bluuebunny/crossref_metadata_2025 huggingface dataset:
Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions
Authors: Landeen Lee K. , Aroonsakool Nakon , Haga Jason H. , Hu Betty S. , Giles Wayne R.
Published: March 2007

The bioactive molecule sphingosine-1-phosphate (S1P) binds with high affinity to five recognized receptors (S1P1–5) to affect various tissues, including cellular responses of cardiac fibroblasts (CFbs) and myocytes. CFbs are essential components of myocardium, and detailed study of their cell signaling and physiology is required for a number of emerging disciplines. Meaningful studies on CFbs, however, necessitate methods for selective, reproducible cell isolations. Macrophages reside within normal cardiac tissues and often are isolated with CFbs. A protocol was therefore developed that significantly reduces macrophage levels and utilizes more CFb-specific markers (discoidin domain receptor-2) instead of, or in addition to, more commonly used cytoskeletal markers. Our results demonstrate that primary isolated, purified CFbs express predominantly S1P1–3; however, the relative levels of these receptor subtypes are modulated with time and by culture conditions. In coculture experiments, macrophages altered CFb S1P receptor levels relative to controls. Further investigations using known macrophage-secreted factors showed that S1P and H2O2 had minimal effects on CFb S1P1–3 expression, whereas transforming growth factor-β1, TNF-α, and PDGF-BB significantly altered all S1P receptor subtypes. Lowering FBS concentrations from 10% to 0.1% increased S1P2, whereas supplementation with either PDGF-BB or Rho-associated protein kinase inhibitor Y-27632 significantly elevated S1P3 levels. S1P2 and S1P3 receptor levels are known to regulate cell migration. Using cells isolated from either normal or S1P3-null mice, we demonstrate that S1P3 is important and necessary for CFb migration. These results highlight the importance of demonstrating CFb culture purity in functional studies of S1P and also identify conditions that modulate S1P receptor expression in CFbs.
Influence of eye colors of Caucasians and Asians on suppression of melatonin secretion by light
Authors: Higuchi Shigekazu , Motohashi Yutaka , Ishibashi Keita , Maeda Takafumi
Published: March 2007

This experiment tested effects of human eye pigmentation depending on the ethnicity on suppression of nocturnal melatonin secretion by light. Ten healthy Caucasian males with blue, green, or light brown irises (light-eyed Caucasians) and 11 Asian males with dark brown irises (dark-eyed Asians) volunteered to participate in the study. The mean ages of the light-eyed Caucasians and dark-eyed Asians were 26.4 ± 3.2 and 25.3 ± 5.7 years, respectively. The subjects were exposed to light (1,000 lux) for 2 h at night. The starting time of exposure was set to 2 h before the time of peak salivary melatonin concentration of each subject, which was determined in a preliminary experiment. Salivary melatonin concentration and pupil size were measured before exposure to light and during exposure to light. The percentage of suppression of melatonin secretion by light was calculated. The percentage of suppression of melatonin secretion 2 h after the start of light exposure was significantly larger in light-eyed Caucasians (88.9 ± 4.2%) than in dark-eyed Asians (73.4 ± 20.0%) ( P < 0.01). No significant difference was found between pupil sizes in light-eyed Caucasians and dark-eyed Asians. These results suggest that sensitivity of melatonin to light suppression is influenced by eye pigmentation and/or ethnicity.
Application of local heat induces capillary recruitment in the Pallid bat wing
Authors: Widmer R. J. , Stewart R. H. , Young M. F. , Laurinec J. E. , Laine G. A. , Quick C. M.
Published: March 2007

Skin blood flow increases in response to local heat due to sensorineural and nitric oxide (NO)-mediated dilation. It has been previously demonstrated that arteriolar dilation is inhibited with NO synthase (NOS) blockade. Flow, nonetheless, increases with local heat. This implies that the previously unexamined nonarteriolar responses play a significant role in modulating flow. We thus hypothesized that local heating induces capillary recruitment. We heated a portion (3 cm2) of the Pallid bat wing from 25°C to 37°C for 20 min, and measured changes in terminal feed arteriole (∼25 μm) diameter and blood velocity to calculate blood flow ( n = 8). Arteriolar dilation was reduced with NOS and sensorineural blockade using a 1% (wt/vol) NG-nitro-l-arginine methyl ester (l-NAME) and 2% (wt/vol) lidocaine solution ( n = 8). We also measured changes in the number of perfused capillaries, and the time precapillary sphincters were open with ( n = 8) and without ( n = 8) NOS plus sensorineural blockade. With heat, the total number of perfused capillaries increased 92.7 ± 17.9% ( P = 0.011), and a similar increase occurred despite NOS plus sensorineural blockade 114.4 ± 30.0% ( P = 0.014). Blockade eliminated arteriolar dilation (−4.5 ± 2.1%). With heat, the percent time precapillary sphincters remained open increased 32.3 ± 6.0% ( P = 0.0006), and this increase occurred despite NOS plus sensorineural blockade (34.1 ± 5.8%, P = 0.0004). With heat, arteriolar blood flow increased (187.2 ± 28.5%, P = 0.00003), which was significantly attenuated with NOS plus sensorineural blockade (88.6 ± 37.2%, P = 0.04). Thus, capillary recruitment is a fundamental microvascular response to local heat, independent of arteriolar dilation and the well-documented sensorineural and NOS mechanisms mediating the response to local heat.
Hypoxic stimulus alters hypothalamic AMP-activated protein kinase phosphorylation concomitant to hypophagia
Authors: Simler N. , Malgoyre A. , Koulmann N. , Alonso A. , Peinnequin A. , Bigard A. X.
Published: March 2007

