bluuebunny/crossref_metadata_2025 huggingface dataset:
Conditional Rhythmicity and Synchrony in a Bilateral Pair of Bursting Motor Neurons inAplysia
Authors: Serrano Geidy E. , Miller Mark W.
Published: June 2006

This investigation examined the activity of a bilateral pair of motor neurons (B67) in the feeding system of Aplysia californica. In isolated ganglia, B67 firing exhibited a highly stereotyped bursting pattern that could be attributed to an underlying TTX-resistant driver potential (DP). Under control conditions, this bursting in the two B67 neurons was infrequent, irregular, and asynchronous. However, bath application of the neuromodulator dopamine (DA) increased the duration, frequency, rhythmicity, and synchrony of B67 bursts. In the absence of DA, depolarization of B67 with injected current produced rhythmic bursting. Such depolarization-induced rhythmic burst activity in one B67, however, did not entrain its contralateral counterpart. Moreover, when both B67s were depolarized to potentials that produced rhythmic bursting, their synchrony was significantly lower than that produced by DA. In TTX, dopamine increased the DP duration, enhanced the amplitude of slow signaling between the two B67s, and increased DP synchrony. A potential source of dopaminergic signaling to B67 was identified as B65, an influential interneuron with bilateral buccal projections. Firing B65 produced bursts in the ipsilateral and contralateral B67s. Under conditions that attenuated polysynaptic activity, firing B65 evoked rapid excitatory postsynaptic potentials in B67 that were blocked by sulpiride, an antagonist of synaptic DA receptors in this system. Finally, firing a single B65 was capable of producing a prolonged period of rhythmic synchronous bursting of the paired B67s. It is proposed that modulatory dopaminergic signaling originating from B65 during consummatory behaviors can promote rhythmicity and bilateral synchrony in the paired B67 motor neurons.
Antiangiogenic Potential of the Mammalian Target of Rapamycin Inhibitor Temsirolimus
Authors: Del Bufalo Donatella , Ciuffreda Ludovica , Trisciuoglio Daniela , Desideri Marianna , Cognetti Francesco , Zupi Gabriella , Milella Michele
Published: June 2006

Mammalian target of rapamycin (mTOR) is increasingly recognized as a master regulator of fundamental cellular functions, whose deregulation may underlie neoplastic transformation and progression. Hence, mTOR has recently emerged as a promising target for therapeutic anticancer interventions in several human tumors, including breast cancer. Here, we investigated the antiangiogenic potential of temsirolimus (also known as CCI-779), a novel mTOR inhibitor currently in clinical development for the treatment of breast cancer and other solid tumors. Consistent with previous reports, sensitivity to temsirolimus-mediated growth inhibition varied widely among different breast cancer cell lines and was primarily due to inhibition of proliferation with little, if any, effect on apoptosis induction. In the HER-2 gene–amplified breast cancer cell line BT474, temsirolimus inhibited vascular endothelial growth factor (VEGF) production in vitro under both normoxic and hypoxic conditions through inhibition of hypoxia-stimulated hypoxia-inducible factor (HIF)-1α expression and transcriptional activation. Interestingly, these effects were also observed in the MDA-MB-231 cell line, independent of its inherent sensitivity to the growth-inhibitory effects of temsirolimus. A central role for mTOR (and the critical regulator of cap-dependent protein translation, eIF4E) in the regulation of VEGF production by BT474 cells was further confirmed using a small interfering RNA approach to silence mTOR and eIF4E protein expression. In addition to its effect on HIF-1α–mediated VEGF production, temsirolimus also directly inhibited serum- and/or VEGF-driven endothelial cell proliferation and morphogenesis in vitro and vessel formation in a Matrigel assay in vivo. Overall, these results suggest that antiangiogenic effects may substantially contribute to the antitumor activity observed with temsirolimus in breast cancer. (Cancer Res 2006; 66(11): 5549-54)
Flavopiridol Targets c-KIT Transcription and Induces Apoptosis in Gastrointestinal Stromal Tumor Cells
Authors: Sambol Elliot B. , Ambrosini Grazia , Geha Rula C. , Kennealey Peter T. , DeCarolis Penelope , O'Connor Rachael , Wu Yuhsin V. , Motwani Monica , Chen Jin-Hong , Schwartz Gary K. , Singer Samuel
Published: June 2006

Gastrointestinal stromal tumors (GIST) are characterized by activating mutations in the c-KIT gene which confers ligand-independent activation of the KIT receptor. Imatinib mesylate has been shown to effectively block constitutively active KIT and delay tumor growth. However, resistance to imatinib mesylate is emerging as a major clinical problem and novel therapies are needed. We report that treatment of GIST cells with the transcriptional inhibitor flavopiridol, initially down-regulates the antiapoptotic proteins bcl-2, mcl-1, and X-linked inhibitor of apoptosis protein which occurs as early as 4 hours after exposure. This is followed at 24 hours by the transcriptional suppression of KIT resulting in poly(ADP-ribose) polymerase cleavage and apoptosis. To separate the apoptotic effect of KIT suppression relative to the down-regulation of antiapoptotic proteins, we used small interfering RNA–directed knockdown of KIT. Results show that focused suppression of KIT alone is sufficient to induce apoptosis in GIST cells, but not to the same extent as flavopiridol. In contrast, imatinib mesylate, which inhibits KIT kinase activity but does not suppress total KIT expression, fails to cause apoptosis. We also show that flavopiridol suppresses KIT mRNA expression through positive transcriptional elongation factor inhibition and decreases KIT promoter activity. This causes a global decrease in the level of functionally mature KIT at the cell surface, resulting in a decrease in autophosphorylation at tyrosine residues 703 and 721, which characterizes activated KIT. Our results indicate that targeting KIT expression and these antiapoptotic proteins with flavopiridol represents a novel means to disrupt GIST cell dependence on KIT signaling and collectively renders these cells sensitive to apoptosis. (Cancer Res 2006; 66(11): 5858-66)
Inhibition of Poly(ADP-Ribose) Polymerase Modulates Tumor-Related Gene Expression, Including Hypoxia-Inducible Factor-1 Activation, during Skin Carcinogenesis
Authors: Martin-Oliva David , Aguilar-Quesada Rocío , O'Valle Francisco , Muñoz-Gámez Jose Antonio , Martínez-Romero Rubén , García del Moral Raimundo , Ruiz de Almodóvar José Mariano , Villuendas Raquel , Piris Miguel Angel , Oliver F. Javier
Published: June 2006

