bluuebunny/crossref_metadata_2025 huggingface dataset:
Anabolic signaling and protein synthesis in human skeletal muscle after dynamic shortening or lengthening exercise
Authors: Cuthbertson Daniel J. , Babraj John , Smith Kenneth , Wilkes Emilie , Fedele Mark J. , Esser Karyn , Rennie Michael
Published: November 2005

We hypothesized a differential activation of the anabolic signaling proteins protein kinase B (PKB) and p70 S6 kinase (p70S6K) and subsequent differential stimulation of human muscle protein synthesis (MPS) after dynamic shortening or lengthening exercise. Eight healthy men [25 ± 5 yr, BMI 26 ± 3 kg/m−2 (means ± SD)] were studied before and after 12 min of repeated stepping up to knee height, and down again, while carrying 25% of their body weight, i.e., shortening exercise with the “up” leg and lengthening exercise with contralateral “down” leg. Quadriceps biopsies were taken before and 3, 6, and 24 h after exercise. After exercise, over 2 h before the biopsies, the subjects ingested 500 ml of water containing 45 g of essential amino acids and 135 g of sucrose. Rates of muscle protein synthesis were determined via incorporation over time of [1-13C]leucine (≤6 h after exercise) or [1-13C]valine (21–24 h after exercise) and phosphorylation of signaling proteins by Western analysis. PKB and p70S6K phosphorylation increased ∼3-fold after 3 h and remained elevated at 6 and 24 h. After exercise, rates of myofibrillar and sarcoplasmic protein synthesis were unchanged over the period including exercise and 3 h of recovery but had increased significantly at 6 (∼3.0- and 2.4-fold, respectively) and 24 h (∼3.2- and 2.0-fold, respectively), independently of the mode of exercise. Short-term dynamic exercise in either shortening or lengthening mode increases MPS at least as much as resistance exercise and is associated with long-term activation of PKB and p70S6K.
Reduced autoregulatory effectiveness in adenosine 1 receptor-deficient mice
Authors: Hashimoto S. , Huang Y. , Briggs J. , Schnermann J.
Published: November 2005

Adjustments of renal vascular resistance in response to changes in blood pressure are mediated by an interplay between the myocyte-inherent myogenic and the kidney-specific tubuloglomerular feedback (TGF) mechanisms. Using mice with deletion of the A1adenosine receptor (A1AR) gene, we tested the prediction that the absence of TGF, previously established to result from A1AR deficiency, is associated with a reduction in the efficiency of autoregulation. In anesthetized wild-type (A1AR+/+) and A1AR-deficient mice (A1AR−/−), glomerular filtration rate (GFR) and renal blood flow (RBF) were determined before and after reducing renal perfusion pressure through a suprarenal aortic clamp. In response to a blood pressure reduction by 15.9 ± 1.34 mmHg in A1AR−/− ( n = 9) and by 14.2 ± 0.9 mmHg in A1AR+/+ mice ( n = 8; P = 0.31), GFR fell by 187.9 ± 37 μl/min and by 72.3 ± 10 μl/min in A1AR−/− and A1AR+/+ mice, respectively ( P = 0.013). Similarly, with pressure reductions of 14.8 ± 1.1 and 13.3 ± 1.5 mmHg in A1AR−/− ( n = 9) and wild-type mice ( n = 8), respectively ( P = 0.43), RBF fell by 0.17 ± 0.02 ml/min in A1AR−/− mice and by only 0.08 ± 0.02 ml/min in wild-type animals ( P = 0.0039). Autoregulatory indexes for both GFR and RBF were significantly higher in A1AR−/− compared with A1AR+/+ mice, indicating reduced regulatory responsiveness in the knockout animals. We conclude that autoregulation of renal vascular resistance is less complete in A1AR-deficient mice, an effect that is presumably related to absence of TGF regulation in these animals.
Coordinate control of prostaglandin E2synthesis and uptake by hyperosmolarity in renal medullary interstitial cells
Authors: Pucci Michael L. , Endo Shinichi , Nomura Teruhisa , Lu Run , Khine Cho , Chan Brenda S. , Bao Yi , Schuster Victor L.
Published: November 2005

During water deprivation, prostaglandin E2(PGE2), formed by renal medullary interstitial cells (RMICs), feedback inhibits the actions of antidiuretic hormone. Interstitial PGE2concentrations represent the net of both PGE2synthesis by cyclooxygenase (COX) and PGE2uptake by carriers such as PGT. We used cultured RMICs to examine the effects of hyperosmolarity on both PG synthesis and PG uptake in the same RMIC. RMICs expressed endogenous PGT as assessed by mRNA and immunoblotting. RMICs rapidly took up [3H]PGE2to a level 5- to 10-fold above background and with a characteristic time-dependent “overshoot.” Inhibitory constants ( Ki) for various PGs and PGT inhibitors were similar between RMICs and the cloned rat PGT. Increasing extracellular hyperosmolarity to the range of 335–485 mosM increased the net release of PGE2by RMICs, an effect that was concentration dependent, maximal by 24 h, reversible, and associated with increased expression of COX-2. Over the same time period, there was decreased cell-surface activity of PGT due to internalization of the transporter. With continued exposure to hyperosmolarity over 7–10 days, PGE2release remained elevated, COX-2 returned to baseline, and PGT-mediated uptake became markedly reduced. Our findings suggest that hyperosmolarity induces coordinated changes in COX-2-mediated PGE2synthesis and PGT-mediated PGE2uptake in RMICs.
Role of Anticipation in Schizophrenia-Related Pursuit Initiation Deficits
Authors: Avila Matthew T. , Hong L. Elliot , Moates Amanda , Turano Kathleen A. , Thaker Gunvant K.
Published: November 2005