Acute exposure to hypobaric hypoxia is known to decrease food intake, but the molecular mechanisms of such alteration in feeding behavior remain unknown. We tested the hypothesis that hypothalamic AMP-activated protein kinase (AMPK) phosphorylation is affected by acute exposure to hypobaric hypoxia and thus would be involved in initial anorexia. To address this issue, male rats weighing 255–270 g were either submitted to hypobaric hypoxia (H, equivalent altitude of 5,500 m), maintained under local barometric pressure conditions (N), or pair-fed an equivalent quantity of food to that consumed by H rats (PF), for 6, 24, or 48 h. Daily food intake dropped by 73% during the first day of hypoxia ( P < 0.01) and remained by 46% lower than in N rats thereafter ( P < 0.01). Hypoxia per se, as estimated by comparing experimental data between the H and PF groups, increased ob gene transcription and plasma leptin concentration. A transient increase in glucose availability occurred in the H group compared with PF animals ( P < 0.05). The hypoxic stimulus led to an early and transient decrease in hypothalamic AMPK and acetyl-CoA carboxylase (ACC) phosphorylation, concomitant with hypophagia and associated alterations in nutrients and hormones. An increase in NPY mRNA levels occurred from day 1, similarly in H and PF rats, and thus mainly related to food restriction alone ( P < 0.05). In conclusion, the present study demonstrates that hypoxia per se inhibited AMPK and ACC phosphorylation in the hypothalamus, concomitant with profound anorexia. A powerful counterregulation occurs rapidly, mediated by NPY and devoted to avoid prolonged anorexia.
Characteristics of skeletal muscle mitochondrial biogenesis induced by moderate-intensity exercise and weight loss in obesity
Authors: Menshikova Elizabeth V. , Ritov Vladimir B. , Ferrell Robert E. , Azuma Koichiro , Goodpaster Bret H. , Kelley David E.
Published: March 2007

There are fewer mitochondria and a reduced oxidative capacity in skeletal muscle in obesity. Moderate-intensity physical activity combined with weight loss increase oxidative enzyme activity in obese sedentary adults; however, this adaptation occurs without a significant increase in mitochondrial DNA (mtDNA), which is unlike the classic pattern of mitochondrial biogenesis induced by vigorous activity. The objective of this study was to examine the hypothesis that the mitochondrial adaptation to moderate-intensity exercise and weight loss in obesity induces increased mitochondrial cristae despite a lack of mtDNA proliferation. Content of cardiolipin and mtDNA and enzymatic activities of the electron transport chain (ETC) and tricarboxylic acid cycle were measured in biopsy samples of vastus lateralis muscle obtained from sedentary obese men and women before and following a 4-mo walking intervention combined with weight loss. Cardiolipin increased by 60% from 47 ± 4 to 74 ± 8 μg/mU CK ( P < 0.01), but skeletal muscle mtDNA content did not change significantly (1,901 ± 363 to 2,169 ± 317 Rc, where Rc is relative copy number of mtDNA per diploid nuclear genome). Enzyme activity of the ETC increased ( P < 0.01); that for rotenone-sensitive NADH-oxidase (96 ± 1%) increased more than for ubiquinol-oxidase (48 ± 6%). Activities for citrate synthase and succinate dehydrogenase increased by 29 ± 9% and 40 ± 6%, respectively. In conclusion, moderate-intensity physical activity combined with weight loss induces skeletal muscle mitochondrial biogenesis in previously sedentary obese men and women, but this response occurs without mtDNA proliferation and may be characterized by an increase in mitochondrial cristae.
Leukemia Inhibitory Factor: A Newly Identified Metastatic Factor in Rhabdomyosarcomas
Authors: Wysoczynski Marcin , Miekus Katarzyna , Jankowski Kacper , Wanzeck Jens , Bertolone Salvatore , Janowska-Wieczorek Anna , Ratajczak Janina , Ratajczak Mariusz Z.
Published: March 2007

Rhabdomyosarcoma frequently infiltrates bone marrow and this process involves the stromal-derived factor-1 (SDF-1)–CXCR4 axis. Because leukemia inhibitory factor (LIF), like SDF-1, is secreted by bone marrow stroma and directs the regeneration of skeletal muscles, we examined whether the LIF–LIF receptor (LIF-R) axis affects the biology of rhabdomyosarcoma cells. We found that in rhabdomyosarcoma cells, LIF stimulates the following: (a) phosphorylation of mitogen-activated protein kinase p42/44, AKT, and signal transducers and activators of transcription 3, (b) adhesion and chemotaxis, and (c) increased resistance to cytostatics. To compare the biological effects of LIF versus SDF-1, we examined the RH30 cell line, which is highly responsive to both ligands, and found that the chemotaxis of these cells is significantly reduced when the inhibitors of both receptors (T140 for CXCR4 and gp190 blocking antibody for LIF-R) are added simultaneously. Subsequently, by using repetitive chemotaxis to LIF or SDF-1, we selected from the RH30 line subpopulations of cells that respond to LIF but not SDF-1 (RH30-L) or to SDF-1 but not LIF (RH30-S). We found that (a) RH30-L cells seed better to the bone marrow, liver, and lymph nodes of immunodeficient mice than RH30-S cells and (b) mice inoculated i.m. with the RH30-L cells had more rhabdomyosarcoma cells in the bone marrow and lung after 6 weeks. Thus, we present the first evidence that the LIF–LIF-R axis may direct rhabdomyosarcoma metastasis. Further, because we showed that the in vivo metastasis of RH30 cells is inhibited by small interfering RNA against LIF-R, molecular targeting of this axis could become a new strategy to control the metastasis of rhabdomyosarcoma. [Cancer Res 2007;67(5):2131–40]
Twist Transcriptionally Up-regulates AKT2 in Breast Cancer Cells Leading to Increased Migration, Invasion, and Resistance to Paclitaxel
Authors: Cheng George Z. , Chan Joseph , Wang Qi , Zhang Weizhou , Sun Calvin D. , Wang Lu-Hai
Published: March 2007

