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R J Coffey

Publications and source records attributed to R J Coffey.

At least 55 records · Page 3Linked to original sources
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

Apical enrichment of human EGF precursor in Madin-Darby canine kidney cells involves preferential basolateral ectodomain cleavage sensitive to a metalloprotease inhibitor.

EGF precursor (proEGF) is a member of the family of membrane-anchored EGF-like growth factors that bind with high affinity to the epidermal growth factor receptor (EGFR). In contrast to human transforming growth factor-alpha precursor (proTGFalpha), which is sorted basolaterally in Madin-Darby canine kidney (MDCK) cells (Dempsey, P., and R. Coffey, 1994. J. Biol. Chem. 269:16878-16889), we now demonstrate that human proEGF overexpressed in MDCK cells is found predominantly at the apical membrane domain under steady-state conditions. Nascent proEGF (185 kD) is not sorted but is delivered equally to the apical and basolateral membranes, where it is proteolytically cleaved within its ectodomain to release a soluble 170-kD EGF form into the medium. Unlike the fate of TGFalpha in MDCK cells, the soluble 170-kD EGF species accumulates in the medium, does not interact with the EGFR, and is not processed to the mature 6-kD peptide. We show that the rate of ectodomain cleavage of 185-kD proEGF is fourfold greater at the basolateral surface than at the apical surface and is sensitive to a metalloprotease inhibitor, batimastat. Batimastat dramatically inhibited the release of soluble 170-kD EGF into the apical and basal medium by 7 and 60%, respectively, and caused a concordant increase in the expression of 185-kD proEGF at the apical and basolateral cell surfaces of 150 and 280%, respectively. We propose that preferential ectodomain cleavage at the basolateral surface contributes to apical domain localization of 185-kD proEGF in MDCK cells, and this provides a novel mechanism to achieve a polarized distribution of cell surface membrane proteins under steady-state conditions. In addition, differences in disposition of EGF and TGFalpha in polarized epithelial cells offer a new conceptual framework to consider the actions of these polypeptide growth factors.

Animals↗
PubMed1997

Targeted disruption of the epidermal growth factor receptor impairs growth of squamous papillomas expressing the v-ras(Ha) oncogene but does not block in vitro keratinocyte responses to oncogenic ras.

We have assessed the role of epidermal growth factor receptor (EGFR) signaling in biological responses to the v-ras(Ha) oncogene using primary keratinocytes from Egfr -/- mice and wild-type littermates. On the basis of several criteria, Egfr -/- keratinocytes were unresponsive to either acute or chronic exposure to several EGFR ligands but were stimulated to proliferate in response to several other mitogens. Although conditioned medium from primary keratinocytes transduced with v-ras(Ha) retrovirus (v-ras(Ha) keratinocytes) was a potent mitogen for wild-type but not Egfr -/- keratinocytes, v-ras(Ha) transduction of primary keratinocytes of either genotype resulted in a strong mitogenic response, arguing against an obligatory role for EGFR activation in v-ras(Ha)-mediated stimulation of keratinocyte proliferation. Infection with high-titer v-ras(Ha) retrovirus altered the keratin expression pattern in keratinocytes of both genotypes, suppressing differentiation-specific keratins K1 and K10 while activating aberrant expression of K8 and K18. In wild-type but not Egfr -/- cultures, K1 and K10 were also suppressed following infection at lower retroviral titers, presumably as a result of paracrine EGFR activation on uninfected cells present in these cultures. Squamous papillomas produced by grafting Egfr -/- v-ras(Ha) keratinocytes onto nude mice were only 21% of the size of wild-type v-ras(Ha) tumors, and a striking redistribution of S-phase cells was detected by immunostaining for bromodeoxyuridine. In Egfr -/- v-ras(Ha) papillomas, the fraction of total labeled nuclei detected in suprabasal layers was increased from 19 to 39%. In contrast, the basal layer labeling index of Egfr -/- papillomas was reduced to 34%, compared to 43% in wild-type tumors. Our results indicate that, although autocrine EGFR signaling is not required for keratinocyte responses to oncogenic ras in culture or benign tumor formation in nude mouse grafts, disruption of this pathway impairs growth of v-ras(Ha) papillomas by a mechanism that may involve alterations in keratinocyte cell cycle progression and/or migration in vivo.

Animals↗
PubMed1997

A raf-independent epidermal growth factor receptor autocrine loop is necessary for Ras transformation of rat intestinal epithelial cells.

