Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A

Dietary iron may contribute to colon cancer risk via production of reactive oxygen species (ROS). The aim of the study was to determine whether physiological ferric/ferrous iron induces oxidative DNA damage in human colon cells. Therefore, differentiated human colon tumour cells (HT29 clone 19A) were incubated with ferric-nitrilotriacetate (Fe-NTA) or with haemoglobin and DNA breaks and oxidised bases were determined by microgelelectrophoresis. The effects of Fe-NTA were measured with additional H(2)O(2) (75microM) and quercetin (25-100microM) treatment. Analytic detection of iron in cell cultures, treated with 250microM Fe-NTA for 15 min to 24h, showed that 48.02+/-5.14 to 68.31+/-2.11% were rapidly absorbed and then detectable in the cellular fraction. Fe-NTA (250-1000microM) induced DNA breaks and oxidised bases, which were enhanced by subsequent H(2)O(2) exposure. Simultaneous incubation of HT29 clone 19A cells with Fe-NTA and H(2)O(2) for 15 min, 37 degrees C did not change the effect of H(2)O(2) alone. The impact of Fe-NTA and H(2)O(2)-induced oxidative damage is reduced by the antioxidant quercetin (75-67% of H(2)O(2)-control). Haemoglobin was as effective as Fe-NTA in inducing DNA damage. From these results we can conclude that iron is taken up by human colon cells and participates in the induction of oxidative DNA damage. Thus, iron or its capacity to catalyse ROS-formation, is an important colon cancer risk factor. Inhibition of damage by quercetin reflects the potential of antioxidative compounds to influence this risk factor. Quantitative data on the genotoxic impact of ferrous iron (e.g. from red meat) relative to the concentrations of antioxidants (from plant foods) in the gut are now needed to determine the optimal balance of food intake that will reduce exposure to this type of colon cancer risk factor.     

2-Dodecylcyclobutanone, a radiolytic product of palmitic acid, is genotoxic in primary human colon cells and in cells from preneoplastic lesions

The irradiation of fat results in the formation of 2-alkylcyclobutanones, a new class of food contaminants. Results of previous in vitro studies with primary human colon cells and in vivo experiments with rats fed with 2-alkylcyclobutanones indicated that these radiolytic derivatives may be genotoxic and enhance the progression of colon tumors. The underlying mechanisms of these effects, however, are not clearly understood. Therefore we performed additional investigations to elucidate the genotoxic potential of 2-dodecylcyclobutanone (2dDCB) that is generated from palmitic acid. In particular, we explored the relative sensitivities of human colon cells, representing different stages of tumor development and healthy colon tissues, respectively. HT29clone19A cells, LT97 adenoma cells and primary human epithelial cells were exposed to 2dDCB (150-2097 microM). We determined cytotoxic effects using trypan blue exclusion. Genotoxicity, reflected as strand breaks, was assessed using the alkaline version of the comet assay and chromosomal abnormalities were investigated by 24-color fluorescence-in-situ-hybridization. 2dDCB was cytotoxic in a time- and dose-dependent manner in LT97 adenoma cells and in freshly isolated primary cells but not in the human colon tumor cell line. Associated with this was a marked induction of DNA damage by 2dDCB in LT97 adenoma cells and in freshly isolated colonocytes, whereas in the HT29clone19A cells no strand breaks were detectable. A long-term incubation of LT97 adenoma cells with lower concentrations of 2dDCB revealed cytogenetic effects. In summary, 2dDCB was clearly genotoxic in healthy human colon epithelial cells and in cells representing preneoplastic colon adenoma. These findings provide additional evidence that this compound may be regarded as a possible risk factor for processes in colon carcinogenesis related to initiation and progression.     

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.     