Poly(ADP-ribose) polymerase (PARP)-1, an enzyme that catalyzes the attachment of ADP ribose to target proteins, acts as a component of enhancer/promoter regulatory complexes. In the present study, we show that pharmacologic inhibition of PARP-1 with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) results in a strong delay in tumor formation and in a dramatic reduction in tumor size and multiplicity during 7,12-dimethylbenz(a)anthracene plus 12-O-tetradecanoylphorbol-13-acetate–induced skin carcinogenesis. This observation was parallel with a reduction in the skin inflammatory infiltrate in DPQ-treated mice and tumor vasculogenesis. Inhibition of PARP also affected activator protein-1 (AP-1) activation but not nuclear factor-κB (NF-κB). Using cDNA expression array analysis, a substantial difference in key tumor-related gene expression was found between chemically induced mice treated or not with PARP inhibitor and also between wild-type and parp-1 knockout mice. Most important differences were found in gene expression for Nfkbiz, S100a9, Hif-1α, and other genes involved in carcinogenesis and inflammation. These results were corroborated by real-time PCR. Moreover, the transcriptional activity of hypoxia-inducible factor-1α (HIF-1α) was compromised by PARP inhibition or in PARP-1–deficient cells, as measured by gene reporter assays and the expression of key target genes for HIF-1α. Tumor vasculature was also strongly inhibited in PARP-1–deficient mice and by DPQ. In summary, this study shows that inhibition of PARP on itself is able to control tumor growth, and PARP inhibition or genetic deletion of PARP-1 prevents from tumor promotion through their ability to cooperate with the activation AP-1, NF-κB, and HIF-1α. (Cancer Res 2006; 66(11): 5744-56)
Mechanism of Apoptosis Induced by the Inhibition of Fatty Acid Synthase in Breast Cancer Cells
Authors: Bandyopadhyay Sucharita , Zhan Rui , Wang Ying , Pai Sudha K. , Hirota Shigeru , Hosobe Sadahiro , Takano Yukio , Saito Ken , Furuta Eiji , Iiizumi Megumi , Mohinta Sonia , Watabe Misako , Chalfant Charles , Watabe Kounosuke
Published: June 2006

Fatty acid synthase (FAS) has been found to be overexpressed in a wide range of epithelial tumors, including breast cancer. Pharmacologic inhibitors of FAS cause apoptosis of breast cancer cells and result in decreased tumor size in vivo. However, how the inhibition of FAS induces apoptosis in tumor cells remains largely unknown. To understand the apoptotic pathway resulting from direct inhibition of FAS, we treated breast tumor cells with or without FAS small interfering RNA (siRNA) followed by a microarray analysis. Our results indicated that the proapoptotic genes BNIP3, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), and death-associated protein kinase 2 (DAPK2) were significantly up-regulated on direct inhibition of the FAS gene. We also found that the knockdown of FAS expression significantly increased ceramide level in the tumor cells, and this increase was abrogated by acetyl-CoA carboxylase inhibitor. In addition, carnitine palmitoyltransferase-1 (CPT-1) inhibitor up-regulated the ceramide and BNIP3 levels in these cells, whereas treatment of tumor cells with FAS siRNA in the presence of a ceramide synthase inhibitor abrogated the up-regulation of BNIP3 and inhibited apoptosis. Furthermore, we found that treatment of cells with BNIP3 siRNA significantly counteracted the effect of FAS siRNA-mediated apoptosis. Consistent with these results, a significant inverse correlation was observed in the expression of FAS and BNIP3 in clinical samples of human breast cancer. Collectively, our results indicate that inhibition of FAS in breast cancer cells causes accumulation of malonyl-CoA, which leads to inhibition of CPT-1 and up-regulation of ceramide and induction of the proapoptotic genes BNIP3, TRAIL, and DAPK2, resulting in apoptosis. (Cancer Res 2006; 66(11): 5934-40)
Dynein Light Chain 1 Contributes to Cell Cycle Progression by Increasing Cyclin-Dependent Kinase 2 Activity in Estrogen-Stimulated Cells
Authors: den Hollander Petra , Kumar Rakesh
Published: June 2006

