Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors.

Malignant pleural mesothelioma (MPM) originates in most of the cases from chronic inflammation of the mesothelium due to exposure to asbestos fibers. Given the limited effect of chemotherapy, a big effort is being made to find new treatment options. The PI3K/mTOR pathway was reported to be upregulated in MPM. We tested the cell growth inhibition properties of two dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 on 19 MPM cell lines. We could identify resistant and sensitive lines; however, there was no correlation to the downregulation of PI3K/mTOR activity markers. As a result of mTOR inhibition, both drugs efficiently induced long-term autophagy but not cell death. Autophagy blockade by chloroquine in combination with the dual PI3K/mTOR inhibitors significantly induced caspase-independent cell death involving RIP1 in the sensitive cell line SPC212. Cell death in the resistant cell line Mero-82 was less pronounced, and it was not induced via RIP1-dependent mechanism, suggesting the involvement of RIP1 downstream effectors. Cell death induction was confirmed in 3D systems. Based on these results, we identify autophagy as one of the main mechanisms of cell death resistance against dual PI3K/mTOR inhibitors in MPM. As PI3K/mTOR inhibitors are under investigation in clinical trials, these results may help interpreting their outcome and suggest ways for intervention.

Searching for targets for the systemic therapy of mesothelioma.

Malignant mesothelioma is an incurable disease associated with asbestos exposure arising in the pleural cavity and less frequently in the peritoneal cavity. Platinum-based combination chemotherapy with pemetrexed is the established standard of care. Multimodality approaches including surgery and radiotherapy are being investigated. Increasing knowledge about the molecular characteristics of mesothelioma had led to the identification of novel potential targets for systemic therapy. Current evidence suggests pathways activated in response to merlin deficiency, including Pi3K/mTOR and the focal adhesion kinase, as well as immunotherapeutic approaches to be most promising. This review elaborates on the rationale behind targeted approaches that have been and are undergoing exploration in mesothelioma and summarizes available clinical results and ongoing efforts to improve the systemic therapy of mesothelioma.

CD74: a new prognostic factor for patients with malignant pleural mesothelioma.

BACKGROUND: The pro-inflammatory cytokine migration inhibitory factor (MIF) and its receptor CD74 have been proposed as possible therapeutic targets in several cancers. We studied the expression of MIF and CD74 together with calretinin in specimens of malignant pleural mesothelioma (MPM), correlating their expression levels with clinico-pathologic parameters, in particular overall survival (OS). METHODS: Migration inhibitory factor, CD74, and calretinin immunoreactivity were investigated in a tissue microarray of 352 patients diagnosed with MPM. Protein expression intensities were semiquantitatively scored in the tumour cells and in the peritumoral stroma. Markers were matched with OS, age, gender, and histological subtype. RESULTS: Clinical data from 135 patients were available. Tumour cell expressions of MIF and CD74 were observed in 95% and 98% of MPM specimens, respectively, with a homogenous distribution between the different histotypes. CD74 (P<0.001) but not MIF overexpression (P=0.231) emerged as an independent prognostic factor for prolonged OS. High expression of tumour cell calretinin correlated with the epithelioid histotype and was also predictive of longer OS (P<0.001). When compared with previously characterised putative epithelial-to-mesenchymal transition markers, CD74 correlated positively with tumoral PTEN and podoplanin expressions, but was inversely related with periostin expression. CONCLUSIONS: High expression of CD74 is an independent prognostic factor for prolonged OS in mesothelioma patients.

Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase.

Inducible nitric oxide synthase (iNOS) expression is regulated at both the transcriptional and post-transcriptional level in epithelial cells. The aim of this study was to characterize the effects of tyrosine phosphorylation on iNOS activity. In a human intestinal epithelial cell line stimulated with cytokines, tyrosine phosphorylation of human iNOS protein was observed after 30 min exposure to pervanadate (PV), an inhibitor of protein tyrosine phosphatases. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor of Src tyrosine kinases, abolished the PV-induced iNOS tyrosine phosphorylation. Cotransfection of Src with iNOS cDNA in human embryonic kidney (HEK) 293 cells resulted in a threefold (P<0.001) increase of iNOS protein levels and tyrosine phosphorylation of iNOS. In the presence of Src, 76% of wild-type (wt) iNOS was redistributed to detergent-insoluble domains and iNOS activity was decreased by 28% (P<0.05) despite increased iNOS protein levels. Analysis of iNOS tyrosine mutants revealed decreased Src-induced effects in Y151F iNOS mutant. Using a GST-fusion protein containing a domain encompassing Y151, we show that Y151 is a direct substrate for active Src in vitro. These findings indicate a role for iNOS tyrosine phosphorylation in the regulation of iNOS activity and the implication of Src tyrosine kinases in this pathway.

