Antioxidants enhance the cytotoxicity of chemotherapeutic agents in colorectal cancer: a p53-independent induction of p21WAF1/CIP1 via C/EBPbeta.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Five-fluorouracil (5FU) remains the single most effective treatment for advanced disease, despite a response rate of only 20%. Herein, we show that the antioxidants pyrrolidinedithiocarbamate and vitamin E induce apoptosis in CRC cells. This effect is mediated by induction of p21WAF1/CIP1, a powerful inhibitor of the cell cycle, through a mechanism involving C/EBPbeta (a member of the CCAAT/enhancer binding protein family of transcription factors), independent of p53. Antioxidants significantly enhance CRC tumor growth inhibition by cytotoxic chemotherapy in vitro (5FU and doxorubicin) and in vivo (5FU). Thus, chemotherapeutic agents administered in the presence of antioxidants may provide a novel therapy for colorectal cancer.

Leukemic HRX fusion proteins inhibit GADD34-induced apoptosis and associate with the GADD34 and hSNF5/INI1 proteins.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.

Antioxidants reduce cyclooxygenase-2 expression, prostaglandin production, and proliferation in colorectal cancer cells.

Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Recent observations suggest that reactive oxygen intermediates play a role in tumor cell growth regulation and expression of the inducible COX, COX-2. We therefore evaluated the effects of various antioxidants on COX expression and cellular growth in the human CRC cell line HCA-7. The antioxidants pyrrolidinedithiocarbamate (PDTC), N-acetylcysteine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and U74006 decreased PG production, intracellular redox status, and cellular growth in a concentration-dependent manner. The decrease in cellular growth was associated with the induction of apoptosis. Unlike the selective COX inhibitors 1-[(4-methylsulfonyl)phenyl]-3-trifluoromethyl-5-[(4-fluoro)phenyl]pyraz ole (SC 58125) and (2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS 398) that inhibit COX-2 catalytic activity, these antioxidants decreased COX-2 expression at the transcriptional level. Combined treatment of HCA-7 cells with PDTC and SC 58125 resulted in an additive decrease in PG levels and anchorage-dependent and -independent growth. Furthermore, whereas antioxidants or SC 58125 reduced tumor growth in vivo, coadministration of PDTC and SC 58125 resulted in actual tumor regression. These results suggest that combined therapy with NSAIDs and antioxidants might be useful in the prevention and/or treatment of CRC.

Prostaglandin J2 and 15-deoxy-delta12,14-prostaglandin J2 induce proliferation of cyclooxygenase-depleted colorectal cancer cells.

Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Nonsteroidal anti-inflammatory agents (NSAIDS) inhibit growth of various CRC cell lines by both COX-dependent and COX-independent pathways. To specifically examine the effect of COX and PGs on proliferation in CRC cells, we introduced an antisense COX-2 cDNA construct under the control of a tetracycline (Tc)-inducible promoter into a CRC cell line, HCA-7, Colony 29 (HCA-7) that expresses COX and produces PGs. In the presence of Tc, PG production in COX-depleted cells was reduced 99.8% compared with either uninduced transfectants or parental HCA-7 cells. This decrease in PG production was associated with a concomitant 60% reduction in DNA replication. Subsequently, we examined the effects of various PGs to modulate cell growth in COX-depleted HCA-7 or COX-null HCT-15 cells by quantifying [3H]thymidine incorporation and/or growth in collagen gels. We report that J-series cyclopentenone PGs, particularly PGJ2 and 15-deoxy-delta12,14-PGJ2, induce proliferation of these cells at nanomolar concentrations. Lipids extracted from parental HCA-7 cell conditioned medium stimulated mitogenesis in COX-depleted HCA-7 cells and COX-null HCT-15 cells. Using chromatographic and mass spectrometric approaches, we were able to detect PGJ2 in conditioned medium from parental HCA-7 cells. Taken together, these findings implicate a role for cyclopentenone PGs in CRC cell proliferation.

8-Cl-adenosine-induced inhibition of colorectal cancer growth in vitro and in vivo.

