bluuebunny/crossref_metadata_2025 huggingface dataset:
Teaching integrative physiology using the quantitative circulatory physiology model and case discussion method: evaluation of the learning experience
Authors: Rodríguez-Barbero A. , López-Novoa J. M.
Published: December 2008

One of the problems that we have found when teaching human physiology in a Spanish medical school is that the degree of understanding by the students of the integration between organs and systems is rather poor. We attempted to remedy this problem by using a case discussion method together with the Quantitative Circulatory Physiology (QCP) program. QCP is a Windows-based computer simulation program that offers almost real-time simulation and allows users to examine the time-dependent interactions of over 750 parameters. We evaluated students' perceptions by an anonymous questionnaire. Teachers' perceptions of this teaching approach were highly positive, as it improved students' perceptions of the complexity of biological processes, their ability to differentiate between acute and chronic responses, and promoted an integrative understanding of human body function. Teachers also identified some problems with the approach, including student difficulties in adopting self-directed learning, a lack of precision in student questions during the discussion sessions, and the lack of a tradition of using several textbooks to explain the changes observed. The results of the student questionnaire revealed that >70% of the students reported that this type of learning gave them a better understanding of the complexity of physiological processes and the role of coordinated actions of several systems in the homeostatic response and enabled them to acquire a better understanding of human body functions. Thus, we conclude that this approach promotes an integrative understanding of cardiovascular and renal functions that is difficult to achieve with other methods.
An overview of undergraduate physiology education in Turkish medical faculties
Authors: Balkanci Z. Dicle , Pehlivanoglu Bilge
Published: December 2008

Physiology education, which occupies an important place in undergraduate medical education, exhibits diversities across the world. Since there was no specific source of information about physiology education in Turkish medical faculties, the authors aimed to evaluate the general status of undergraduate physiology teaching of medical students in Turkey. A questionnaire designed for the program used for medical students was sent to the physiology departments of 38 faculties that had academic personnel and had carried out medical education for at least 3 years. It questioned the educational load, content, and duration of the lectures, written materials, techniques, assessment methods, basic equipments, and subjects used in practical sessions. All 38 departments answered the questionnaire. This study investigating 38 faculties showed that the content and time devoted to lectures and practical sessions (169 and 35 h) differed, as it does throughout the world, and teaching laboratories constituting 17% of total physiology education were performed and assessed by all of the departments. The practical hours correlated with the number of teaching staff. Our results indicated an insufficient number of teaching staff with a heavy educational load. This survey showed that the number of teaching staff is critical for practical sessions. Considering that the actual number of medical schools is 61 schools, with some established but not yet admitting students and educating with their own staff, if the requirements for teaching staff are not met, physiology education in Turkey will face important problems in the coming years.
The use of formative online quizzes to enhance class preparation and scores on summative exams
Authors: Dobson John L.
Published: December 2008

Online quizzes were introduced into an undergraduate Exercise Physiology course to encourage students to read ahead and think critically about the course material before coming to class. The purpose of the study was to determine if the use of the online quizzes was associated with improvements in summative exam scores and if the online quizzes were valid predictors of summative exam performance. A retrospective analysis was performed on the course scores from three different groups of Exercise Physiology students. Students in group 1 completed the original version of the course, those in group 2 completed an updated version of the course that included more rigorous exam questions, and those in group 3 completed the same updated version of the course but with the addition of 10 required online quizzes. Results showed that the overall mean summative exam score from group 3 was significantly higher than that from group 2 (81.79 ± 8.26 and 78.72 ± 9.61, respectively). A significant positive correlation ( r = 0.50) was also found between individual mean online quiz scores and individual mean exam scores for those students in group 3. It was concluded that the formative online quizzes did enhance summative exam performance and that the online quizzes were valid predictors of exam performance.
X-ray powder diffraction data for new ternary phases in the Al–Ti–Pt system
Authors: Zaikina O. , Pavlyuchkov D. , Khorujaya V. , Velikanova T.
Published: December 2008

Two ternary phases, designated τ4 and τ5, were revealed in Al–Ti–Pt. The τ4-phase with equiatomic composition (i.e., AlTiPt) was found to have a hexagonal structure with a=4.3908(9) Å and c=5.4823(10) Å (space group P63/mmc), and the τ5-phase, forming in a compositional range between
∼
Al14Ti58Pt28 and
Al21Ti63Pt16, has a tetragonal structure (possible space groups P42nm, P-4n2, and P42/mnm). The refined lattice parameters for
Al15Ti60Pt25 are a=9.7019(20) Å and
c=5.0231
(13)
Å.
Defining the Cooperative Genetic Changes That Temporally Drive Alveolar Rhabdomyosarcoma
Authors: Naini Sarasija , Etheridge Katherine T. , Adam Stacey J. , Qualman Stephen J. , Bentley Rex C. , Counter Christopher M. , Linardic Corinne M.
Published: December 2008

