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Link: http://www.washingtonpost.com/wp-dyn/content/article/2009/05/13/AR2009051301751.html?hpid=topnews
President Obama will oppose the release of several dozen photos depicting abuse of detainees held in U.S. military custody abroad, reversing his previous position on the grounds that the pictures could inflame anti-American sentiment and endanger U.S. troops. In announcing the shift today, the White House said in a statement that Obama strongly believes that the release of these photos, particularly at this time, would only serve the purpose of inflaming the theaters of war, jeopardizing US forces, and making our "job" more difficult in places like Iraq and Afghanistan. [Cheney & Bush couldn't have said it better. Obama would have opposed the release the old ones too and for the same phoney reasons...] WikiPedia: Abu Ghraib torture and prisoner abuse. The National Expositor/The People's Voice: Obama to Ignore CIA War Crimes.
Link: http://www.wsws.org/articles/2009/may2009/trib-m13.shtml
The administration's apparent decision to resuscitate the military commissions is based on its fear that should the trials be heard in civilian court, many would result in the dismissal of charges. This is because the evidence against the accused is based largely on torture and forms of hearsay inadmissible in normal proceedings. The proposed changes aim to lend an air of legitimacy to the military tribunals—which legal experts and human rights groups have criticized for failing to meet minimum norms of judicial fairness and which have damaged the credibility of the US internationally—while still controlling the court process to secure guilty verdicts where it is deemed politically necessary. The People's Voice: Guantanamo Under Obama.
Link: http://uk.reuters.com/article/worldNews/idUKTRE54C1R920090513
Ninety-three children and 25 adult women are among a list of 140 names of Afghans who villagers say were killed in a battle and U.S. air strikes last week, causing a crisis between Washington and its Afghan allies. The list, obtained by Reuters, bears the endorsement of seven senior provincial and central government officials, including an Afghan two-star general who headed a task force dispatched by the government to investigate the incident. Titled "list of the martyrs of the bombardment of Bala Boluk district of Farah Province", it includes the name, age and father's name of each
alleged victim. Yahoo: Probe finds 140 Afghan civilians killed in strikes. Lew Rockwell: Barack Obama – Mass Murderer.
Link: http://www.wsws.org/articles/2009/may2009/mili-m13.shtml
Touting the abilities of McChrystal, Gates spoke of his "unique skill set in counterinsurgency." These "unique" skills consist largely of deploying assassination squads to behead insurgencies opposing US occupation. He directed such a program as an essential part of the so-called "surge" initiated by the Bush administration in Iraq. Also included in the "skill set" employed by the units under McChrystal's command in Iraq was torture. A Special Operations unit known as Task Force 6-26 ran a detention center in which prisoners were subjected to Abu Ghraib-style abuse that was so extreme that the CIA ordered its personnel not to enter the facility. It appears that the Obama administration wants to replicate these methods in Afghanistan, launching its own "surge," which will entail a dramatic escalation in the bloodshed there.
Link: http://www.salon.com/opinion/feature/2009/05/13/cheney/
Out of office, he continues to push his tortured version of reality -- and his vision of an imperial presidency -- and there are signs he is succeeding. Dick Cheney is out there. He is defending torture, dissing Colin Powell, and genuflecting before radio personality Rush Limbaugh as the high priest of what's left of conservatism. His refusal to go quietly, unlike his much-reviled boss, is risky. He was a laugh line more than once at Saturday's White House Correspondents' Dinner. But the media's focus on the sheer spectacle of the ex-veep's antics, and on the Republican vs. Democrat feud he's stoking, underestimates the way Cheney's principles still inform many of the country's most crucial policies.
Link: http://zerohedge.blogspot.com/2009/05/federal-reserve-can-not-account-for-9.html
This video is a must watch for anyone who wants to understand just how "effective" the Fed is at safeguarding taxpayer money. Apparently nobody at the Federal Reserve has any clue where the trillions of dollars that have come from the Fed's expanded balance sheet have gone. Additionally, nobody there seems to have any idea what the losses on the Fed's $2 trillion portfolio really are. As for the pittance of $9 trillion in Fed off-balance sheet transactions over the past 8 months, well, yeah, that's also somewhere out there... Just don't ask the Federal Reserve where. Rep. Alan Grayson summarizes it best "I am shocked to find out that nobody at the Federal Reserve is keeping track of anything."
(P.S. Zero Hedge uses the term "anyone" generically, with the presumption that the Fed's Inspector General should traditionally receive most memos on memorandum items that deal with a dollar sign and +/- 12 zeros after it). [High Quality Video HERE]
Link: http://www.youtube.com/watch?v=cI4Z65qa7mY
More proof vaccines are dangerous as all get out. I would advise you NOT to VACCINATE your children. Pentagon uses Marines as lab rats. Ohhhh the government loves you soo much.
Link: http://www.presstv.ir/detail.aspx?id=94534§ionid=351020201
The Iraqi parliament is urging the cabinet to ask Turkey, Syria, and Iran for a larger share of water from the Tigris and Euphrates rivers. Water levels have fallen to dangerous levels. The three countries are using up much of the water from the two rivers and their tributaries leaving very little for Iraq.
Link: http://news.antiwar.com/2009/05/12/israel-finally-supplies-maps-of-cluster-bomb-sites/
Nearly three years after the end of Israel's 2006 invasion of Lebanon, the Israeli military is finally providing the United Nations with maps and technical data regarding locations which were attacked with cluster munitions. The data has been requested essentially since the war ended. Israel's use of the munitions in populated areas was roundly condemned by human rights groups, as was the United States for expediting the shipment of bombs to Israel so they could be used in the waning hours of the war.
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Co-expression of several receptor tyrosine kinases (RTKs), including erbB2 and erbB3, is frequently identified in breast cancers. A member of the RTK family, the kinase-deficient erbB3 can activate downstream signaling via heterodimer formation with erbB2. We studied the expression of RTK receptors in mammary tumors from the wild-type (wt) rat c-neu transgenic model. We hypothesized that physical and functional interactions between the wt rat neu/ErbB2 transgene and mouse ErbB3-encoded proteins could occur, activating downstream signaling and promoting mammary oncogenesis.
Immunohistochemical and Western blot analyses were performed to study the expression of rat c-neu/ErbB2 and mouse erbB3 in mammary tumors and tumor-derived cell lines from the wt rat c-neu transgenic mice. Co-immunoprecipitation methods were employed to quantitate heterodimerization between the transgene-encoded protein erbB2 and the endogenous mouse erbB3. Tumor cell growth in response to growth factors, such as Heregulin (HRG), epidermal growth factor (EGF), or insulin-like growth factor-1 (IGF-1), was also studied. Post-HRG stimulation, activation of the RTK downstream signaling was determined by Western blot analyses using antibodies against phosphorylated Akt and mitogen-activated protein kinase (MAPK), respectively. Specific inhibitors were then used with cell proliferation assays to study the phosphoinositide-3 kinase (PI-3K)/Akt and MAPK kinase (MEK)/MAPK pathways as possible mechanisms of HRG-induced tumor cell proliferation.
Mammary tumors and tumor-derived cell lines frequently exhibited elevated co-expression of erbB2 and erbB3. The transgene-encoded protein erbB2 formed a stable heterodimer complex with endogenous mouse erbB3. HRG stimulation promoted physical and functional erbB2/erbB3 interactions and tumor cell growth, whereas no response to EGF or IGF-1 was observed. HRG treatment activated both the Akt and MAPK pathways in a dose- and time-dependent manner. Both the PI-3K inhibitor LY 294002 and MEK inhibitor PD 98059 significantly decreased the stimulatory effect of HRG on tumor cell proliferation.
The co-expression of wt rat neu/ErbB2 transgene and mouse ErbB3, with physical and functional interactions between these two species of RTK receptors, was demonstrated. These data strongly suggest a role for erbB3 in c-neu (ErbB2)-associated mammary tumorigenesis, as has been reported in human breast cancers.
The erbB or epidermal growth factor receptor (EGFR) family forms subclass I of the receptor tyrosine kinase (RTK) superfamily. Type I RTKs are expressed by epithelial, mesenchymal and neural tissues to regulate cell proliferation, differentiation and other important biological functions critical to species development [1]. Dysregulated expression of erbB receptors or mutational events thereof have been implicated in diverse types of human cancers [1,2]. Members of the family include: ErbB1 (also known as EGFR), ErbB2 (also known as Her-2 or neu), ErbB3 (or Her-3) and ErbB4 (or Her-4) [3-7]. erbB2 is an orphan receptor whereas other family members directly bind ligands (like the epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) for EGFR, and HRG for erbB3 and erbB4) to initiate intracellular signaling [8].
ErbB2 may be activated via either ligand-dependent heterodimeric, or ligand-independent homodimeric processes. In the former, erbB2 is the preferred heterodimerization partner for other erbB family receptors with bound ligand [9]. In ligand-independent signaling, erbB2 may be upregulated as a result of gene amplification, promoting homodimerization, or be activated through mutational events. ErbB2 amplification with enhanced protein expression is noted in approximately one-third of invasive human breast cancers [10]. Selected heterodimers may enhance receptor activation and downstream signaling as compared with homodimers [1,11,12]. Although erbB3 lacks a functional kinase to initiate cell signaling [13,14], the erbB2/erbB3 heterodimer complex is believed to be the most biologically active and pro-tumorigenic form of these receptor complexes [15,16].
The erbB receptors and their respective ligands influence a wide range of cellular processes such as proliferation, maturation, survival, apoptosis and angiogenesis [11,17-19]. In general, activated RTKs add phosphorylated tyrosine residues to downstream signaling molecules, such as the p85 subunit of phosphatidylinositol 3-kinase (PI-3K), Shc and/or Grb2 of the mitogen-activated protein kinase (MAPK) pathway. However, because of the complexity of RTK ligand-dependent and -independent mechanisms, the downstream signaling effects may be highly diverse and interactive. RTK-induced signaling is also influenced by, and may modulate, other molecular factors and signaling pathways.
The ErbB2 gene-encoded protein is over-expressed in 25 to 30% of invasive breast and ovarian cancers and has been associated with a poor clinical outcome [20-25].
Evidence of a causal relationship in human breast cancer has been derived from numerous prognostic studies and clinical trials. In vivo and in vitro model systems including transgenic mouse models support a relationship between erbB2 alterations and mammary tumorigenesis. Overexpression of erbB3 is also frequently reported in erbB2 altered breast, ovarian and bladder cancers [23,26,27]. Human breast cancer cell lines commonly co-overexpress both erbB2 and erbB3, further supporting their role in breast carcinogenesis [2,11].
