Tumor Cell Lines HT-29 Research Articles

DSLIM Immunomodulators Induce Anti-Tumor Responses Both In Vitro and In Vivo.

Cytosine-guanine (CpG) motifs containing oligonucleotides (ODN) are commonly used for immunomodulatory purpose in cancer therapy and for the treatment of allergic diseases since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response with secretion of IL-12 and IFN-γ, In addition their broad potential includes activation of B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6, and stimulation of plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. Usually phosphorothioate (PS) modifications are to enhance the stability, but these are leading to several side-effects, like severe organ enlargements, morphological changes and immunosuppression in mice. We designed immunomodulatory molecules based on short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. To evaluate the anti-tumor effect of the dSLIM molecules we developed an in vitro anti-tumor assay. This assay uses supernatant from dSLIM-activated human PBMCs for incubation with tumor cells in vitro. We observed increased apoptosis and necrosis by the HT-29 tumor cell line after incubation with supernatant from dSLIM-treated PBMC which was significantly higher than the effect of supernatant from non-treated PBMC. In addition, supernatant from dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor cells, a pre-requisite for tumor cell recognition by the immune system. These effects were confirmed with human HEK293 and murine Renca cell lines. Analyzing the effect with neutralizing antibodies to various apoptosis-related cytokines, we observed a crucial role of IFN-γ but not IFN-α or TNFα. To investigate the anti-tumor effects of dSLIM in vivo, we employed a SKH1 murine model which is prone to spontaneous development of papillomas. Using chemicals for initiation and weekly promotion of de novo papilloma development we compared groups of weekly s.c. or i.p. dSLIM injections, respectively, with the PBS control group. The number of papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced by approximately 50%. In conclusion, we showed that dSLIM immunomodulators exhibit potent anti-tumor effects in vitro and in vivo.

Synthesis, solution equilibria and antiproliferative activity of copper(II) aminomethyltriazole and aminomethylthioxotriazoline complexes

The aim of the present study was the synthesis, the determination of formation constants, and the evaluation of the antiproliferative activity of two copper(II) complexes formed with triazole-type ligands. The synthesis of the unsymmetrical triazole ligand 4-amino-3-aminomethyl-5-methyl-1,2,4-triazole (L 1), and its copper(II) complex is reported. The ligand was prepared by functionalization of the carboxylate function of tert-butyloxycarbonyl (BOC) protected glycine O-methyl ester. All intermediates and final products were isolated and characterized with IR, 1H NMR, and elemental analysis. X-ray structures of the ligand as a sulfate salt ((H 2L 1) 2SO 4 · H 2O) and the copper(II) complex [CuCl 2(L 1) 2] are described. The ligand forms a ( N, N) bidentate chelate with the amino group and one triazole nitrogen atom. The tetragonally distorted octahedral coordination of Cu II results from two axially coordinated chloride ions. Protonation constants for L 1 and speciation of the Cu II/L 1 system were determined in 0.1 M aqueous KCl solution at 25 °C. Complexes formed in solution were also characterized by visible spectrophotometry. Ligand substitution competition between L 1 and glycine has also been studied using potentiometric titrations. Antiproliferative activities of ([CuCl 2(L 1) 2]) and [CuCl 2(H 2L 2)]Cl, where HL 2 is the 5-thioxo analog of L 1, against human tumor cell lines HT1080 and HT29 as well as normal human fibroblasts (HF) are presented along with the antiproliferative activities of L 1, CuCl 2, and cisplatin. Activity of these two complexes are discussed and compared with the activity of analogous compounds reported in the literature which contain pyridyl groups in place of the aminomethyl group. In particular, it is suggested that a lypophilic residue such as a pyridyl group is important for antiproliferative activity of this class of compounds.

Iso-, Nor-, and Dinor-Spiculoic Acids A, Polyketides from the Marine Sponge Plakortis zyggompha

The new iso-, nor-, and dinor-spiculoic acids A (1, 2, and 3, respectively) with a rare spiculane skeleton were isolated from the marine sponge Plakortis zyggompha, collected in the waters south of Martinique. The structural determination of the compounds was based on 1D and 2D NMR studies and mass spectral determinations. Compounds 1 and 2 showed weak cytotoxicity against the two tumor cell lines A549 and HT29.