Progression of hormone-responsive cancers is characterized by deregulation of the cell cycle and cytoskeleton signaling. In addition, development of breast and endometrial cancer is influenced by the stimulatory action of estrogen. Up-regulation of dynein light chain 1 (DLC1), a component of cytoskeleton signaling, was recently found to promote tumorigenesis. The purpose of our study was to determine the role that DLC1 up-regulation plays in cell cycle progression. To achieve this goal, we used human breast ductal carcinoma ZR-75 cells overexpressing DLC1 as a model system. We found that ZR-75 cells with up-regulated DLC1 were hypersensitive to estrogen-dependent growth stimulation and that DLC1 had an accelerating effect on the G1-S transition and stimulated cyclin-dependent kinase 2 (Cdk2) activity. To better understand the promotion of the G1-S transition by DLC1, we sought to identify new DLC1-interacting proteins with roles in cell cycle regulation. Using a modified proteomic strategy, we identified two such DLC1-interacting proteins: Cdk2 and Cip-interacting zinc finger protein 1 (Ciz1). DLC1 was verified to interact with Cdk2 and Ciz1 in vivo. We also showed that down-regulation of DLC1 and Ciz1 reduced both Cdk2 activity and cell cycle progression of breast cancer ZR-75 and MCF-7 and endometrial Ishikawa cancer cells. Further, we showed that overexpression of DLC1 is accompanied by a reduction of nuclear p21WAF1. These findings suggest that interactions among DLC1, Cdk2, and Ciz1 play a regulatory role in cell cycle progression of cancer cells presumably by influencing the levels of nuclear p21WAF1. (Cancer Res 2006; 66(11): 5941-9)
Dominant-Negative Histone H3 Lysine 27 Mutant Derepresses Silenced Tumor Suppressor Genes and Reverses the Drug-Resistant Phenotype in Cancer Cells
Authors: Abbosh Phillip H. , Montgomery John S. , Starkey Jason A. , Novotny Milos , Zuhowski Eleanor G. , Egorin Merrill J. , Moseman Annie P. , Golas Adam , Brannon Kate M. , Balch Curtis , Huang Tim H.M. , Nephew Kenneth P.
Published: June 2006

Histone modifications and DNA methylation are epigenetic phenomena that play a critical role in many neoplastic processes, including silencing of tumor suppressor genes. One such histone modification, particularly at H3 and H4, is methylation at specific lysine (K) residues. Whereas histone methylation of H3-K9 has been linked to DNA methylation and aberrant gene silencing in cancer cells, no such studies of H3-K27 have been reported. Here, we generated a stable cell line overexpressing a dominant-negative point mutant, H3-K27R, to examine the role of that specific lysine in ovarian cancer. Expression of this construct resulted in loss of methylation at H3-K27, global reduction of DNA methylation, and increased expression of tumor suppressor genes. One of the affected genes, RASSF1, was shown to be a direct target of H3-K27 methylation–mediated silencing. By increasing DNA-platinum adduct formation, indicating increased access of the drug to target DNA sequences, removal of H3-K27 methylation resensitized drug-resistant ovarian cancer cells to the chemotherapeutic agent cisplatin. This increased platinum-DNA access was likely due to relaxation of condensed chromatin. Our results show that overexpression of mutant H3-K27 in mammalian cells represents a novel tool for studying epigenetic mechanisms and the Histone Code Hypothesis in human cancer. Such findings show the significance of H3-K27 methylation as a promising target for epigenetic-based cancer therapies. (Cancer Res 2006; 66(11): 5582-91)
Assembly of Mutant-Template Telomerase RNA into Catalytically Active Telomerase Ribonucleoprotein That Can Act on Telomeres Is Required for Apoptosis and Cell Cycle Arrest in Human Cancer Cells
Authors: Goldkorn Amir , Blackburn Elizabeth H.
Published: June 2006

The telomerase ribonucleoprotein is a promising target for cancer therapy, as it is highly active in many human malignancies. A novel telomerase targeting approach combines short interfering RNA (siRNA) knockdown of endogenous human telomerase RNA (hTer) with expression of a mutant-template hTer (MT-hTer). Such combination MT-hTer/siRNA constructs induce a rapid DNA damage response, telomere uncapping, and inhibition of cell proliferation in a variety of human cancer cell lines. We tested which functional aspects of the protein catalytic component of telomerase [human telomerase reverse transcriptase (hTERT)] are required for these effects using human LOX melanoma cells overexpressing various hTERTs of known properties. Within 3 days of MT-hTer/siRNA introduction, both growth inhibition and DNA damage responses were significantly higher in the setting of wild-type hTERT versus catalytically dead hTERT or mutant hTERT that is catalytically competent but unable to act on telomeres. These effects were not attenuated by siRNA-induced knockdown of the telomeric protein human Rap1 and were additive with knockdown of the telomere-binding protein TRF2. Hence, the effects of MT-hTer/siRNA require a telomerase that is both catalytically competent to polymerize DNA and able to act on telomeres in cells. (Cancer Res 2006; 66(11): 5763-71)
Lymphatic Zip Codes in Premalignant Lesions and Tumors
Authors: Zhang Lianglin , Giraudo Enrico , Hoffman Jason A. , Hanahan Douglas , Ruoslahti Erkki
Published: June 2006

Blood vessels in tumors are morphologically and functionally distinct from normal resting blood vessels. We probed lymphatic vessels in premalignant lesions and tumors by in vivo screening of phage-displayed peptide libraries, asking whether they too have distinctive signatures. The resulting peptides begin to define such signatures. One peptide identified the lymphatics in a human melanoma xenograft. Another recognized the lymphatics in prostate cancers but not in premalignant prostate lesions; this peptide similarly identifies human prostate cancer lymphatics. A third was selective for the lymphatics in the premalignant prostate lesions. A fourth identified the lymphatics in dysplasias and squamous carcinomas of the cervix and skin. None recognize lymphatics in normal tissues. Thus, tumor development is associated with organ- and stage-specific changes in lymphatics. Systemic treatment of mice with fusions of a lymphatic homing peptide and a proapoptotic motif reduced the number of tumor lymphatics in prostate tumor and melanoma, forecasting future lymphatic targeting agents for detection and therapeutic intervention. (Cancer Res 2006; 66(11): 5696-706)
Immunologic and Clinical Responses after Vaccinations with Peptide-Pulsed Dendritic Cells in Metastatic Renal Cancer Patients
Authors: Wierecky Jan , Müller Martin R. , Wirths Stefan , Halder-Oehler Edith , Dörfel Daniela , Schmidt Susanne M. , Häntschel Maik , Brugger Wolfram , Schröder Stephen , Horger Marius S. , Kanz Lothar , Brossart Peter
Published: June 2006

