PubMed Health⌕ Search

Biomedical subjects

John M Daly

Publications and source records attributed to John M Daly.

At least 19 recordsLinked to original sources
PubMed2007

Field electroantennogram and trap assessments of aerosol pheromone dispensers for disrupting mating in Epiphyas postvittana.

An electronically controlled aerosol system for mating disruption was evaluated against Epiphyas postvittana Walker in apple orchards in New Zealand. The area in which male moths were affected by the aerosol system was examined using catches in traps radiating from a central single-point source of either one aerosol can dispenser or 100 polyethylene tubing dispensers, compared with catches in orchard plots without pheromone dispensers. Both pheromone dispensing systems decreased moth catch to similar levels at 5-10 m from the central release point, but there was 5.5-fold more pheromone released from aerosol cans than from polyethylene dispensers over a 24 h period. Trap catches were reduced by about 90% in plots treated with either five aerosol cans per hectare or uniform deployment of polyethylene dispensers. Recordings of electroantennograms in open grassed plots and orchards indicated that the treated cotton pad of an aerosol dispenser and a point source of 100 polyethylene tubing dispensers produced similar electroantennogram recordings. Electroantennogram recordings provided evidence that pheromone plume detection from a single-point source was maintained over a range of 5-40 m downwind in the orchard. On present evidence, aerosol pheromone dispensers could not be recommended for further testing towards control of E. postvittana under New Zealand conditions owing to their higher cost of purchase and operation.

Aerosols↗
PubMed2005

Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system.

Reporter assays are widely used in applications that require measurement of changes in gene expression over time (e.g. drug screening). With standard reporter vectors, the measurable effect of a treatment or compound (altered reporter activity) is substantially diluted and delayed, compared with its true effect (altered transcriptional activity). This problem is caused by the relatively long half-lives of both the reporter protein and its mRNA. As a result, the activities of compounds, ligands or treatments that have a relatively minor effect, or a substantial but transient effect, often remain undetected. To circumvent this problem, we introduced modular protein- and mRNA-destabilizing elements into a range of commonly used reporters. Our data show that both elements are required for maximal responses to both increases and decreases in transcriptional activity. The double-destabilized reporter vectors showed markedly improved performance in drug screening, kinetic assays and dose-response titrations.

Amino Acid Motifs↗
PubMed2005

Lysis-deficient bacteriophage therapy decreases endotoxin and inflammatory mediator release and improves survival in a murine peritonitis model.

BACKGROUND: Lysis-deficient (LyD) bacteriophages (phages) kill bacteria without endotoxin (Et) release. This may minimize systemic cytokine responses and limit inflammation in bacterial sepsis. We determined the effects of t amber A3 T4 LyD and virulent wild-type (WT) phages on mouse bacterial peritonitis. METHODS: Balb/c mice were injected with B40sul Escherichia coli, treated intraperitoneally with LyD, WT, or a beta-lactam antibiotic [latamoxef sodium (LMOX)], and followed for survival. We measured Et release, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, as well as bacterial counts and peritoneal exudative cells (PECs) in peritoneal lavage fluid at 6 and 12 hours after infection. RESULTS: LyD mice showed significantly greater survival compared with other groups. Et levels were significantly lower in the LyD mice at 6 and 12 hours after infection. TNF-alpha and IL-6 levels were lower in LyD mice compared with control (untreated) mice at 12 hours. Compared with controls, bacteria counts in peritoneal lavage fluid were lower in all treatment groups (LyD, WT, or LMOX) at 6 and 12 hours. PEC counts were highest in LyD mice at 6 hours but significantly lower than that in WT phage- and LMOX-treated mice at 12 hours. CONCLUSIONS: LyD phage therapy significantly improves survival and attenuates the systemic effects of bacterial sepsis by minimizing Et release and pro-inflammatory mediators in murine bacterial peritonitis. Further studies may find phage therapy useful in treating peritonitis and multidrug-resistant bacterial infections.

Animals↗
PubMed2004

NS-398 inhibits tumor growth and liver metastasis of colon cancer through induction of apoptosis and suppression of the plasminogen activation system in a mouse model.

