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Biomedical subjects

R Chinery

Publications and source records attributed to R Chinery.

At least 19 recordsLinked to original sources
PubMed2001

A phase I study of vitamin E, 5-fluorouracil and leucovorin for advanced malignancies.

Six patients with incurable malignancies were originally treated with vitamin E, 3200 IU/day for fourteen days, followed by the same dose of vitamin E daily plus LCV (20 mg/m2 i.v. bolus daily x 5) with 5FU (425 mg/m2 i.v. bolus immediately following LCV). The same schedule of LCV and 5FU was repeated 4 weeks later, then every 5 weeks indefinitely. When 3 of the first 6 had grade 3/4 toxicity, six more patients were treated on the identical drugs and schedule. Seven of twelve total patients had one or more grade 3/4 toxicities. Neutropenia, abdominal pain, and diarrhea were most common. No patient had a documented response, though seven patients did have stable disease. Though the combination of vitamin E and chemotherapy was toxic, this trial demonstrated maximal therapeutic doses of vitamin E can be combined with standard 5FU and LCV, without significantly increasing the side effects of the chemotherapy itself.

Abdominal Pain↗
PubMed2001

Thermal injury effects on intestinal crypt cell proliferation and death are cell position dependent.

We examined the effects of thermal injury on intestinal epithelial cell proliferation and death. We recorded histologically identifiable mitotic and apoptotic crypt cells in relation to cell position after a 60% full thickness cutaneous thermal injury in the rat. The injury significantly reduced mitosis (0.53 +/- 0.11 vs. 1. 50 +/- 0.70, P < 0.05) at cell positions 4-6, stem cells, 6 h after injury. A similar reduction in mitosis (1.13 +/- 0.59 vs. 3.50 +/- 0. 80, P < 0.05) was observed at higher cell positions 7-9 12 h after injury, indicating a positional cell shift. In addition, a significant increase in the number of apoptotic bodies occurred at cell positions 7-9 (2.32 +/- 0.87 vs. 0.13 +/- 0.22, P < 0.05) and 10-12 (2.2 +/- 0.12 vs. 0.00, P < 0.05) 6 h after injury. Thermal injury-induced alterations in mitotic and apoptotic activities were transient since crypts recovered with a moderate increase in mitotic activity 24 h after injury. In control and thermal-injury rats 24 h after injury, crypt cell mitosis and apoptosis did not differ significantly. This demonstrates that cutaneous thermal injury causes a transient suppression of mitosis as well as induction of apoptosis in a cell position-dependent manner in the small intestinal crypt.

Animals↗
PubMed2000

Pyrrolidinedithiocarbamate increases the therapeutic index of 5-fluorouracil in a mouse model.

BACKGROUND & AIMS: The thiol-containing antioxidant pyrrolidinedithiocarbamate (PDTC) enhances the cytotoxic efficacy of 5-fluorouracil (5-FU) against human colorectal cancer cell lines in vitro and in vivo. This process appears to be mediated by a sustained increase in p21 expression, independent of p53 function, resulting in growth arrest and apoptosis. We determined whether PDTC augmented 5-FU intestinal toxicity in non-tumor-bearing mice. METHODS: Apoptotic and mitotic indices were measured in the small and large intestine on a cell positional basis at intervals throughout the 72-hour period after administration of 5-FU (40 mg/kg) and PDTC (250 mg/kg). The proportion of crypts regenerating after 5-FU (600-1200 mg/kg) and PDTC (500 mg/kg) was also measured. RESULTS: 5-FU therapy induces substantial apoptotic cell death with simultaneous inhibition of mitotic activity within the small and large intestinal epithelium. PDTC reduces 5-FU-induced apoptotic events in the colon by 49%, predominantly among clonogenic stem and transit cells while promoting the early recovery of mitotic activity. As a consequence, PDTC increased the proportion of regenerating colonic crypts after 5-FU therapy. PDTC did not, however, significantly modulate 5-FU toxicity in the small intestine. CONCLUSIONS: PDTC does not augment the intestinal toxicity of 5-FU and actually protects the colonic mucosa. These results support further investigation of PDTC and related compounds as treatments for colorectal cancer.

Animals↗
PubMed1999

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.

Anti-Bacterial Agents↗
PubMed1999

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.

Antigens, Differentiation↗
PubMed1998

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.

