bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation discrimination interference and comodulation masking release as a function of the number and spectral placement of narrow-band noise modulators
Authors: Moore Brian C. J. , Jorasz Urszula
Published: October 2005

The discrimination of the depth of amplitude modulation (AM) of a target carrier can be adversely affected by the presence of other modulated carriers (flankers), an effect called modulation discrimination interference (MDI). Conversely, when the task is to detect a sinusoidal signal added to a modulated carrier of the same frequency, the presence of comodulated flankers (with the same modulation as the target carrier) can improve performance (comodulation masking release: CMR). This paper examines how CMR and MDI are influenced by the number and placement of the flankers. Flankers were spaced at 2-ERB intervals from the target, and their number was manipulated by adding flankers that were more remote from the target. Narrow-band noise (centered at 10 Hz) was used as the modulator for all carriers. In experiment 1, the MDI task required detection of a decrease in modulation depth (m) of the target from a reference value of 0.7. Unmodulated flankers did not affect thresholds. Modulated flankers (m=0.15) produced MDI, which increased as more flankers were added. MDI was generally slightly greater for comodulated than for noncomodulated flankers. The CMR task required detection of a sinusoidal signal added (in 90° phase) to the target carrier (m=0.7). Unmodulated flankers or flankers that were noncomodulated with the target had no effect on threshold. Comodulated flankers (m=0.7) gave CMR, which generally increased as more flankers were added. This effect was greater for flankers below the target frequency than for flankers above. Experiment 2 examined MDI using similar stimuli to experiment 1, except that the task was to detect an increase in m from a reference value of 0.1. Even unmodulated flankers elevated thresholds, and this effect increased as more flankers were added, especially on the high-frequency side of the target. Modulated flankers (m=0.2) produced additional increases in threshold, and modulation thresholds generally increased as more flankers were added. However, the amount of MDI (the difference between thresholds with modulated and unmodulated flankers) did not vary systematically with the number of flankers and did not show consistent differences between comodulated and noncomodulated flankers. The results are interpreted in terms of the mechanisms underlying MDI and CMR, and especially perceptual grouping processes.
Effects of phase and level on vowel identification: Data and predictions based on a nonlinear basilar-membrane model
Authors: Alcántara Joseph I. , Holube Inga , Moore Brian C. J.
Published: October 2005

This paper examines the role of component phase and level on vowel identification and interprets the results in terms of the shapes of the waveforms occurring at the outputs of the filters in a nonlinear basilar-membrane model. Four normally hearing subjects were asked to identify which of six possible vowel-like harmonic complexes was presented on each trial. The stimuli were complex tones containing the first 35 harmonics of a 100-Hz fundamental. All of the harmonics below 3000 Hz were equal in amplitude except for three pairs of successive harmonics, at frequencies corresponding to the first three formants of six vowels, which were incremented in level relative to the background harmonics by 1, 2, 4, 8, and 16 dB. The components in the harmonic complexes were added in four different starting phase relationships; cosine, random, Schroeder positive, and Schroeder negative. The stimuli were presented at three overall levels; 85, 65, and 45 dB SPL. Performance was similar for the random and Schroeder-negative phases and did not vary as a function of level. Performance for the cosine- and Schroeder-positive-phase conditions was better than for the other two phase conditions, but decreased as the level was reduced. Performance for all four phase conditions was equivalent for the lowest level. The variation in performance as a function of level and component phase is explained in terms of the shapes of the temporal waveforms that would occur at the output of nonlinear ‘‘basilar-membrane filters’’ [H. W. Strube, J. Acoust. Soc. Am. 79, 1511–1518 (1986)], with asymmetric phase responses about the center frequency.
Evaluating the articulation index for auditory–visual consonant recognition
Authors: Grant Ken W. , Walden Brian E.
Published: October 2005

Adequacy of the ANSI standard for calculating the articulation index (AI) [ANSI S3.5-1969 (R1986)] was evaluated by measuring auditory (A), visual (V), and auditory–visual (AV) consonant recognition under a variety of bandpass-filtered speech conditions. Contrary to ANSI predictions, filter conditions having the same auditory AI did not necessarily result in the same auditory–visual AI. Low-frequency bands of speech tended to provide more benefit to AV consonant recognition than high-frequency bands. Analyses of the auditory error patterns produced by the different filter conditions showed a strong negative correlation between the degree of A and V redundancy and the amount of benefit obtained when A and V cues were combined. These data indicate that the ANSI auditory–visual AI procedure is inadequate for predicting AV consonant recognition performance under conditions of severe spectral shaping.
Infant vocalizations in response to speech: Vocal imitation and developmental change
Authors: Kuhl Patricia K. , Meltzoff Andrew N.
Published: October 2005

