bluuebunny/crossref_metadata_2025 huggingface dataset:
Prediction of Radiation Sensitivity Using a Gene Expression Classifier
Authors: Torres-Roca Javier F. , Eschrich Steven , Zhao Haiyan , Bloom Gregory , Sung Jimmy , McCarthy Susan , Cantor Alan B. , Scuto Anna , Li Changgong , Zhang Suming , Jove Richard , Yeatman Timothy
Published: September 2005

The development of a successful radiation sensitivity predictive assay has been a major goal of radiation biology for several decades. We have developed a radiation classifier that predicts the inherent radiosensitivity of tumor cell lines as measured by survival fraction at 2 Gy (SF2), based on gene expression profiles obtained from the literature. Our classifier correctly predicts the SF2 value in 22 of 35 cell lines from the National Cancer Institute panel of 60, a result significantly different from chance (P = 0.0002). In our approach, we treat radiation sensitivity as a continuous variable, significance analysis of microarrays is used for gene selection, and a multivariate linear regression model is used for radiosensitivity prediction. The gene selection step identified three novel genes (RbAp48, RGS19, and R5PIA) of which expression values are correlated with radiation sensitivity. Gene expression was confirmed by quantitative real-time PCR. To biologically validate our classifier, we transfected RbAp48 into three cancer cell lines (HS-578T, MALME-3M, and MDA-MB-231). RbAp48 overexpression induced radiosensitization (1.5- to 2-fold) when compared with mock-transfected cell lines. Furthermore, we show that HS-578T-RbAp48 overexpressors have a higher proportion of cells in G2-M (27% versus 5%), the radiosensitive phase of the cell cycle. Finally, RbAp48 overexpression is correlated with dephosphorylation of Akt, suggesting that RbAp48 may be exerting its effect by antagonizing the Ras pathway. The implications of our findings are significant. We establish that radiation sensitivity can be predicted based on gene expression profiles and we introduce a genomic approach to the identification of novel molecular markers of radiation sensitivity.
Expanding the Use of Magnetic Resonance in the Assessment of Tumor Response to Therapy: Workshop Report
Authors: Evelhoch Jeffrey , Garwood Michael , Vigneron Daniel , Knopp Michael , Sullivan Daniel , Menkens Anne , Clarke Laurence , Liu Guoying
Published: September 2005

Although dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and magnetic resonance spectroscopy (MRS) have great potential to provide routine assessment of cancer treatment response, their widespread application has been hampered by a lack of standards for use. Thus, the National Cancer Institute convened a workshop to assess developments and applications of these methods, develop standards for methodology, and engage relevant partners (drug and device industries, researchers, clinicians, and government) to encourage sharing of data and methodologies. Consensus recommendations were reached for DCE-MRI methodologies and the focus for initial multicenter trials of MRS. In this meeting report, we outline the presentations, the topics discussed, the ongoing challenges identified, and the recommendations made by workshop participants for the use of DCE-MRI and 1H MRS in the clinical assessment of antitumor therapies.
Characterization of DNA Demethylation Effects Induced by 5-Aza-2′-Deoxycytidine in Patients with Myelodysplastic Syndrome
Authors: Mund Cora , Hackanson Björn , Stresemann Carlo , Lübbert Michael , Lyko Frank
Published: September 2005

Azanucleoside drugs such as 5-azacytidine (Vidaza) and 5-aza-2′-deoxycytidine (decitabine, Dacogen) function as DNA methyltransferase inhibitors in vitro and represent promising new drugs for the treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia. In this study, we aimed to determine the effect of decitabine on the genomic methylation level in MDS patients. Comparison of different assays established micellar electrokinetic chromatography as a reliable method for the analysis of genomic methylation levels. When used for the determination of DNA methylation levels in bone marrow DNA from MDS patients during various time points of decitabine treatment, the results revealed a significant (up to 70%) demethylation in five of seven patients. Interestingly, genome-wide demethylation appeared after karyotype normalization, which suggests demethylation of nonclonal cells. Drug-induced demethylation dynamics were also confirmed by bisulfite sequencing of pericentromeric satellite elements. Our results are the first to show a genome-wide demethylating activity of decitabine in tumor material. In addition, our data uncovers novel targets of decitabine-mediated demethylation that are important for the refinement of treatment schedules with demethylating drugs.
Low-dose Metronomic Combined with Intermittent Bolus-dose Cyclophosphamide Is an Effective Long-term Chemotherapy Treatment Strategy
Authors: Shaked Yuval , Emmenegger Urban , Francia Giulio , Chen Limor , Lee Christina R. , Man Shan , Paraghamian Armen , Ben-David Yaacov , Kerbel Robert S.
Published: September 2005

