P2Y12 inhibitor use predicts hematoma expansion in patients with intracerebral hemorrhage.

OBJECTIVE: Hematoma expansion (HE) predicts disability and death after acute intracerebral hemorrhage (ICH). Aspirin and anticoagulants have been associated with HE. We tested the hypothesis that P2Y12 inhibitors predict subsequent HE in patients. We explored laboratory measures of P2Y12 inhibition and dual antiplatelet therapy with aspirin (DAPT). METHODS: We prospectively identified patients with ICH. Platelet activity was measured with the VerifyNow-P2Y12 assay. Hematoma volumes for initial and follow-up CTs were calculated using a validated semi-automated technique. HE was defined as the difference between hematoma volumes on the initial and follow-up CT scans. Nonparametric statistics were performed with Kruskal-Wallis H, and correction for multiple comparisons performed with Dunn's test. RESULTS: In 194 patients, 15 (7.7%) were known to take a P2Y12 inhibitor (clopidogrel in all but one). Patients taking a P2Y12 inhibitor had more HE compared to patients not taking a P2Y12 inhibitor (3.5 [1.2-11.9] vs. 0.1 [-0.8-1.4] mL, p = 0.004). Patients taking DAPT experienced the most HE (7.2 [2.6-13.8] vs. 0.0 [-1.0-1.1] mL, p = 0.04). The use of P2Y12 inhibitors was associated with less P2Y12 activity (178 [149-203] vs. 288 [246-319] P2Y12 reaction units, p = 0.005). INTERPRETATION: Patients taking a P2Y12 inhibitor had more HE and less P2Y12 activity. The effect was most pronounced in patients on DAPT, suggesting a synergistic effect of P2Y12 inhibitors and aspirin with respect to HE. Acute reversal of P2Y12 inhibitors in acute ICH requires further study.

Role of Viscoelastic and Conventional Coagulation Tests for Management of Blood Product Replacement in the Bleeding Patient.

An important aim of viscoelastic testing (VET) is to implement transfusion algorithms based on coagulation test results to help reduce transfusion rates and improve patient outcomes. Establishing a rapid diagnosis and providing timely treatment of coagulopathy is the cornerstone of management of severely bleeding patients in trauma, postpartum hemorrhage, and major surgery. As the nature of acute bleeding and trauma leads to an unstable and tenuous physiologic state, conventional coagulation tests (CCTs) are too slow to diagnose, manage, and also course correct any hemostatic abnormalities that accompany an acute critical illness. Viscoelastic point-of-care tests strongly correlate with results from standard laboratory tests but are designed to enable clinicians to make timely, informed bleeding management decisions when time to intervene is critical. These assays provide an individualized and goal-oriented approach to patient blood management and are increasingly becoming involved in transfusion algorithms. The scope of this review aims to evaluate the current literature on VETs and their impact on actionable outputs in clinical decision making and their relationship to CCT.

Thrombosis Risk in Cancer Patients Receiving Red Blood Cell Transfusions.

Patients with cancer have increased risk of thrombosis and often need red blood cell (RBC) transfusions. However, RBC transfusions may also promote thrombosis because of raised hematocrit and viscosity, storage-related RBC damage, and exposure to thrombogenic mediators from obsolescent RBCs. The authors conducted a literature survey for studies examining whether RBC transfusions were associated with increased risk of venous thromboembolism (VTE) in cancer patients. In perioperative cancer surgery patients with categorical comparisons of any versus no RBC transfusion, increased risk of VTE with RBC transfusion was found in 11 of 31 studies, 5 by univariate correlation only and 6 in multivariate analysis. All six multivariate-positive studies had intermediate overall rates of thrombosis (1.4-6.0%), and three were in urological surgery series. In the larger studies of > 2,000 patients (range: 2,219-44,656), the maximum odds ratio among the multivariate-positive studies was 1.3. Perioperative RBC transfusion volume was more strongly associated with VTE risk, with a positive association in six of seven studies. One large registry-based study of hospitalized cancer patients, not restricted to the perioperative setting, found an adjusted odds ratio of 1.60 (95% confidence interval: 1.53-1.67) for VTE risk in patients receiving RBCs compared with nontransfused patients.