BACKGROUND: Cyclooxygenase-2 (COX-2) is overexpressed in colon cancers. The plasminogen activation (PA) system relates to cancer invasion and metastasis through the degradation of the extracellular matrix. COX-2 also relates to degradation of the extracellular matrix, but the relationship between COX-2 and the plasminogen activator system is unclear. STUDY DESIGN: In vivo: Colon 38 (G0) primary and (G5) metastatic cell lines were implanted in C57BL/6 mice treated with or without COX-2 inhibitor (NS-398). Animal survival and tumor growth were measured. On day 19, tumors were excised and tumor cell apoptosis measured. For metastasis, G5 cells were injected into the spleen, and, after 23 days, liver metastasis was determined. In vitro: G0 or G5 cells were treated with NS-398. Supernatant prostaglandin E2 and mRNA expressions of COX-2, vascular endothelial growth factor (VEGF), urokinase-type plasminogen activator (u-PA), u-PA receptor, plasminogen activator inhibitor type-1 (PAI-1), and PAI-2 were measured. Tumor cell proliferation was also determined. RESULTS: In vivo: Mean survival of NS-398-treated animals was higher than controls for both G5 and G0 (G5: p < 0.003, G0: p < 0.02). G5 tumors grew faster than G0 tumors (p < 0.001) and NS-398 significantly inhibited tumor growth (p < 0.001), induced tumor cell apoptosis (p < 0.001), and significantly reduced metastasis (p < 0.003) in G5 animals. In vitro: PGE(2) production was higher in G5 than G0 cells (p < 0.001); NS-398 significantly reduced prostaglandin E(2) levels in G5 cells (p < 0.001). mRNA expression of COX-2, vascular endothelial growth factor, and u-PA receptor was higher in G5 than G0 cells, and NS-398 significantly inhibited u-PA mRNA expression in G5 cells. NS-398 significantly reduced proliferation in G5 cells (p < 0.05). CONCLUSIONS: COX-2 inhibition significantly decreases tumor growth in this model by inducing apoptosis and blocking u-PA production in G5 colon cancer cells, which is associated with significant inhibition of liver metastases.

Animals↗
PubMed2004

Gender affects macrophage cytokine and prostaglandin E2 production and PGE2 receptor expression after trauma.

BACKGROUND: Gender influences morbidity and mortality after injury. Hormonal differences are important; however, the role of prostaglandins as mediators in immune dysfunction relating to gender differences after trauma is unclear. We hypothesized that gender-dependent differences in PGE(2) receptor expression and signaling may be involved in immune-related differences. This study determined prostaglandin receptor subtype (EP1-EP4) expression following injury and determined whether gender differences influence EP receptor expression. MATERIALS AND METHODS: BALB/c male and female mice (estrus and pro-estrus) (n = 6 per group) were subjected to femur fracture and 40% hemorrhage (trauma) or sham injury (anesthesia). Seven days later, the splenic macrophages were harvested and stimulated with lipopolysaccharide (Escherichia coli serotype O55:B5). After 6 h mRNA samples were collected for EP receptor mRNA expression and at 24 h supernatants were collected for PGE(2), TNF-alpha, and IL-6 production. RESULTS: The expression of EP2-4 receptors was higher in female pro-estrus mice compared with male mice. EP1 receptor expression was higher in males than pro-estrus females. There was decreased expression of all four receptors after trauma in female estrus compared with control estrus mice. Macrophage PGE(2), TNF-alpha, and IL-6 production was significantly increased in injured female mice compared with female controls but there were no differences in injured male mice compared with male controls. PGE(2) and TNF-alpha production by traumatized male mice were significantly less than that produced by traumatized pro-estrus females. CONCLUSIONS: These data suggest gender-related differences in response to traumatic injury and that alterations in specific EP receptor subtypes may be involved in immune dysfunction after injury. Studies to evaluate targeted modulation of these receptor subtypes may provide further insights to gender-specific differences in the immune response after injury.

Animals↗
PubMed2004

Neuroendocrine responses mediate macrophage function after trauma.