Metronomic chemotherapy refers to the close, regular administration of comparatively low doses of cytotoxic drugs, with minimal or no drug-free breaks, over prolonged periods. It is thought to have an antiangiogenic basis. However, whereas surprisingly durable and potent tumor responses have been observed in a number of preclinical tumor models, relapses usually eventually occur using this type of treatment strategy. We therefore decided to test modified metronomic chemotherapy regimens that might significantly delay such relapses, but still maintain modest and acceptable toxicity profiles. Here, we show that repeated administration of bolus doses (BDs) of cyclophosphamide every 3 or 6 weeks, combined with a daily oral low-dose metronomic (LDM) regimen (20 mg/kg/d cyclophosphamide), improves efficacy and significantly delays progression of transplanted PC-3 human prostate cancer xenografts, syngeneic transplanted EMT-6 breast tumors, and “spontaneous” murine erythroleukemia. Efficacy was superior whereas toxicity was mild and comparable to the LDM regimen, the latter assessed by body weight, neutrophil, lymphocyte, and total white blood counts. Antiangiogenic activity, measured by reduction in circulating endothelial precursor cells, revealed that the greatest degree of suppression occurred using the combination treatment. Overall, our results indicate that the administration of intermittent BD combined with chronic oral LDM cyclophosphamide is a potent treatment regimen for controlling tumor growth, which has a low toxicity profile, over prolonged periods of time.
Galectin-3 Regulates a Molecular Switch from N-Ras to K-Ras Usage in Human Breast Carcinoma Cells
Authors: Shalom-Feuerstein Ruby , Cooks Tomer , Raz Avraham , Kloog Yoel
Published: September 2005

Galectin-3 (Gal-3), a pleiotropic carbohydrate-binding protein, is a selective binding partner of activated K-Ras-GTP. Because both proteins are antiapoptotic and associated with cancer progression, we questioned the possible functional role of Gal-3 in K-Ras activation. We found that overexpression of Gal-3 in human breast cancer cells (BT-549/Gal-3) coincided with a significant increase in wild-type (wt) K-Ras-GTP coupled with loss in wt N-Ras-GTP, whereas the nononcogenic Gal-3 mutant proteins [Gal-3(S6E) and Gal-3(G182A)] failed to induce the Ras isoform switch. Only wt Gal-3 protein coimmunoprecipitated and colocalized with oncogenic K-Ras, resulting in its activation with radical alterations in Ras signaling pathway, whereby the activation of AKT and Ral was suppressed and shifted to the activation of extracellular signal-regulated kinase (ERK). Specific inhibitors for Ras or mitogen-activated protein/ERK kinase (farnesylthiosalicylic acid and UO126, respectively) inhibited Gal-3–mediated apoptotic resistance and anchorage-independent growth functions. In conclusion, this study shows that Gal-3 confers on BT-549 human breast carcinoma cells several oncogenic functions by binding to and activation of wt K-Ras, suggesting that some of the molecular functions of Gal-3 are, at least in part, a result of K-Ras activation.
FTY720 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Drug Resistance
Authors: Yasui Hiroshi , Hideshima Teru , Raje Noopur , Roccaro Aldo M. , Shiraishi Norihiko , Kumar Shaji , Hamasaki Makoto , Ishitsuka Kenji , Tai Yu-Tzu , Podar Klaus , Catley Laurence , Mitsiades Constantine S. , Richardson Paul G. , Albert Rainer , Brinkmann Volker , Chauhan Dharminder , Anderson Kenneth C.
Published: September 2005

The novel immunomodulator FTY720 down-modulates sphingosine-1-phosphate receptor 1 on lymphocytes at low nanomolar concentrations, thereby inhibiting sphingosine-1-phosphate receptor 1–dependent egress of lymphocytes from lymph nodes into efferent lymphatics and blood. At high micromolar concentration, FTY720 has been shown to induce growth inhibition and/or apoptosis in human cancer cells in vitro. In this study, we investigated the biological effects of FTY720 on multiple myeloma cells. We found that FTY720 induces potent cytotoxicity against drug-sensitive and drug-resistant multiple myeloma cell lines as well as freshly isolated tumor cells from multiple myeloma patients who do not respond to conventional agents. FTY720 triggers activation of caspase-8, -9, and -3, followed by poly(ADP-ribose) polymerase cleavage. Interestingly, FTY720 induces alterations in mitochondrial membrane potential (ΔΨm) and Bax cleavage, followed by translocation of cytochrome c and Smac/Diablo from mitochondria to the cytosol. In combination treatment studies, both dexamethasone and anti-Fas antibodies augment anti–multiple myeloma activity induced by FTY720. Neither interleukin-6 nor insulin-like growth factor-I, which both induce multiple myeloma cell growth and abrogate dexamethasone-induced apoptosis, protect against FTY720-induced growth inhibition. Importantly, growth of multiple myeloma cells adherent to bone marrow stromal cells is also significantly inhibited by FTY720. Finally, it down-regulates interleukin-6–induced phosphorylation of Akt, signal transducers and activators of transcription 3, and p42/44 mitogen-activated protein kinase; insulin-like growth factor-I–triggered Akt phosphorylation; and tumor necrosis factor α–induced IκBα and nuclear factor-κB p65 phosphorylation. These results suggest that FTY720 overcomes drug resistance in multiple myeloma cells and provide the rationale for its clinical evaluation to improve patient outcome in multiple myeloma.
Heterozygous ATR Mutations in Mismatch Repair–Deficient Cancer Cells Have Functional Significance
Authors: Lewis Kriste A. , Mullany Sally , Thomas Bijoy , Chien Jeremy , Loewen Ralitsa , Shridhar Viji , Cliby William A.
Published: September 2005