Infants’ development of speech begins with a language-universal pattern of production that eventually becomes language specific. One mechanism contributing to this change is vocal imitation. The present study was undertaken to examine developmental change in infants’ vocalizations in response to adults’ vowels at 12, 16, and 20 weeks of age and test for vocal imitation. Two methodological aspects of the experiment are noteworthy: (a) three different vowel stimuli (/a/, /i/, and /u/) were videotaped and presented to infants by machine so that the adult model could not artifactually influence infant utterances, and (b) infants’ vocalizations were analyzed both physically, using computerized spectrographic techniques, and perceptually by trained phoneticians who transcribed the utterances. The spectrographic analyses revealed a developmental change in the production of vowels. Infants’ vowel categories become more separated in vowel space from 12 to 20 weeks of age. Moreover, vocal imitation was documented. Infants listening to a particular vowel produced vocalizations resembling that vowel. A hypothesis is advanced extending Kuhl’s native language magnet (NLM) model to encompass infants’ speech production. It is hypothesized that infants listening to ambient language store perceptually derived representations of the speech sounds they hear which in turn serve as targets for the production of speech utterances. NLM unifies previous findings on the effects of ambient language experience on infants’ speech perception and the findings reported here that short-term laboratory experience with speech is sufficient to influence infants’ speech production.
Why stress position bias?
Authors: van Heuven Vincent J. , Menert Ludmila
Published: October 2005

There is ample evidence in the literature that English and Dutch listeners tend to perceive stress on the word-initial syllable. This bias is most easily seen in the perception of (nonsense) words containing repetitions of identical syllables. In four experiments the possible causes of this bias are investigated. The results show that the bias disappears when (i) words are preceded by a spoken context, when (ii) the voice source is replaced by noise (whisper), or when (iii) the fundamental frequency level of the utterance as a whole is lowered. The data are best explained by assuming that the listener interprets the onset of voicing of an isolated word as a (silent) pitch rise from the bottom of the speaker’s pitch range.
Spectral balance as an acoustic correlate of linguistic stress
Authors: Sluijter Agaath M. C. , van Heuven Vincent J.
Published: October 2005

Although intensity has been reported as a reliable acoustical correlate of stress, it is generally considered a weak cue in the perception of linguistic stress. In natural speech stressed syllables are produced with more vocal effort. It is known that, if a speaker produces more vocal effort, higher frequencies increase more than lower frequencies. In this study, the effects of lexical stress on intensity are examined in the abstraction from the confounding accent variation. A production study was carried out in which ten speakers produced Dutch lexical and reiterant disyllabic minimal stress pairs spoken with and without an accent in a fixed carrier sentence. Duration, overall intensity, formant frequencies, and spectral levels in four contiguous frequency bands were measured. Results revealed that intensity differences as a function of stress are mainly located above 0.5 kHz, i.e., a change in spectral balance emphasizing higher frequencies for stressed vowels. Furthermore, we showed that the intensity differences in the higher regions are caused by an increase in physiological effort rather than by shifting formant frequencies due to stress. The potential of each acoustic correlate of stress to differentiate between initial- and final-stressed words was examined by linear discriminant analysis. Duration proved the most reliable correlate of stress. Overall intensity and vowel quality are the poorest cues. Spectral balance, however, turned out to be a reliable cue, close in strength to duration.
The β-agonist isoproterenol attenuates EGF-stimulated wound closure in human airway epithelial cells
Authors: Schnackenberg Bradley J. , Jones Stacie M. , Pate Crystal , Shank Brian , Sessions Laura , Pittman Laura M. , Cornett Lawrence E. , Kurten Richard C.
Published: October 2005

Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although β-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that β-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the β-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing β2-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or to an alteration of the ERK/MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways.
Natural antisense transcripts: sound or silence?
Authors: Werner Andreas , Berdal Ariane
Published: October 2005

Antisense RNA was a rather uncommon term in a physiology environment until short interfering RNAs emerged as the tool of choice to knock down the expression of specific genes. As a consequence, the concept of RNA having regulatory potential became widely accepted. Yet, there is more to come. Computational studies suggest that between 15 and 25% of mammalian genes overlap, giving rise to pairs of sense and antisense RNAs. The resulting transcripts potentially interfere with each other’s processing, thus representing examples of RNA-mediated gene regulation by endogenous, naturally occurring antisense transcripts. Concerns that the large-scale antisense transcription may represent transcriptional noise rather than a gene regulatory mechanism are strongly opposed by recent reports. A relatively small, well-defined group of antisense or noncoding transcripts is linked to monoallelic gene expression as observed in genomic imprinting, X chromosome inactivation, and clonal expression of B and T leukocytes. For the remaining, much larger group of bidirectionally transcribed genes, however, the physiological consequences of antisense transcription as well as the cellular mechanism(s) involved remain largely speculative.
Detection of Early Antiangiogenic Effects in Human Colon Adenocarcinoma Xenografts:In vivoChanges of Tumor Blood Volume in Response to Experimental VEGFR Tyrosine Kinase Inhibitor
Authors: Kim Young Ro , Yudina Anna , Figueiredo JoseLuis , Reichardt Wilfried , Hu-Lowe Dana , Petrovsky Alexander , Kang Hye Won , Torres Denise , Mahmood Umar , Weissleder Ralph , Bogdanov Alexei A.
Published: October 2005