Mixing Studies in Patients With Prolonged Activated Partial Thromboplastin Time or Prothrombin Time.

BACKGROUND: Patients presenting for surgery may have isolated or combined prolonged activated partial thromboplastin time (aPTT) and/or prothrombin time (PT). In patients not receiving anticoagulants or with no identifiable cause for abnormal clot formation, a mixing study is performed. The index of circulating anticoagulant (ICA) has been used to predict the presence of an inhibitor, usually a lupus anticoagulant. METHODS: We retrospectively reviewed the results of mixing studies performed at Northwestern Memorial Hospital, between January 1, 2010 and February 29, 2012. We determined the number of samples that normalized or remained prolonged, the clotting factors associated with prolonged test results, and the presence of coagulation inhibitors. We calculated the ICA in the samples with prolonged aPTT and PT to determine its ability to predict a lupus anticoagulant. The primary comparison of interest was the diagnostic utility of the ICA at cutoff values of 11% for predicting the presence of lupus anticoagulant. RESULTS: There were 269 mixing studies performed: 131 samples with prolonged aPTT; 95 with prolonged PT; and 43 with both prolonged aPTT and prolonged PT. Of the samples with a prolonged aPTT, 55 of 131 (42%) normalized, 36 of 131 (27%) partially corrected, and 40 of 131 (31%) remained prolonged. Thirty-three of 95 samples (35%) with prolonged PT normalized, while 62 of 95 (65%) remained prolonged. Eight of 43 (19%) mixing studies of patients with prolonged PT and aPTT normalized; the aPTT normalized, but the PT remained prolonged in 17 of 43 (39%); the PT normalized, but the aPTT remained prolonged in 7 of 43 (16%); and both tests remained prolonged in 11 of 43 (26%) samples. Prolongations in the aPTT were primarily associated with low activities of CF XII, while the majority of the prolongations in PT were secondary to low activities in CF VII. Combined prolongations were secondary to deficiencies in both the intrinsic and extrinsic as well as the common pathways. An ICA >11% had 100% (95% CI, 59%-100%) sensitivity, 53% (95% CI, 35%-70%) specificity, and 77% (95% CI, 62%-92%) accuracy in predicting the presence of lupus anticoagulant in patients with prolonged aPTT. CONCLUSIONS: Normalization of the aPTT and PT in a mixing study was associated with low clotting factor activity. The ICA may be helpful in predicting the presence of a lupus anticoagulant. As anesthesiologists take ownership of the perioperative surgical home, we need to understand the clinical implications of the results of mixing studies.

Fibrin and Fibrinolysis in Cancer.

In 1878, Billroth discovered that tumor cells invest themselves in a fibrin thrombus, and he hypothesized that fibrin promotes tumor growth and invasion. Since then, many observations have supported this concept, showing that many hemostatic factors including fibrinogen, fibrin, and components of the fibrinolytic system have indeed a complex interaction with cancer growth and metastasis. Fibrin promotes cell migration by providing a matrix for tumor cell migration and by interactions with adhesive molecules and integrins. Fibrin-containing vascular endothelial growth factor promotes angiogenesis. Fibrin interacts with platelets and leukocytes, and promotes their respective carcinogenic properties. Fibrinolytic components exert different effects on tumors. Plasmin activates latent growth factors, and breaks down extracellular matrix (ECM), while urokinase plasminogen activator (uPA) and the uPA receptor (uPAR) form complexes with vitronectin and integrins to promote tumor cells to adhere to the ECM. This complex also binds the epidermal growth factor receptor on the tumor cell membrane, and signals the RAF-MEK-ERK pathway. The complex also binds to the G protein-coupled receptors leading to cell proliferation. Plasminogen activator inhibitor 1 (PAI-1) inhibits apoptosis, and increases tumor cell survival. PAI-1 also enhances cell senescence, leading to production of tumorigenic cytokines by the senescence secretome. The presence of uPA/uPAR and PAI-1 represents a strong biomarker for tumor aggressiveness and poor prognosis. Multiple attempts by blocking various carcinogenic steps have shown tumor-suppressing effects in experimental animals, but human responses are uncertain without clinical trials.