BACKGROUND: Clearly understanding the interactions between macrophage (M phi)-generated inflammatory mediators and the neuroendocrine system in regulating immune function after traumatic injury may aid in reversing trauma-mediated immune dysfunction and diminish the incidence and severity of infection in the traumatized patient. METHODS: Trauma consisted of an open femur fracture and 40% retro-orbital hemorrhage (Trauma) or anesthesia alone (Control). Female Balb/C mice (6-8 weeks) with intact adrenal glands (Intact) or a bilateral adrenalectomy (ADX) were used. For glucocorticoid studies, corticosterone or a vehicle was administered via intraperitoneal (ip) injection 2 hours before the trauma. Splenic M phis were harvested and prostaglandin E(2) (PGE(2)) and interleukin-6 (IL-6) production, and mRNA, cyclooxygenase-2 (COX-2) protein, and nuclear factor kappa B (NF-kappa B) activity were measured. RESULTS: M phi, PGE(2) and IL-6 production in Trauma+Intact mice was significantly increased compared with Control+Intact mice. Adrenalectomy decreased these levels to Control levels. Similar changes were observed for COX-2 and IL-6 expression. M phi nuclear NF-kappa B levels were increased in Trauma+Intact mice compared with controls. Adrenalectomy abrogated this increase. Treating Trauma+Intact mice with RU-486 did not restore PGE(2) and IL-6 production or COX-2 and IL-6 messenger RNA to control levels. Administering exogenous glucocorticoid to Intact mice did not increase PGE(2) and IL-6 production or COX-2 and IL-6 mRNA to Trauma levels. CONCLUSIONS: The neuroendocrine system upregulates certain M phi inflammatory mediators, including PGE(2), IL-6, and NF-kappa B, after trauma. This upregulation does not seem to be mediated via glucocorticoids and possibly may be mediated via catecholamines. Elucidation of the interactions between the neuroendocrine system, the immune system, and inflammatory mediator secretion might provide novel therapeutic strategies for the injured patient.

Animals↗
PubMed2004

Glucocorticoid pretreatment induces cytokine overexpression and nuclear factor-kappaB activation in macrophages.

BACKGROUND: Glucocorticoids are widely used in treating inflammatory diseases. The contribution of adrenal glucocorticoids to inflammatory regulation is unknown. Endogenous glucocorticoids, as distinct from synthetic analogues, not only suppress but also enhance immune functions. Elevated circulating cortisol levels are characteristic of injured patients. In a model of trauma, an early glucocorticoid surge occurs concomitantly with decreased cellular cytokine responses. Cytokine production elevated late after injury is associated with increased mortality. We hypothesized that this glucocorticoid surge mediates the later heightened macrophage responses. MATERIALS AND METHODS: The murine macrophage like cells RAW 264.7 were incubated with corticosterone (35 ng/mL), or vehicle control, for 1 h, after which the cells were washed and corticosterone-free medium added. At 0, 3, 6, 12, and 24 h after removal of the corticosterone, the cells were stimulated with lipopolysaccharide (LPS) and interferon-gamma. Supernatant tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and nitrite levels were measured. In separate experiments the effect of pretreatment with corticosterone on TNF-alpha, IL-6, and nitrite mRNA expression as well as nuclear factor-kappaB and glucocorticoid receptor activity was determined. CD14 receptor expression was determined by flow cytometry. RESULTS: Glucocorticoid pretreatment caused significantly increased RAW 264.7 cell production of nitrite, IL-6 and TNF-alpha. mRNA for these inflammatory mediators was induced 6 h after the corticosterone pretreatment, and was associated with activation of nuclear factor-kappaB in the presence of activated glucocorticoid receptor. Cell surface-expression of CD14 was likewise increased. CONCLUSIONS: The results of this study demonstrate a novel role for glucocorticoids and provide a mechanism for the late upregulation in macrophage function after injury.

Animals↗
PubMed2004

Altered cyclooxygenase-2 expression and nitric oxide metabolism following major elective surgery.