To investigate the role of RTKs in mammary tumorigenesis, transgenic mice bearing the wild-type (wt) or mutated, activated rat c-neu (ErbB2) were generated, and have been widely studied [28-31]. Transgenic mice expressing the activated rat c-neu (with deletion mutations) bear mammary tumors with elevated co-expression of the mutant c-neu/ErbB2 and the endogenous mouse ErbB3-encoded protein [32]. Functional and physical interactions between these two cross-species receptors have not been reported, although interactions have been widely speculated. Transgenic mice bearing the wt-rat c-neu, under control of the mouse mammary tumor virus promoter (MMTV-LTR), typically develop unifocal, well-circumscribed, low-grade tumors after a long latency [29]. In addition to transgene expression and, in some cases, mutation, upregulation of EGFR and p53 have been reported in derived tumors [33,34].
We have used the wt-erbB2 transgenic mouse model to study the effects of exogenous pharmacological or dietary estrogens and anti-estrogens. In particular, we have studied interactions between RTK-associated mammary tumorigenesis and steroid hormones. From the derived mouse tumors, we have established over 150 novel murine cell lines which have proven useful for in vitro studies [35,36]. Most tumor-derived cell lines express significant mouse ErbB3-encoded protein, in addition to high levels of the rat c-neu/ErbB2 transgene. These are also typically negative for ERα but show ERβ protein expression. A similar pattern of receptor expression has also been detected in the mouse mammary tumors.
The co-expression of erbB3 with erbB2 in both the activated and wt-neu/ErbB2 transgenic model systems suggested a biological role for erbB3 in mammary tumor pathogenesis. We hypothesized that physical and functional interactions between these RTK receptors should occur, despite their cross-species molecular structures. Signaling initiated by activated erbB2/erbB3 heterodimers should provide a more potent oncogenic signal than erbB2 homodimers alone. This would require ligand binding, most likely HRG, to activate erbB3. To test this hypothesis, we studied the responsiveness of tumor-derived cell lines to growth factors, including HRG, EGF and insulin-like growth factor-1 (IGF-1); we evaluated the effects of ligand stimulation and heterodimer formation on downstream signaling activation; and we sought evidence of physical interactions between the wt-rat c-neu/erbB2 and the endogenous mouse erbB3.
Materials and methods
Cells and cell culture
Human breast cancer cell lines SKBR-3 and BT-474 were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in DMEM and Ham's F-12 medium (1:1, v/v) (Invitrogen Corp, Grand Island, NY, USA) supplemented with 10% FBS (Invitrogen Corp). These cell lines were cultured in a 37°C humidified atmosphere containing 95% air and 5% CO2 and were split twice a week. These human breast cancer cells were used primarily as controls.
Establishment of novel, mouse mammary tumor cell lines
Mammary tumors were obtained from the transgenic mice by surgical removal immediately following euthanasia, according to our approved IACUC protocol. The histological pattern and tumor diagnoses were confirmed by microscopic analysis. These methods have been previously described in detail [35], although the specific cell lines described in this work have not been previously published. In brief, solid tumor tissue was transferred into a tissue culture dish containing PBS. After removal of mammary fat and connective tissues, tumors were minced into small pieces and treated with 0.25% trypsin-EDTA (Invitrogen Corp) at 37°C for 30 min. Cells were subsequently centrifuged at 1,200 rpm for 5 min. After discarding supernatant, cells were suspended in DMEM/F12 medium supplemented with 10% FBS and 1% antibiotics and antimycotics (Invitrogen Corp). These mammary tumor cells (~1.0 × 106 cells/plate) were seeded in tissue culture dishes and kept in a 37°C humidified atmosphere containing 95% air and 5% CO2. The media was changed twice a week to maintain cells in culture. Each line was passaged approximately 20 times before stability was assumed.
Soft agar cloning assays
Soft agar cloning was performed as described previously [35] with some modification. The bottom agar was prepared with a mixture of 1.6 ml of 1 × DMEM/F12 (complete medium), 3.2 ml of 2 × DMEM/F12 (complete medium), and 3.2 ml of 1.25% Noble agar (Sigma Co, St Louis, MO, USA) and maintained at 42°C. From this, 2 ml was pipetted into each well of six-well cell culture plates and agar was allowed harden in the hood. For each well, top agar was a mixture of 0.2 ml of 1 × DMEM/F12, 0.4 ml of 2 × DMEM/F12, and 0.4 ml of 0.95% Noble agar. Five thousand cells (in 80 μl complete medium) were added into the top agar mixture. After vortexing gently, the cell containing top agar was added in a drop-wise fashion onto the bottom agar containing six-well plates (in triplicate per cell line). After resting for 10 min in the hood, the six-well plates were cultured in a 37°C incubator for 3 weeks. Colony counts were obtained under an inverted microscope, from three wells per cell line counting all colonies >50 μM in diameter.
Doubling time in culture
Measurement of cell growth rate in culture was determined using sulforhodamine B (SRB; Sigma Co) assays as previously described [35]. Two thousand cells were seeded into each well of a six-well plate with complete medium. Cells were fixed with 50% trichloroacetic acid (TCA) at 24 h intervals for 3 days. TCA-fixed cells were then stained with 0.4% SRB for 30 min followed by three washes. Protein-bound dye was dissolved in 10 mM Tris base. Plates were read at 565 nM using a micro-plate reader. Cell-doubling time was calculated based on proliferation curves that resulted from changes in SRB absorbance over time. Data represent the means of at least three independent experiments.
Cell proliferation assay
A CellTiter96™ AQ non-radioactive cell proliferation kit (Promega Corp, Madison, WI, USA) was used to determine the responsiveness of cells to various growth factors. Cells were plated onto 96-well plates, 5,000 cells/well for each cell line. Twenty-four hours later, the culture media were replaced by 0.5% FBS in DMEM/F12 fresh medium or the same medium containing 25 ng/ml HRG (R&D Systems, Inc, Minneapolis, MN, USA), 10 ng/ml EGF (Sigma Chemical Co), or 40 ng/ml IGF-1 (R&D Systems, Inc) for another 72 h incubation with 5% CO2 at 37°C. After reading at 490 nM with the micro-plate reader, the percentages of viable cells were determined by reduction of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; inner salt) relative to controls. Data reflect the means of at least three independent experiments.
RT-PCR and DNA sequencing analysis
RT-PCR analyses were performed as previously described [37]. The primers specific for rat neu were synthesized according to the literature [38]. Forward primer AB2913, 5'-CGG AAC CCA CAT CAG GCC-3' and reverse primer AB1310, 5'-TTT CCT GCA GCA GCC TAC GC-3' amplify the region corresponding to nucleotides 1492 to 2117 of rat neu cDNA [38]. The PCR products purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen, Inc, San Valencia, CA, USA) were submitted to the core facility at the Oklahoma Medical Research Foundation for direct sequencing.
Immunohistochemical staining of mammary tumor tissues was performed as previously described [39]. Briefly, after deparaffinization and rehydration, tissue sections were steamed in a 10 mM citrate buffer, pH 6.0, for 30 min. Non-specific reactivity was blocked with 0.3% H2O2 in buffer. For erbB3 immunoassays, CAS Block (Zymed Laboratories, Inc, South San Francisco, CA, USA) and 10% normal horse serum (Vector Laboratories, Inc, Burlingame, CA, USA) were used sequentially. For phospho-Akt immunostaining, we used 1% H2O2 and 5% normal goat serum (Vector Laboratories, Inc) sequentially. Primary antibodies included an anti-erbB2 (reactive with rat c-neu/erbB2 rabbit polyclonal, dilution 1:1000 (Dako, Carpinteria, CA, USA) for 2 h incubation at room temperature), anti-erbB3 (cross-reacts with mouse and human, mouse monoclonal, dilution 1:50 (NeoMarkers, Inc, Fremont, CA, USA), overnight incubation at 4°C), anti-phospho-Akt (rabbit polyclonal, diluted in 5% normal goat serum 1:12.5 (Cell Signaling Technology, Beverly, MA, USA), overnight at 4°C), or anti-phospho-MAPK (E10 monoclonal antibody, diluted in 5% normal goat serum 1:25 (Cell Signaling Technology), overnight at 4°C). After multiple washes with buffer, tissue sections were sequentially incubated for 30 min at room temperature with diluted biotinylated secondary antibody (1:500; Dako) and VECTASTAIN Elite ABC reagent (Vector Laboratories, Inc) diluted in PBS. After reaction with diaminobenzidine (Dako) and counterstaining with hematoxylin, tumors were individually examined. Each slide was evaluated in its entirety for antigen expression, cell type and histopathological diagnoses.
Immunoprecipitation and Western blot analysis
Immunoprecipitation and Western blot assays were performed as previously described [40]. Briefly, cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 μg/ml leupeptin, 25 μg/ml aprotinin). The supernatants were cleared by centrifugation. Protein concentrations were measured using the Coomassie plus protein assay reagent (Pierce Chemical Co, Rockford, IL, USA). Total cell lysates containing 200 μg of protein were subjected to immunoprecipitation in the presence of 1 μg anti-erbB2 antibody (mouse monoclonal antibody, Ab-4; Oncogene Science Products, Cambridge, MA, USA) for 2 h at 4°C, followed by incubation with immobilized protein A-agarose (Roche Diagnostics Corp, Indianapolis, IN, USA) at 4°C overnight with rotation. For Western blot analyses, the immunoprecipitates or equal amounts of crude extracts were boiled in Laemmli SDS-sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose (Bio-Rad Laboratories, Hercules, CA, USA), and probed with different primary antibodies. After the blots were incubated for another 1 h at room temperature with horseradish peroxidase-labeled secondary antibody (goat anti-rabbit IgG or goat anti-mouse IgG; Perkin Elmer, Boston, MA, USA), the signals were detected using the Enhanced Chemiluminance assay (Amersham Life Science Inc., Arlington Heights, IL, USA) according to the manufacturer's instructions.
Co-expression of erbB2 and erbB3 protein in tumor-derived cell lines and tumors
Western blot analyses were used to determine erbB2 and erbB3 protein expression in tumor-derived cell lines (and the control SKBR-3 human breast cancer cell line). The majority of tumor-derived cell lines expressed moderate to high levels of both erbB3 and erbB2 (Fig. 1). In general, lines with the highest erbB2 expression showed the highest levels of erbB3 protein. Tyrosine phosphorylation (activation) of these receptors was examined by Western blots using antibodies specific for phophorylated erbB2 (P-erbB2) or phosphorylated erbB3 (P-erbB3). Tumor lines with co-overexpression of both proteins showed higher P-erbB2 and P-erbB3 levels (Fig. 1). The intensity of P-erbB2 and P-erbB3 signals did not necessarily correlate with their corresponding protein levels. The expression of either receptor protein was undetectable in only one of our novel, derived tumor cell lines (78423). AIB-1 (also called SRC3, RAC3, ACTR and p/CIP), a co-activator of estrogen receptor commonly amplified in breast cancer cells [41], was used as a loading control. Expression of AIB-1 further established the origin of these cells as mammary-derived.
