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PubMed2006

The range of optimal concentration and mechanisms of paclitaxel in radio-enhancement in gastrointestinal cancer cell lines.

AIM: This study was performed to investigate the range of optimal concentration and mechanisms of paclitaxel (PXL) radio-enhancement in gastrointestinal cancer cell lines HT29 and MKN45. METHODS: Cell growth inhibition by PXL pretreatment at various concentrations (0-10 microM) followed by irradiation was investigated using a modified MTT assay. To investigate the mechanisms of the observed radio-enhancement, flow cytometry was conducted to define the cell cycle distributions. Furthermore, the alterations in expression of a DNA repair molecule [excision repair cross-complementation group1 (ERCC1)] and an angiogenesis factor [vascular endothelial growth factor (VEGF)] induced by PXL were investigated. RESULTS: Cytotoxic concentrations of PXL (0.1-10 microM) that cause accumulation of cells in the G2/M phase have strong radio-enhancing effects and inhibit total cell growth. The maximal non-cytotoxic concentration of PXL (0.01 microM) also had a radio-enhancing effect. The expression of the genes of ERCC1 and VEGF induced by radiation was suppressed by PXL pretreatment. The protein secretion of VEGF induced by radiation was suppressed at cytotoxic doses of PXL, and the induced protein secretion of ERCC1 was also suppressed even at maximal non-cytotoxic doses of PXL. CONCLUSION: The range of optimal concentration for PXL pretreatment was 0.01-0.1 microM in these cells. Two major mechanisms of radio-enhancement are suggested: (1) PXL induces G2/M arrest leading to increased DNA damage after radiation, which results in mitotic death, and (2) PXL suppresses the expression of radiation-induced DNA repair molecules and angiogenesis factors, resulting in inhibition of cell growth and cell death.

Adenocarcinoma↗
PubMed1993

Mitotic arrest-associated enhancement of O-linked glycosylation and phosphorylation of human keratins 8 and 18.

Arrest of the human colonic cell line HT29 at the G2/M phase of the cell cycle resulted in changes in keratin assembly that were coupled with a significant increase in the O-linked glycosylation and serine phosphorylation of keratin polypeptides 8 and 18 (K8/18). With mitotic arrest, enhanced keratin phosphorylation occurred preferentially on K8, whereas K18 showed a higher glycosylation level than K8. Removal of the arresting agent allowed cells to proceed through the cell cycle with a concomitant decrease in K8/18 glycosylation. In contrast, keratins isolated from S phase-enriched cells, obtained after synchronization with aphidicolin, did not show enhanced glycosylation. Tryptic peptide analysis of keratins in G2/M-arrested cells showed changes in the glycopeptide pattern of K8 and in the phosphopeptide patterns of K8 and K18. Labeling of K8/18 immunoprecipitates, isolated from G2/M-arrested cells, with [3H]galactose followed by beta-elimination showed that K8/18 glycosylation consisted of single N-acetylglucosamine residues. Threonine was identified as the site of glycosylation after comparing acid hydrolysis products of beta-eliminated and non-beta-eliminated K8 and K18. Specific cleavage at tryptophan residues indicated that K18 glycosylation and phosphorylation were restricted to the head and proximal rod domains, whereas K8 did not show the same restriction. Our results show a unique association of the single O-linked N-acetylglucosamine type of modification of keratins with mitotic arrest in HT29 cells. There was no reciprocal relationship between K8/18 glycosylation and phosphorylation, and each keratin showed a preferential G2/M cell cycle-associated increase in either serine phosphorylation or threonine glycosylation.

Aphidicolin↗
PubMed1990

Selective immunosuppressive action of a factor produced by colon cancer cells.