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of RMS known as alveolar (aRMS) have a 5-year survival rate of <30%. aRMS tissues exhibit a number of genetic changes, including loss-of-function of the p53 and Rb tumor suppressor pathways, amplification of MYCN, stabilization of telomeres, and most characteristically, reciprocal translocation of loci involving the PAX and FKHR genes, generating the PAX7-FKHR or PAX3-FKHR fusion proteins. We previously showed that PAX3-FKHR expression in primary human myoblasts, cells that can give rise to RMS, cooperated with loss of p16INK4A to promote extended proliferation. To better understand the genetic events required for aRMS formation, we then stepwise converted these cells to their transformed counterpart. PAX3-FKHR, the catalytic unit of telomerase hTERT, and MycN, in cooperation with down-regulation of p16INK4A/p14ARF expression, were necessary and sufficient to convert normal human myoblasts into tumorigenic cells that gave rise to aRMS tumors. However, the order of expression of these transgenes was critical, as only those cells expressing PAX3-FKHR early could form tumors. We therefore suggest that the translocation of PAX3 to FKHR drives proliferation of myoblasts, and a selection for loss of p16INK4A/p14ARF. These early steps, coupled with MycN amplification and telomere stabilization, then drive the cells to a fully tumorigenic state. [Cancer Res 2008;68(23):9583–8]
Interstitial Doppler Optical Coherence Tomography as a Local Tumor Necrosis Predictor in Photodynamic Therapy of Prostatic Carcinoma: AnIn vivoStudy
Authors: Standish Beau A. , Lee Kenneth K.C. , Jin Xiao , Mariampillai Adrian , Munce Nigel R. , Wood Michael F.G. , Wilson Brian C. , Vitkin I. Alex , Yang Victor X.D.
Published: December 2008

We have tested the feasibility of real-time localized blood flow measurements, obtained with interstitial (IS) Doppler optical coherence tomography (DOCT), to predict photodynamic therapy (PDT)-induced tumor necrosis deep within solid Dunning rat prostate tumors. IS-DOCT was used to quantify the PDT-induced microvascular shutdown rate in s.c. Dunning prostate tumors (n = 28). Photofrin (12.5 mg/kg) was administered 20 to 24 hours before tumor irradiation, with 635 nm surface irradiance of 8 to 133 mWcm−2 for 25 minutes. High frequency ultrasound and calipers were used to measure the thickness of the skin covering the tumor and the location of the echogenic IS probe within it. A two-layer Monte Carlo model was used to calculate subsurface fluence rates within the IS-DOCT region of interest (ROI). Treatment efficacy was estimated by percent tumor necrosis within the ROI, as quantified by H&E staining, and correlated to the measured microvascular shutdown rate during PDT treatment. IS-DOCT measured significant PDT-induced vascular shutdown within the ROI in all tumors. A strong relationship (R2 = 0.723) exists between the percent tumor necrosis at 24 hours posttreatment and the vascular shutdown rate: slower shutdown corresponded to higher treatment efficacy, i.e., more necrosis. Controls (needle + light, no drug, n = 3) showed minimal microvascular changes or necrosis (4% ± 1%). This study has correlated a biological end point with a direct and localized measurement of PDT-induced microvascular changes, suggesting a potential clinical role of on-line, real-time microvascular monitoring for optimizing treatment efficacy in individual patients. [Cancer Res 2008;68(23):9987–95]
Effects of Dasatinib on Src Kinase Activity and Downstream Intracellular Signaling in Primitive Chronic Myelogenous Leukemia Hematopoietic Cells
Authors: Konig Heiko , Copland Mhairi , Chu Su , Jove Richard , Holyoake Tessa L. , Bhatia Ravi
Published: December 2008

Bcr-Abl tyrosine kinase inhibitors (TKI) are effective in inducing remissions in chronic myelogenous leukemia (CML) patients but do not eliminate primitive CML hematopoietic cells. There is a need to identify mechanisms that contribute to retention of CML progenitors. Src family tyrosine kinases have been identified as potential mediators of Bcr-Abl–induced leukemogenesis. Dasatinib (BMS-354825) is a potent dual Abl/Src kinase inhibitor approved for clinical use in CML patients. We evaluated Src activity in primitive human CML progenitors from different stages of disease and investigated effects of Dasatinib on Src activity and downstream signaling pathways. P-Src expression was increased in CD34+ cells and CD34+CD38− cells in all phases of CML. Dasatinib showed potent Src inhibitory activity in CML progenitors, inhibiting both Bcr-Abl–dependent and –independent Src activity. In contrast, Imatinib inhibited only Bcr-Abl–dependent Src activity. Dasatinib inhibited P–mitogen-activated protein kinase (MAPK), P-Akt, and P-STAT5 levels in CML progenitors in the absence of growth factors but not in the presence of growth factors. A marked increase in P-MAPK levels seen in the presence of growth factors with Imatinib was much less prominent with Dasatinib. Dasatinib significantly suppressed CML colony-forming cells and long-term culture-initiating cells but did not significantly alter the level of apoptosis-regulating proteins in CML CD34+ cells. Our results indicate that Dasatinib, in addition to potent anti–Bcr-Abl kinase activity, effectively inhibits Src kinase activity and downstream signaling pathways in CML progenitors but does not induce a strong proapoptotic response. These observations argue against a prominent role for Src kinases in persistence of primitive CML cells in TKI-treated patients. [Cancer Res 2008;68(23):9624–33]
Global Hypomethylation of Genomic DNA in Cancer-Associated Myofibroblasts
Authors: Jiang Le , Gonda Tamas A. , Gamble Mary V. , Salas Martha , Seshan Venkatraman , Tu Shuiping , Twaddell William S. , Hegyi Peter , Lazar Gyorgy , Steele Islay , Varro Andrea , Wang Timothy C. , Tycko Benjamin
Published: December 2008

Global hypomethylation has long been recognized as a feature of the malignant epithelial component in human carcinomas. Here we show evidence for this same type of epigenetic alteration in cancer-associated stromal myofibroblasts. We used methylation-sensitive SNP array analysis (MSNP) to profile DNA methylation in early-passage cultures of stromal myofibroblasts isolated from human gastric cancers. The MSNP data indicated widespread hypomethylation in these cells, with rare focal gains of methylation, conclusions that were independently validated by bisulfite sequencing and by a methylation-sensitive cytosine incorporation assay. Immunohistochemistry with anti–5-methylcytosine (anti–5-methyl-C) in a series of gastrectomy specimens showed frequent loss of methylation in nuclei of both the malignant epithelial cells and α-smooth muscle actin (ASMA)–positive stromal myofibroblasts of both intestinal-type and diffuse carcinomas. We confirmed this phenomenon and established its onset at the stage of noninvasive dysplastic lesions by immunohistochemistry for anti–5-methyl-C in a transgenic mouse model of multistage gastric carcinogenesis. These findings indicate similar general classes of epigenetic alterations in carcinoma cells and their accompanying reactive stromal cells and add to accumulating evidence for biological differences between normal and cancer-associated myofibroblasts. [Cancer Res 2008;68(23):9900–8]
Glutathione Peroxidase 2 Inhibits Cyclooxygenase-2–Mediated Migration and Invasion of HT-29 Adenocarcinoma Cells but Supports Their Growth as Tumors in Nude Mice
Authors: Banning Antje , Kipp Anna , Schmitmeier Stephanie , Löwinger Maria , Florian Simone , Krehl Susanne , Thalmann Sophie , Thierbach René , Steinberg Pablo , Brigelius-Flohé Regina
Published: December 2008

The selenoprotein gastrointestinal glutathione peroxidase 2 (GPx2) is up-regulated in a variety of cancer cells with thus far unknown consequences. Therefore, two clones of a human colon cancer cell line (HT-29) in which GPx2 was stably knocked down by small interfering RNA (siRNA; siGPx2) were used to test whether cancer-relevant processes are affected by GPx2. The capacity to grow anchorage independently in soft agar was significantly reduced in siGPx2 cells when compared with controls (i.e., HT-29 cells stably transfected with a scramble siRNA). The weight of tumors derived from siGPx2 cells injected into nude mice was lower in 9 of 10 animals. In contrast, in a wound-healing assay, wound closure was around 50% in controls and 80% in siGPx2 cells, indicating an enhanced capacity of the knockdown cells to migrate. Similarly, invasion of siGPx2 cells in a Transwell assay was significantly increased. Migration and invasion of siGPx2 cells were inhibited by celecoxib, a cyclooxygenase-2 (COX-2)–specific inhibitor, but not by α-tocopherol. Selenium supplementation of cell culture medium did not influence the results obtained with siGPx2 cells, showing that none of the other selenoproteins could replace GPx2 regarding the described effects. The data show that GPx2 inhibits malignant characteristics of tumor cells, such as migration and invasion, obviously by counteracting COX-2 expression but is required for the growth of transformed intestinal cells and may, therefore, facilitate tumor cell growth. The data also shed new light on the use of selenium as a chemopreventive trace element: a beneficial effect may depend on the stage of tumor development. [Cancer Res 2008;68(23):9746–53]
Tubulin-Targeted Drug Action: Functional Significance of Class II and Class IVb β-Tubulin inVincaAlkaloid Sensitivity
Authors: Gan Pei Pei , Kavallaris Maria
Published: December 2008

Aberrant expression of β-tubulin isotypes is frequently described in tumor tissues and tubulin-binding agent (TBA)–resistant cell lines. There is limited understanding of the role of specific β-tubulin isotypes in cellular sensitivity to TBAs, and to gain insights into the functional role of βII- and βIVb-tubulin, we examined these isotypes in lung cancer cell lines NCI-H460 (H460) and Calu-6. Drug-treated clonogenic assays revealed that small interfering RNA–mediated knockdown of either βII- or βIVb-tubulin hypersensitized the lung cancer cell lines to Vinca alkaloids, with the effects more pronounced following βIVb-tubulin knockdown. In contrast, there was no change in paclitaxel sensitivity following knockdown of either isotype. Cell cycle analysis revealed a greater propensity for the βII- and βIVb-tubulin knockdown cells to undergo G2-M cell cycle block following 5 nmol/L vincristine treatment, with the βIVb knockdown cells being more sensitive than the βII-tubulin knockdown cells compared with control. In contrast to βII-tubulin knockdown, βIVb-tubulin knockdown cells showed a significant increase in the sub-G1 population (cell death) following treatment with both 5 and 40 nmol/L of vincristine compared with controls. Importantly, βIVb-tubulin knockdown in H460 cells caused a significant dose-dependent increase in Annexin V staining in response to vincristine but not paclitaxel. Therefore, increased sensitivity to induction of apoptosis is one mechanism underlying the Vinca alkaloid hypersensitivity. This study provides direct evidence that βII- or βIVb-tubulins have functionally distinct roles and expression of these isotypes may serve as strong predictors of Vinca alkaloid response and resistance. [Cancer Res 2008;68(23):9817–24]
Activation of Phosphatidylinositol 3-Kinase by Membrane Localization of p110α Predisposes Mammary Glands to Neoplastic Transformation
Authors: Renner Oliver , Blanco-Aparicio Carmen , Grassow Maja , Cañamero Marta , Leal Juan F.M. , Carnero Amancio
Published: December 2008

Phosphatidylinositol 3-kinases (PI3K) constitute important regulators of various signaling pathways with relevance in cancer. Enhanced activation of p110α, the catalytic subunit of PI3K, was found in a high proportion of many human tumor types. We generated a mouse model in which PI3K is activated by forced recruitment of p110α to the membrane. Different transgenic lines expressing myristoylated p110α protein under the control of the epithelial-specific mouse mammary tumor virus promoter were selected according to different levels of PI3K activity and characterized. Delayed mammary gland involution and morphologic changes of the mammary ducts could be detected in young transgenic female mice. These changes were more pronounced in old animals, especially in mutiparous females, in which we observed increased ductal branching, alveolar hyperplasia, and intraductal neoplasia. We also observed a small percentage of mammary tumors. Crosses of myrp110α transgenic mice with heterozygous p53(+/−) knockout mice resulted in neither enhanced tumorigenesis nor in a stronger mammary gland phenotype. However, the CDK4 activating mutation (R24C) lead to increased tumorigenesis in transgenic myrp110α mice, emphasizing the postulated perturbation of the interaction of the CDK4/Rb/E2F cascade and the PI3K signaling in many human cancers. Interestingly, in tumors of myrp110α transgenic mice, we observed an increased phosphorylation of the estrogen receptor-α, a typical feature of human breast cancer. The model presented here will help to discover additional factors which influence the progression of preneoplastic lesions to tumors in the mammary gland and to explore antitumor therapies based on PI3K or estrogen receptor-α pathway inhibition. [Cancer Res 2008;68(23):9643–53]
Differential p53 Engagement in Response to Oxidative and Oncogenic Stresses in Fanconi Anemia Mice
Authors: Rani Reena , Li Jie , Pang Qishen
Published: December 2008

Members of the Fanconi anemia (FA) protein family are involved in repair of genetic damage caused by DNA cross-linkers. It is not clear whether the FA proteins function in oxidative DNA damage and oncogenic stress response. Here, we report that deficiency in the Fanca gene in mice elicits a p53-dependent growth arrest and DNA damage response to oxidative DNA damage and oncogenic stress. Using a Fanca−/−Trp53−/− double knockout model and a functionally switchable p53 retrovirus, we define the kinetics, dependence, and persistence of p53-mediated response to oxidative and oncogenic stresses in Fanca−/− cells. Notably, oxidative stress induces persistent p53 response in Fanca−/− cells, likely due to accumulation of unrepaired DNA damage. On the other hand, whereas wild-type cells exhibit prolonged response to oncogene activation, the p53-activating signals induced by oncogenic ras are short-lived in Fanca−/− cells, suggesting that Fanca may be required for the cell to engage p53 during constitutive ras activation. We propose that the FA proteins protect cells from stress-induced proliferative arrest and tumor evolution by acting as a modulator of the signaling pathways that link FA to p53. [Cancer Res 2008;68(23):9693–702]
Nuclear Bile Acid Receptor FXR Protects against Intestinal Tumorigenesis
Authors: Modica Salvatore , Murzilli Stefania , Salvatore Lorena , Schmidt Daniel R. , Moschetta Antonio
Published: December 2008

Bile acids have been considered intestinal tumor promoters, and because they are natural ligands for the nuclear receptor FXR, we examined the role of FXR in intestinal tumorigenesis. Using gain- and loss-of-function studies, we found that FXR suppresses intestinal tumorigenesis in vivo. Loss of FXR in the ApcMin/+ and in the chronic colitis mouse models of intestinal tumorigenesis resulted in early mortality and increased tumor progression via promotion of Wnt signaling by infiltrating neutrophils and macrophages and tumor necrosis factor α production. Treatment with the bile acid binding resin cholestyramine did not modify the intestinal tumor susceptibility of FXR−/− mice, indicating that loss of FXR and not merely elevated bile acid concentrations increases susceptibility to tumorigenesis. Activation of FXR induced a proapoptotic program in the differentiated normal colonic epithelium as well as transformed colonocytes. Our data suggest that it is unlikely that the tumor-promoting activity of bile acids occurs as a function of their ability to activate FXR. However, FXR activity is relevant to the pathogenesis of intestinal cancer. When FXR is absent in the intestine, there is a promotion of Wnt signaling with expansion of the basal proliferative compartment, and a concomitant reduction in the apical differentiated apoptosis-competent compartment. When FXR is activated in the intestine and in colon cancer cells, there is an induction of apoptosis and removal of genetically altered cells, which may otherwise progress to complete transformation. Thus, from a therapeutic standpoint, strategies aimed at reactivating FXR expression in colon tumors might be useful in treatment of colon cancer. [Cancer Res 2008;68(23):9589–94]
Ornithine Decarboxylase Inhibition by α-Difluoromethylornithine Activates Opposing Signaling Pathways via Phosphorylation of Both Akt/Protein Kinase B and p27Kip1 in Neuroblastoma
Authors: Koomoa Dana-Lynn T. , Yco Lisette P. , Borsics Tamas , Wallick Christopher J. , Bachmann André S.
Published: December 2008

Ornithine decarboxylase (ODC) is a key enzyme in mammalian polyamine biosynthesis that is up-regulated in various types of cancer. We previously showed that treating human neuroblastoma (NB) cells with the ODC inhibitor α-difluoromethylornithine (DFMO) depleted polyamine pools and induced G1 cell cycle arrest without causing apoptosis. However, the precise mechanism by which DFMO provokes these changes in NB cells remained unknown. Therefore, we further examined the effects of DFMO, alone and in combination with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or Akt/protein kinase B (PKB) inhibitor IV, on the regulation of cell survival and cell cycle–associated pathways in LAN-1 NB cells. In the present study, we found that the inhibition of ODC by DFMO promotes cell survival by inducing the phosphorylation of Akt/PKB at residue Ser473 and glycogen synthase kinase-3β at Ser9. Intriguingly, DFMO also induced the phosphorylation of p27Kip1 at residues Ser10 (nuclear export) and Thr198 (protein stabilization) but not Thr187 (proteasomal degradation). The combined results from this study provide evidence for a direct cross-talk between ODC-dependent metabolic processes and well-established cell signaling pathways that are activated during NB tumorigenesis. The data suggest that inhibition of ODC by DFMO induces two opposing pathways in NB: one promoting cell survival by activating Akt/PKB via the PI3K/Akt pathway and one inducing p27Kip1/retinoblastoma-coupled G1 cell cycle arrest via a mechanism that regulates the phosphorylation and stabilization of p27Kip1. This study presents new information that may explain the moderate efficacy of DFMO monotherapy in clinical trials and reveals potential new targets for DFMO-based combination therapies for NB treatment. [Cancer Res 2008;68(23):9825–31]
Inhibition of Repair of Radiation-Induced DNA Damage Enhances Gene Expression from Replication-Defective Adenoviral Vectors
Authors: Hingorani Mohan , White Christine L. , Merron Andrew , Peerlinck Inge , Gore Martin E. , Slade Andrew , Scott Simon D. , Nutting Christopher M. , Pandha Hardev S. , Melcher Alan A. , Vile Richard G. , Vassaux Georges , Harrington Kevin J.
Published: December 2008

Radiation has been shown to up-regulate gene expression from adenoviral vectors in previous studies. In the current study, we show that radiation-induced dsDNA breaks and subsequent signaling through the mitogen-activated protein kinase (MAPK) pathway are responsible, at least in part, for this enhancement of transgene expression both in vitro and in vivo. Inhibitors of ataxia-telangiectasia–mutated, poly(ADP-ribose) polymerase–mutated, and DNA-dependent protein kinase (DNA-PK)–mediated DNA repair were shown to maintain dsDNA breaks (γH2AX foci) by fluorescence-activated cell sorting and microscopy. Inhibition of DNA repair was associated with increased green fluorescent protein (GFP) expression from a replication-defective adenoviral vector (Ad-CMV-GFP). Radiation-induced up-regulation of gene expression was abrogated by inhibitors of MAPK (PD980059 and U0126) and phosphatidylinositol 3-kinase (LY294002) but not by p38 MAPK inhibition. A reporter plasmid assay in which GFP was under the transcriptional control of artificial Egr-1 or cytomegalovirus promoters showed that the DNA repair inhibitors increased GFP expression only in the context of the Egr-1 promoter. In vivo administration of a water-soluble DNA-PK inhibitor (KU0060648) was shown to maintain luciferase expression in HCT116 xenografts after intratumoral delivery of Ad-RSV-Luc. These data have important implications for therapeutic strategies involving multimodality use of radiation, targeted drugs, and adenoviral gene delivery and provide a framework for evaluating potential advantageous combinatorial effects. [Cancer Res 2008;68(23):9771–8]
N-Myc Regulates a Widespread Euchromatic Program in the Human Genome Partially Independent of Its Role as a Classical Transcription Factor
Authors: Cotterman Rebecca , Jin Victor X. , Krig Sheryl R. , Lemen Jessica M. , Wey Alice , Farnham Peggy J. , Knoepfler Paul S.
Published: December 2008

Myc proteins have long been modeled to operate strictly as classic gene-specific transcription factors; however, we find that N-Myc has a robust role in the human genome in regulating global cellular euchromatin, including that of intergenic regions. Strikingly, 90% to 95% of the total genomic euchromatic marks histone H3 acetylated at lysine 9 and methylated at lysine 4 is N-Myc–dependent. However, Myc regulation of transcription, even of genes it directly binds and at which it is required for the maintenance of active chromatin, is generally weak. Thus, Myc has a much more potent ability to regulate large domains of euchromatin than to influence the transcription of individual genes. Overall, Myc regulation of chromatin in the human genome includes both specific genes, but also expansive genomic domains that invoke functions independent of a classic transcription factor. These findings support a new dual model for Myc chromatin function with important implications for the role of Myc in cancer and stem cell biology, including that of induced pluripotent stem cells. [Cancer Res 2008;68(23):9654–62]
Overcoming Immunoescape Mechanisms of BCL1 Leukemia and Induction of CD8+ T-Cell–Mediated BCL1-Specific Resistance in Mice Cured by Targeted Polymer-Bound Doxorubicin
Authors: Kovar Marek , Tomala Jakub , Chmelova Helena , Kovar Lubomir , Mrkvan Tomas , Joskova Radka , Zakostelska Zuzana , Etrych Tomas , Strohalm Jiri , Ulbrich Karel , Sirova Milada , Rihova Blanka
Published: December 2008

BALB/c mice bearing syngeneic BCL1 leukemia, a mouse model of human chronic lymphocytic leukemia, were treated with polymer-bound doxorubicin conjugate targeted with BCL1-specific monoclonal antibody. Such treatment can cure up to 100% of mice and the cured mice show long-lasting resistance to BCL1 leukemia. We show that both CD4+ and CD8+ T cells are required for establishment of the resistance, but only CD8+ T cells are necessary for its maintenance. BCL1 cells express MHC class I and II and also costimulatory molecules CD80 and CD86, which can aid eliciting of antitumor response. On the other hand, BCL1 cells also use several immunoescape mechanisms, such as expression of PD-L1, PD-L2, and interleukin-10. BCL1 cells thus can be recognized by BCL1-specific T cells, but instead of effective priming, such T cells are anergized or deleted by apoptosis. Moreover, BCL1 leukemia progression is accompanied by robust expansion of CD4+CD25+Foxp3+ regulatory T (Treg) cells. Although it has been shown that depletion of Treg cells in tumor-bearing mice can retard tumor growth, direct evidence that expansion of Treg cells can promote tumor growth was lacking. In this study, we provide first direct evidence that expanded Treg cells can indeed promote tumor progression by using mice with selectively expanded Treg cells before inoculation of BCL1 leukemia. Finally, we have also shown that elimination of some immunoescape mechanism (e.g., deletion of Treg) can significantly improve the therapeutic outcome of chemotherapy. [Cancer Res 2008;68(23):9875–83]
Requirement for Estrogen Receptor α in a Mouse Model for Human Papillomavirus–Associated Cervical Cancer
Authors: Chung Sang-Hyuk , Wiedmeyer Kerri , Shai Anny , Korach Kenneth S. , Lambert Paul F.
Published: December 2008

The majority of human cervical cancers are associated with the high-risk human papillomaviruses (HPV), which encode the potent E6 and E7 oncogenes. On prolonged treatment with physiologic levels of exogenous estrogen, K14E7 transgenic mice expressing HPV-16 E7 oncoprotein in their squamous epithelia succumb to uterine cervical cancer. Furthermore, prolonged withdrawal of exogenous estrogen results in complete or partial regression of tumors in this mouse model. In the current study, we investigated whether estrogen receptor α (ERα) is required for the development of cervical cancer in K14E7 transgenic mice. We show that exogenous estrogen fails to promote either dysplasia or cervical cancer in K14E7/ERα−/− mice despite the continued presence of the presumed cervical cancer precursor cell type, reserve cells, and evidence for E7 expression therein. We also observed that cervical cancers in our mouse models are strictly associated with atypical squamous metaplasia (ASM), which is believed to be the precursor for cervical cancer in women. Consistently, E7 and exogenous estrogen failed to promote ASM in the absence of ERα. We conclude that ERα plays a crucial role at an early stage of cervical carcinogenesis in this mouse model. [Cancer Res 2008;68(23):9928–34]
Chromosome 12p Deletions inTEL-AML1Childhood Acute Lymphoblastic Leukemia Are Associated with Retrotransposon Elements and Occur Postnatally
Authors: Wiemels Joseph L. , Hofmann Jerry , Kang Michelle , Selzer Rebecca , Green Roland , Zhou Mi , Zhong Sheng , Zhang Luoping , Smith Martyn T. , Marsit Carmen , Loh Mignon , Buffler Patricia , Yeh Ru-Fang
Published: December 2008

TEL-AML1 (ETV6-RUNX1) is the most common translocation in the childhood leukemias, and is a prenatal mutation in most children. This translocation has been detected at a high rate among newborns (∼1%); therefore, the rate-limiting event for leukemia seems to be secondary mutations. One such frequent mutation in this subtype is partial deletion of chromosome 12p, trans from the translocation. Nine del(12p) breakpoints within six leukemia cases were sequenced to explore the etiology of this genetic event, and most involved cryptic sterile translocations. Twelve of 18 del(12p) parent sequences involved in these breakpoints were located in repeat regions (8 of these in long interspersed nuclear elements). This stands in contrast with TEL-AML1, in which only 21 of 110 previously assessed breakpoints (19%) occur in DNA repeats (P = 0.0001). An exploratory assessment of archived neonatal blood cards revealed significantly more long interspersed nuclear element CpG methylations in individuals at birth who were later diagnosed with TEL-AML1 leukemia, compared with individuals who did not contract leukemia (P = 0.01). Nontemplate nucleotides were also more frequent in del(12p) than in TEL-AML1 junctions (P = 0.004), suggesting formation by terminal deoxynucleotidyl transferase. Assessment of six archived neonatal blood cards indicated that no del(12p) rearrangements backtracked to birth, although two of these patients were previously positive for TEL-AML1 using the same assay with comparable sensitivity. These data are compatible with a two-stage natural history: TEL-AML1 occurs prenatally, and del(12p) occurs postnatally in more mature cells with a structure that suggests the involvement of retrotransposon instability. [Cancer Res 2008;68(23):9935–44]
Do Placental Genes Affect Maternal Breast Cancer? Association between Offspring's CGB5 and CSH1 Gene Variants and Maternal Breast Cancer Risk
Authors: Chen Yu , Kibriya Muhammad G. , Jasmine Farzana , Santella Regina M. , Senie Ruby T. , Ahsan Habibul
Published: December 2008