The cAMP analogue 8-Cl-cAMP induces apoptosis and inhibits proliferation of a wide variety of malignancies in vitro and in vivo with relatively little toxicity. The antitumor effects of this compound are thought to involve its ability to modulate type I protein kinase A (PKAI). However, a nontoxic metabolite of 8-Cl-cAMP, 8-Cl-adenosine, with no known activity against PKAI, exerts growth inhibitory effects in breast, ovary, pancreas, and colorectal cancer cells in vitro and accumulates in xenografted tumors after 8-Cl-cAMP treatment in vivo. To characterize further the antitumor effects of 8-Cl-adenosine in colorectal cancer, we examined its effects on cell growth in vitro (cell number, 3H-thymidine incorporation, and soft agar colony formation) using the isogenically matched colorectal cancer cell lines HCT116, HCT116-E6 (p53-depleted), and 80S14 (p21WAF1/Cip1-null). 8-Cladenosine inhibited cell growth by 89%, 74%, and 79%, respectively in HCT116, HCT116-E6, and 80S14 cells after a 72-hour exposure. Growth inhibition coincided with DNA endoreduplication and subsequent apoptosis. Furthermore, nontoxic doses of 8-Cl-adenosine administered i.p. twice weekly for 4 weeks to athymic mice suppressed growth of HCT116-derived xenografts by 50%. These results show that 8-Cl-adenosine exerts antitumor activity against colorectal cancer independent of p53 and p21WAF1/Cip1.

The gene encoding mouse intestinal trefoil factor: structural organization, partial sequence analysis and mapping to murine chromosome 17q.

Trefoil peptides, a growing family of secretory molecules, have been identified mainly in the gastrointestinal tract of humans, rodents and amphibians. In the present study, the nucleotide sequence of a large portion (81%) of the gene encoding murine intestinal trefoil factor (mITF) and its whole genomic organization were determined. The mITF gene contains three exons distributed over 5 kb of genomic DNA. The genomic sequence is highly conserved, as compared with that of the rat and human ITF, and contains several AP-1-binding sites, the consensus binding site for the transcription factor Sp1, and a sequence homologous to a heat-shock element. Fluorescence in situ hybridization was used to assign ITF to chromosome 17 of the murine genome, a region syntenic with the trefoil gene cluster on human chromosome 21q22.3.

Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers.

A bacterial recombinant expression system was established to produce biologically active rat Intestinal Trefoil Factor (rITF). Characterisation of purified rITF shows that both monomers and dimers can be observed under reducing and non-reducing conditions, respectively. Site-directed mutagenesis studies show that Cys57 is necessary for rITF dimer formation. Samples of human gastrointestinal tissue following biopsy also demonstrated the presence of reducible human pS2 and ITF covalent dimers. Three-dimensional models for pS2 and ITF support the hypothesis that both pS2 and ITF can exist as disulphide-linked dimers in vivo and that any proposed function for these peptides must take dimer formation into account.

Modulation of epidermal growth factor effects on epithelial ion transport by intestinal trefoil factor.

1. The direct epithelial effects of epidermal growth factor (EGF) and its modulation by intestinal trefoil factor (ITF) have been studied in a human colonic adenocarcinoma cell line called Colony-29 (Col-29). 2. When grown in culture as confluent monolayers and voltage-clamped in Ussing chambers, these epithelia responded with an increase in short circuit current (SCC) to basolateral as well as to apically applied EGF although the latter responses (at 10 nM) were only 25% of those observed following basolateral peptide. 3. Recombinant rat ITF (added to the basolateral surface) did not alter basal SCC levels, but it did enhance the electrogenic effects of basolateral EGF. The EC50 values for EGF-induced ion transport were 0.25 nM in control, and 0.26 nM in ITF pretreated Col-29 epithelia. A significant increase in the size of EGF responses (0.1 nM-10 nM) was observed in the presence of 10 nM ITF and the half-maximal concentration for this modulatory effect of ITF was 7.6 nM. 4. The EGF-induced increases in SCC were partially inhibited (50%) by piretanide pretreatment, indicating that Cl- secretion is involved. EGF responses either in the presence or absence of ITF were also significantly reduced (84% and 66% respectively) by the cyclo-oxygenase inhibitor, piroxicam, therefore implicating prostaglandins as mediators of EGF-stimulated anion secretion. 5. We conclude that in confluent Col-29 epithelia, basolateral EGF stimulates a predominantly prostaglandin-dependent increase in Cl- secretion that is enhanced by basolateral ITF, and that these two peptides may interact in normal and damaged mucosa to alter the local apical solute and fluid environment.

Immunoprecipitation and characterization of a binding protein specific for the peptide, intestinal trefoil factor.