A soluble substance (HT29 factor) produced by HT29 colon cancer cells markedly suppresses mitogen-induced T-cell proliferation and interleukin-2 production. In this study the range of T-cell functions susceptible to the inhibitory effects of the HT29 factor was evaluated. Serum-free conditioned medium was collected from confluent cultures of HT29 cancer cells, concentrated, and subjected to gel filtration chromatography and anion exchange chromatography, resulting in 24.4-fold purification of the HT29 factor with 31% yield. This factor abolished the development of lymphokine-activated killer cells when present during activation of peripheral blood lymphocytes by interleukin-2 but did not affect the lysis of target cells by normal effectors when added in the lysis stage only. The HT29 factor did not affect the generation of concanavalin A-induced suppressor lymphocytes, even though it markedly inhibited synthesis of DNA, RNA, and protein as well as expression of the CD25 (Tac) antigen during mitogen activation of T-cells. The HT29 factor itself did not induce suppressor cells. These results indicate that the immunosuppressive action of the HT29 factor is selective.

Colonic Neoplasms↗
PubMed1999

Tumor necrosis factor-alpha and interferon-gamma induce expression of functional Fas ligand on HT29 and MCF7 adenocarcinoma cells.

Tumor cells develop diverse mechanisms to escape from immune surveillance, including expression of Fas ligand (FasL), a 40-kDa type II transmembrane protein that mediates apoptosis by binding to its cognate receptor Fas (APO-1/CD95). Upon activation, T lymphocytes and natural killer (NK) cells express Fas and thus become sensitive to FasL-mediated apoptosis. Here we show that tumor necrosis factor-alpha (TNF-alpha) in addition to interferon-gamma (IFN-gamma) induces cell surface expression of functional FasL in human HT29 colon and MCF7 breast adenocarcinoma cells that constitutively lack cell surface expression of FasL. These cells, expressing FasL, are capable of inducing apoptosis in Fas-positive Jurkat T cells mediated by plasma membrane-bound FasL and soluble forms of FasL. By contrast, mutational deletion of Fas cell surface expression as well as inhibition of caspase family proteases involved in Fas signaling and monoclonal antibodies neutralizing FasL protect Jurkat T cells from apoptosis caused by the FasL-expressing HT29 or MCF7 cells. These results demonstrate that TNF-alpha and IFN-gamma induce expression of functional FasL in adenocarcinoma cells which thereby can kill T lymphocytes by the FasL/Fas pathway.

Adenocarcinoma↗
PubMed1995

In vivo adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma-derived tumors induces chemosensitivity to 5-fluorocytosine.

To evaluate the concept that in vivo transfer of the Escherichia coli cytosine deaminase gene will confer sensitivity of a solid tumor to the prodrug 5-fluorocytosine (5FC), we constructed an adenovirus vector (AdCMV.CD) carrying the cytosine deaminase gene driven by the cytomegalovirus (CMV) promoter, infected HT29 colon carcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CD produced a functional cytosine deaminase protein in HT29 cells in vitro as evidenced by the ability of lysates from the infected cells to convert [3H]5FC to its active metabolite 5-fluorouracil (5FU). The AdCMV.CD vector effectively suppressed HT29 cell growth in vitro in the presence of 5FC in a dose-dependent manner. Infection with AdCMV.CD, when as few as 10% of cells expressed the cytosine deaminase gene, was associated with a bystander effect when combined with 5FC in cell mixing studies. Further, this bystander effect was not dependent on cell-to-cell contact as demonstrated by suppression of [3H]thymidine incorporation in HT29 cells when supernatant from AdCMV.CD-infected cells treated with 5FC was transferred cells. Consistent with these in vitro observations, when AdCMV.CD was directly injected into established subcutaneous HT29 tumors in nude mice receiving 5FC, there was a four-fold reduction in tumor size at day 15 compared to controls, and a five-fold reduction at day 28. These observations suggest that adenovirus-mediated gene transfer of the E. coli cytosine deaminase gene and concomitant administration of 5FC may have potential as a strategy for local control of the growth of tumor cells susceptible to 5FU.

Adenoviridae↗
PubMed2002

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 gamma-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.

Adaptation, Physiological↗
PubMed2006

A recombinant human RNASET2 glycoprotein with antitumorigenic and antiangiogenic characteristics: expression, purification, and characterization.