A phase I trial was conducted to evaluate the feasibility, safety, and efficacy of a dendritic cell–based vaccination in patients with metastatic renal cell carcinoma (RCC). Autologous mature dendritic cells derived from peripheral blood monocytes were pulsed with the HLA-A2-binding MUC1 peptides (M1.1 and M1.2). For the activation of CD4+ T-helper lymphocytes, dendritic cells were further incubated with the PAN-DR-binding peptide PADRE. Dendritic cell vaccinations were done s.c. every 2 weeks for four times and repeated monthly until tumor progression. After five dendritic cell injections, patients additionally received three injections weekly of low-dose interleukin-2 (1 million IE/m2). The induction of vaccine-induced T-cell responses was monitored using enzyme-linked immunospot and Cr release assays. Twenty patients were included. The treatment was well tolerated with no severe side effects. In six patients, regression of the metastatic sites was induced after vaccinations with three patients achieving an objective response (one complete response, two partial responses, two mixed responses, and one stable disease). Additional four patients were stable during the treatment for up to 14 months. MUC1 peptide–specific T-cell responses in vivo were detected in the peripheral blood mononuclear cells of the six patients with objective responses. Interestingly, in patients responding to the treatment, T-cell responses to antigens not used for vaccinations, such as adipophilin, telomerase, or oncofetal antigen, could be detected, indicating that epitope spreading might occur. This study shows that MUC1 peptide–pulsed dendritic cells can induce clinical and immunologic responses in patients with metastatic RCC. (Cancer Res 2006; 66(11): 5910-8)
Inhibition of Glutathione Synthesis Overcomes Bcl-2-Mediated Topoisomerase Inhibitor Resistance and Induces Nonapoptotic Cell Death via Mitochondrial-Independent Pathway
Authors: Yoshida Akira , Takemura Haruyuki , Inoue Hitoshi , Miyashita Toshiyuki , Ueda Takanori
Published: June 2006

Bcl-2 protein plays a critical role in inhibiting anticancer drug–induced apoptosis. We found that Bcl-2 overexpression is associated with a nearly 3-fold increase in cellular glutathione levels and with increased resistance to cell death after treatment with etoposide or SN-38, a derivative of camptothecin, in leukemia 697 cells with wild-type p53. Treatment of Bcl-2-overexpressing 697 cells (697-Bcl-2) with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, reduced cellular glutathione levels and completely abolished Bcl-2-mediated drug resistance. Morphologic studies revealed that nonapoptotic cell death was induced in 697-Bcl-2 cells after treatment with BSO plus etoposide or SN-38. Activation of caspase-3/7 and cytochrome c release could not be detected in 697-Bcl-2 cells after these drug treatments. Notably, we showed that proteasome-mediated down-regulation of Puma and Noxa proteins occurs in 697-Bcl-2 cells after treatment with BSO plus topoisomerase inhibitor, although there is an increase in the protein levels of p53 in these 697-Bcl-2 cells. In contrast, parental 697 cells underwent typical apoptosis with up-regulation of Puma and Noxa proteins, followed by cytochrome c release and caspase-3/7 activation after treatment with topoisomerase inhibitor in the presence or absence of BSO. Our data suggest that BSO may possess a unique activity to overcome Bcl-2-mediated drug resistance by stimulating the signals that can bypass mitochondrial process in Bcl-2-overexpressing cells. (Cancer Res 2006; 66(11): 5772-80)
Androgen-Dependent Regulation of Her-2/neu in Prostate Cancer Cells
Authors: Berger Raanan , Lin Douglas I. , Nieto Maria , Sicinska Ewa , Garraway Levi A. , Adams Heiner , Signoretti Sabina , Hahn William C. , Loda Massimo
Published: June 2006

The mechanisms underlying the progression of prostate cancer to a state of resistance to hormone ablation remain poorly understood. Here, we have investigated the relationship between androgen receptor (AR) and Her-2/neu in prostate cancer cells. Overexpression of Her-2/neu (c-ErbB2) activates the AR pathway and confers a survival and growth advantage to prostate cancer cells in an androgen-deficient milieu. In vitro, the absence of androgens or AR blockade induced Her-2/neu protein expression and phosphorylation. In contrast, upon readministration of androgens, Her-2/neu mRNA, protein, and phosphorylation levels decreased linearly with increasing concentrations of dihydrotestosterone as LNCaP cells reentered the cell cycle. In vivo, induction of Her-2/neu by castration in orthotopically injected LNCaP cells resulted in a progressive increase in prostate-specific antigen secretion into the mouse serum, indicating that Her-2/neu-mediated, AR-dependent transcription occurs following castration and results in tumor cell growth. Finally, selection of LNCaP cells stably transfected with short hairpin RNA specific for AR resulted in Her-2/neu overexpression. Similarly, knockdown of Her-2/neu led to induction of AR. However, when Her-2/neu and AR were simultaneously targeted, we observed cell death, whereas surviving cells retained low level expression of Her-2/neu. Thus, induction and activation of Her-2/neu occurs in an androgen-depleted environment or as a result of AR inactivation, promoting ablation-resistant survival of prostate cancer cells. These data provide the biochemical rationale to target Her-2/neu in hormone-refractory prostate cancer. (Cancer Res 2006; 66(11): 5723-8)
Conditional Deletion ofc-mycDoes Not Impair Liver Regeneration
Authors: Li Feng , Xiang Yan , Potter James , Dinavahi Ramani , Dang Chi V. , Lee Linda A.
Published: June 2006