Schizophrenia patients exhibit several smooth pursuit abnormalities including poor pursuit initiation. Velocity discrimination is also impaired and is correlated with pursuit initiation performance—suggesting that pursuit deficits are related to impairments in processing velocity information. Studies suggest that pursuit initiation is influenced by prior target motion information and/or expectations and that this is likely caused by expectation-based changes in the perceptual inputs to the pursuit system. We examined whether poor pursuit initiation in schizophrenia results from inaccurate encoding of immediate velocity signals, or whether these deficits reflect a failure to use prior target motion information to “optimize” the response. Twenty-eight patients and 24 controls performed an adapted version of a “remembered pursuit task.” Trials consisted of a series of target motions, the first of which occurred unexpectedly, followed by four to seven identical targets each preceded by an auditory cue and a “catch target” in which a cue was given followed by target extinction. Initiation eye velocity in response to unexpected, first targets was similar in the patient and control groups. In contrast, patients showed lower eye velocity in response to repeated, cued targets compared with controls. Patients also showed reduced eye velocity in response to catch targets. Reduction in pursuit latency across repeated targets was less robust in patients. Results suggest that processing of immediate velocity information is unaffected in schizophrenia and that pursuit initiation deficits reflect an inability to accurately generate, store, and/or access “remembered” velocity signals.
Glucocorticoid regulation of genes in the amiloride-sensitive sodium transport pathway by semicircular canal duct epithelium of neonatal rat
Authors: Pondugula Satyanarayana R. , Raveendran Nithya N. , Ergonul Zuhal , Deng Youping , Chen Jun , Sanneman Joel D. , Palmer Lawrence G. , Marcus Daniel C.
Published: November 2005

The lumen of the inner ear has an unusually low concentration of endolymphatic Na+, which is important for transduction processes. We have recently shown that glucocorticoid receptors (GR) stimulate absorption of Na+by semicircular canal duct (SCCD) epithelia. In the present study, we sought to determine the presence of genes involved in the control of the amiloride-sensitive Na+transport pathway in rat SCCD epithelia and whether their level of expression was regulated by glucocorticoids using quantitative real-time RT-PCR. Transcripts were present for α-, β-, and γ-subunits of the epithelial sodium channel (ENaC); the α1-, α3-, β1-, and β3-isoforms of Na+-K+-ATPase; inwardly rectifying potassium channels [IC50of short circuit current ( Isc) for Ba2+: 210 μM] Kir2.1, Kir2.2, Kir2.3, Kir2.4, Kir3.1, Kir3.3, Kir4.1, Kir4.2, Kir5.1, and Kir7.1; sulfonyl urea receptor 1 (SUR1); GR; mineralocorticoid receptor (MR); 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2; serum- and glucocorticoid-regulated kinase 1 (Sgk1); and neural precursor cell-expressed developmentally downregulated 4-2 (Nedd4-2). On the other hand, transcripts for the α4-subunit of Na+-K+-ATPase, Kir1.1, Kir3.2, Kir3.4, Kir6.1, Kir6.2, and SUR2 were found to be absent, and Iscwas not inhibited by glibenclamide. Dexamethasone (100 nM for 24 h) not only upregulated the transcript expression of α-ENaC (∼4-fold), β2-subunit (∼2-fold) and β3-subunit (∼8-fold) of Na+-K+-ATPase, Kir2.1 (∼5-fold), Kir2.2 (∼9-fold), Kir2.4 (∼3-fold), Kir3.1 (∼ 3- fold), Kir3.3 (∼2-fold), Kir4.2 (∼3-fold ), Kir7.1 (∼2-fold), Sgk1 (∼4-fold), and Nedd4-2 (∼2-fold) but also downregulated GR (∼3-fold) and 11β-HSD1 (∼2-fold). Expression of GR and 11β-HSD1 was higher than MR and 11β-HSD2 in the absence of dexamethasone. Dexamethasone altered transcript expression levels (α-ENaC and Sgk1) by activation of GR but not MR. Proteins were present for the α-, β-, and γ-subunits of ENaC and Sgk1, and expression of α- and γ-ENaC was upregulated by dexamethasone. These findings are consistent with the genomic stimulation by glucocorticoids of Na+absorption by SCCD and provide an understanding of the therapeutic action of glucocorticoids in the treatment of Meniere's disease.
Proliferation of Human Neuroblastomas Mediated by the Epidermal Growth Factor Receptor
Authors: Ho Ruth , Minturn Jane E. , Hishiki Tomoro , Zhao Huaqing , Wang Qun , Cnaan Avital , Maris John , Evans Audrey E. , Brodeur Garrett M.
Published: November 2005

