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B M Evers

Publications and source records attributed to B M Evers.

At least 19 recordsLinked to original sources
PubMed2000

Human colorectal cancers express a constitutively active cholecystokinin-B/gastrin receptor that stimulates cell growth.

Although ectopic expression of the cholecystokinin B/gastrin receptor (CCK-BR) is widely reported in human colorectal cancers, its role in mediating the proliferative effects of gastrin1-17 (G-17) on these cancers is unknown. Here we report the isolation of a novel splice variant of CCK-BR that exhibits constitutive (ligand-independent) activation of pathways regulating intracellular free Ca(2+) ([Ca(2+)](i)) and cell growth. The splice variant (designated CCK-BRi4sv for intron 4-containing splice variant) is expressed in colorectal cancers but not in normal colonic mucosa adjacent to the cancer. Balb3T3 cells expressing CCK-BRi4sv exhibited spontaneous, ligand-independent, oscillatory increases in [Ca(2+)](i), whereas cells expressing wild-type CCK-BR did not. Primary cultures of cells isolated from resected colorectal cancers also exhibited a similar pattern of spontaneous [Ca(2+)](i) oscillations. For both Balb3T3 and primary tumor cells, application of G-17 (10 and 200 nm, respectively) caused an increase in [Ca(2+)](i). Selective CCK-BR antagonists blocked the G-17-stimulated Ca(2+) responses but not the spontaneous [Ca(2+)](i) oscillations. Cells expressing CCK-BRi4sv exhibited an increased growth rate ( approximately 2.5-fold), in the absence of G-17, compared with cells expressing wild-type CCK-BR. The selective pattern of expression, constitutive activity, and trophic action associated with CCK-BRi4sv suggest that this variant may regulate colorectal cancer cell proliferation though a gastrin-independent mechanism.

3T3 Cells↗
PubMed2000

Isolation and molecular characterization of the 5'-upstream region of the human TRAIL gene.

TRAIL, a novel member of the TNF family, acts through membrane receptors to induce apoptosis of activated T lymphocytes and may represent a mechanism for the "immune escape" of certain cancers. Various cytokines appear to increase expression of other TNF family members; however, the regulation of TRAIL has not been defined. The purpose of this study was to assess molecular mechanisms regulating TRAIL gene expression in human colon cancers. In this study, we have cloned the human TRAIL (hTRAIL) promoter ( approximately 1.6 kb) and identified a number of putative transcription factor binding sites such as NFAT, AP-1 and Sp1 sequences which are important for the expression of other TNF family members. Transient transfections of 5'-deletion promoter constructs into either Caco-2 or HT29 colon cancer cells identified TRAIL promoter regions critical for both basal and interferon-gamma (IFN-gamma)-mediated induction. Furthermore, induction of TRAIL mRNA levels was demonstrated in HT29 and Caco-2 cells with IFN-gamma treatment suggesting an important role for this cytokine in TRAIL expression.

Apoptosis Regulatory Proteins↗
PubMed2000

Activation of NF-kappaB binding in HT-29 colon cancer cells by inhibition of phosphatidylinositol 3-kinase.

The ubiquitous transcription factor NF-kappaB, which is activated in cells by diverse stimuli including phosphatidylinositol 3-kinase (PI3-kinase), is a critical factor for cell survival and growth. Inhibition of PI3-kinase enhances enterocyte-like differentiation of the human colon cancer cell line HT-29. The purpose of our study was to determine whether PI3-kinase alters NF-kappaB in HT-29 cells. Wortmannin, a specific PI3-kinase inhibitor, stimulated NF-kappaB binding activity in HT-29 cells by 4 h after treatment. Activation of NF-kappaB occurred without degradation of IkappaBalpha, a protein that sequesters NF-kappaB in the cytosol. In addition to increasing NF-kappaB binding, either wortmannin or cotransfection with a dominant negative mutant of the p85 regulatory subunit of PI3-kinase (Deltap85) induced NF-kappaB transactivation. Taken together, these results suggest that inhibition of PI3-kinase in HT-29 cells results in induction of NF-kappaB binding activity and transactivation which is independent of IkappaBalpha degradation.

Colonic Neoplasms↗
PubMed2000

Neurotensin-mediated activation of MAPK pathways and AP-1 binding in the human pancreatic cancer cell line, MIA PaCa-2.