The oncogene c-myc encodes a transcription factor that has long been considered essential to liver regeneration, the process by which fully differentiated hepatocytes proliferate in an attempt to maintain a normal functional mass in response to hepatic injury. Experimental liver regeneration can be induced upon 70% partial hepatectomy and is accompanied by an increase in c-myc expression accompanying the synchronous entry of remaining hepatocytes into the cell cycle. Because liver regeneration is an essential process for achieving liver homeostasis, therapies directed at reducing MYC expression in hepatocellular carcinoma are fraught with the theoretical possibility of injuring adjacent noncancerous liver cells, thereby restricting the liver's normal regenerative response to injury. To determine if intact c-myc is required for liver regeneration, we reduced hepatic c-myc in c-mycfl/fl mice using an adenoviral vector that expresses Cre recombinase. Despite a 90% decrease in hepatic expression of c-myc, restoration of liver mass 7 days later was not compromised. Reconstituted liver retained the same decrease in hepatic c-myc, indicating that hepatocytes deficient in c-myc were able to proliferate in response to partial hepatectomy. Although c-myc is required for embryonic development, our findings indicate that it is not required for the maintenance of the adult liver. (Cancer Res 2006; 66(11): 5608-12)
Regression of Latent Endometrial Precancers by Progestin Infiltrated Intrauterine Device
Authors: Ørbo Anne , Rise Cecil E. , Mutter George L.
Published: June 2006

PTEN tumor suppressor inactivation is the earliest step in endometrial carcinogenesis, occurring in morphologically unremarkable endometrial glands in half of normal women. We test the hypothesis that sex hormones positively or negatively select for these “latent precancers” by examining their emergence, persistence, and regression rates under differing hormonal conditions. Perimenopausal and postmenopausal women had an intake endometrial biopsy and underwent hormonal therapy with progestin-impregnated intrauterine device (IUD; n = 21), cyclic oral progestins (n = 28), or surveillance only (n = 22) with follow-up biopsies. For comparison, premenopausal naturally cycling endometrial biopsies were studied as single time points in 87 patients and multiple surveillance time points in 34 patients. Biopsies in which any PTEN protein-null glands were found by immunohistochemistry were scored as containing a latent endometrial precancer. All groups had a similar proportion of latent precancers at intake but differed after therapy. Emergence rates were highest (21%) for the naturally cycling premenopausal group compared with just 9% for untreated perimenopausal women. The IUD group had the highest rate of regression, with a 62% pretherapy and 5% post-therapy rate of latent precancers. This contrasted to nonsignificant changes for the oral progestin and untreated control groups. Delivery of high doses of progestins locally to the endometrium by IUD leads to ablation of preexisting PTEN-inactivated endometrial latent precancers and is a possible mechanism for reduction of long-term endometrial cancer risk known to occur in response to this hormone. (Cancer Res 2006; 66(11): 5613-7)
Mutation of Tumor Suppressor Gene Men1 Acutely Enhances Proliferation of Pancreatic Islet Cells
Authors: Schnepp Robert W. , Chen Ya-Xiong , Wang Haoren , Cash Tim , Silva Albert , Diehl J. Alan , Brown Eric , Hua Xianxin
Published: June 2006

Multiple endocrine neoplasia type 1 (MEN1), an inherited tumor syndrome affecting endocrine organs including pancreatic islets, results from mutation of the tumor suppressor gene Men1 that encodes protein menin. Although menin is known to be involved in regulating cell proliferation in vitro, it is not clear how menin regulates cell cycle and whether mutation of Men1 acutely promotes pancreatic islet cell proliferation in vivo. Here we show that excision of the floxed Men1 in mouse embryonic fibroblasts (MEF) accelerates G0/G1 to S phase entry. This accelerated S-phase entry is accompanied by increased cyclin-dependent kinase 2 (CDK2) activity as well as decreased expression of CDK inhibitors p18Ink4c and p27Kip1. Moreover, Men1 excision results in decreased expression of p18Ink4c and p27Kip1 in the pancreas. Furthermore, complementation of menin-null cells with wild-type menin represses S-phase entry. To extend the role of menin in repressing cell cycle in cultured cells to in vivo pancreatic islets, we generated a system in which floxed Men1 alleles can be excised in a temporally controllable manner. As early as 7 days following Men1 excision, pancreatic islet cells display increased proliferation, leading to detectable enlargement of pancreatic islets 14 days after Men1 excision. These observations are consistent with the notion that an acute effect of Men1 mutation is accelerated S-phase entry and enhanced cell proliferation in pancreatic islets. Together, these results suggest a molecular mechanism whereby menin suppresses MEN1 tumorigenesis at least partly through repression of G0/G1 to S transition. (Cancer Res 2006; 66(10): 5707-15)
Emodin Inhibits Tumor Cell Adhesion through Disruption of the Membrane Lipid Raft-Associated Integrin Signaling Pathway
Authors: Huang Qing , Shen Han-Ming , Shui Guanghou , Wenk Markus R. , Ong Choon-Nam
Published: June 2006

Cell adhesion and spreading is a crucial step in the metastatic cascade of cancer cells, and interruption of this step is considered to be a logical strategy for prevention and treatment of tumor metastasis. Emodin is the major active component of the rhizome of Rheum palmatum L., with known anticancer activities. Here, we first found that emodin significantly inhibited cell adhesion of various human cancer cells. This inhibition was achieved through suppressing the recruitment of focal adhesion kinase (FAK) to integrin β1 as well as the phosphorylation of FAK followed by the decreased formation of focal adhesion complex (FAC). In understanding the underlying mechanisms, we found that emodin inhibited the lipid raft clustering and subsequent colocalization of integrin β1 and FAC proteins within lipid rafts. Lipid profile analysis revealed significant decrease of cholesterol and sphingolipids in raft fraction after emodin treatment. Cholesterol replenishment abolished the adverse effect of emodin on the translocation of integrin β1 and FAC proteins into the lipid raft fraction and cell adhesion. Therefore, data from this study provide novel evidence that emodin inhibits cell adhesion and spreading through disruption of the membrane lipid raft-associated integrin signaling pathway. (Cancer Res 2006; 66(11): 5807-15)
Generation of Antitumor Responses by Genetic Modification of Primary Human T Cells with a Chimeric NKG2D Receptor
Authors: Zhang Tong , Barber Amorette , Sentman Charles L.
Published: June 2006