Animals↗
PubMed1998

Intestinal trefoil factor controls the expression of the adenomatous polyposis coli-catenin and the E-cadherin-catenin complexes in human colon carcinoma cells.

Intestinal trefoil factor 3 (TFF3) is a member of the trefoil family of peptides, small molecules constitutively expressed in epithelial tissues, including the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance mucosal healing and epithelial restitution in vivo. In this study, we evaluated the effect of recombinant TFF3 (rTFF3) stimulation on the expression and cellular localization of the epithelial (E)-cadherin-catenin complex, a prime mediator of Ca2+ dependent cell-cell adhesion, and the adenomatous polyposis coli (APC)-catenin complex in HT29, HCT116, and SW480 colorectal carcinoma cell lines. Stimulation by rTFF3 (10(-9) M and 10(-8) M) for 20-24 hr led to cell detachment and to a reduction in intercellular adhesion in HT29 and HCT116 cells. In both cell lines, E-cadherin expression was down-regulated. The expression of APC, alpha-catenin and beta-catenin also was decreased in HT29 cells, with a translocation of APC into the nucleus. No change in either cell adhesion or in the expression of E-cadherin, the catenins, and APC was detected in SW480 cells. In addition, TFF3 induced DNA fragmentation and morphological changes characteristic of apoptosis in HT29. Tyrphostin, a competitive inhibitor of protein tyrosine kinases, inhibited the effects of TFF3. Our results indicate that by perturbing the complexes between E-cadherin, beta-catenin, and associated proteins, TFF3 may modulate epithelial cell adhesion, migration, and survival.

Adenomatous Polyposis Coli Protein↗
PubMed1997

Antioxidant-induced nuclear translocation of CCAAT/enhancer-binding protein beta. A critical role for protein kinase A-mediated phosphorylation of Ser299.

Alterations in intracellular oxidative status activate several signal transduction pathways resulting in distinct patterns of gene expression. Treatment of colorectal cancer cells with antioxidants can lead to apoptosis by induction of p21 through a mechanism involving CCAAT/enhancer-binding protein beta (C/EBPbeta). Herein, we demonstrate that the antioxidant pyrrolidinedithiocarbamate activates cAMP-dependent protein kinase (PKA) in a colorectal cancer cell line DKO-1. Activation of PKA phosphorylates Ser299 within C/EBPbeta, which is essential for protein translocation to the nucleus. Pharmacological inhibition of PKA and mutation of Ser299 to alanine blocks C/EBPbeta nuclear translocation and induction of p21. Our results indicate that a cAMP-dependent phosphorylation of C/EBPbeta at Ser299 is critical for nuclear translocation of this protein and its subsequent transactivation of genes in response to antioxidant treatment.

Antioxidants↗
PubMed1997

Epidermal growth factor receptor activation induces nuclear targeting of cyclooxygenase-2, basolateral release of prostaglandins, and mitogenesis in polarizing colon cancer cells.

Nonsteroidal antiinflammatory drugs reduce the risk of colon cancer, possibly via cyclooxygenase (COX) inhibition. The growth factor-inducible COX-2, which is overexpressed in neoplastic colonic tissue, is an attractive target to mediate this effect. Herein we have exploited the ability of a human colon cancer cell line, HCA-7 Colony 29, to polarize when cultured on Transwell (Costar) filters to study COX-2 production and the vectorial release of prostaglandins (PGs). Administration of type alpha transforming growth factor to the basolateral compartment, in which the epidermal growth factor receptor (EGFR) resides, results in a marked induction of COX-2 immunoreactivity at the base of the cells and the unexpected appearance of COX-2 in the nucleus. The increase in COX-2 protein is associated with a dose- and time-dependent increase in PG levels in the basolateral, but not apical, medium. Amphiregulin is the most abundantly expressed EGFR ligand in these cells, and the protein is present at the basolateral surface. EGFR blockade reduces baseline COX-2 immunoreactivity, PG levels, and mitogenesis in a concentration-dependent manner. Two specific COX-2 inhibitors, SC-58125 and NS 398, also, in a dose-dependent manner, attenuate baseline and type alpha transforming growth factor-stimulated mitogenesis, although PG levels are decreased > 90% at all concentrations of inhibitor tested. These findings show that activation of the EGFR stimulates COX-2 production and its translocation to the nucleus, vectorial release of PGs, and mitogenesis in polarized HCA-7 Colony 29 cells.