Neuroblastoma is a common solid tumor of childhood that is derived from the neural crest. Expression of epidermal growth factor (EGF) receptors (EGFRs) has been associated with enhanced cell growth and aggressive behavior in other tumors. Here, we examined the expression profile of EGFRs in neuroblastoma cell lines and primary tumors. We found that all 13 neuroblastoma cell lines examined expressed EGFR1 (HER1), most at readily detectable levels. Low levels of other human EGFR family receptors were also detected in almost all cell lines. All primary tumors examined expressed readily detectable levels of HER1 and HER3 and lower levels of HER2 and HER4. EGF had a significant effect on the proliferation of neuroblastoma cell lines in vitro. EGF treatment (100 ng/mL) of the cell lines SY5Y and NLF significantly increased cell number (P < 0.01). EGF stimulated more cells to enter S and G2-M phase, as suggested by flow cytometry, indicating that EGF increases cell number by increasing proliferation, with no appreciable change in apoptosis. EGF exposure resulted in receptor autophosphorylation and activation of both the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. Exposure to 0.5 μmol/L ZD1839, a HER1-specific inhibitor, caused a 40% to 50% reduction in the number of SY5Y and NLF cells grown in medium containing 10% fetal bovine serum (P < 0.01). Even at 0.01 μmol/L, ZD1839 inhibited autophosphorylation of HER1 by EGF. At 0.1 μmol/L, it also blocked phosphorylation of AKT, but not MAPK, in NLF cells. Additional studies showed that the PI3K/AKT–specific inhibitor LY294002 had a more profound effect than the MAPK-specific inhibitor U0126 in blocking EGF-induced cell proliferation. This suggests that the PI3K/AKT pathway is the main signaling pathway responsible for the proliferation effects of EGF in neuroblastomas. Our results also indicate that ZD1839 is a potent inhibitor of neuroblastoma cell proliferation; therefore, it may be a useful, biologically based therapeutic agent for these tumors.
Cyclin E Overexpression Obstructs Infiltrative Behavior in Breast Cancer: A Novel Role Reflected in the Growth Pattern of Medullary Breast Cancers
Authors: Berglund Pontus , Stighall Maria , Jirström Karin , Borgquist Signe , Sjölander Anita , Hedenfalk Ingrid , Landberg Göran
Published: November 2005

Cell cycle deregulation is a prerequisite in tumor development and overexpression of cyclin E, a major G1-S regulator, is often observed in breast cancer and is further linked to poor prognosis. By overexpressing cyclin E in a retinoblastoma-inactivated breast cancer cell line, we induced significant alterations in the expression of genes associated with proliferation and cell adhesion. Rearrangements of the actin cytoskeleton in addition to increased adhesive properties, decreased motility, and invasive potential in functional assays, indicated an overall abrogated mobility. Consistent in vivo findings were obtained upon investigation of 985 primary breast cancers, where cyclin E–high tumors predominantly (67%) displayed a low infiltrative, pushing growth pattern. Furthermore, medullary breast cancers, a subtype defined by its pushing, delimited growth, exhibited a remarkable frequency of cyclin E deregulation (87%) compared with other histologic subtypes (5-20%). Taken together, our results suggest the novel role of cyclin E in modeling infiltrative behavior. The consequences of cyclin E overexpression in breast cancer seems to be multiple, including effects on proliferation as well as growth patterns, a scenario that is indeed observed in the archetype of cyclin E–overexpressing medullary breast cancers.
Pericytes and Endothelial Precursor Cells: Cellular Interactions and Contributions to Malignancy
Authors: Bagley Rebecca G. , Weber William , Rouleau Cecile , Teicher Beverly A.
Published: November 2005

Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function–associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cell-based models for tumor vascular development and selection of agents that may provide therapeutic benefit.
Maternal Embryonic Leucine Zipper Kinase/Murine Protein Serine-Threonine Kinase 38 Is a Promising Therapeutic Target for Multiple Cancers
Authors: Gray Daniel , Jubb Adrian M. , Hogue Deborah , Dowd Patrick , Kljavin Noelyn , Yi Sothy , Bai Wei , Frantz Gretchen , Zhang Zemin , Koeppen Hartmut , de Sauvage Frederic J. , Davis David P.
Published: November 2005

To identify genes that could serve as targets for novel cancer therapeutics, we used a bioinformatic analysis of microarray data comparing gene expression between normal and tumor-derived primary human tissues. From this approach, we have found that maternal embryonic leucine zipper kinase (Melk), a member of the AMP serine/threonine kinase family, exhibits multiple features consistent with the potential utility of this gene as an anticancer target. An oligonucleotide microarray analysis of multiple human tumor samples and cell lines suggests that Melk expression is frequently elevated in cancer relative to normal tissues, a pattern confirmed by quantitative reverse transcription-PCR and Western blotting of selected primary tumor samples. In situ hybridization localized Melk expression to malignant epithelial cells in 96%, 23%, and 13% of colorectal, lung, and ovarian tissue tumor samples, respectively. Expression of this gene is also elevated in spontaneous tumors derived from the ApcMin and Apc1638N murine models of intestinal tumorigenesis. To begin addressing whether Melk is relevant for tumorigenesis, RNA interference–mediated silencing within human and murine tumor cell lines was done. We show that Melk knockdown decreases proliferation and anchorage-independent growth in vitro as well as tumor growth in a xenograft model. Together, these results suggest that Melk may provide a growth advantage for neoplastic cells and, therefore, inactivation may be therapeutically beneficial.
Expression of Frzb/Secreted Frizzled-Related Protein 3, a Secreted Wnt Antagonist, in Human Androgen-Independent Prostate Cancer PC-3 Cells Suppresses Tumor Growth and Cellular Invasiveness
Authors: Zi Xiaolin , Guo Yi , Simoneau Anne R. , Hope Christopher , Xie Jun , Holcombe Randall F. , Hoang Bang H.
Published: November 2005