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide.

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1-5 mM), or NO donor (DETA NONOate 10-100 microM). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 microM had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 microM decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.

Oxidative stress in gastric mucosa of asymptomatic humans infected with Helicobacter pylori: effect of bacterial eradication.

BACKGROUND: Inducible nitric oxide synthase (iNOS) and interleukin 8 (IL-8) are positive in approximately 50% of Helicobacter pylori-related diseases but it is not clear whether oxidative stress is also present in H. pylori asymptomatic humans. Our aim was to study the expression of iNOS, superoxide dismutase, catalase and IL-8 production in H. pylori-infected asymptomatic humans, and to investigate the effect of eradication of H. pylori. MATERIALS AND METHODS: Biopsies of corpus and antrum of asymptomatic H. pylori positive and negative humans served for determination of the gastritis score and H. pylori status; iNOS was measured by reverse transcriptase polymerase chain reaction and immunohistochemistry and superoxide dismutase and catalase by immunohistochemistry. IL-8 in biopsies was assessed by enzyme-linked immunosorbent assay. RESULTS: Immunostaining of iNOS, catalase and superoxide dismutase was significantly associated with H. pylori infection and was localized to inflammatory cells. IL-8 concentrations were greater in the H. pylori positive than H. pylori negative group and decreased after bacterial eradication. A decrease in staining for iNOS and catalase was observed after H. pylori eradication. CONCLUSIONS: INOS and antioxidant enzymes are present in gastric biopsies of asymptomatic H. pylori positive humans. Eradication caused a significant decrease in staining for iNOS and catalase. These results indicate that oxidative stress occurs in asymptomatic patients and can be modulated by H. pylori eradication.

L-tyrosine and nitric oxide synergize to prevent cytotoxic effects of superoxide.

We found previously that the nitric oxide donor DEA/NO enhanced lipid peroxidation, DNA fragmentation, and cytotoxicity in human bronchial epithelial cells (BEAS-2B) when they were cultured in LHC-8 medium containing the superoxide-generating system hypoxanthine/xanthine oxidase (HX/XO). We have now discovered that DEA/NO's prooxidant action can be reversed by raising the L-tyrosine concentration from 30 to 400 microM. DEA/NO also protected the cells when they were cultured in Dulbecco's Modified Eagle's Medium (DMEM), whose standard concentration of L-tyrosine is 400 microM. Similar trends were seen with the colon adenoma cell line CaCo-2. Since HPLC analysis of cell-free DMEM or LHC-8 containing 400 microM L-tyrosine, DEA/NO, and HX/XO revealed no evidence of L-tyrosine nitration, our data suggest the existence of an as-yet uncharacterized mechanism by which L-tyrosine can influence the biochemical and toxicological effects of reactive nitrogen species.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Transition mutation in codon 248 of the p53 tumor suppressor gene induced by reactive oxygen species and a nitric oxide-releasing compound.

Exposing the human bronchial epithelial cell line BEAS-2B to the nitric oxide (NO) donor sodium 1-(N,N-diethylamino)diazen-1-ium-1, 2-diolate (DEA/NO) at an initial concentration of 0.6 mM while generating superoxide ion at the rate of 1 microM/min with the hypoxanthine/xanthine oxidase (HX/XO) system induced C:G-->T:A transition mutations in codon 248 of the p53 gene. This pattern of mutagenicity was not seen by 'fish-restriction fragment length polymorphism/polymerase chain reaction' (fish-RFLP/PCR) on exposure to DEA/NO alone, however, exposure to HX/XO led to various mutations, suggesting that co-generation of NO and superoxide was responsible for inducing the observed point mutation. DEA/NO potentiated the ability of HX/XO to induce lipid peroxidation as well as DNA single- and double-strand breaks under these conditions, while 0.6 mM DEA/NO in the absence of HX/XO had no significant effect on these parameters. The results show that a point mutation seen at high frequency in certain common human tumors can be induced by simultaneous exposure to reactive oxygen species and a NO source.

