bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic DNA-Chip Hybridization Reveals a Higher Incidence of Genomic Amplifications in Pancreatic Cancer than Conventional Comparative Genomic Hybridization and Leads to the Identification of Novel Candidate Genes
Authors: Holzmann Karlheinz , Kohlhammer Holger , Schwaenen Carsten , Wessendorf Swen , Kestler Hans A. , Schwoerer Alexandra , Rau Bettina , Radlwimmer Bernd , Döhner Hartmut , Lichter Peter , Gress Thomas , Bentz Martin
Published: September 2005

Genomic analyses aimed at the detection of high-level DNA amplifications were performed on 13 widely used pancreatic cancer cell lines and 6 pancreatic tumor specimens. For these analyses, array-based comparative genomic hybridization (Matrix-CGH) onto dedicated microarrays was used. In comparison with chromosomal CGH (eight amplifications), a >3-fold number of DNA amplifications was detected (n = 29). The most frequent amplifications mapped to 7p12.3 (three pancreatic cancer cell lines and three pancreatic tumor specimens), 8q24 (four pancreatic cancer cell lines and one pancreatic tumor specimen), 11q13 (three pancreatic cancer cell lines and three pancreatic tumor specimens), and 20q13 (four pancreatic cancer cell lines and three pancreatic tumor specimens). Genes contained in the consensus regions were MYC (8q24), EGFR (7p12.3), and FGF3 (11q13). In six of seven pancreatic cancer cell lines and pancreatic tumor specimens with 20q13 amplifications, the novel candidate gene NFAT C2, which plays a role in the activation of cytokines, was amplified. Other amplifications also affected genes for which a pathogenetic role in pancreatic carcinoma has not been described, such as BCL10 and BCL6, two members of the BCL family. A subset of amplified genes was checked for overexpression by means of real-time PCR, revealing the highest expression levels for BCL6 and BCL10. Thus, Matrix-CGH allows the detection of a high number of amplifications, resulting in the identification of novel candidate genes in pancreatic cancer.
Boron-Containing Nucleosides as Potential Delivery Agents for Neutron Capture Therapy of Brain Tumors
Authors: Barth Rolf F. , Yang Weilian , Al-Madhoun Ashraf S. , Johnsamuel Jayaseharan , Byun Youngjoo , Chandra Subhash , Smith Duane R. , Tjarks Werner , Eriksson Staffan
Published: September 2005

The purpose of the present study was to evaluate both in vitro and in vivo a series of boron-containing nucleosides that potentially could be used as delivery agents for neutron capture therapy. The rationale for their synthesis was based on the fact that proliferating neoplastic cells have increased requirements for nucleic acid precursors, and, therefore, they should preferentially localize in the tumor. A series of 3-carboranlyalkyl thymidine analogs has been synthesized and a subset, designated N4, N5, and N7, and the corresponding 3-dihydroxypropyl derivatives, designated N4–2OH, N5–2OH, and N7–2OH, have been selected for evaluation. Using these compounds as substrates for recombinant human thymidine kinase-1 and the mitochondrial isoenzyme thymidine kinase-2, the highest phosphorylation levels relative to thymidine were seen with N5 and the corresponding dihydroxypropyl analog N5–2OH. In contrast, N4, N4-OH, N7, and N7-OH had substantially lower phosphorylation levels. To compare compounds with high and low thymidine kinase-1 substrate activity, N5 and N7 and the corresponding dihydroxypropyl derivatives were selected for evaluation of their cellular toxicity, uptake and retention by the F98 rat glioma, human MRA melanoma, and murine L929 cell lines, all of which are thymidine kinase-1(+), and a mutant L929 cell line that is thymidine kinase-1(−). N5–2OH was the least toxic (IC50, 43–70 μm), and N7 and N7–2OH were the most toxic (IC50, 18–49 μm). The highest boron uptake was seen with N7–2OH by the MRA 27 melanoma and L929 wild-type (wt) cell lines. The highest retention was seen with L929 (wt) cells, and this ranged from 29% for N5–2OH to 46% for N7. Based on the in vitro toxicity and uptake data, N5–2OH was selected for in vivo biodistribution studies either in rats bearing intracerebral implants of the F98 glioma or in mice bearing either s.c. or intracerebral implants of L929 (wt) tumors. At 2.5 hours after convection-enhanced delivery, the boron values for the F98 glioma and normal brain were 16.2 ± 2.3 and 2.2 μg/g, respectively, and the tumor to brain ratio was 8.5. Boron values at 4 hours after convection-enhanced delivery of N5–2OH to mice bearing intracerebral implants of L929 (wt) or L929 thymidine kinase-1(−) tumors were 39.8 ± 10.8 and 12.4 ± 1.6 μg/g, respectively, and the corresponding normal brain values were 4.4 and 1.6 μg/g, thereby indicating that there was selective retention by the thymidine kinase-1(+) tumors. Based on these favorable in vitro and in vivo data, neutron capture therapy studies will be initiated using N5–2OH in combination with two non-cell cycle dependent boron delivery agents, boronophenylalanine and sodium borocaptate.
17β-Hydroxysteroid Dehydrogenase Type 1 Is an Independent Prognostic Marker in Breast Cancer
Authors: Oduwole Olayiwola O. , Li Yan , Isomaa Veli V. , Mäntyniemi Anne , Pulkka Anitta E. , Soini Ylermi , Vihko Pirkko T.
Published: September 2005