Inter- and intra-individual variation of faecal water - genotoxicity in human colon cells

Exogenous nutritional factors modulate the faecal contents leading to an enhanced or reduced burden with toxic and cancerogenic factors. These factors are thought to contribute to colon cancer by inducing mutations or enhancing proliferation in colon cells. Faecal water more or less causes these effects in model systems and thus could be the basis for valuable biomarker approaches. Our investigations are aimed at determining geno- and cytotoxicity of faecal water in human colon cell lines in vitro. We are developing techniques for their applicability as biomarker tests during dietary intervention studies. Faecal water is isolated by centrifugation of the faeces at 25000xg and added to cultured human colon cells (HT29). Membrane damage as assessed by trypan blue exclusion is determined as a measure for cytotoxicity. Semiquantitative analysis of inducible DNA damage (breaks and alkali labile sites) are analysed with the single cell microgelelectrophoresis assay (comet-assay) and oxidised DNA bases by the additional use of repair specific enzymes. We have now determined baseline toxic activities and calculated inter- and intra-individual and -experimental coefficients of variation for faecal water from different subjects consuming similar or different diets. Most faecal water induced DNA damage and oxidised DNA bases in HT29 clone 19a cells (0.9-9.14 fold and 1.7-4.9 fold, respectively in comparison to the NaCl controls). Intra- and inter-experimental coefficients (CV) of variation, were in a similar order of magnitude and ranged from 6.9 to 31.4. In contrast both intra- and inter-individual variability were considerably higher (CV-ranges of 29.7-76.6 and 21.3-64.0, respectively). Interestingly, these inter-individual values were not lowered when subjects consumed identical diets (CV-ranges of 28.4-126.0). However, following intervention with certain protective dietary regimens (e.g. lignan containing bread) significant reductions of faecal water-induced genotoxicity can be observed. Therefore, in spite of the expected and observed degrees of variation in this methodology, effective experimental protocols may still lead to detectable modulations of the level of toxic and genotoxic effects.     

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology.

Methods We have measured H₂O₂-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H₂O₂ and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H₂O₂-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage.

Results A dose-response relationship was found for the H₂O₂-induced dye decomposition in NIH3T3 cells (7.8-125 μM H₂O₂) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62-1000 μM H₂O₂). Fluorescence was significantly increased at 62 μM H₂O₂ in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H₂O₂ than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells.

Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H₂O₂-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H₂O₂.     

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology.

Methods We have measured H₂O₂-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H₂O₂ and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H₂O₂-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage.

Results A dose-response relationship was found for the H₂O₂-induced dye decomposition in NIH3T3 cells (7.8–125 μM H₂O₂) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62–1000 μM H₂O₂). Fluorescence was significantly increased at 62 μM H₂O₂ in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H₂O₂ than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells.

Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H₂O₂-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H₂O₂.     

Transferrin receptors and iron utilization in DMSO-inducible and -uninducible friend erythroleukemia cells

Dimethylsulfoxide (DMSO) induces hemoglobin synthesis and erythroid differentiation of Friend erythroleukemia cells in vitro. Induction is accompanied by increased transferrin-binding activity which is necessary for the cellular acquisition of iron from transferrin for hemoglobin synthesis. There are Friend cell variants in which hemoglobin synthesis is not induced by DMSO unless exogenous hemin is also present. In this study we have compared the inducibility of transferrin receptors and iron incorporation in DMSO-inducible (745) and -uninducible (M-18 and TG-13) Friend cell lines. Cellular transferrin-binding sites were estimated by Scatchard analysis of data obtained from specific binding of [125I]transferrin by the cells. Our results show that unlike 745, DMSO treatment of the variant cell lines M-18 and TG-13 does not result in increased transferrin-binding activity. The number of transferrin-binding sites and the rate of iron uptake is similar in uninduced 745 and DMSO-treated M-18 and TG-13 cells. Although exposure of M-18 cells to DMSO and hemin induces hemoglobinization, this treatment does not cause induction of transferrin receptors. These results indicate that the primary defect in M-18 cells may be the uninducibility of transferrin receptors. We have also shown that exposure of 745 cells to hemin during DMSO treatment prevents the induction of transferrin receptors, suggesting that hemin may control the expression of transferrin receptors in erythroid cells.     

