Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Initial Transformations in the Biodegradation of Benzothiazoles by Rhodococcus Isolates

Benzothiazole-2-sulfonate (BTSO₃) is one of the side products occurring in 2-mercaptobenzothiazole (MBT) production wastewater. We are the first to isolate an axenic culture capable of BTSO₃ degradation. The isolate was identified as a Rhodococcus erythropolis strain and also degraded 2-hydroxybenzothiazole (OBT) and benzothiazole (BT), but not MBT, which was found to inhibit the biodegradation of OBT, BT, and BTSO₃. In anaerobic resting cell assays, BTSO₃ was transformed into OBT in stoichiometric amounts. Under aerobic conditions, OBT was observed as an intermediate in BT breakdown and an unknown compound transiently accumulated in several assays. This product was identified as a dihydroxybenzothiazole. Benzothiazole degradation pathways seem to converge into OBT, which is then transformed further into the dihydroxy derivative.     

Microbial transformations of 2-substituted benzothiazoles

The occurrence of benzothiazoles in the environment seems to be restricted to aquatic compartments and is mainly associated with the manufacture and use of the rubber additive 2-mercaptobenzothiazole (MBT) and its derivatives. Although data on benzothiazole biotransformations in natural environments at ppb and ppt levels are scarce, the unsubstituted benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) are generally considered to be biodegradable, whereas 2-methylthiobenzothiazole is recalcitrant. The fungicide 2-thiocyanomethylthiobenzothiazole is assumed to be hydrolysed to MBT, which is then further methylated. At higher concentration levels, similar conclusions can generally be drawn. In addition, BT, MBT, 2-aminobenzothiazole and benzothiazole-2-sulphonate can be biodegraded, although side- and end-products may form. For BT and MBT, threshold concentration were reported above which inhibitory effects on biological treatment processes occur. Due to the limited availability of axenic bacterial cultures capable of benzothiazole mineralization, only the initial steps of the degradation pathways have been elucidated so far.     

Molecular genotyping of Mycobacterium tuberculosis strains isolated from patients in the Samara region by the restriction DNA fragment length polymorphism

The typing of 106 M. tuberculosis (MBT) strains isolated from patients in the Samara region by the restriction DNA fragment length polymorphism (RFLP) IS6110 revealed that most of the strains (71.7%) belonged to the W family, 5 MBT strains (4.7%) belonged to the AI family, one culture was the mixture of two strains, AI and W. In addition, 24 MBT strains (22.6%) classified with other genotypes were detected. The analysis of the sensitivity of the MBT strains to rifampicin and isoniazid, with the method of absolute concentrations and by point mutations, demonstrated that 29 MBT strains (27.3%) were sensitive to rifampicin and isoniazid and 56 MBT strains (52.9%) were resistant to rifampicin and isoniazid simultaneously. Among the MBT strains of different RFLP families, strains both sensitive and resistant to these two preparations could be detected, but strains with multiple drug resistance prevailed in the W family (61.8%).     

Psychological pain treatment in fibromyalgia syndrome: efficacy of operant behavioural and cognitive behavioural treatments

The present study focused on the evaluation of the effects of operant behavioural (OBT) and cognitive behavioural (CBT) treatments for fibromyalgia syndrome (FMS). One hundred and twenty-five patients who fulfilled the American College of Rheumatology criteria for FMS were randomly assigned to OBT (n = 43), CBT (n = 42), or an attention-placebo (AP) treatment (n = 40) that consisted of discussions of FMS-related problems. Assessments of physical functioning, pain, affective distress, and cognitive and behavioural variables were performed pre-treatment and post-treatment as well as 6 and 12 months post-treatment. Patients receiving the OBT or CBT reported a significant reduction in pain intensity post-treatment (all Fs > 3.89, all Ps < 0.01). In addition, the CBT group reported statistically significant improvements in cognitive (all Fs > 7.95, all P < 0.01) and affective variables (all Fs > 2.99, all Ps < 0.02), and the OBT group demonstrated statistically significant improvements in physical functioning and behavioural variables (all Fs > 5.99, all Ps < 0.001) compared with AP. The AP group reported no significant improvement but actually deterioration in the outcome variables. The post-treatment effects for the OBT and CBT groups were maintained at both the 6- and 12-month follow-ups. These results suggest that both OBT and CBT are effective in treating patients with FMS with some differences in the outcome measures specifically targeted by the individual treatments compared with an unstructured discussion group. The AP group showed that unstructured discussion of FMS-related problems may be detrimental.     