BACKGROUND: Human RNASET2 is a T2-RNase glycoprotein encoded by the RNASET2 gene, which is located on chromosome 6 (6q27). Deletion in 6q27 is associated with several human malignancies. METHODS: A synthetic RNASET2 gene that was optimized for expression in the yeast Pichia pastoris was designed according to the cDNA sequence and was cloned under the control of the methanol-induced promoter fused to the alpha-mating secretion peptide. The recombinant protein was purified from the culture supernatant of transformed P. pastoris through an affinity Sepharose-concanavalin A column. Actin-binding activity was examined by membrane blotting using monoclonal mouse antiactin immunoglobulin M and by cross-linking in solution to G-actin using 1-[3-(dimethylamino)propyl]-3-ethyl-carboimide methiodide. The antiangiogenic activity of RNASET2 (from 0.5 microM to 10 microM) was assessed by a human umbilical vein endothelial (HUVE) cell assay in the presence of 1 microg/mL angiogenin, basic fibroblast growth factor (bFGF), or recombinant human vascular endothelial growth factor (VEGF). Cell colony formation was examined in human colon HT29 cancer cells to assess the antitumorigenic activity of RNASET2 or the enzymatic-inactivated RNASET2 (EI-RNASET2) (1 microM each). In an athymic mouse xenograft model, LS174T human cancer cells were injected subcutaneously. When tumors were palpable, the mice were treated for 3 weeks with RNASET2 (1 mg/kg), paclitaxel (10 mg/kg or 15 mg/kg), or a combination of the 2 drugs. RESULTS: The recombinant RNASET2 was identified as a 27-kilodalton glycoprotein that possessed the ability to bind actin in vitro. RNASET2 significantly inhibited clonogenicity in HT29 cells. EI-RNASET2 produced a similar effect, suggesting that its antitumorigenic activity is unrelated to its RNase activity. In HUVE cells, RNASET2 inhibited angiogenin-, bFGF-, and VEGF-induced tube formation in a dose-dependent manner. In athymic mice, RNASET2 inhibited the development of an LS174T-derived xenograft by 40%. A synergistic effect was obtained with combined RNASET2 and paclitaxel treatments. CONCLUSIONS: The current results suggested that RNASET2 represents a new class of antitumorigenic and antiangiogenic drugs, and the findings of this study emphasize the advantage of using agents like RNASET2 in combined therapy.

Angiogenesis Inhibitors↗
PubMed2005

Inhibitors of the epidermal growth factor receptor in apple juice extract.

The polyphenol-rich extract of a consumer-relevant apple juice blend was found to potently inhibit the growth of the human colon cancer cell line HT29 in vitro. The epidermal growth factor receptor (EGFR) and its subsequent signaling cascade play an important role in the regulation of cell proliferation in HT29 cells. The protein tyrosine kinase activity of an EGFR preparation was effectively inhibited by the polyphenol-rich apple juice extract. Treatment of intact cells with this extract resulted in the suppression of the subsequent mitogen-activated protein kinase cascade. Amongst the so far identified apple juice constituents, the proanthocyanidins B1 and B2 as well as quercetin-3-glc (isoquercitrin) and quercetin-3-gal (hyperoside) were found to possess substantial EGFR-inhibitory properties. However, as to be expected from the final concentration of these potential EGFR inhibitors in the original polyphenol-rich extract, a synthetic mixture of the apple juice constituents identified and available so far, including both proanthocyanidins and the quercetin glycosides, showed only marginal inhibitory effects on the EGFR. These results permit the assumption that yet unknown constituents contribute substantially to the potent EGFR-inhibitory properties of polyphenol-rich apple juice extract. In summary, the polyphenol composition of apple juice possesses promising growth-inhibitory properties, affecting proliferation-associated signaling cascades in colon tumor cells.

Beverages↗
PubMed2001

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.

Carbohydrate Metabolism↗
PubMed2000

Sik (BRK) phosphorylates Sam68 in the nucleus and negatively regulates its RNA binding ability.