Metastasis, the cardinal feature of malignant tumors, is an important clinical variable in patient prognosis. To understand the basis for metastasis, we systematically selected for highly invasive cells from breast cancer cell lines, MCF7 and MDA-MB-453, with moderate to low invasive ability using Boyden chamber invasion assay. The four-cycle selected invasive lines, named MCF7-I4 and MDA-MB-453-I4, respectively, displayed epithelial-mesenchymal transition (EMT) and dramatically enhanced invasive ability. EMT changes were corroborated with decreased level of E-cadherin and increased vimentin, fibronectin, and β1 integrin. Twist, a basic helix-loop-helix transcription factor, and AKT2, a known proto-oncogene, were found to be elevated in the invasive cells compared with the parental. Ectopic expression and knockdown of Twist by short interference RNA resulted in significant increase and reduction, respectively, of AKT2 protein and mRNA expression. Twist bound to E-box elements on AKT2 promoter and enhanced its transcriptional activity. Moreover, silencing AKT2 decreased Twist-promoted migration, invasion, and paclitaxel resistance. Reintroducing AKT2 largely rescued the phenotype resulted from knockdown of Twist in I4 cells, suggesting that AKT2 is a downstream target and functional mediator of Twist. Finally, we observed a 68.8% correlation of elevated Twist and AKT2 expression in late-stage breast cancers as oppose to 13% in early-stage breast cancers. Our study identifies Twist as a positive transcriptional regulator of AKT2 expression, and Twist-AKT2 signaling is involved in promoting invasive ability and survival of breast cancer cells. [Cancer Res 2007;67(5):1979–87]
Elevated Expression of the Oncogene c-fms and Its Ligand, the Macrophage Colony-Stimulating Factor-1, in Cervical Cancer and the Role of Transforming Growth Factor-β1 in Inducing c-fms Expression
Authors: Kirma Nameer , Hammes Luciano S. , Liu Ya-Guang , Nair Hareesh B. , Valente Philip T. , Kumar Shantha , Flowers Lisa C. , Tekmal Rajeshwar Rao
Published: March 2007

Cervical cancer is the third most common gynecologic cancer in the United States. The presence and possible involvement of several cytokines have been studied in cervical cancer; however, very little data, if any, are available on whether cervical tumors are responsive to stimulation by the macrophage colony-stimulating factor-1 (CSF-1). Given the involvement of c-fms and its ligand CSF-1 in gynecologic cancers, such as that of the uterus and the ovaries, we have examined the expression of c-fms and CSF-1 in cervical tumor (n = 17) and normal cervix (n = 8) samples. The data show that c-fms and its ligand are significantly higher in cervical carcinomas compared with normal samples. Immunohistochemistry not only showed that tumor cells expressed significantly higher levels of c-fms but also c-fms levels were markedly higher in tumor cells than tumor-associated stromal cells. Blocking c-fms activity in cervical cancer cells, which express CSF-1 and c-fms, resulted in increased apoptosis and decreased motility compared with control, suggesting that CSF-1/c-fms signaling may be involved in enhanced survival and possibly invasion by cervical cancer cells via an autocrine mechanism. Combined, the data show for the first time the induction of CSF-1 and c-fms in cervical carcinomas and suggest that c-fms activation may play a role in cervical carcinogenesis. Additionally, our data suggest that transforming growth factor-β1 may be a factor in inducing the expression of c-fms in cervical cancer cells. The data suggest that c-fms may be a valuable therapeutic target in cervical cancer. [Cancer Res 2007;67(5):1918–26]
CD28 Costimulation Overcomes Transforming Growth Factor-β–Mediated Repression of Proliferation of Redirected Human CD4+ and CD8+ T Cells in an Antitumor Cell Attack
Authors: Koehler Heike , Kofler David , Hombach Andreas , Abken Hinrich
Published: March 2007

The T-cell–mediated antitumor immune response is frequently repressed in the tumor environment by an immunologic barrier, the predominant mediators of which are thought to be interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). We explored the effect of these cytokines on the individual T-cell effector functions on antigen engagement during an antitumor cell attack. Isolated CD4+ and CD8+ T cells were antigen-specifically redirected toward carcinoembryonic antigen (CEA)-positive tumor cells by expression of a recombinant T-cell receptor (immunoreceptor), which triggers T-cell activation via CD3ζ on binding to CEA. Immunoreceptor-activated T cells secrete IFN-γ, proliferate, and lyse CEA+ but not CEA− tumor cells. Whereas IL-10 has no direct effect on immunoreceptor-triggered effector functions, TGF-β represses proliferation of both CD4+ and CD8+ T cells but neither IFN-γ secretion nor specific cytolytic activities. CD28 costimulation, however, overcomes TGF-β–mediated repression in T-cell proliferation. Consequently, T cells redirected by a combined CD28-CD3ζ signaling immunoreceptor are largely resistant to TGF-β–mediated repression. This is reflected in vivo by a more pronounced antitumor activity of T cells against TGF-β–secreting tumors when redirected by a costimulatory CD28-CD3ζ than by a CD3ζ signaling immunoreceptor. [Cancer Res 2007;67(5):2265–73]
Metabolomics by Proton Nuclear Magnetic Resonance Spectroscopy of the Response to Chloroethylnitrosourea Reveals Drug Efficacy and Tumor Adaptive Metabolic Pathways
Authors: Morvan Daniel , Demidem Aicha
Published: March 2007