Parameters affecting the degradation of benzothiazoles and benzimidazoles in activated sludge systems

It was found that benzothiazole, 2-oxybenzothiazole and 2-benzothiazolesulphonate were degraded in activated sludge systems. 2-Mercaptobenzothiazole (MBT) was more resistant, although the first step in MBT degradation seemed to be transformation to the sulphonate form. At higher MBT concentrations, it was transformed into a disulphide, which accumulated in the sludge. MBT was also found to be mainly responsible for the toxicity of rubber chemical waste-water towards activated sludges. It inhibited the degradation of the other hetrocycles. Only at concentrations of around 20 ppm was MBT degraded. Mercaptobenzimidazole ranked second in resistance to degradation. Correspondence to: H. Verachtert     

古田山亚热带常绿阔叶林树皮厚度的变异特征

树皮是木本植物茎干最外层结构,具有保护茎干、养分储存与运输等重要作用。因此,树皮厚度是一项非常重要的功能性状,其变异不仅影响树皮的各种生态功能,还能影响群落构建与物种共存。然而,以往对树皮厚度的研究集中于火灾易发生态系统,对火灾不易发生的亚热带常绿阔叶林的研究仍较缺乏。测量了古田山国家级自然保护区亚热带常绿阔叶林内树种的树皮厚度,并检验了总树皮厚度、内树皮厚度与外树皮厚度在各分类群间以及功能群间的差异。结果发现：1）39个树种807个个体的总树皮厚度、内树皮厚度与外树皮厚度均值分别为1.90 mm、1.38 mm和0.54 mm。漆树科（Anacardiaceae）、杨梅科（Myricaceae）以及亚热带常绿阔叶林代表类群壳斗科（Fagaceae）、山茶科（Theaceae）的树皮厚度较大。短柄枹（Quercus serrata）、木荷（Schima superba）、小叶青冈（Cyclobalanopsis myrsinifolia）等树种的树皮厚度较大。2）种间、科间的各树皮厚度差异均显著。不同功能类群间,乔木类群的各树皮厚度均较灌木类群大,常绿类群的各树皮厚度均较落叶类群大（内树皮厚度除外）。本次研究结果表明,相对于火灾易发生态系统中的树皮厚度,古田山亚热带常绿阔叶林群落内的树皮厚度相对较薄,表明这些森林树种对当地湿润气候的适应性。同时,树皮厚度在各种分类水平与功能群水平间的显著变异,反映了群落内不同生态策略的共存。     

Membrane permeability transition promoted by phosphate enhances 1-anilino-8-naphthalene sulfonate fluoresence in calcium-loaded liver mitochondria

Phosphate and a number of other compounds induce membrane permeability transition (MBT) in Ca²⁺-loaded mitochondria. 1-Anilino-8-naphthalene sulfonate (ANS) was used as a fluorescent probe to investigate perturbations on the inner membrane during MBT. Induction of MBT caused ANS fluoresence enhancement with a biphasic rate that reached a plateau. The enhancement is analogous to that reported for de-energization of mitochondria. The fluoresence level was independent of whether ANS was added before or at different times after phosphate. In the absence of ANS, fluorescence was low and remained unchanged. The initial time course of MBT, as followed by large-amplitude swelling, was similar to that of fluorescence enhancement. Ruthenium red, EGTA, ADP, and cyclosporin A inhibited the enhancement. Only EGTA + ADP (or ATP) reversed the enhancement when added after phosphate. Efflux of matrix Ca²⁺ by sodium acetate or A23187 did not alter ANS fluoresence. The binding parameters (K _d and number of binding sites) were not significantly different, but the fluorescence maximum was more than doubled after MBT. Although the flourescence of bound ANS showed a nonlinear relationship, it was always higher (73.0 +/- 19.0%) after reaching the plateau. Since ANS binding to membranes is nonspecific, the exact mechanism of the enhanced fluorescence is not apparent. The dependence of the initial rate of fluorescence enhancement on Ca²⁺ concentration was nonlinear, with 45 µM at half-maximal rate. The dependence on phosphate was hyperbolic with 0.7 mM at half-maximal rate, which is close to theK _m value of phosphate carrier. The kinetics is compatible with Ca²⁺ binding to some membrane component(s) during MBT and cause ANS fluorescence enhancement. It is suggested that the bilayer-nonbilayer (hexagonal₁₁) transition consequent to Ca²⁺ binding to proteinphospholipid domains containing cardiolipin may play a role in fluorescence enhancement and MBT.     