To create more effective T cells against human tumors, we have designed a strategy to allow T cells to recognize tumor cells using natural killer (NK) cell receptors but retain the effector responses of T lymphocytes. NKG2D is an activating cell surface receptor expressed on NK cells and on some T-cell subsets. Its ligands are primarily expressed on tumor cells. We have shown that by linking mouse NKG2D to the CD3ζ chain, it was possible to generate a chimeric NKG2D (chNKG2D) receptor that allowed activation of murine T cells on engagement with NKG2D ligand-positive tumor cells leading to antitumor responses in mice. In this study, a human version of the chNKG2D receptor was expressed on primary human T cells, and antitumor responses were determined. Human peripheral blood mononuclear cell–derived T cells were retrovirally transduced with a human chNKG2D receptor gene. These chNKG2D-bearing human T cells responded to NKG2D ligand-positive tumor cells by producing T-helper 1 cytokines, proinflammatory chemokines, and significant cellular cytotoxicity. This response could be blocked by anti-NKG2D antibodies, and it was dependent on NKG2D ligand expression on the target cells but not on expression of MHC molecules. In addition, the activity of chNKG2D-bearing T cells remained unimpaired after exposure to a soluble NKG2D ligand, soluble MICA, at concentrations as high as 1.5 μg/mL. These data indicate the feasibility of using chNKG2D receptors in primary human T cells and suggest that this approach may be a promising means for cancer immunotherapy. (Cancer Res 2006; 66(11): 5927-33)
Identification of Specific Gene Copy Number Changes in Asbestos-Related Lung Cancer
Authors: Nymark Penny , Wikman Harriet , Ruosaari Salla , Hollmén Jaakko , Vanhala Esa , Karjalainen Antti , Anttila Sisko , Knuutila Sakari
Published: June 2006

Asbestos is a well-known lung cancer-causing mineral fiber. In vitro and in vivo experiments have shown that asbestos can cause chromosomal damage and aberrations. Lung tumors, in general, have several recurrently amplified and deleted chromosomal regions. To investigate whether a distinct chromosomal aberration profile could be detected in the lung tumors of heavily asbestos-exposed patients, we analyzed the copy number profiles of 14 lung tumors from highly asbestos-exposed patients and 14 matched tumors from nonexposed patients using classic comparative genomic hybridization (CGH). A specific profile could lead to identification of the underlying genes that may act as mediators of tumor formation and progression. In addition, array CGH analyses on cDNA microarrays (13,000 clones) were carried out on 20 of the same patients. Classic CGH showed, on average, more aberrations in asbestos-exposed than in nonexposed patients, and an altered region in chromosome 2 seemed to occur more frequently in the asbestos-exposed patients. Array CGH revealed aberrations in 18 regions that were significantly associated with either of the two groups. The most significant regions were 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2, and 19p13.1-p13.3 (P < 0.005). Furthermore, 11 fragile sites coincided with the 18 asbestos-associated regions (P = 0.08), which may imply preferentially caused DNA damage at these sites. Our findings are the first evidence, indicating that asbestos exposure may produce a specific DNA damage profile. (Cancer Res 2006; 66(11): 5737-43)
Specific Recruitment of CC Chemokine Receptor 4–Positive Regulatory T Cells in Hodgkin Lymphoma Fosters Immune Privilege
Authors: Ishida Takashi , Ishii Toshihiko , Inagaki Atsushi , Yano Hiroki , Komatsu Hirokazu , Iida Shinsuke , Inagaki Hiroshi , Ueda Ryuzo
Published: June 2006

Hodgkin lymphoma (HL) is characterized by the presence of a small number of tumor cells in a rich background of inflammatory cells, but the contribution of the abundant nontumor cells to HL pathogenesis is poorly understood. We showed that migratory CD4+ cells induced by HL cells were hyporesponsive to T-cell receptor stimulation and suppressed the activation/proliferation of the effector CD4+ T cells in an autologous setting. We further showed that HL cells in the affected lymph nodes were surrounded by a large number of lymphocytes expressing both CC chemokine receptor 4 (CCR4) and FOXP3. These findings indicate that the migratory cells induced by HL cells function as regulatory T (Treg) cells so that these cells create a favorable environment for the tumor cells to escape from host immune system. In addition, we showed that a chimeric anti-CCR4 monoclonal antibody (mAb) could deplete CCR4+ T cells and inhibit the migration of CD4+CD25+ T cells in vitro. Recognition of the importance of CCR4+ Treg cells in the pathogenesis of HL will allow rational design of more effective treatments, such as use of an anti-CCR4 mAb, to overcome the suppressive effect of CCR4+ Treg cells on the host immune response to tumor cells. (Cancer Res 2006; 66(11): 5716-22)
Identification of Interleukin-13 Receptor α2 Peptide Analogues Capable of Inducing Improved Antiglioma CTL Responses
Authors: Eguchi Junichi , Hatano Manabu , Nishimura Fumihiko , Zhu Xinmei , Dusak Jill E. , Sato Hidemitsu , Pollack Ian F. , Storkus Walter J. , Okada Hideho
Published: June 2006