Influence of iron-overload on DNA damage and its repair in human leukocytes in vitro

Iron is an important element that modulates the production of reactive oxygen species, which are thought to play a causative role in biological processes such as mutagenesis and carcinogenesis. The potential genotoxicity of dietary iron has been seldom studied in human leukocyte and only few reports have investigated in human colon tumor cells. Therefore, DNA damage and repair capacity of human leukocytes were examined using comet assay for screening the potential toxicity of various iron-overloads such as ferric-nitrilotriacetate (Fe-NTA), FeSO(4), hemoglobin and myoglobin, and compared with 200μM of H(2)O(2) and HNE. The iron-overloads tested were not cytotoxic in the range of 10-1000 microM by trypan blue exclusion assay. The exposure of leukocytes to Fe-NTA (500 and 1000 microM), FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) for 30 min induced significantly higher DNA damage than NC. Treatment with 500 and 1000 microM of Fe-NTA showed a similar genotoxic effect to H(2)O(2), and a significant higher genotoxic effect than HNE. The genotoxicity of FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) was not significantly different from that of H(2)O(2) and HNE. Iron-overloads generated DNA strand break were rejoined from the first 1h. Their genotoxic effect was not observed at 24h. These data from this study provide additional information on the genotoxicity of iron-overloads and self-repair capacity in human leukocytes.     

The role of heme in the regulation of the late program of Friend cell erythroid differentiation.

The addition of a chemical inducer, such as dimethylsulfoxide (DMSO), to cultures of mouse Friend erythroleukemic cells results in the induction of a number of late erythroid events, including the accumulation of globin mRNA, the inducation of hemoglobin synthesis, the appearance of erythrocyte membrane antigens (EMA), and the cessation of cell division. The experiments presented in this study demonstrate that heme is necessary but not sufficient for the loss of proliferative capacity associated with DMSO-induced Friend cell differentiation, whereas the accumulation of globin mRNA and EMA can occur in the absence of heme synthesis or heme itself. These conclusions were reached by selectively inhibiting heme synthesis in DMSO-treated cells in two independent ways: (i) Inducible cells were treated with 3-amino-1,2,4-triazole (AT), a drug which inhibits the induction of heme synthesis in Friend cells in a dose-dependent manner. Treatment of inducible Friend cells with 1.5% DMSO for five days caused the plating efficiency in methyl cellulose to decrease to 1% of that in untreated cultures. However, treatment of the cells with DMSO plus AT almost totally prevented this decrease in plating efficiency. The addition of exogenous hemin, which alone had no significant effect on plating efficiency, largely reversed the effect of AT in DMSO-treated cells, reducing the plating efficiency to below 5%. In contrast to the marked effects of AT on the proliferative capacity of differentiating Friend cells, the levels of globin mRNA and EMA were only partially decreased in cells treated with DMSO plus AT, compared to cells treated with DMSO alone. (ii) The relationship between heme synthesis, terminal cell division, and the induction of globin mRNA was investigated further through the use of non-inducible Friend cell variant clones. One such non-inducible clone, M18, appears to be a phenotypic analog of inducible cells treated with DMSO plus AT. Clone M18 did not accumulate heme or hemoglobin, as detected by benzidine staining, nor lose its proliferative capacity in response to DMSO. However, globin mRNA was induced by DMSO in this clone. Treatment of clone M18 with DMSO plus hemin overcame the block in hemoglobin accumulation suggesting that M18 has a defect in the induction of heme biosynthesis. In addition, exposure of M18 cells to DMSO plus hemin caused a gradual decrease in plating efficiency which was not due to non-specific toxicity. Prior incubation of M18 cells in DMSO for three to five days was necessary before hemin caused a rapid loss of proliferative capacity. Thus, these results, in agreement with the AT studies on inducible Friend cells and previous studies on the induction of EMA in clone M18, indicate that there may be both heme-dependent and heme-independent events in the program of Friend cell differentiation.     