Figure 1. Protein expression of erbB2, P-erbB2, erbB3, P-erbB3 and AIB1 in breast tumor cell lines. The cell lysates from human breast cancer cell line SKBR-3 and the six novel, mammary tumor-derived cell lines were prepared as described in Materials and methods. 50 μg total cell lysates were used for Western blot analyses with antibodies directed against erbB2 (c-neu Ab-3, Oncogene Research Products), P-erbB2 (clone PN2A, NeoMarkers, Inc), erbB3 (Ab-7, NeoMarkers, Inc), P-erbB3 (clone 21D3, Cell Signaling Technology) and AIB1 (clone 34, BD Biosciences Pharmingen, San Diego, CA, USA).
To confirm the transformed characteristics of these lines, soft agar cloning assays (which quantitate anchorage-independent cloning capability) were used. All six tumor-derived cell lines formed colonies in soft agar. Colony formation was variable when comparing one cell line with another (range 17–180, Table 1). There was no correlation between the ability of a cell line to form anchorage-independent clones and the expression levels of erbB2 or erbB3.
Table 1. Anchorage-independent cloning of mammary tumor-derived cell lines
Immunohistochemical methods were used to visualize RTK expression and downstream signaling (protein activation) by tumors in situ. Tumors showed strong and typically diffuse co-expression of both erbB2 and erbB3. The only exception to this was the mammary tumor 78423 R1, the progenitor of the cell line that did not co-express erbB2 and erbB3 discussed above. We also studied RTK signaling activation in situ, using phosphospecific antibodies. Phosphorylated-Akt (P-Akt) showed cytoplasmic and membranous staining, which was less diffuse than the erbB-2 expression. Phosphorylated-MAPK (P-MAPK) was the most selectively expressed, typically expressed by clustered or isolated tumor cells as shown in Fig. 2 (left panel) with tumor 78617 R3. The majority of tumor cells from 78423 R1 were erbB3 negative, although some cells showed weak erbB2 protein expression. In this later tumor, P-Akt staining was weak with clustered or isolated tumor cells and no staining for P-MAPK was observed (Fig. 2, right panel). The histological, cytological and biological features of these tumors have been reported elsewhere [36,42]. As a control, we also studied cytokeratin expression and all tumors were positive. This confirmed the epithelial nature of these tumors (data not shown).
Figure 2. Immunohistochemical staining for erbB2, erbB3, phospho-Akt (P-Akt) and phospho-MAPK (P-MAPK) in mammary tumor tissues. Representative photomicrographs were taken from the similar area of 78617 R3 and 78423 R1 mammary tumor sections (40X).
Sequencing analyses of the transgene neu in established mammary tumor-derived cell lines
As alluded to above, in-frame deletions of 7–12 amino acids have been reported in the extracellular region of the transgene, proximal to the transmembrane domain [38]. To study the mutational status of tumor-derived lines, we performed RT-PCR amplification of exactly the same region followed by direct sequencing analysis. The PCR primers used were specific for rat neu and were designed to amplify the 603 bp extracellular region [38]. Of six tumor-derived cell lines used in this manuscript and therefore studied for mutation, only four showed PCR gene amplification (Fig. 3a). Of these, the strongest PCR signal was seen in 85819 cells. These data are consistent with our Western blot results that showed overexpression of the rat neu/erbB2 in only the four PCR-positive lines (Fig. 1). Direct sequencing of the PCR products revealed no deletion mutations in the amplified product. Sequencing showed three of the four were wt rat neu cDNA sequence. Sequencing data from the 83923 cells indicated a mixture of two kinds of neu cDNA. Using a reverse primer, we verified that both wt and point mutation neu transcripts co-existed in 83923 cells (Fig. 3b). This suggests biclonal populations or a heterozygous mutation. Further studies and sub-cloning are in process.
Figure 3. RT-PCR and sequencing analyses of partial extracellular domain of rat neu in the tumor-derived cell lines. (a) RT-PCR analyses of rat neu. Total RNA isolated from the indicated tumor cells by the TRIZOL reagent was analyzed with electrophoresis using a 1% agarose gel containing ethidium bromide and visualized under UV light (bottom). First-strand cDNA was synthesized using a kit from Roche Diagnostics Corp. The partial extracellular domain of rat neu was amplified with specific primers. The PCR products were separated on a 1.2% agarose gel containing ethidium bromide and visualized under UV light (top). (b) Partial sequencing of the PCR product from 83923 cells with reverse primer AB1310. The mixture of wt nucleotide T (red) and mutant nucleotide A (green) is indicated by an arrow.
Mammary tumor cell response to growth factors corresponds with erbB receptor data
To study the functionality and interactions of the erbB receptors, 78423 and other three representative mouse mammary tumor-derived lines with the highest expression of wt erbB2 and co-expression of erbB3 were chosen for further study. Baseline proliferation was determined using monolayer culture conditions and the SRB assay (Fig. 4a). Some variability in the basal doubling time was observed between these cell lines. The mouse mammary tumor cell lines 78423, 78617, 85815 and 85819 showed population doubling times of 15.15 ± 1.10, 16.25 ± 1.40, 30.85 ± 2.31 and 20.35 ± 1.89 h, respectively. Using an MTS assay, we then tested the response of these lines to EGF, HRG and insulin-like growth factor (IGF)-1 (Fig. 4b). HRG strongly stimulated the proliferation of three of the four mouse mammary tumor cell lines (78617, 85815, 85819) with overexpression of both erbB2 and erbB3. Proliferation was not induced by EGF or IGF-1, which bind to EGFR and IGF-1 receptor, respectively. HRG also promoted the growth of SKBR-3 and BT-474 human breast cancer cells (controls). These data strongly support a functional interaction between the wt-rat neu/ErbB2 and endogenous mouse erbB3.
Figure 4. Proliferation of the tumor-derived cell lines and their responsiveness to growth factors. (a) The tumor-derived cell lines 78423, 78617, 85815 and 85819 were subjected to SRB assays for measurement of cell growth rate as described in Materials and methods. The means of at least three independent experiments were plotted. SD for each point was less than 10%. (b) The indicated breast cancer cells (5 × 103) in 0.1 ml culture media were plated onto 96-well plates. After 24 h incubation, cells were grown in either 0.1 ml fresh medium with 0.5% FBS as control, or 0.1 ml same medium containing either 25 ng/ml HRG or 10 ng/ml EGF, and 40 ng/ml IGF-1. Cells were incubated at 37°C with 5% CO2 for another 72 h, and the percentages of surviving cells from each group relative to controls, defined as 100% survival, were determined by reduction of MTS. Data reflect the means of at least three independent experiments.
HRG activation of PI-3K/Akt and MAPK kinase (MEK)/MAPK signaling promotes mammary tumor cell growth
It is well documented that the MEK/MAPK and PI-3K/Akt pathways are the two major signal transduction pathways downstream of the erbB receptors [11,17-19]. To determine which signaling pathways were activated in the mouse-derived mammary tumor cells exposed to HRG, we performed Western blots to detect P-MAPK or P-Akt. With 2 h of HRG treatment, both P-Akt and P-MAPK increased in the 85815 and 85819 mouse mammary tumor cell lines (Fig. 5a). This study included a series of HRG concentrations, and stimulation was maximal at a concentration of 2.5 ng/ml. Next, we performed a time-course analysis to further verify these results. HRG stimulated both Akt and MAPK in 85815 and 85819 cells, whereas it had no effect on Akt or MAPK activation in the 78423 cells (Fig. 5b). These data were consistent with the results of minimal stimulation by HRG in this cell line (Fig. 4b). In aggregate, these data suggest that HRG induces activation of both MEK/MAPK and PI-3K/Akt signaling transduction pathways in mammary tumor cells with elevated expression levels of both the transgene rat c-neu/ErbB2 and the endogenous mouse ErbB3 gene. This activation was both dose- and time-dependent.
Figure 5. Effects of HRG on the phosphorylation of Akt and MAPK in tumor-derived cells. (a) 85815 and 85819 cells were cultured overnight in medium containing 0.5% FBS before being exposed to HRG at the indicated concentrations for 2 h. Cells were harvested and 50 μg total cell lysates were subjected to Western blot analysis for total Akt, phosphorylated Akt, total ERK2 (polyclonal antibody C-14; Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), and phosphorylated MAPK (E10 mAb; Cell Signaling Technology) expression. β-actin was used as loading control. (b) 78423, 85815 and 85819 cells were cultured overnight in medium containing 0.5% FBS before being exposed to 2.5 ng/ml HRG for the indicated time intervals. At each time point, cells were harvested and 50 μg total cell lysates were subjected to Western blot analysis for total Akt, phosphorylated Akt, total ERK2, and phosphorylated MAPK expression. β-actin was used as loading control.
To study cross-species functional interactions between the rat c-neu/ErbB2 transgene and mouse ErbB3, we evaluated tumor and tissue expression in vivo, ligand-associated interactions, and signaling in vitro. Immunohistochemical studies showed cytoplasmic P-Akt and P-MAPK expression in tumor cells with erbB2 and erbB3 co-expression, predominantly a perivascular distribution. In rare tumors without erbB2 and erbB3 expression (e.g. 78423 R1), the perivascular distribution was not identified and only rare cells showed immunoreactivity. This evidence of perivascular pathway activation suggests that ligand-associated signaling via erbB3 may be involved. Ligand-associated signaling probably provides enhanced growth or pro-tumorigenic signaling, in addition to ligand-independent, transgene activation. Our data, and those from others showing frequent erbB3 upregulation in transgenic mice bearing activated neu/ErbB2, suggest that the concomitant upregulation of erbB3 and ligand-associated signaling may be an important additional factor in both wt and activated neu/ErbB2-associated mammary tumor development. To further define the role of HRG (ligand)-associated signaling, we utilized derived cell lines and specific inhibitors in vitro. The PI-3K inhibitor LY294002 was significantly more potent than the MEK inhibitor PD98059 in blocking the stimulatory effects of HRG (Fig. 7). Hence, while the MEK/MAPK and PI-3K/Akt signaling cascades both contribute HRG induced proliferation, the PI-3K/Akt pathway appears to provide the dominant response.
Figure 7. Inhibitory effects of PD98059 and LY294002 blocking HRG-mediated tumor cell proliferation. The indicated breast cancer cells (5 × 103) in 0.1 ml culture media were plated onto 96-well plates. After 24 h incubation, cells were grown in either 0.1 ml fresh medium with 0.5% FBS as control, or 0.1 ml same medium containing PD98059 (6.7 μM for 78617 and 85815, 5 μM for 85819), or LY294002 (3.3 μM for 78617 and 85815, 2.5 μM for 85819), or 25 ng/ml HRG alone or in combination of the same concentrations of HRG and PD98059, or HRG and LY294002. Cells were incubated at 37°C with 5% CO2 for another 72 h, and the percentages of surviving cells from each group relative to controls, defined as 100% survival, were determined by reduction of MTS. Data reflect the means of at least three independent experiments.