The ability of Frzb/secreted Frizzled-related protein 3 (sFRP3) to inhibit Wnt signaling and the localization of Frzb/sFRP3 on chromosome 2q to a region frequently deleted in cancers have led some investigators to hypothesize that Frzb/sFRP3 is a tumor suppressor gene. Here, we examined the biological effects of Frzb/sFRP3 on an androgen-independent prostate cancer cell model. We showed that expression of Frzb/sFRP3 in PC-3 cells resulted in decreased colony formation in soft agar and a dramatic inhibition of tumor growth in a xenograft mouse model. When cellular morphology was examined, PC-3 cells expressing Frzb/sFRP3 exhibited an increase in cell-cell contact formation accompanied by a pronounced induction of epithelial markers E-cadherin and keratin-8 and down-regulation of mesenchymal markers N-cadherin, fibronectin, and vimentin. This phenomenon suggested a reversal of epithelial-to-mesenchymal transition and a less invasive phenotype. Indeed, further in vitro studies with a Matrigel assay showed that Frzb/sFRP3 decreased the invasive capacity of PC-3 cells. These changes in the biology of PC-3 cells are associated with a decrease in the expression and activities of both matrix metalloproteinase (MMP)-2 and MMP-9 as well as decreases in AKT activation, cytosolic β-catenin levels, T-cell factor transcription activity, and expression of Slug and Twist. In addition, transfection of PC-3 with a dominant-negative low-density lipoprotein receptor–related protein 5 (DN-LRP5) coreceptor showed similar biological effects as Frzb/sFRP3 transfection. Together, these data suggest that Frzb/sFRP3 and DN-LRP5 exhibit antitumor activity through the reversal of epithelial-to-mesenchymal transition and inhibition of MMP activities in a subset of prostate cancer.
Suberoylanilide Hydroxamic Acid Enhances Gap Junctional Intercellular Communication via Acetylation of Histone Containing Connexin 43 Gene Locus
Authors: Ogawa Takahiko , Hayashi Tomonori , Tokunou Masahide , Nakachi Kei , Trosko James E. , Chang Chia-Cheng , Yorioka Noriaki
Published: November 2005

A histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induces apoptosis in neoplastic cells, but its effect on gap junctional intercellular communication in relation to apoptosis was unclear. Therefore, we carried out a comparative study of the effects of two HDAC inhibitors, SAHA and trichostatin-A, on gap junctional intercellular communication in nonmalignant human peritoneal mesothelial cells (HPMC) and tumorigenic ras oncogene–transformed rat liver epithelial cells (WB-ras) that showed a significantly lower level of gap junctional intercellular communication than did HPMC. Gap junctional intercellular communication was assessed by recovery rate of fluorescence recovery after photobleaching. Treatment of HPMC with SAHA at nanomolar concentrations caused a dose-dependent increase of recovery rate without inducing apoptosis. This effect was accompanied by enhanced connexin 43 (Cx43) mRNA and protein expression and increased presence of Cx43 protein on cell membrane. Trichostatin-A induced apoptosis in HPMC but was less potent than SAHA in enhancing the recovery rate. In contrast, treatment of WB-ras cells with SAHA or trichostatin-A induced apoptosis at low concentrations, in spite of smaller increases in recovery rate, Cx43 mRNA, and protein than in HPMC. Chromatin immunoprecipitation analysis revealed that SAHA enhanced acetylated histones H3 and H4 in the chromatin fragments associated with Cx43 gene in HPMC. These results indicate that SAHA at low concentrations selectively up-regulates Cx43 expression in normal human cells without induction of apoptosis, as a result of histone acetylation in selective chromatin fragments, in contrast to the apoptotic effect observed in tumorigenic WB-ras cells. These results support a cancer therapeutic and preventive role for specific HDAC inhibitors.
Constitutive Activation of Akt by Flt3 Internal Tandem Duplications Is Necessary for Increased Survival, Proliferation, and Myeloid Transformation
Authors: Brandts Christian H. , Sargin Bülent , Rode Miriam , Biermann Christoph , Lindtner Beate , Schwäble Joachim , Buerger Horst , Müller-Tidow Carsten , Choudhary Chunaram , McMahon Martin , Berdel Wolfgang E. , Serve Hubert
Published: November 2005

Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, encoding a receptor tyrosine kinase. These mutations induce constitutive tyrosine kinase activity in the absence of the natural Flt3 ligand and confer growth factor independence, increased proliferation, and survival to myeloid precursor cells. The signaling pathways and downstream nuclear targets mediating leukemic transformation are only partly identified. Here, we show that the presence of Flt3-ITD constitutively activates Akt (PKB), a key serine-threonine kinase within the phosphatidylinositol 3-kinase pathway. Constitutive activation of Akt phosphorylated and inhibited the transcription factor Foxo3a. Restored Foxo3a activity reversed Flt3-ITD–mediated growth properties and dominant-negative Akt prevented Flt3-ITD–mediated cytokine independence. Conditional Akt activation targeted to the cell membrane induced cytokine-independent survival, cell cycle progression, and proliferation. Importantly, Akt activation was sufficient to cause in vitro transformation of 32D myeloid progenitor cells and in vivo promoted the development of a leukemia-like myeloid disease. Akt phosphorylation was found in myeloid blasts of 86% of AML patients, suggesting an important role in leukemogenesis. In summary, Akt is necessary for increased survival, proliferation, and leukemic transformation by Flt3-ITD, possibly by inactivation of Foxo transcription factors. These findings indicate that Akt and Foxo transcription factors are attractive targets for therapeutic intervention in AML.
Copy Number Aberrations in Mouse Breast Tumors Reveal Loci and Genes Important in Tumorigenic Receptor Tyrosine Kinase Signaling
Authors: Hodgson J. Graeme , Malek Tiffany , Bornstein Sophia , Hariono Sujatmi , Ginzinger David G. , Muller William J. , Gray Joe W.
Published: November 2005