脓肿分枝杆菌MBT结构鉴定与比较演化分析

【背景】作为临床最常见的非结核条件致病分枝杆菌,脓肿分枝杆菌(Mycobacteroides abscessus)因其天然、多耐药等特性成为目前临床治疗的一大挑战。作为分枝杆菌限制性营养元素——铁摄取的关键系统,分枝杆菌素(mycobactin,MBT)、羧基分枝杆菌素(carboxymycobactin,cMBT)与病原分枝杆菌的毒力、耐药等密切相关。【目的】丰富分枝杆菌MBT、cMBT结构数据,探究MBT在致病分枝杆菌起源过程中的演化规律。【方法】在MALDI-TOF-MS与FT-MS/MS解析脓肿MBT、cMBT结构的基础上,进一步开展其活性分析与生物合成基因簇比较基因组分析。【结果】虽然脓肿分枝杆菌MBT、cMBT母核修饰模式与海洋分枝杆菌最相似,R1、R2、R3、R5等位置的修饰完全相同,而且脂肪酸链均位于R4位置;但脂肪酸链长度不同[C10-17 (MBT)、C4-8 (cMBT)],为新结构。Fe-cMBT不仅以浓度依赖方式促进脓肿分枝杆菌生长,而且利用效率显著高于FeCl₃,相关结果表明MBT-cMBT是脓肿分枝杆菌高效获取铁元素的关键系统。与MBT结构结果一致,mbt-1基因簇共线性分析及mbt-1、mbt-2系统发育分析结果均表明脓肿分枝杆菌与海洋分枝杆菌(M.marinum)亲缘关系最近,而非结核分枝杆菌(M.tuberculosis)或耻垢分枝杆菌(M.smegmatis) (基于16S rRNA基因序列分析)。进一步分析发现,M.marinum、M.tuberculosis、M.bovis等病原分枝杆菌脂肪酸链长度变化范围仅4 C,而M.abscessus、M.fortuitum、M.avium和M.smegmatis等条件致病与非致病菌的脂肪酸链长度变化范围为7-11 C,暗示MBT同系物脂肪酸链长度变化范围与分枝杆菌不同生活方式、环境之间可能存在关联。【结论】作为获取铁元素的关键系统,具有独特结构的脓肿分枝杆菌MBT-cMBT在致病、耐药等方面的作用及起源、演化规律值得深入研究。     

Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol-14-demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol-14-demethylase herbicide inhibitor

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to γ-ketotriazole (γ-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of γ-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective. Further analysis of the transgenic calli grown in the presence of γ-kt showed that their Δ⁵-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substrate obtusifoliol. In contrast, control calli when treated with γ-kt, displayed a sterol content strongly enriched in 14α-methyl sterols and depressed in physiological Δ⁵-sterols. When the transgenic calli were cultured in mixtures of γ-kt and LAN14DM inhibitors sterol compositions enriched in 14α-methyl sterols were obtained, reflecting a strong inhibition of both ‘endogenous’ OBT14DM and ‘exogenous’ LAN14DM. Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.     

Benzothiazole degradation by Rhodococcus pyridinovorans strain PA: evidence of a catechol 1,2-dioxygenase activity

The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ (1)H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional (1)H-(13)C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by (1)H and (19)F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.     

Plant sterol biosynthesis: novel potent and selective inhibitors of cytochrome P450-dependent obtusifoliol 14 alpha-methyl demethylase.

The R-(-) isomer of methyl 1-(2,2-dimethylindan-1-yl)imidazole-5-carboxylate (CGA 214372; 2) strongly inhibited P450-dependent obtusifoliol 14 alpha-demethylase (P450OBT.14DM) (I50 = 8 x 10(-9) M, I50/Km = 5 x 10(-5) in a maize (Zea mays) microsomal preparation. Kinetic studies indicated uncompetitive inhibition with respect to obtusifoliol. The corresponding S-(+) isomer was a 20-fold weaker inhibitor for P450OBT.14DM. The molecular features of a variety of analogues of 2 were related to their potency as inhibitors of P450OBT.14DM in vitro, allowing delineation of the key structural requirements governing inhibition of the demethylase. CGA 214372 proved to have a high degree of selectivity for P450OBT.14DM. This allowed easy distinction of this activity from other P450-dependent activities present in the maize microsomal preparation and gave strong evidence that P450OBT.14DM is a herbicidal target. Microsomal maize P450OBT.14DM and yeast P450LAN.14DM, the only known examples of P450-dependent enzymes carrying out an identical metabolic function in different eukaryotes, showed distinct inhibition patterns with CGA 214372 and ketoconazole, a substituted imidazole anti-mycotic.     