Physical interaction between wt-rat-c-neu/ErbB2 and endogenous mouse erbB3
The erbB2/erbB3 complex is believed to be the most biologically active erbB heterodimer [43,44], with potent activation of the downstream signaling cascade [13,14]. Since both erbB2 and erbB3 were highly expressed by our mammary tumor cell lines and HRG-promoted tumor cell proliferation, we sought physical evidence that the wt-rat-neu/ErbB2 could form a complex with the endogenous mouse erbB3. Immunoprecipitation of erbB2, followed by Western blot analysis for erbB2 and erbB3 (Fig. 8a) showed a low level of complex formation between these receptors in untreated cell lines. HRG treatment significantly increased the physical interaction between the rat transgene and mouse erbB3 in two out of three cell lines. The antibody we used for immunoprecipitation (c-neu Ab-4) appeared to be wt-rat-neu/ErbB2-specific, because human erbB2 was not immunoprecipitated from SKBR-3 cell lysates (Fig. 8a, upper panel), although it was expressed by SKBR-3 cells (Fig. 8b, Western blot analysis with c-neu Ab-3). HRG treatment (for 2 h) did not increase the total protein levels of erbB2 or erbB3 as compared with untreated cell lines (Fig. 8b).
Figure 8. Increased physical associations between wt-rat c-neu protein and mouse erbB3 by HRG treatment. (a) The indicated breast cancer cells were cultured overnight in medium containing 0.5% FBS, and then incubated with or without 2.5 ng/ml HRG for another 2 h. Cell lysates were prepared and 200 μg of total protein was subjected to immunoprecipitation with anti-erbB2 specific antibody (c-neu Ab-4), followed by Western blot analysis for erbB2 (c-neu Ab-3) and erbB3. The bar graph underneath was obtained by densitometry analysis. The relative signal intensities of erbB3 were measured by EAGLE EYE™ II (Stratagene, La Jolla, CA, USA). (b) The same bath of 50 μg total cell lysates was subjected to Western blot analysis with antibodies directed against erbB2 and erbB3.
We have shown that transgenic mice bearing the wt-rat c-neu gene, under control of the MMTV promoter, develop mammary tumors that overexpress the rat c-neu transgene [45,46] and the endogenous mouse erbB3 protein, in the vast majority of cases. We have shown a functional interaction between these two important RTK receptors and a role for ligand-induced signaling in vitro and in vivo. While others have reported that transgenic mice bearing activated forms of rat c-neu/erbB2 have co-expression of erbB2 and endogenous erbB3 in mammary tumors [32], direct physical and functional interactions between these two species receptors have not previously been reported.
Deletion mutants of the neu oncogene have been reported in two out of three of the mammary tumors derived from this wt-rat c-neu transgenic model [38]. We did not find the same mutation rate or type in selected tumor-derived cell lines. However, we have identified a potential point mutation in 83923 cells (Fig. 3). This missense mutation is located inside the same extracellular region of neu where the deletion mutations have been reported. This particular mutation changes the amino acid 654 serine (codon AGC) into cysteine (codon TGC). It is different from the active neu mutation G664V reported in the transmembrane domain [47]. The biological significance of the newly discovered S654C mutant neu is not yet known.
Using ligand stimulation with or without specific inhibitors, we have studied RTK-induced signaling in response to HRG and have shown activation of both PI-3K/Akt and the MEK/MAPK signal transduction pathways. A greater role for PI-3K/Akt signaling was suggested in response to HRG treatment (Fig. 7). PI-3K/Akt signaling is known to be regulated by erbB2-mediated tyrosine kinase activity. This pathway plays a crucial role in cell proliferation and survival [18] and has been associated with the pathogenesis of human breast cancers. PI-3K/Akt activation has also been cited as a key pathway that influences chemo-resistance patterns [48,49]. Akt is frequently upregulated in ErbB2 amplified or overexpressing human breast cancer cells. These similarities between our transgenic model and human breast carcinogenesis suggest that the model and derived tumor cell lines may be a useful resource to study ligand dependent and independent RTK signaling in vivo and in vitro.
As a major ligand for erbB3, HRG is known to bind to erbB3, foster heterodimer complex formation and promote potent downstream signaling [12]. HRG can thus promote mammary tumorigenesis, cell growth, differentiation and phenotypic aggression [50]. Our immunohistochemical studies of tumors for phosphorylated proteins facilitated studies of the cellular location and architectural context of signaling. We noted enhanced phosphorylated Akt and MAPK in a perivascular distribution in mammary tumors, with overexpression of both erbB2 and erbB3 (Fig. 6), suggesting that circulating HRG may enhance the physical and functional erbB2/erbB3 interactions in vivo, similar to what we observe in vitro. This study has focused primarily on erbB3, whereas others have demonstrated upregulation of EGFR in tumors (by immunohistochemistry and Western blot) in the same model system [33]. Low and variable expression of EGFR has also been found in mammary tumors that develop in transgenic mice bearing activated forms of rat c-neu/ErbB2 [32]. Using in vitro analyses of the tumor-derived cell lines, we have found no significant physical or functional interaction between EGFR (erbB1) and erbB2 in the presence of EGF (data not shown). However, by immunohistochemical study, we also detected erbB1 expression at the tumor periphery as reported by DiGiovanna [33]. These data suggest to us that erbB3 plays a more significant role in tumorigenesis than erbB1 in this model system.
Figure 6. Immunohistochemical staining for phospho-Akt (P-Akt) and phospho-MAPK (P-MAPK) in 78617 R3 and 78423 R1 mammary tumor tissues. Procedure used was similar to Fig. 2. Representative photomicrographs were taken from perivascular areas of the tumor sections (20X).
These data and this model probably have relevance to human breast cancer biology and treatment strategies. We have reported that only a minority of erbB2-altered invasive human breast cancers have overexpression of erbB1 (EGFR) and activation of erbB2 [51]. Given the complexity of the RTK receptors, various ligands and downstream signaling, it is likely that combinations of these factors including erbB3 contribute to cell signaling, biological behavior and treatment response [52,53]. To date, the role of erbB3 in human breast carcinogenesis is not well defined, although many investigators have suggested that HRG-associated signaling may be important. In view of these complexities, it is not surprising that erbB2 aberrant breast cancers have shown variable responses to anti-erbB2 therapeutics [52,53]. It is widely believed that co-expression of other erbB RTK family members may be one mechanism of Herceptin resistance [54]. Ligand-induced heterodimerization between erbB3 and erbB2, the most potent signaling complex amongst the various heterodimers, is one likely mechanism of Herceptin resistance [55]. More detailed investigations using banks of human tumors and clinical trial-associated specimens, to define the incidence of erbB3 abnormalities, functional complex formation and downstream signaling, may provide important new clues regarding these interactions and their role in breast carcinogenesis.
Our results indicate that over-expression of endogenous mouse erbB3 plays an important role in the development and progression of mammary tumors that arise in mice bearing the wt-rat c-neu transgene. The functional and physical interactions between these important cross-species erbB receptors result in activation of both PI-3K/Akt and MEK/MAPK signaling. These data support the concept that ligand-dependent and -independent signaling through erbB2 may promote mammary tumorigenesis in these transgenic mice, similar to what is observed in human breast cancers.
DMEM = Dulbecco's modified Eagle's medium; EGF = epidermal growth factor; EGFR = EGF receptor; ER = estrogen receptor; FBS = fetal bovine serum; HRG = heregulin; IGF-1 = insulin-like growth factor-1; mAb = monoclonal antibody; MAPK = mitogen-activated protein kinase; MEK = MAPK kinase; MMTV = mouse mammary tumor virus; PBS = phosphate-buffered saline; RTK = receptor tyrosine kinase; PI-3K = phosphoinositide 3-kinase; RT-PCR = reverse transcription-polymerase chain reaction; SRB = sulforhodamine B; TCA = trichloroacetic acid; TGF-α = transforming growth factor-α.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
The authors' contributions to this research work are reflected in the order shown, with the exception of ADT who supervised the research and finalized the report. AK, BL and DOE carried out most of the experiments. AK and BL drafted the manuscript. KMA collected mammary tumors from the transgenic mice. LDJ performed immunohistochemistry analysis. CM maintained tumor cell culture. SME, XY and ADT conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.
This work was supported by NIH 1RO1 CA82848 and 1P50 CA89018 to ADT.
Olayioye MA, Neve RM, Lane HA, Hynes NE: The ErbB signaling network: receptor heterodimerization in development and cancer.
EMBO J 2000, 19:3159-3167. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
deFazio A, Chiew YE, Sini RL, Janes PW, Sutherland RL: Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines.
Int J Cancer 2000, 87:487-498. PubMed Abstract | Publisher Full Text
Coussens L, Yang-Feng TL, Liao YC, Chen E, Gray A, McGrath J, Seeburg PH, Libermann TA, Schlessinger J, Francke U, et al.: Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene.
Science 1985, 230:1132-1139. PubMed Abstract
King CR, Kraus MH, Aaronson SA: Amplification of a novel v-erbB-related gene in a human mammary carcinoma.
Science 1985, 229:974-976. PubMed Abstract
Plowman GD, Culouscou JM, Whitney GS, Green JM, Carlton GW, Foy L, Neubauer MG, Shoyab M: Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family.
Proc Natl Acad Sci USA 1993, 90:1746-1750. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kraus MH, Issing W, Miki T, Popescu NC, Aaronson SA: Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors.
Proc Natl Acad Sci USA 1989, 86:9193-9197. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Plowman GD, Whitney GS, Neubauer MG, Green JM, McDonald VL, Todaro GJ, Shoyab M: Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.
Proc Natl Acad Sci USA 1990, 87:4905-4909. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Riese DJ 2nd, Stern DF: Specificity within the EGF family/ErbB receptor family signaling network.
Bioessays 1998, 20:41-48. PubMed Abstract | Publisher Full Text
Graus-Porta D, Beerli RR, Daly JM, Hynes NE: ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.
EMBO J 1997, 16:1647-1655. PubMed Abstract | Publisher Full Text
Penuel E, Akita RW, Sliwkowski MX: Identification of a region within the ErbB2/HER2 intracellular domain that is necessary for ligand-independent association.
J Biol Chem 2002, 277:28468-28473. PubMed Abstract | Publisher Full Text
Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF 3rd, Hynes NE: The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation.
Proc Natl Acad Sci USA 2003, 100:8933-8938. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Tzahar E, Waterman H, Chen X, Levkowitz G, Karunagaran D, Lavi S, Ratzkin BJ, Yarden Y: A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.
Mol Cell Biol 1996, 16:5276-5287. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Harari D, Yarden Y: Molecular mechanisms underlying ErbB2/HER2 action in breast cancer.
Oncogene 2000, 19:6102-6114. PubMed Abstract | Publisher Full Text
Yarden Y, Sliwkowski MX: Untangling the ErbB signalling network.