Sik (mouse Src-related intestinal kinase) and its orthologue BRK (human breast tumor kinase) are intracellular tyrosine kinases that are distantly related to the Src family and have a similar structure, but they lack the myristoylation signal. Here we demonstrate that Sik and BRK associate with the RNA binding protein Sam68 (Src associated during mitosis, 68 kDa). We found that Sik interacts with Sam68 through its SH3 and SH2 domains and that the proline-rich P3 region of Sam68 is required for Sik and BRK SH3 binding. In the transformed HT29 adenocarcinoma cell cell line, endogenous BRK and Sam68 colocalize in Sam68-SLM nuclear bodies (SNBs), while transfected Sik and Sam68 are localized diffusely in the nucleoplasm of nontransformed NMuMG mammary epithelial cells. Transfected Sik phosphorylates Sam68 in SNBs in HT29 cells and in the nucleoplasm of NMuMG cells. In functional studies, expression of Sik abolished the ability of Sam68 to bind RNA and act as a cellular Rev homologue. While Sam68 is a substrate for Src family kinases during mitosis, Sik/BRK is the first identified tyrosine kinase that can phosphorylate Sam68 and regulate its activity within the nucleus, where it resides during most of the cell cycle.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Edible mushroom (Agaricus bisporus) lectin, which reversibly inhibits epithelial cell proliferation, blocks nuclear localization sequence-dependent nuclear protein import.

The Galbeta1-3GalNAcalpha (TF antigen)-binding lectin (ABL) from the common edible mushroom (Agaricus bisporus) has a potent anti-proliferative effect without any apparent cytotoxicity. This unusual combination of properties prompted investigation of its mechanism of action. In contrast to soluble lectin, agarose-immobilized, and hence noninternalizable ABL had no effect on proliferation of HT29 colon cancer cells. Electron microscopy of HT29 cells incubated with fluorescein- and gold-conjugated ABL showed internalization of the lectin into endocytotic vesicles and multivesicular bodies. Confocal microscopy showed perinuclear accumulation of fluorescein isothiocyanate-conjugated lectin, which also inhibits HT29 cell proliferation, raising the possibility that the lectin might interfere with nuclear pore function. Transport of heat shock protein 70 into the nucleus in response to heat shock was blocked by preincubation of HT29 cells for 6 h with 40 micrograms/ml ABL. In digitonin-permeabilized cells, nuclear uptake of bovine albumin conjugated to a nuclear localization sequence (NLS)-containing peptide was also inhibited by a 15-min preincubation with 40-100 micrograms/ml ABL. In contrast, serum-stimulated nuclear translocation of mitogen-activated protein kinase, which is NLS-independent, was not affected by pretreatment of cells with the lectin. These results suggest that the anti-proliferative effect of ABL is likely to be a consequence of the lectin trafficking to the nuclear periphery, where it blocks NLS-dependent protein uptake into the nucleus.

Adult↗
PubMed2003

Serine/threonine protein phosphatase inhibition enhances the effect of thymidylate synthase inhibition.

PURPOSE: The serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) are key enzymes in regulating entry into the cell cycle, mitosis and apoptosis. Inhibition of PP1 and PP2A is associated with enhanced S-phase entry culminating in G(2)/M arrest and apoptotic cell death. Thymidylate synthase (TS) is a key regulatory enzyme in DNA synthesis, inhibition of which is often a first-line treatment for colorectal carcinoma. In this study the effect of combining PP inhibition with TS inhibition in two colorectal cell lines was examined. METHODS: Cantharidin and nolatrexed were used to inhibit PP and TS activity, respectively. The MTT cytotoxicity assay and cell cycle analysis were performed following single-drug treatment of HT29 and HCT116 colorectal cell lines. The median effect method was used to determine a combination index (CI), where drug antagonism was indicated by a CI>1.1, additivity by a CI between 0.9 and 1.1, and synergism by a CI<0.9. RESULTS: Both cell lines were equally sensitive to cantharidin alone (GI(50) values 5.4 and 7.3 micro M), which induced a significant increase in the S-phase population of both cell lines within 6 h with a concomitant increase in DNA synthesis. This response culminated in G(2)/M cell cycle arrest within 24 h and subsequent cell death. In response to nolatrexed alone, HT29 cells were more sensitive than HCT116 cells (GI(50) 1.9 micro M vs 9.8 micro M), with G(1)/S-phase cell cycle arrest occurring within 24 h in both cell lines. In HT29 cells, this was followed by cell death, whereas in HCT116 cells, a proportion of cells died following arrest but the predominant event was re-entry into the cell cycle. The simultaneous exposure of HT29 cells to the combination of nolatrexed and cantharidin in drug molar ratios of 1:1 and 1:2.5 for 72 h was synergistic producing composite CIs of 0.88 and 0.87, respectively. The sequence of nolatrexed followed by cantharidin 24 h later resulted in greater synergism (CI values of 0.75, 0.52, 0.55, 0.68 for molar ratios of 10:1, 1:1, 1:2.5, 1:10), whereas the reverse sequence was antagonistic, suggesting that the point of interaction is downstream of TS inhibition. In HCT116 cells only additive and antagonistic interactions were observed for any of the treatment combinations. The lack of synergism in these cells may be caused by the reduced sensitivity of these cells to nolatrexed as a single agent. CONCLUSION: The effect of TS inhibition can be enhanced by the inhibition of serine/threonine protein phosphatases.