Evidence that hydrogen sulfide is a genotoxic agent

Hydrogen sulfide (H2S) produced by commensal sulfate-reducing bacteria, which are often members of normal colonic microbiota, represents an environmental insult to the intestinal epithelium potentially contributing to chronic intestinal disorders that are dependent on gene-environment interactions. For example, epidemiologic studies reveal either persistent sulfate-reducing bacteria colonization or H2S in the gut or feces of patients suffering from ulcerative colitis and colorectal cancer. However, a mechanistic model that explains the connection between H2S and ulcerative colitis or colorectal cancer development has not been completely formulated. In this study, we examined the chronic cytotoxicity of sulfide using a microplate assay and genotoxicity using the single-cell gel electrophoresis (SCGE; comet assay) in Chinese hamster ovary (CHO) and HT29-Cl.16E cells. Sulfide showed chronic cytotoxicity in CHO cells with a %C1/2 of 368.57 micromol/L. Sulfide was not genotoxic in the standard SCGE assay. However, in a modified SCGE assay in which DNA repair was inhibited, a marked genotoxic effect was observed. A sulfide concentration as low as 250 micromol/L (similar to that found in human colon) caused significant genomic DNA damage. The HT29-Cl.16E colonocyte cell line also exhibited increased genomic DNA damage as a function of Na2S concentration when DNA repair was inhibited, although these cells were less sensitive to sulfide than CHO cells. These data indicate that given a predisposing genetic background that compromises DNA repair, H2S may lead to genomic instability or the cumulative mutations found in adenomatous polyps leading to colorectal cancer.     

Beta-carotene inhibits growth of human colon carcinoma cells in vitro by induction of apoptosis

Epidemiological studies suggest that beta-carotene is able to modulate the risk of cancer. A number of in vitro studies reported that beta-carotene inhibits the growth of cancer cells; however, so far little is known about the molecular mechanisms of the antiproliferative effect of beta-carotene. Here we have investigated the effects of two beta-carotene preparations, (i) beta-carotene dissolved in tetrahydrofuran (final concentration in cell culture medium: 0.5%) and (ii) beta-carotene incorporated in a water dispersible bead form, on cultured human colon carcinoma cells HT29. The treatment of cells with beta-carotene up to 30 microM for 72 h led to a significant increase in the cellular beta-carotene concentration and formation of retinol. Beta-Carotene showed only low cytotoxicity for confluent cells tested up to 30 microM, but at dietary relevant concentrations for the intestinal tract (10, 30 microM) beta-carotene was strongly cytotoxic for growing cells and induced apoptosis in HT29 cells as assessed by the Annexin-V assay (the maximal effect was observed 15 h after treatment with beta-carotene). Exposure of cells to retinol at concentrations yielding cellular retinol levels similar to those observed by beta-carotene treatment had no antiproliferative or cytotoxic effect. Furthermore, beta-carotene did not affect the activation of the extracellular signal-regulated kinases (ERK1 and ERK2) that are essential for cellular growth. In summary, beta-carotene can inhibit growth of human colon carcinoma cells in vitro by induction of apoptosis in proliferating cells.     

Quinone-induced apoptosis in human colon adenocarcinoma cells via DT-diaphorase mediated bioactivation

DT-diaphorase (DTD) activity has been related to bioactivation and cytotoxicity of antitumor quinones. A pair of human colon adenocarcinoma cell lines, HT29 and BE, were used in this study to examine the role of DTD in antitumor quinone induced apoptosis. HT29 cells have elevated levels of DTD whereas BE cells lack functional DTD due to a point mutation which results in a complete lack of DTD activity. MeDZQ, a quinone that is efficiently bioactivated by DTD, induced apoptosis both in HT29 and BE cells, but with a much higher incidence in HT29, as assessed by morphological criteria and the formation of oligonucleosomal fragments of DNA. Two other quinone compounds which are also substrates for DTD, i.e. streptonigrin and mitomycin C, also preferentially induced apoptosis in HT29 cells, which could be inhibited by dicoumarol. Our data suggest that bioreductive activation of antitumor quinones by DTD results in induction of apoptosis in human colon carcinoma cells.     

Effects of Hemin and Nitrite on Intestinal Tumorigenesis in the A/J Min/+ Mouse Model

Red and processed meats are considered risk factors for colorectal cancer (CRC); however, the underlying mechanisms are still unclear. One cause for the potential link between CRC and meat is the heme iron in red meat. Two pathways by which heme and CRC promotion may be linked have been suggested: fat peroxidation and N-nitrosation. In the present work we have used the novel A/J Min/+ mouse model to test the effects of dietary hemin (a model of red meat), and hemin in combination with nitrite (a model of processed meat) on intestinal tumorigenesis. Mice were fed a low Ca²⁺ and vitamin D semi-synthetic diet with added hemin and/or nitrite for 8 weeks post weaning, before termination followed by excision and examination of the intestinal tract. Our results indicate that dietary hemin decreased the number of colonic lesions in the A/J Min/+ mouse. However, our results also showed that the opposite occurred in the small intestine, where dietary hemin appeared to stimulate tumor growth. Furthermore, we find that nitrite, which did not have an effect in the colon, appeared to have a suppressive effect on tumor growth in the small intestine.     