BACKGROUND AND AIMS: Postoperative variation in immune function leads to increased susceptibility to infections. Cyclooxygenase-2 (COX-2)-generated Prostaglandin-E(2) (PGE(2)), which signals through the PGE(2) receptor (EP receptor), as well as nitric oxide metabolites (NOx), appear to be important in postoperative immune dysfunction. It is unclear, however, how these substrates and receptors change over time. This study was conducted to evaluate postoperative changes in inflammatory mediator production and monocyte COX-2 and EP receptor expression. MATERIALS AND METHODS: Nineteen patients had blood drawn preoperatively and up to 1 week postoperatively. Plasma NOx levels were measured. Peripheral blood mononuclear cell (PBMC) COX-2 and EP receptor mRNA expression were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). PBMC PGE(2), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and IL-10 productions were evaluated by enzyme-linked immunosorbent assay (ELISA) kits. Statistical analyses were by ANOVA and Student's t tests. RESULTS: Postoperatively, PBMC mean PGE(2) and IL-6 productions were significantly increased at all time points. Mean TNF-alpha production was maximal on postoperative day 2, while mean IL-10 production was unchanged. Mean circulating NOx levels demonstrated a biphasic response decreasing early postoperatively and normalizing at postoperative day (POD) 7. PBMC COX-2 enzyme and EP receptor mRNA expression were unchanged. CONCLUSIONS: Altered PBMC PGE(2) production and plasma NOx levels support a role for altered macrophage activity, which may contribute to immune dysfunction in the postoperative period.

Adult↗
PubMed2004

Decreased peritoneal macrophage NF-kappaB translocation to the nucleus in protein energy malnutrition--a role for the glucocorticoid response?

BACKGROUND & AIMS: Protein energy malnutrition (PEM) induces immune suppression leading to increased morbidity and mortality. The mechanism(s) underlying PEM-mediated immune suppression remain unclear. Plasma glucocorticoid levels are elevated in PEM and it has been postulated that these increased levels may mediate macrophage (MØ) dysfunction in PEM. We have previously shown that nuclear factor kappa B (NF-kappaB) activation in response to LPS stimulation is diminished in peritoneal macrophages (PMØs) from PEM mice. We hypothesized that decreased NF-kappaB activation in lipopolysaccharide (LPS)-stimulated PMØs in PEM is mediated through increased circulating glucocorticoid levels. METHODS: Mice were randomized to six groups of n = 15 each as follows: (1) control diet (24% casein) (C); (2) protein-free diet (PF); (3) mice with subcutaneously implanted corticosterone pellet on the control diet; (4) mice with subcutaneously implanted placebo pellet on the control diet; (5) adrenalectomized mice on the control diet; (6) adrenalectomized mice on the PF diet. Within each group, the mice were pair-fed for 7 days. At the end of the experimental time period, PMØs were harvested and NF-kappaB activation determined by electrophoretic mobility shift assay (EMSA). RESULTS: Elevated circulating glucocorticoids diminished NF-kappaB activation but adrenalectomy failed to restore this diminution in a murine model of PEM. CONCLUSION: NF-kappaB translocation to the nucleus in PEM is independent of elevated circulating glucocorticoid levels.

Adrenalectomy↗
PubMed2004

Laparoscopic esophagomyotomy for achalasia: does anterior hemifundoplication affect clinical outcome?

OBJECTIVE: To determine whether the addition of anterior hemifundoplication to laparoscopic esophagomyotomy for achalasia yields better clinical outcomes than laparoscopic esophagomyotomy alone. SUMMARY BACKGROUND DATA: Although hemifundoplication may prevent gastroesophageal reflux after esophagomyotomy for achalasia, it may also lead to persistent dysphagia in these patients with esophageal aperistalsis. METHODS: This is a retrospective study of 51 consecutive patients (mean age 47.5 +/- 12.6 years) who had laparoscopic esophagomyotomy for achalasia by our group between August 1995 and January 2001. In 29 patients (57%) an anterior hemifundoplication was added to the esophagomyotomy. In 22 patients (43%), no wrap was added. Patients scored (0 = none; 1 = mild; 2 = moderate; 3 = severe) symptom severity (dysphagia, regurgitation, heartburn, chest pain) preoperatively and postoperatively. Weight gain, use of gastrointestinal (GI) medication, tolerance to food, and patient satisfaction were also assessed. RESULTS: Mean patient follow-up was 33 months, and there were no operative deaths. Four patients were converted to open operation (8%). The wrap and no wrap groups were similar in terms of esophageal dilation, preoperative symptom severity and duration (5.7 +/- 7.1 versus 6.1 +/- 7.0 years), and preoperative weight loss (18 +/- 15 versus 20 +/- 20 pounds). Both groups had similar improvement in symptom grade postoperatively and equivalent satisfaction rates (86%). Postoperative weight gain, GI medication use, and food intolerance was also similar. Postoperatively, patients in the wrap group did not have higher dysphagia scores or lower heartburn scores than the no wrap group. CONCLUSION: The addition of anterior hemifundoplication to esophagomyotomy for achalasia does not improve or worsen clinical results.