Neurotensin (NT), a gastrointestinal (GI) hormone, binds its receptor (NTR) to stimulate proliferation of normal and neoplastic GI tissues; the molecular mechanisms remain largely undefined. Mitogen-activated protein kinases (MAPKs) are a family of intracellular kinases that transmit mitogenic signals by translocating to the nucleus and activating transcription factors. The purposes of this study were: (1) to identify whether the MAPKs (ERK1/2 and JNK) are activated by NT and (2) to determine the effect of NT on downstream transcription factors using the human pancreatic adenocarcinoma cell line, MIA PaCa-2, which possesses high-affinity NTR. Both ERK and JNK activity were stimulated within 3-6 min by treatment with NT (10 nM); steady-state levels of ERK and JNK protein were unchanged. Moreover, NT treatment resulted in increased AP-1 binding activity as determined by gel shift analysis. Delineating the signal transduction mechanisms regulating the cellular effects of NT will provide important insights into the molecular pathways responsible for NT-mediated effects on both normal and neoplastic cells.

Adenocarcinoma↗
PubMed2000

Assessment of differential gene expression patterns in human colon cancers.

OBJECTIVE: To use a novel genomic approach to determine differential gene expression patterns in colon cancers of different metastatic potential. SUMMARY BACKGROUND DATA: Colorectal cancer is the third leading cause of cancer deaths in the United States; despite aggressive treatment strategies, the 5-year survival rate for metastatic cancer has not changed in 50 years. The analysis of changes in gene expression patterns associated with metastasis may provide new treatment strategies. METHODS: Human colon cancer cells KM12C (derived from a Dukes B colon cancer), KML4A (a metastatic variant derived from KM12C), and KM20 (derived from a Dukes D colon cancer) were extracted for RNA. In addition, RNA was extracted from normal colon, primary cancer, and liver metastasis in a patient with metastatic colon cancer. Gene expression patterns for approximately 1,200 human genes were analyzed and compared by cDNA array techniques. RESULTS: Of the roughly 1,200 genes assessed in the KM cell lines, 9 genes were noted to have a more than threefold change in expression (either increased or decreased) in the more metastatic KML4A and KM20 cells compared with KM12C. Assessment of tissues from a patient with metastatic colon cancer demonstrated a more than threefold change in the expression of 14 genes in the primary cancer and liver metastasis compared with normal mucosa. CONCLUSIONS: Using cDNA expression array technology, the authors identified genes with expression levels that are altered with metastasis. The ability to analyze and compare the expression patterns of multiple genes simultaneously provides a powerful technique to identify potential molecular targets for novel therapeutic strategies.

Blotting, Northern↗
PubMed2000

Acute pancreatitis results in induction of heat shock proteins 70 and 27 and heat shock factor-1.

Heat shock proteins (HSPs) 70 and 27 are stress-responsive proteins that are important for cell survival after injury; the expression of these HSPs is regulated primarily by the transcription factor heat shock factor-1 (HSF-1). The purpose of this study was to determine the effect of acute pancreatitis on pancreatic HSPs (70, 27, 60, and 90) expression and to assess potential mechanisms for HSP induction using a murine model of cerulein-induced pancreatitis. We found an increase of both HSP70 and HSP27 levels with expression noted throughout the pancreas after induction of pancreatitis. HSP60 and HSP90 levels were constitutively expressed in the pancreas and did not significantly change with acute pancreatitis. HSF-1 DNA binding activity increased in accordance with increased HSP expression. We conclude that acute pancreatitis results in a marked increase in the expression of HSP70 and HSP27. Furthermore, the induction of HSP70 and HSP27 expression was associated with a concomitant increase in HSF-1 binding activity. The increased expression of both HSP70 and HSP27 noted with pancreatic inflammation may confer a protective effect for the remaining acini after acute pancreatitis.

Acute Disease↗
PubMed2000

Characterization and regulation of E2F activity during Caco-2 cell differentiation.

The specific mechanisms controlling intestinal cell differentiation remain largely undefined. The retinoblastoma (Rb) proteins (pRb, p130, and p107) appear crucial to the terminal differentiation process of certain cells through their association and repression of E2F transcription factors. We have examined the expression of pRb-related proteins p130 and p107 as well as the regulation of E2F during spontaneous differentiation of the Caco-2 intestinal cell line. Nuclear protein levels of p130 and p107 were increased with Caco-2 differentiation. Induction of a slower-migrating E2F complex was noted in postconfluent (i.e., differentiated) Caco-2 cells; p130 protein was the predominant component of this E2F complex with a minor contribution from cyclin-dependent kinase-2. A small component of p107 binding was identified by deoxycholate release gel shift assays. In contrast, no pRb binding to E2F was noted in Caco-2 cells. In addition to increased association with p130, E2F-4 phosphorylation was markedly decreased in differentiated Caco-2 cells, whereas E2F protein levels remained unchanged. Taken together, our findings suggest that the regulation of E2F function may be an important contributing factor in the cell cycle block and spontaneous differentiation of Caco-2 cells. This regulation of E2F occurs most likely through its increased association with p130 as well as decreased phosphorylation.

