Laura E. Gordon

RASSF6 is a novel member of the RASSF family of tumor suppressors

RASSF family proteins are tumor suppressors that are frequently downregulated during the development of human cancer. The best-characterized member of the family is RASSF1A, which is downregulated by promoter methylation in 40–90% of primary human tumors. We now identify and characterize a novel member of the RASSF family, RASSF6. Like the other family members, RASSF6 possesses a Ras Association domain and binds activated Ras. Exogenous expression of RASSF6 promoted apoptosis, synergized with activated K-Ras to induce cell death and inhibited the survival of specific tumor cell lines. Suppression of RASSF6 enhanced the tumorigenic phenotype of a human lung tumor cell line. Furthermore, RASSF6 is often downregulated in primary human tumors. RASSF6 shares some similar overall properties as other RASSF proteins. However, there are significant differences in biological activity between RASSF6 and other family members including a discrete tissue expression profile, cell killing specificity and impact on signaling pathways. Moreover, RASSF6 may play a role in dictating the degree of inflammatory response to the respiratory syncytial virus. Thus, RASSF6 is a novel RASSF family member that demonstrates the properties of a Ras effector and tumor suppressor but exhibits biological properties that are unique and distinct from those of other family members.

NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

NORE1A (RASSF5) is a proapoptotic Ras effector that is frequently inactivated by promoter methylation in human tumors. It is structurally related to the RASSF1A tumor suppressor and is itself implicated as a tumor suppressor. In the presence of activated Ras, NORE1A is a potent inducer of apoptosis. However, when expressed at lower levels in the absence of activated Ras, NORE1A seems to promote cell cycle arrest rather than apoptosis. The mechanisms underlying NORE1A action are poorly understood. We have used microarray analysis of an inducible NORE1A system to screen for physiologic signaling targets of NORE1A action. Using this approach, we have identified several potential signaling pathways modulated by NORE1A. In particular, we identify the cyclin-dependent kinase inhibitor p21(CIP1) as a target for NORE1A activation and show that it is a vital component of NORE1A-mediated growth inhibition. In primary human hepatocellular carcinomas (HCC), loss of NORE1A expression is frequent and correlates tightly with loss of p21(CIP1) expression. NORE1A down-regulation in HCC also correlates with poor prognosis, enhanced proliferation, survival, and angiogenic tumor characteristics. Experimental inactivation of NORE1A results in the loss of p21(CIP1) expression and promotes proliferation. The best characterized activator of p21(CIP1) is the p53 master tumor suppressor. Further experiments showed that NORE1A activates p21(CIP1) via promoting p53 nuclear localization. Thus, we define the molecular basis of NORE1A-mediated growth inhibition and implicate NORE1A as a potential component of the ill-defined connection between Ras and p53.

Endotoxin and muramyl dipeptide modulate surface receptor expression on human mononuclear cells

Endotoxin (lipopolysaccharide (LPS), 100 ng/ml) and muramyl dipeptide (MDP 100 ng/ml), two immunomodulatory bacterial cell wall products, were incubated with human whole blood, and the expression of receptors involved in antigen presentation, costimulation, and cell activation was investigated by use of flow cytometry. On monocytes, LPS and MDP increased surface expression of human leukocyte antigen-DR (HLA-DR), CD18, CD54 (intercellular adhesion molecule-1, ICAM-1), and CD86 (B7-2). On lymphocytes, LPS but not MDP increased HLA-DR expression after 18 h. The expression of CD28, CD49d/CD29, and CD106 (vascular cell adhesion molecule-1, VCAM-1) remained unchanged on both monocytes and lymphocytes. The early increase (1-6 h) of CD18 and ICAM-1 expression led us to hypothesize that CD18-dependent costimulatory signals were involved in the later (6 h) increase of monocyte HLA-DR expression. However, blocking studies using monoclonal antibodies against CD18 (IB4, 15 microg/ml) demonstrated that the LPS- and MDP-induced increase of HLA-DR and ICAM-1 expression on monocytes was not mediated through CD18. LPS induced the expression of the early activation marker CD69 by a CD14-dependent but CD18-independent mechanism, whereas MDP did not induce CD69 expression. Analysis of leukocyte subsets demonstrated that CD4(+) T-cells, CD8(+) T-cell, CD19(+) B-cells, CD56(+) natural killer (NK)-cells, and CD14(+) monocytes increased the expression of CD69 after stimulation with LPS. Collectively, these data demonstrate a stronger immunomodulatory effect of LPS compared with MDP which may, in part, explain the established difference of toxicity between these two bacterial cell wall products.