Transcriptional control of TFF3 (intestinal trefoil factor) via promoter binding sites for the nuclear factor κB and C/EBPβ

The acute phase response is strictly connected with modulation of gene expression. Transcriptional control of many genes is mediated by binding of diverse transcription factors to cis-acting DNA motifs in the respective promoter sequence. We now describe such regulatory elements for the TFF3 gene coding for a peptide involved in response to gut irritation. TNF-α stimulation, which induces NF-κB activation, evoked up to 10-fold reduction of TFF3 expression in the colon tumour cell line HT-29. Several consensus binding sites for members of the NF-κB transcription factor subunits are located in the 5′-flanking region of TFF3. Mutating these sites was aimed at discovering which one is responsible for NF-κB binding and thus regulation of TFF3 expression.

New lignans and cytotoxic constituents from Wikstroemia lanceolata.

Two new lignans, (-)-aptosimon ( 1) and (-)-diasesamin-di-gamma-lactone ( 2), together with twenty-two known compounds, have been isolated from the stems and roots of Wikstroemia lanceolata. The structures of the new compounds were determined through spectral analyses. The proton and carbon assignments of the known sesquiterpenoid, 1alpha,7alpha,10alpha H-guaia-4,11-dien-3-one ( 24) were revised by 2D NMR techniques. Among the isolates, three compounds, (+)-hinokinin ( 6), 2,6-dimethoxy- p-benzoquinone ( 17), and 1alpha,7alpha,10alpha H-guaia-4,11-dien-3-one ( 24) exhibited effective cytotoxicities (ED50 values < 4.0 microg mL-1) against P-388 and HT-29 tumor cell lines.

Hormonemate, a new cytotoxic and apoptosis-inducing compound from the endophytic fungus Hormonema dematioides. I. Identification of the producing strain, and isolation and biological properties of hormonemate.

In a search for new fungal compounds inducing apoptosis of the colon cancer derived cell line COLO-320, hormonemate (1) was purified from fermentations of an endophytic fungus isolated from living needles of a Pinus species. The producing strain was identified as Hormonema dematioides by microscopy and ITS rDNA sequence analysis. The structure of hormonemate was determined by spectroscopic methods. The compound exhibited cytotoxic effects against the human colon tumor cell lines COLO-320, DLD-1 and HT-29 as well as five other human cell lines (HL-60, JURKAT, HEP-G2, MCF-7, HeLa S3). It also induced apoptosis in COLO-320 cells as detected by a caspase-activity assay and morphological changes, and it triggered morphological and physiological differentiation of HL-60 cells into granulocytes, which subsequently died by apoptosis.

Pharmacological characteristics of the specific transporter for the endogenous cell growth inhibitor agmatine in six tumor cell lines.

This study examined agmatine transport into six human intestinal tumor cell lines and compared the pharmacological properties of this transporter with those of the agmatine carrier previously characterized in human glioblastoma cells. Carrier-mediated uptake was determined as specific accumulation of [(14)C]agmatine in the cells. The changes in intracellular agmatine concentration in the tumor cells after 24 h incubation with 1 mM agmatine was analyzed by high-performance liquid chromatography. Specific [(14)C]agmatine accumulation was found in the six human intestinal tumor cell lines Caco2, Cx1, Colo320, HT29, Colo205E, and SW480. Specific [(14)C]agmatine accumulation was inhibited by phentolamine, putrescine, spermine, clonidine, and decynium-22 but not by corticosterone, O-methylisoprenaline, or l-carnitine. Incubation with exogenous agmatine for 24 h increased intracellular agmatine content in all cell lines by a multiple of the basal endogenous content. Transfection of HEK293 cells with cDNA encoding either hOCT1, hOCT2, or hOCT3 did not enhance [(14)C]agmatine accumulation compared to nontransfected cells. All intestinal tumor cell lines investigated express a functional specific agmatine transporter which exhibit pharmacological characteristics similar to those of the agmatine transporter in glioblastoma cells. This agmatine carrier is not identical with any so far known organic cation transport system.

Growth-inhibitory effects of the chemopreventive agent indole-3-carbinol are increased in combination with the polyamine putrescine in the SW480 colon tumour cell line.