Blotting, Western↗
PubMed2000

Signaling mechanisms regulating bombesin-mediated AP-1 gene induction in the human gastric cancer SIIA.

The hormone bombesin (BBS) and its mammalian equivalent gastrin-releasing peptide (GRP) act through specific GRP receptors (GRP-R) to affect multiple cellular functions in the gastrointestinal tract; the intracellular signaling pathways leading to these effects are not clearly defined. Previously, we demonstrated that the human gastric cancer SIIA possesses GRP-R and that BBS stimulates activator protein-1 (AP-1) gene expression. The purpose of our present study was to determine the signaling pathways leading to AP-1 induction in SIIA cells. A rapid induction of c-jun and jun-B gene expression was noted after BBS treatment; this effect was blocked by specific GRP-R antagonists, indicating that BBS is acting through the GRP-R. The signaling pathways leading to increased AP-1 gene expression were delineated using phorbol 12-myristate 13-acetate (PMA), which stimulates protein kinase C (PKC)-dependent pathways, by forskolin (FSK), which stimulates protein kinase A (PKA)-dependent pathways, and by the use of various protein kinase inhibitors. Treatment with PMA stimulated AP-1 gene expression and DNA binding activity similar to the effects noted with BBS; FSK stimulated jun-B expression but produced only minimal increases of c-jun mRNA and AP-1 binding activity. Pretreatment of SIIA cells with either H-7 or H-8 (primarily PKC inhibitors) inhibited the induction of c-jun and jun-B mRNAs in response to BBS, whereas H-89 (PKA inhibitor) exhibited only minimal effects. Pretreatment with tyrphostin-25, a protein tyrosine kinase (PTK) inhibitor, attenuated the BBS-mediated induction of c-jun and jun-B, but the effect was not as pronounced as with H-7. Collectively, our results demonstrate that BBS acts through its receptor to produce a rapid induction of both c-jun and jun-B mRNA and AP-1 DNA binding activity in the SIIA human gastric cancer. Moreover, this induction of AP-1, in response to BBS, is mediated through both PKC- and PTK-dependent signal transduction pathways with only minimal involvement of PKA.

Bombesin↗
PubMed2000

Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers.

The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in approximately 25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from -373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.

Adult↗
PubMed1999

Functional genomics: clinical effect and the evolving role of the surgeon.

The genetic and molecular revolution that has occurred over the last 2 decades has dramatically increased our understanding of basic disease processes and will undoubtedly lead to improved detection methods and treatment. This will occur at an even more rapid rate after the completion of the Human Genome Project in the next 2 to 3 years. While these remarkable technological advances offer great hopes for novel therapeutic modalities, complicated medical, ethical, and legal issues will need to be addressed. This article briefly describes the advances that have occurred and their future ramifications for the field of surgery. Most assuredly, we will all be affected by these changes. Surgeons have the opportunity to be active participants and real leaders in the research and complex decisions regarding the optimal treatment of patients. However, formal training in these techniques and their potential applications will be required. Surgeons, as well as all physicians, must rise to the occasion or, otherwise, we will be relegated to a bystander status with clinical and moral decisions being made by nonclinicians.

Ethics, Medical↗
PubMed1999

Enterocyte-like differentiation of the Caco-2 intestinal cell line is associated with increases in AP-1 protein binding.

The nuclear factor AP-1, a large family of transcription factors composed of the Jun and Fos protein families, plays a role in the differentiation of various cells; the role of the AP-1 factors in intestinal differentiation is not known. Members of the AP-1 family can be activated by the Ras pathway and, in addition, Ras appears to be important for gut differentiation. The purpose of this study was to determine whether AP-1 activity is altered in the Caco-2 cell line, which spontaneously differentiates to a small bowel phenotype after confluency, and the Caco-2-ras cell line, which exhibits differentiated properties regardless of culture conditions. AP-1 binding activity, consisting of c-Jun, JunD, c-Fos and Fra-2 proteins, was increased in Caco-2 cells at 3 days postconfluency, a time point associated with G1 block and cessation of proliferation. Steady state levels of JunD were increased at day 3 postconfluency as determined by Western blot. Furthermore, AP-1 binding was increased in preconfluent Caco-2-ras cells compared with parental Caco-2 cells, suggesting that AP-1 induction may be mediated by the Ras pathway. The early induction of AP-1 binding activity suggests a role for these proteins in the differentiation of the Caco-2 intestinal cell line.