Recombinant rat intestinal trefoil factor (rITF) and human spasmolytic polypeptide (hSP) were irreversibly cross-linked to specific binding sites in solubilized rat intestinal epithelial membranes and human adenocarcinoma cells. Analysis of the immunoprecipitates by immunoblotting identified a cross-linked protein complex of approximately 45 kDa, which under reducing conditions appeared as a approximately 28-kDa band and the latter displayed ligand-stimulated phosphorylation of a tyrosine, but not a threonine or serine, residue in the binding complex. [125I]rITF was used to localize binding sites by autoradiography of frozen sections from rat gastrointestinal tissues. A high density of specific [125I]rITF binding sites was present within gastric, colonic, and jejunal mucosal glands. Unlabeled hSP partially inhibited [125I]rITF binding at a concentration of 1 microM when compared with the same concentration of unlabeled rITF. These studies support earlier observations for the existence of trefoil binding sites in the gastrointestinal tract and further suggest that hSP has affinity for the mucosal rITF binding site.

Soy polysaccharide in an enteral diet: Effects on rat intestinal cell proliferation, morphology and metabolic function.

The present study was conducted to compare the effect of soy polysaccharide (SP)-supplemented and fibre-free enteral diets and a normal chow diet on intestinal and colonic adaptation of rat gastrointestinal morphometrics and cytokinetics. Results showed that the fibre-free diet caused a significant decrease in various gut parameters, such as cell proliferation, tissue wet weight, and intestinal brush border enzymes, when compared to the normal gut of chow-fed rats. However the SP-supplemented enteral diet resulted in a significant improvement in several parameters in most regions along the gastrointestinal tract, when compared to orally-fed animals. These studies demonstrate that significant changes occur in the intestine as a result of enteral diets, with regional variation. Furthermore, the results demonstrate that SP stimulates crypt cell proliferation which could, in part, hasten recovery from intestinal mucosal damage. The addition of SP to enteral diets seems potentially advantageous.

Trefoil peptide expression in intestinal adaptation and renewal.

There are many avenues where molecular biology is important in studying the gut, and here we explore methods for defining expression of a new gene family in the gut. We have defined the pattern of trefoil peptide gene expression in the ulceration-associated cell lineage (UACL) and in the nearby mucosa in Crohn's disease. In the UACL, human spasmolytic polypeptide mRNA and peptide are expressed in the acinar and proximal duct cells, whereas pS2 mRNA and peptide are found in the distal duct cells and in the surface cells. In adjacent mucosa, pS2 mRNA and protein are expressed ectopically by goblet cells. Ultrastructural immunolocalisation showed the pS2 to be co-packaged in the mucous cell granules. pS2 peptide was demonstrated in local neuroendocrine cells and was also co-packaged with the neuroendocrine granules. The crypts associated with the UACL also showed marked neuroendocrine cell hyperplasia. We have also cloned the newest trefoil peptide intestinal trefoil factor from human and rat intestinal mucosa and shown its co-expression with mucus by normal intestinal goblet cells. The co-packaging of the same secretory protein in both mucous and neuroendocrine granules, which have different functions, is unusual and indicates an important role for pS2 in the secretory process itself or as a ligand delivered to its receptor via multiple routes. We conclude that the trefoil peptides are widely distributed in the intestine in inflammatory bowel disease and are of considerable potential functional importance.

A compartmental model for the prediction of breath concentration and absorbed dose of chloroform after exposure while showering.

In order to predict the exhaled breath concentration of chloroform in individuals exposed to chloroform while showering, an existing physiologically based pharmacokinetic (PB-PK) model was modified to include a multicompartment, PB-PK model for the skin and a completely mixed shower exposure model. The PB-PK model of the skin included the stratum corneum as the principal resistance to absorption and a viable epidermis which is in dynamic equilibrium with the skin microcirculation. This model was calibrated with measured exhaled breath concentrations of chloroform in individuals exposed while showering with and without dermal absorption. The calibration effort indicated that the expected value of skin-blood partitioning coefficient would be 1.2 when the degree of transfer of chloroform from shower water into shower air was 61%. The stratum corneum permeability coefficient for chloroform was estimated to be within the range of 0.16-0.36 cm/hr and the expected value was 0.2 cm/hr. The estimated ratio of the dermally and inhaled absorbed doses ranged between 0.6 and 2.2 and the expected value was 0.75. These results indicate that for the purposes of risk assessment for dermal exposure to chloroform, a simple steady-state model can be used to predict the degree of dermal absorption and that a reasonable value of skin permeability coefficient for chloroform used in this model would be 0.2 cm/hr. Further research should be conducted to compare the elimination of chloroform via exhaled breath when different exposure routes are being compared. The model results from this study suggest that multiple measurements of exhaled breath concentrations after exposure may be necessary when making comparisons of breath concentrations that involve different exposure routes.