Metabolomics of tumors may allow discovery of tumor biomarkers and metabolic therapeutic targets. Metabolomics by two-dimensional proton high-resolution magic angle spinning nuclear magnetic resonance spectroscopy was applied to investigate metabolite disorders following treatment by chloroethylnitrosourea of murine B16 melanoma (n = 33) and 3LL pulmonary carcinoma (n = 31) in vivo. Treated tumors of both types resumed growth after a delay. Nitrosoureas provoke DNA damage but the metabolic consequences of genotoxic stress are little known yet. Although some differences were observed in the metabolite profile of untreated tumor types, the prominent metabolic features of the response to nitrosourea were common to both. During the growth inhibition phase, there was an accumulation of glucose (more than ×10; P < 0.05), glutamine (×3 to 4; P < 0.01), and aspartate (×2 to 5; P < 0.01). This response testified to nucleoside de novo synthesis down-regulation and drug efficacy. However, this phase also involved the increase in alanine (P < 0.001 in B16 melanoma), the decrease in succinate (P < 0.001), and the accumulation of serine-derived metabolites (glycine, phosphoethanolamine, and formate; P < 0.01). This response witnessed the activation of pathways implicated in energy production and resumption of nucleotide de novo synthesis, thus metabolic pathways of DNA repair and adaptation to treatment. During the growth recovery phase, it remained polyunsaturated fatty acid accumulation (×1.5 to 2; P < 0.05) and reduced utilization of glucose compared with glutamine (P < 0.05), a metabolic fingerprint of adaptation. Thus, this study provides the proof of principle that metabolomics of tumor response to an anticancer agent may help discover metabolic pathways of drug efficacy and adaptation to treatment. [Cancer Res 2007;67(5):2150–9]
Inactivation of the Candidate Tumor Suppressor Par-4 in Endometrial Cancer
Authors: Moreno-Bueno Gema , Fernandez-Marcos Pablo J. , Collado Manuel , Tendero Mercedes J. , Rodriguez-Pinilla Socorro M. , Garcia-Cao Isabel , Hardisson David , Diaz-Meco Maria T. , Moscat Jorge , Serrano Manuel , Palacios Jose
Published: March 2007

Recently, it has been shown that mice deficient in the proapoptotic protein prostate apoptosis response 4 (Par-4) are specifically prone to develop endometrial carcinomas. Based on this, we have examined here the possible role of Par-4 as a tumor suppressor gene in human endometrial cancer. Using cDNA arrays, quantitative reverse transcription-PCR, and immunohistochemistry, we detected Par-4 down-regulation in ∼40% of endometrial carcinomas. This alteration was not associated with phosphatase and tensin homologue (PTEN), K-RAS, or β-catenin mutations, but was more frequent among tumors showing microsatellite instability (MSI) or among tumors that were estrogen receptor positive. Mutational analysis of the complete coding sequence of Par-4 in endometrial cancer cell lines (n = 6) and carcinomas (n = 69) detected a mutation in a single carcinoma, which was localized in exon 3 [Arg (CGA) 189 (TGA) Stop]. Interestingly, Par-4 promoter hypermethylation was detected in 32% of the tumors in association with low levels of Par-4 protein and was more common in MSI-positive carcinomas. Par-4 promoter hypermethylation and silencing was also detected in endometrial cancer cell lines SKUT1B and AN3CA, and reexpression was achieved by treatment with the demethylating agent 5′-aza-2′-deoxycytidine. Together, these data show that Par-4 is a relevant tumor suppressor gene in human endometrial carcinogenesis. [Cancer Res 2007;67(5):1927–34]
Engagement of Collagen-Binding Integrins Promotes Matrix Metalloproteinase-9–Dependent E-Cadherin Ectodomain Shedding in Ovarian Carcinoma Cells
Authors: Symowicz Jaime , Adley Brian P. , Gleason Kara J. , Johnson Jeffrey J. , Ghosh Supurna , Fishman David A. , Hudson Laurie G. , Stack M. Sharon
Published: March 2007

Reversible modulation of cell-cell adhesion, cell-matrix adhesion, and proteolytic activity plays a critical role in remodeling of the neoplastic ovarian epithelium during metastasis, implicating cadherins, integrins, and proteinases in i.p. metastatic dissemination of epithelial ovarian carcinoma (EOC). Aberrant epithelial differentiation is an early event in ovarian carcinogenesis; thus, in contrast to most carcinomas that lose E-cadherin expression with progression, E-cadherin is abundant in primary EOC. Metastasizing EOCs engage in integrin-mediated adhesion to submesothelial interstitial collagens and express matrix metalloproteinases (MMP) that facilitate collagen invasion, thereby anchoring secondary lesions in the submesothelial matrix. As metalloproteinases have also been implicated in E-cadherin ectodomain shedding, the current study was undertaken to model the effects of matrix-induced integrin clustering on proteinase-catalyzed E-cadherin ectodomain shedding. Aggregation of collagen-binding integrins induced shedding of an 80-kDa E-cadherin ectodomain [soluble E-cadherin (sE-cad)] in a MMP- and Src kinase-dependent manner, and sE-cad was prevalent in ascites from ovarian cancer patients. Expression of MMP-9 was elevated by integrin aggregation, integrin-mediated ectodomain shedding was inhibited by a MMP-9 function blocking antibody, and incubation of cells with exogenous MMP-9 catalyzed E-cadherin ectodomain shedding. In contrast to other tumors wherein sE-cad is released into the circulation, EOC tumors maintain direct contact with sE-cad–rich ascites at high concentration, and incubation of EOC cells with physiologically relevant concentrations of recombinant sE-cad disrupted adherens junctions. These data support a novel mechanism for posttranslational modification of E-cadherin function via MMP-9 induction initiated by cell-matrix contact and suggest a mechanism for promotion of EOC metastatic dissemination. [Cancer Res 2007;67(5):2030–9]
Homeodomain-Interacting Protein Kinase 2 Is the Ionizing Radiation–Activated p53 Serine 46 Kinase and Is Regulated by ATM
Authors: Dauth Ilka , Krüger Jana , Hofmann Thomas G.
Published: March 2007