Nat Rev Mol Cell Biol 2001, 2:127-137. PubMed Abstract | Publisher Full Text
Alimandi M, Romano A, Curia MC, Muraro R, Fedi P, Aaronson SA, Di Fiore PP, Kraus MH: Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas.
Oncogene 1995, 10:1813-1821. PubMed Abstract
Wallasch C, Weiss FU, Niederfellner G, Jallal B, Issing W, Ullrich A: Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3.
EMBO J 1995, 14:4267-4275. PubMed Abstract
Chang HW, Aoki M, Fruman D, Auger KR, Bellacosa A, Tsichlis PN, Cantley LC, Roberts TM, Vogt PK: Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase.
Science 1997, 276:1848-1850. PubMed Abstract | Publisher Full Text
Hermanto U, Zong CS, Wang LH: ErbB2-overexpressing human mammary carcinoma cells display an increased requirement for the phosphatidylinositol 3-kinase signaling pathway in anchorage-independent growth.
Oncogene 2001, 20:7551-7562. PubMed Abstract | Publisher Full Text
Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH: Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions.
Endocr Rev 2001, 22:153-183. PubMed Abstract | Publisher Full Text
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
Science 1987, 235:177-182. PubMed Abstract
Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, et al.: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.
Science 1989, 244:707-712. PubMed Abstract
Paterson MC, Dietrich KD, Danyluk J, Paterson AH, Lees AW, Jamil N, Hanson J, Jenkins H, Krause BE, McBlain WA, et al.: Correlation between c-erbB-2 amplification and risk of recurrent disease in node-negative breast cancer.
Cancer Res 1991, 51:556-567. PubMed Abstract
Gullick WJ, Love SB, Wright C, Barnes DM, Gusterson B, Harris AL, Altman DG: c-erbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes.
Br J Cancer 1991, 63:434-438. PubMed Abstract
Andrulis IL, Bull SB, Blackstein ME, Sutherland D, Mak C, Sidlofsky S, Pritzker KP, Hartwick RW, Hanna W, Lickley L, et al.: neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group.
J Clin Oncol 1998, 16:1340-1349. PubMed Abstract | Publisher Full Text
Thor AD, Liu S, Edgerton S, Moore D 2nd, Kasowitz KM, Benz CC, Stern DF, DiGiovanna MP: Activation (tyrosine phosphorylation) of ErbB-2 (HER-2/neu): a study of incidence and correlation with outcome in breast cancer.
J Clin Oncol 2000, 18:3230-3239. PubMed Abstract | Publisher Full Text
Bieche I, Onody P, Tozlu S, Driouch K, Vidaud M, Lidereau R: Prognostic value of ERBB family mRNA expression in breast carcinomas.
Int J Cancer 2003, 106:758-765. PubMed Abstract | Publisher Full Text
Chow NH, Chan SH, Tzai TS, Ho CL, Liu HS: Expression profiles of ErbB family receptors and prognosis in primary transitional cell carcinoma of the urinary bladder.
Clin Cancer Res 2001, 7:1957-1962. PubMed Abstract | Publisher Full Text
Bouchard L, Lamarre L, Tremblay PJ, Jolicoeur P: Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV/c-neu oncogene.
Cell 1989, 57:931-936. PubMed Abstract | Publisher Full Text
Guy CT, Webster MA, Schaller M, Parsons TJ, Cardiff RD, Muller WJ: Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease.
Proc Natl Acad Sci USA 1992, 89:10578-10582. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Muller WJ, Sinn E, Pattengale PK, Wallace R, Leder P: Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene.
Cell 1988, 54:105-115. PubMed Abstract | Publisher Full Text
Andrechek ER, Hardy WR, Siegel PM, Rudnicki MA, Cardiff RD, Muller WJ: Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis.
Proc Natl Acad Sci USA 2000, 97:3444-3449. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Siegel PM, Ryan ED, Cardiff RD, Muller WJ: Elevated expression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer.
EMBO J 1999, 18:2149-2164. PubMed Abstract | Publisher Full Text
DiGiovanna MP, Lerman MA, Coffey RJ, Muller WJ, Cardiff RD, Stern DF: Active signaling by Neu in transgenic mice.
Oncogene 1998, 17:1877-1884. PubMed Abstract | Publisher Full Text
Li B, Rosen JM, McMenamin-Balano J, Muller WJ, Perkins AS: neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice.
Mol Cell Biol 1997, 17:3155-3163. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Jeruss JS, Liu NX, Chung Y, Magrane G, Waldman F, Edgerton S, Yang X, Thor AD: Characterization and chromosomal instability of novel derived cell lines from a wt-erbB-2 transgenic mouse model.
Carcinogenesis 2003, 24:659-664. PubMed Abstract | Publisher Full Text
Yang X, Edgerton SM, Kosanke SD, Mason TL, Alvarez KM, Liu N, Chatterton RT, Liu B, Wang Q, Kim A, et al.: Hormonal and dietary modulation of mammary carcinogenesis in mouse mammary tumor virus-c-erbB-2 transgenic mice.
Cancer Res 2003, 63:2425-2433. PubMed Abstract | Publisher Full Text
Liu B, Edgerton SM, Yang X, Kim A, Ordonez-Ercan D, Mason TL, Alvarez KM, McKimmey C, Liu N, Thor AD: Low dose dietary phytoestrogen abrogates tamoxifen associated mammary tumor prevention.
Cancer Res 2005, 65:879-886. PubMed Abstract | Publisher Full Text
Siegel P, Dankort D, Hardy W, Muller W: Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors.
Mol Cell Biol 1994, 14:7068-7077. PubMed Abstract | PubMed Central Full Text
Thor AD, Berry DA, Budman DR, Muss HB, Kute T, Henderson IC, Barcos M, Cirrincione C, Edgerton S, Allred C, et al.: erbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer [comment].
J Natl Cancer Inst 1998, 90:1346-1360. PubMed Abstract | Publisher Full Text
Liu B, Fang M, Lu Y, Mendelsohn J, Fan Z: Fibroblast growth factor and insulin-like growth factor differentially modulate the apoptosis and G1 arrest induced by anti-epidermal growth factor receptor monoclonal antibody.
Oncogene 2001, 20:1913-1922. PubMed Abstract | Publisher Full Text
Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS: AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer.
Science 1997, 277:965-968. PubMed Abstract | Publisher Full Text
Kosanke S, Edgerton SM, Moore D 2nd, Yang X, Mason T, Alvarez K, Jones L, Kim A, Thor AD: Mammary tumor heterogeneity in wt-ErbB-2 transgenic mice.
Comp Med 2004, 54:280-287. PubMed Abstract
Riese DJ 2nd, van Raaij TM, Plowman GD, Andrews GC, Stern DF: The cellular response to neuregulins is governed by complex interactions of the erbB receptor family.
Mol Cell Biol 1995, 15:5770-5776.
[Erratum Mol Cell Biol 1996, 16:735]PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Pinkas-Kramarski R, Soussan L, Waterman H, Levkowitz G, Alroy I, Klapper L, Lavi S, Seger R, Ratzkin BJ, Sela M, Yarden Y: Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.
EMBO J 1996, 15:2452-2467. PubMed Abstract
Sacco MG, Gribaldo L, Barbieri O, Turchi G, Zucchi I, Collotta A, Bagnasco L, Barone D, Montagna C, Villa A, et al.: Establishment and characterization of a new mammary adenocarcinoma cell line derived from MMTV neu transgenic mice.
Breast Cancer Res Treat 1998, 47:171-180. PubMed Abstract | Publisher Full Text
Campbell MJ, Wollish WS, Lobo M, Esserman LJ: Epithelial and fibroblast cell lines derived from a spontaneous mammary carcinoma in a MMTV/neu transgenic mouse.
In Vitro Cell Dev Biol Anim 2002, 38:326-333. PubMed Abstract | Publisher Full Text
Bargmann CI, Hung MC, Weinberg RA: Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185.
Cell 1986, 45:649-657. PubMed Abstract | Publisher Full Text
Bacus SS, Altomare DA, Lyass L, Chin DM, Farrell MP, Gurova K, Gudkov A, Testa JR: AKT2 is frequently upregulated in HER-2/neu-positive breast cancers and may contribute to tumor aggressiveness by enhancing cell survival.
Oncogene 2002, 21:3532-3540. PubMed Abstract | Publisher Full Text
Knuefermann C, Lu Y, Liu B, Jin W, Liang K, Wu L, Schmidt M, Mills GB, Mendelsohn J, Fan Z: HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells.
Oncogene 2003, 22:3205-3212. PubMed Abstract | Publisher Full Text
Lupu R, Cardillo M, Cho C, Harris L, Hijazi M, Perez C, Rosenberg K, Yang D, Tang C: The significance of heregulin in breast cancer tumor progression and drug resistance.
Breast Cancer Res Treat 1996, 38:57-66. PubMed Abstract | Publisher Full Text
DiGiovanna MP, Stern DF, Edgerton S, Whalen SG, Moore D 2nd, Thor AD: Relationship of epidermal growth factor receptor expression to ErbB2-signaling activity and prognosis in breast cancer patients.
J Clin Oncol 2005, 23:1152-1160. PubMed Abstract | Publisher Full Text
Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Dantis L, Sklarin NT, Seidman AD, Hudis CA, Moore J, et al.: Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer [comment].
J Clin Oncol 1996, 14:737-744. PubMed Abstract | Publisher Full Text
Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, Wolter JM, Paton V, Shak S, Lieberman G, Slamon DJ: Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease.
J Clin Oncol 1999, 17:2639-2648. PubMed Abstract | Publisher Full Text
Cardoso F, Piccart MJ, Durbecq V, Di Leo A: Resistance to trastuzumab: a necessary evil or a temporary challenge?
Clin Breast Cancer 2002, 3:247-257. PubMed Abstract | Publisher Full Text
Agus DB, Akita RW, Fox WD, Lewis GD, Higgins B, Pisacane PI, Lofgren JA, Tindell C, Evans DP, Maiese K, et al.: Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth [comment].
Cancer Cell 2002, 2:127-137. PubMed Abstract | Publisher Full Text

subset: commoncrawl


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I saw this today on the M40, driven by a dude with a duckface and sunnies sat on his head rather than nose.
He must be being ironic with this...
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Posted 04 October 2013 - 07:11 PM
I saw this today on the M40, driven by a dude with a duckface and sunnies sat on his head rather than nose.
He must be being ironic with this...
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Posted 07 October 2013 - 02:05 PM
lmfao, I would get a white marker and write "SERIOUSLY DONT" in between "you" and "like"
Posted 07 October 2013 - 03:28 PM
I saw this today on the M40, driven by a dude with a duckface and sunnies sat on his head rather than nose.
He must be being ironic with this...