ATR (ataxia telangiectasia and Rad3-related) function is necessary for the proper response to commonly used chemotherapeutic agents. Heterozygous truncating mutations in exon 10 of the ATR gene have been described in numerous cancers exhibiting microsatellite instability. We show that truncating mutations of ATR are capable of acting in a dominant-negative manner to abrogate ATR-dependent Chk1 phosphorylation and cell-cycle arrests after DNA damage. In addition, endometrial cell lines harboring ATR mutations are defective for ATR-dependent responses. These findings imply that ATR mutations play an important role in the development and clinical behavior of a subset of microsatellite instability–positive endometrial, colon, and stomach cancers.
Expression Profile of Malignant and Nonmalignant Lesions of Esophagus and Stomach: Differential Activity of Functional Modules Related to Inflammation and Lipid Metabolism
Authors: Gomes Luciana I. , Esteves Gustavo H. , Carvalho Alex F. , Cristo Elier B. , Hirata Roberto , Martins Waleska K. , Marques Sarah M. , Camargo Luiz P. , Brentani Helena , Pelosof Adriane , Zitron Cláudia , Sallum Rubens A. , Montagnini André , Soares Fernando A. , Neves E. Jordão , Reis Luiz F.L.
Published: September 2005

Adenocarcinomas of stomach and esophagus are frequently associated with preceding inflammatory alterations of the normal mucosa. Whereas intestinal metaplasia of the gastric mucosa is associated with higher risk of malignization, Barrett's disease is a risk factor for adenocarcinoma of the esophagus. Barrett's disease is characterized by the substitution of the squamous mucosa of the esophagus by a columnar tissue classified histopathologically as intestinal metaplasia. Using cDNA microarrays, we determined the expression profile of normal gastric and esophageal mucosa as well as intestinal metaplasia and adenocarcinomas from both organs. Data were explored to define functional alterations related to the transformation from squamous to columnar epithelium and the malignant transformation from intestinal metaplasia to adenocarcinomas. Based on their expression profile, adenocarcinomas of the esophagus showed stronger correlation with intestinal metaplasia of the stomach than with Barrett's mucosa. Second, we identified two functional modules, lipid metabolism and cytokine, as being altered with higher statistical significance. Whereas the lipid metabolism module is active in samples representing intestinal metaplasia and inactive in adenocarcinomas, the cytokine module is inactive in samples representing normal esophagus and esophagitis. Using the concept of relevance networks, we determined the changes in linear correlation of genes pertaining to these two functional modules. Exploitation of the data presented herein will help in the precise molecular characterization of adenocarcinoma from the distal esophagus, avoiding the topographical and descriptive classification that is currently adopted, and help with the proper management of patients with Barrett's disease.
Claudin-3 and Claudin-4 Expression in Ovarian Epithelial Cells Enhances Invasion and Is Associated with Increased Matrix Metalloproteinase-2 Activity
Authors: Agarwal Rachana , D'Souza Theresa , Morin Patrice J.
Published: September 2005

Claudin proteins form a large family of integral membrane proteins crucial for tight junction formation and function. Our previous studies have revealed that claudin-3 and claudin-4 proteins are highly overexpressed in ovarian cancer. To clarify the roles of claudins in ovarian tumorigenesis, we have generated human ovarian surface epithelial (HOSE) cells constitutively expressing wild-type claudin-3 and claudin-4. Expression of these claudins in HOSE cells increased cell invasion and motility as measured by Boyden chamber assays and wound-healing experiments. Conversely, small interfering RNA (siRNA)–mediated knockdown of claudin-3 and claudin-4 expression in ovarian cancer cell lines reduced invasion. Claudin expression also increased cell survival in HOSE cells but did not significantly affect cell proliferation. Moreover, the claudin-expressing ovarian epithelial cells were found to have increased matrix metalloproteinase-2 (MMP-2) activity indicating that claudin-mediated increased invasion might be mediated through the activation of MMP proteins. However, siRNA inactivation of claudins in ovarian cancer cell lines did not have a significant effect on the high endogenous MMP-2 activity present in these cells, showing that malignant cells have alternative or additional pathways to fully activate MMP-2. Taken together, our results suggest that claudin overexpression may promote ovarian tumorigenesis and metastasis through increased invasion and survival of tumor cells.
An Alternative Inhibitor Overcomes Resistance Caused by a Mutation of the Epidermal Growth Factor Receptor
Authors: Kobayashi Susumu , Ji Hongbin , Yuza Yuki , Meyerson Matthew , Wong Kwok-Kin , Tenen Daniel G. , Halmos Balázs
Published: September 2005

Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in non–small cell lung cancer specimens from patients responding to anilinoquinazoline EGFR inhibitors. However, clinical resistance to EGFR inhibitor therapy is commonly observed. Previously, we showed that such resistance can be caused by a second mutation of the EGFR gene, leading to a T790M amino acid change in the EGFR tyrosine kinase domain and also found that CL-387,785, a specific and irreversible anilinoquinazoline EGFR inhibitor, was able to overcome this resistance on the biochemical level. Here, we present the successful establishment of a stable Ba/F3 cell line model system for the study of oncogenic EGFR signaling and the functional consequences of the EGFR T790M resistance mutation. We show the ability of gefitinib to induce growth arrest and apoptosis in cells transfected with wild-type or L858R EGFR, whereas the T790M mutation leads to high-level functional resistance against gefitinib and erlotinib. In addition, CL-387,785 is able to overcome resistance caused by the T790M mutation on a functional level, correlating with effective inhibition of downstream signaling pathways. Similar data was also obtained with the use of the gefitinib-resistant H1975 lung cancer cell line. The systems established by us should prove useful for the large-scale screening of alternative EGFR inhibitor compounds against the T790M or other EGFR mutations. These data also support the notion that clinical investigations of compounds similar to CL-387,785 may be useful as a treatment strategy for patients with resistance to EGFR inhibitor therapy caused by the T790M mutation.
Suppression of p53 by Notch in Lymphomagenesis: Implications for Initiation and Regression
Authors: Beverly Levi J. , Felsher Dean W. , Capobianco Anthony J.
Published: September 2005

Aberrant Notch signaling contributes to more than half of all human T-cell leukemias, and accumulating evidence indicates Notch involvement in other human neoplasms. We developed a tetracycline-inducible mouse model (Top-Notchic) to examine the genetic interactions underlying the development of Notch-induced neoplastic disease. Using this model, we show that Notch suppresses p53 in lymphomagenesis through repression of the ARF-mdm2-p53 tumor surveillance network. Attenuation of Notch expression resulted in a dramatic increase in p53 levels that led to tumor regression by an apoptotic program. This shows that continued Notch activity is required to maintain the disease state. However, all tumors relapsed with rapid kinetics, most of which, by reactivation of Notch expression. Furthermore, by directly inhibiting the mdm2-p53 interaction by using either ionizing radiation or the novel small molecule therapeutic Nutlin, p53 can be activated and cause tumor cell death, even in the presence of sustained Notch activity. Therefore, it is the suppression of p53 that provides the Achilles heel for Notch-induced tumors, as activation of p53 in the presence of Notch signaling drives tumor regression. Our study provides proof-of-principle for the rational targeting of therapeutics against the mdm2-p53 pathway in Notch-induced neoplasms. Furthermore, we propose that suppression of p53 by Notch is a key mechanism underlying the initiation of T-cell lymphoma.
Ser392 Phosphorylation Regulates the Oncogenic Function of Mutant p53
Authors: Yap Damian B. S. , Hsieh Jung-Kuang , Zhong Shan , Heath Vicky , Gusterson Barry , Crook Tim , Lu Xin
Published: September 2005

Despite the wealth of information on the regulation of wild-type p53 function by phosphorylation, nothing is known about the biological effect of phosphorylation on mutant p53. Here we show that p53H175 is phosphorylated like wild-type p53 in cells of the same background. Ser392 nonphosphorylatable p53 mutants p53H175A392 and p53W248A392 more potently transformed rat embryo fibroblasts in cooperation with the ras oncogene than p53H175S392 and p53W248S392. p53H175A392 also had an enhanced ability to confer cellular resistance to the cytotoxic effect of cisplatin and UV radiation. This correlated with p53H175A392 being a more potent dominant negative mutant than p53H175 in inhibiting the apoptotic functions of wild-type p53. Moreover, p53H175E392, which mimics the phosphorylated form of p53H175, was less able to confer cellular resistance to DNA-damaging agents. p53H175 and p53W248 are phosphorylated like wild-type p53 in cells of the same background. Ser392 nonphosphorylated p53 was present in human breast tumors expressing mutant p53 including p53H175. Together, these results demonstrated a novel function of Ser392 phosphorylation in regulating the oncogenic function of mutant p53.
p53-Induced Up-Regulation of MnSOD and GPx but not Catalase Increases Oxidative Stress and Apoptosis
Authors: Hussain S. Perwez , Amstad Paul , He Peijun , Robles Ana , Lupold Shawn , Kaneko Ichiro , Ichimiya Masato , Sengupta Sagar , Mechanic Leah , Okamura Shu , Hofseth Lorne J. , Moake Matthew , Nagashima Makoto , Forrester Kathleen S. , Harris Curtis C.
Published: September 2005

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca2+ from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.
Microenvironmental Regulation of Proliferation in Multicellular Spheroids Is Mediated through Differential Expression of Cyclin-Dependent Kinase Inhibitors
Authors: LaRue Karen E. A. , Khalil Mona , Freyer James P.
Published: September 2005

Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18INK4c, p21waf1/cip1, and p27Kip1 all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18INK4c and p27Kip1 expression positively correlate with growth arrest, whereas p21waf1/cip1 is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18INK4c is found in increasing association with CDK6, whereas p27Kip1 associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growth-conducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.
Reversible Regulation of the Transformed Phenotype of Ornithine Decarboxylase- and Ras-Overexpressing Cells by Dominant-Negative Mutants of c-Jun
Authors: Kielosto Mari , Nummela Pirjo , Katainen Riikka , Leaner Virna , Birrer Michael J. , Hölttä Erkki
Published: September 2005

c-Jun is an oncogenic transcription factor involved in the regulation of cell proliferation, apoptosis and transformation. We have previously reported that cell transformations induced by ornithine decarboxylase (ODC) and c-Ha-ras oncogene, commonly activated in various cancer cells, are associated with constitutively increased phosphorylation of c-Jun on Ser residues 63 and 73. In the present study, we examined the significance of c-Jun phosphorylation and activation on the phenotype of the ODC- and ras-transformants, by using specific inhibitors and dominant-negative (DN) mutants to c-Jun NH2-terminal kinase (JNK) and its upstream kinase, SEK1/MKK4 (mitogen-activated protein kinase kinase 4), and to c-Jun. The transformed morphology of both the ODC- and ras-expressing cells was reversed partially by JNK inhibitors and DN JNK1, more effectively by DN SEK1/MKK4 and phosphorylation-deficient c-Jun mutants (c-JunS63,73A, c-JunS63,73A,T91,93A) and most potently by a transactivation domain deletion mutant of c-Jun (TAM67). Moreover, tetracycline-inducible TAM67 expression in ODC- and ras-transformed cells showed that the transformed phenotype of the cells is reversibly regulatable. TAM67 also inhibited the tumorigenicity of the cells in nude mice. These inducible cell lines, together with their parental cell lines, provide good models to identify the genes and proteins relevant to cellular transformation.
Binding and Cytotoxicity of Conjugated and Recombinant Fusion Proteins Targeted to the Gonadotropin-Releasing Hormone Receptor
Authors: Qi Lin , Nett Terry M. , Allen Matthew C. , Sha Xiaoming , Harrison Gail S. , Frederick Barbara A. , Crawford E. David , Glode L. Michael
Published: September 2005

Pokeweed antiviral protein (PAP) is a plant-derived, highly potent ribosome inactivating protein that causes inhibition of protein translation and rapid cell death. We and others have delivered this protein to various cell types, including cancer cells, using hormones to specifically target cells bearing the hormone receptor. Here, we compare binding and cytotoxicity of GnRH-PAP hormonotoxins prepared either by protein conjugation (GnRH-PAP conjugate) or through recombinant DNA technology (GnRH-PAP fusion). Although GnRH-PAP conjugate protein bound specifically to and caused cell death in cells bearing the gonadotropin-releasing hormone (GnRH) receptor, we could not detect binding or cytotoxicity using two different versions of the fusion protein in receptor-positive cells. We conclude that generation of an active GnRH-PAP fusion protein may not be feasible either because both ends of the GnRH molecule are required for receptor binding, but only the NH2 terminus is free in the fusion protein and/or that more potent analogues of GnRH (inclusion of which is not feasible in the fusion protein) are needed for efficient targeting. In contrast, the GnRH-PAP conjugate shows promise as a novel anticancer agent, capable of targeting cancer cells expressing the GnRH receptor such as prostate, breast, ovarian, endometrial, and pancreatic cells. It may also be useful as a therapeutic agent to eliminate pituitary gonadotrophs, eliminating the need for chronic GnRH analogue administration to treat hormone-sensitive diseases.
Ex VivoExpansion of CD8+CD56+ and CD8+CD56− Natural Killer T Cells Specific for MUC1 Mucin
Authors: Wajchman Howard J. , Pierce Carl W. , Varma Vijay A. , Issa Muta M. , Petros John , Dombrowski Kenneth E.
Published: September 2005

Prostate cancers express MUC1, but nearly all metastatic cells lack HLA class I molecules. Thus, a lymphocyte population that can sense its antigenic environment, while also able to react to stimuli of natural killer (NK) cells, may be a more versatile effector cell population for antitumor immune responses. Herein, we report that tumor-specific MUC1 peptide, interleukin 2, and interleukin 12 act synergistically to stimulate the ex vivo expansion of CD8+CD56− T cells and CD8+CD56+ natural killer T (NKT) cells from the peripheral blood mononuclear cells of prostate cancer patients, as well as healthy male and female donors. Both the CD56+ NKT cells and CD56− T cells lysed allogeneic mucin-bearing target cells, as well as NK target cells, but not lymphokine-activated killer target cells. However, the CD56+ NKT cells displayed a 2-fold greater cytolytic activity than the CD56− T cells. The mucin-specific cytolytic activity and NK cytolytic activities for both lymphocyte populations were independent of HLA class I and CD1 molecules. The CD56− T cells up-regulated CD56 with continued antigenic stimulation in the presence of interleukin 12, suggesting that CD8+CD56− T cells are NKT cells. However, CD56+ NKT cells expand poorly to continued stimulation. All mucin-stimulated NKT cells exhibited the activated/memory CD45RO phenotype. The NKT cell lines express the α/β T-cell receptor (TCR). The TCR repertoire was limited and varied with cell line, but was not the Vα24Vβ11 TCR typically associated with NKT cells. Whereas CD161 is generally considered a marker of NKT cells, the mucin-stimulated NKT cells did not express this marker. Thus, we have described two phenotypically distinct NKT types that do not display a biased TCR repertoire, but do display specificity for a tumor-specific peptide antigen (CTL-like activity), as well as HLA class I-deficient target cells (NK-like activity).
Antiangiogenic and Antitumor Efficacy of EphA2 Receptor Antagonist
Authors: Dobrzanski Pawel , Hunter Kathryn , Jones-Bolin Susan , Chang Hong , Robinson Candy , Pritchard Sonya , Zhao Hugh , Ruggeri Bruce
Published: September 2005