Desferrioxamine protects human red blood cells from hemin-induced hemolysis

Hemin binding to red cell membranes, its effect on red cell hemolysis, and it interaction with desferrioxamine (DFO) in these processes were investigated. DFO interacted with hemin via the iron moiety. Blockage of the binding groups in DFO prevented interaction of DFO with hemin, implying the importance of the hydroxamic acid groups in DFO-hemin interactions. Since hemolysis is a result of hemin association with the membrane components, its binding in the presence and absence of DFO was studied. DFO strongly inhibited hemin-induced lysis in a concentration-dependent manner. With 50 microM hemin, 1 mM DFO completely inhibited lysis. Preincubation of ghost membranes with DFO (1 mM) inhibited binding of hemin (50 microM) to membranes by 42%. After ghost membranes were preincubated with hemin (50 microM), the addition of DFO (1 mM) removed 20% of the membrane-bound hemin. It is suggested that DFO may have an important role in alleviating the hemin-induced deleterious effects on the red cell membrane, especially in hemolytic anemias associated with unstable, autoxidized hemoglobins.     

Methemoglobin is a supplement for in vitro culture of human nasopharyngeal epithelial cells transformed by human Papillomavirus type 16 DNA

NPC-N cells were normal human nasopharvngeal epithelial cells transformed by transfection with human papillomavirus type 16 deoxyribonucleic acid. Bovine pituitary extract (BPE) was one of the indispensable ingredients for in vitro culture of NPC-N cells in a serum-free medium. Chromatographic fractionation of BPE and subsequent immunoblotting analyses identified the hemoglobin growth-stimulating factor. Methemoglobin (metHb) was then synthesized, and also found to be growth stimulating. The growth-stimulating effect of metHb was abolished when NPC-N cells were cultured in a medium that also contained haptoglobin, a molecule that binds to hemoglobin. A defined medium consisting of insulin and metHb was then developed for optimal growth of NPC-N cells. MetHb kept under the conditions identical to those of cell culture released hemin which also enhanced the cell growth. Though all the degradation products of hemin are currently known to be physiologically significant. only ferric iron derived from metHb or hemin could stimulate the growth of NPC-N cells. Abnormal vasculature showing leaky walls and hemorrhage is a common feature of malignant tumors. Hemoglobin originating from extravasated red blood cells and subsequently oxidized to metHb because of the presence of activated inflammatory cells might contribute to the increased proliferation of cancerous cells.     

Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation

A clone HT29-18 has been isolated from the parent cell line HT-29, which derived from a human colon adenocarcinoma (Fogh, J., and G. Trempe, 1975, Human Tumor Cells in Vitro, J. Fogh, editor, Plenum Publishing Corp., New York, 115-141). This clone is able to differentiate as the parent cell line does. Differentiation occurs when glucose is replaced by galactose in the culture medium (Pinto, M., M.D. Appay, P. Simon-Assman, G. Chevalier, N. Dracopoli, J. Fogh, and A. Zweibaum, 1982, Biol. Cell., 44:193-196). We demonstrate here that the differentiated cloned population HT29-18/gal is heterogenous: although 90% of the cells show morphological characteristics of "absorptive cells", only 20-30% of them display sucrase-isomaltase in their apical microvillar membranes. About 10% of the entire cell population consists of cells containing mucous granules similar to intestinal goblet cells. We have isolated two subclones, HT29-18-C1 and HT29-18-N2, from the differentiated HT29-18/gal cells. HT29-18-C1 cells show morphological characteristics of polarized absorptive cells, when growing either in glucose- or in galactose-containing media, but the sucrase-isomaltase is not expressed in the cells grown in glucose-containing medium. The clone HT29-18-N2 is also polarized in both culture conditions and is similar to globlet cells in vivo. It grows as a monolayer, exhibits tight junctions, and contains numerous mucous granules whose exocytosis can be triggered by carbachol, a parasympathomimetic drug. We conclude that the clone HT29-18 first isolated was a multipotent cell population from which we isolated several subclones that differentiate either as absorptive (HT29-18-C1) or as mucous (HT29-18-N2) cells. In contrast to the parent HT-29 cell line, the subclones retain most of their differentiated properties in glucose-containing medium.     

