A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm.

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

Transcriptional suppression of BACH2 by the Bcr-Abl oncoprotein is mediated by PAX5.

Bach2 is a lymphoid-specific transcription factor with a prominent role in B-cell development and apoptosis-induction in response to oxidative stress. We previously showed that Bach2 is downregulated in chronic myeloid leukaemia (CML), and here we demonstrate the mechanism by which Bcr-Abl mediates this phenomenon. We have cloned a 3.9 Kb genomic DNA fragment upstream of the transcription initiation site, and delineated the core and proximal BACH2 promoter regions. Transient BCR-ABL expression led to significant reduction in BACH2 promoter activity and this effect was dependent on the kinase function of the oncoprotein. Sequential deletions disclosed several regulatory elements within the promoter region, as well as within BACH2 exonic sequences. Analysis of these elements and transient transfection assays led to the identification of the Pax5 transcription factor as a potent trans-activator of BACH2, whose effect is predominantly mediated through occupation of a binding site on the BACH2 promoter, as demonstrated by both in vitro and in vivo experiments. Overall, our data show that Pax5 functions as an intermediate effector in the Bcr-Abl-mediated transcriptional repression of BACH2. The current results, combined with previous reports, establish Pax5 and Bach2 as transcriptional targets of Bcr-Abl, whose downregulation may contribute to lymphoid blast crisis of CML.

Increased endothelin-1 reactivity and endothelial dysfunction in carotid arteries from rats with hyperhomocysteinemia.

BACKGROUND AND PURPOSE: There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries. EXPERIMENTAL APPROACH: Vascular reactivity to ET-1 and ET(A) and ET(B) receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ET(A) and ET(B) receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [(125)I]-ET-1. KEY RESULTS: HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ET(A) or ET(B) receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ET(A) but not of ET(B) receptors abolished enhancement in HHcy tissues. ET(A) and ET(B) receptor gene expressions were not up-regulated. ET(A) receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A(2) receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO. CONCLUSIONS AND IMPLICATIONS: Increased ET(A) receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ET(A) receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility.

Transcriptional up-regulation of PHLDA1 by 17beta-estradiol in MCF-7 breast cancer cells.

Most breast cancer risk factors are associated with prolonged exposure of the mammary gland to high levels of estrogens. The actions of estrogens are predominantly mediated by two receptors, ERalpha and ERbeta, which act as transcription factors binding with high affinity to estrogen response elements in the promoter region of target genes. However, most target genes do not contain the consensus estrogen response elements, but rather degenerated palindromic sequences showing one or more mutations and other ER-binding sites such as AP-1 and SP-1. Using the differential display reverse transcription-polymerase chain reaction technique, our group identified several genes differentially expressed in normal tissue and in ER-positive and ER-negative primary breast tumors. One of the genes shown to be down-regulated in breast tumors compared to normal breast tissue was the PHLDA1 (Pleckstrin homology-like domain, family A, member 1). In the present study, we investigated the potential of PHLDA1 to be regulated by estrogen via ER in MCF-7 breast cancer cells. The promoter region of PHLDA1 shows an imperfect palindrome, an AP-1- and three SP-1-binding sites potentially regulated by estrogens. We also assessed the effects of 17beta-estradiol on PHLDA1 mRNA expression in MCF-7 breast cancer cells. MCF-7 cells exposed to 10 nM 17beta-estradiol showed more than 2-fold increased expression of the PHLDA1 transcripts compared to control cells (P = 0.05). The anti-estrogen ICI 182,780 (1 microM) inhibited PHLDA1 mRNA expression and completely abolished the effect of 10 nM 17beta-estradiol on PHLDA1 expression (P < 0.05), suggesting that PHLDA1 is regulated by estrogen via ER.

Transcriptional up-regulation of PHLDA1 by 17â-estradiol in MCF-7 breast cancer cells

Most breast cancer risk factors are associated with prolonged exposure of the mammary gland to high levels of estrogens. The actions of estrogens are predominantly mediated by two receptors, ERá and ERâ, which act as transcription factors binding with high affinity to estrogen response elements in the promoter region of target genes. However, most target genes do not contain the consensus estrogen response elements, but rather degenerated palindromic sequences showing one or more mutations and other ER-binding sites such as AP-1 and SP-1. Using the differential display reverse transcription-polymerase chain reaction technique, our group identified several genes differentially expressed in normal tissue and in ER-positive and ER-negative primary breast tumors. One of the genes shown to be down-regulated in breast tumors compared to normal breast tissue was the PHLDA1 (Pleckstrin homology-like domain, family A, member 1). In the present study, we investigated the potential of PHLDA1 to be regulated by estrogen via ER in MCF-7 breast cancer cells. The promoter region of PHLDA1 shows an imperfect palindrome, an AP-1- and three SP-1-binding sites potentially regulated by estrogens. We also assessed the effects of 17â-estradiol on PHLDA1 mRNA expression in MCF-7 breast cancer cells. MCF-7 cells exposed to 10 nM 17â-estradiol showed more than 2-fold increased expression of the PHLDA1 transcripts compared to control cells (P = 0.05). The anti-estrogen ICI 182,780 (1 µM) inhibited PHLDA1 mRNA expression and completely abolished the effect of 10 nM 17â-estradiol on PHLDA1 expression (P < 0.05), suggesting that PHLDA1 is regulated by estrogen via ER.

Inhibition of tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression in diabetic rats: role of insulin.

OBJECTIVE: The effects of insulin on intercellular adhesion molecule (ICAM)-1-mediated leukocyte adhesion and migration were investigated. METHODS: Diabetic rats (alloxan, 42 mg/kg, i. v., 42 days), matching controls, and insulin (NPH, 2 IU/day for 12 days) treated diabetic rats were used. The internal spermatic fascia of the animals was used for direct vital microscopy of the microcirculation, and for quantitation of ICAM-1 expression by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Experiments were performed 2 h after the local injection of recombinant rat tumor necrosis factor-alpha (5 ng). RESULTS: Relative to controls (C), diabetic (D) rats exhibited a reduced number of adhered (D: 2.2 +/- 0.4 and C: 14.1 +/- 0.6 cells/100 microm venule length, P < 0.001) and migrated leukocytes (D: 1.1 +/- 0.3 and C: 6.3 +/- 0.6 cells/1,000 microm (2), P < 0.001) accompanied by low expression of ICAM-1 in postcapillary venules (D: 18 +/- 4 and C: 51 +/- 7 arbitrary units, P < 0.001). There were no differences in ICAM-1 mRNA levels (D: 1.01 +/- 0.05 and C: 1.18 +/- 0.09 ICAM-1/GAPDH ratio, P > 0.05). Treatment of diabetic rats with insulin restored the number of adhered (10.9 +/- 1.2 cells/100 microm venule length), and migrated leukocytes (4.0 +/- 0.3 cells/1,000 microm (2)) as well as ICAM-1 expression (45 +/- 3 arbitrary units). Levels of mRNA for ICAM-1 remained unchanged after treatment (1.15 +/- 0.04 ICAM-1/GAPDH ratio). CONCLUSION: Insulin modulates TNF-alpha-induced ICAM-1 expression on microvascular endothelium controlling, therefore, leukocyte adhesion and migration.