Fatal massive hemolysis caused by immunoglobulin M anti-c antibody in a patient with newly diagnosed B-cell acute lymphoblastic leukemia: a case report.

Delayed hemolytic transfusion reactions (DHTRs) occur secondary to slow, mild IgG-mediated processes against minor red blood cell antigens. Herein, we report the case of a rapidly fatal alloimmune anti-c IgM-mediated hemolysis, a rare, previously undescribed, pathophysiologic scenario. Early recognition of such phenomena can expedite supportive measures and optimize patient outcomes.

Blood Bank Specimen Mislabeling: A College of American Pathologists Q-Probes Study of 41 333 Blood Bank Specimens in 30 Institutions.

CONTEXT: -Incorrectly labeled patient blood specimens create opportunities for laboratory testing personnel to mistake one patient's specimen for a specimen from a different patient. Transfusion of blood that is typed on specimens that are mislabeled can result in acute hemolytic transfusion reactions. OBJECTIVE: -To assess the rates of blood bank ABO typing specimens that are mislabeled and/or contain blood belonging to another patient (so-called wrong blood in tube [WBIT]), and to compare these rates with those determined in a similar study performed in 2007. DESIGN: -Participants enrolled in this College of American Pathologists Q-Probes study for the first quarter of 2015 tallied the number of mislabeled and WBIT ABO blood typing specimens. Outcome measurements were the number of mislabeled and WBIT instances per 1000 specimens. We also evaluated the effects of various practice characteristics, in particular the use of bar coding, on the outcome measurements. RESULTS: -A total of 30 institutions submitting data on 41 333 ABO blood typing specimens recorded aggregate rates of 7.4 instances of mislabeling (306 specimens) and 0.43 instances of WBIT (10 of 23 234) per 1000 specimens submitted. Mislabeling rates were lower in institutions requiring that specimens be labeled with patients' birth dates than those that did not. The rates of specimen mislabeling and WBIT were otherwise unassociated with any of the other practice variables evaluated. CONCLUSIONS: -The rates of ABO blood typing specimen mislabeling and WBIT are not statistically different from those determined in a similar study performed in 2007 (P = .94 and P = .10). The use of bar coding was not associated with lower mislabeling (P = .80) or WBIT rates (P = .79).

LPA Increases Tumor Growth and Bone Destruction Through Enhancement of Osteoclastogenic Cytokines.

BACKGROUND: Lysophosphatidic acid (LPA) production in osteoblasts has multiple effects on osteoclast formation and function and raises the possibility that LPA may serve as a signaling molecule for the reciprocal conversation of both osteoblasts and osteoclasts within the tumor-bone microenvironment for bone resorption. However, little is known on the effect of LPA in regulating the function of both cancer cells and osteoclasts in the bone microenvironment. MATERIALS AND METHODS: PC-3 tumor growth and bone destruction upon LPA administration were observed in a mouse calvarium xenograft. The osteoclastogenic cytokines produced by LPA-stimulated prostate cancer cells were also defined. RESULTS: LPA administration was found to increase PC-3 tumor growth and bone destruction in a mouse calvarium xenograft. Using a cytokine antibody array, LPA highly stimulated the expression and release of osteoclastogenic cytokines from PC-3 cells. Conditioned medium from LPA-stimulated PC-3 cells containing enhanced levels of osteoclastogenic cytokines facilitated osteoclast formation. Histopathologically, LPA administration supports the erosive type of bone destruction by PC-3 prostate cancer cells. CONCLUSION: LPA is a critical regulator in the tumor-bone microenvironment and may be a therapeutic target for patients with prostate cancer. In addition, LPA-enhanced osteoclastogenic cytokines are critical to therapeutic strategies targeting osteolytic prostate cancer.

Lysophosphatidic acid activates the RhoA and NF-κB through Akt/IκBα signaling and promotes prostate cancer invasion and progression by enhancing functional invadopodia formation.