We recently have shown that activated Ras, but not Raf, causes transformation of intestinal (RIE-1, IEC-6) epithelial cells, whereas both activated Ras and Raf transform NIH 3T3 fibroblasts (Oldham, S. M., Clark, G. J., Gangarosa, L. M., Coffey, R. J., and Der, C. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6924-6928). The observations that conditioned medium from Ras-, but not Raf-, transfected RIE-1 cells, as well as exogenous transforming growth factor alpha (TGFalpha), promoted morphological transformation of parental RIE-1 cells prompted us to identify epidermal growth factor (EGF) receptor (EGFR) ligands produced by Ras-transformed RIE-1 cells responsible for this autocrine effect. Since studies in fibroblasts have shown that v-Src is transforming, we also determined if v-Src could transform RIE-1 cells. H- or K-Ras-transformed cells secreted significant amounts of TGFalpha protein, and mRNA transcripts for TGFalpha, amphiregulin (AR), and heparin-binding EGF-like growth factor (HB-EGF) were induced. Like Ras, v-Src caused morphological and growth transformation of parental RIE-1 cells. However, TGFalpha protein was not secreted by RIE-1 cells stably expressing v-Src or activated Raf, and only minor increases in EGFR ligand mRNA expression were detected in these cells. A selective EGFR tyrosine kinase inhibitor PD153035 attenuated the Ras-, but not Src-, transformed phenotype. Taken together, these observations provide a mechanistic and biochemical basis for the ability of activated Ras, but not activated Raf, to cause transformation of RIE-1 cells. Finally, we suggest that an EGFR-dependent mechanism is necessary for Ras, but not Src, transformation of these intestinal epithelial cells.

Animals↗
PubMed1997

Stereotactic radiosurgery for glomus jugulare tumors: a preliminary report.

PURPOSE: This study was conducted to evaluate the toxicity and efficacy of stereotactic radiosurgery treatment of glomus jugulare tumors. METHODS AND MATERIALS: Between March 1990 and January 1995, nine patients underwent stereotactic radiosurgery with the Leksell Gamma Knife Unit for glomus jugulare tumors. Previous treatment had failed in four patients. The seven female and two male patients had a median age of 67 years. RESULTS: The median time from stereotactic radiosurgery to the most recent clinical follow-up was 20 months (range 7-65 months). Subjectively, 7 of 9 patients noted a decrease in the intensity of their symptoms. Objectively, 8 of 9 tumors remained stable in size by serial magnetic resonance imaging scans and one was smaller. There was no acute or chronic toxicity. CONCLUSION: This early experience suggests that stereotactic radiosurgery is a promising treatment for glomus jugulare tumors.

Adult↗
PubMed1997

Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2.

A considerable amount of evidence collected from several different experimental systems indicates that cyclooxygenase-2 (COX-2) may play a role in colorectal tumorigenesis. Large epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in persons taking aspirin or other nonsteroidal antiinflammatory drugs on a regular basis. One property shared by all of these drugs is their ability to inhibit COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. In this study, we selected two transformed human colon cancer cell lines for studies on the role of COX-2 in intestinal tumorigenesis. We evaluated HCA-7 cells which express high levels of COX-2 protein constitutively and HCT-116 cells which lack COX-2 protein. Treatment of nude mice implanted with HCA-7 cells with a selective COX-2 inhibitor (SC-58125), reduced tumor formation by 85-90%. SC-58125 also inhibited colony formation of cultured HCA-7 cells. Conversely, SC-58125 had no effect on HCT-116 implants in nude mice or colony formation in culture. Here we provide evidence that there may be a direct link between inhibition of intestinal cancer growth and selective inhibition of the COX-2 pathway.

Animals↗
PubMed1997

Mob-1, a Ras target gene, is overexpressed in colorectal cancer.

Mutations in the ras oncogenes have been linked to many different cancers. In contrast to the extensive body of knowledge related to the genetics of ras activation, relatively little is known of the transcriptional events triggered by ras. In previous work we have used differential display to identify Mob-1, a member of alpha-chemokine family, as one of the immediate transcriptional targets following Ras activation. Here, we provide additional experimental evidence to support this finding by the use of an inducible H-ras expression system, the treatment of Ras farnesyl transferase inhibitor and activation of endogenous Ras by serum growth factors. We further demonstrate that IP-10, the human homolog of Mob-1, is overexpressed in the majority of colorectal cancers.

Alkyl and Aryl Transferases↗
PubMed1997

Targeting transforming growth factor alpha expression to discrete loci of the neuroendocrine brain induces female sexual precocity.

Precocious puberty of cerebral origin is a poorly understood disorder of human sexual development, brought about by the premature activation of those neurons that produce luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation. An increased production of transforming growth factor alpha (TGF alpha) in the hypothalamus has been implicated in the mechanism underlying both normal and precocious puberty. We have now used two gene delivery systems to target TGF alpha overexpression near LHRH neurons in immature female rats. Fibroblasts infected with a retroviral construct in which expression of the human TGF alpha gene is constitutively driven by the phosphoglycerate kinase promoter, or transfected with a plasmid in which TGF alpha expression is controlled by an inducible metallothionein promoter, were transplanted into several regions of the hypothalamus. When the cells were in contact with LHRH nerve terminals or in the vicinity of LHRH perikarya, sexual maturation was accelerated. These results suggest that precocious puberty of cerebral origin may result from a focal disorder of TGF alpha production within the confines of the LHRH neuron microenvironment.