How pathetic
Posted 23 October 2013 - 12:17 PM
Just to show how corsa drivers are inconsiderate c***s
Coming up to some lights in the left hand lane, and I needed to get into the right hand one so I could go straight on. Some idiot in a red corsa to the side of me, so I slowed down to let him go in front, then I would slip in behind him. What did he do? Slow right down. So I speeded up to get in front, he did the same. It was obvious he was doing it on purpose. A Fiesta driver would never do that.
Then he looked at me as if it was my fault.
If I'd had the power to catch him up I would have told him what a right !Removed! he is
Oh and I like how the spell check doesn't recognise corsa but it does fiesta
Posted 23 October 2013 - 02:05 PM
Thats because the digital world doesnt like the corsa, and its learned from reality that its the same point of view from everywhere else!
Posted 24 October 2013 - 01:34 PM
Terrifying Vauxhall Corsa Moment
This is the terrifying moment a lorry driver came face-to-face with a motorist driving the wrong way down a dual-carriageway.
The female driver of the Vauxhall Corsa is seen joining the busy A90 in Scotland via the exit slip road. She then swerves to avoid one lorry leaving the road and ends up heading straight into the firing line of the other juggernaut.
The Corsa driver then turns away from the 44-tonne truck and collides with the first lorry.
The driver of the second lorry loses control of his truck which rolls onto its side, spewing cement bags all over the road.
Amazingly, no one was injured when the incident took place last Tuesday outside Aberdeen, with the whole event captured on the lorry drivers Road hawk GPS camera system.
Grampian Police shut the road for six hours while highway workers cleared
(source http://www.lsg4drive....co.uk/news.htm)
Posted 24 October 2013 - 01:40 PM
Vauxhall corsa drivers round my way
Posted 24 October 2013 - 01:42 PM
Posted 24 October 2013 - 01:55 PM
That is a crazy story for the Corsa, glad no one was injured, but glad the corsa was its own casualty!
Posted 24 October 2013 - 02:04 PM
Posted 24 October 2013 - 02:16 PM
That is unfortunately the devastating part of this issue. I dread to think the cost of the cleanup operation and insurance claims after!
Posted 26 October 2013 - 05:04 PM
Posted 26 October 2013 - 05:11 PM
Posted 02 November 2013 - 12:58 AM
Posted 02 November 2013 - 01:09 AM
Well I've bashed Corsa's enough now and one has bashed me
Parked up dealing with drunks in clubland and some jerk delivering kebabs has decided to jam his crapmobile into the back of my patrol car.
Couple of scratches and a tiny crack in the bumper.
When he realised he'd hit a police vehicle he proceeded to go back into the takeaway and scarper out of the back door.
No licence or insurance, vehicle not registered to him either.
Pity his home address was in his satnav though.
One in custody, job a good'un.
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I love really good, authentic Chinese food- don't you? As with all restaurants that aren't committed to serving halal dishes, if you dine at one you'll have to ask a zillion questions about the ingredients to find out if there are any questionable or clearly non-halal ingredients. If you only eat dhabiha (slaughtered according to Islamic requirements) meats and their ...Read More »
Thai Beef Curry Stew
This recipe comes from Derya Singhsumalee, a former teacher colleague of mine who is such a fun and interesting person. She is very well-traveled, is of Turkish ethnicity and married to a man from a Thai Muslim family. In the school lunch room we used to talk quite a bit about cultural diversity and, of course, food. She was very familiar ...Read More »
Halal Korean Cuisine Made Delicious and Convenient
When I first came to Chicago over thirteen ago, the last type of cuisine I ever thought I would fall in love with is Korean, not for any other reason than the fact that I had no idea there was such a large population of Koreans living in and around the city. The first way I found out was by ...Read More »
Ramen Noodle Soup with Lamb, Bok Choy and Mushrooms
Here in the U.S., the Thanksgiving holiday and Black Friday shopping extravaganzas are both over. The big whole turkey meal and turkey leftovers are gone for most people and perhaps the mere thought of anything made with it like turkey soup, turkey hash, turkey burgers, turkey lasagna, and turkey sandwiches are making people seriously tired of the same ingredient on ...Read More »
Korean Slow Cooked Beef & Vegetables
Since we've moved into our house and begun construction, it's the simple things in life that have kind of gone straight out the window- like an organized closet, knowing where all my cooking spices are, and the luxury (because it truly is) of having a working stove hooked up to a gas line, right next to a working sink where ...Read More »
Malaysian Nasi Lemak with Sambal Ikan Bilis (Coconut Milk Rice with Sambal Anchovies)
At the very first Halal Food Tour by Sameer's Eats that took place in Irvine, CA I met some of the most amazing and humble people who I liked the instant I met them. The night before the tour, Sameer invited all of the volunteers and myself to dinner at MAS Islamic Chinese Restaurant in Anaheim. I had no idea ...Read More »
Icelandic Haddock in Lemongrass Basil Simmer Sauce
Unless you have an amazing Asian store nearby that sells fresh ingredients from the East, it's pretty hard to find things like lemongrass, real coconut and interesting chili peppers to cook with, unfortunate because when combined they make for such an exotic and flavorful combination like what's been prepared for us in one of the latest simmer sauces by Saffron Road ...Read More »
French Beans with Garlic, Cumin and Toasted Sesame
French green beans are one of my favorite side dishes. They're light, go well with most foods and make me feel content that I'm getting a healthy vegetable on my plate. I usually make them Italian style with garlic, tomatoes and olive oil, but this time I opted to satisfy my craving for something Asian. I had some lovely fresh ...Read More »
Cooking with Sadaf Syed of iCOVER: A Day in the Life of a Muslim-American COVERed Girl
I recently had the great pleasure of meeting with the very lovely, down-to-earth and talented young photographer, Sadaf Syed, a Chicago-based woman who literally wrote the book on how Muslim women in America wear the hijab (head covering). I sat down for coffee then later made no-cook appetizers with her and her kids to talk about the beautiful photodocumentary she ...Read More »
Rice Paper Rolls with Mango & Shrimp Stuffing and Orange-Lime Asian Sauce for Dipping
This recipe is unbelievably simple and there's absolutely no cooking involved- a perfect crowd pleaser for these hot summer days. Use the freshest ingredients, including oranges and limes, instead of juices or concentrates which really makes a difference in the resulting taste. Makes 10 rolls (20 servings) Rice Paper Roll Ingredients 2 ounces rice vermicelli noodles ½ lb. tiny cooked shrimp or ...Read More »


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DENNIS — Veterinarian Betsy Powers will offer a class for pet owners on what to do when their animals have emergencies.
The CPR and first aid training class is scheduled for 6 p.m. April 1 in the Dennis Police Department community room.
Powers said she will talk about ways to treat bee stings and insect bites, burns, dehydration, heat stroke, seizures and other ailments in animals. She also will show participants how to administrator CPR to their pets.
The class is sponsored by the Dennis Animal Control Department and Klassy Kanine Inc.
A donation of $15 per person is requested at the door, to go to the MSPCA Cape Cod Animal Care and Adoption Center or New England Society for Abandoned Animals. Please do not bring pets to the class.
Location, ST | website.com


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As of Friday, February 22, 2013
© Copyright 2015 Clayton News Daily
MORROW A proposed movie soundstage designed to bring stars to Clayton County could be the catalyst needed to get a long-dormant development off the ground.
The development, once known as Gateway Village, was originally intended to house a conference center, resort hotel and a golf course. More recently, it was to be home to a business class hotel and a hub for genealogy-related business because it is located across Hwy. 54 from the national and state archives.
But the newly renamed development will now have a balance of different aspects. County officials still plan to attract a business-class hotel and make genealogy tourism a key component of the development, but they hope to add hubs for business incubation and area filmmakers.
That diversification of University Station is what Clayton County Economic Development Director Grant Wainscott believes will make it a success, after years of planning with little physical progress at the site.
"You've got to diversify and prove you're not a one-pony town," Wainscott said.
A key to the revamping of University Station will be the former Ingles grocery store on Hwy. 54. The old storefront was separate from Gateway Village for years, but it was merged into the development in recent months.
Wainscott and his staff are planning to renovate it to be a center of filmmaking activity for Clayton County.
The old grocery store sat vacant for a decade, but served as a temporary soundstage last fall for some scenes involving Lindsay Lohan and Charlie Sheen from the upcoming Scary Movie 5. Plans call for much of the store to be renovated into a permanent 60,000-square foot film soundstage, with a screening room for independent filmmakers and business space for the county's film office.
Wainscott said film scouts and agents have expressed interest in getting office space in University Station.
"We're still learning what we need to do," Wainscott said. "We're taking advice from the state's film office, from trade associations and from production companies and filmmakers to see what they need from us.
"We need to be responsive to the industry and what the industry is looking for."
But, creating a hub for film productions at University Station may have a ripple effect that reaches beyond the development's borders. Wainscott explained that's because businesses that provide tools and services needed for film productions might be interested in relocating to the surrounding areas in Forest Park, Lake City and Morrow.
Those businesses could range from catering services, to hardware stores. Joel Lanken, the city manager for Lake City, said that is something that could benefit the three cities located on that stretch of Hwy. 54.
"It's going to impact this entire tri-city area by having a residual effect on the businesses around it," Lanken said.
Economic Development, Chamber of Commerce under one roof?
In addition to the film industry, talks are under way to bring what Clayton County chamber of Commerce President Yulonda Beauford called a "one-stop shop" for business development at University Station.
She and Wainscott said the talks are still in the early stages, but they confirmed discussions have taken place to house the economic development department, Chamber of Commerce, Clayton County Convention and Visitor's Bureau and Clayton State University's Small Business Development Center in one four-story building at the site.
Wainscott said the facility would be a place where prospective businesses could come to meet with county officials and "see Clayton County is serious about economic development."
Beauford said the chamber of commerce is committed to staying in its current location on Mount Zion Road — at least until the end of its lease, which is approximately a year away. However, she said the group is open to listening to county officials about making a move to University Station after the lease ends.
"We're very optimistic about the potential at University Station, and when you think about it, it just makes perfect sense to have all of us together in one location," Beauford said. "But, it's just in the very early discussion stages right now."
OscarKnight 2 years, 9 months ago
....."Built It, and They will Come"
......Just like The Southlake Mall, The Olde Towne Morrow, and the other closed businesses in Clayton County.
OscarKnight 2 years, 9 months ago
....."Built It, and They will Come"
......Just like The Southlake Mall, The Olde Towne Morrow, and the other closed businesses in Clayton County.
Robert 2 years, 9 months ago
Well, I have to say that it's creative thinking. I wish them, and us, the best of luck.
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More like this story
- BOC okays Hollywood film to shoot in Clayton ( August 15, 2012 )
- Three cities unite ( October 11, 2013 )
- Three Clayton cities announce join development effort ( October 11, 2013 )
- 'Scary Movie' marks county sound stage's movie debut ( April 12, 2013 )
- Clayton County Economic Development Director Grant Wainscott leaving to take Metro Atlanta Chamber of Commerce position ( December 30, 2014 )


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He was originally charged with murder, but now a grand jury has indicted Timothy Geneve for manslaughter.