Antiangiogenesis is emerging as efficient strategy for targeting and potentially eliminating neoplastic tumor vessels. The main goal of this study was to establish whether absolute tumor blood volume (Vb) change could be used as an early predictor of antiangiogenesis in ectopic and orthotopic colon carcinomas. To assess therapy-induced changes of Vb, we did comparative analysis of signal intensities in tumors and muscle using steady-state magnetic resonance imaging (MRI) assisted with an intravascular paramagnetic contrast agent [gadolinium-labeled protected graft copolymer (PGC-Gd)]. Athymic mice with implanted human MV522 tumors were treated with vascular endothelial growth factor type 2 receptor tyrosine kinase inhibitor (VEGFR2-TKI) that has been shown to inhibit VEGFR2 phosphorylation and tumor growth in vivo. Animals were imaged either after a single day or after a 1-week course of treatments. The measured Vb in ectopic tumors was 2.5 ± 1.5% of total tissue volume 1 week after the implantation (n = 8). Two doses of VEGFR2-TKI (25 mg/kg, p.o., b.i.d.) resulted in a decrease of Vb to 1.3 ± 0.3%. In orthotopic tumors, the measured Vb was initially higher (11.9 ± 2.0%); however, VEGFR2-TKI treatment also resulted in a statistically significant decrease of Vb. The absolute Vb was not affected in the muscle as a result of treatments. MRI measurements were corroborated by using isotope and correlative histology experiments. Our results show that steady-state MRI is highly sensitive to early antiangiogenic effects caused by small molecule drugs.
Vascular Endothelial Growth Factor-A Promotes Peritumoral Lymphangiogenesis and Lymphatic Metastasis
Authors: Björndahl Meit A. , Cao Renhai , Burton Jeremy B. , Brakenhielm Ebba , Religa Piotr , Galter Dagmar , Wu Lily , Cao Yihai
Published: October 2005

Metastases are commonly found in the lymphatic system. The molecular mechanism of lymphatic metastasis is, however, poorly understood. Here we report that vascular endothelial growth factor (VEGF)-A stimulated lymphangiogenesis in vivo and that overexpression of VEGF-A in murine T241 fibrosarcomas induced the growth of peritumoral lymphatic vessels, which occasionally penetrated into the tumor tissue. As a result of peritumoral lymphangiogenesis, metastases in lymph nodes of mice were detected. VEGF-A–overexpressing tumors contained high numbers of infiltrating inflammatory cells such as macrophages, which are known to express VEGF receptor (VEGFR)-1. It seemed that in the mouse cornea, VEGF-A stimulated lymphangiogenesis through a VEGF-C/-D/VEGFR-3–independent pathway as a VEGFR-3 antagonist selectively inhibited VEGF-C–induced, but not VEGF-A–induced, lymphangiogenesis. Our data show that VEGF-A contributes to lymphatic mestastasis. Thus, blockage of VEGF-A–induced lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic metastasis.
Tissue Inhibitor of Metalloproteinase-3 Expression from an Oncolytic Adenovirus Inhibits Matrix Metalloproteinase Activity In vivo without Affecting Antitumor Efficacy in Malignant Glioma
Authors: Lamfers Martine L.M. , Gianni Davide , Tung Ching-Hsuan , Idema Sander , Schagen Frederik H.E. , Carette Jan E. , Quax Paul H.A. , Van Beusechem Victor W. , Vandertop W. Peter , Dirven Clemens M.F. , Chiocca E. Antonio , Gerritsen Winald R.
Published: October 2005

Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdΔ24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdΔ24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdΔ24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2–activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdΔ24TIMP-3–treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87δEGFR) tumors with AdΔ24TIMP-3 and AdΔ24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdΔ24 did not significantly affect the antitumor efficacy of this oncolytic agent.
Cross Talk between Apoptosis and Invasion Signaling in Cancer Cells through Caspase-3 Activation
Authors: Mukai Mutsuko , Kusama Toshiyuki , Hamanaka Yukou , Koga Takumi , Endo Hiroko , Tatsuta Masaharu , Inoue Masahiro
Published: October 2005