3T3 Cells↗
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

A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells.

BACKGROUND & AIMS: Constitutive expression of cyclooxygenase 2 (COX-2) has been found in 85% of colorectal cancers. Ras mutations are found in 50% of colorectal adenocarcinomas. The aim of this study was to determine the role of COX-2 in ras-induced transformation in rat intestinal epithelial (RIE) cells. METHODS: Cell growth was determined by cell counts. The expression of COX-2 was examined by Northern and Western analyses. For tumorigenicity assays, cells were inoculated into dorsal subcutaneous tissue of athymic nude mice. DNA-fragmentation assays were performed to detect apoptosis. RESULTS: The expression of COX-2 was increased in RIE-Ras cells at both messenger RNA (9-fold) and protein (12-fold) levels. Prostaglandin I2 levels were elevated 2.15-fold in RIE-Ras cells. Serum deprivation further increased COX-2 expression 3.8-fold in RIE-Ras cells. Treatment with a selective COX-2 antagonist (SC58125) inhibited the growth of RIE-Ras cells through inhibition of cell proliferation and by induction of apoptosis. SC-58125 treatment reduced the colony formation in Matrigel by 83.0%. Intraperitoneal administration of SC-58125 suppressed RIE-Ras tumor growth in nude mice by 60.3% in 4 weeks. SC-58125 treatment also induced apoptosis in RIE-Ras cells as indicated by increased DNA fragmentation. CONCLUSIONS: Overexpression of COX-2 may contribute to tumorigenicity of ras-transformed intestinal epithelial cells. Selective inhibition of COX-2 activity inhibits growth of ras-transformed intestinal epithelial cells and induces apoptosis.

Animals↗
PubMed1997

Transforming growth factor-alpha (TGF-alpha) levels in human proximal gastrointestinal epithelium. Effect of mucosal injury and acid inhibition.

TGF-alpha inhibits gastric acid secretion and may play an important role in epithelial repair. We quantitated regional levels of TGF-alpha in the human proximal gastrointestinal tract and determined whether they are affected by acid suppression or aspirin-induced injury. Ten healthy volunteers were studied. After baseline endoscopy with biopsy, five randomly received no treatment, aspirin, omeprazole, or cimetidine for one week. Endoscopy was repeated and prior unhealed biopsy sites quantitated. TGF-alpha levels were measured by RIA. Five additional subjects then completed an extended protocol of three weeks duration. All subjects were free of H. pylori infection. TGF-alpha levels in the antrum, 34.76 +/- 5.54 pg TGF-alpha/micrograms DNA were threefold higher than in the gastric body and duodenum (11.03 +/- 2.60 and 10.41 +/- 1.64 respectively, P < 0.01). The number of unhealed sites in the aspirin group was significantly greater than in the control or acid inhibition groups; however, TGF-alpha levels were not different from-the surrounding mucosa. TGF-alpha increased in the controls after biopsy; the increase was significant in the body at week 2 only. Aspirin significantly increased TGF-alpha levels in the gastric body and duodenum after one week. The rise in antral TGF-alpha appeared delayed and blunted by the aspirin treatment compared to control. There was no relationship between the number of visible biopsy sites, degree of aspirin-induced injury, and the TGF-alpha level. Acid suppression was associated with a significant increase in TGF-alpha in the gastric body and antrum at one week. Immunochemical staining did not demonstrate differences in proliferation in any treatment group compared to controls. TGF-alpha levels vary by location in the proximal gastrointestinal tract, with significantly greater levels in the antrum. After biopsy, TGF-alpha levels increase; short-term aspirin and acid inhibitors modulate this effect. Aspirin significantly impaired the healing of endoscopic biopsies in the antrum; however, this was not associated with changes in TGF-alpha levels. TGF-alpha levels did not change in response to acid secretory state. Further studies of mucosal levels of TGF-alpha in response to aspirin-induced injury in humans appear warranted.

Adult↗
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↗
PubMed1996

Gamma knife for glioma: selection factors and survival.