Antimetabolites, Antineoplastic↗
PubMed2001

Interaction between bacterial peptides, neutrophils and goblet cells: a possible mechanism for neutrophil recruitment and goblet cell depletion in colitis.

Goblet cell depletion occurs in various forms of colitis, but its mechanism is unknown. We have investigated two linked hypotheses: (i) that bacterial peptides, such as formyl-methionyl-leucyl-phenylalanine (fMLP), interact with epithelial cells inducing the release of chemokines, including interleukin-8 (IL-8), which in turn leads to the recruitment of neutrophils which release mucin secretagogues; (ii) that fMLP acts directly on epithelial cells to cause mucus secretion. Studies were performed to measure the effects of fMLP on the synthesis and secretion of IL-8 and mucus by the goblet cell differentiated colon cancer cell lines HT29-MTX (methotrexate-conditioned HT29 colonic adenocarcinoma cell line) and LS174T, and to assess the effects of neutrophil-derived secretagogues on goblet cell secretion in these cell lines. fMLP (0.1 microM) increased the secretion of IL-8 by 105% (P<0.0001) in HT29-MTX cells and by 401% (P<0.0001) in LS174T cells. fMLP also increased the synthesis and secretion of mucins by these cell lines, with maximal effects of 65% above control values for synthesis (P<0.01) and 73% for secretion (P<0.01). A dose-related increase (up to 67%; P<0.01) in mucin secretion was demonstrated in HT29-MTX cells in response to incubation with supernatant from activated neutrophils. This effect was largely (83%; P<0.02) inhibited by ICI 200,355, a specific inhibitor of neutrophil elastase. In conclusion, the bacterial peptide fMLP and neutrophil elastase are both potent mucus secretagogues for colon epithelial cells. fMLP also elicits release of the potent neutrophil chemoattractant IL-8 from colon epithelial cells. These findings support the hypothesis that the mucosal inflammation and mucus depletion seen in ulcerative colitis could result from interaction between bacterial peptides and the mucosa.

Bacterial Proteins↗
PubMed1989

The effect of modifying the culture medium on cell polarity in a human colon carcinoma cell line.

The human colonic cancer cell line HT29, when grown in DMEM, forms a morphologically unpolarized cell culture in which the cells are covered with irregular microvilli and devoid of belt zonula occludens type tight junctions. However, by modifying the culture medium and growing the cells in RPMI, a different morphology was obtained. A large number of intracellular luminae appeared and at late confluency 90-95% of cells exhibited an apical brush border after four subsequent passages. Junctional complexes and a well developed zonula occludens were revealed under the apical brush border. Immuno-electron microscopical localization of specific markers, sucrase isomaltase (SI), secretory components (SC) and beta 2 microglobulin (beta 2M) showed that SI was limited to the apical surface, whereas 2M and SC were located at the basolateral surfaces. These results indicate that modification of culture conditions affects the ability of HT29 cells to express epithelial cell polarity.

Carcinoma↗
PubMed1992

The effect of the hypoxic cell drug SR-4233 alone or combined with the ionizing radiations on two human tumor cell lines having different radiosensitivity.