In solid tumors, cancer cells are exposed to various microenvironmental stresses such as hypoxia, nutritional depletion, and low pH. Cancer cells adapt to these stresses and circumvent cell death. When the antiapoptotic signals overcome the stress, cancer cells might acquire physiologic functions, such as invasiveness, instead of cell death. Here, we report that tumor cells acquire an invasive capacity from apoptotic signals through caspase activation. We treated rat ascites hepatoma MM1 cells with an apoptosis-inducing drug, etoposide, or hypoxia, and assessed the invasion capacity with an in vitro bioassay. Although MM1 cells hardly showed invasiveness in serum-free medium, under stress conditions, invasive capacity accompanied with morphologic change was induced with caspase-3 activation. Such stress-induced invasion as well as morphologic change was suppressed by blocking caspase-3 activity with caspase inhibitors or by RNA interference of caspase-3. In contrast, lysophosphatidic acid–induced invasiveness was not affected by caspase-3 inhibition. These results suggest that caspase-3 activation contributes to the stress-induced invasive capacity of these cancer cells.
Cyclin D1 Induction through IκB Kinase β/Nuclear Factor-κB Pathway Is Responsible for Arsenite-Induced Increased Cell Cycle G1-S Phase Transition in Human Keratinocytes
Authors: Ouyang Weiming , Ma Qian , Li Jingxia , Zhang Dongyun , Liu Zheng-gang , Rustgi Anil K. , Huang Chuanshu
Published: October 2005

Environmental and occupational exposure to arsenite is associated with an increased risk of human cancers, including skin, urinary bladder, and respiratory tract cancers. Although much evidence suggests that alterations in cell cycle machinery are implicated in the carcinogenic effect of arsenite, the molecular mechanisms underlying the cell cycle alterations are largely unknown. In the present study, we observed that exposure of human keratinocyte HaCat cells to arsenite resulted in the promotion of cell cycle progression, especially G1-S transition. Further studies found that arsenite exposure was able to induce cyclin D1 expression. The induction of cyclin D1 by arsenite required nuclear factor-κB (NF-κB) activation, because the inhibition of IκB phosphorylation by overexpression of the dominant-negative mutant, IKKβ-KM, impaired arsenite-induced cyclin D1 expression and G1-S transition. The requirement of IκB kinase β (IKKβ) for cyclin D1 induction was further confirmed by the findings that arsenite-induced cyclin D1 expression was totally blocked in IKKβ knockout (IKKβ−/−) mouse embryo fibroblasts. In addition, knockdown of cyclin D1 expression using cyclin D1–specific small interference RNA significantly blocked arsenite-induced cell cycle progression in HaCat cells. Taken together, our results show that arsenite-induced cell cycle from G1 to S phase transition is through IKKβ/NF-κB/cyclin D1–dependent pathway.
Regulation of Dendritic Cell Differentiation and Antitumor Immune Response in Cancer by Pharmacologic-Selective Inhibition of the Janus-Activated Kinase 2/Signal Transducers and Activators of Transcription 3 Pathway
Authors: Nefedova Yulia , Nagaraj Srinivas , Rosenbauer Amsler , Muro-Cacho Carlos , Sebti Said M. , Gabrilovich Dmitry I.
Published: October 2005

Abnormal dendritic cell differentiation and accumulation of immunosuppressive myeloid cells in cancer is one of the major factors of tumor nonresponsiveness. We have previously shown that hyperactivation of the Janus-activated kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) induced by tumor-derived factors (TDF) is responsible for abnormal dendritic cell differentiation. Here, using a novel selective inhibitor of JAK2/STAT3 JSI-124, we investigated the possibility of pharmacologic regulation of dendritic cell differentiation in cancer. Our experiments in vitro have shown that JSI-124 overcomes the differentiation block induced by TDF and promotes the differentiation of mature dendritic cells and macrophages. JSI-124 significantly reduced the presence of immature myeloid cells in vivo and promoted accumulation of mature dendritic cells. In addition to a direct antitumor effect in several animal models, JSI-124 significantly enhanced the effect of cancer immunotherapy. This indicates that pharmacologic inhibition of the JAK2/STAT3 pathway can be an important new therapeutic strategy to enhance antitumor activity of cancer immunotherapy.
Transactivation of Epidermal Growth Factor Receptor Is Involved in Leptin-Induced Activation of Janus-Activated Kinase 2 and Extracellular Signal–Regulated Kinase 1/2 in Human Gastric Cancer Cells
Authors: Shida Dai , Kitayama Joji , Mori Ken , Watanabe Toshiaki , Nagawa Hirokazu
Published: October 2005