Receptor tyrosine kinase (RTK) signaling plays a key role in the development of breast cancer. Defining the genes and pathways in the RTK signaling network that are important regulators of tumorigenesis in vivo will unveil potential candidates for targeted therapeutics. To this end, we used microarray comparative genomic hybridization to identify and compare copy number aberrations in five mouse models of breast cancer induced by wild-type and mutated forms of oncogenic ErbB2 or the polyomavirus middle T antigen (PyMT). We observed distinct genomic alterations among the various models, including recurrent chromosome 11 amplifications and chromosome 4 deletions, syntenic with human 17q21-25 and 1p35-36, respectively. Expression of oncogenic Erbb2 (NeuNT) under control of the endogenous Erbb2 promoter results in frequent (85%) amplification at the Erbb2 locus with striking structural similarity to the human amplicon, resulting in overexpression of at least two of the genes, Erbb2 and Grb7. Chromosome 11 amplicons distal to Erbb2 arise in a model (DB) overexpressing a mutant variant of PyMT (Y315/322F) unable to activate phosphatidylinositol 3-kinase. These amplicons are not observed in DB hyperplasias or in tumors overexpressing wild-type PyMT and result in overexpression of Grb2 and Itgb4. Distal chromosome 4 deletions occur in a significantly higher proportion of Erbb2 than PyMT tumors and encompass 14-3-3σ (Stratifin), which is expressed at low or undetectable levels in the majority of NeuNT tumors. Our studies highlight loci and genes important in the regulation of tumorigenic RTK signaling in mammary epithelial cells in vivo.
Adenovirus-Mediated Overexpression of REIC/Dkk-3 Selectively Induces Apoptosis in Human Prostate Cancer Cells through Activation of c-Jun-NH2-Kinase
Authors: Abarzua Fernando , Sakaguchi Masakiyo , Takaishi Mikiro , Nasu Yasutomo , Kurose Kyouhei , Ebara Shin , Miyazaki Masahiro , Namba Masayoshi , Kumon Hiromi , Huh Nam-ho
Published: November 2005

Alteration in genes which takes place during malignant conversion and progression could be potential targets for gene therapy. We previously identified REIC/Dkk-3 as a gene whose expression is reduced in many human cancers. Here, we showed that expression of REIC/Dkk-3 was consistently reduced in human prostate cancer tissues in a stage-dependent manner. Forced expression of REIC/Dkk-3 induced apoptosis in human prostate cancer cell lines lacking endogenous REIC/Dkk-3 expression but not in REIC/Dkk-3-proficient normal prostate epithelial and stromal cells. The apoptosis involved c-Jun-NH2-kinase activation, mitochondrial translocation of Bax, and reduction of Bcl-2. A single injection of an adenovirus vector carrying REIC/Dkk-3 showed a dramatic antitumor effect on a xenotransplanted human prostate cancer. Thus, REIC/Dkk-3 could be a novel target for gene-based therapy of prostate cancer.
Tumor-Secreted Vascular Endothelial Growth Factor-C Is Necessary for Prostate Cancer Lymphangiogenesis, but Lymphangiogenesis Is Unnecessary for Lymph Node Metastasis
Authors: Wong Sunny Y. , Haack Herbert , Crowley Denise , Barry Marc , Bronson Roderick T. , Hynes Richard O.
Published: November 2005

Dissemination to draining lymph nodes is a frequent first step in prostate cancer metastasis. Although tumors metastasize to lymph nodes via the lymphatics, the importance of lymphangiogenesis in mediating the process remains controversial. Here, we inhibit intratumoral lymphangiogenesis in s.c. and surgical orthotopic implantation mouse models of human prostate cancer using several strategies. Stable expression of small interfering RNAs (siRNA) targeted against human vascular endothelial growth factor-C (VEGF-C) in PC-3 cells reduced intratumoral lymphatics by 99% in s.c. tumors, indicating that tumor-secreted VEGF-C is necessary for lymphangiogenesis. Expression of siRNAs against human VEGF-A somewhat reduced tumor lymphangiogenesis. Secretion of a soluble VEGF receptor-3/Flt4 fusion protein by PC-3 cells reduced intratumoral lymphatics by 100% in s.c. tumors. Combination of soluble Flt4 and VEGF-C siRNA yielded >92% reduction of intratumoral lymphatics in orthotopic prostate tumors. However, metastasis to lymph nodes was not significantly affected regardless of intratumoral lymphatic vessel density. The abundance of marginal lymphatics at the tumor-stromal interface was unchanged in orthotopic tumors whose intratumoral lymphatics were inhibited, suggesting that these marginal vessels could be sufficient for lymph node metastasis. Hematogenous metastasis (blood tumor burden, lung metastasis) correlated with degree of lymph node invasion. We also analyzed the lymphatics in spontaneous transgenic adenocarcinomas of the mouse prostate which metastasize to lymph nodes. Progression from well-differentiated prostate intraepithelial neoplasia to metastatic, undifferentiated adenocarcinoma was accompanied by loss of lymphatics. These results suggest that tumor-secreted VEGF-C and, to a lesser extent, VEGF-A, are important for inducing prostate cancer intratumoral lymphangiogenesis but are unnecessary for lymph node metastasis.
Telomerase Expression in Noncancerous Bronchial Epithelia Is a Possible Marker of Early Development of Lung Cancer
Authors: Miyazu Yuka Matsuoka , Miyazawa Teruomi , Hiyama Keiko , Kurimoto Noriaki , Iwamoto Yasuo , Matsuura Hiroo , Kanoh Koji , Kohno Nobuoki , Nishiyama Masahiko , Hiyama Eiso
Published: November 2005