Tumor-associated angiogenesis is critical for tumor growth and metastasis and is controlled by various pro- and antiangiogenic factors. The Eph family of receptor tyrosine kinases has emerged as one of the pivotal regulators of angiogenesis. Here we report that interfering with EphA signaling resulted in a pronounced inhibition of angiogenesis in ex vivo and in vivo model systems. Administration of EphA2/Fc soluble receptors inhibited, in a dose-dependent manner, microvessel formation in rat aortic ring assay, with inhibition reaching 76% at the highest dose of 5000 ng/ml. These results were further confirmed in vivo in a porcine aortic endothelial cell-vascular endothelial growth factor (VEGF)/basic fibroblast growth factor Matrigel plug assay, in which administration of EphA2/Fc soluble receptors resulted in 81% inhibition of neovascularization. The additive effects of simultaneous inhibition of VEGF receptor 2 and EphA signaling pathways in aortic ring assay and antiangiogenic efficacy of EphA2/Fc soluble receptors against VEGF/basic fibroblast growth factor-mediated neovascularization in vivo indicated a critical and nonredundant role for EphA signaling in angiogenesis. Furthermore, in two independent experiments, we demonstrated that EphA2/Fc soluble receptors strongly (by ∼50% versus controls) suppressed growth of ASPC-1 human pancreatic tumor s.c. xenografts. Inhibition of tumor growth was due to decreased proliferation of tumor cells. In an orthotopic pancreatic ductal adenocarcinoma model in mice, suppression of EphA signaling by i.p. administration of EphA2/Fc (30 μg/dose, three times a week for 56 days) profoundly inhibited the growth of primary tumors and the development of peritoneal, lymphatic, and hepatic metastases. These data demonstrate a critical role of EphA signaling in tumor growth and metastasis and provide a strong rationale for targeting EphA2 receptors for anticancer therapies.
Inhibition of Connective Tissue Growth Factor (CTGF/CCN2) Expression Decreases the Survival and Myogenic Differentiation of Human Rhabdomyosarcoma Cells
Authors: Croci Stefania , Landuzzi Lorena , Astolfi Annalisa , Nicoletti Giordano , Rosolen Angelo , Sartori Francesca , Follo Matilde Y. , Oliver Noelynn , De Giovanni Carla , Nanni Patrizia , Lollini Pier-Luigi
Published: September 2005

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.
β-Catenin Up-Regulates the Expression of the Urokinase Plasminogen Activator in Human Colorectal Tumors
Authors: Hiendlmeyer Elke , Regus Susanne , Wassermann Stella , Hlubek Falk , Haynl Angela , Dimmler Arno , Koch Claudia , Knoll Claudia , van Beest Moniek , Reuning Ute , Brabletz Thomas , Kirchner Thomas , Jung Andreas
Published: September 2005

Expression of the urokinase plasminogen activator (uPA) increases during the progression of colorectal tumors from adenomas to carcinomas. The highest amounts of uPA are found at the invasion front of carcinomas, which also displays a strong expression of nuclear β-catenin and is therefore a region expressing β-catenin target genes at high levels. Here we show that β-catenin contributes to the transactivation of uPA. Therefore, β-catenin might have an impact on the capacity of colorectal tumors for invasion and metastasis, as well as dormancy, which are hallmarks of cancer.
Association of SULT1A1 Phenotype and Genotype with Prostate Cancer Risk in African-Americans and Caucasians
Authors: Nowell Susan , Ratnasinghe D. Luke , Ambrosone Christine B. , Williams Suzanne , Teague-Ross Terri , Trimble Lyndsey , Runnels Gail , Carrol Alindria , Green Bridgett , Stone Angie , Johnson Don , Greene Graham , Kadlubar Fred F. , Lang Nicholas P.
Published: September 2005

