripts was demonstrated. In most patients, expression of the variant transcript (lacking exon 4) remained constant, while that of the full-length mRNA was elevated. The extent of induction also showed interindividual variability. In one patient, while both transcripts were present at baseline, expression of the variant transcript disappeared almost completely after MMC treatment. To analyze these events under more controlled conditions, we examined the effects of MMC treatment on two human colon tumor cell lines. MMC treatment induced expression of the full-length mRNA but did not influence the abundance of the variant transcript. We then performed single-strand conformational polymorphism analysis of genomic DNA from the 16 patients to investigate the potential role of cis-acting factors in the variable splicing responses. Two patients demonstrated sequence differences in the region spanning exon 4, but in neither was the change in a region critical to splicing regulation. These data demonstrate that the expression of DT-diaphorase in hyman cells is polymorphic, and that the levels of individual transcripts can be regulated by exogenous factors. The findings support a role for alternative splicing in the control of DT-diaphorase gene expression.

Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines.

We have shown previously that tumor cell resistance to cisplatin is associated with elevated intracellular levels of glutathione, which is accomplished at least in part by increased expression of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS). To investigate the mechanism by which gamma-GCS expression is elevated, we have examined four related human ovarian cancer cell lines with increasing cisplatin resistance. Relative amounts of steady-state gamma-GCS mRNA in CP70, C30, and C200 were 4.8, 6.0, and 10.6, respectively, compared to the parental A2780 cell line, and a proportional increase in the transcriptional rate but not RNA stability was demonstrated. In contrast, no increase in mRNA for the gamma-GCS light subunit was found. To determine the mechanism of upregulation of this mRNA, we have cloned the promoter of the gene that encodes the heavy subunit of gamma-GCS. This region contains AP-1, NF-kappa B, XRE, AP-2, EpRE, CAAT, and TATA box elements upstream of the transcription initiation site and two MREs between this site and the start codon for the protein. Using gel mobility shift assays, we have found nuclear extract binding activity to the AP-1 response element to be closely associated with the level of gamma-GCS gene expression. A supershift assay showed that the AP-1 DNA-binding complexes are predominantly formed by dimers of JUN family members. Consistent with this finding, the expression of c-JUN was found to be elevated in the resistant cells. In contrast to AP-1 binding, AP-2 and NF-kappa B binding were inversely related to resistance. Furthermore, we have examined a partial revertant of the C200-resistant cells, which shows lower glutathione levels, and found decreased gamma-GCS expression associated with decreased AP-1 binding activity.

Modulation of glutathione and related enzymes in reversal of resistance to anticancer drugs.

Eukaryotic cells have evolved several mechanisms to protect cellular constituents, especially DNA, from highly reactive molecules entering from without. The greater affinity of electrophiles for thiol groups than for hydroxyl or amine groups provides a teleologic rationale that the availability of high concentrations of thiol could be protective of these other important entities. The major intracellular nonprotein thiol is the tripeptide glutathione.

Involvement of NF-kappa B in the induction of NAD(P)H:quinone oxidoreductase (DT-diaphorase) by hypoxia, oltipraz and mitomycin C.

The activity of the two-electron bioreductive enzyme DT-diaphorase (DTD) is induced by heat shock, hypoxic stress, oltipraz, and mitomycin C (MMC). Transcriptional induction is associated with nuclear factor binding to elements mediating immediate early response including AP-1, though the DTD mRNA peaks at 24 hr. Electrophoretic mobility shift assays revealed that nuclear protein extracts from hypoxia-, oltipraz-, and MMC-treated cells bound a specific oligonucleotide probe corresponding to the NF-kappa B transcriptional binding site in two human cancer cell lines, HT29 and HepG2. The binding activity for the NF-kappa B site was induced with a time-course similar to that of the induction of DTD, and was delayed in comparison to the induction of AP-1 binding proteins. The time-courses of the NF-kappa B binding response to MMC, oltipraz and hypoxic treatment were similar, and binding was most pronounced at 24 hr. All three stimuli were associated with the late appearance of a higher molecular weight complex in HT29 but not in HepG2 cells, suggestive of the participation of additional rel family proteins in DNA binding in this cell line. Competition experiments indicated that the bound protein complex was specific for the NF-kappa B binding site. An immunodepletion assay showed that in each case the bound complex consisted of a heterodimer of the NF-kappa B proteins p50 and p65. These data suggest that hypoxia, oltipraz and MMC may each induce the overexpression of DTD through a mechanism involving the NF-kappa B response element in the DTD 5'-flanking region, and support a role for this element in the control of detoxication responses to environmental changes.