Adult↗
PubMed2003

Gallbladder cancer in the twentieth century: single institution's experience.

The purpose of this study was to understand trends in the presentation, management, and outcome of patients of patients with gallbladder cancer treated over a period of 85 years at a single institution. We analyzed patients with gallbladder carcinoma treated at our institution between 1990 and 2000 (n=66). Data from this series were analyzed in the context of previously reported series from our institution (beginning in 1915) to understand trends in the presentation, management, and outcome of patients with gallbladder carcinoma. The mean age of patients has increased from 53.6 years (1915-1932) to 65.0 years (1990-2000). The gender (73% female) distribution of patients and most the common presenting symptoms (abdominal pain, weight loss, jaundice, nausea, abdominal mass) have not changed over the 85 years. More extensive surgery is being performed on patients with gallbladder carcinoma. The mean survival of patients with gallbladder cancer has increased from 3.6 months (1915-1932) to 10.0 months (1990-2000). The presentation of patients with gallbladder cancer has not changed over the 85 years. Most patients still present with advanced disease. The overall survival of patients with gallbladder cancer is poor, but it has improved since 1915.

Adult↗
PubMed2003

Influence of surgical subspecialty training on in-hospital mortality for gastrectomy and colectomy patients.

OBJECTIVE: This study examined the relationship of surgeon subspecialty training and interests to in-hospital mortality while controlling for both hospital and surgeon volume. SUMMARY BACKGROUND DATA: The relationship between volume of surgical procedures and in-hospital mortality has been studied and shows an inverse relationship. METHODS: A large Statewide Planning and Research Cooperative System was used to identify all 55,016 inpatients who underwent gastrectomy (n = 6434) or colectomy (n = 48,582) between January 1, 1998 and December 31, 2001. Surgical subspecialty training and interest was defined as surgeons who were members of the Society of Surgical Oncology (training/interest; n = 68) or the Society of Colorectal Surgery (training; n = 61) during the study period. The association of in-hospital mortality and subspecialty training/interest was examined using a logistic regression model, adjusting for demographics, comorbidities, insurance status, and hospital and surgeon volume. RESULTS: Overall mortality for colectomy patients was 4.6%; the adjusted mortality rate for subspecialty versus nonsubspecialty-trained surgeons was 2.4% versus 4.8%, respectively (adjusted odds ratio [OR] = 0.45; 95% confidence interval [CI] = 0.34, 0.60; P < 0.0001). Gastrectomy patients experienced an overall mortality rate of 8.4%; the adjusted mortality rate for patients treated by subspecialty trained surgeons was 6.5%, while the adjusted mortality rate for nonsubspecialty trained surgeons was 8.7% (adjusted OR = 0.70; 95% CI = 0.46, 1.08; P = 0.10). CONCLUSIONS: For gastrectomies and colectomies, risk-adjusted mortality is substantially lower when performed by subspecialty interested and trained surgeons, even after accounting for hospital and surgeon volume and patient characteristics. These findings may have implications for surgical training programs and for regionalization of complex surgical procedures.

Aged↗
PubMed2003

Comparison of linear array endoscopic ultrasound and helical computed tomography for the staging of periampullary malignancies.

BACKGROUND: The purpose of this study was to compare linear array endoscopic ultrasound (EUS) and helical computed tomography (CT) scan in the preoperative local staging evaluation of patients with periampullary tumors. METHODS: Patients evaluated with EUS and CT for suspected periampullary malignancies from 1996 to 2000 were analyzed. Surgical/pathology staging results were the reference standard. RESULTS: Forty-eight patients (28 men and 20 women; mean age, 62 +/- 4.9 years; range, 18-90 years) were identified. Malignancy was histologically confirmed in 44 patients. Parameters evaluated included tumor size, lymph node metastases, and major vascular invasion. EUS was significantly more sensitive (100%), specific (75%), and accurate (98%) than helical CT (68%, 50%, and 67%, respectively) for evaluation of the periampullary mass (P <.05). In addition, EUS detected regional lymph node metastases in more patients than helical CT. Sensitivity, specificity, and accuracy of EUS were 61%, 100%, and 84%, in comparison to 33%, 92%, and 68%, respectively, with CT. Major vascular involvement was noted in 9 of 44 patients. EUS correctly identified vascular involvement in 100% compared with 45% with CT (P <.05). CONCLUSIONS: Linear array EUS was consistently superior to helical CT in the preoperative local staging of periampullary malignancies.