Exposure to heterocyclic amines may increase prostate cancer risk. Human sulfotransferase 1A1 (SULT1A1) is involved in the bioactivation of some dietary procarcinogens, including the N-hydroxy metabolite of the food-borne heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo(4,5-b) pyridine. This study compares a polymorphism in the SULT1A1 gene, SULT1A1 enzyme activity, meat consumption, and the risk of prostate cancer in a population based case-control study. Prostate cancer patients (n = 464) and control individuals (n = 459), frequency matched on age and ethnicity, provided informed consent, answered a survey, and provided a blood sample. Platelets were isolated for phenotype analysis, and DNA was isolated from lymphocytes for genotype determination. Meat consumption was assessed using a dietary questionnaire. Caucasians homozygous for the SULT1A1*1 high activity allele were at increased risk for prostate cancer [odds ratio (OR), 1.68; 95% confidence interval (CI), 1.05–2.68] compared with individuals homozygous for the low-activity allele. The association between SULT1A1 genotype and prostate cancer risk in African-Americans did not reach significance (OR, 1.60; 95% CI, 0.46–5.62). When SULT1A1 activity was considered, there was a strong association between increased SULT1A1 activity and prostate cancer risk in Caucasians (OR, 3.04; 95% CI, 1.8–5.1 and OR, 4.96; 95% CI, 3.0–8.3, for the second and third tertiles of SULT1A1 activity, respectively) compared with individuals in the low enzyme activity tertile. A similar association was also found in African-American patients, with ORs of 6.7 and 9.6 for the second and third tertiles of SULT1A1 activity (95% CI, 2.1–21.3 and 2.9–31.3, respectively). When consumption of well-done meat was considered, there was increased risk of prostate cancer (OR, 1.42; 95% CI, 1.01–1.99 and OR, 1.68; 95% CI, 1.20–2.36 for the second and third tertiles, respectively). When SULT1A1 activity was stratified by tertiles of meat consumption, there was greater risk of prostate cancer in the highest tertile of meat consumption. These results indicate that variations in SULT1A1 activity contributes to prostate cancer risk and the magnitude of the association may differ by ethnicity and be modified by meat consumption.
MTHFR
C677T
and
A1298C
Polymorphisms
Authors: Curtin Karen , Bigler Jeannette , Slattery Martha L. , Caan Bette , Potter John D. , Ulrich Cornelia M.
Published: September 2005

5,10-methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism, diverting metabolites toward methylation reactions or nucleotide synthesis. Using data from an incident case-control study (1608 cases and 1972 controls) we investigated two polymorphisms in the MTHFR gene, C677T and A1298C, and their associations with risk of colon cancer. All of the combined genotypes were evaluated separately, and the 1298AA/677CC (wild-type/wild-type) group was considered the reference group. Among both men and women, the 677TT/1298AA (variant/wild-type) genotype was associated with a small reduction in risk [men: odds ratio (OR), 0.7, 95% confidence interval (CI), 0.5–1.0; women: OR, 0.8, 95% CI, 0.5–1.2]. However, the 677CC/1298CC (wild-type/variant) genotype was associated with a statistically significant lower risk among women (OR, 0.6; 95% CI, 0.4–0.9) but not men. When the polymorphisms were considered individually, for A1298C a significant risk reduction associated with the homozygous variant CC genotype was seen among women only (OR, 0.6; 95% CI, 0.5–0.9), and nonstatistically significant reduced risks were observed for the variant 677 TT genotypes among both men and women. Stratification by nutrient intakes showed inverse associations with higher intakes of folate, vitamin B2, B6, B12, and methionine among women with the MTHFR 677CC/1298AA genotypes, but not those with 677TT/1298AA. We observed opposite risk trends for both MTHFR variants, depending on whether women used hormone-replacement therapy or not (P for interaction = <.01).

In summary, this study supports recent findings that the MTHFR A1298C polymorphism may be a predictor of colon cancer risk and have functional relevance. The possible interaction with hormone-replacement therapy warrants additional investigation.
Inverse Association between Prostate Cancer and the Use of Calcium Channel Blockers
Authors: Debes Jose D. , Roberts Rosebud O. , Jacobson Debra J. , Girman Cynthia J. , Lieber Michael M. , Tindall Donald J. , Jacobsen Steven J.
Published: September 2005

Calcium channel blockers block calcium signal-mediated apoptosis. It is hypothesized that the use of these drugs may be associated with the development of cancer. This study investigated the association between daily use of calcium channel blockers and prostate cancer in a community-based cohort of men who participated in a longitudinal study of lower urinary tract symptoms. Study subjects were men ages 40 to 79 years by January 1, 1990, and were randomly selected from Olmsted County in Minnesota. At baseline, participants underwent an interview to determine all medications taken on a daily basis, including calcium channel blockers and to elicit a family history of prostate cancer. During follow-up, all men with a histological diagnosis of prostate cancer were identified through patient self-report and by a review of the complete medical record. Over 12,668 person years of follow-up, 15 (6.8%) of 220 calcium channel blocker users and 120 (10.5%) of 1142 nonusers developed prostate cancer (P = 0.09; odds ratio, 0.62; 95% confidence interval, 0.36–1.10). With adjustment for age and family history of prostate cancer, the risk (odds ratio, 95% confidence interval) of prostate cancer was 0.55 (0.31–0.97) in calcium channel blocker users compared with nonusers. In analyses stratified by family history of prostate cancer, the risk of prostate cancer was 0.45 (0.23–0.88) in men without a family history and 2.64 (0.82–8.47) in men with a family history of prostate cancer (P = 0.006). These findings suggest an association between prostate cancer and daily use of calcium channel blockers that varies by family history of prostate cancer.
Exon 5 Polymorphisms in the
O
6-Alkylguanine DNA Alkyltransferase Gene and Lung Cancer Risk in Non–Smokers Exposed to Second-Hand Smoke
Authors: Cohet Catherine , Borel Stephane , Nyberg Fredrik , Mukeria Anush , Brüske-Hohlfeld Irene , Constantinescu Vali , Benhamou Simone , Brennan Paul , Hall Janet , Boffetta Paolo
Published: September 2005

Purpose: The objective of the study was to examine the association of three exon 5 variants in the O6-alkylguanine DNA alkyltransferase (AGT) gene involved in the repair of the mutagenic DNA lesion O6-alkylguanine formed by nitrosamines, with lung cancer risk in never-smokers.