BackgroundMany tumours undergo disregulation of polyamine homeostasis and upregulation of ornithine decarboxylase (ODC) activity, which can promote carcinogenesis. In animal models of colon carcinogenesis, inhibition of ODC activity by difluoromethylornithine (DFMO) has been shown to reduce the number and size of colon adenomas and carcinomas. Indole-3-carbinol (I3C) has shown promising chemopreventive activity against a range of human tumour cell types, but little is known about the effect of this agent on colon cell lines. Here, we investigated whether inhibition of ODC by I3C could contribute to a chemopreventive effect in colon cell lines.MethodsCell cycle progression and induction of apoptosis were assessed by flow cytometry. Ornithine decarboxylase activity was determined by liberation of CO2 from 14C-labelled substrate, and polyamine levels were measured by HPLC.ResultsI3C inhibited proliferation of the human colon tumour cell lines HT29 and SW480, and of the normal tissue-derived HCEC line, and at higher concentrations induced apoptosis in SW480 cells. The agent also caused a decrease in ODC activity in a dose-dependent manner. While administration of exogenous putrescine reversed the growth-inhibitory effect of DFMO, it did not reverse the growth-inhibition following an I3C treatment, and in the case of the SW480 cell line, the effect was actually enhanced. In this cell line, combination treatment caused a slight increase in the proportion of cells in the G2/M phase of the cell cycle, and increased the proportion of cells undergoing necrosis, but did not predispose cells to apoptosis. Indole-3-carbinol also caused an increase in intracellular spermine levels, which was not modulated by putrescine co-administration.ConclusionWhile indole-3-carbinol decreased ornithine decarboxylase activity in the colon cell lines, it appears unlikely that this constitutes a major mechanism by which the agent exerts its antiproliferative effect, although accumulation of spermine may cause cytotoxicity and contribute to cell death. The precise mechanism by which putrescine enhances the growth inhibitory effect of the agent remains to be elucidated, but does result in cells undergoing necrosis, possibly following accumulation in the G2/M phase of the cell cycle.

5-(2'-oxoheptadecyl)-resorcinol and 5-(2'-oxononadecyl)-resorcinol, cytotoxic metabolites from a wood-inhabiting basidiomycete.

5-(2'-oxoheptadecyl)-resorcinol [structure: see text] and 5-(2'-oxononadecyl)-resorcinol [structure: see text] were isolated from fermentations of an imperfect basidiomycete. The structures of the compounds were determined by spectroscopic techniques. Both compounds exhibit cytotoxic effects against the human colon tumor cell lines COLO-320, DLD-1 and HT-29 and the human promyeloid leukemia cell line HL-60, the human leukemia T cell JURKAT, the human hepatocellular carcinoma cell line HEP-G2 as well as the J774 mouse macrophage cell line. The compounds induce morphological and physiological differentiation of HL-60 cells into granulocytes, which subsequently die by apoptosis. Both compounds show no antibacterial and antifungal activity.

Enhanced antitumor activity and selectivity of lactoferrin-derived peptides.

A number of peptide analogs derived from the N-terminal alpha-helical region of bovine lactoferrin (LFB 14-31), were designed in order to investigate how deviating numbers and positions of positively charged residues and numbers of aromatic residues affected their activity against prokaryotic, normal and transformed eukaryotic cells. Most of the LFB derivatives were highly active against both Escherichia coli and Staphylococcus aureus. The peptides were more active against the tumor cell lines MethA, HT-29 and MT-1 than normal eukaryotic cells. The peptides that were most active against the tumor cell lines had all cationic residues concentrated in one sector of the helical structure. These peptides were less selective against the tumor cell lines than against normal fibroblasts. Quantitative structure-activity relationship studies showed that certain structural parameters affected toxicity against the tumor cell lines more than against fibroblasts. Peptides encompassing these parameters were slightly less active against tumor cells, but gained significant selectivity.

Comparative analysis of metastatic variants from the colorectal tumor cell line HT-29

OBJECTIVE: To analyze the relationship between metastatic potential and related facters of colorectal tumor cell lines. METHODS: The variants HT-29c and HT-29d cell lines derived from the selection of HT-29 cells were injected into nude rats and the metastatic potential of the two tumor cell variants was analyzed. Expression of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were measured with ELISA in vitro in colorectal carcinoma cell lines WiDr, HT-29 and HT-29d. Expression of carcinoembryonic antigen (CEA) and phosphoinositide 3-kinase (PI3-Kinase) were determined with immunohistochemistry, (IHC) in vitro and in vivo in WiDr, HT-29 and HT-29d cell lines. In addition, CEA expression was demonstrated with fluorescence activated cell sorter (FACS) in vitro. RESULTS: The liver metastasis rate of the variant HT-29d (with 4 cycles of selection), increased as compared with that of parental HT-29 cells and that of variant HT-29b cells (with 2 cycle of selection). The uPA concentration of variant HT-29d cell line was significantly higher than that of the non metastatic WiDr and the low metastatic HT-29 cells (P<0.05). The variant HT-29d cells produced stronger PI3-kinase expression as compared with the non-anetastatic WiDr cells and the low metastatic HT-29 cells in vivo. CONCLUSION: The selected variant cell lines can exhibit an enhanced metastatic potential. The level of uPA and PAI-1 are positively correlated with the metastatic capacity of tumor cells. The expression of PI3 kinasecorrelates with tumor development and metastasis.