Binding, Competitive↗
PubMed1999

Characterization of early developmental pattern of expression of neurotensin/neuromedin N gene in foregut and midgut.

The gut endocrine gene, neurotensin (NT/N), is expressed in a strict temporally and spatially specific pattern. Utilizing a sensitive reverse transcription-polymerase chain reaction procedure, we analyzed foregut and midgut organs for NT/N expression, determined the earliest apparent time point that NT/N is expressed in the gastrointestinal tract and compared the temporal relationship of NT/N with other genes. NT/N expression was detected in the fetal and early postnatal stomach. In the small bowel, NT/N was expressed at the earliest fetal time (14 days) that the small bowel could be reliably delineated from other gut organs; in contrast, expression of sucrase-isomaltase was only apparent at 28 days after birth. NT/N was expressed in the fetal (12, 14 and 16 days) liver and then again on days 3 and 7 after birth; however, NT/N was detected only in the fetal (14 and 16 days) pancreas. Finally, NT/N expression was first detected at approximately 12 days gestation in the primitive foregut and midgut, thus occurring significantly earlier than actual intestinal cytodifferentiation. NT/N is widely expressed in the gastrointestinal tract during fetal development, suggesting the presence of a shared ancestral stem cell; NT/N expression is then restricted to the small bowel of the adult. The determination of the cellular factors regulating the expression of NT/N will provide a better understanding of the mechanisms responsible for the strict patterning of gene expression in the gastrointestinal tract and possibly gut differentiation.

Animals↗
PubMed1999

Characterization of two novel proabsorptive peptide YY analogs, BIM-43073D and BIM-43004C.

Effective clinical therapy to augment intestinal absorption of water and electrolytes does not exist; the gut hormone, peptide YY (PYY), is a potent proabsorptive agent in animal models. The purpose of our study was to evaluate the effects of two novel PYY analogs, BIM-43073D and BIM-43004C, on intestinal absorption. Dogs with ileal Thiry-Vella fistulae (TVF) were treated with either PYY, BIM-43073D, or BIM-43004C. Administration of BIM-43073D significantly increased water and sodium absorption over baseline and maintained this level of increased absorption for a longer duration than an equimolar dose of PYY. Administration of BIM-43004C significantly increased sodium and water absorption over baseline at a level equal to that of PYY. The novel PYY analogs, BIM-43073D and BIM-43004C, are effective proabsorptive agents with BIM-43073D producing more sustained effects than PYY. These compounds may be clinically useful in the treatment of gut malabsorption in conditions such as cholera, Crohn's disease, and the short-bowel syndrome.

Animals↗
PubMed1999

Intestinal adaptation and enterocyte apoptosis following small bowel resection is p53 independent.

Adaptation following small bowel resection (SBR) signals enterocyte proliferation and apoptosis. Because p53-induced p21(waf1/cip1) may be important for apoptosis in many cells, we hypothesized that these genes are required for increased enterocyte apoptosis during adaptation. Male C57BL/6 (wild-type) or p53-null mice underwent 50% proximal SBR or sham operation (bowel transection-reanastomosis). Adaptation (DNA-protein content, villus height-crypt depth, enterocyte proliferation), appearance of apoptotic bodies, and p53 and p21(waf1/cip1) protein expression were measured in the ileum after 5 days. Adaptation was equivalent after SBR in both wild-type and p53-null mice as monitored by significantly increased ileal DNA-protein content, villus height, and enterocyte proliferation. The number of crypt apoptotic bodies increased significantly after SBR evenly in both wild-type and p53-null mice. In the p53-null mice, SBR substantially induced the expression of p21(waf1/cip1) protein in villus enterocytes. The p53-independent induction of p21(waf1/cip1) may account for the similar intestinal response to SBR between wild-type and p53-null mice. Intestinal adaptation and increased enterocyte apoptosis following intestinal resection occur via a p53-independent mechanism.

Adaptation, Physiological↗
PubMed1999

Mitochondrial DNA damage and altered membrane potential (delta psi) in pancreatic acinar cells induced by reactive oxygen species.