The influence of the hypoxic cell drug, SR-4233, alone and/or combined with ionizing radiations on the survival of two human cell lines having very different intrinsic radiosensitivity was analysed. The killing effect of SR-4233 in hypoxia was similar for both cell lines. Twenty microM SR-4233 under hypoxic conditions had a killing effect equivalent to 6.6 Gy for the poorly sensitive cell line (HT29) and equivalent to 3.9 Gy for the highly sensitive cell line (SW48). The effect of SR-4233 and ionizing radiations on these two cell lines was tested in vitro: the cells were incubated for one hour in hypoxia with or without 20 microM SR-4233 and then irradiated in air. The HT29 cells that survived treatment with SR-4233 are more radiosensitive than untreated cells. However, their radiosensitivity is similar to that of cells that have been given a dose of 6.6 Gy. This suggests that SR-4233 acts additively, rather than as a radiosensitiser. As SR-4233 acts selectively in hypoxia, these results appear encouraging for the treatment of poorly-radiosensitive human tumors.

Cell Hypoxia↗
PubMed2001

In vitro induction of PDT resistance in HT29, HT1376 and SK-N-MC cells by various photosensitizers.

Our approach to examine the mechanism(s) of action for photodynamic therapy (PDT) has been via the generation of PDT-resistant cell lines. In this study we used three human cell lines, namely, human colon adenocarcinoma (HT29), human bladder carcinoma and human neuroblastoma. The three photosensitizers used were Photofrin, Nile Blue A and aluminum phthalocyanine tetrasulfonate. The protocol for inducing resistance consisted of repeated in vitro photodynamic treatments with a photosensitizer to the 1-10%-survival level followed by regrowth of single surviving colonies. Varying degrees of resistance were observed. The three induced variants of the HT29 cell line were the most extensively studied. Their ratios of increased survival at the LD90 level range between 1.5- and 2.62-fold more resistant.

Cell Survival↗
PubMed2004

A new source of hepatocytes for transplantation.

INTRODUCTION: The most effective treatment for acute or chronic liver failure is orthotopic liver transplantation. Worldwide there is a shortage of organs for transplantation. This shortage has called for research into new treatments for management of patients with liver failure. One such treatment is hepatocyte transplantation. During liver resections considerable amounts of normal liver are unavoidably resected. We aim to harvest these hepatocytes and to filter the tumor cells from them to provide a source for transplantation. MATERIALS AND METHODS: After liver resection, the largest vessel at the resected liver edge was identified and cannulated. Seglen's two-stage technique of perfusing the liver with EDTA and collagenase was performed to harvest the hepatocytes. Ep-CAM Ags are consistently present on the surface of epithelial cells and in particular in colorectal cancer cells. Therefore, MOC31 antibodies (selective Abs for Ep-CAM) attached to magnetic beads were used to target the tumor cells. These tumor cells are selectively removed using a magnet. CEA staining was then used to ensure the hepatocyte collection was tumor cell free. Five million hepatocytes were rosetted with one million HT29 CRC cells to assess the immunomagnetic filtration technique. RESULTS: The hepatocyte harvesting resulted in 864,000 viable hepatocytes to be harvested per gram of liver. Histochemical staining using CEA demonstrated 75% of the HT29 cells in the hepatocyte collection were removed after one use of magnetic beads. CONCLUSION: We have demonstrated the successful initial stages of harvesting tumor-free hepatocytes from liver resected for malignancy.

Aged↗
PubMed2000

Regulation of [Ca(2+)](i) homeostasis in MRP1 overexpressing cells.

Regulation of capacitative Ca(2+) entry was studied in two different multidrug resistance (MDR) protein (MRP1) overexpressing cell lines, HT29(col) and GLC4/ADR. MRP1 overexpression was accompanied by a decreased response to thapsigargin. Moreover, inhibition of capacitative Ca(2+) entry by D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was abolished in MRP1 overexpressing cells. Both PDMP and the MRP1 inhibitor MK571 greatly reduced InsP(3)-mediated (45)Ca(2+) release from intracellular stores in HT29 cells. Again, these effects were virtually abolished in HT29(col) cells. Our results point to a modulatory role of MRP1 on intracellular calcium concentration ([Ca(2+)](i)) homeostasis which may contribute to the MDR phenotype.

ATP-Binding Cassette Transporters↗