APC-dependent regulation of ornithine decarboxylase in human colon tumor cells.

Mutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, we used the human colon tumor cell line HT29 (wtAPC-/-), which has been stably transfected with a zinc-inducible wild-type APC gene. The addition of ZnCl(2) to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein and decreased levels of c-myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the beta-galactosidase gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. When the e-box domain in the 5' flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c-myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results demonstrated that wild-type APC suppressed c-myc and activated Mad1 expression in HT29 colon-derived cells. These proteins, in turn, regulated the transcription of target genes, including ODC. The data presented indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues.

Comprehensive karyotyping of the HT-29 colon adenocarcinoma cell line.

The tumor cell line HT-29 was derived from a primary adenocarcinoma of the rectosigmoid colon. HT-29 is hypertriploid (3n+) and has accumulated numerous chromosomal structural aberrations. To identify material involved in chromosome rearrangements, we performed a comprehensive cytogenetic analysis using G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). The combination of molecular cytogenetic techniques enabled us to define the first comprehensive karyotype for HT-29. Seventeen marker chromosomes were found in 75-100% of metaphase cells, generally in a single copy per cell. We confirmed the composition of eight previously described markers, refined the classification of seven others, and identified two novel marker chromosomes. Notable aberrations included a reciprocal translocation between chromosomes 6 and 14 and an unusual, large derivative chromosome 8 composed entirely of 8q material. The telomere status, evaluated by FISH, revealed telomeric signals at the termini of all chromosomes. No interstitial telomeric sequences were observed in any cell. Although numerous chromosomal aberrations are present in HT-29, the cell line appears to have retained a high level of genomic stability during passage in culture since undergoing transformation. The excellent resolving power of SKY, coupled with additional information obtained from molecular cytogenetic analyses, will improve our ability to identify genetic lesions characteristic of cancer.

Anti-tumour Screening of Brazilian Plants

Organic and aqueous extracts of 145 Brazilian plants (538 extracts) from 34 families were evaluated for anti-tumour activity against the human tumour cell lines HT29 and NCIH460. Of the extracts tested, 117 (22%) demonstrated cytotoxicity against one or both of the cell lines at a concentration of 100µg/ml. Of special interest are the families Anacardiaceae, Annonaceae, Asteraceae, Celestraceae, Leguminosae (Fabaceae), Meliaceae and Myrtaceae, which contain a high proportion of active species. On the basis of these results we are further examining the cytotoxic species, with the objective of isolating and identifying the active phytochemicals. These results also confirm the continuing importance of natural product screening models, alongside targeted drug development, in the discovery of new anti-neoplastic pharmacophores.

Implication of PBP74/mortalin/GRP75 in the radio-adaptive response

Purpose : To investigate the relationship between expression of the human peptide-binding protein PBP74 and the occurrence of an adaptive response to ionizing radiation. Materials and methods : Human tumour cell lines HT29 and MCF-7 were transfected with a PBP74 or PBP74 antisense construct. For demonstration of an adaptive response, cells lines were irradiated with a conditioning dose of 0.25 Gy cobalt-60 γ-rays followed by a second dose of 4.0 Gy after an interval of 4.5 h. Response was measured in terms of clonogenic survival. Results : Transfection of a PBP74 plasmid caused transient overexpression of PBP74 mRNA in both cell lines. The optimal dose for the induction of PBP74 in the cell lines investigated was 0.1-0.25Gy and PBP74 induction occurred within 30 min of irradiation. For both cell lines, the adaptive response was repressed when cells were transfected with the anti-PBP plasmid. However, the converse, an enhancement of the adaptive response in cell lines transfected with the PBP74 construct, was seen only for HT29 cells under certain experimental conditions. Conclusions : The results support the view that while PBP74 is necessary to the adaptive response, it may not by itself be sufficient for the adaptive response to occur.

Natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) is not impaired in patients suffering from chronic hepatitis C

Background/Aims : Recent observations suggest that natural killer (NK) cell activity might be impaired in chronic hepatitis C. However, to date antibody-dependent cellular cytotoxicity (ADCC) has not been studied in chronic hepatitis C in detail. Methods : Therefore, we investigated spontaneous and cytokine-induced (interleukin-2 and interferon- γ) natural cytotoxicity and ADCC in 29 patients suffering from chronic hepatitis C and 19 healthy controls. Cytotoxicity was determined with a flow-cytometric assay, which can also assess monocyte cytotoxicity. As target cells we used the colorectal tumor cell line HT29 and the lymphoma cell line Raji. Results : We found no significant differences with respect to spontaneous cytotoxicity (HCV versus healthy controls (32 vs. 46%) and 17-1A specific ADCC (59 vs. 48%), even if isolated monocytes or NK cells were studied. Preincubation and stimulation of effector cells with cytokines increased both natural cytotoxicity and ADCC by 20–30%. However, natural cytotoxicity and ADCC after stimulation did not differ between the two groups. Conclusions : Our data obtained with a long-term cytotoxicity assay do not reveal impaired cytolytic capacity of the innate immune system in chronic hepatitis C, even when isolated monocytes and NK cells were studied as effector cells.

Design and optimization of 20-O-linked camptothecin glycoconjugates as anticancer agents.

To improve the biological profile of 20(S)-camptothecin, a novel class of 20-O-linked camptothecin glycoconjugates has been designed for preferential cellular uptake into tumor cells by an active transport mechanism. Such conjugates have been optimized for enhanced solubility, stabilization of the camptothecin lactone ring, sufficient hydrolytic and proteolytic stability, and for an overall improvement in tumor selectivity. The constitution of the peptide spacer has a major impact on stability and biological activity of the conjugates both in vitro and in vivo. Glycoconjugates 17-22 with valine residues at the linkage position to camptothecin are sufficiently stable and show good antitumor activity in vitro against HT29 and other tumor cell lines. Fluorescence microscopy and flow cytometry experiments indicate that glycoconjugates such as 19 are taken up into lysosomal compartments of the tumor cell line HT29 by an active transport mechanism. The steric configuration of the particular amino acid residues linked to the camptothecin moiety has a major impact on the in vivo activity of the corresponding glycoconjugates in the breast cancer xenograft MX-1 model. Inhibiting tumor growth by >96%, the glycoconjugates 19 and 21 show the best activity in this particular model and have been investigated more extensively. The glycoconjugate 19 compares favorably to topotecan 4 and glycoconjugate 21 with respect to toxicity against hematopoietic stem cells and hepatocytes. Based on its profile, 19 has been selected for clinical trials.

Butyrate Induces Glutathione S-Transferase in Human Colon Cells and Protects From Genetic Damage by 4-Hydroxy-2-Nonenal

Butyrate, one of the major products of gut fermentation, is known to inhibit proliferation, induce apoptosis and differentiation, and increase phase II enzyme activities in tumor cells, whereas little information is available on protective effects in less-transformed colon cells. The aim of this study was to investigate whether the chemoprotective mechanism of glutathione S-transferase (GST) induction by butyrate could also play a role in earlier stages of colon carcinogenesis and whether chemoresistance of cells toward the endogenous genotoxic risk factor 4-hydroxy-2-nonenal (HNE) could be a consequence of butyrate treatment. As cell models, we used the human tumor cell lines HT29 and HT29 clone 19A, a differentiated subclone with properties resembling primary colon cells. We determined the expression of GSTP1 protein (enzyme-linked immunosorbent assay), the major GST in HT29, GSTP1 mRNA (Northern blotting), GST activity, intracellular glutathione, and total protein. The genotoxic impact of HNE (100-200 μM) was compared in butyrate-treated and nontreated cells using single-cell microgel electrophoresis. Our results show that GSTP1 mRNA, GSTP1 protein, GST activity, and total protein were increased (1.2- to 2.5-fold) and glutathione levels were maintained after 24- 72 h of incubation with 4 mM butyrate. Moreover, a marked reduction of HNE-induced genotoxicity was caused by preincubation with butyrate. Butyrate also induced the phosphorylation of extracellular signal-regulated kinases (ERK1/2, Western blotting) after 5-30 min, which indicates a regulation of GST expression by this signal pathway. Most effects were greater in HT29 parent cells than in clone cells. In conclusion, butyrate enhances expression of GST and other proteins in both cell lines, which leads to an enhanced chemoprotection, reducing the impact of HNE genotoxicity. Thus butyrate could play a role in early and later stages of cancer prevention by reducing exposure to relevant risk factors.

Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer.

Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo.

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon alpha and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells.

Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon gamma (IFNgamma), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3+CD56+ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNalpha but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis(Y) antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors.