bluuebunny/crossref_metadata_2025 huggingface dataset:
Eye, Head, and Body Coordination During Large Gaze Shifts in Rhesus Monkeys: Movement Kinematics and the Influence of Posture
Authors: McCluskey Meaghan K. , Cullen Kathleen E.
Published: January 2007

Coordinated movements of the eye, head, and body are used to redirect the axis of gaze between objects of interest. However, previous studies of eye-head gaze shifts in head-unrestrained primates generally assumed the contribution of body movement to be negligible. Here we characterized eye-head-body coordination during horizontal gaze shifts made by trained rhesus monkeys to visual targets while they sat upright in a standard primate chair and assumed a more natural sitting posture in a custom-designed chair. In both postures, gaze shifts were characterized by the sequential onset of eye, head, and body movements, which could be described by predictable relationships. Body motion made a small but significant contribution to gaze shifts that were ≥40° in amplitude. Furthermore, as gaze shift amplitude increased (40–120°), body contribution and velocity increased systematically. In contrast, peak eye and head velocities plateaued at velocities of ∼250–300°/s, and the rotation of the eye-in-orbit and head-on-body remained well within the physical limits of ocular and neck motility during large gaze shifts, saturating at ∼35 and 60°, respectively. Gaze shifts initiated with the eye more contralateral in the orbit were accompanied by smaller body as well as head movement amplitudes and velocities were greater when monkeys were seated in the more natural body posture. Taken together, our findings show that body movement makes a predictable contribution to gaze shifts that is systematically influenced by factors such as orbital position and posture. We conclude that body movements are part of a coordinated series of motor events that are used to voluntarily reorient gaze and that these movements can be significant even in a typical laboratory setting. Our results emphasize the need for caution in the interpretation of data from neurophysiological studies of the control of saccadic eye movements and/or eye-head gaze shifts because single neurons can code motor commands to move the body as well as the head and eyes.
Role of Membrane Potential in Calcium Signaling During Rhythmic Bursting in Tritonia Swim Interneurons
Authors: Hill Evan S. , Katz Paul S.
Published: January 2007

Rhythmic bursting in neurons is accompanied by dynamic changes in intracellular Ca2+ concentration. These Ca2+ signals may be caused by membrane potential changes during bursting and/or by synaptic inputs. We determined that membrane potential is responsible for most, if not all, of the cytoplasmic Ca2+ signal recorded during rhythmic bursting in two neurons of the escape swim central pattern generator (CPG) of the mollusk, Tritonia diomedea: ventral swim interneuron B (VSI) and cerebral neuron 2 (C2). Ca2+ signals were imaged with a confocal laser scanning microscope while the membrane potential was recorded at the soma. During the swim motor pattern (SMP), Ca2+ signals in both neurons transiently increased during each burst of action potentials with a more rapid decay in secondary than in primary neurites. VSI and C2 were then voltage-clamped at the soma, and each neuron's own membrane potential waveform recorded during the SMP was played back as the voltage command. In all regions of VSI, this completely reproduced the amplitude and time course of Ca2+ signals observed during the SMP, but in C2, the amplitude was lower in the playback experiments than during the SMP, possibly due to space clamp problems. Therefore in VSI, the cytoplasmic Ca2+ signal during the SMP can be accounted for by its membrane potential excursions, whereas in C2 the membrane potential excursions can account for most of the SMP Ca2+ signal.
Radioiodinated versus Radiometal-Labeled Anti–Carcinoembryonic Antigen Single-Chain Fv-Fc Antibody Fragments: Optimal Pharmacokinetics for Therapy
Authors: Kenanova Vania , Olafsen Tove , Williams Lawrence E. , Ruel Nora H. , Longmate Jeffrey , Yazaki Paul J. , Shively John E. , Colcher David , Raubitschek Andrew A. , Wu Anna M.
Published: January 2007

Antibody fragments with optimized pharmacokinetic profiles hold potential for detection and therapy of tumor malignancies. We studied the behavior of three anti–carcinoembryonic antigen (CEA) single-chain Fv-Fc (scFv-Fc) variants (I253A, H310A, and H310A/H435Q; Kabat numbering system) that exhibited differential serum persistence. Biodistribution studies done on CEA-positive tumor xenografted mice revealed that the 111In-labeled I253A fragment with the slowest clearance kinetics (T1/2β, 27.7 h) achieved the highest tumor uptake (44.6% ID/g at 24 h), whereas the radiometal-labeled H310A/H435Q fragment with the most rapid elimination (T1/2β, 7.05 h) reached a maximum of 28.0% ID/g at 12 h postinjection. The H310A protein was characterized by both intermediate serum half-life and tumor uptake. The 111In-based biodistribution studies showed that all three fragments were eliminated primarily through the liver, and hepatic radiometal activity correlated with the rate of fragment clearance. The 111In-labeled H310A/H435Q protein exhibited the highest liver uptake (23.5% ID/g at 24 h). Metabolism of the 125I-labeled scFv-Fc proteins resulted in low normal organ activity. Finally, the 125I/111In biodistribution data allowed for dose estimations, which suggest the 131I-labeled scFv-Fc H310A/H435Q as a promising candidate for radioimmunotherapy. [Cancer Res 2007;67(2):718–26]
Suppression of Mammary Carcinoma Cell Growth by Retinoic Acid: the Cell Cycle Control Gene Btg2 Is a Direct Target for Retinoic Acid Receptor Signaling
Authors: Donato Leslie J. , Suh Jean H. , Noy Noa
Published: January 2007

The anticarcinogenic activities of retinoic acid (RA) are believed to be mediated by the nuclear RA receptor (RAR) and by the RA-binding protein cellular RA-binding protein-II (CRABP-II). In MCF-7 mammary carcinoma cells, growth inhibition by RA entails an early cell cycle arrest followed by induction of apoptosis. Here, we aimed to obtain insights into the initial cell cycle response. We show that a 3- to 5-h RA pulse is sufficient for inducing a robust growth arrest 2 to 4 days later, demonstrating inhibition of the G1-S transition by RA is triggered by immediate-early RAR targets and does not require the continuous presence of the hormone throughout the arrest program. Expression array analyses revealed that RA induces the expression of several genes involved in cell cycle regulation, including the p53-controlled antiproliferative gene B-cell translocation gene, member 2 (Btg2) and the BTG family member Tob1. We show that induction of Btg2 by RA does not require de novo protein synthesis and is augmented by overexpression of CRABP-II. Additionally, we identify a RA response element in the Btg2 promoter and show that the element binds retinoid X receptor/RAR heterodimers in vitro, is occupied by the heterodimers in cells, and can drive RA-induced activation of a reporter gene. Hence, Btg2 is a novel direct target for RA signaling. In concert with the reports that Btg2 inhibits cell cycle progression by down-regulating cyclin D1, induction of Btg2 by RA was accompanied by a marked decrease in cyclin D1 expression. The observations thus show that the antiproliferative activity of RA in MCF-7 cells is mediated, at least in part, by Btg2. [Cancer Res 2007;67(2):609–15]
NKX3.1 Homeodomain Protein Binds to Topoisomerase I and Enhances Its Activity
Authors: Bowen Cai , Stuart August , Ju Jeong-Ho , Tuan Jenny , Blonder Josip , Conrads Thomas P. , Veenstra Timothy D. , Gelmann Edward P.
Published: January 2007

The prostate-specific homeodomain protein NKX3.1 is a tumor suppressor that is commonly down-regulated in human prostate cancer. Using an NKX3.1 affinity column, we isolated topoisomerase I (Topo I) from a PC-3 prostate cancer cell extract. Topo I is a class 1B DNA-resolving enzyme that is ubiquitously expressed in higher organisms and many prokaryotes. NKX3.1 interacts with Topo I to enhance formation of the Topo I-DNA complex and to increase Topo I cleavage of DNA. The two proteins interacted in affinity pull-down experiments in the presence of either DNase or RNase. The NKX3.1 homeodomain was essential, but not sufficient, for the interaction with Topo I. NKX3.1 binding to Topo I occurred independently of the Topo I NH2-terminal domain. The binding of equimolar amounts of Topo I to NKX3.1 caused displacement of NKX3.1 from its cognate DNA recognition sequence. Topo I activity in prostates of Nkx3.1+/− and Nkx3.1−/− mice was reduced compared with wild-type mice, whereas Topo I activity in livers, where no NKX3.1 is expressed, was independent of Nkx3.1 genotype. Endogenous Topo I and NKX3.1 could be coimmunoprecipitated from LNCaP cells, where NKX3.1 and Topo I were found to colocalize in the nucleus and comigrate within the nucleus in response to either γ-irradiation or mitomycin C exposure, two DNA-damaging agents. This is the first report that a homeodomain protein can modify the activity of Topo I and may have implications for organ-specific DNA replication, transcription, or DNA repair. [Cancer Res 2007;67(2):455–64]
Merlin/Neurofibromatosis Type 2 Suppresses Growth by Inhibiting the Activation of Ras and Rac
Authors: Morrison Helen , Sperka Tobias , Manent Jan , Giovannini Marco , Ponta Helmut , Herrlich Peter
Published: January 2007

The small G-protein Ras is a tightly controlled regulator of cell fate. Prolonged or persistent arrest in the activated GTP-loaded state by mutation of Ras as in lung cancer or in a Ras–GTPase-activating protein as in neurofibromatosis type 1 promotes tumorigenesis. We now show that the tumor-suppressor protein merlin (mutated in neurofibromatosis type 2) also controls Ras activity. Systematic analysis of growth factor signaling located the step of merlin interference to the activation of Ras and Rac. Merlin independently uncouples both Ras and Rac from growth factor signals. In the case of Ras, merlin acts downstream of the receptor tyrosine kinase-growth factor receptor binding protein 2 (Grb2)-SOS complex. However, merlin does not bind either SOS or Ras, but it counteracts the ERM (ezrin, radixin, moesin)–dependent activation of Ras, which correlates with the formation of a complex comprising ERM proteins, Grb2, SOS, Ras, and filamentous actin. Because efficient signaling from Ras requires Rac-p21-activated kinase–dependent phosphorylations of Raf and mitogen-activated protein/extracellular signal-regulated kinase kinase, merlin can also inhibit signal transfer from dominantly active Ras mutants. We propose that the interference of merlin with Ras- and Rac-dependent signal transfer represents part of the tumor-suppressive action of merlin. [Cancer Res 2007;67(2):520–7]
Distinguishing Cancer-Associated Missense Mutations from Common Polymorphisms
Authors: Kaminker Joshua S. , Zhang Yan , Waugh Allison , Haverty Peter M. , Peters Brock , Sebisanovic Dragan , Stinson Jeremy , Forrest William F. , Bazan J. Fernando , Seshagiri Somasekar , Zhang Zemin
Published: January 2007

Missense variants are commonly identified in genomic sequence but only a small fraction directly contribute to oncogenesis. The ability to distinguish those missense changes that contribute to cancer progression from those that do not is a difficult problem usually only accomplished through functional in vivo analyses. Using two computational algorithms, Sorting Intolerant from Tolerant (SIFT) and the Pfam-based LogR.E-value method, we have identified features that distinguish cancer-associated missense mutations from other classes of missense change. Our data reveal that cancer mutants behave similarly to Mendelian disease mutations, but are clearly distinct from either complex disease mutations or common single-nucleotide polymorphisms. We show that both activating and inactivating oncogenic mutations are predicted to be deleterious, although activating changes are likely to increase protein activity. Using the Gene Ontology and data from the SIFT and LogR.E-value metrics, a classifier was built that predicts cancer-associated missense mutations with a very low false-positive rate. The classifier does remarkably well in a number of different experiments designed to distinguish polymorphisms from true cancer-associated mutations. We also show that recurrently observed mutations are much more likely to be predicted to be cancer-associated than rare mutations, suggesting that our classifier will be useful in distinguishing causal from passenger mutations. In addition, from an expressed sequence tag–based screen, we identified a previously unknown germ line change (P1104A) in tumor tissues that is predicted to disrupt the function of the TYK2 protein. The data presented here show that this novel bioinformatics approach to classifying cancer-associated variants is robust and can be used for large-scale analyses. [Cancer Res 2007;67(2):465–73]
Synuclein-γ Targeting Peptide Inhibitor that Enhances Sensitivity of Breast Cancer Cells to Antimicrotubule Drugs
Authors: Singh Vinay K. , Zhou Yue , Marsh Joseph A. , Uversky Vladimir N. , Forman-Kay Julie D. , Liu Jingwen , Jia Zongchao
Published: January 2007

Synuclein-γ (SNCG) plays oncogenic roles in breast carcinogenesis. Although the expression of SNCG is abnormally high in advanced and metastatic breast carcinomas, SNCG is not expressed in normal or benign breast tissues. SNCG is an intrinsically disordered protein known to interact with BubR1, a mitotic checkpoint kinase. The SNCG-BubR1 interaction inhibits mitotic checkpoint control upon spindle damage caused by anticancer drugs, such as nocodazole and taxol. Antimicrotubule drugs that cause mitotic arrest and subsequent apoptosis of cancer cells are frequently used to treat breast cancer patients with advanced or metastatic diseases. However, patient response rates to this class of chemotherapeutic agents vary significantly. In this study, we have designed a novel peptide (ANK) and shown its interaction with SNCG using fluorometry, surface plasmon resonance, and isothermal titration calorimetry. Binding of the ANK peptide did not induce folding of SNCG, suggesting that SNCG can function biologically in its intrinsically disordered state. Microinjection of the ANK peptide in breast cancer cell line overexpressing SNCG (MCF7-SNCG) exhibited a similar cell killing response by nocodazole as in the SNCG-negative MCF7 cells. Overexpression of enhanced green fluorescent protein–tagged ANK reduces SNCG-mediated resistance to paclitaxel treatment by ∼3.5-fold. Our coimmunoprecipitation and colocalization results confirmed the intracellular association of the ANK peptide with SNCG. This is likely due to the disruption of the interaction of SNCG with BubR1 interaction. Our findings shed light on the molecular mechanism of the ANK peptide in releasing SNCG-mediated drug resistance. [Cancer Res 2007;67(2):626–33]
IFN-α–Stimulated Genes and Epstein-Barr Virus Gene Expression Distinguish WHO Type II and III Nasopharyngeal Carcinomas
Authors: Pegtel D. Michiel , Subramanian Aravind , Meritt David , Tsai Ching-Hwa , Sheen Tzung-Shiahn , Golub Todd R. , Thorley-Lawson David A.
Published: January 2007

Nonkeratinizing nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr Virus (EBV) and divided into two subtypes (WHO types II and III) based on histology. We tested whether these subtypes can be distinguished at the molecular genetic level using an algorithm that analyzes sets of related genes (gene set enrichment analysis). We found that a class of IFN-stimulated genes (ISG), frequently associated with the antiviral response, was significantly activated in type III versus type II NPC. Consistent with this, replication of the endogenous EBV was suppressed in type III. A strong association was also seen with a subset of ISGs previously identified in systemic lupus erythematosus, another disease in which ‘normal’ EBV biology is deregulated, suggesting that this pattern of ISG expression may be linked to the increased EBV activity in both diseases. In contrast, unsupervised hierarchical clustering of the complete expression profiles failed to distinguish the two subsets. These results suggest that type II and III NPC have not originated from obviously distinct epithelial precursors; rather, the histologic differences may be a consequence of a differential antiviral response, involving IFNs, to chronic EBV infection. [Cancer Res 2007;67(2):474–81]
RUNX1 DNA-Binding Mutants, Associated with Minimally Differentiated Acute Myelogenous Leukemia, Disrupt Myeloid Differentiation
Authors: Cammenga Jörg , Niebuhr Birte , Horn Stefan , Bergholz Ulla , Putz Gabriele , Buchholz Frank , Löhler Jürgen , Stocking Carol
Published: January 2007

Mutations in the RUNX1 gene are found at high frequencies in minimally differentiated acute myelogenous leukemia. In addition to null mutations, many of the mutations generate Runx1 DNA-binding (RDB) mutants. To determine if these mutants antagonize wild-type protein activity, cDNAs were transduced into murine bone marrow or human cord blood cells using retroviral vectors. Significantly, the RDB mutants did not act in a transdominant fashion in vivo to disrupt Runx1 activity in either T-cell or platelet development, which are highly sensitive to Runx1 dosage. However, RDB mutant expression impaired expansion and differentiation of the erythroid compartment in which Runx1 expression is normally down-regulated, showing that a RDB-independent function is incompatible with erythroid differentiation. Significantly, both bone marrow progenitors expressing RDB mutants or deficient for Runx1 showed increased replating efficiencies in vitro, accompanied by the accumulation of myeloblasts and dysplastic progenitors, but the effect was more pronounced in RDB cultures. Disruption of the interface that binds CBFβ, an important cofactor of Runx1, did not impair RDB mutant replating activity, arguing against inactivation of Runx1 function by CBFβ sequestration. We propose that RDB mutants antagonize Runx1 function in early progenitors by disrupting a critical balance between DNA-binding–independent and DNA-binding–dependent signaling. [Cancer Res 2007;67(2):537–45]
Cytochrome P450 1B1–Mediated Estrogen Metabolism Results in Estrogen-Deoxyribonucleoside Adduct Formation
Authors: Belous Alexandra R. , Hachey David L. , Dawling Sheila , Roodi Nady , Parl Fritz F.
Published: January 2007

The oxidative metabolism of estrogens has been implicated in the development of breast cancer; yet, relatively little is known about the mechanism by which estrogens cause DNA damage and thereby initiate mammary carcinogenesis. To determine how the metabolism of the parent hormone 17β-estradiol (E2) leads to the formation of DNA adducts, we used the recombinant, purified phase I enzyme, cytochrome P450 1B1 (CYP1B1), which is expressed in breast tissue, to oxidize E2 in the presence of 2′-deoxyguanosine or 2′-deoxyadenosine. We used both gas and liquid chromatography with tandem mass spectrometry to measure E2, the 2- and 4-catechol estrogens (2-OHE2, 4-OHE2), and the depurinating adducts 4-OHE2-1(α,β)-N7-guanine (4-OHE2-N7-Gua) and 4-OHE2-1(α,β)-N3-adenine (4-OHE2-N3-Ade). CYP1B1 oxidized E2 to the catechol 4-OHE2 and the labile quinone 4-hydroxyestradiol-quinone to produce 4-OHE2-N7-Gua and 4-OHE2-N3-Ade in a time- and concentration-dependent manner. Because the reactive quinones were produced as part of the CYP1B1-mediated oxidation reaction, the adduct formation followed Michaelis-Menten kinetics. Under the conditions of the assay, the 4-OHE2-N7-Gua adduct (Km, 4.6 ± 0.7 μmol/L; kcat, 45 ± 1.6/h) was produced 1.5 times more efficiently than the 4-OHE2-N3-Ade adduct (Km, 4.6 ± 1.0 μmol/L; kcat, 30 ± 1.5/h). The production of adducts was two to three orders of magnitude lower than the 4-OHE2 production. The results present direct proof of CYP1B1-mediated, E2-induced adduct formation and provide the experimental basis for future studies of estrogen carcinogenesis. [Cancer Res 2007;67(2):812–7]
Lenalidomide and CC-4047 Inhibit the Proliferation of Malignant B Cells while Expanding Normal CD34+ Progenitor Cells
Authors: Verhelle Dominique , Corral Laura G. , Wong Kevin , Mueller Jessica H. , Moutouh-de Parseval Laure , Jensen-Pergakes Kristen , Schafer Peter H. , Chen Roger , Glezer Emilia , Ferguson Gregory D. , Lopez-Girona Antonia , Muller George W. , Brady Helen A. , Chan Kyle W.H.
Published: January 2007

Clinical studies involving patients with myelodysplastic syndromes or multiple myeloma have shown the efficacy of lenalidomide by reducing and often eliminating malignant cells while restoring the bone marrow function. To better understand these clinical observations, we investigated and compared the effects of lenalidomide and a structurally related analogue, CC-4047, on the proliferation of two different human hematopoietic cell models: the Namalwa cancer cell line and normal CD34+ progenitor cells. Both compounds had antiproliferative effects on Namalwa cells and pro-proliferative effects on CD34+ cells, whereas p21WAF-1 expression was up-regulated in both cell types. In Namalwa cells, the up-regulation of p21WAF-1 correlated well with the inhibition of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 activity leading to pRb hypophosphorylation and cell cycle arrest, whereas in CD34+ progenitor cells the increase of p21WAF-1 did not inhibit proliferation. Similarly, antiproliferation results were observed in two B lymphoma cell lines (LP-1 and U266) but interestingly not in normal B cells where a protection of apoptosis was found. Finally, CC-4047 and lenalidomide had synergistic effects with valproic acid [a histone deacetylase (HDAC) inhibitor] by increasing the apoptosis of Namalwa cells and enhancing CD34+ cell expansion. Our results indicate that lenalidomide and CC-4047 have opposite effects in tumor cells versus normal cells and could explain, at least in part, the reduction of malignant cells and the restoration of bone marrow observed in patients undergoing lenalidomide treatment. Moreover, this study provides new insights on the cellular pathways affected by lenalidomide and CC-4047, proposes new potential clinical uses, such as bone marrow regeneration, and suggests that the combination of lenalidomide or CC-4047 with certain HDAC inhibitors may elevate the therapeutic index in the treatment of hematologic malignancies. [Cancer Res 2007;67(2):746–55]
Sphingosine-Induced Apoptosis in Rhabdomyosarcoma Cell Lines Is Dependent on Pre-Mitochondrial Bax Activation and Post-Mitochondrial Caspases
Authors: Phillips Darren C. , Martin Sophie , Doyle Belinda T. , Houghton Janet A.
Published: January 2007

Sphingolipids is the collective term ascribed to components of the sphingomyelin cycle. Modulation of the cellular levels of individual sphingolipids can induce a diverse range of cellular responses including apoptosis, proliferation, and cell cycle arrest. We present data showing that rhabdomyosarcoma cell lines, independent of lineage (alveolar rhabdomyosarcoma and embryonal rhabdomyosarcoma), are particularly sensitive to the induction of apoptosis as a result of an elevation in the cellular levels of sphingosine (d-erythro-sphingosine). Sphingosine-mediated apoptosis does not require its metabolism to the related proapoptotic molecule ceramide and is stereospecific because exposure of the rhabdomyosarcoma cell line RD to the l-erythro and dl-threo isoforms of sphingosine did not induce apoptosis. Importantly, for efficient induction of apoptosis, sphingosine required Bax activation and consequential translocation to the mitochondria. This resulted in selective mitochondrial release of cytochrome c and Smac/Diablo but not other mitochondrial related factors (apoptosis-inducing factor, endonuclease G, and HtrA2/Omi). Using small interfering RNA, reduced Bax expression conferred the impaired release of mitochondrial cytochrome c to the cytoplasm following sphingosine exposure, inhibiting the induction of apoptosis. Furthermore, dissipation of the inner mitochondrial membrane potential and enhanced production of reactive oxygen species were not observed. Bax activation and cytochrome c release were independent of caspases; however, caspase-3 and caspase-9 activity distal to the mitochondria was essential for the execution of apoptosis. [Cancer Res 2007;67(2):756–64]
Human prx1 Gene Is a Target of Nrf2 and Is Up-regulated by Hypoxia/Reoxygenation: Implication to Tumor Biology
Authors: Kim Yun-Jeong , Ahn Ji-Yeon , Liang Ping , Ip Clement , Zhang Yuesheng , Park Young-Mee
Published: January 2007

Peroxiredoxin 1 (Prx1) has been found to be elevated in several human cancers. The cell survival–enhancing function of Prx1 is traditionally attributed to its reactive oxygen species–removing capacity, although the growth-promoting role of Prx1 independent of this antioxidant activity is increasingly gaining attention. Although much progress has been made in understanding the behavior of Prx1, little information is available on the mechanism responsible for the abnormal elevation of Prx1 level in cancer. We hypothesized that the hypoxic and unstable oxygenation microenvironment of a tumor might be crucial for prx1 up-regulation. In this study, we cloned the human prx1 promoter and identified nuclear factor (erythroid-derived 2)–related factor 2 (Nrf2) as a key transcription factor. Hypoxia/reoxygenation, an in vitro condition suited to mimic changes of oxygenation, increased Nrf2 nuclear localization and its binding to the electrophile-responsive elements located at the proximal (−536 to −528) and distal (−1429 to −1421) regions of the prx1 promoter. A significant reduction of both steady-state and hypoxia/reoxygenation–mediated prx1 gene expression was shown in Nrf2 knock-out cells. Our results indicated that decreased Kelch-like ECH-associated protein, Keap1, might be an important mechanism for the increased nuclear translocation and activation of Nrf2 in response to hypoxia/reoxygenation. A constitutive elevation of prx1 mRNA and protein was observed in Keap1 knock-out cells. The above information suggests that the Nrf2-Prx1 axis may be a fruitful target for intervention with respect to inhibiting the malignant progression and/or reducing the treatment resistance of cancer cells. [Cancer Res 2007;67(2):546–53]
PRL-1 Tyrosine Phosphatase Regulates c-Src Levels, Adherence, and Invasion in Human Lung Cancer Cells
Authors: Achiwa Hiroyuki , Lazo John S.
Published: January 2007

Phosphatases of regenerating liver (PRL) constitute a subfamily of the protein tyrosine phosphatases that are implicated in oncogenic and metastatic phenotypes. In this study, we evaluated the role of PRL-1 in cell proliferation and metastatic processes in human lung cancer cells. We stably transfected human A549 lung cancer cells with several short hairpin RNAs for PRL-1 and found decreased invasive activity in the resulting clones compared with control cells. In addition, cells with suppressed PRL-1 exhibited greater adherence and cell spreading on fibronectin and a decreased proliferation rate compared with control cells. To address possible mechanisms for the altered phenotypes, we examined known biochemical regulators of adhesion and invasion. Inhibition of PRL-1 decreased c-Src and p130Cas expression and Rac1 and Cdc42 activation without any apparent modification of focal adhesion kinase (FAK) expression. Total tyrosine FAK phosphorylation and Tyr397 phosphorylation levels were continuously elevated in PRL-1 knockdown cells plated on fibronectin. In immunofluorescence studies, reduction in PRL-1 seemed to decrease cell membrane protrusions with a reduction in actin fiber extensions in spite of continuous phosphorylation of Tyr397 FAK, which could reflect reduced adhesion turnover. Our data implicate PRL-1 in the fundamental process of cell adhesion and migration in human lung cancer cells by affecting Rac1, Cdc42, and c-Src activation. These results support the hypothesis that PRL-1 plays an important role in maintaining the malignant phenotype by exploiting Src activation processes, and that PRL-1 could be a promising therapeutic target for cancer metastasis and cell growth. [Cancer Res 2007;67(2):643–50]
Epigallocatechin-3-Gallate Enhances CD8+ T Cell–Mediated Antitumor Immunity Induced by DNA Vaccination
Authors: Kang Tae Heung , Lee Jin Hyup , Song Chung Kil , Han Hee Dong , Shin Byung Cheol , Pai Sara I. , Hung Chien-Fu , Trimble Cornelia , Lim Jong-Seok , Kim Tae Woo , Wu T-C.
Published: January 2007

Immunotherapy and chemotherapy are generally effective against small tumors in animal models of cancer. However, these treatment regimens are generally ineffective against large, bulky tumors. We have found that a multimodality treatment regimen using DNA vaccination in combination with chemotherapeutic agent epigallocatechin-3-gallate (EGCG), a compound found in green tea, is effective in inhibiting large tumor growth. EGCG was found to induce tumor cellular apoptosis in a dose-dependent manner. The combination of EGCG and DNA vaccination led to an enhanced tumor-specific T-cell immune response and enhanced antitumor effects, resulting in a higher cure rate than either immunotherapy or EGCG alone. In addition, combined DNA vaccination and oral EGCG treatment provided long-term antitumor protection in cured mice. Cured animals rejected a challenge of E7-expressing tumors, such as TC-1 and B16E7, but not a challenge of B16 7 weeks after the combined treatment, showing antigen-specific immune responses. These results suggest that multimodality treatment strategies, such as combining immunotherapy with a tumor-killing cancer drug, may be a more effective anticancer strategy than single-modality treatments. [Cancer Res 2007;67(2):802–11]
Hypoxia-Inducible Factor-1α Is a Key Regulator of Metastasis in a Transgenic Model of Cancer Initiation and Progression
Authors: Liao Debbie , Corle Courtney , Seagroves Tiffany N. , Johnson Randall S.
Published: January 2007

Adaptation to hypoxia is a critical step in tumor progression and is, in part, regulated by the transcription factor hypoxia-inducible factor-1α (HIF-1α). Xenograft models have been extensively used to characterize the role of HIF-1α in experimental cancers. Although these models provide an understanding of tumor growth at terminal stages of malignancy, they do not address tumor initiation or metastatic progression. To elucidate these roles, HIF-1α was conditionally deleted in the mammary epithelium of a transgenic mouse model for metastatic breast cancer. Conditional deletion of HIF-1α in the mammary epithelium resulted in delayed tumor onset and retarded tumor growth; this was correlated with decreased tumor cell proliferation. Tumors with conditional deletion of HIF-1α were also less vascular during early tumor progression. Perhaps most surprisingly, deletion of HIF-1α in the mammary epithelium resulted in decreased pulmonary metastasis. These results show that whereas HIF-1α is not required for the initiation of breast tumor growth or tumor cell metastasis, the transcriptional activity of HIF-1α is a significant positive regulator of tumor progression and metastatic potential. [Cancer Res 2007;67(2):563–72]
Nutlin3 Blocks Vascular Endothelial Growth Factor Induction by Preventing the Interaction between Hypoxia Inducible Factor 1α and Hdm2
Authors: LaRusch Gretchen A. , Jackson Mark W. , Dunbar James D. , Warren Robert S. , Donner David B. , Mayo Lindsey D.
Published: January 2007

Hdm2 is elevated in numerous types of malignancies and is thought to impede the function of wild-type p53. Reactivation of p53 by disrupting the association with Hdm2 was the impetus for the development of Nutlin3. Although regulation of p53 has been the central focus of Hdm2 activity, it also binds other proteins through its p53-binding domain. Here, we show that hypoxia-inducible factor 1α (HIF1α) binds to Hdm2 in the domain designated to bind p53. HIF1α and p53 share a conserved motif that is required to bind Hdm2. Distinct complexes form between Hdm2-HIF1α and Hdm2-p53 as determined by immunoprecipitation of nuclear extracts and in vitro. The Hdm2 antagonist Nutlin3 prevents the association between Hdm2 and HIF1α. The vascular endothelial growth factor (VEGF) gene is a transcriptional target of HIF1α, and under normoxic or hypoxic conditions, Hdm2 increases HIF1α activity to induce VEGF production. Blocking the association of Hdm2 and HIF1α by Nutlin3, or ablating Hdm2 expression, diminished the level of VEGF under conditions of normoxia or hypoxia. Our findings establish a unique role for Nutlin3 in attenuating VEGF induction by preventing the association of Hdm2 with HIF1α. [Cancer Res 2007;67(2):450–4]
An Antagonist of Dishevelled Protein-Protein Interaction Suppresses β-Catenin–Dependent Tumor Cell Growth
Authors: Fujii Naoaki , You Liang , Xu Zhidong , Uematsu Kazutsugu , Shan Jufang , He Biao , Mikami Iwao , Edmondson Lillian R. , Neale Geoffrey , Zheng Jie , Guy R. Kiplin , Jablons David M.
Published: January 2007

Recent progress in the development of inhibitors of protein-protein interactions has opened the door for developing drugs that act by novel and selective mechanisms. Building on that work, we designed a small-molecule inhibitor of the Wnt signaling pathway, which is aberrantly activated across a wide range of human tumors. The compound, named FJ9, disrupts the interaction between the Frizzed-7 Wnt receptor and the PDZ domain of Dishevelled, down-regulating canonical Wnt signaling and suppressing tumor cell growth. The antitumorigenic effects of FJ9 were pronounced, including induction of apoptosis in human cancer cell lines and tumor growth inhibition in a mouse xenograft model. FJ9 is thus among the first non-peptide inhibitors to show therapeutic efficacy through disruption of PDZ protein-protein interactions. [Cancer Res 2007;67(2):573–9]
Blocking CXCR4-Mediated Cyclic AMP Suppression Inhibits Brain Tumor GrowthIn vivo
Authors: Yang Lihua , Jackson Erin , Woerner B. Mark , Perry Arie , Piwnica-Worms David , Rubin Joshua B.
Published: January 2007

The chemokine CXCL12 and its cognate receptor CXCR4 regulate malignant brain tumor growth and are potential chemotherapeutic targets. However, the molecular basis for CXCL12-induced tumor growth remains unclear, and the optimal approach to inhibiting CXCR4 function in cancer is unknown. To develop such a therapeutic approach, we investigated the signaling pathways critical for CXCL12 function in normal and malignant cells. We discovered that CXCL12-dependent tumor growth is dependent upon sustained inhibition of cyclic AMP (cAMP) production, and that the antitumor activity of the specific CXCR4 antagonist AMD 3465 is associated with blocking cAMP suppression. Consistent with these findings, we show that pharmacologic elevation of cAMP with the phosphodiesterase inhibitor Rolipram suppresses tumor cell growth in vitro and, upon oral administration, inhibits intracranial growth in xenograft models of malignant brain tumors with comparable efficacy to AMD 3465. These data indicate that the clinical evaluation of phosphodiesterase inhibitors in the treatment of patients with brain tumors is warranted. [Cancer Res 2007;67(2):651–8]
Small Ubiquitin-Related Modifier Pathway Is a Major Determinant of Doxorubicin Cytotoxicity in Saccharomyces cerevisiae
Authors: Huang Ruea-Yea , Kowalski David , Minderman Hans , Gandhi Nishant , Johnson Erica S.
Published: January 2007

Development of drug resistance is a major challenge in cancer chemotherapy using doxorubicin. By screening the collection of Saccharomyces cerevisiae deletion strains to identify doxorubicin-resistant mutants, we have discovered that the small ubiquitin-related modifier (SUMO) pathway is a major determinant of doxorubicin cytotoxicity in yeast. Mutants lacking UBA2 (SUMO activating enzyme; E1), UBC9 (conjugating enzyme; E2), and ULP1 and ULP2 (desumoylation peptidases) are all doxorubicin resistant, as are mutants lacking MLP1, UIP3, and NUP60, which all interact with ULP1. Most informatively, mutants lacking the SUMO E3 ligase Siz1 are strongly doxorubicin resistant, whereas mutants of other SUMO ligases are either weakly resistant (siz2) or hypersensitive (mms21) to doxorubicin. These results suggest that doxorubicin cytotoxicity is regulated by Siz1-dependent sumoylation of specific proteins. Eliminating SUMO attachment to proliferating cell nuclear antigen or topoisomerase II does not affect doxorubicin cytotoxicity, whereas reducing SUMO attachment to the bud neck–associated septin proteins has a modest effect. Consistent with these results, doxorubicin resistance in the siz1Δ strain does not seem to involve an effect on DNA repair. Instead, siz1Δ cells accumulate lower intracellular levels of doxorubicin than wild-type (WT) cells, suggesting that they are defective in doxorubicin retention. Although siz1Δ cells are cross-resistant to daunorubicin, they are hypersensitive to cisplatin and show near WT sensitivity to other drugs, suggesting that the siz1Δ mutation does not cause a general multidrug resistance phenotype. Cumulatively, these results reveal that SUMO modification of proteins mediates the doxorubicin cytotoxicity in yeast, at least partially, by modification of septins and of proteins that control the intracellular drug concentration. [Cancer Res 2007;67(2):765–72]
Emerging Roles of MUC4 in Cancer: A Novel Target for Diagnosis and Therapy
Authors: Singh Ajay P. , Chaturvedi Pallavi , Batra Surinder K.
Published: January 2007

The MUC4 mucin is a transmembrane glycoprotein that is implicated in the pathogenesis of pancreatic cancer and is aberrantly expressed in many other epithelial carcinomas. Recent studies suggest its significant potential as a clinical tool for cancer diagnosis and prognosis. MUC4 modulates HER2/ErbB2 signaling and is a determinant of therapeutic outcome of Herceptin-based therapy, which further indicates its prospective usefulness in cancer therapy and treatment planning. [Cancer Res 2007;67(2):433–6]
Angiogenic Response Caused by Oncolytic Herpes Simplex Virus–Induced Reduced Thrombospondin Expression Can Be Prevented by Specific Viral Mutations or by Administering a Thrombospondin-Derived Peptide
Authors: Aghi Manish , Rabkin Samuel D. , Martuza Robert L.
Published: January 2007

Wild-type (WT) herpes simplex virus (HSV) causes some pathology, such as ocular keratitis, by increasing infected tissue vascularity, possibly reflecting altered angiogenic factor expression in infected cells. Oncolytic HSVs possess specific mutations enabling selective replication in tumor cells. We investigated whether this ability to enhance infected tissue vascularity is retained in oncolytic HSV, which could be an undesirable effect of oncolytic HSVs that may need to be addressed when treating tumors with oncolytic HSVs. s.c. tumors derived from U87 human glioma cells in athymic mice were treated with oncolytic HSVs G207 or G47Δ in the presence or absence of a recombinant protein composed of the three type-1 repeats (3TSR) of thrombospondin-1 (TSP-1). Real-time reverse transcription-PCR and Western blot of infected cultured cells measured angiogenic factor expression. Microvessel density was assessed using immunofluorescence. G207-treated U87 s.c. tumors had elevated microvessel densities compared with saline- and G47Δ-treated tumors, and G207 treatment caused delayed tumor growth resumption. G207-infected U87 and U373 cells exhibited reduced protein, not mRNA, expression of angiogenesis inhibitors TSP-1 and thrombospondin-2 (TSP-2). 3TSR restored the G207-treated tumor microvessel density to the low level of G47Δ-treated tumors and prevented delayed growth resumption. Oncolytic HSV G207 thus retains the ability of WT HSV to increase infected tissue vascularity. In infected tumors, this increased vascularity is mediated by reduced TSP-1 and TSP-2 levels and causes delayed tumor growth resumption. Incorporating viral mutations, such as those seen in G47Δ or administering thrombospondin-derived peptides, counteracts the angiogenic effect of oncolytic HSV and should be considered when designing oncolytic HSV therapies. [Cancer Res 2007;67(2):440–4]
Novel Chemical Enhancers of Heat Shock Increase Thermal Radiosensitization through a Mitotic Catastrophe Pathway
Authors: Sekhar Konjeti R. , Sonar Vijayakumar N. , Muthusamy Venkatraj , Sasi Soumya , Laszlo Andrei , Sawani Jamil , Horikoshi Nobuo , Higashikubo Ryuji , Bristow Robert G. , Borrelli Michael J. , Crooks Peter A. , Lepock James R. , Roti Roti Joseph L. , Freeman Michael L.
Published: January 2007

Radiation therapy combined with adjuvant hyperthermia has the potential to provide outstanding local-regional control for refractory disease. However, achieving therapeutic thermal dose can be problematic. In the current investigation, we used a chemistry-driven approach with the goal of designing and synthesizing novel small molecules that could function as thermal radiosensitizers. (Z)-(±)-2-(1-Benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol was identified as a compound that could lower the threshold for Hsf1 activation and thermal sensitivity. Enhanced thermal sensitivity was associated with significant thermal radiosensitization. We established the structural requirements for activity: the presence of an N-benzenesulfonylindole or N-benzylindole moiety linked at the indolic 3-position to a 2-(1-azabicyclo[2.2.2]octan-3-ol) or 2-(1-azabicyclo[2.2.2]octan-3-one) moiety. These small molecules functioned by exploiting the underlying biophysical events responsible for thermal sensitization. Thermal radiosensitization was characterized biochemically and found to include loss of mitochondrial membrane potential, followed by mitotic catastrophe. These studies identified a novel series of small molecules that represent a promising tool for the treatment of recurrent tumors by ionizing radiation. [Cancer Res 2007;67(2):695–701]
Deletion of Chromosome 11q Predicts Response to Anthracycline-Based Chemotherapy in Early Breast Cancer
Authors: Climent Joan , Dimitrow Peter , Fridlyand Jane , Palacios Jose , Siebert Reiner , Albertson Donna G. , Gray Joe W. , Pinkel Daniel , Lluch Ana , Martinez-Climent Jose A.
Published: January 2007