Construction and evaluation of self‐cloning bottom‐fermenting yeast with high SSU1 expression

Aim: To construct a self‐cloning brewer’s yeast that can minimize the unfavourable flavours caused by oxidation and certain kinds of sulfur compounds. Methods and Results: DNA fragments of a high‐expression promoter from the TDH3 gene originating from Saccharomyces cerevisiae were integrated into the promoter regions of the S. cerevisiae‐type and Saccharomyces bayanus‐type SSU1 genes of bottom‐fermenting brewer’s yeast. PCR and sequencing confirmed the TDH3 promoter was correctly introduced into the SSU1 regions of the constructed yeasts, and no foreign DNA sequences were found. Using the constructed yeasts, the concentration of sulfite in fermenting wort was higher when compared with the parent strain. In addition, the concentrations of hydrogen sulfide, 3‐methyl‐2‐buten‐1‐thiol (MBT) and 2‐mercapto‐3‐methyl‐1‐butanol (2M3MB) were lower when compared with the parent strain. Conclusion: We successfully constructed a self‐cloning brewer’s yeast with high SSU1 expression that enhanced the sulfite‐excreting ability and diminished the production ability of hydrogen sulfide, MBT and 2M3MB. Significance and Impact of the Study: The self‐cloning brewer’s yeast with high SSU1 expression would contribute to the production of superior quality beer with a high concentration of sulfite and low concentrations of hydrogen sulfide, MBT and 2M3MB.     

Toxicity of 2-mercaptobenzothiazole towards bacterial growth and respiration

Active sludge systems containing benzothiazoles may be intoxicated by 2-mercaptobenzothiazole (MBT). This toxicity towards several bacteria is now confirmed and is situated at around 100 mg MBT l^–1. Octanol-water partition coefficients indicated that MBT might interact with membrane-bound systems. This was confirmed through experiments showing that bacterial cell respiration was inhibited using lactate or succinate as substrates. Using these substrates and also NADH, it was found that their oxidation was also inhibited using isolated membrane fragments of Escherichia coli and Paracoccus denitrificans. Methylene blue reduction was also found to be inhibited. The oxidation of ascorbate was not inhibited in P. denitrificans. From these results it is suggested that MBT might interact with the respiratorychain at the level of flavoproteins or quinones and Fe-S clusters.     

Wee1 kinase alters cyclin E/Cdk2 and promotes apoptosis during the early embryonic development of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Background  

The cell cycles of the Xenopus laevis embryo undergo extensive remodeling beginning at the midblastula transition (MBT) of early development. Cell divisions 2–12 consist of rapid cleavages without gap phases or cell cycle checkpoints. Some remodeling events depend upon a critical nucleo-cytoplasmic ratio, whereas others rely on a maternal timer controlled by cyclin E/Cdk2 activity. One key event that occurs at the MBT is the degradation of maternal Wee1, a negative regulator of cyclin-dependent kinase (Cdk) activity.     

Phosphorylation of Claspin is triggered by the nucleocytoplasmic ratio at the Xenopus laevis midblastula transition

At the Xenopus midblastula transition (MBT), cell cycles lengthen, and checkpoints that respond to damaged or unreplicated DNA are established. The MBT is triggered by a critical nucleocytoplasmic (N/C) ratio; however, the molecular basis for its initiation remains unknown. In egg extracts, activation of Chk1 checkpoint kinase requires the adaptor protein Claspin, which recruits Chk1 for phosphorylation by ATR. At the MBT in embryos, Chk1 is transiently activated to lengthen the cell cycle. We show that Xenopus Claspin is phosphorylated at the MBT at both DNA replication checkpoint-dependent and -independent sites. Further, in egg extracts, Claspin phosphorylation depends on a threshold N/C ratio, but occurs even when ATR is inhibited. Not all phosphorylation that occurs at the MBT is reproduced in egg extracts. Our results identify Claspin as the most upstream molecule in the signaling pathway that responds to the N/C ratio and indicate that Claspin may also respond to an independent timer to trigger the MBT and activation of cell cycle checkpoints.     