Leptin is known to act as a growth factor through the Janus-activated kinase (JAK)/signal transducer and activator of transcription signaling pathway as well as the mitogen-activated protein kinase pathway. In this study, we showed a novel signal transduction pathway using two human gastric cancer cell lines, MKN28 and MKN74. Both gastric cancer cells expressed leptin and its receptors (Ob-R) at the protein level. We found that leptin, even at as low as 0.1 ng/mL, induced significant tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Time-course experiments revealed that phosphorylation was maximal after 5 minutes of stimulation and declined thereafter. We also revealed that tyrosine phosphorylation of EGFR induced by leptin was significantly attenuated by two inhibitors, an EGFR tyrosine kinase inhibitor, AG1478, and a broad-spectrum matrix metalloproteinase inhibitor, GM6001. This indicates that the pathway of EGFR transactivation induced by leptin is dependent on proteolytically released EGFR ligands. Leptin induced JAK2 activation and extracellular signal–regulated kinase (ERK) 1/2 activation in these gastric cancer cells, both of which occurred after the peak of EGFR transactivation. Pretreatment of gastric cancer cells with AG1478 significantly reduced the degree of phosphorylation of both JAK2 and ERK1/2. These findings indicate the involvement of EGFR transactivation in the activation of JAK2 and ERK1/2. Our results reveal that EGFR transactivation is involved in the leptin signaling pathway in gastric cancer cells, which extends the physiologic action of leptin beyond its central effects in the hypothalamus to regulate body weight.
Insulin Receptor Substrate Is a Mediator of Phosphoinositide 3-Kinase Activation in Quiescent Pancreatic Cancer Cells
Authors: Asano Takayuki , Yao Yixin , Shin Sonyo , McCubrey James , Abbruzzese James L. , Reddy Shrikanth A.G.
Published: October 2005

Phosphoinositide 3-kinase (PI3K) is activated in pancreatic cancer cells and plays a central role in their proliferation, survival, and drug resistance. Although the mechanism is unclear, PI3K activation in these cells could be due to physical interaction between its regulatory subunit (p85) and specific tyrosine kinases or their mediators. Consistent with this possibility, PI3K was precipitated with anti-phosphotyrosine antibodies and Akt phosphorylation was blocked by the tyrosine kinase inhibitors SU6656 and PD158780 in quiescent pancreatic cancer cells. Pull-down assays with a fusion protein (GST-p85NC-SH2), and coimmunoprecipitation studies, indicated that the insulin receptor substrate (IRS), and not the epidermal growth factor and insulin-like growth factor receptors or the Src tyrosine kinase, was physically associated with PI3K in these cells. Our data also indicated that SU6656 and PD158780 inhibited Akt activation in pancreatic cancer cells by interfering with the ability of IRS-1 to recruit PI3K. Furthermore, IRS-1 was phosphorylated on a p85-binding site (Y612), and IRS-specific small interfering RNA potently inhibited activation of PI3K and Akt in transfected cells. Taken together, these observations indicate that IRS is a mediator of PI3K activation in quiescent pancreatic cancer cells.
Decoy Receptor 2 (DcR2) Is a p53 Target Gene and Regulates Chemosensitivity
Authors: Liu Xiangguo , Yue Ping , Khuri Fadlo R. , Sun Shi-Yong
Published: October 2005

Decoy receptor 2 (DcR2) is one of the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptors and suppresses TRAIL-induced apoptosis. Its expression, like the other three TRAIL receptors (i.e., DR4, DR5, and DcR1), is regulated by p53. Here, we report that DcR2 is a p53 target gene and regulates chemosensitivity. In this study, we identified a p53-binding site (p53BS) in the first intron of the DcR2 gene. This p53BS is almost identical to the ones found in the first introns of other three TRAIL receptor genes. By a chromatin immunoprecipitation assay, we detected that the p53 protein bound to the DcR2 p53BS in intact cells. Subcloning of the DcR2 p53BS into a luciferase reporter vector driven by a SV40 promoter exhibited enhanced luciferase activity when transiently cotransfected with a wild-type (wt) p53 expression vector in p53-null cell lines or stimulated with DNA-damaging agents in cell lines having wt p53. Moreover, when the DcR2 p53BS, together with its own corresponding promoter regions, was subcloned into a basic luciferase vector without a promoter element, its transcriptional activities were strikingly increased by cotransfection of the wt p53 gene. However, when this p53BS was deleted from the construct, wt p53 failed to transactivate this reporter construct. Collectively, we conclude that p53 directly regulates the DcR2 gene expression via an intronic p53BS. In addition, overexpression of DcR2 conferred resistance to TRAIL-mediated apoptosis and attenuated cell response to DNA-damaging agents, whereas silencing of DcR2 expression enhanced chemotherapeutic agent–induced apoptosis. These results suggest that DcR2 regulates chemosensitivity.
Low-Level Expression of Proapoptotic Bcl-2–Interacting Mediator in Leukemic Cells in Patients with Chronic Myeloid Leukemia: Role of BCR/ABL, Characterization of Underlying Signaling Pathways, and Reexpression by Novel Pharmacologic Compounds
Authors: Aichberger Karl J. , Mayerhofer Matthias , Krauth Maria-Theresa , Vales Anja , Kondo Rudin , Derdak Sophia , Pickl Winfried F. , Selzer Edgar , Deininger Michael , Druker Brian J. , Sillaber Christian , Esterbauer Harald , Valent Peter
Published: October 2005