Despite the recent consensus on the eligibility of adjuvant systemic therapy in patients with lymph node–negative breast cancer (NNBC) based on clinicopathologic criteria, specific biological markers are needed to predict sensitivity to the different available therapeutic options. We examined the feasibility of developing a genomic predictor of chemotherapy response and recurrence risk in 185 patients with NNBC using assembled arrays containing 2,460 bacterial artificial chromosome clones for scanning the genome for DNA copy number changes. After surgery, 90 patients received anthracycline-based chemotherapy, whereas 95 did not. Tamoxifen was administered to patients with hormone receptor–positive tumors. The association of genomic and clinicopathologic data and outcome was computed using Cox proportional hazard models and multiple testing adjustment procedures. Analysis of NNBC genomes revealed a common genomic signature. Specific DNA copy number aberrations were associated with hormonal receptor status, but not with other clinicopathologic variables. In patients treated with chemotherapy, none of the genomic changes were significantly correlated with recurrence. In patients not receiving chemotherapy, deletion of eight bacterial artificial chromosome clones clustered to chromosome 11q was independently associated with relapse (disease-free survival at 10 years ± SE, 40% ± 14% versus 86% ± 6%; P < 0.0001). The 54 patients with deletion of 11q (29%) did not present more aggressive clinicopathologic features than those without 11q loss. The adverse influence of 11q deletion on clinical outcome was confirmed in an independent validation series of 88 patients with NNBC. Our data suggests that patients with NNBC with the 11q deletion might benefit from anthracycline-based chemotherapy despite other clinical, pathologic, or genetic features. However, these initial findings should be evaluated in randomized clinical trials. [Cancer Res 2007;67(2):818–26]
Vascular Endothelial Growth Factor Receptor 3 Is Involved in Tumor Angiogenesis and Growth
Authors: Laakkonen Pirjo , Waltari Marika , Holopainen Tanja , Takahashi Takashi , Pytowski Bronislaw , Steiner Philipp , Hicklin Daniel , Persaud Kris , Tonra James R. , Witte Larry , Alitalo Kari
Published: January 2007

Vascular endothelial growth factor receptor 3 (VEGFR-3) binds VEGF-C and VEGF-D and is essential for the development of the lymphatic vasculature. Experimental tumors that overexpress VEGFR-3 ligands induce lymphatic vessel sprouting and enlargement and show enhanced metastasis to regional lymph nodes and beyond, whereas a soluble form of VEGFR-3 that blocks receptor signaling inhibits these changes and metastasis. Because VEGFR-3 is also essential for the early blood vessel development in embryos and is up-regulated in tumor angiogenesis, we wanted to determine if an antibody targeting the receptor that interferes with VEGFR-3 ligand binding can inhibit primary tumor growth. Our results show that antibody interference with VEGFR-3 function can inhibit the growth of several human tumor xenografts in immunocompromised mice. Immunohistochemical analysis showed that the blood vessel density of anti-VEGFR-3–treated tumors was significantly decreased and hypoxic and necrotic tumor tissue was increased when compared with tumors treated with control antibody, indicating that blocking of the VEGFR-3 pathway inhibits angiogenesis in these tumors. As expected, the anti-VEGFR-3–treated tumors also lacked lymphatic vessels. These results suggest that the VEGFR-3 pathway contributes to tumor angiogenesis and that effective inhibition of tumor progression may require the inhibition of multiple angiogenic targets. [Cancer Res 2007;67(2):593–9]
Depletion of the Ras Association Domain Family 1, Isoform A–Associated Novel Microtubule-Associated Protein, C19ORF5/MAP1S, Causes Mitotic Abnormalities
Authors: Dallol Ashraf , Cooper Wendy N. , Al-Mulla Fahd , Agathanggelou Angelo , Maher Eamonn R. , Latif Farida
Published: January 2007

Ras association domain family 1, isoform A (RASSF1A) is a novel tumor suppressor gene that is found to be inactivated in more than 40 types of sporadic cancers. In addition, mouse Rassf1a knockout models have an increased frequency of spontaneous and induced tumors. The mechanisms by which RASSF1A exerts its tumor suppression activities or the pathways it can regulate are not yet fully understood. Using yeast two-hybrid system, we have previously identified C19ORF5/MAP1S as the major RASSF1A-interacting protein. C19ORF5 has two conserved microtubule-associated regions and may function to anchor RASSF1A to the centrosomes. In this study, we have analyzed the cellular functions of C19ORF5. By using small interfering RNA–mediated depletion and time-lapse video microscopy, we show that C19ORF5 knockdown causes mitotic abnormalities that consist of failure to form a stable metaphase plate, premature sister chromatid separation, lagging chromosomes, and multipolar spindles. We also show that a fraction of C19ORF5 localizes to the spindle microtubules. Additionally, we show here that C19ORF5 localizes to the microtubule-organizing centers during microtubule regrowth after nocodazole washout. Knockdown of C19ORF5 disrupts the microtubule-organizing center and results in microtubule nucleation from several sites. Whereas the localization of pericentrin is not affected, α- and γ-tubulin localization and sites of nucleation are greatly altered by C19ORF5 depletion. This may indicate that C19ORF5 plays a role in anchoring the microtubule-organizing center to the centrosomes. In addition, we show that the NH2 terminus of C19ORF5 is essential for this process. This novel role for C19ORF5 could explain the resulting mitotic abnormalities that occur on its depletion and can potentially provide an underlying mechanism for the frequent centrosome and microtubule abnormalities detected in several cancers. [Cancer Res 2007;67(2):492–500]