Restricted and high-level expression of interleukin-13 receptor α2 (IL-13Rα2) in a majority of human malignant gliomas makes this protein an attractive vaccine target. We have previously described the identification of the IL-13Rα2345-353 peptide as a human leukocyte antigen-A2 (HLA-A2)–restricted CTL epitope. However, as it remains unclear how efficiently peptide-based vaccines can induce specific CTLs in patients with malignant gliomas, we have examined whether analogue epitopes could elicit heteroclitic antitumor T-cell responses versus wild-type peptides. We have created three IL-13Rα2 analogue peptides by substitutions of the COOH-terminal isoleucine (I) for valine (V) and the NH2-terminal tryptophan (W) for either alanine (A), glutamic acid (E), or nonsubstituted (W; designated as 1A9V, 1E9V, and 9V, respectively). In comparison with the native IL-13Rα2 epitope, the analogue peptides 9V and 1A9V displayed higher levels of binding affinity and stability in HLA-A2 complexes and yielded an improved stimulatory index for patient-derived, specific CTLs against the native epitope expressed by HLA-A2+ glioma cells. In HLA-A2-transgenic HHD mice, immunization with the peptides 9V and 1A9V induced enhanced levels of CTL reactivity and protective immunity against an intracranial challenge with IL13Rα2-expressing syngeneic tumors when compared with vaccines containing the native IL-13Rα2 epitope. These findings indicate highly immunogenic IL-13Rα2 peptide analogues may be useful for the development of vaccines capable of effectively expanding IL-13Rα2-specific, tumor-reactive CTLs in glioma patients. (Cancer Res 2006; 66(11): 5883-91)
Developmental Exposure to Estradiol and Bisphenol A Increases Susceptibility to Prostate Carcinogenesis and Epigenetically Regulates Phosphodiesterase Type 4 Variant 4
Authors: Ho Shuk-Mei , Tang Wan-Yee , Belmonte de Frausto Jessica , Prins Gail S.
Published: June 2006

Early developmental perturbations have been linked to adult-onset prostate pathology, including excessive exposure to estrogenic compounds; however, the molecular basis for this imprinting event is not known. An important and controversial health concern is whether low-dose exposures to hormonally active environmental estrogens, such as bisphenol A, can promote human diseases, including prostate cancer. Here, we show that transient developmental exposure of rats to low, environmentally relevant doses of bisphenol A or estradiol increases prostate gland susceptibility to adult-onset precancerous lesions and hormonal carcinogenesis. We found permanent alterations in the DNA methylation patterns of multiple cell signaling genes, suggesting an epigenetic basis for estrogen imprinting. For phosphodiesterase type 4 variant 4 (PDE4D4), an enzyme responsible for cyclic AMP breakdown, a specific methylation cluster was identified in the 5′-flanking CpG island that was gradually hypermethylated with aging in normal prostates, resulting in loss of gene expression. Early and prolonged hypomethylation at this site following neonatal estradiol or bisphenol A exposure resulted in continued, elevated PDE4D4 expression. Cell line studies confirmed that site-specific methylation is involved in transcriptional silencing of the PDE4D4 gene and showed hypomethylation of this gene in prostate cancer cells. Importantly, the PDE4D4 alterations in the estrogen-exposed prostates were distinguishable before histopathologic changes of the gland, making PDE4D4 a candidate molecular marker for prostate cancer risk assessment as a result of endocrine disruptors. In total, these findings indicate that low-dose exposures to ubiquitous environmental estrogens affect the prostate epigenome during development and, in so doing, promote prostate disease with aging. (Cancer Res 2006; 66(11): 5624-32)
Mechanistic Investigation and Implications of Photodynamic Therapy Induction of Vascular Endothelial Growth Factor in Prostate Cancer
Authors: Solban Nicolas , Pål Selbo K. , Alok Sinha K. , Sung Chang K. , Hasan Tayyaba
Published: June 2006

Photodynamic therapy (PDT) is now an approved therapeutic modality, and induction of vascular endothelial growth factor (VEGF) following subcurative PDT is of concern as VEGF may provide a survival stimulus to tumors. The processes that limit the efficacy of PDT warrant investigation so that mechanism-based interventions may be developed. This study investigates VEGF increase following subcurative PDT using the photosensitizer benzoporphyrin derivative (BPD) both in an in vitro and in an orthotopic model of prostate cancer using the human prostate cancer cell line LNCaP. The two subcurative doses used, 0.25 and 0.5 J/cm2, mimicked subcurative PDT and elicited a 1.6- and 2.1-fold increase, respectively, in secreted VEGF 24 hours following PDT. Intracellular VEGF protein measurement and VEGF mRNA showed a 1.4- and 1.6-fold increase only at 0.5 J/cm2. In vivo subcurative PDT showed an increase in VEGF by both immunohistochemistry and ELISA. In vitro analysis showed no activation of hypoxia-inducible factor-1α (HIF-1α) or cyclooxygenase-2 (COX-2) following subcurative PDT; furthermore, small interfering RNA inhibition of HIF-1α and COX-2 inhibitor treatment had no effect on PDT induction of VEGF. PDT in the presence of phosphatidylinositol 3-kinase/AKT inhibitor or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor still induced VEGF. However, subcurative PDT increased phosphorylated p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase. The p38 MAPK inhibitor abolished PDT induction of VEGF. The results establish the importance of VEGF in subcurative BPD-PDT of prostate cancer and suggest possible molecular pathways for its induction. These findings should provide the basis for the development of molecular-based interventions for enhancing PDT and merit further studies. (Cancer Res 2006; 66(11):5633-40)
Activation of Abl Tyrosine Kinases Promotes Invasion of Aggressive Breast Cancer Cells
Authors: Srinivasan Divyamani , Plattner Rina
Published: June 2006