Centrally located lung cancers in smokers frequently associated with subsequent primary tumors. We evaluated the telomerase expression chronologically in noncancerous epithelia as a risk factor of susceptibility to lung cancer development. Telomerase protein expression was examined in situ by immunohistochemistry in 26 noncancerous bronchial epithelia adjacent to centrally located early-stage lung cancers in sequential 23 patients treated by photodynamic therapy or surgery among 206 patients who underwent autofluorescence bronchoscopy from 1997 to 2003. Among the 15 lesions in 12 patients treated by photodynamic therapy alone, 11 lesions achieved complete remission after photodynamic therapy, and none of their noncancerous bronchial epithelia was telomerase positive. On the contrary, in the remaining four lesions, either recurrence or secondary lung cancer developed adjacent to the successfully treated primary cancer within 26 months, and the telomerase protein expression in noncancerous epithelia was detected before the secondary cancer development (P < 0.001). The overall relationship of human telomerase reverse transcriptase positivity in noncancerous epithelia and subsequent lung cancer development, including patients treated by radiation or surgery, showed higher significance (P < 0.0001). Histologically “normal” bronchial epithelia in smokers may unphysiologically express telomerase as a field, and such epithelia are likely susceptible to develop lung cancer. We propose that ectopic expression of telomerase in bronchial epithelia may precede transformation in human lung cancer development and that detection of telomerase protein in noncancerous bronchial epithelia will become a useful marker detecting high-risk patients for lung cancer development.
Mitochondrially Targeted p53 Has Tumor Suppressor Activities In vivo
Authors: Talos Flaminia , Petrenko Oleksi , Mena Patricio , Moll Ute M.
Published: November 2005

Complex proapoptotic functions are essential for the tumor suppressor activity of p53. We recently described a novel transcription-independent mechanism that involves a rapid proapoptotic action of p53 at the mitochondria and executes the shortest known circuitry of p53 death signaling. Here, we examine if this p53-dependent mitochondrial program could be exploited for tumor suppression in vivo. To test this, we engage Eμ-Myc transgenic mice, a well-established model of p53-dependent lymphomagenesis. We show that exclusive delivery of p53 to the outer mitochondrial membrane confers a significant growth disadvantage on Eμ-Myc–transformed B-cells of p53-deficient or alternate reading frame–deficient genotypes, resulting in efficient induction of apoptosis and impinged proliferation. Conversely, normal cells from thymus, spleen, and bone marrow showed poor infectivity with these viruses. This proof-of-principle experiment shows that exclusive reliance on the direct mitochondrial program exerts a significant tumor suppressor activity in vivo. Our in vivo data on the direct mitochondrial apoptotic p53 program lays the groundwork to further investigate its efficacy and safety and to address its possible therapeutic value in the future.
Up-regulation of CXCR4 Expression in PC-3 Cells by Stromal-Derived Factor-1α (CXCL12) Increases Endothelial Adhesion and Transendothelial Migration: Role of MEK/ERK Signaling Pathway–Dependent NF-κB Activation
Authors: Kukreja Promil , Abdel-Mageed Asim B. , Mondal Debasis , Liu Kai , Agrawal Krishna C.
Published: November 2005

The chemokine stromal-derived factor-1α (SDF-1α/CXCL-12) and its receptor, CXCR4, play a crucial role in adhesion and transendothelium migration (TEM) of prostate cancer cells. We tested the hypothesis that enhanced expression of CXCR4 in prostate cancer cells is dependent upon SDF-1α-mediated activation of nuclear factor-κB (NF-κB). SDF-1α increased the CXCR4 mRNA and protein expression in PC-3 cells but not in LNCaP cells. Similarly, SDF-1α enhanced the NF-κB-dependent transcriptional activity in PC-3 cells but not in LNCaP cells. SDF-1α increased PC-3 cell adhesion to the human umbilical vein endothelial cell monolayer and enhanced TEM, which was abrogated with anti-CXCR4 monoclonal antibody (mAb). Suppression of NF-κB activity in PC-3 cells by a mutant IκBα super-repressor adenoviral vector decreased the CXCR4 mRNA expression and inhibited adhesion and TEM. Transient overexpression of p65 subunit of NF-κB in PC-3 cells up-regulated CXCR4 receptor expression and increased the adhesion and TEM of these cells in response to SDF-1α gradient. Treatment of PC-3 cells with SDF-1α leads to nuclear translocation of NF-κB protein within 15 to 30 minutes, which correlated with IκBα phosphorylation. A p42/44 mitogen-activated protein kinase [MAPK, extracellular signal regulated kinase-1/2 (ERK-1/2)] biphasic activation pattern was observed in these cells at 15 minutes and 3 hours after SDF-1α treatment. Phosphorylation of IκB kinase α was observed within 30 minutes, which was blocked by PD98059 [MAPK kinase (MEK) inhibitor]. PD98059 cotreatment significantly inhibited SDF-1α-induced NF-κB reporter activity and CXCR4 receptor expression as shown by flow cytometry. These data suggest that SDF-1α-induced expression of CXCR4 in PC-3 cells is dependent on MEK/ERK signaling cascade and NF-κB activation.
Protein Transduction of Dendritic Cells for NY-ESO-1-Based Immunotherapy of Myeloma
Authors: Batchu Ramesh B. , Moreno Amberly M. , Szmania Susann M. , Bennett Grant , Spagnoli Guilio C. , Ponnazhagan Selvarangan , Barlogie Bart , Tricot Guido , van Rhee Frits
Published: November 2005

Myeloma vaccines, based on dendritic cells pulsed with idiotype or tumor lysate, have been met with limited success, probably in part due to insufficient cross-priming of myeloma antigens. A powerful method to introduce myeloma-associated antigens into the cytosol of dendritic cells is protein transduction, a process by which proteins fused with a protein transduction domain (PTD) freely traverse membrane barriers. NY-ESO-1, an immunogenic antigen by itself highly expressed in 60% of high-risk myeloma patients, was purified to near homogeneity both alone and as a recombinant fusion protein with a PTD, derived from HIV-Tat. Efficient entry of PTD-NY-ESO-1 into dendritic cells, confirmed by microscopy, Western blotting, and intracellular flow cytometry, was achieved without affecting dendritic cell phenotype. Experiments with amiloride, which inhibits endocytosis, and N-acetyl-l-leucinyl-l-norleucinal, a proteasome inhibitor, confirmed that PTD-NY-ESO-1 entered dendritic cells by protein transduction and was degraded by the proteasome. Tetramer analysis indicated superior generation of HLA-A2.1, CD8+ T lymphocytes specific for NY-ESO-1157-165 with PTD-NY-ESO-1 compared with NY-ESO-1 control protein (44% versus 2%, respectively). NY-ESO-1-specific T lymphocytes generated with PTD-NY-ESO-1 secreted IFN-γ indicative of a Tc1-type cytokine response. Thus, PTD-NY-ESO-1 accesses the cytoplasm by protein transduction, is processed by the proteasome, and NY-ESO-1 peptides presented by HLA class I elicit NY-ESO-1-specific T lymphocytes.
Focal Adhesion Kinase Regulates Syndecan-2–Mediated Tumorigenic Activity of HT1080 Fibrosarcoma Cells
Authors: Park Haein , Han Innoc , Kwon Ho Jeong , Oh Eok-Soo
Published: November 2005