The 24-year old is accused of driving drunk and hitting a man as he walked out the door of his sister's Lexington home. The accident happened on Patchen Drive in August.
Police tell us Geneve lost control of his pick-up truck, side swiped several parked cars and took out some trees. He then drove into a yard where he hit 29-year old Charlie Johnson.
Johnson was killed from the impact. Johnson was an attorney in Bourbon County.
Geneve will be arraigned in court next Monday.
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Coffman speaking to
Civil War Roundtable
Edward Coffman, Ph.D., will speak at a meeting of the Frankfort Civil War Roundtable on the topic, "Thomas Hines, Confederate Spy and Special Operations Officer" at 6:30 p.m. Monday at the Paul Sawyier Library, 319 Wapping St. The presentation is free and open to the public. For more information contact Charles Bogart at 502-227-2436.
Garden club council
plans 'meet and greet'
The Franklin County Council of Garden Clubs will host a "Meet & Greet" event from 5-7 p.m. Tuesday in the Community Room of the Paul Sawyier Public Library.
The event is to celebrate National Garden Club Week and will be an opportunity to meet Adam Leonberger, the new Franklin County Extension horticulture agent, view educational exhibits and participate in a hands-on youth activity. Refreshments will be served.
Grand's film series begins Friday
The Grand Theatre's Classic Film Series begins at 7:30 p.m. Friday, with the romantic comedy "When Harry Met Sally."
Throughout the summer, seven movies will be shown, selected from a survey conducted by the Grand. A season ticket covering admission to all seven movies costs $30, a savings of nearly 30 percent over the individual ticket price of $6.
The remaining movies in the series are: "Pulp Fiction," June 20; "Blazing Saddles," July 4; "O Brother, Where Art Thou," July 11; "Four Weddings and a Funeral," July 25; "Fast Times at Ridgemont High," Aug. 8; and "Animal House," Aug. 22.
Season tickets are available at the Grand Theatre ticket office, 312 W. Main St., weekdays between 10 a.m. and 3 p.m. or by calling 502-352-7469. Individual tickets for "When Harry Met Sally" will be available beginning Monday. The same schedule will hold true for the remaining movies, with tickets being sold starting the Monday prior to the showing. Tickets will also be sold at the door.
Civil War event set for Ft. Hill
"Civil War Sesquicentennial, The Confederate Assault on Frankfort" is from 10 a.m.-5 p.m. Saturday at Leslie Morris Park on Fort Hill, 400 Clifton Ave.
Admission is free and the event includes guided tours, living history camp, artillery salute, talks about the Civil War in Frankfort and more. For more information call 502-696-0607 or visit www.frankfortparksandrec.com
Band festival this week in Danville
DANVILLE — The 25th Great American Band Festival is Thursday through next Sunday. Nearly all events of this salute to brass band music are free and open to the public. For a complete schedule visit www.gabbf.org.
Pioneer Playhouse
set to open season
DANVILLE — Pioneer Playhouse opens its 2014 season Friday with "Whodunnit, Darling?" The play continues through June 21. For complete information about the season and/or to reserve tickets, visit pioneerplayhouse.com or call 866-597-5297 or 859-236-2747.
Shrine Club plans
pancake breakfast
The Capital City Shrine Club will host a pancake breakfast from 7-10 a.m. Saturday at VFW Post 4075, 104 E. Second St. The cost is $5 for all-you-can-eat sausage, eggs, pancakes, biscuits and gravy, orange juice, coffee and milk. All proceeds will benefit Shrine Club operations.
'Steel Magnolias' opens Friday
SHELBYVILLE — Tickets are on sale now for Shelby County Community Theatre's production of "Steel Magnolias," by Robert Harling. Performances are June 6-7, 13-14 at 7:30 p.m., and June 8 and 15 at 2:30 p.m. All performances are at the theatre, 801 Main St.
Tickets are $15 for adults, $12 for seniors, and $10 for students. Tickets to the opening night dinner June 6 are an additional $20 each. For reservations, call 502-633-0242. Rick Reinle directs the play.
'Living in History'
June 14
The Garden Club of Frankfort and the Frankfort/Franklin County Tourism Commission are sponsoring the eighth annual Living in History Home and Garden Tour June 14 from 10 a.m.-5 p.m.
Also included in the day's events are a luncheon and boutique and antique appraisal fair at the Frankfort Country Club, 101 Duntreath. The luncheon is from 11 a.m.-2 p.m. and the boutique and antique appraisal from 10-3.
The proceeds from these three events go to help fund Frankfort beautification projects such as the Wilkinson Boulevard median improvements and the Frankfort Cemetery Chapel.
Tickets for the tour of homes and gardens are $15 and the lunch is $15. They are available at any of the homes, the Country Club, or at the Frankfort Tourism Center, 100 Capital Ave.
VFW Auxiliary sets Saturday yard sale
The Ladies Auxiliary VFW 4075 will have a yard sale beginning at 8 a.m. Saturday in the parking lot of the Board of Tourism, 100 Capital Ave. The event will include a bake sale and all proceeds from the yard and bake sale will be given for cancer research.
In case of rain the event will be cancelled. For more information call Margaret Logan at 502-229-1762.
Club meetings
Capital City Toastmasters Club, noon-1 p.m., lower level of John Watts Building, 330 West Broadway. For more information or to join, contact Jeff Barr, 502-777-9555.
Silent Workers Circle of The King's Daughter, noon luncheon at Frankfort Regional Medical Center.
Frankfort Optimist Club, noon, Office Pub and Deli. Program: Debbie Bell of the Franklin County Health Department will talk about the "Longest Day of Play."
Frankfort Rotary Club, 11:45 a.m., Capital Plaza Hotel.


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LANCASTER — A Pleasantville couple accused of locking their 4-year-old child in a bed after deputies discovered the childs bed wrapped in steel mesh and equipped with a sliding latch were sentenced Monday to 180 days in the county jail.
Fairfield County Common Pleas Judge Richard Berens first imposed 78-month prison sentences before suspending them in lieu of five years community control and the 180 days in jail.
The jail sentences will be monitored and possibly modified, Berens said. Both were issued jail time credits.
In August, Russell D. Terry, 34, and Jamie M. Curnell, 32, each pleaded guilty to three counts of child endangering, third-degree felonies. The couple originally was charged with 10 counts each of abduction, all third-degree felonies; 10 counts each of kidnapping, all first-degree felonies; and 10 counts each of endangering children, all third-degree felonies.
The remaining charges were dismissed as a part of a negotiated plea agreement.
The enclosure was discovered July 10, 2012, after Fairfield County Sheriffs Office deputies responded to a home along the 8000 block of Lancaster-Thornville Road for a domestic violence call.
Deputies said the child was kept locked in the bottom bed of a bunk bed because she would get up during the night. Fairfield County Sheriff Dave Phalen said the parents told deputies they had kept the child in the enclosure during the night for the past three months.
Terrys attorney, Aaron Conrad, said the couple have undergone counseling and parent education classes. Conrad said the couple constructed the enclosure to prevent their child from wandering.
It was done for the safety of the child, he said.
Currently, the couple are allowed supervised visits with their four children. The children are living with family members.
Both Terry and Russell testified during the contested sentencing hearing Monday. Terry said he made a poor decision.
I realize I made a poor decision. I was trying to accomplish protecting my daughter, he said.
Curnells attorney, Kristen Burkett, said her client was happy with the decision and is looking forward to being reunited with her children.
Fairfield County Prosecutor Gregg Marx said he was confident that Judge Berens made a fairly based decision.
He said the Child Protective Services is working on some type of reunification plan for the family.


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I find it interesting that presidential candidate Ron Paul is introducing legislation (H.R> 1897) to abolish federal gun restrictions in National Parks.Link here: http://www.ronpaul2008.com/issues/second-amendment/Without getting into a tit-for-tat discussion over broad spectrum gun ownership rights, I was curious as to what others think about this proposal?
Firearms aren't allowed in National Parks for the general public, with an incredibly small number of exceptions, and this won't change. This is a non-issue, not to mention that Ron Paul is a non-entity in the primary.
Edited by Bearpaw on 02/05/2008 19:30:18 MST.
I respectfully disagree that this is a non-issue. Explain how this is a non-issue with 47 Senators signing a letter urging the Bush administration to lift regulations?http://ap.google.com/article/ALeqM5irWgvJzXz3UFknRjrJgjierKwnFwD8U38K7O0Ron Paul will not win the presidency, but that is not the same as saying his run will not have an impact. He's resounded with a segment of voters, who, in a tight contest, will be courted by candidates.Personally, I have no attachment to the man or his campaign. I am concerned that this rule could be changed.
Were the ruling to apply only to the back-country, I could live with it. I certainly don't see much need for it though; and don't think it is likely to happen. But the thought of swarms of armed front-country tourists in the Grand Canyon or Yosemite sure gives me the willies.
In all fairness, I am much more troubled by the urging of 47 Senators than I am by the platform of Ron Paul. Shawn is correct, Paul isn't gong to win the presidency and is a fringe candidate. It's just that park service is a highly political body, at the behest of the Senate for funding.Introducing guns into the front-country, as Charles suggested, is a bad idea. Accidents will happen. Also, where does someone place their gun in a campground? Their car? Their tent? And inevitably, someone will say they "felt threatened" by some animal and shoot it. Now, this is where it gets tricky. How does one judge the extent of the threat? What happens if they have no experience with said animal or have no idea of what constitutes a threatening animal. I remember the first time I saw a bear while hiking: it was a thrill but at the same time, kind of scary. But I had read up on bears enough to know that if I gave it space, it was likely not a threat. In some parks there are typically enough people around that taking a shot in the front-country places others at risk.Overwhelmingly, the greatest threat in any wilderness situation comes from the very person who put us there: yourself. Poor judgment (I'm guilty), getting over your head (guilty again), unwillingness to change the plan despite the obvious danger imposed by conditions (guilty)and lack of preparedness (guilty) are responsible for the overwhelming majority of incidents. Certainly bad things do happen in the National Parks. Things that had a gun been available may have ended differently and a tragedy averted. However, introducing guns would, in my estimation, would introduce new and unacceptable risks.
Edited by dirk9827 on 02/06/2008 11:06:26 MST.