The Abl family of nonreceptor tyrosine kinases consists of two related proteins, c-Abl and Abl-related gene (Arg). Activated forms of the Abl kinases (BCR-Abl, Tel-Abl, and Tel-Arg) induce the development of human leukemia; it is not known, however, whether Abl kinases are activated in solid tumors or whether they contribute to tumor development or progression. Previously, we showed that Abl kinases are activated downstream of growth factor receptors, Src family kinases, and phospholipase Cγ1 (PLCγ1) in fibroblasts and influence growth factor–mediated proliferation, membrane ruffling, and migration. Growth factor receptors, Src kinases, and PLCγ1 are deregulated in many solid tumors and drive tumor invasion and metastasis. In this study, we found that Abl kinases are constitutively activated, in highly invasive breast cancer cell lines, downstream of deregulated ErbB receptors and Src kinases. Furthermore, activation of Abl kinases promotes breast cancer cell invasion, as treatment of cells with the Abl kinase inhibitor, STI571, or silencing c-Abl and Arg expression with RNA interference dramatically inhibits Matrigel invasion. This is the first evidence that (a) Abl kinases are deregulated and activated in a nonhematopoietic cancer, (b) activation of Abl kinases in breast cancer cells occurs via a novel mechanism, and (c) constitutive activation of Abl kinases promotes invasion of breast cancer cells. These data suggest that pharmacologic inhibitors targeted against Abl kinases could potentially be useful in preventing breast cancer progression in tumors harboring activated Abl kinases. (Cancer Res 2006; 66(11): 5648-55)
Insulin-Like Growth Factor I Receptor Pathway Inhibition by ADW742, Alone or in Combination with Imatinib, Doxorubicin, or Vincristine, Is a Novel Therapeutic Approach in Ewing Tumor
Authors: Martins Ana S. , Mackintosh Carlos , Martín David Herrero , Campos Maria , Hernández Teresa , Ordóñez Jose-Luis , de Alava Enrique
Published: June 2006

Purpose: Ewing tumor cell survival and proliferation depends on several autocrine loops. Targeting these loops is a promising therapeutic approach. We recently showed the cytostatic role of imatinib, an inhibitor of the SCF-KIT loop, on Ewing tumor cells, and in this study, we intend to analyze the inhibition of the insulin-like growth factor I receptor (IGF1R) loop.

Experimental Design: We analyzed IGF1R blockade by ADW742, a small molecule specific for this receptor, alone and in combination with imatinib, vincristine, and doxorubicin on Ewing tumor cell lines. We studied the effect on proliferation, apoptosis, cell cycle, pathway phosphorylation, soft-agar growth, motility, and vascular endothelial growth factor expression levels.

Results: Treatment with ADW742 induced down-regulation of IGF1R/AKT/mammalian target of rapamycin (mTOR) phosphorylation, which was deeper in cell lines having higher IGF1R activation levels. Treatment also induced dose-dependent inhibition of cell proliferation (IC50 = 0.55-1.4 μmol/L), inducing a G1 phase blockage and apoptosis. Addition of imatinib to ADW742 synergistically augmented these effects and was especially effective in inhibiting AKT/mTOR phosphorylation and reducing vascular endothelial growth factor expression in cell lines having high IGF1R activation levels. Combination with usual chemotherapeutic agents vincristine and doxorubicin showed synergistic interactions.

Conclusions: Inhibition of Ewing tumor cell proliferation by ADW742 is mediated through blockade of IGF1R signaling. Combination of ADW742 with imatinib, vincristine, and doxorubicin induces a significant reduction of tumor cell growth, mainly by the increase in apoptosis with a pattern depending on IGF1R activation levels. This study supports a potential role for ADW742 in the treatment of Ewing tumor and AKT/mTOR as a possible surrogate marker of response to therapy.
Sulindac Sulfide and Exisulind Inhibit Expression of the Estrogen and Progesterone Receptors in Human Breast Cancer Cells
Authors: Lim Jin T.E. , Joe Andrew K. , Suzui Masumi , Shimizu Masahito , Masuda Muneyuki , Weinstein I. Bernard
Published: June 2006

In previous studies, we found that sulindac sulfide and exisulind (sulindac sulfone, Aptosyn) cause growth inhibition, arrest cells in the G1 phase of the cell cycle, and induce apoptosis in human breast cancer cell lines. These effects were associated with decreased expression of cyclin D1. The present study focuses on the effects of sulindac sulfide and exisulind on hormone signaling components in breast cancer cells. We found that estrogen receptor (ER)–positive and progesterone receptor (PR)–positive T47D breast cancer cells were somewhat more sensitive to growth inhibition by sulindac sulfide or exisulind than ER-negative PR-negative MB-MDA-468 breast cancer cells. Further studies indicated that sulindac sulfide and exisulind caused marked down-regulation of expression of the ER and PR-A and PR-B in T47D cells. However, neither compound caused a major change in expression of the retinoic acid receptor α (RARα), RARβ, or RARα in T47D cells. Sulindac sulfide and exisulind also caused a decrease in expression of the ER in estrogen-responsive MCF-7 breast cancer cells. Both compounds also markedly inhibited estrogen-stimulated activation of an estrogen-responsive promoter in transient transfection reporter assays. Treatment of T47D cells with specific protein kinase G (PKG) activators did not cause a decrease in ER or PR expression. Therefore, although sulindac sulfide and exisulind can cause activation of PKG, the inhibitory effects of these two compounds on ER and PR expression does not seem to be mediated by PKG. Our findings suggest that the growth inhibition by sulindac sulfide and exisulind in ER-positive and PR-positive human breast cancer cells may be mediated, in part, by inhibition of ER and PR signaling. Thus, these and related compounds may provide a novel approach to the prevention and treatment of human breast cancers, especially those that are ER positive.