Expression of syndecan-2, a transmembrane heparan sulfate proteoglycan, is crucial for the tumorigenic activity in colon carcinoma cells. However, despite the high-level expression of syndecan-2 in mesenchymal cells, few studies have addressed the function of syndecan-2 in sarcoma cells. In HT1080 fibrosarcoma cells, we found that syndecan-2 regulated migration, invasion into Matrigel, and anchorage-independent growth but not cell-extracellular matrix adhesion or proliferation, suggesting that syndecan-2 plays different functional roles in fibrosarcoma and colon carcinoma cells. Consistent with the increased cell migration/invasion of syndecan-2–overexpressing HT1080 cells, syndecan-2 overexpression increased phosphorylation and interaction of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), membrane localization of T-lymphoma invasion and metastasis gene-1 (Tiam-1), and activation of Rac. Syndecan-2–mediated cell migration/invasion of HT1080 cells was diminished when (a) cells were cotransfected with nonphosphorylatable mutant FAK Y397F or with other FAK mutants lacking PI3K interactions, (b) cells were treated with a specific PI3K inhibitor, or (c) levels of Tiam-1 were knocked down with small interfering RNAs. Furthermore, expression of several FAK mutants inhibited syndecan-2–mediated enhancement of anchorage-independent growth in HT1080 cells. Taken together, these data suggest that syndecan-2 regulates the tumorigenic activities of HT1080 fibrosarcoma cells and that FAK is a key regulator of syndecan-2–mediated tumorigenic activities.
Novel Pheochromocytoma Susceptibility Loci Identified by Integrative Genomics
Authors: Dahia Patricia L.M. , Hao Ke , Rogus John , Colin Christian , Pujana Miguel A.G. , Ross Ken , Magoffin Danielle , Aronin Neil , Cascon Alberto , Hayashida César Y. , Li Cheng , Toledo Sérgio P.A. , Stiles Charles D. ,   for the Familial Pheochromocytoma Consortium
Published: November 2005

Pheochromocytomas are catecholamine-secreting tumors that result from mutations of at least six different genes as components of distinct autosomal dominant disorders. However, there remain familial occurrences of pheochromocytoma without a known genetic defect. We describe here a familial pheochromocytoma syndrome consistent with digenic inheritance identified through a combination of global genomics strategies. Multipoint parametric linkage analysis revealed identical LOD scores of 2.97 for chromosome 2cen and 16p13 loci. A two-locus parametric linkage analysis produced maximum LOD score of 5.16 under a double recessive multiplicative model, suggesting that both loci are required to develop the disease. Allele-specific loss of heterozygosity (LOH) was detected only at the chromosome 2 locus in all tumors from this family, consistent with a tumor suppressor gene. Four additional pheochromocytomas with a similar genetic pattern were identified through transcription profiling and helped refine the chromosome 2 locus. High-density LOH mapping with single nucleotide polymorphism–based array identified a total of 18 of 62 pheochromocytomas with LOH within the chromosome 2 region, which further narrowed down the locus to <2 cM. This finding provides evidence for two novel susceptibility loci for pheochromocytoma and adds a recessive digenic trait to the increasingly broad genetic heterogeneity of these tumors. Similarly, complex traits may also be involved in other familial cancer syndromes.
Addressing the “Fas Counterattack” Controversy: Blocking Fas Ligand Expression Suppresses Tumor Immune Evasion of Colon CancerIn vivo
Authors: Ryan Aideen E. , Shanahan Fergus , O'Connell Joe , Houston Aileen M.
Published: November 2005

Fas ligand (FasL/CD95L) is a transmembrane protein belonging to the tumor necrosis factor superfamily that can trigger apoptotic cell death following ligation to its receptor, Fas (CD95/APO-1). Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of antitumor immune effector cells—the “Fas counterattack.” However, the ability of FasL to mediate tumor immune privilege is controversial due to studies that indicate FasL has both pro- and anti-inflammatory activities. To resolve this controversy and functionally define the role of FasL in tumor immune evasion, we investigated if suppression of endogenously expressed FasL in colon tumor cells resulted in reduced tumor development and improved antitumor immune challenge in vivo. Specifically, FasL expression in CMT93 colon carcinoma cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression had no effect on tumor growth in vitro but significantly reduced tumor development in syngeneic immunocompetent mice in vivo. Tumor size was also significantly decreased. Reduced FasL expression by tumor cells led to increased lymphocyte infiltration. The overall level of neutrophils present in all of the tumors examined was low, with no difference between the tumors, irrespective of FasL expression. Thus, down-regulation of FasL expression by colon tumor cells results in an improved antitumor immune challenge in vivo, providing functional evidence in favor of the “Fas counterattack” as a mechanism of tumor immune evasion.
The Role of DOC-2/DAB2 in Modulating Androgen Receptor–Mediated Cell Growth via the Nongenomic c-Src–Mediated Pathway in Normal Prostatic Epithelium and Cancer
Authors: Zhoul Jian , Hernandez Gina , Tu Szu-Wei , Huang Chien-Ling , Tseng Ching-Ping , Hsieh Jer-Tsong
Published: November 2005