PURPOSE: To determine factors associated with survival differences in patients treated with radiosurgery for glioma. METHODS AND MATERIALS: We analyzed 189 patients treated with Gamma Knife radiosurgery for primary or recurrent glioma World Health Organization (WHO) Grades 1-4. RESULTS CONCLUSION: The median minimum tumor dose was 16 Gy (8-30 Gy) and the median tumor volume was 5.9 cc (1.3-52 cc). Brachytherapy selection criteria were satisfied in 65% of patients. Median follow-up of all surviving patients was 65 weeks after radiosurgery. For primary glioblastoma patients, median survival from the date of pathologic diagnosis was 86 weeks if brachytherapy criteria were satisfied and 40 weeks if they were not (p = 0.01), indicating that selection factors strongly influence survival. Multivariate analysis showed that increased survival was associated with five variables: lower pathologic grade, younger age, increased Karnofsky performance status (KPS), smaller tumor volume, and unifocal tumor. Survival was not found to be significantly related to radiosurgical technical parameters (dose, number of isocenters, prescription isodose percent, inhomogeneity) or extent of preradiosurgery surgery. We developed a hazard ratio model that is independent of the technical details of radiosurgery and applied it to reported radiosurgery and brachytherapy series, demonstrating a significant correlation between survival and hazard ratio. CONCLUSIONS: Survival after radiosurgery for glioma is strongly related to five selection variables. Much of the variation in survival reported in previous series can be attributed to differences in distributions of these variables. These variables should be considered in selecting patients for radiosurgery and in the design of future studies.

Adolescent↗
PubMed1996

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.

3T3 Cells↗
PubMed1996

Overexpression of transforming growth factor-alpha alters differentiation of gastric cell lineages.

Overexpression of transforming growth factor-alpha (TGF-alpha) in the gastric fundic mucosa of metallothionein promoter/enhancer-TGF-alpha(MT-TGF-alpha) transgenic mice produces a phenotype of foveolar hyperplasia similar to that observed in Ménétrier's disease. We have investigated the dynamics involved in the alterations of gastric mucosal morphology in the MT-TGF-alpha mouse model. The fundic mucosa of MT-TGF-alpha mice and nontransgenic littermates was evaluated in animals treated with cadmium sulfate. To mark the mucosal proliferative zone, 8-bromodeoxyuridine (BrdU) was administered 2 hr prior to killing. Gastric mucosa was examined by diastase-resistant, periodic acid-Schiff-positive (DR-PAS) staining and immunohistochemistry for H/K-ATPase an BrdU. MT-TGF-alpha mice demonstrated increased numbers of DR-PAS-staining mucous cells and lower parietal cell numbers per gland unit. While the proliferative zone in nontransgenic mice was located in the upper half of the gland, the zone in MT-TGF-alpha mice was located in the basal region. Overexpression of TGF-alpha in MT-TGF-alpha mice leads to an alteration in the development of mucosal lineages from the fundic progenitor zone, which is biased towards the predominant differentiation of foveolar mucous cells.

Animals↗
PubMed1996

Transforming growth factor-alpha.

Major advances in understanding growth factor biology, especially in epithelial cells, have resulted from work with TGF-alpha over the past decade. It is clear that TGF-alpha is a potent epithelial oncoprotein, but equally important biological activities in normal epithelial homeostasis have been described. A number of major challenges lie ahead. Foremost is the formidable task of dissecting out the individual contributions of each EGF-related peptide in the biological response to stimulation of the EGFR. Appreciation of the complexity of heterodimerization of receptors within the EGFR family will be equally important in the final analysis. These considerations assure the continued vitality and productivity of investigation of the EGF-related peptide/EGFR axis.

Amino Acid Sequence↗
PubMed1996

Increased gastroduodenal concentrations of transforming growth factor alpha in adaptation to aspirin in monkeys and rats.

BACKGROUND & AIMS: The mechanism by which gastric mucosa becomes more resistant to damage by repeated aspirin administration is not known. Transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) prevent drug-induced gastric injury. The aim of this study was to determine whether gastroduodenal tissue levels of TGF-alpha and EGF protein were altered during adaptation to aspirin-induced injury in monkeys and rats in vivo. METHODS: Animals were given aspirin daily for up to 28 days. Gross mucosal injury was assessed by computerized image analysis in rats and by endoscopy in monkeys. Mucosal concentrations of TGF-alpha and EGF were quantitated by radioimmunoassays from endoscopic biopsy samples in monkeys and from scraped mucosa in rats. RESULTS: Long-term administration of aspirin caused a significant increase in gastric and duodenal tissue levels of TGF-alpha in monkeys and rats; the increased levels of TGF-alpha significantly correlated with the decrease in aspirin-induced injury. No change in the gastroduodenal tissue levels of EGF was observed. Adaptation was not associated with any significant change in basal gastric acid secretion in monkeys and occurred despite a significant decrease in gastric mucin in rats. CONCLUSIONS: Adaptation of the gastric mucosa to the damaging effect of aspirin is associated with a significant and specific increase in TGF-alpha protein in the gastroduodenum.

Adaptation, Physiological↗