Comprehensive pharmacological profiling of neurofibromatosis cell lines.

Patients with Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are predisposed to tumors of the nervous system. NF1 patients predominantly develop neurofibromas, and Malignant Peripheral Nerve Sheath Tumors (MPNST) while NF2 patients develop schwannomas and meningiomas. Here we quantified the drug sensitivities of NF1 and NF2 tumor cell lines in a high throughput platform. The platform contained a comprehensive collection of inhibitors of MEK, RAF, RAS, farnesyl transferase, PAK and ERK, representative drugs against many other cancer pathways including Wnt, Hedgehog, p53, EGF, HDAC, as well as classical cytotoxic agents recommended for treating MPNST, such as doxorubicin and etoposide. We profiled seven NF1-associated MPNST cell lines (ST88-14, ST88-3, 90-8, sNF02.2, T265, S462TY, SNF96.2), one sporadic MPNST cell line (STS26), one schwannoma from a NF2 patient (HEI193), one NF2-deficient malignant meningioma (KT21-MG-Luc5D), one mouse NF2 schwannoma (SC4) and one sporadic rat schwannoma (RT4-67 or RT4). NF1 cells were primarily distinguished from NF2 cells and the sporadic MPNST cell line by their sensitivity to MEK and ERK inhibitors, and to a smaller extent their sensitivity to BH3 mimetics and farnesyl transferase inhibitors. The platform was highly successful in predicting the effects of clinical trials for Neurofibromas.

Hemin upregulates Egr-1 expression in vascular smooth muscle cells via reactive oxygen species ERK-1/2-Elk-1 and NF-kappaB.

Reactive oxygen species (ROS) and oxidant stress are important mediators of cardiovascular pathologies including atherosclerosis. One source of ROS in the vasculature is free heme released from hemoglobin. Because Egr-1, the regulator of cell proliferation and apoptosis, is also induced by oxidant stress and is likewise implicated in atherosclerosis, we examined the regulation of Egr-1 by heme in vascular smooth muscle cells (SMCs). Hemin increased Egr-1 expression (mRNA, protein) within 30 minutes and ERK-1/2 phosphorylation and nuclear translocation within 5 minutes. Inhibiting hemin-induced ERK-1/2 activation by U0126 (MAPK-inhibitor), the antioxidant N-acetyl cysteine, the NADPH oxidase inhibitors apocynin and diphenyleneiodonium chloride, the superoxide scavenger tiron, or tricarbonyldichlororuthenium(II)-dimer (carbon-monoxide donor; CORM-2) blocked hemin-induced Egr-1 expression. Hemin activated Elk-1, SRF, and NF-kappaB and promoted their interaction with the Egr-1 promoter. Downregulating Elk-1 (via siRNA) or blocking NF-kappaB activation (via BAY-11-7082) abolished hemin induction of Egr-1. Finally, hemin-induced Egr-1 bound the promoters of tissue factor (TF), Plasminogen Activator Inhibitor (PAI)-1, and NGF-1A Binding (NAB)-2, upregulating their expression, and increased the biochemical activity of TF and PAI-1. Upregulation of Egr-1 and its target genes by heme-induced oxidant stress may be an important event in the initiation and progression of inflammatory vascular diseases such as atherosclerosis.

Differential regulation of early growth response gene-1 expression by insulin and glucose in vascular endothelial cells.

OBJECTIVE: Early growth response gene (Egr)-1 is a key transcription factor involved in vascular pathophysiology. Its role in diabetic vascular complications, however, remains unclear. Because hyperinsulinemia and hyperglycemia are major risk factors leading to diabetic vascular complications, we examined the effect of insulin and glucose on Egr-1 expression in murine glomerular vascular endothelial cells. METHODS AND RESULTS: Insulin or glucose, when added separately, increased egr-1 mRNA levels and promoter activity, as well as Egr-1 protein levels in nuclear extracts. When insulin was added to cells preincubated with glucose, the two had an additive effect on Egr-1 expression. Furthermore, vascular endothelial growth factor receptor-1 (flt-1) and plasminogen activator inhibitor-1, two known Egr-1-responsive genes, were also upregulated in the presence of insulin or glucose. An investigation into the underlying molecular mechanisms demonstrated that insulin, but not glucose, increased Egr-1 expression through extracellular signal-regulated kinase 1/2 activation, which is consistent with our previous reports. In contrast, inhibition of protein kinase C by phorbol ester or by the specific protein kinase C inhibitor chelerythrine chloride downregulated glucose-induced, but not insulin-induced, Egr-1 expression. CONCLUSIONS: Differential regulation of Egr-1 expression by insulin and glucose in vascular cells may be one of the initial key events that plays a crucial role in the development of diabetic vascular complications.

The control of the yeast H2O2 response by the Msn2/4 transcription factors.

We have analysed the contribution of the Msn2/4 transcription factors and the Ras-cAMP-protein kinase A (PKA) pathway to the control of the yeast H2O2 response. Strains deleted for MSN2 and MSN4 are hypersensitive to H2O2, although they can still adapt to this oxidant. They are also unable to induce 27 proteins of the H2O2 stimulon as shown by quantitative two-dimensional gel analysis. This peculiar H2O2 tolerance defect, the nature of the proteins of the Msn2/4 regulon, and the partial overlap of this regulon with the Yap1 H2O2-response regulon, suggest an independent and distinctive role of these two H2O2 stress response pathways. A strain lacking PDE2, and therefore carrying high intracellular cAMP levels, is also hypersensitive to H2O2. In the presence of exogenous cAMP, this strain does not induce the entire H2O2 Msn2/4 regulon and some other proteins. This, and the normal H2O2 induction of a gene reporter under control of the Yap1 regulator when intracellular cAMP level are high, demonstrate that the Ras-cAMP pathway negatively affects the H2O2 stress response through Msn2/4. However, the high H2O2 sensitivity of a strain lacking the PKA-negative regulatory subunit Bcy1, is not only the consequence of the inhibition of Msn2/4 but also of Yap1 through a yet undefined mechanism.

Hyperphosphorylation of Msn2p and Msn4p in response to heat shock and the diauxic shift is inhibited by cAMP in Saccharomyces cerevisiae.

In response to various stresses, as well as during the diauxic transition, the Msn2p and Msn4p transcription factors of Saccharomyces cerevisiae are activated and induce a large set of genes. This activation is inhibited by the Ras/cAMP/PKA (cAMP-dependent protein kinase) pathway. Here we show by immunoblotting experiments that Msn2p and Msn4p are phosphorylated in vivo during growth on glucose, and become hyperphosphorylated at the diauxic transition and upon heat shock. This hyperphosphorylation is correlated with activation of Msn2/4p-dependent transcription. An increased level of cAMP prevents and reverses these hyperphosphorylations, indicating that kinases other than PKA are involved. These results suggest that PKA and stress-activated kinases control Msn2/4p activity by antagonistic phosphorylation. It was also noted that Msn4p is transiently increased at the diauxic transition. Msn2p and Msn4p present different hyperphosphorylation patterns in response to different stresses.