The administration of cobra venom factor reduces post-ischemic cerebral injury in adult and neonatal rats

The role of complement in post-ischemic cerebral injury is incompletely understood. Therefore, experiments were designed to test the effect of complement depletion on cerebral infarct volume in adult rats and cerebral atrophy in neonatal rats. Cerebral infarcts were induced in adult rats by transient filamentous occlusion of the right middle cerebral artery (MCAO). Cerebral atrophy was induced by subjecting 7-day-old rats to ligation of the right common carotid artery followed by 2.5h of hypoxia (8% O2). Forty-eight hours after MCAO, coronal sections of adult brains were obtained and stained with 2,3,5-triphenyl tetrazolium chloride. The infant rat brains were removed for analysis 6 weeks after the hypoxic-ischemic insult. Volumes of infarcts and normal hemispheric parenchyma were quantified by computer-based planimetry. Twenty-four hours prior to MCAO (adults) or hypoxia-ischemia (neonates), each animal received an i.p. injection of either 1 mcg/g body weight cobra venom factor (CVF; adult n=11; neonatal n=20) or normal saline (adult n=12; neonatal n=24). In the neonates, a second dose of CVF or saline was administered 2 days after hypoxia-ischemia. The administration of CVF significantly reduced: (1) post-ischemic cerebral infarct volume in the adults and (2) post-hypoxic-ischemic cerebral atrophy in the neonates. Therefore, complement activation augmented post-ischemic cerebral injury in adult and neonatal rats. Complement depletion induced by CVF significantly reduced post-ischemic cerebral infarct volume and atrophy in adult and neonatal rats.

Heparin and enoxaparin enhance endotoxin-induced tumor necrosis factor-α production in human monocytes

OBJECTIVE To determine whether heparin or the low-molecular-weight heparin enoxaparin alter lipopolysaccharide (LPS)-induced monocyte activation. SUMMARY BACKGROUND DATA Heparin is widely used in clinical practice to inhibit the coagulation cascade. However, heparin also is a naturally occurring glucosaminoglycan and a pleiotropic immunomodulator that binds to a variety of proteins. LPS is a component of gram-negative bacteria and is thought to be responsible for many of the deleterious effects seen in sepsis. The binding of LPS to CD14 induces a signaling cascade that results in the release of many inflammatory mediators, including tumor necrosis factor-alpha (TNF-alpha). METHODS Monocytes from healthy volunteers were isolated and cultured in the presence of saline, LPS (10 ng/ml), heparin (0.1 to 1000 microg/ml), or enoxaparin (0.1 to 1000 microg/ml). In blocking experiments, cells were pretreated for 60 minutes with the monoclonal anti-CD14 antibody MY4 (10 microg/ml) or with isotype-matched control IgG2 (10 microg/ml). TNF-alpha values were measured with enzyme-linked immunosorbent assay. Significance was assessed with analysis of variance. RESULTS Heparin (10 to 1000 microg/ml) and enoxaparin (1000 microg/ml) significantly enhanced LPS-induced TNF-alpha release. Heparin (1000 microg/ml) or enoxaparin (1000 microg/ml) did not produce TNF-alpha in the absence of LPS. Blockade of CD14 abrogated both LPS-induced TNF-alpha release and the effect of heparin or enoxaparin to enhance LPS-induced TNF-alpha release. CONCLUSIONS The effect of heparin to enhance LPS-induced TNF-alpha release is a biologic phenomenon that reveals a novel and potentially important host defense mechanism during endotoxemia and sepsis. Binding of LPS to CD14 is necessary to induce this phenomenon, suggesting that both heparin and enoxaparin induce signaling mechanisms that are downstream from the initial binding of LPS on CD14.

Early elevation of soluble CD14 may help identify trauma patients at high risk for infection.

BACKGROUND Elevated levels of soluble CD14 (sCD14) have been implicated in both gram-positive and gram-negative sepsis, and it has been associated with high mortality in trauma patients who become infected. METHODS Eleven healthy volunteers and 25 adult trauma patients with multiple injuries and a mean Injury Severity Score of 32 participated. Whole blood was obtained at intervals. Immunohistochemistry was used to quantify membrane CD14 (mCD14), by flow cytometry and plasma levels of sCD14 by enzyme-linked immunosorbent assay. Analysis of variance and Students T test with Mann-Whitney posttest were used to determine significance at p < 0.05. RESULTS On posttrauma day 1, sCD14 was significantly different in the plasma of infected patients compared with normal controls (7.16 +/- 1.87 microg/mL vs. 4.4 +/- 0.92 microg/mL, p < 0.01), but not significantly different from noninfected patients. The percentage of monocytes expressing mCD14 in trauma patients did not differentiate them from normal controls; however, mCD14 receptor density did demonstrate significance in septic trauma patients (n = 15) versus normal controls on posttrauma day 3 (p = 0.0065). CONCLUSION On the basis of our data, mCD14 did not differentiate infected and noninfected trauma patients, although trauma in general reduced mCD14 and elevated sCD14. Interestingly, 100% of patients who exceeded plasma levels of 8 microg/mL of sCD14 on day 1 after injury developed infections. Therefore, early high expressers of sCD14 may be at higher risk for infectious complications after trauma.

Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells.

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-a-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of 14C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.

Salvador Protein Is a Tumor Suppressor Effector of RASSF1A with Hippo Pathway-independent Functions

The RASSF1A tumor suppressor binds and activates proapoptotic MST kinases. The Salvador adaptor protein couples MST kinases to the LATS kinases to form the hippo pathway. Upon activation by RASSF1A, LATS1 phosphorylates the transcriptional regulator YAP, which binds to p73 and activates its proapoptotic effects. However, although serving as an adaptor for MST and LATS, Salvador can also bind RASSF1A. The functional role of the RASSF1A/Salvador interaction is unclear. Although Salvador is a novel tumor suppressor in Drosophila and mice, its role in human systems remains largely unknown. Here we show that Salvador promotes apoptosis in human cells and that Salvador inactivation deregulates the cell cycle and enhances the transformed phenotype. Moreover, we show that although the salvador gene is seldom mutated or epigenetically inactivated in human cancers, it is frequently down-regulated posttranscriptionally. Surprisingly, we also find that although RASSF1A requires the presence of Salvador for full apoptotic activity and to activate p73, this effect does not require a direct interaction of RASSF1A with MST kinases or the activation of the hippo pathway. Thus, we confirm a role for Salvador as a human tumor suppressor and RASSF1A effector and show that Salvador allows RASSF1A to modulate p73 independently of the hippo pathway.

Free hemoglobin enhances tumor necrosis factor-alpha production in isolated human monocytes.

BACKGROUND A systemic inflammatory response (SIR) is seen in approximately 75% of patients with complex blunt liver injuries treated nonoperatively. Many feel this response is caused by blood, bile, and necrotic tissue accumulation in the peritoneal cavity. Our current treatment for these patients is a delayed laparoscopic washout of the peritoneal cavity, resulting in a dramatic resolution of the SIR. Spectrophotometric analysis of the intraperitoneal fluid has confirmed the presence of high concentrations of free hemoglobin (Hb). We hypothesize that free Hb enhances the local peritoneal response by increasing tumor necrosis factor-alpha (TNF-alpha) production by monocytes, contributing to the local inflammatory response and SIR. METHODS Monocytes from five healthy volunteers were isolated and cultured in RPMI-1640 for 24 hours. Treatment groups included saline controls, lipopolysaccharide ([LPS], 10 ng/mL, from Escherichia coli), human Hb (25 microg/mL), and Hb + LPS. Supernatants were analyzed by enzyme-linked immunosorbent assay. Students t test with Mann-Whitney posttest was used for statistical analysis with p < or = 0.05 considered significant. RESULTS Free Hb significantly increased TNF-alpha production 915 +/- 223 pg/mL versus saline (p = 0.02). LPS and Hb + LPS further increased TNF-alpha production (2294 pg/mL and 2501 pg/mL, respectively, p < 0.001) compared with saline controls. CONCLUSION These data confirm that free Hb is a proinflammatory mediator resulting in the production of significant amounts of TNF-alpha. These in vitro findings support our clinical data in which timely removal of intraperitoneal free hemoglobin helps prevent its deleterious local and systemic inflammatory effects in patients with complex liver injuries managed nonoperatively.

The Ras effector RASSF2 controls the PAR-4 tumor suppressor.

ABSTRACT RASSF2 is a novel proapoptotic effector of K-Ras. Inhibition of RASSF2 expression enhances the transforming effects of K-Ras, and epigenetic inactivation of RASSF2 is frequently detected in mutant Ras-containing primary tumors. Thus, RASSF2 is implicated as a tumor suppressor whose inactivation facilitates transformation by disconnecting apoptotic responses from Ras. The mechanism of action of RASSF2 is not known. Here we show that RASSF2 forms a direct and endogenous complex with the prostate apoptosis response protein 4 (PAR-4) tumor suppressor. This interaction is regulated by K-Ras and is essential for the full apoptotic effects of PAR-4. RASSF2 is primarily a nuclear protein, and shuttling of PAR-4 from the cytoplasm to the nucleus is essential for its function. We show that RASSF2 modulates the nuclear translocation of PAR-4 in prostate tumor cells, providing a mechanism for its biological effects. Thus, we identify the first tumor suppressor signaling pathway emanating from RASSF2, we identify a novel mode of action of a RASSF protein, and we provide an explanation for the extraordinarily high frequency of RASSF2 inactivation we have observed in primary prostate tumors.