Phosphorylation of p53 at Ser46 is important to activate the apoptotic program. The protein kinase that phosphorylates p53 Ser46 in response to DNA double-strand breaks is currently unknown. The identification of this kinase is of particular interest because it may contribute to the outcome of cancer therapy. Here, we report that ionizing radiation (IR) provokes homeodomain-interacting protein kinase 2 (HIPK2) accumulation, activation, and complex formation with p53. IR-induced HIPK2 up-regulation strictly correlates with p53 Ser46 phosphorylation. Down-regulation of HIPK2 by RNA interference specifically inhibits IR-induced phosphorylation of p53 at Ser46. Moreover, we show that HIPK2 activation after IR is regulated by the DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM). Cells from ataxia telangiectasia patients show defects in HIPK2 accumulation. Concordantly, IR-induced HIPK2 accumulation is blocked by pharmacologic inhibition of ATM. Furthermore, ATM down-regulation by RNA interference inhibited IR-induced HIPK2 accumulation, whereas checkpoint kinase 2 deficiency showed no effect. Taken together, our findings indicate that HIPK2 is the IR-activated p53 Ser46 kinase and is regulated by ATM. [Cancer Res 2007;67(5):2274–9]
Fractalkine (CX3CL1)– and Interleukin-2–Enriched Neuroblastoma Microenvironment Induces Eradication of Metastases Mediated by T Cells and Natural Killer Cells
Authors: Zeng Yan , Huebener Nicole , Fest Stefan , Weixler Silke , Schroeder Ulrike , Gaedicke Gerhard , Xiang Rong , Schramm Alexander , Eggert Angelika , Reisfeld Ralph A. , Lode Holger N.
Published: March 2007

Fractalkine (FKN) is a unique CX3C chemokine (CX3CL1) known to induce both adhesion and migration of leukocytes mediated by a membrane-bound and a soluble form, respectively. Its function is mediated through CX3C receptor (CX3CR), which is expressed by TH1 immune cells including T cells and natural killer (NK) cells. FKN was shown to be expressed in >90% of 68 neuroblastoma samples as determined by cDNA microarray analysis. Here, we characterized the effect of FKN in the neuroblastoma microenvironment using a syngeneic model genetically engineered to secrete FKN. We show FKN-mediated migration, adhesion, and IFN-γ secretion of immune effector cells, but limited antineuroblastoma activity, in vitro and in vivo. Therefore, we tested the hypothesis that a combined increase of FKN and interleukin-2 (IL-2) in the neuroblastoma microenvironment induces an effective antitumor immune response. For this purpose, IL-2 was targeted to ganglioside GD2, which is highly expressed on neuroblastoma tissue, using an anti-GD2 antibody IL-2 immunocytokine (ch14.18-IL-2). Only mice bearing FKN- and IL-2–enriched neuroblastoma tumors exhibited a reduction in primary tumor growth and a complete eradication of experimental liver metastases. The depletion of T cells and NK cells in vivo abrogated the effect, and these effector cells showed the highest cytolytic activity in vitro. Finally, only the FKN- and IL-2–enriched neuroblastoma microenvironment resulted in T-cell activation and the release of proinflammatory cytokines. In summary, we showed for the first time the immunologic mechanisms by which targeted IL-2 treatment of neuroblastoma with an FKN-rich microenvironment induces an effective antitumor response. [Cancer Res 2007;67(5):2331–8]
Targeting the Oligomerization Domain of ETO Interferes with RUNX1/ETO Oncogenic Activity in t(8;21)-Positive Leukemic Cells
Authors: Wichmann Christian , Chen Linping , Heinrich Markus , Baus Daniela , Pfitzner Edith , Zörnig Martin , Ottmann Oliver G. , Grez Manuel
Published: March 2007

About 12% of all de novo acute myeloid leukemias are characterized by the translocation t(8;21), which generates the oncogenic fusion protein RUNX1/ETO. RUNX1/ETO has a modular structure and contains several docking sites for heterologous proteins, including transcriptional corepressors like N-CoR, SMART, and mSIN3A. RUNX1/ETO is found in high molecular weight complexes, which are crucial for the block in myeloid differentiation observed in RUNX1/ETO–transformed cells. Essential for high molecular weight complex formation is the nervy homology region 2 (NHR2) within ETO, which serves as interacting surface for oligomerization as well as association with members of the ETO protein family. Here, we show that the expression of a fusion peptide consisting of 128 amino acids (NC128), including the entire NHR2 domain of ETO, disrupts the stability of the RUNX1/ETO high molecular weight complexes, restores transcription of RUNX1/ETO target genes, and reverts the differentiation block induced by RUNX1/ETO in myeloid cells. In the presence of NC128, RUNX1/ETO–transformed cells lose their progenitor cell characteristics, are arrested in cell cycle progression, and undergo cell death. Our results indicate that selective interference with the oligomerization domain of ETO could provide a promising strategy to inhibit the oncogenic properties of the leukemia-associated fusion protein RUNX1/ETO. [Cancer Res 2007;67(5):2280–9]
The p53 Isoform Δp53 Lacks Intrinsic Transcriptional Activity and Reveals the Critical Role of Nuclear Import in Dominant-Negative Activity
Authors: Chan Wan Mui , Poon Randy Y.C.
Published: March 2007