We have demonstrated previously that increased RhoA and nuclear factor (NF)-κB activities are associated with increased PC-3 prostate cancer cell invasion and that lysophosphatidic acid (LPA) significantly increases cancer invasion through RhoA and NF-κB activation. In this study, we identified the intermediate signaling molecules and specialized cell structures which are activated by LPA, resulting in enhanced cellular invasion. LPA-induced Akt and IκBα signaling pathways were necessary for RhoA and NF-κB activation, and these LPA effects were abolished by RhoA inhibition. Mice injected with PC-3 cells expressing dominant-negative RhoA N19 developed significantly less tumor growth compared with those injected with control (pcDNA 3.1). In addition, LPA treatment increased functional invadopodia formation. Activation of RhoA and NF-κB through the Akt and IκBα signaling pathway was required for LPA-stimulated gelatin degradation activity. LPA administration increased tumor growth and osteolytic lesions in a mouse xenograft model. These results indicate that LPA promotes PC-3 cell invasion by increasing functional invadopodia formation via upregulating RhoA and NF-κB signaling which contributes to prostate cancer progression. Therefore, the LPA and RhoA-NF-κB signaling axis may represent key molecular targets to inhibit prostate cancer invasion and progression.

Monocyte-Induced Prostate Cancer Cell Invasion is Mediated by Chemokine ligand 2 and Nuclear Factor-κB Activity.

STUDY BACKGROUND: The tumor microenvironment contains inflammatory cells which can influence cancer growth and progression; however the mediators of these effects vary with different cancer types. The mechanisms by which prostate cancer cells communicate with monocytes to promote cancer progression are incompletely understood. This study tested prostate cancer cell and monocyte interactions that lead to increased prostate cancer cell invasion. METHODS: We analyzed the prostate cancer cell invasion and NF-κB activity and cytokine expression during interaction with monocyte-lineage cells in co-cultures. The roles of monocyte chemotactic factor (MCP-1/CCL2) and NF-κB activity for co-culture induced prostate cancer invasion were tested. Clinical prostate cancer NF-κB expression was analyzed by immunohistochemistry. RESULTS: In co-cultures of prostate cancer cell lines with monocyte-lineage cells, (C-C motif) ligand 2 (CCL2) levels were significantly increased when compared with monocytes or cancer cells cultured alone. Prostate cancer cell invasion was induced by recombinant CCL2 in a dose dependent manner, similar to co-cultures with monocytes. The monocyte-induced prostate cancer cell invasion was inhibited by CCL2 neutralizing antibodies and by the CCR2 inhibitor, RS102895. Prostate cancer cell invasion and CCL2 expression induced in the co-cultures was inhibited by Lactacystin and Bay11-7082 NF-κB inhibitors. Prostate cancer cell NF-κB DNA binding activity depended on CCL2 dose and was inhibited by CCL2 neutralizing antibodies. Clinical prostate cancer NF-κB expression correlated with tumor grade. CONCLUSIONS: Co-cultures with monocyte-lineage cell lines stimulated increased prostate cancer cell invasion through increased CCL2 expression and increased prostate cancer cell NF-κB activity. CCL2 and NF-κB may be useful therapeutic targets to interfere with inflammation-induced prostate cancer invasion.

Constitutive and Inducible Expression of Invasion-related Factors in PC-3 Prostate Cancer Cells.

BACKGROUND: Tumor growth and invasion are interconnected with the tumor microenvironment. Overexpression of genes that regulate cancer cell invasion by growth factors, cytokines, and lipid factors can affect cancer aggressiveness. A comparative gene expression analysis between highly invasive and low invasive cells revealed that various genes are differentially expressed in association with invasive potential. In this study, we selected variant PC-3 prostate cancer cell sublines and discovered critical molecules that contributed to their invasive potential. METHODS: The high invasive and low invasive variant PC-3 cell sublines were obtained by serial selection following Matrigel-coated Transwell invasion and were characterized by Transwell invasion, luciferase reporter assay, and Rhotekin pull-down assay. Lysophosphatidic acid (LPA) was added to the cultures to observe the response to this extracellular stimulus. The essential molecules related with cancer invasiveness were detected with Northern blotting, quantitative reverse transcription-polymerase chain reaction, and cDNA microarray. RESULTS: Highly invasive PC-3 cells showed higher nuclear factor kappa B (NF-κB), activator protein 1 (AP-1) and RhoA activities than of low invasive PC-3 cells. LPA promoted cancer invasion through NF-κB, AP-1, and RhoA activities. Thrombospondin-1, interleukin-8, kallikrein 6, matrix metalloproteinase-1, and tissue factor were overexpressed in the highly invasive PC-3 variant cells and further upregulated by LPA stimulation. CONCLUSIONS: The results suggest that the target molecules are involved in invasiveness of prostate cancer. These molecules may have clinical value for anti-invasion therapy by serving as biomarkers for the prediction of aggressive cancers and the detection of pharmacological inhibitors.

Three-stage ex vivo expansion of high-ploidy megakaryocytic cells: toward large-scale platelet production.

Hematopoietic stem and progenitor cells (HSPCs) have been cultured using a wide variety of cytokines to promote differentiation into megakaryocytic cells (Mks), the precursors to platelets. Greater Mk DNA content, or ploidy, has been correlated with increased platelet release. Gradients of pH, pO2, and signaling factors regulate megakaryopoiesis in the bone marrow niche. In this study, we demonstrate that a 3-phase culture process with increasing pH and pO2 and different cytokine cocktails greatly increases megakaryocyte production. CD34(+) HSPCs were first cultured at 5% O2 and pH 7.2 with a cytokine cocktail previously shown to promote Mk progenitor production. At day 5, cells were shifted to 20% O2 and pH 7.4 and maintained in 1 of 17 cytokine cocktails identified using a 2(4) factorial design of experiments method to evaluate the effects of interleukin (IL)-3, IL-6, IL-9, and high- or low-dose stem cell factor (SCF), in conjunction with thrombopoietin (Tpo) and IL-11, on expansion of mature Mks from progenitors. The combination of Tpo, high-dose SCF, IL-3, IL-9, and IL-11 best promoted Mk expansion. IL-3 greatly increased total cell fold expansion, but this was partially offset by lower Mk purity. IL-9 promoted CD41 and CD42b expression. High-dose (100 ng/mL) SCF increased Mk production and ploidy. Different commercial media and IL-3 sources substantially impacted differentiation, and X-VIVO 10 serum-free media best supported mature Mk expansion. Shifting from pH 7.4 to pH 7.6 at day 7 increased Mk production by 30%. Treatment with nicotinamide at day 7 or day 8 more than doubled the fraction of high-ploidy (>4N) Mks. Ultimately, the 3-phase culture system gave rise to 44.5±8.1 Mks and 8.5±3.1 high-ploidy Mks per input HSPC. Further optimization was required to improve platelet production. Using Iscove's modified Dulbecco's medium (IMDM)+20% BSA, insulin and transferin (BIT) 9500 Serum Substitute greatly improved the frequency and quality of Mk proplatelet extensions without affecting Mk expansion, commitment, or polyploidization in the 3-phase process. Mks cultured in IMDM+20% BIT 9500 gave rise to platelets with functional activity similar to that of fresh platelets from normal donors, as evidenced by basal tubulin distribution and the expression of surface markers and spreading in response to platelet agonists.

Role of monocyte-lineage cells in prostate cancer cell invasion and tissue factor expression.

BACKGROUND: Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocyte-lineage cells in prostate cancer cell TF expression and cell invasion. METHODS: Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression. RESULTS: Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in co-cultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 ± 53/mm(2)) than benign prostate (108 ± 31/mm(2)). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium. CONCLUSIONS: This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.

Lysophosphatidic acid stimulates PC-3 prostate cancer cell Matrigel invasion through activation of RhoA and NF-kappaB activity.

This study was performed to determine the relationship of lysophosphatidic acid (LPA) stimulation and increased Ras homolog A (RhoA) activity to nuclear factor kappa B (NF-kappaB) activity, and the role of these factors in regulating prostate cancer cell invasion. PC-3 high invasive cells demonstrated constitutively increased RhoA, NF-kappaB, and in vitro Matrigel invasion which were further induced by LPA stimulation or transfection with constitutively active RhoA Q63E mutant. LPA treatment rapidly and transiently induced RhoA activity followed by maximally increased DNA binding of NF-kappaB at 1 h and AP-1 at 4 h. The LPA-induced NF-kappaB DNA binding was preceded by transient IkappaBalpha phosphorylation, and decreased total IkappaBalpha levels. Further demonstrating the relationship between RhoA and NF-kappaB activation, PC-3 cells stably transfected with constitutively active RhoA Q63E demonstrated constitutively increased phospho-IkappaBalpha, while PC-3 cells transfected with dominant negative RhoA N19 exhibited decreased phospho-IkappaBalpha levels. The LPA-induced Matrigel invasion and NF-kappaB DNA binding activity were both inhibited by expression of the RhoA inhibitor C3 exoenzyme or dominant negative mutant NF-kappaB inhibitor IkappaBalpha S32/36A. Similarly, transfection with dominant negative IkappaBalpha S32/36A inhibited PC-3 RhoA Q63E cell in vitro invasion. Treatment of PC-3 high invasive and RhoA Q63E cells with sodium salicylate or lactacystin inhibited NF-kappaB and invasion, while pyrrolidine dithiocarbamate (PDTC) treatment of PC-3 high invasive cells inhibited NF-kappaB only. Each inhibitor blocked LPA-induced invasion while PDTC inhibited LPA-induced NF-kappaB and invasion to the greatest extent. These results point to a model where LPA stimulates RhoA and increased PC-3 prostate cancer cell invasion activity through an NF-kappaB-dependent pathway.

Colon biopsies for evaluation of acute graft-versus-host disease (A-GVHD) in allogeneic bone marrow transplant patients.

BACKGROUND: Proper histomorphological interpretation of intestinal acute graft versus host disease (A-GVHD) associated with allogeneic bone marrow transplantation (BMT) is critical for clinical management. However, studies methodically evaluating different histomorphological features of A-GVHD are rare. METHODS: Colonic biopsies from 44 allogeneic BMT patients having biopsy-proven cutaneous A-GVHD were compared with colon biopsies from 48 negative controls. RESULTS: A-GVHD showed intra-cryptal apoptosis in 91% and pericryptal apoptosis in adjacent lamina propria in 70% (p < 0.002). Nonspecific apoptosis along the surface epithelium was observed in all groups with comparable frequency. The number of apoptotic cells in mucosa were approximately four times (5.3 per 10 HPF) the negative controls (p < 0.002) in A-GVHD group. 48% of cases with A-GVHD showed decreased number of lymphocytes in lamina propria. Some features, including intraepithelial lymphocytes in surface or crypt epithelium; and neutrophils, eosinophils, and edema in lamina propria, did not demonstrate significant difference in A-GVHD and negative controls. Pericryptal apoptosis, dilated crypts, irregular distribution of crypts, decreased lymphocytes, increased microvessel network, focal fibrosis, presence of muciphages, reactive changes in surface epithelium with mucin depletion, mucosal ulceration, and/or reduced mucosal thickness showed higher association with A-GVHD group. CONCLUSIONS: Intracyptal apoptosis is a reliable indicator of A-GVHD. Its diagnostic significance was improved if intracyptal apoptosis was associated with features which were observed more frequently in A-GVHD group as mentioned above.

Requirement of RhoA activity for increased nuclear factor kappaB activity and PC-3 human prostate cancer cell invasion.

To determine the molecular mechanisms of aggressive prostate cancer behavior, we studied RhoGTPases in high and low invasive variants of PC-3 prostate cancer cells. Prior studies with these cells revealed that elevated nuclear factor kappaB (NF-kappaB) expression and activity were necessary for the highly invasive phenotype. In the current study, increased RhoA expression was found in the PC-3 highly invasive cells as compared with the PC-3 low invasive cells through cDNA array and Western blot analyses. Similarly, RhoA activity, as measured by the Rhotekin binding assay, was elevated in the PC-3 highly invasive cells. Transfection of these highly invasive cells with dominant negative RhoA N19 or treatment with 1.0 micro g/ml RhoA inhibitor C3 exoenzyme demonstrated that RhoA activity was necessary for both NF-kappaB activity and cellular invasion of a Matrigel reconstituted basement membrane. Furthermore, stable transfection of the PC-3 highly invasive cells with constitutively active RhoA Q63L resulted in activation of NF-kappaB activity and Matrigel invasion, effects reversed by treatment of the cells with C3 exoenzyme. RhoA was also shown to act through the motility component of the invasion process. RhoA activity was therefore both necessary and sufficient for the elevated NF-kappaB, invasion, and motility activities of the PC-3 highly invasive cells. These findings suggest molecular targets to control cancer cell invasion and aid in the development of definitive tools for predicting the invasive and metastatic potential of cancer cells.

Requirement of cyclooxygenase-2 expression and prostaglandins for human prostate cancer cell invasion.

The PC-3 Low Invasive cells and the PC-3 High Invasive cells were used to investigate the correlation of the COX-2 expression and its arachidonic acid metabolites, prostaglandins, with their invasiveness through Matrigel using a Boyden chamber assay. The COX-2 expression in PC-3 High Invasive cells was approximately 3-fold higher than in PC-3 Low Invasive cells while the COX-1 expression was similar in both cell sublines. When incubated with arachidonic acid, PGE2 was the major prostaglandin produced by these cells. PC-3 High Invasive cells produced PGE2 approximately 2.5-fold higher than PC-3 Low Invasive cells. PGD2 was the second most abundant prostaglandin produced by these cells. Both indomethacin (a nonspecific COX inhibitor) and NS-398 (a specific COX-2 inhibitor) inhibited the production of prostaglandins and the cell invasion. PGE2 alone did not induce the cell invasion of PC-3 Low Invasive cells. However, PGE2 reversed the inhibition of cell invasion by NS-398 and enhanced the cell invasion of the PC-3 High Invasive cells. In contrast, PGD2 slightly inhibited the cell invasion. These results suggest that in the PC-3 Low Invasive cells, COX-2-derived PGE2 may not be sufficient to induce cell invasion while in the PC-3 High Invasive cells, PGE2 may be sufficient to act as an enhancer for the cell invasion. Further, PGD2 may represent a weak inhibitor and counteracts the effect of PGE2 in the cell invasion.

Transfection of human endothelial cells with HIV-1 tat gene activates NF-kappa B and enhances monocyte adhesion.

Human immunodeficiency virus (HIV)-1 Tat released from HIV-1-infected monocytes is believed to enter other cells via an integrin-facilitated pathway, resulting in altered gene expression. Indeed, exogenous Tat protein can increase cell adhesion molecule gene expression in human endothelial cells. Signaling pathways initiated by Tat in endothelial cells are not known. We evaluated the ability of endogenous tat to stimulate monocyte adhesion via activation of nuclear factor-kappaB (NF-kappaB) within human umbilical vein endothelial cells. Transfection with pcTat, but not control vector DNA, increased NF-kappaB binding activity, NF-kappaB luciferase reporter activity, and monocyte adhesion. pcTat also increased kappaB-dependent HIV-1-LTR-CAT reporter activity 28-fold compared with a 3-fold increase produced by transfection with an equivalent amount of pcTax (from human leukemia virus). The pcTat-induced increase in pNF-kappaB-Luc activity and monocyte adhesion to endothelial cells was blocked by cotransfection with dominant-negative mutant IkappaBalpha and by incubation with 10 mM aspirin. We conclude that monocyte adhesion to human endothelial cells stimulated by pcTat is mediated via an NF-kappaB-dependent mechanism. Furthermore, inhibition studies using aspirin suggest that pcTat-stimulated NF-kappaB activation and monocyte adhesion occur via a redox-sensitive mechanism.