Amphiregulin↗
PubMed1997

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.

Acetylcysteine↗
PubMed1997

Phosphorylation of beta-catenin and epidermal growth factor receptor by intestinal trefoil factor.

Intestinal trefoil factor (TFF3) is a member of the trefoil family of peptides, which are constitutively expressed in the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance epithelial restitution in vivo. In the present study, we show that the stimulatory effect of TFF3 on the migration of HT29 colonic carcinoma cells requires the perturbation of E-cadherin function, a calcium-dependent cell-cell adhesion molecule in epithelia. A rapid (< 1 minute) and specific tyrosine phosphorylation of beta-catenin and epidermal growth factor receptor was detected in cells treated with recombinant rat TFF3. No phosphorylation of E-cadherin or alpha-catenin was detected. Tyrosine phosphorylation of beta-catenin was associated with reduced membranous E-cadherin expression, perturbation of intercellular adhesion, and promotion of cell motility. These results suggest that TFF3 enhances cell migration through modulation of E-cadherin/catenin complex function. Tyrosine phosphorylation of beta-catenin and epidermal growth factor receptor seems to be involved in this process.

Animals↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

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.

Chromosome Mapping↗
PubMed1996

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.

Animals↗
PubMed1996

The role of transforming growth factor alpha in the enterochromaffin-like cell tumor autonomy in an African rodent mastomys.

BACKGROUND & AIMS: Gastric carcinoids evolved from enterochromaffin-like (ECL) cell hyperplasia are usually associated with high pH and hypergastrinemia. The Mastomys species exhibits a genetic propensity to gastric carcinoid formation that can be accelerated by acid inhibition-induced hypergastrinemia. Although gastrin is critical in the initiation of the ECL cell transformation, the role of other growth factors involved in the evolution of the tumor autonomy has not been established. The aim of this study was to evaluate the role of transforming growth factor (TGF) alpha in the regulation of ECL cell transformation. METHODS: Mastomys were orally administered an irreversible H2-receptor antagonist loxtidine for 0, 8, and 16 weeks, and ECL cell transformation was monitored by assessing gastrin levels, mucosal histamine content, and chromogranin immunoreactivity. The ECL cells were purified, and cell proliferation at each stage in response to gastrin and TGF alpha was measured by bromodeoxyuridine uptake. TGF-alpha expression was evaluated by radioimmunoassay and Northern blot, and epidermal growth factor (EGF) receptor expression was determined by Western blot, immunoprecipitation, and immunocytochemistry. RESULTS: Although the response to gastrin decreased during hypergastrinemia, the proliferative effect of TGF-alpha on ECL cells was specifically amplified during the development of hyperplasia. TGF-alpha and EGF receptor expression increased steadily in the transformed cells. CONCLUSIONS: During low acid-induced hypergastrinemia, the expression of TGF-alpha and EGF receptor may constitute an autocrine regulatory mechanism in ECL cell tumor transformation.

Animals↗
PubMed1996

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.

Adult↗
PubMed1996

Downregulation of E-cadherin in the reparative epithelium of the human gastrointestinal tract.

E-cadherin, an epithelial adhesion molecule, is critical for the maintenance of cell polarity and differentiation. We studied the distribution of E-cadherin in normal gut and in enteric ulceration to test the hypothesis that the motility of regenerative epithelium over ulcers is associated with a decrease in E-cadherin expression. Sections of normal stomach, small intestine, and colon were examined for E-cadherin distribution using the antibody HECD-1 and compared with the pattern seen in peptic ulceration and Crohn's disease. A subset was examined by in situ hybridization using 35S radiolabeled E-cadherin riboprobes. A wounding system employing the HT-29 cell line was used as an in vitro model of early healing. In the normal gut uniform strong basolateral staining was seen. Areas of ulceration showed a patchy reduction in membrane localized E-cadherin in regenerating epithelium, even though E-cadherin mRNA was demonstrable in this population. In wounded confluent HT29 layers, migrating cells also showed reduced E-cadherin immunostaining. These data support the notion that the motility of restitutive epithelial cells may relate to altered patterns of E-cadherin and that this may play an important role in the reconstitution of epithelial integrity after mucosal injury.

Cadherins↗