Experimental Design: Exon 5 of the AGT gene was sequenced in genomic DNA from 136 cases and 133 hospital- or population-based controls for whom questionnaire information on second-hand smoke and diet was available to determine the frequencies of the Gly160Arg, Ile143Val, and Lys178Arg variant alleles.

Results: No codon 160Arg variant alleles were found in the study population. The codon 143Val and 178Arg variant alleles, present at allele frequencies of 0.07, showed 100% linkage. The odds ratio (OR) of lung cancer for these variant carriers was 2.05 [95% confidence interval (CI) 1.03–4.07]. The risk varied between the different lung cancer pathologies with an increased risk for adenocarcinoma (OR 2.67, 95% CI 1.21–5.87) or small cell carcinoma (OR 4.83, 95% CI 0.91–25.7) but not for squamous cell carcinoma (OR 1.07, 95% CI 0.27–4.18). Compared with individuals carrying the mutant alleles unexposed to second-hand smoke, the OR for exposed variant carriers was 1.95 (95% CI 0.53–1.15); a similar interaction, although not significative, was observed for low consumption of cruciferous vegetables and for green vegetables and tomatoes.

Conclusions: These results point toward a role of AGT polymorphisms in lung cancer susceptibility among never-smokers, in particular among subjects exposed to environmental carcinogens.
Effects of a High-Selenium Yeast Supplement on Celecoxib Plasma Levels
Authors: Frank Denise H. , Roe Denise J. , Chow H-H. Sherry , Guillen Jose M. , Choquette Karin , Gracie Debra , Francis Jennifer , Fish Airley , Alberts David S.
Published: September 2005

A combination of celecoxib and selenium was used in a randomized double-blind Phase II trial as a preliminary study to a multicenter Phase III colorectal cancer chemoprevention trial using these two agents together. The purpose of this trial was to determine whether high-selenium baker’s yeast [(Saccharomyces cerevisiae) 200 μg once daily] in combination with celecoxib (400 mg once daily) altered the steady-state plasma concentration of celecoxib or produced clinically significant toxicities. Seventy-three healthy subjects (ages 40–75 years) were recruited to the 6-week study from the general local population and were randomized to either the celecoxib plus selenized baker’s yeast group or the celecoxib plus placebo group after a 2-week run in period of celecoxib only. Blood samples were taken at baseline (to document that there was no evidence of celecoxib intake), after the 2-week run-in period on celecoxib to verify steady-state blood levels of this agent, and at end of study (4 weeks postrandomization). Toxicities were monitored at 2 weeks after initiation of celecoxib, at 4 weeks after initiation, and at the end of the study. Blood level concentrations of celecoxib did not differ between the two groups as determined by high-performance liquid chromatography analysis nor were there significant differences in blood chemistry values between the two groups. Subjects’ self-report of general physical toxicities was uncommon and limited to National Cancer Institute toxicity grade 2 or less; however, 2 female participants (3%) were removed from the study medications because of grade 2 edema and significant weight gain after 2 and 2.5 weeks of celecoxib administration. In conclusion, high-selenium yeast and celecoxib can be taken at the described doses with minimum short-term negative effects. In future Phase III chemoprevention trials of celecoxib, weight gain should be carefully monitored, and participants should be made aware of this potential side effect before study entry.
BRCA1 and BRCA2 Mutations in Women from Shanghai China
Authors: Suter Nicola M. , Ray Roberta M. , Hu Yong Wei , Lin Ming Gang , Porter Peggy , Gao Dao Li , Zaucha Renata E. , Iwasaki Lori M. , Sabacan Leah P. , Langlois Mariela C. , Thomas David B. , Ostrander Elaine A.
Published: September 2005

Little is known about the frequency of germ-line mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 among Asian populations. We investigated the distribution of BRCA1 and BRCA2 germ-line mutations and polymorphisms in a cohort of women from Shanghai, China.

Study subjects totaled 1306, and included 645 women with breast cancer, 342 women with benign breast disease, and 319 unaffected controls, born between 1924 and 1958, selected from women enrolled in a randomized trial of Breast Self-Examination in Shanghai, China. Women were selected without regard to family history of breast or ovarian cancer. All of the coding regions and exon-intron boundaries were screened. Data were analyzed with respect to age at diagnosis, and family history of breast and ovarian cancer.

The prevalence of known disease-associated mutations in women with breast cancer was 1.1% each, for BRCA1 and BRCA2. Among breast cancer cases with a family history of breast or ovarian cancer, 8.1% and 2.7% carried likely BRCA1 and BRCA2 disease-associated mutations, respectively.

Overall, these results suggest that inherited susceptibility to breast cancer due to germ-line BRCA1/2 mutations among women with a family history of breast cancer is comparable between women from Shanghai and Caucasian women of Western European descent. Most alterations observed appear unique to the Chinese population, suggesting a resource that will be useful for assessing risk among both Chinese women and United States women of Chinese descent.