CD4 molecules are restricted to the basolateral membrane domain of in vitro differentiated human colon cancer cells (HT29-D4)

The CD4 glycoprotein serves as a receptor for the human immunodeficiency virus HIV, the etiologic agent of acquired immunodeficiency syndrome (AIDS). We have examined the expression of CD4 molecules in a clone (HT29-D4) derived from a human colon adenocarcinoma cell line. HT29-D4 cells synthesized a 60 kDa polypeptide immunoprecipitated with two anti-CD4 monoclonal antibodies after metabolic or cell surface labeling. This 60 kDa polypeptide was also immunodetected using the same antibodies in human acute lymphoblastic leukemia cells CEM which are known to express CD4. HT29-D4 cells can be induced to differentiate into enterocyte-like cells by removing glucose from the culture medium. Under these conditions, HT29-D4 cells form a polarized epithelial monolayer in which tight junctions separate the plasma membrane in an apical and a basolateral domain. The localization of CD4 molecules in differentiated HT29-D4 cells was exclusively restricted to the basolateral membrane domain as demonstrated by radioimmunoassay and indirect immunofluorescence studies. Therefore the HT29-D4 clonal cell line represents a unique model for polarized HIV infection of colonic epithelial cells and may be useful to understand some of the gastrointestinal disorders occurring in AIDS patients.     

Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein

Knowledge regarding the expression of the recently cloned estrogen receptor beta (ERbeta) in colonic mucosa is limited. In this study, we demonstrated that five human colon cancer cell lines, HT29, Colo320, Lovo, SW480, and HCT116, expressed ERbeta mRNA, but lacked ERalpha mRNA. Results from a cell growth assay demonstrated that these colon cancer cells were not influenced by estrogen, while genistein possessed slight growth inhibitory effects on HT29, Colo320 and Lovo cells at 10 microM, at which concentration is stimulated the growth of ERalpha-positive human breast cancer MCF-7 cells. Tamoxifen inhibited the growth of HT29 and Colo320 cells, dose-dependently, as well as MCF-7 cells. A transfected reporter plasmid containing a vitellogenin estrogen response element could be activated by estradiol in Colo320 cells. Taken together with previous reports, these data suggest that ERalpha and ERbeta may have different biological functions in colon cells.     

Sodium 5,6‐benzylidene‐L‐ascorbate induces oxidative stress,autophagy, and growth arrest in human colon cancer HT‐29 cells

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT‐29 cells with SAA and SBA, and found a significant exposure time‐dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC₅₀ = 5 mM) than SBA (IC₅₀ = 10 mM). A short‐term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase‐dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21^+/+ and p21^?/?), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co‐incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N‐acetyl‐L‐cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3‐II were found in SBA‐treated HT‐29 cells; implicating that SBA induced AKT phosphorylation‐autophagy and p21‐growth arrest in colon cancer HT‐29 cells through an NAC‐inhibitable oxidative stress pathway. J. Cell. Biochem. 111: 412–424, 2010. © 2010 Wiley‐Liss, Inc.     

The effect of hemin in vitro and in vivo on human erythroid progenitor cells

Hemin stimulates erythropoiesis and hemoglobin synthesis in vitro. We cultured erythroid progenitor cells from normal individuals, patients with sickle cell anemia, and a patient with acute variegate porphyria who received intravenous hemin treatment, with 0-800 microM hemin added in vitro. Fifty to 200 microM hemin consistently stimulated colony growth from normal donors 2- to 8-fold, while concentrations of up to 400 microM were stimulatory in cultures from donors with sickle cell anemia. In vivo hemin decreased the number of blood BFU-e in the patient with porphyria, but did not abrogate the in vitro stimulatory effect of hemin. Hemin concentrations which increased colony numbers increased gamma globin synthesis in some studies and decreased it in others. Hemin thus has clearcut erythroid growth-potentiating activity, although a consistent effect on globin chain regulation is not apparent.