The transcription factor p53 is one of the most frequently mutated tumor suppressors. Recent progress has unraveled several novel isoforms of p53. Intriguingly, one of the p53 isoform, Δp53, which lacks part of the DNA binding domain, was reported to be transcriptionally active toward some p53 target genes and is critical for the intra–S phase checkpoint. Here, we show that, in contrast to full-length p53, ectopically expressed Δp53 neither transactivated the promoters of p21CIP1/WAF1 or murine double minute-2 (MDM2) nor repressed the cyclin B1 promoter in unstressed H1299 cells. Due to the deletion of a nuclear localization signal, Δp53 was not imported into the nucleus. Engineering of nuclear localization signals to Δp53 restored nuclear accumulation. However, the nuclear-targeting Δp53 remained inactive, indicating that the lack of intrinsic activity of Δp53 was not simply due to subcellular localization but to its incomplete DNA binding domain. Similar to p53, Δp53 was subjected to MDM2-mediated ubiquitination/proteolysis. The cytoplasmic localization of Δp53 correlated with the instability of the protein because forcing Δp53 into the nucleus increased its stability. Although Δp53 could form a complex with p53 and stimulated the cytoplasmic retention of p53, it was not a robust inhibitor of p53. Targeting Δp53 into the nucleus enhanced the dominant-negative activity of Δp53. These observations underscore the critical role of subcellular localization in the dominant-negative action of p53. [Cancer Res 2007;67(5):1959–69]
Osteopontin Promotes Integrin Activation through Outside-In and Inside-Out Mechanisms: OPN-CD44V Interaction Enhances Survival in Gastrointestinal Cancer Cells
Authors: Lee Jia-Lin , Wang Mei-Jung , Sudhir Putty-Reddy , Chen Gen-Der , Chi Chin-Wen , Chen Jeou-Yuan
Published: March 2007

Osteopontin (OPN) and splice variants of CD44 (CD44V) have independently been identified as markers for tumor progression. In this study, we show that both OPN and CD44V are frequently overexpressed in human gastric cancer and that OPN-engaged CD44V ligation confers cells an increased survival mediated through integrin activation. First, we show that OPN treatment confers cells an increased resistance to UV-induced apoptosis. The OPN-mediated antiapoptosis is dependent on the expression of the variant exon 6 (V6)- or V7-containing CD44 as shown by overexpression of individual CD44V in gastric AZ521 cells that express no or very low level of endogenous CD44 and by knockdown of the constitutively expressed V6-containing CD44 isoforms in colon HT29 cells. Although OPN also interacts with RGD integrins, OPN-RGD sequence is dispensable for OPN-mediated antiapoptosis. OPN-induced antiapoptosis is mainly attributed to the engagement of CD44V isoforms and the relay of an inside-out signaling via Src activity, leading to robust integrin activation. Furthermore, OPN-elicited antiapoptosis was observed when cells were plated on fibronectin but not on poly-d-lysin, and preincubation of cells with anti-integrin β1 antibody to block integrin-extracellular matrix (ECM) interaction or ectopic expression of the dominant-negative forms of focal adhesion kinase to block ECM-derived signal abolished OPN-induced survival, suggesting that OPN-elicited antiapoptotic function is propagated from matrix transduced by integrin. Taken together, we showed that OPN-CD44V interaction promotes ECM-derived survival signal mediated through integrin activation, which may play an important role in the pathogenic development and progression of gastric cancer. [Cancer Res 2007;67(5):2089–97]
Identification of Candidate Molecular Markers Predicting Sensitivity in Solid Tumors to Dasatinib: Rationale for Patient Selection
Authors: Huang Fei , Reeves Karen , Han Xia , Fairchild Craig , Platero Suso , Wong Tai W. , Lee Francis , Shaw Peter , Clark Edwin
Published: March 2007

Dasatinib is a multitargeted kinase inhibitor that was recently approved for the treatment of chronic myelogenous leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia with resistance or intolerance to prior therapy. It is also in clinical trials for treating patients with solid tumors. The identification of molecular markers predictive of response to dasatinib could assist in clinical development by selecting patients most likely to derive clinical benefit. Using baseline gene expression profiling of a panel of 23 breast cancer cell lines, we identified genomic signatures highly correlated with in vitro sensitivity to dasatinib. The ability of these signatures to predict dasatinib sensitivity was further confirmed and validated in independent test cell lines. A six-gene model was used to correctly predict dasatinib sensitivity in 11 out of 12 (92%) additional breast and 19 out of 23 (83%) lung cancer cell lines. Quantitative real-time PCR and immunohistochemical assays further confirmed the differential expression pattern of selected markers. Finally, these gene signatures were observed in a subset of primary breast, lung, and ovarian tumors suggesting potential utility in patient selection. The subset of breast cancer patients expressing the dasatinib-sensitive signature includes a distinct clinical and molecular subgroup: the so-called “triple negative” (i.e., estrogen receptor–negative, progesterone receptor–negative, and HER2-negative) or “basal” breast cancer subtype. This patient population has a poor prognosis and currently has few effective treatment options. Our results implicate that dasatinib may represent a valuable treatment option in this difficult-to-treat population. To test this hypothesis, clinical studies are now under way to determine the activity of dasatinib in these patients. [Cancer Res 2007;67(5):2226–38]
The Possible Role of Cytokeratin 8 in Cadmium-Induced Adaptation and Carcinogenesis
Authors: Lau Andy T.Y. , Chiu Jen-Fu
Published: March 2007