Adolescent↗
PubMed2003

Cyclooxygenase-2 inhibition improves macrophage function in melanoma and increases the antineoplastic activity of interferon gamma.

BACKGROUND: Melanoma inhibits macrophage tumoricidal activity and increases the expression of cyclooxygenase-2 (COX-2). In this study, we sought to determine whether inhibition of COX-2 could restore macrophage function and hence maximize the antitumor activity of the immune stimulant interferon gamma (IFN gamma). METHODS: Peritoneal macrophages were exposed to B16 melanoma-conditioned medium for 24 hours with or without the COX-2 inhibitor NS-398 and then were stimulated with lipopolysaccharide and IFN gamma. Cytotoxic activity, nitrite production, and cytokine production by the stimulated macrophages were measured. In addition, B16 melanoma cells were implanted intradermally into mice treated with IFN gamma (14,000 U on alternate days) alone or with a combination of IFN gamma and a COX-2 inhibitor (NS-398 or nimesulide). Mice were assessed for tumor growth and survival. RESULTS: Macrophage cytotoxicity and nitrite production were significantly suppressed by melanoma-conditioned medium (P <.01). This was prevented by 200 micro M of NS-398 (P <.05). In vivo, combined treatment with IFN gamma and a COX-2 inhibitor caused a significant inhibition of tumor growth (P <.01) and improved survival (P =.02) compared with controls. CONCLUSIONS: COX-2 inhibition reversed melanoma-induced suppression of macrophage function, and combined treatment of IFN gamma plus a COX-2 inhibitor was maximally effective in reducing tumor growth and improving survival.

Animals↗
PubMed2003

Renal cell carcinoma induces prostaglandin E2 and T-helper type 2 cytokine production in peripheral blood mononuclear cells.

BACKGROUND: Patients with renal cell carcinoma (RCC) do not develop an effective antitumor immune response, despite significant infiltration by lymphocytes. Tumor production of immunosuppressive factors may account for this failure. The object of this study was to investigate the production of immunosuppressive mediators, especially prostaglandin E(2) (PGE(2)), by RCC. METHODS: Peripheral blood mononuclear cells (PBMC) were cocultured with conditioned medium (CM) from human RCC cell lines in the presence or absence of NS-398, a selective cyclooxygenase 2 (COX-2) inhibitor. Supernatants were analyzed for levels of PGE(2), interleukin (IL)-10, IL-6, IL-2, interferon-gamma, and IL-12. The effects of RCC CM on PBMC proliferation were also examined. The expression of basal and stimulated COX-2 messenger RNA in the cell lines was assessed by reverse transcriptase-polymerase chain reaction. RESULTS: RCC CM significantly increased PGE(2) production by PBMC. T-helper type 2 (Th2) cytokine production was also significantly increased. Th1 cytokines were unchanged or decreased. RCC CM increased proliferation of PBMC. Coculture with NS-398 reduced PBMC PGE(2) production to below control levels and significantly decreased IL-6 production and PBMC proliferation. NS-398 had no effect on cellular production of IL-10 or Th1 cytokines. CONCLUSIONS: Human RCC inhibits the host antitumor immune response by promoting PGE(2) production and Th2 cytokines in PBMC. Selective inhibition of COX-2 may have a role in abrogating this effect.

Carcinoma, Renal Cell↗
PubMed2002

The 3'-untranslated region of p21WAF1 mRNA is a composite cis-acting sequence bound by RNA-binding proteins from breast cancer cells, including HuR and poly(C)-binding protein.

Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21(WAF1) 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21(WAF1) 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21(WAF1) protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21(WAF1) protein expression is mediated largely by stabilization of p21(WAF1) mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21(WAF1) expression or growth inhibition in this system. CP1 is a novel p21(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21(WAF1) mRNA stability.

3' Untranslated Regions↗