The protective effect of full-term pregnancy against breast cancer is thought to be induced by two placental hormones: human chorionic gonadotropin and human chorionic somatotropin hormone (CSH) produced by the placental trophoblastic cells. We hypothesized that variants in placental genes encoding these hormones may alter maternal breast cancer risk subsequent to pregnancy. We conducted a case-control study to examine the association between polymorphisms in a woman's placental (i.e., her offspring's) homologous chorionic gonadotrophin β5 (CGB5) and CSH1 genes and her post-pregnancy breast cancer risk. A total of 293 breast cancer cases and 240 controls with at least one offspring with available DNA were selected from the New York site of the Breast Cancer Family Registry. Three single nucleotide polymorphisms (SNP) in CGB5 and CSH1 genes were genotyped for 844 offspring of the cases and controls. Overall, maternal breast cancer risk did not significantly differ by the offspring's carrier status of the three SNPs. Among women with an earlier age at childbirth (younger than the median age of 26 years), those with a child carrying the variant C allele of CGB5 rs726002 SNP had an elevated breast cancer risk [odds ratio (OR), 2.09; 95% confidence interval (95% CI), 1.17–3.73]. Among women with a later age at childbirth, breast cancer risk did not differ by offspring's carrier status of CGB5 rs726002 SNP (OR, 0.90; 95% CI, 0.53–1.51; P for interaction = 0.04). The findings suggest that placental CGB5 genotype may be predictive of maternal post-pregnancy breast cancer risk among women who give birth early in life. [Cancer Res 2008;68(23):9729–34]
Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression
Authors: Aleku Manuela , Schulz Petra , Keil Oliver , Santel Ansgar , Schaeper Ute , Dieckhoff Britta , Janke Oliver , Endruschat Jens , Durieux Birgit , Röder Nadine , Löffler Kathrin , Lange Christian , Fechtner Melanie , Möpert Kristin , Fisch Gerald , Dames Sibylle , Arnold Wolfgang , Jochims Karin , Giese Klaus , Wiedenmann Bertram , Scholz Arne , Kaufmann Jörg
Published: December 2008

We have previously described a small interfering RNA (siRNA) delivery system (AtuPLEX) for RNA interference (RNAi) in the vasculature of mice. Here we report preclinical data for Atu027, a siRNA-lipoplex directed against protein kinase N3 (PKN3), currently under development for the treatment of advanced solid cancer. In vitro studies revealed that Atu027-mediated inhibition of PKN3 function in primary endothelial cells impaired tube formation on extracellular matrix and cell migration, but is not essential for proliferation. Systemic administration of Atu027 by repeated bolus injections or infusions in mice, rats, and nonhuman primates results in specific, RNAi-mediated silencing of PKN3 expression. We show the efficacy of Atu027 in orthotopic mouse models for prostate and pancreatic cancers with significant inhibition of tumor growth and lymph node metastasis formation. The tumor vasculature of Atu027-treated animals showed a specific reduction in lymph vessel density but no significant changes in microvascular density. [Cancer Res 2008;68(23):9788–98]
Chemotherapy Induces Tumor Clearance Independent of Apoptosis
Authors: Guerriero Jennifer L. , Ditsworth Dara , Fan Yongjun , Zhao Fangping , Crawford Howard C. , Zong Wei-Xing
Published: December 2008

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. The ultimate goal of cancer treatment is to selectively induce cancer cell death and overcome drug resistance. A deeper understanding of how a given chemotherapy affects tumor cell death is needed to develop strategically designed anticancer agents. Here, we use a xenograft mouse tumor system generated from genetically defined cells deficient in apoptosis to examine the involvement of multiple forms of cell death induced by cyclophosphamide (CP), a DNA alkylating agent commonly used in chemotherapy. We find that although apoptosis facilitates tumor regression, it is dispensable for complete tumor regression as other forms of cell death are activated. Sporadic necrosis is observed in both apoptosis-competent and deficient tumors evident by tumor cell morphology, extracellular release of high mobility group box 1 protein, and activation of innate immune cells in CP-treated tumors. Our findings indicate that in apoptosis-deficient tumors, necrosis may play a fundamental role in tumor clearance by stimulating the innate immune response. [Cancer Res 2008;68(23):9595–600]
F14512, a Potent Antitumor Agent Targeting Topoisomerase II Vectored into Cancer Cells via the Polyamine Transport System
Authors: Barret Jean-Marc , Kruczynski Anna , Vispé Stéphane , Annereau Jean-Philippe , Brel Viviane , Guminski Yves , Delcros Jean-Guy , Lansiaux Amélie , Guilbaud Nicolas , Imbert Thierry , Bailly Christian
Published: December 2008

The polyamine transport system (PTS) is an energy-dependent machinery frequently overactivated in cancer cells with a high demand for polyamines. We have exploited the PTS to selectively deliver a polyamine-containing drug to cancer cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine tail supports three complementary functions: (a) facilitate formulation of a water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II inhibition, and (c) facilitate selective uptake by tumor cells via the PTS. F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210 leukemia cells to F14512 was observed in the presence of putrescine, spermidine, and spermine. In parallel, the spermine moiety considerably enhances the drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II. The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA anchor, and this property translates at the cellular level into a distinct pharmacologic profile. Twenty-nine human solid or hematologic cell lines were used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18 μmol/L). Finally, the potent antitumor activity of F14512 in vivo was evidenced with a MX1 human breast tumor xenograft model, with partial and complete tumor regressions. This work supports the clinical development of F14512 as a novel targeted cytotoxic drug and sheds light on the concept of selective delivery of drugs to tumor cells expressing the PTS. [Cancer Res 2008;68(23):9845–53]
The Role of CD133 in Normal Human Prostate Stem Cells and Malignant Cancer-Initiating Cells
Authors: Vander Griend Donald J. , Karthaus Wouter L. , Dalrymple Susan , Meeker Alan , DeMarzo Angelo M. , Isaacs John T.
Published: December 2008