Prostate cancer is initially responsive to androgen ablation, but prostate cancer tumors invariably progress to an androgen-independent state that is ultimately lethal. The onset of the androgen-independent prostate cancer is often associated with up-regulation of the androgen receptor that can cause antagonists to exhibit agonistic activity, which could lead to the failure of androgen ablation therapy. We describe a unique protein—DOC-2/DAB2 (differentially expressed in ovarian cancer-2/disabled 2)—that antagonizes androgen receptor–mediated cell growth in prostate cancer cells via interaction with c-Src protein. This interaction causes inactivation of Erk and Akt proteins critical for proliferation and survival of prostate cancer cells. However, DOC-2/DAB2 does not change the capacity of androgen receptor to regulate the transcription of androgen-responsive reporter genes, indicating that DOC-2/DAB2 selectively inhibits androgen receptor–mediated cell growth in androgen-independent prostate cancer by disrupting the androgen receptor/c-Src complex. In normal prostatic epithelia, DOC-2/DAB2 protein levels are more abundant than androgen receptor protein levels and reduced endogenous DOC-2/DAB2 protein levels in these cells by DOC-2/DAB2 RNA interference result in enhancing androgen receptor–mediated cell growth. We conclude that DOC-2/DAB2 can modulate androgen receptor–mediated cell growth in both normal and malignant prostatic epithelial cells and the outcome of this study could evolve into a new therapeutic strategy of prostate cancer.
Fine Mapping Reveals Multiple Loci and a Possible Epistatic Interaction within the Mammary Carcinoma Susceptibility Quantitative Trait Locus, Mcs5
Authors: Samuelson David J. , Aperavich Beth A. , Haag Jill D. , Gould Michael N.
Published: November 2005

To identify high-frequency, low-penetrance breast cancer modifier genes, we have developed a rat genetic model that uses the Wistar-Kyoto (WKy) inbred strain, resistant to developing 7,12-dimethylbenz[a]anthracene–induced mammary carcinogenesis, as a congenic donor and the susceptible Wistar-Furth (WF) strain as the recipient. Here, data from congenic rat lines containing smaller WKy genomic intervals of the Mcs5 quantitative trait locus region are presented to fine map three independently acting Mcs5 subloci. WKy-homozygous females from congenic lines defining Mcs5a, Mcs5b, and Mcs5c averaged, respectively, 4.0 ± 0.4, 11.6 ± 0.6, and 3.5 ± 0.4 mammary carcinomas per rat. These phenotypic values are statistically different from the WF-homozygous phenotype value of 8.0 ± 0.4, which is the baseline phenotype used for these experiments. We identified a likely Mcs5a × Mcs5b epistatic interaction that results in masking the increased susceptibility effect of the Mcs5b WKy allele by the Mcs5a WKy allele. We also provide evidence for a Mcs5a × Mcs5c interaction that is synergistic to decrease mammary carcinoma susceptibility below the additive effects of WKy alleles at each locus independently. The Mcs5 subloci are currently localized to 1.0, 7.5, and 4.5 Mb of rat chromosome 5, and the orthologous regions are on human chromosome 9 and mouse chromosome 4. These loci will provide unbiased candidate gene loci for evaluation in human case-control association studies.
A Network of Genetic Events Sufficient to Convert Normal Human Cells to a Tumorigenic State
Authors: Kendall S. DiSean , Linardic Corinne M. , Adam Stacey J. , Counter Christopher M.
Published: November 2005

Although great progress has been made at identifying and characterizing individual genes involved in cancer, less is known about how the combination of such genes collaborate to form tumors in humans. To this end, we sought to genetically recreate tumorigenesis in normal human cells using genes altered in human cancer. We now show that expression of mammalian proteins that inactivate the tumor suppressors Rb and p53 in conjunction with the oncoproteins Ras and Myc and the telomerase subunit hTERT is sufficient to drive a number of normal human somatic cells to a tumorigenic fate. This provides a blueprint of the events that lead to human cancer, allowing different cancers to be genetically modeled from normal human cells.
High Correlation of Whole-Body Red Fluorescent Protein Imaging and Magnetic Resonance Imaging on an Orthotopic Model of Pancreatic Cancer
Authors: Bouvet Michael , Spernyak Joseph , Katz Matthew H. , Mazurchuk Richard V. , Takimoto Shinako , Bernacki Ralph , Rustum Youcef M. , Moossa Abdool R. , Hoffman Robert M.
Published: November 2005

We have developed genetically fluorescent orthotopic models of human pancreatic cancer. In these models, noninvasive fluorescent protein imaging (FPI) of internal primary tumors and metastatic deposits has been carried out. Whole-body tumor images are easily and inexpensively obtained using FPI, permitting both detection and quantification of tumor load. In this study, we simultaneously compared single mice with a highly fluorescent, red fluorescent protein–expressing orthotopic pancreatic cancer xenografts with both FPI and high-resolution magnetic resonance imaging (MRI). Images were acquired at multiple time points after tumor implantation in the pancreas. Indwelling pancreatic primary tumors and metastatic foci were detected by both FPI and MRI. Moreover, a strong correlation existed between images taken with these two technologies. FPI permitted rapid, high-throughput imaging without the need for either anesthesia or contrast agents. Both FPI and MRI enabled accurate imaging of tumor growth and metastasis, although MRI enabled tissue structure to be visualized as well. FPI has high resolution and is exceedingly rapid with instant image capture. We suggest a complimentary role for these two imaging modalities.