Combined intestinal trefoil factor and epidermal growth factor is prophylactic against indomethacin-induced gastric damage in the rat.

1. The availability of recombinant epidermal growth factor provides a potentially exciting development for the treatment of gastrointestinal ulceration. However, because of its potent mitogenic activity, there is a need for strategies which reduce the dose required. Intestinal trefoil factor stimulates mucosal healing without increasing proliferation. Studies were undertaken to examine the biological effects of rat intestinal trefoil factor and/or human epidermal growth factor upon gastrointestinal epithelial cell functions pertinent to mucosal protection, using two wounding models. 2. The study of epithelial restitution in vitro demonstrated a marked synergistic effect on the rate of migration of the wound edge when intestinal trefoil factor was used in combination with epidermal growth factor. There was no increased cellular proliferation due to the addition of intestinal trefoil factor to the cells when given alone, or to the stimulatory effect of cells treated with epidermal growth factor. In the rat model of gastric ulceration, the presence of both epidermal growth factor and intestinal trefoil factor protected against the development of indomethacin-induced gastric lesions. 3. We conclude that combination therapy of epidermal growth factor with intestinal trefoil factor could provide a more potent, safer approach to the treatment of human gastrointestinal ulceration.

The gene encoding human intestinal trefoil factor (TFF3) is located on chromosome 21q22.3 clustered with other members of the trefoil peptide family.

The gene coding for human intestinal trefoil factor (hITF), a recently described cellular motogen produced by gastrointestinal goblet cells and epithelia elsewhere, is a member of the rapidly growing trefoil peptide family. In a rodent-human somatic cell hybrid panel, the hITF (HGMW-approved symbol TFF3) genomic locus segregated with human chromosome 21q. Fluorescence in situ hybridization with a 2.1-kb genomic probe of the hITF gene mapped this locus more precisely to the q22.3 region. Triple fluorescence in situ hybridization, together with physical mapping of human genomic DNA using pulsed-field gel electrophoresis, revealed that the hITF gene is tightly linked to those encoding the other known human trefoil peptides, namely the breast cancer estrogen-inducable gene pS2 (BCEI) and human spasmolytic polypeptide (hSP/SML1). This gene family could become a useful marker for the genetic and physical mapping of chromosome 21 and for a better definition of the region involved in the clinical phenotype of several genetic diseases.

p53 expression in cultured cells following radioisotope labelling.

p53 inhibits division following cellular damage. Cultured cells were found to express p53 protein following pulse labelling with radioisotopes, even at low doses normally used for growth and metabolic labelling studies. Some stem cells are exquisitely sensitive to radiation and thus p53 may have evolved as a major regulator of stem cell function. Therefore any genetic damage may be able to induce p53 expression, which in turn will affect the biochemical outcome of many experiments by both cell cycle arrest and other mechanisms. In some cases the use of radioisotopes may directly change the results of the experiment. This will require a careful re-evaluation of the current literature and experimental protocols utilising radioisotopes.

Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites.

The cysteine-rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270-bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a beta-galactosidase collagen-hinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil-motif-containing fusion protein. Purification of the rITF moiety was achieved by p-aminophenyl-thio-beta-D-galactoside(APTG)-affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the beta-galactosidase-hinge molecule by a further APTG-affinity step. It was demonstrated that intrachain disulphide-bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil-motif-containing protein were recognised by an antibody raised against the chemically synthesised peptide. The trefoil motif present in the fusion protein was used to localise putative trefoil-binding sites in sections of frozen rat tissue. Binding was demonstrated using the beta-galactosidase portion of the fusion protein as a reporter moiety, either directly with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, or indirectly using a monoclonal antibody to beta-galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of beta-galactosidase fusion proteins for histochemical localisation of peptide-binding sites should prove more generally useful.

The production and characterization of a new monoclonal antibody to the trefoil peptide human spasmolytic polypeptide.