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting–purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (ΔNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor–positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires “stem-like activity” and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs. [Cancer Res 2008;68(23):9703–11]
In vivo Expansion, Persistence, and Function of Peptide Vaccine–Induced CD8 T Cells Occur Independently of CD4 T Cells
Authors: Assudani Deepak , Cho Hyun-Il , DeVito Nicholas , Bradley Norma , Celis Esteban
Published: December 2008

Significant efforts are being devoted toward the development of effective therapeutic vaccines against cancer. Specifically, well-characterized subunit vaccines, which are designed to generate antitumor cytotoxic CD8 T-cell responses. Because CD4 T cells participate at various stages of CD8 T-cell responses, it is important to study the role of CD4 T cells in the induction and persistence of antitumor CD8 T-cell responses by these vaccines. Recent evidence points to the requirement of CD4 T cells for the long-term persistence of memory CD8 T cells, which in the case of cancer immunotherapy would be critical for the prevention of tumor recurrences. The purpose of the present study was to assess whether CD4 T cells are necessary for the generation and maintenance of antigen-specific CD8 T cells induced by subunit (peptide or DNA) vaccines. We have used a vaccination strategy that combines synthetic peptides representing CD8 T-cell epitopes, a costimulatory anti-CD40 antibody and a Toll-like receptor agonist (TriVax) to generate large numbers of antigen-specific CD8 T-cell responses. Our results show that the rate of decline (clonal contraction) of the antigen-specific CD8 T cells and their functional state is not affected by the presence or absence of CD4 T cells throughout the immune response generated by TriVax. We believe that these results bear importance for the design of effective vaccination strategies against cancer. [Cancer Res 2008;68(23):9892–9]
Centromere-Associated Protein E: A Motor That Puts the Brakes on the Mitotic Checkpoint
Authors: Wood Kenneth W. , Chua Penelope , Sutton David , Jackson Jeffrey R.
Published: December 2008

Cell cycle checkpoints have long been recognized as important nodes for regulating cell proliferation and maintaining genomic integrity. These checkpoints are often altered in cancer and represent promising points for therapeutic intervention. Until recently, direct targeting of the mitotic checkpoint has been an untapped area for cancer drug discovery. Regulation of the mitotic checkpoint is complex, but many of the critical players have been identified and functionally characterized. A substantial number of these proteins can be localized to the kinetochore, a structure located at the centromeric region of each mitotic chromosome. The kinetochore mediates chromosome attachment to spindle microtubules and subsequent chromosome movement. The mitotic checkpoint monitors microtubule attachment and chromosome position on the mitotic spindle, inhibiting progression into anaphase until proper attachment and metaphase positioning is achieved. Centromere-associated protein E is a kinesin microtubule motor protein that plays an essential role in integrating the mechanics of microtubule-chromosome interactions with mitotic checkpoint signaling, and has emerged as a novel target for cancer therapy.
Epigenetic Inactivation of Galanin Receptor 1 in Head and Neck Cancer
Authors: Misawa Kiyoshi , Ueda Yo , Kanazawa Takeharu , Misawa Yuki , Jang Ilwhan , Brenner John Chadwick , Ogawa Tetsuya , Takebayashi Satoru , Grenman Reidar A. , Herman James G. , Mineta Hiroyuki , Carey Thomas E.
Published: December 2008

Purpose: One copy of the galanin receptor 1 (GALR1) locus on 18q is often deleted and expression is absent in some head and neck squamous cell carcinoma (HNSCC) cell lines. To determine if loss of heterozygosity and hypermethylation might silence the GALR1 gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors.

Experimental Design: Promoter methylation of GALR1 in 72 cell lines and 100 primary tumor samples was analyzed using methylation-specific PCR. GALR1 expression and methylation status were analyzed further by real-time PCR and bisulfite sequencing analysis.

Results: The GALR1 promoter was fully or partially methylated in 38 of 72 (52.7%) HNSCC cell lines but not in the majority 18 of 20 (90.0%) of nonmalignant lines. GALR1 methylation was also found in 38 of 100 (38%) primary tumor specimens. Methylation correlated with decreased GALR1 expression. In tumors, methylation was significantly correlated with increased tumor size (P = 0.0036), lymph node status (P = 0.0414), tumor stage (P = 0.0037), cyclin D1 expression (P = 0.0420), and p16 methylation (P = 0.0494) and survival (P = 0.045). Bisulfite sequencing of 36 CpG sites upstream of the transcription start site revealed that CpG methylation within transcription factor binding sites correlated with complete suppression of GALR1 mRNA. Treatment with trichostatin A and 5-azacytidine restored GALR1 expression. In UM-SCC-23 cells that have total silencing of GALR1, exogenous GALR1 expression and stimulation with galanin suppressed cell proliferation.

Conclusions: Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after reexpression support the hypothesis that GALR1 is a tumor suppressor gene in HNSCC.