This looks like it is going to pass. See below.All gun free zones do is keep the legal citizen from protecting themselves. The criminals don't follow the rules. Sure it makes more sense in the back country, but we can not choose when/where violence will occur.What legitimate reason is there to limit law abiding citizens from their right to defend themselves in a public place? It's not like you're bringing a gun to Riker's or something. Oh wait... No crimes ever happen in federal parks or buildings. I forgot.Look at the safe zones on college campus's. They are well meaning but it is a fantasy to think they are effective.Interior Department To Propose New Rules Regarding Right-to-Carry In National ParksIt would be great if all guns disappeared overnight, but that is not going to happen. Usually the only way to stop violent wacks is with overwhelming force.Friday, February 22, 2008After nearly five years of effort by NRA-ILA, the U.S. Department of the Interior has finally responded to the many requests for a change in its policy on carrying and transporting firearms in national parks and wildlife refuges. In a letter hand-delivered today to the U.S. Senators who wrote him asking for this policy change, Interior Secretary Dirk Kempthorne has directed Lyle Laverty, Assistant Secretary for Fish, Game and Parks, to "develop and propose for public comment by April 30, Federal regulations that will update firearms policies on these lands to reflect existing Federal laws (such as those prohibiting weapons in Federal buildings) and the laws by which the host States govern transporting and carrying of firearms on their analogous public lands.""This announcement represents an important and overdue change to now-outdated federal regulations imposed 25 years ago. Once this new federal regulation is proposed, published for public comment and then finalized, law-abiding citizens will have their right to self-defense restored and be able to legally carry and transport firearms through national parks and wildlife refuges for a variety of lawful purposes -- just like in national forests and BLM lands," said NRA-ILA Executive Director Chris W. Cox.The move for regulatory change by the Administration will restore the rights of law-abiding gun owners who wish to transport and carry firearms for lawful purposes in most national park lands and wildlife refuges and will make the laws consistent with state law where these lands are located. Fifty-one U.S. Senators from both parties sent a letter to the Department of Interior late last year supporting the move to make state firearm laws applicable to national park lands and wildlife refuges. (Click here to read the letters: Letter 1, Letter 2)"These changes will respect the Second Amendment rights of honest citizens, and we look forward to the issuance of a final rule this year," concluded Cox.To read the NRA Press Release on this development, please click here.For more information about this issue and the efforts behind it, please visit: www.nraila.org/Legislation/Read.aspx?ID=3529 .
"Without getting into a tit-for-tat discussion over broad spectrum gun ownership rights, I was curious as to what others think about this proposal?"My only question is how you expect folks to share their thoughts about this GUN proposal -- without getting into a tit-for-tat discussion??? :)
You do it with lots of smileys ;?)
I found this interesting...Did not even realize hunting of any kind was allowed!! I wonder if this is hunting with rifles, shotguns, or ? guns?!! Perhaps it is limited to bows and arrows...WOW and here I thought I was safe in my little U.L. Single walled tent LOLhttp://www.blm.gov/nhp/news/regulatory/6300etal/6300f.html#6302.20Another comment asserted that hunting is a competitive event that BLM should prohibit. In general, hunting is not a competitive sport, but the regulations do prohibit organized competitive hunting events. The regulations treat orienteering in the same way--prohibiting it only if competitive. One comment suggested that commercial hunting be prohibited. We assume the comment refers to commercial guiding and outfitting for hunters. Commercial outfitters often serve as guides for hunters, and this activity is considered among the recreational purposes contemplated in the Wilderness Act.
I think the person painting the picture of hundreds of tourists armed to the teeth is really unrealistic. There are at least a dozen states that have unregulated open carry laws, that means ANYONE who is not a felon can legally open carry almost anywhere in public. Arizona is probably the most open and tolerant of open carry laws. I lived there for 4 years. I would see someone with a gun about 3-4 times a month. Thats it. Out of the thousand of people I passed on the streets, malls, grocery stores, and restaurants, a very very small part of the population actually chose to open carry a gun. So why would we think that people would show up by the thousands armed in national parks if the ban was lifted? It is simply just not gonna happen that way.
Disclaimer: I am a strict-constructionist 2nd Amendment guy, so I believe that the phrase "the people" in the Second Amendment means "every individual," just like it does everywhere else in the Constitution. (And the Supreme Court agrees with me.)However, I will say that I have very little confidence in the judgment and gun-handling skills of the vast majority of Americans, and the thought of running into one armed in the back-country chills my spine. Perhaps I could distract him by tossing a microwaveable burrito into the underbrush and running the other way.On the other hand, I can think of precisely (only) one good reason to carry a firearm on a hiking trip in North America: Bears. And even that is a stretch. 99.9999999% of bear encounters are non-threatening, I know. That said, if I run into bear number 0.00000001 I want to know that I can take him with me.I was recently planning an Alaskan trip and was very uneasy about not being able to carry a rifle into a national park. I know that Alaskan natives are not shy about carrying firearms in bear country. Bear spray is good for morale, but discounting individual testaments when one looks at the well-constructed studies no one is really sure how well it works. At all. Rarely, some bears are even attracted to the smell. The Alaskan, B.C., and Yukon natives I talked to seemed to think bear spray is charming, but not very practical.So I changed my trip to one where I could carry my rifle, even though I had to jump through hoops to do it (because part of the trip was in Canada). Never did see a bear. But that's the thing about guns: You'll probably never need it, but if you do you'll need it VERY BADLY.But back-country Alaska is a special circumstance, isn't it? As I said, I'm a 2nd Amendment guy, but from an environmental viewpoint I just don't trust most beer-swilling Americans. Thus I also have worries about some camper-cowboys gunning down elk "that were about to charge." Honestly, how can we expect an urbane retired New Yorker who has barely ever left the city to make informed judgments about when an elk constitutes a threat? There are too many people in many (not all) of our national parks as it is, dropping litter and stomping on the undergrowth, and I really don't want those teeming masses armed. They have driven me into the back-country already.I admit I'm being elitist, here, in that I have no objections to ME going armed. I know what I'm doing. (Seriously.)Anyway, wouldn't the people who really need a gun in a national park probably be those back-country campers, not the drones there to see Old Faithful? I haven't heard of many bear maulings in the vicinity of Old Faithful. The tourists there are all either in the lodge or camped in massively populated campgrounds. Perhaps a back-country pass could act as a gun permit as well? I think that would be ideal.Rats, I'm getting really conflicted now, because I just remembered some ruminations of mine about an Appalachian Trail hike. There have been several well-publicized murders on the trail (rare though they are). AT through-hikers make excellent targets because they can't carry a gun for self-defense, since they travel through several areas where that is illegal. I'm not a big guy, so I'm pretty certain that if I ever through-hiked the AT I'd commit at least one felony, because I wouldn't do it without a derringer (American Derringer Co. "Lightweight", 7.5oz not counting ammunition). The most dangerous animal on Earth is man, after all.
Edited by acrosome on 04/04/2008 03:02:00 MDT.
How could anyone possibly consider this a non-issue. Anything even REMOTELY related to your constitutional rights should be taken with such seriousness you should be willing to lay down your life for it. Whether or not you would use this right, you must ask yourself "Will this law unnecessarily infringe on the rights of others?" In this case, yes it would. Carrying a gun is a personal choice. I have friends who don't care to carry, but they care when they hear their right to choose could be taken away.
Guns are already in the parks. It's just not the norm.http://www.nps.gov/biso/planyourvisit/hunting.htm
I agree that anything in the Constitution is a big issue. Personally, I'm not a gun owner. I've never carried a gun, I've never shot a gun, and I know little to nothing about gun types, etc. If you wanna carry a gun, I believe that's your choice, and I support individual choices of freedom. Strange how that works...
Christopher,True, but the website you linked admits that "Big South Fork is one of only a FEW National Park Service units legislated to allow hunting." (My emphasis added.) And although administered by NPS, I don't think it is technically a national park. I thought it was a "National River and Recreation Unit," whatever that means. And you can hunt in national forests, too.Also, while the debate so far has included hunting use, most of it seems to concern concealed carry for personal defense from animals (especially humans). Does Big South Fork allow concealed carry? I'll bet it doesn't.
Edited by acrosome on 04/04/2008 03:35:01 MDT.
Well, it seems that the saga of guns in National Parks continues....News story from Associated Press here --- http://tinyurl.com/48qfbgQuoted from story ----WASHINGTON (AP) — Interior Secretary Dirk Kempthorne proposed new regulations Wednesday that would allow people to carry a concealed weapon in some national parks and wildlife refuges.The new rules would allow someone to carry a loaded weapon in a park or wildlife refuge only if the person has a permit for a concealed weapon and the state where the park or refuge is located allows guns in parks, Kempthorne said.----- End of Quote from StoryLeave it to the Bush Administration to not only sponsor an unnecessary rule, but to implement in a way that is completely flawed.I cannot understand the rationale to place National Parks under the purview of state gun regulations. Regardless of your views toward guns, the fact is that National Parks are among the safest places in the United States. The "personal protection" angle is, at best, a tepid argument for the inclusion of firearms.Leaving it up to states will further burden and complicate the rules and regulations faced by our underpaid park personnel. And frankly, I can't imagine a more delightful situation than dealing with some yahoo who pulls out his handgun the minute a bear walks through the campground.
I propose a Guns-for-Pepper-Spray exchange program, to be meted out at the National Park entrance stations. Official slogan of the program, "Give a hoot, spray don't shoot."When it comes to upholding the auspices of our increasingly antiquated national heritage, it seems the constitutional right to bare arms trumps our god-given right to clear skies, clean rivers, and uncluttered horizons. From ego and fear, we posture a strong defense, with increasingly little left to defend.
Amen Brett. I'm a gun owner, and possess them for sport (target shooting) and defense. That being said, there are FAR bigger issues to be tackled on the public land management front than firearms regulations. The greatest dangers to most recreational users stem from abuse, neglect, and the budgetary shortfalls that create them. I'd like to see 47 members of our Senate send a letter to the Secretary of the Interior expressing outrage at the assualts to our public lands over the eight years of the Bush administration. Nature needs to be defended from humans far more than humans need defending from nature.
This would be a good first step into incorporating the second amendment under the equal protection clause of the 14th amendment. States and the federal government recognize my state issued drivers license and marriage license, but not a gun carry license. Even though that license entails a federal FBI background check and fingerprinting. The way I see it this has more to do with the concept of equality under the law more than any descent into pragmatism or paranoid strawmen whipping boys. I mean, it's not as if they have metal detectors and x-ray inspections at the park entrances to keep the dishonest and the predatory from carrying all manner of intimidating and concealable weapons. Forcing permit holders to keep their sidearms disassembled and locked in a car at the trailhead only invites theft. It also sets up our federal servants as our masters. The traditional name for the disarmed class is slave. Either we are equal or we are not.
You know those crazy people that everyone is worried about carrying in NPs and not knowing how to be responsible and shooting things "willy-nilly", well, they ALREADY carry in National Parks illegally. We call them criminals. By allowing law-abiding citizens to carry in NPs isn't going to decrease your safety in the front-country. Also remember that BLM and National Forests allow guns. Law-abiding citizens carry there all the time. Why don't we hear much about these crazy people shooting up wildlife (apart from legal hunting)? Do you limit your outdoor experience to front-country National Park visits because it is the only "safe" gun free area with only criminals packing? Law-abiding citizens are responsible gun owners and should be allowed to lawfully carry on National Parks.
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