Chronic myeloid leukemia (CML) is a myeloproliferative disease in which BCR/ABL enhances survival of leukemic cells through modulation of proapoptotic and antiapoptotic molecules. Recent data suggest that proapoptotic Bcl-2–interacting mediator (Bim) plays a role as a tumor suppressor in myeloid cells, and that leukemic cells express only low amounts of this cell death activator. We here show that primary CML cells express significantly lower amounts of bim mRNA and Bim protein compared with normal cells. The BCR/ABL inhibitors imatinib and AMN107 were found to promote expression of Bim in CML cells. To provide direct evidence for the role of BCR/ABL in Bim modulation, we employed Ba/F3 cells with doxycycline-inducible expression of BCR/ABL and found that BCR/ABL decreases expression of bim mRNA and Bim protein in these cells. The BCR/ABL-induced decrease in expression of Bim was found to be a posttranscriptional event that depended on signaling through the mitogen-activated protein kinase pathway and was abrogated by the proteasome inhibitor MG132. Interestingly, MG132 up-regulated the expression of bim mRNA and Bim protein and suppressed the growth of Ba/F3 cells containing wild-type BCR/ABL or imatinib-resistant mutants of BCR/ABL. To show functional significance of “Bim reexpression,” a Bim-specific small interfering RNA was applied and found to rescue BCR/ABL-transformed leukemic cells from imatinib-induced cell death. In summary, our data identify BCR/ABL as a Bim suppressor in CML cells and suggest that reexpression of Bim by novel tyrosine kinase inhibitors, proteasome inhibition, or by targeting signaling pathways downstream of BCR/ABL may be an attractive therapeutic approach in imatinib-resistant CML.
In vivo Induction of Resistance to Gemcitabine Results in Increased Expression of Ribonucleotide Reductase Subunit M1 as the Major Determinant
Authors: Bergman Andries M. , Eijk Paul P. , Ruiz van Haperen Veronique W.T. , Smid Kees , Veerman Gijsbert , Hubeek Isabelle , van den IJssel Paul , Ylstra Bauke , Peters Godefridus J.
Published: October 2005

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.
Multiple Roles for the Receptor Tyrosine Kinase Axl in Tumor Formation
Authors: Holland Sacha J. , Powell Mark J. , Franci Christian , Chan Emily W. , Friera Annabelle M. , Atchison Robert E. , McLaughlin John , Swift Susan E. , Pali Erlina S. , Yam George , Wong Stephen , Lasaga Joe , Shen Mary R. , Yu Simon , Xu Weiduan , Hitoshi Yasumichi , Bogenberger Jakob , Nör Jacques E. , Payan Donald G. , Lorens James B.
Published: October 2005

A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA–mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development.
Therapeutic Cure against Human Tumor Xenografts in Nude Mice by a Microtubule Stabilization Agent, Fludelone, via Parenteral or Oral Route
Authors: Chou Ting-Chao , Dong Huajin , Zhang Xiuguo , Tong William P. , Danishefsky Samuel J.
Published: October 2005

Epothilones, 16-membered macrolides isolated from a myxobacterium in soil, exert their antitumor effect, like Taxol, by induction of microtubule polymerization and microtubule stabilization. They are effective against tumor cells that are resistant to Taxol or vinblastine. We recently designed, via molecular editing and total synthesis, a new class of epothilones represented by 26-trifluoro-(E)-9,10-dehydro-12,13-desoxy-epothilone B (Fludelone), which has emerged as a lead candidate for clinical development. Treatment of nude mice bearing MX-1 human mammary carcinoma xenografts (as large as 3.4% body weight) with Fludelone (6-hour i.v. infusion, 25 mg/kg, q3d × 5, q3d × 4) led to complete disappearance and de facto “cure” (i.e., remission without a relapse for over 15% of the average life span of 2 years). The toxicities induced by bolus i.v. injection could be avoided through prolonged i.v. infusion, which allowed for a 10-fold increase in maximal tolerated dose. Complete remission of MX-1 xenografts was achieved with only one third of this maximal tolerated dose. Parallel studies with Taxol and Fludelone [20 mg/kg, 6-hour i.v. infusion (q2d × 4) ×3] against HCT-116 human colon carcinoma xenografts revealed that both drugs achieved tumor remission; however, all Taxol-treated mice relapsed in ∼1.3 months, whereas the Fludelone-treated mice were cured without any relapse for over 7 months. Furthermore, tumor remission was achieved by Fludelone against SK-OV-3 (ovary), PC-3 (prostate), and the Taxol-resistant CCRF-CEM/Taxol (leukemia) xenograft tumors. Most remarkably, p.o. administration of Fludelone (30 mg/kg, q2d × 7, q2d × 9, q2d × 5) against MX-1 xenografts achieved a nonrelapsing cure for as long as 8.4 months. The above results indicate that Fludelone is a highly promising compound for cancer chemotherapeutics.
Lack of Expression of the EP2 but not EP3 Receptor for Prostaglandin E2 Results in Suppression of Skin Tumor Development
Authors: Sung You Me , He Guobin , Fischer Susan M.
Published: October 2005