Human spasmolytic polypeptide (hSP) is a member of the growing family of trefoil peptides which are expressed in discrete regions of the body, most notably the gastrointestinal tract. Much of the research into the localization of the spasmolytic polypeptide has relied on hybridization in situ to detect its mRNA, due to the absence of a suitable antibody. The aim of the present study was to develop and characterize a monoclonal antibody against the human spasmolytic polypeptide, using a combination of immunohistochemistry and hybridization in situ. After immunoblotting, the antibody detected a 14 kDa protein in gastrointestinal tissue extracts from the stomach and small intestine only. Using immunohistochemistry, human spasmolytic polypeptide showed a distinctive staining pattern in the duodenum which co-localized with its mRNA. The co-localization of the immunoreactive peptide with its mRNA provides good evidence that the antibody truly recognized human spasmolytic polypeptide.

Regulation of mastomys ECL cell function by transforming growth factor alpha.

Little progress has been made in the understanding of the pathobiology of gastric neoplasia over the past 4 decades. This reflects the paucity of information available regarding the biology of gastric mucosal cell proliferation. More recently it has become apparent that growth factor regulation of cell proliferation is of considerable relevance in initiating mucosal mitogenesis. We have recently identified the histamine secreting enterochromaffin-like (ECL) cell as a pivotal cellular regulator of gastric acid secretion. In addition to its critical role in initiating acid secretion, we have proposed that the ECL cell may produce agents responsible for the regulation of mucosal cell proliferation. We have therefore hypothesized that such a function may be subserved by production of transforming growth factor alpha (TGFalpha). TGFalpha is known to play a significant role both in normal physiology and in the transformation of naive cells into a neoplastic form. We therefore proposed that increased levels of gastrin induced by low acid states might stimulate TGFalpha secretion and that this agent might be capable of regulating ECL cell DNA synthesis and cell proliferation. We used the mastomys rodent to generate an in vivo hypergastrinemia model using long-term histamine-2 receptor blockade (loxtidine 1 mg/kg/day). In order to evaluate the cell-specific effects, we developed a pure isolated ECL cell system from the mastomys stomach. This utilized pronase digestion (1.0 mg/ml) and EDTA exposure (1 mM) of the mucosa followed by particle size separation with countercurrent elutriation and density purification on a Nycodenz step gradient. ECL cells were obtained with a purity of 90-95%. Histamine secretion from ECL cells was measured by radioimmunoassay (RIA). TGFalpha content was measured by RIA, and TGFalpha expression was measured by RNAse probe protection assay. DNA synthesis was quantified by measuring bromo-deoxyuridine (BrdU) incorporation into cultured cells. TGFalpha levels were increased in fundic mucosa after 16 weeks of hypergastrinemia from 4.3 +/- 0.6 to 32.6 +/- 2.6 fmole/mg protein, P < 0.05). TGFalpha message was identified in the ECL cells by RNAse probe protection assay, and was fourfold amplified in ECL cell tumors after 16 weeks of exposure to hypergastrinemia. Gastrin stimulated (10 nM) histamine secretion in isolated naive ECL cells was inhibited by TGFalpha (IC50 5 x 10 (-9) M). DNA synthesis was stimulated by gastrin (EC50 2 X 10 M) and TGFalpha (EC50 5 x 10(-9) M). These data are consistent with the proposal that elevated gastrin levels are associated with ECL cell TGFalpha production and that TGFalpha stimulates ECL cell DNA synthesis.

Distribution and expression of pancreatic secretory trypsin inhibitor and its possible role in epithelial restitution.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent serine protease inhibitor that prevents excessive digestion of the gastrointestinal mucus but may also directly affect epithelial function. We therefore examined the distribution of PSTI in the human adult and fetus using immunohistochemistry and in situ hybridization and examined its effects on cell proliferation and migration in vitro. PSTI peptide and mRNA were found in the exocrine pancreas, mucus-producing cells of the normal gastrointestinal tract, acinar component of the normal breast, and surface epithelial cells at the edge of benign gastric ulcers. Peptide, but not message, was identified in the renal proximal tubule, probably reflecting reabsorption of filtered peptide. Purified human PSTI did not affect proliferation of the human colonic cell line HT-29 but caused a threefold increase in the rate of migration in an in vitro wounding model of restitution. This effect was inhibited by co-administering a PSTI-neutralizing antibody, a transforming growth factor-beta-neutralizing antibody, or an epidermal growth factor receptor-blocking antibody. As PSTI is widely distributed in several human organ systems and stimulates cell migration in vitro, we conclude that PSTI is likely to have additional roles to that of preserving the gastrointestinal mucous layer from excessive digestion.