Chronic exposures to cadmium compounds are carcinogenic. It was hypothesized that the development of resistance to cadmium may drive carcinogenesis. This is achieved by selection of resistant cells in which the apoptotic response is significantly attenuated. The induction of cadmium resistance in rat lung epithelial cells (LEC) was used to explore the mechanisms of cadmium-induced adaptation and carcinogenesis. Our previous results showed that LECs developed resistance to apoptosis during cadmium adaptation possibly due to perturbation of the c-Jun NH2-terminal kinase pathway. Here, we further study these cells by comparative proteomics. Interestingly, we showed that two intermediate filament proteins, cytokeratin 8 (CK8) and cytokeratin 14 (CK14), were increased significantly and stably maintained only in the adapted cells but not in cadmium-treated parental cells. It has been documented that CK8/cytokeratin 18 provided resistance to tumor necrosis factor (TNF)–induced apoptosis and CK14 may function as an inhibitor of TNF-TNF receptor 1 (TNFR1) signaling through an association with TNFR1-associated death domain protein, suggesting that up-regulation of CK8 and CK14 may be responsible for apoptotic resistance. Finally, we showed that small interfering RNA–specific knockdown of CK8 in cadmium-adapted cells attenuated the cadmium resistance, indicating the potential role of CK8 in cadmium resistance. This acquired self-resistance to apoptosis could account for cadmium-induced carcinogenesis, as this promotes neoplastic cell survival as well as subsequent clonal expansion and then progression of tumor development. Thus, increased expression of these cytokeratins represents an adaptive survival mechanism that resists cadmium-induced apoptosis and it is unprecedented that cells respond to long-term cadmium exposure by modulating keratin dynamics. [Cancer Res 2007;67(5):2107–13]
Notch Signaling, γ-Secretase Inhibitors, and Cancer Therapy
Authors: Shih Ie-Ming , Wang Tian-Li
Published: March 2007

The Notch signaling pathway represents a critical component in the molecular circuits that control cell fate during development. Aberrant activation of this pathway contributes to tumorigenesis. The role of Notch in human cancer has been highlighted recently by the presence of activating mutations and amplification of Notch genes in human cancer and by the demonstration that genes in the Notch signaling pathway could be potential therapeutic targets. It has become clear that one of the major therapeutic targets in the Notch pathway are the Notch receptors, in which γ-secretase inhibitors prevent the generation of the oncogenic (intracellular) domain of Notch molecules and suppress the Notch activity. This review article summarizes the biological roles of Notch molecules in cancer development with special emphasis on the promise and challenges in applying γ-secretase inhibitors as a new line of targeted therapeutic agents. [Cancer Res 2007;67(5):1879–82]
Haplotypes, Loss of Heterozygosity, and Expression Levels of Glycine N-Methyltransferase in Prostate Cancer
Authors: Huang Yu-Chuen , Lee Cheng-Ming , Chen Marcelo , Chung Ming-Yi , Chang Yen-Hwa , Huang William Ji-Shian , Ho Donald Ming-Tak , Pan Chin-Chen , Wu Tony T. , Yang Stone , Lin Ming-Wei , Hsieh Jer-Tsong , Chen Yi-Ming Arthur
Published: March 2007

Purpose: Glycine N-methyltransferase (GNMT) affects genetic stability by regulating DNA methylation and interacting with environmental carcinogens. In a previous study, we showed that GNMT acts as a susceptibility gene for hepatocellular carcinoma. Here, we report on our efforts to characterize the haplotypes, loss of heterozygosity (LOH), and expression levels of the GNMT in prostate cancer.

Experimental Design: Peripheral blood mononuclear cell DNA collected from 326 prostate cancer patients and 327 age-matched controls was used to determine GNMT haplotypes. Luciferase reporter constructs were used to compare the promoter activity of different GNMT haplotypes. GNMT LOH rates in tumorous specimens were investigated via a comparison with peripheral blood mononuclear cell genotypes. Immunohistochemical staining was used to analyze GNMT expression in tissue specimens collected from 5 normal individuals, 33 benign prostatic hyperplasia patients, and 45 prostate cancer patients.

Results: Three major GNMT haplotypes were identified in 92% of the participants: A, 16GAs/DEL/C (58%); B, 10GAs/INS/C (19.9%); and C, 10GAs/INS/T (14.5%). Haplotype C carriers had significantly lower risk for prostate cancer compared with individuals with haplotype A (odds ratio, 0.68; 95% confidence interval, 0.48-0.95). Results from a phenotypic analysis showed that haplotype C exhibited the highest promoter activity (P < 0.05, ANOVA test). In addition, 36.4% (8 of 22) of the prostatic tumor tissues had LOH of the GNMT gene. Immunohistochemical staining results showed abundant GNMT expression in normal prostatic and benign prostatic hyperplasia tissues, whereas it was diminished in 82.2% (37 of 45) of the prostate cancer tissues.

Conclusions: Our findings suggest that GNMT is a tumor susceptibility gene for prostate cancer.
Antibody-Dependent Cellular Cytotoxicity Mediated by Cetuximab against Lung Cancer Cell Lines
Authors: Kurai Jun , Chikumi Hiroki , Hashimoto Kiyoshi , Yamaguchi Kosuke , Yamasaki Akira , Sako Takanori , Touge Hirokazu , Makino Haruhiko , Takata Miyako , Miyata Masanori , Nakamoto Masaki , Burioka Naoto , Shimizu Eiji
Published: March 2007

Purpose: Epidermal growth factor receptor (EGFR) is commonly overexpressed in lung cancer. Cetuximab is a chimeric mouse-human antibody targeted against EGFR. Compared with its inhibitory properties, its immunologic mechanisms have not been well studied. In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab against lung cancer cell lines.

Experimental Design: We studied the correlation between EGFR expression in lung cancer cell lines and the ADCC activity of cetuximab as well as the influence of interleukin-2 and chemotherapy on the ADCC activity. EGFR expression was measured by a quantitative flow cytometric analysis and immunohistochemistry. The ADCC activity was assessed by a 4-h 51Cr release assay. Peripheral blood mononuclear cells, purified T cells, natural killer (NK) cells, and monocytes from healthy donors or lung cancer patients were used as effector cells.

Results: Fresh peripheral blood mononuclear cells exhibited cetuximab-mediated ADCC activity against lung cancer cell lines at a low concentration of cetuximab (0.25 μg/mL). A logarithmic correlation was observed between the number of EGFRs and ADCC activity. Even low EGFR expression, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity, and further increases in EGFR expression on the target cells had no further effect on the ADCC activity. In addition, ADCC activity was enhanced by interleukin-2 mainly through activation of NK cells and was less susceptible to immunosuppression by chemotherapy than NK activity in lung cancer patients.

Conclusions: These observations suggest the importance of ADCC activity as an immunologic mechanism of cetuximab in biological therapy for lung cancer patients.
Epigenetic Silencing of the Candidate Tumor Suppressor Gene Per1 in Non–Small Cell Lung Cancer
Authors: Gery Sigal , Komatsu Naoki , Kawamata Norihiko , Miller Carl W. , Desmond Julian , Virk Renu K. , Marchevsky Alberto , Mckenna Robert , Taguchi Hirokuni , Koeffler H. Phillip
Published: March 2007

Purpose: Epigenetic events are a critical factor contributing to cancer development. The purpose of this study was to identify tumor suppressor genes silenced by DNA methylation and histone deacetylation in non–small cell lung cancer (NSCLC).

Experimental Design: We used microarray analysis to screen for tumor suppressor genes.

Results: We identified Per1, a core circadian gene, as a candidate tumor suppressor in lung cancer. Although Per1 levels were high in normal lung, its expression was low in a large panel of NSCLC patient samples and cell lines. Forced expression of Per1 in NSCLC cell lines led to significant growth reduction and loss of clonogenic survival. Recent studies showed that epigenetic regulation, particularly histone H3 acetylation, is essential for circadian function. Using bisulfite sequencing and chromatin immunoprecipitation, we found that DNA hypermethylation and histone H3 acetylation are potential mechanisms for silencing Per1 expression NSCLC.

Conclusions: These results support the hypothesis that disruption of circadian rhythms plays an important role in lung tumorigenesis. Moreover, our findings suggest a novel link between circadian epigenetic regulation and cancer development.
Tumor Growth Inhibition with Cetuximab and Chemotherapy in Non–Small Cell Lung Cancer Xenografts Expressing Wild-type and Mutated Epidermal Growth Factor Receptor
Authors: Steiner Philipp , Joynes Christopher , Bassi Rajiv , Wang Su , Tonra James R. , Hadari Yaron R. , Hicklin Daniel J.
Published: March 2007

Purpose: Targeting the epidermal growth factor receptor (EGFR) is a validated approach to treat cancer. In non–small cell lung cancer (NSCLC), EGFR contains somatic mutations in 10% of patients, which correlates with increased response rates to small molecule inhibitors of EGFR. We analyzed the effects of the monoclonal IgG1 antibody Erbitux (cetuximab) in NSCLC xenografts with wild-type (wt) or mutated EGFR.

Experimental Design: NSCLC cell lines were grown s.c. in nude mice. Dose-dependent efficacy was established for cetuximab. To determine whether combination therapy produces tumor regressions, cetuximab was dosed at half-maximal efficacy with chemotherapy used at maximum tolerated dose.

Results: Cetuximab showed antitumor activity in wt (A549, NCI-H358, NCI-H292) and mutated [HCC-827 (delE746-A750), NCI-H1975 (L858R, T790M)] EGFR-expressing xenografts. In the H292 model, cetuximab and docetaxel combination therapy was more potent to inhibit tumor growth than cetuximab or docetaxel alone. Cisplatin augmented efficacy of cetuximab to produce 6 of 10 regressions, whereas 1 of 10 regressions was found with cetuximab and no regression was found with cisplatin. Using H1975 xenografts, gemcitabine increased efficacy of cetuximab resulting in 12 of 12 regressions. Docetaxel with cetuximab was more efficacious with seven of nine regressions compared with single treatments. Cetuximab inhibited autophosphorylation of EGFR in both H292 and H1975 tumor lysates. Exploring the underlying mechanism for combination effects in the H1975 xenograft model, docetaxel in combination with cetuximab added to the antiproliferative effects of cetuximab but was the main component in this drug combination to induce apoptosis.

Conclusions: Cetuximab showed antitumor activity in NSCLC models expressing wt and mutated EGFR. Combination treatments increased the efficacy of cetuximab, which may be important for the management of patients with chemorefractory NSCLC.
Longitudinal Assessment of Genetic and Epigenetic Markers in Oligodendrogliomas
Authors: Lavon Iris , Zrihan Daniel , Zelikovitch Bracha , Fellig Yakov , Fuchs Dana , Soffer Dov , Siegal Tali
Published: March 2007

Purpose: Because little is known about the evolution of genetic and epigenetic changes that occur during tumor progression in oligodendrogliomas, we evaluated these changes in paired early and progressive oligodendrogliomas.

Experimental Design: 1p36, 19q13, 10q22-26, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status were assessed in 46 paired early and progressive oligodendrogliomas from 23 patients.

Results: In early tumors, 60.8% were of low grade compared with only 17% low-grade tumors at recurrence. Of 17 early tumors described as pure oligodendrogliomas, 76.5% remained in this lineage, regardless of their grade, whereas others changed to astrocytic tumors. Oligoastrocytic tumors had a significantly higher tendency to transform to astrocytic tumors. All pure oligodendrogliomas with 1p/19q codeletions remained phenotypically unchanged, unlike mixed tumors with codeletions, of which 83% changed their cell lineage. Of tumors with early 1p deletion, 80% remained oligodendroglial at progression, whereas 75% of tumors with an intact 1p changed to astrocytic phenotype. 10q loss was uncommon in both early and progressive tumors. The proportional gain in methylation at progression was 31% for tumors with early 1p deletion, unlike tumors with an intact 1p, which had an 87.5% gain of methylation at progression.

Conclusions: Pure oligodendrogliomas with 1p/19q deletion tend to retain their cell phenotype and genetic profile unlike tumors with no deletions or mixed histology. MGMT promoter methylation is more pronounced at tumor progression, particularly in tumors with an intact 1p. These observations suggest that MGMT promoter methylation is a late event in progressive oligodendrogliomas, and therefore, their chemosensitivity is not necessarily related to MGMT methylation status.