The EP2 receptor for prostaglandin E2 (PGE2) is a membrane receptor that mediates at least part of the action of PGE2. It has been shown that EP2 plays a critical role in tumorigenesis in mouse mammary gland and colon. However, the possibility that the EP2 receptor is involved in the development of skin tumors was unknown. The purpose of this study was to investigate the role of the EP2 receptor in mouse skin carcinogenesis. Unlike EP3 knockout mice, the EP2 knockout mice produced significantly fewer tumors and reduced tumor incidence compared with wild type (WT) mice in a 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) two-stage carcinogenesis protocol. EP2 knockout mice had significantly reduced cellular proliferation of mouse skin keratinocytes in vivo and in vitro compared with that in WT mice. In addition, the epidermis of EP2 knockout mice 48 hours after topical TPA treatment was significantly thinner compared with that of WT mice. The inflammatory response to TPA was reduced in EP2 knockout mice, based on a reduced number of macrophages in the dermis and a reduced level of interleukin-1α mRNA expression, compared with WT mice. EP2 knockout mice also had significantly reduced epidermal cyclic AMP levels after PGE2 treatment compared with WT mice. Tumors from WT mice produced more blood vessels and fewer apoptotic cells than those of EP2 knockout mice as determined by immunohistochemical staining. Our data suggest that the EP2 receptor plays a significant role in the protumorigenic action of PGE2 in skin tumor development.
Regulation of Krüppel-like Factor 6 Tumor Suppressor Activity by Acetylation
Authors: Li Dan , Yea Steven , Dolios Georgia , Martignetti John A. , Narla Goutham , Wang Rong , Walsh Martin J. , Friedman Scott L.
Published: October 2005

Krüppel-like factor 6 (KLF6) is a zinc finger transcription factor and tumor suppressor that is inactivated in a number of human cancers by mutation, allelic loss, and/or promoter methylation. A key mechanism of growth inhibition by wild-type KLF6 is through p53-independent up-regulation of p21WAF1/cip1 (CDKN1A), which is abrogated in several tumor-derived mutants. Here we show by chromatin immunoprecipitation that transactivation of p21WAF1/cip1 by KLF6 occurs through its direct recruitment to the p21WAF1/cip1 promoter and requires acetylation by histone acetyltransferase activity of either cyclic AMP–responsive element binding protein–binding protein or p300/CBP-associated factor. Direct lysine acetylation of KLF6 peptides can be shown by mass spectrometry. A single lysine-to-arginine point mutation (K209R) derived from prostate cancer reduces acetylation of KLF6 and abrogates its capacity to up-regulate endogenous p21WAF1/cip1 and reduce cell proliferation. These data indicate that acetylation may regulate KLF6 function, and its loss in some tumor-derived mutants could contribute to its failure to suppress growth in prostate cancer.
Polymorphisms in Cytokine and Cellular Adhesion Molecule Genes and Susceptibility to Hematotoxicity among Workers Exposed to Benzene
Authors: Lan Qing , Zhang Luoping , Shen Min , Smith Martyn T. , Li Guilan , Vermeulen Roel , Rappaport Stephen M. , Forrest Matthew S. , Hayes Richard B. , Linet Martha , Dosemeci Mustafa , Alter Blanche P. , Weinberg Rona S. , Yin Songnian , Yeager Meredith , Welch Robert , Waidyanatha Suramya , Kim Sungkyoon , Chanock Stephen , Rothman Nathaniel
Published: October 2005

Benzene is a recognized hematotoxin and leukemogen but its mechanism of action and the role of genetic susceptibility are still unclear. Cytokines, chemokines, and cellular adhesion molecules are soluble proteins that play an important regulatory role in hematopoiesis. We therefore hypothesized that variation in these genes could influence benzene-induced hematotoxicity. We analyzed common, well-studied single-nucleotide polymorphisms (SNPs) in 20 candidate genes drawn from these pathways in a study of 250 workers exposed to benzene and 140 unexposed controls in China. After accounting for multiple comparisons, SNPs in five genes were associated with a statistically significant decrease in total WBC counts among exposed workers [IL-1A (−889C>T), IL-4 (−1098T>G), IL-10 (−819T>C), IL-12A (8685G>A), and VCAM1 (−1591T>C)], and one SNP [CSF3 (Ex4−165C>T)] was associated with an increase in WBC counts. The adhesion molecule VCAM1 variant was particularly noteworthy as it was associated with a decrease in B cells, natural killer cells, CD4+ T cells, and monocytes. Further, VCAM1 (−1591T>C) and CSF3 (Ex4−165C>T) were associated, respectively, with decreased (P = 0.041) and increased (P = 0.076) CFU-GEMM progenitor cell colony formation in 29 benzene-exposed workers. This is the first report to provide evidence that SNPs in genes that regulate hematopoiesis influence benzene-induced hematotoxicity.
Dissecting the Role of Matrix Metalloproteinases (MMP) and Integrin αvβ3 in AngiogenesisIn vitro: Absence of Hemopexin C Domain Bioactivity, but Membrane-Type 1-MMP and αvβ3 Are Critical
Authors: Nisato Riccardo E. , Hosseini Ghamartaj , Sirrenberg Christian , Butler Georgina S. , Crabbe Thomas , Docherty Andrew J.P. , Wiesner Matthias , Murphy Gillian , Overall Christopher M. , Goodman Simon L. , Pepper Michael S.
Published: October 2005