Anti-tubercular Activity of Ruthenium (II) Complexes with Polypyridines

A series of nine polypyridyl-ruthenium (II) complexes (N-ligands = 2,2′-bipyridines; 2,2′-6′,2′-terpyridines, di-alkyloxy-2,2′-6,2-bipyridine-3,3′-di-carboxylates), were tested against Mycobacterium tuberculosis (MBT). The complex (11) showed remarkable activity against MBT as compared to other complexes, (1–10). The aquo ligand of complex (11), as opposed to other chloro and acetonitrile derivatives, appears to play a key role in the antitubercular potency of this new class of metal-based compounds.     

Some biochemical properties of higher plant tubulins

Tubulin was isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose) and ion exchange (DEAE-Sephacel) chromatography from mung bean and cultured carrot suspension cells. SDS-PAGE (Blose 1981) of mung bean tubulin has shown it to consist of two major subunits (MBT1 and MBT2) and a minor subunit (MBT3). Tubulin isolated from carrot cells was resolved into only two bands on SDS-PAGE (slow moving subunit was named CT1). However, the faster moving subunit on SDS-PAGE was resolved into two bands (CT2 and CT3) on SDS-4M urea-PAGE. On SDS-4M urea-PAGE, CT1 migrated faster than CT2, CT3. By contrast in SDS-4M urea-PAGE, mung bean tubulin remains unresolved. Mammalian tubulin could be resolved into alpha and beta-subunits in both electrophoretic systems. Monoclonal antibodies to mammalian alpha and beta-tubulin subunits (MCA-T alpha and MCA-T beta, respectively) and Western blot analysis clearly demonstrated a cross-reactivity of MCA-T alpha with MBT2, MBT3, CT2 and CT3, while MCA-T beta showed cross-reactivity with MBT1 and CT1. Although MBT2, MBT3, CT2 and CT3 are immunologically related to the alpha-subunit of mammalian tubulin, their migration on SDS-PAGE was reversed with respect to MBT1 or CT1, which were immunologically related to the beta-subunit of mammalian tubulin. Peptide mapping patterns also supported above the results.     

Epigenetic modulation of BRCA-1 and MGMT genes,and histones H4 and H3 are associated with breast tumors

Aberrant patterns in promoter methylation of tumor-suppressor genes and posttranslational modifications of histone proteins are considered as major features of malignancy. In this study, we aimed to investigate promoter methylation of three tumor-suppressor genes (BRCA-1, MGMT, and P16) and three histone marks (H3K9ac, H3K18ac, and H4K20me3) in patients with breast tumors. This case-control study included 27 patients with malignant breast tumors (MBT) and 31 patients with benign breast tumors (BBT). The methylation-specific PCR was used for determining promoter methylation of BRCA-1, MGMT, and P16 genes. Western blot analysis was performed to detect histone lysine acetylation (H3K9ac and H3K18ac) and lysine methylation (H4K20me3). BRCA-1 promoter methylation was detected in 44.4% of the MBT whereas this alteration was found in 9.7% of BBT (P = 0.005). The Kaplan-Meier analysis indicated that hypermethylation in BRCA-1 promoter was significantly associated with poor overall survival of patients with breast cancer (P = 0.039). MGMT promoter methylation was identified in 18.5% of MBT and 0.0% of the BBT (P = 0.01). The frequency of P16 promoter methylation was 25.8% in BBT and 11.1% in MBT (P = 0.12). As compared with BBT, MBT samples displayed the aberrant patterns of histones marks with hypomethylation of H4K20 and hypoacetylation of H3K18 (P = 0.03 and P = 0.04, respectively). There was a negative significant correlation between H3K9ac levels and tumor size in MBT group (r = −0.672; P = 0.008). The present findings suggest that promoter hypermethylation of MGMT and BRCA-1 genes along with alterations in H3K18ac and H4K20me3 levels may have prognostic values in patients with breast cancer. Moreover, the detection of these epigenetic modifications in breast tumors could be helpful in finding new methods for breast cancer therapy.     

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8⁺ T or CD4⁺ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18. Received: 7 May 1998 / Accepted: 27 May 1999