Information transfer between large and small two-dimensional polyacrylamide gel electrophoresis.

To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.

The nitric oxide pathway in pig isolated calyceal smooth muscle.

In pig and humans, whose kidneys have a multi-calyceal collecting system, the initiation of ureteral peristalsis takes place in the renal calyces. In the pig and human ureter, recent evidence suggests that nitric oxide (NO) is an inhibitory mediator that may be involved in the regulation of peristalsis. This study was designed to assess whether the NO synthase/NO/cyclic GMP pathway modulates the motility of pig isolated calyceal smooth muscle. Immunohistochemistry revealed a moderate overall innervation of the smooth muscle layer, and no neuronal or inducible NO synthase (NOS) immunoreactivities. Endothelial NOS immunoreactivities were observed in the urothelium and vascular endothelium, and numerous cyclic GMP-immunoreactive (-IR) calyceal smooth muscle cells were found. As measured by monitoring the conversion of L-arginine to L-citrulline, Ca(2+)-dependent NOS activity was moderate. Assessment of functional effects was performed in tissue baths and showed that NO and SIN-1 decreased spontaneous and induced contractions of isolated preparations in a concentration-dependent manner. In strips exposed to NO, there was a 10-fold increase of the cyclic GMP levels compared with control preparations (P < 0.01). It is concluded that a non-neuronal NOS/NO/cyclic GMP pathway is present in pig calyces, where it may influence motility. The demonstration of cyclic GMP-IR smooth muscle cells suggests that NO acts directly on these cells. This NOS/NO/cyclic GMP pathway may be a target for drugs inhibiting peristalsis of mammalian upper urinary tract. Neurourol. Urodynam. 18:673-685, 1999.

Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production.

Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under similar conditions, human macrophages produce low levels or no NO at all. Although human macrophages can express iNOS mRNA and protein on activation, whether they possess the complete machinery necessary for NO synthesis remains controversial. To define the conditions necessary for human monocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, following infection with a retroviral expression vector containing human hepatic iNOS (DFGiNOS). Northern blot and Western blot analysis confirmed the expression of iNOS in transfected U937 cells both at the RNA and protein levels. NOS enzymatic activity was demonstrated in cell lysates by the conversion of L-[3H]arginine into L-[3H]citrulline and the production of NO by intact cells was measured by nitrite and nitrate accumulation in culture supernatants. When expressing functional iNOS, U937 cells were capable of releasing high levels of NO. NO production was strictly dependent on supplementation of the culture medium with tetrahydrobiopterin (BH4) and was not modified by stimulation of the cells with different cytokines. These observations suggest that (1) human monocytic U937 cells contain all the cofactors necessary for NO synthesis, except BH4 and (2) the failure to detect NO in cytokine-stimulated untransfected U937 cells is not due to the presence of a NO-scavenging molecule within these cells nor to the destabilization of iNOS protein. DFGiNOS U937 cells represent a valuable human model to study the role of NO in immunity toward tumors and pathogens.

Role of nitric oxide in genotoxicity: implication for carcinogenesis.

Reactive oxygen species can initiate carcinogenesis by virtue of their capacity to react with DNA and cause mutations. Recently, it has been suggested that nitric oxide (NO) and its derivatives produced in inflamed tissues could contribute to the carcinogenesis process. Genotoxicity of NO follows its reaction with oxygen and superoxide. It can be due either to direct DNA damage or indirect DNA damage. Direct damage includes DNA base deamination, peroxynitrite-induced adducts formation and single strand breaks in the DNA. Indirect damage is due to the interaction of NO reactive species with other molecules such as amines, thiols and lipids. The efficiency of one pathway or another might depend on the cellular antioxidant status or the presence of free metals.

Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression.

An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ.

Decreased activity of inducible nitric oxide synthase type 2 and modulation of the expression of glutathione S-transferase alpha, bcl-2, and metallothioneins during the differentiation of CaCo-2 cells.

Reactive oxygen species modulate the cell growth of a wide variety of mammalian cells. To determine whether oxidative metabolism is altered during the differentiation process, we studied the expression of pro- and antioxidant proteins in proliferating and differentiated CaCo-2 cells, a human colon adenocarcinoma cell line. Nitric oxide synthase type 2 (iNOS) produces nitric oxide (NO). Depending on its rate of synthesis, NO may either promote cellular and DNA damage or reduce the ability of other free radicals to induce cell injury. Using Western and Northern blot analysis and arginine conversion assay, we demonstrate that the expression of iNOS decreases when cells undergo differentiation. This biological event entails a diminished production of NO metabolites and correlates with the loss of activation of soluble guanylate cyclase activity. In differentiated cells, a 2-fold down-regulation of the nuclear factor kappa B activity was observed, suggesting that nuclear factor kappa B could be one of the iNOS gene regulatory factors in the CaCo-2 model. In parallel, we studied the expression of other antioxidant proteins including glutathione S-transferase alpha (GST alpha), bcl-2, and the metallothioneins (MTs). We show that the protein levels of GST alpha and MT increase during the differentiation of CaCo-2 cells, whereas bcl-2 levels decrease. Our investigation indicates that the expression of iNOS, GST alpha, bcl-2, and MT is associated with the enterocytic differentiation. The shift in the expression of specific antioxidant genes during CaCo-2 cell differentiation may occur to avoid alterations in the cell redox potential.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Nitric oxide and ethylnitrosourea: relative mutagenicity in the p53 tumor suppressor and hypoxanthine-phosphoribosyltransferase genes.

Nitric oxide (NO) is a cellular messenger which is mutagenic in bacteria and human TK6 cells and induces deamination of 5-methylcytosine (5meC) residues in vitro. The aims of this study were: (i) to investigate whether NO induces 5meC deamination in codon 248 of the p53 gene in cultured human bronchial epithelial cells (BEAS-2B); and (ii) to compare NO mutagenicity to that of ethylnitrosourea (ENU), a strong mutagen. Two approaches were used: (i) a novel genotypic assay, using RFLP/PCR technology on purified exon VII sequence of the p53 gene; and (ii) a phenotypic (HPRT) mutation assay using 6-thioguanine selection. BEAS-2B cells were either exposed to 4 mM DEA/NO (Et2N[N2O2]Na, an agent that spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation assay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU induces detectable numbers of G --> A transitions in codon 248 of p53 while 5-methylcytosine deamination was not detected in either iNOS-transfected cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose-responsively mutagenic in the phenotypic HPRT assay, reaching mutation frequencies of 24 and 96 times that of untreated control cells at ENU concentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO induced no detectable mutations in this assay, nor were any observed in cells transfected with murine iNOS. We conclude that if NO is at all promutagenic in these cells, it is significantly less so than the ethylating mutagen, ENU.

Involvement of a transforming-growth-factor-beta-like molecule in tumor-cell-derived inhibition of nitric-oxide synthesis in cerebral endothelial cells.

Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat-brain-microvessel-derived endothelial cell line, produced NO through cytokine-inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derived NO-inhibitory factor was characterized. Northern-blot analysis demonstrated that the induction of iNOS mRNA in cytokine-activated EC219 cells was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Sepharose or heparin-Sepharose, the NO-inhibitory activity was found only in Con A-unbound or heparin-unbound fractions, respectively, indicating that the PROb-derived inhibitory factor was likely to be a non-glycosylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb-cell supernatant with anti-TGF-beta neutralizing antibody completely blocked the DHD/PROb-derived inhibition of NO production by EC219 cells. Addition of exogenous TGF-beta 1 dose-dependently inhibited NO release by EC219 cells. The presence of active TGF-beta in the DHD/PROb cell supernatant was demonstrated using a growth-inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF-beta, increased both TGF-beta activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta.