Dolichol-phosphate-mannose-3 (DPM3)/prostin-1 is a novel phospholipase C-gamma regulated gene negatively associated with prostate tumor invasion.

The most ominous development in tumor progression is the transition to an invasive and metastatic phenotype. Little is known, however, about the molecular alterations that cause a tumor to become invasive. In view of this, we have used microarray expression analysis to evaluate the expression profiles of a unique panel of human DU145 prostate cancer sublines that vary in their invasive potential. The three DU145 sublines expressed epidermal growth factor (EGF) receptors that differed in their ability to activate phospholipase C-gamma (PLC gamma). Three-way analyses yielded 11 genes out of 4608 genes screened that associated directly or inversely with invasive potential. The gene whose expression correlated most strongly with lack of invasion was identified as a potential invasion suppressor and called prostin-1. Pharmacological inhibition of PLC gamma (U73122) confirmed that PLC gamma signaling suppressed prostin-1 in that U73122 treatment caused induction of prostin-1 in PLC gamma competent cells. The prostin-1 gene, conserved through phylogeny, is induced by androgen in LNCaP cells and encodes a 92 amino acid protein. The protein shares no extensive homologies with other known genes, yet was recently identified as a small stabilizer subunit of the dolichol-phosphate-mannose (DPM) synthase complex. That DPM3/prostin-1 might suppress tumor progression was supported by the finding that exogenous expression in COS cells leads to apoptosis. These findings support the use of model cell lines to identify putative tumor suppressors and promoters.

Assessment of tumor necrosis factor receptor and Fas signaling pathways by transcriptional profiling.

Apoptosis, or programmed cell death, is an important mechanism by which cells are eliminated during immune regulation and embryonic development. Aberrations in the signaling pathways leading to apoptosis may result in cancer, autoimmune diseases, or inflammatory disorders. In view of this, an understanding of the signaling capabilities of apoptosis-inducing or death receptors is essential to understanding their roles in biology and disease. We used cDNA microarrays to examine the downstream transcriptional effects of two members of the tumor necrosis factor (TNF) family of death receptor ligands. We compared the transcriptional responses of a model colon cancer cell line, HT29, to TNF-alpha and anti-Fas activating antibody. Both ligands induced a subset of genes characteristic of activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Follow-up analyses demonstrated that, although TNF-alpha activated NF-kappaB through IkappaB-alpha degradation, alpha-Fas treatment led to NF-kappaB activation through a mechanism distinct from IkappaB-alpha degradation.

Inhibition of MAP kinase kinase (MEK) results in an anti-inflammatory response in vivo.

The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.

Identification of a novel inhibitor of mitogen-activated protein kinase kinase.

The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.

MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products.

In search of antiinflammatory drugs with a new mechanism of action, U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with an IC50 of 0.07 microM for MEK 1 and 0.06 microM for MEK 2. U0126 can undergo isomerization and cyclization reactions to form a variety of products, both chemically and in vivo, all of which exhibit less affinity for MEK and lower inhibition of AP-1 activity than parent, U0126.

The p38 mitogen-activated protein kinase pathway in activated and anergic Th1 cells.

Stimulation of T cells through the TCR leads to activation of the mitogen-activated protein kinase (MAPK) family members ERK (extracellular signal-regulated kinase) and JNK (jun NH2-terminal kinase). These kinases act in synergy to increase the activity of the transcription factor AP-1 which is involved in the transcriptional upregulation of IL-2. Recently a third MAPK member, p38, has been identified. The effects of T cell activation on this pathway have not yet been elucidated. Using two murine Th1 clones, we demonstrate that the p38 pathway is induced upon anti-CD3 plus anti-CD28 crosslinking or PMA plus ionomycin stimulation. p38 activity was induced fully by anti-CD3 or PMA alone and is not enhanced by costimulation even at low levels of TCR signaling. p38 activity peaked at 20 min and was significantly decreased by 2 hr. Anergic (tolerant) Th1 cells showed decreased p38 activity as well as decreased ERK and JNK activities even though levels of these proteins remained unchanged.

Low molecular weight heparin versus regular heparin or aspirin in the treatment of unstable angina and silent ischemia.

OBJECTIVES: This study was designed to test the hypothesis that low molecular weight heparin may lessen the severity of ischemic events in patients with unstable angina. BACKGROUND: Unstable angina is a thrombotic process that requires intensive medical treatment. Although current treatments can reduce the number of complications, serious bleeding continues to occur. Nadroparin calcium, a low molecular weight heparin, seems to be a safe therapeutic agent that does not require laboratory monitoring. METHODS: A total of 219 patients with unstable angina entered the study at a mean time of 6.17 h after the last episode of rest pain. Patients were randomized to receive aspirin (200 mg/day [group A]), aspirin plus regular heparin (400 IU/kg body weight per day intravenously and titered by activated partial thromboplastin time [group B]) and aspirin plus low molecular weight heparin (214 UIC/kg anti-Xa twice daily subcutaneously [group C]). The major end points determined for the in-hospital period were 1) recurrent angina, 2) myocardial infarction, 3) urgent revascularization, 4) major bleeding, and 5) death. Minor end points were 1) silent myocardial ischemia, and 2) minor bleeding. Event rates were tested by chi-square analysis. RESULTS: Recurrent angina occurred in 37%, 44% and 21% of patients in groups A, B and C, respectively, and was significantly less frequent in group C than in either group A (odds ratio 2.26, 95% confidence interval [CI] 1 to 5.18, p = 0.03) or group B (odds ratio, 3.07, 95% CI 1.36 to 7.00, p = 0.002). Nonfatal myocardial infarction was present in seven patients in group A, four in group B and none in group C (group B vs. A, p = 0.5; group C vs. A, p = 0.01). Urgent revascularization was performed in nine patients in group A, seven in group B and one in group C (C vs. A, p = 0.01). Two episodes of major bleeding occurred in group B. Silent myocardial ischemia was present in 38%, 41% and 25% of patients in groups A, B and C, respectively, and was significantly less frequent in group C than group B (odds ratio 2.12, 95% CI 0.97 to 4.69, p = 0.04). Minor bleeding was detected in 10 patients in group B, 1 patient in group C (B vs. C, p = 0.01) and no patient in group A (A vs. B, p = 0.003). CONCLUSIONS: In this study, treatment with aspirin plus a high dose of low molecular weight heparin during the acute phase of unstable angina was significantly better than treatment with aspirin alone or aspirin plus regular heparin.