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin αvβ3–mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti–membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin αvβ3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin αvβ3. Moreover, no specific binding of pro-MMP-2 to integrin αvβ3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin αvβ3, and this in a PEX-independent manner. Likewise, integrin αvβ3–expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and αvβ3- dependent but MMP-2 independent and does not support a role for PEX in αvβ3 integrin binding or in modulating angiogenesis in this system.
Increases of Amphiregulin and Transforming Growth Factor-α in Serum as Predictors of Poor Response to Gefitinib among Patients with Advanced Non–Small Cell Lung Cancers
Authors: Ishikawa Nobuhisa , Daigo Yataro , Takano Atsushi , Taniwaki Masaya , Kato Tatsuya , Hayama Satoshi , Murakami Haruyasu , Takeshima Yukio , Inai Kouki , Nishimura Hitoshi , Tsuchiya Eiju , Kohno Nobuoki , Nakamura Yusuke
Published: October 2005

Serum levels of amphiregulin and transforming growth factor-α (TGF-α), which were identified previously to be expressed at high levels in non–small cell lung cancer (NSCLC) with poor response to gefitinib, were examined by ELISA using blood samples taken from 50 patients with advanced NSCLCs. Of 14 cases that revealed above the cutoff line for amphiregulin in serum, 12 responded poorly to gefitinib, whereas 18 of the 36 cases showing below the cutoff revealed partial response (PR) or stable disease (SD; P = 0.026). Thirteen of 15 patients who were positive for TGF-α responded poorly to gefitinib, whereas 18 of the 35 patients with negative TGF-α levels turned out to be relatively good responders (P = 0.014). Of 22 patients with positive values for either or both markers, 19 were poor responders. On the other hand, among 28 patients negative for both markers, 17 were classified into the PR or SD groups (P = 0.001). Gefitinib-treated NSCLC patients whose serum amphiregulin or TGF-α was positive showed a poorer tumor-specific survival (P = 0.037 and 0.002, respectively, by univariate analysis) compared with those whose serum amphiregulin or TGF-α concentrations were negative. Multivariate analysis showed an independent association between positivity for TGF-α and shorter survival times among NSCLC patients treated with gefitinib (P = 0.034). Amphiregulin or TGF-α positivity in NSCLC tissues was significantly higher in male, nonadenocarcinomas, and smokers. Our data suggest that the status of amphiregulin and TGF-α in serum can be an important predictor of the resistance to gefitinib among patients with advanced NSCLC.
Low-Density Lipoprotein Receptor–Related Protein Contributes to the Antiangiogenic Activity of Thrombospondin-2 in a Murine Glioma Model
Authors: Fears Constance Y. , Grammer J. Robert , Stewart Jerry E. , Annis Douglas S. , Mosher Deane F. , Bornstein Paul , Gladson Candece L.
Published: October 2005

Host antiangiogenesis factors defend against tumor growth. The matricellular protein, thrombospondin-2 (TSP-2), has been shown to act as an antiangiogenesis factor in a carcinogen-induced model of skin cancer. Here, using an in vivo malignant glioma model in which the characteristics of the tumors formed after intracerebral implantation of GL261 mouse glioma cells are assessed, we found that tumor growth and microvessel density were significantly enhanced in tumors propagated in TSP-2−/− mice. Mechanistically, matrix metalloproteinase (MMP)-2 has been associated with neoangiogenesis and it has been proposed that the levels of available MMP-2 may be down-regulated by formation of a complex with TSP-2 that is internalized by low-density lipoprotein receptor–related protein 1 (LRP1). We found elevated expression of MMP-2 and MMP-9 in tumors propagated in TSP-2−/− mice, with a preferential localization in the microvasculature. In wild-type mice, MMP-2 was coexpressed with TSP-2 in the tumor microvasculature. In vitro, addition of recombinant (rec) TSP-2 to mouse brain microvessel endothelial cells reduced MMP-2 levels and invasion through mechanisms that could be inhibited by a competitive inhibitor of ligand binding to LRP1 or by siLRP1. Thus, the antiangiogenic activity of TSP-2 is capable of inhibiting the growth of gliomas in part by reducing the levels of MMP-2 in the tumor microvasculature. This mechanism is mediated by LRP1.