BACKGROUND: Reactive oxygen species (ROS) have been implicated in the induction of acute pancreatitis. Mitochondria possess a distinct genome (mtDNA) that is more susceptible to ROS-induced damage than nuclear DNA (nDNA). The purpose of our study was to determine the effect of ROS on mitochondrial function and membrane potential (delta psi mt), to identify signal transduction mechanisms activated by ROS, and to quantify damage to mtDNA in an in vitro pancreatitis model. METHODS: Pancreatic acinar cells, AR4-2J, were treated with saline solution (control) or hydrogen peroxide (H2O2), a representative ROS. Mitochondrial function was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; to determine delta psi mt, rhodamine-123 uptake was measured. Intracellular calcium levels and c-Jun N-terminal kinase activity was determined; gel mobility shift assays were performed to assess induction of the transcription factor NF-kappa B. To quantitate DNA damage, a novel polymerase chain reaction-based procedure was performed. RESULTS: Mitochondrial function and delta psi mt were significantly decreased with oxidative damage. H2O2 treatment resulted in increased intracellular calcium levels, activation of c-Jun N-terminal kinase, and induction of NF-kappa B DNA binding. Treatment of AR4-2J cells with H2O2 resulted in selective mtDNA damage; nDNA was not affected. CONCLUSIONS: Our data demonstrate that pancreatic mtDNA is more susceptible to oxidative damage than nDNA; this damage is associated with decreases in mitochondrial function and delta psi mt and activation of downstream signal transduction pathways. Mitochondrial damage mediated by ROS may play a central role in pancreatic cell injury associated with acute pancreatitis.

Animals↗
PubMed1999

Inhibition of gastric cancer by camptothecin involves apoptosis and multiple cellular pathways.

BACKGROUND: The prognosis for gastric cancer remains dismal; novel agents that target specific molecular pathways are needed as adjuvant therapy. Camptothecin (CPT), on inhibitor of topoisomerase I, is effective in the treatment of certain solid tumors; its effects on gastric cancer are largely undefined. The purpose of this study was to (1) characterize the effects of CPT on the growth of a human gastric cancer and (2) assess potential cellular mechanisms responsible for CPT-mediated growth inhibition. METHODS: The human gastric cancer SIIA was transplanted subcutaneously into athymic nude mice. After tumors reached approximately 100 mm2, mice were randomized into 3 groups to receive either CPT (5 or 10 mg/kg) or vehicle (control) intraperitoneally 3 days per week for 3 weeks; tumor size was measured biweekly. To assess potential mechanisms of CPT-mediated inhibition, SIIA cells were treated with CPT (20 mumol/L) and cells were counted over a time course; apoptosis was assessed by Hoechst stain and DNA laddering. Expression of p53 (a tumor suppressor), p21Waf1 and p27Kip1 (cell cycle inhibitors), and Bcl-2 and Bcl-XL (antiapoptotic proteins) was determined. RESULTS: CPT (5 and 10 mg/kg) significantly inhibited tumor growth of SIIA gastric cancers compared with controls. CPT-mediated inhibition of SIIA cell proliferation was associated with an increase in apoptosis. Moreover, CPT treatment resulted in induction of p53, p21Waf1, and p27Kip1 and a decrease in Bcl-2 and Bcl-XL RNA and protein levels. CONCLUSIONS: Treatment with CPT effectively inhibited the growth of the human gastric cancer SIIA; the mechanism involved was induction of apoptosis mediated by up-regulation of p53, p21Waf1/Cip1, and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL. Novel agents such as CPT, which target specific molecular pathways, may prove clinically useful in the adjuvant treatment of gastric cancers.

Adenocarcinoma↗
PubMed1998

Inhibition of heat-shock protein 70 induction in intestinal cells overexpressing cyclooxygenase 2.

BACKGROUND & AIMS: The cyclooxygenase (COX) enzymes catalyze the initial step of prostaglandin formation; the inducible form, COX-2, plays a role in inflammation. Heat-shock protein 70 (hsp70) is a stress-responsive gene important for cell survival; induction of hsp70 appears to be mediated, in part, by the prostaglandin pathway. We determined the effect of COX-2 overexpression on hsp70 induction in rat intestinal epithelial (RIE) cells. METHODS: RIE cells transfected with COX-2 complementary DNA oriented in the sense (RIE-S) or antisense (RIE-AS) direction were subjected to a heat shock; RNA and protein were harvested and analyzed by Northern and Western blots, respectively. Gel shift assays were performed to assess DNA binding. RESULTS: Both hsp70 messenger RNA and HSP70 protein levels were increased in the RIE-AS cells, whereas induction was markedly inhibited in the RIE-S cells after heat shock. Inhibition of heat-shock factor binding was noted in RIE-S cells, suggesting that heat-shock transcription factor regulation may explain the inhibition of hsp70. The COX-2 selective inhibitor, NS-398, reversed the effects of COX-2 overexpression. CONCLUSIONS: The results support a functional role for the prostaglandin/COX pathway in the induction of hsp70. The findings underscore a potential regulatory mechanism involving an inverse relationship between COX-2 expression and hsp70 induction.

Animals↗