Estrogens have an important role in the development and progression of breast cancer. 17β-Hydroxysteroid dehydrogenase type 1 (17HSD1), type 2 (17HSD2), and type 5 (17HSD5) are associated with sex steroid metabolism in normal and cancerous breast tissue. The mRNA expressions of the 17HSD1, 17HSD2, and 17HSD5 enzymes were analyzed in 794 breast carcinoma specimens by using tissue microarrays and normal histologic sections. The results were correlated with the estrogen receptor α (ER-α) and β (ER-β), progesterone receptor, Ki67, and c-erbB-2 expressions analyzed by immunohistochemical techniques and with the Tumor-Node-Metastasis classification, tumor grade, disease-free interval, and survival of the patients. Signals for 17HSD1 mRNA were detected in 16%, 17HSD2 in 25%, and 17HSD5 in 65% of the breast cancer specimens. No association between the 17HSD1, 17HSD2, and 17HSD5 expressions was detected. A significant association was observed between ER-α and ER-β (P = 0.02; odds ratio, 1.96) expressions. There was also a significant inverse association between ER-α and 17HSD1 (P = 0.04; odds ratio, 0.53), as well as ER-α and 17HSD5 (P = 0.001; odds ratio, 0.35). Patients with tumors expressing 17HSD1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients (P = 0.0010 and 0.0134, log rank). The expression of 17HSD5 was significantly higher in breast tumor specimens than in normal tissue (P = 0.033; odds ratio, 5.56). The group with 17HSD5 overexpression had a worse prognosis than the other patients (P = 0.0146). ER-α also associated with survival (P = 0.045). Cox multivariate analyses showed that 17HSD1 mRNA, tumor size, and ER-α had independent prognostic significance.
Gene Expression Profiling of Gliomas Strongly Predicts Survival
Authors: Freije William A. , Castro-Vargas F. Edmundo , Fang Zixing , Horvath Steve , Cloughesy Timothy , Liau Linda M. , Mischel Paul S. , Nelson Stanley F.
Published: September 2005

In current clinical practice, histology-based grading of diffuse infiltrative gliomas is the best predictor of patient survival time. Yet histology provides little insight into the underlying biology of gliomas and is limited in its ability to identify and guide new molecularly targeted therapies. We have performed large-scale gene expression analysis using the Affymetrix HG U133 oligonucleotide arrays on 85 diffuse infiltrating gliomas of all histologic types to assess whether a gene expression-based, histology-independent classifier is predictive of survival and to determine whether gene expression signatures provide insight into the biology of gliomas. We found that gene expression-based grouping of tumors is a more powerful survival predictor than histologic grade or age. The poor prognosis samples could be grouped into three different poor prognosis groups, each with distinct molecular signatures. We further describe a list of 44 genes whose expression patterns reliably classify gliomas into previously unrecognized biological and prognostic groups: these genes are outstanding candidates for use in histology-independent classification of high-grade gliomas. The ability of the large scale and 44 gene set expression signatures to group tumors into strong survival groups was validated with an additional external and independent data set from another institution composed of 50 additional gliomas. This demonstrates that large-scale gene expression analysis and subset analysis of gliomas reveals unrecognized heterogeneity of tumors and is efficient at selecting prognosis-related gene expression differences which are able to be applied across institutions.
Role of p53 in Sensing Oxidative DNA Damage in Response to Reactive Oxygen Species-Generating Agents
Authors: Achanta Geetha , Huang Peng
Published: September 2005

The tumor suppressor p53 plays an important role in the regulation of cellular response to DNA damage. Recent studies suggest that p53 is able to bind DNA with certain structural alterations in a sequence-independent manner and to interact with several molecules involved in DNA repair. This study was undertaken to test the hypothesis that p53 may participate in sensing oxidative DNA damage, the most frequently occurring spontaneous DNA lesion, and modulate its repair by the base excision repair (BER) machinery. Using synthetic DNA containing 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxoG), we showed that p53 was pulled down together with two BER proteins, human 8-oxoguanine glycosylase (hOGG1) and AP endonuclease (APE). Functional analysis showed that p53 significantly enhanced the sequential activities of hOGG1 and APE in excising the 8-oxoG nucleotide from DNA in vitro. The ability of p53 to enhance the removal of oxidized DNA bases was further demonstrated in vivo using a pair of p53 isogenic lines. HCT116 p53+/+ cells exhibit a more rapid removal of 8-oxoG from DNA than p53−/− cells exposed to the same levels of reactive oxygen species (ROS) stress. Together, these results suggest that p53 participates in sensing oxidative DNA damage and modulates BER function in response to persistent ROS stress.
Robust Classification of Renal Cell Carcinoma Based on Gene Expression Data and Predicted Cytogenetic Profiles
Authors: Furge Kyle A. , Lucas Kerry A. , Takahashi Masayuki , Sugimura Jun , Kort Eric J. , Kanayama Hiro-omi , Kagawa Susumu , Hoekstra Philip , Curry John , Yang Ximing J. , Teh Bin T.
Published: September 2005

Renal cell carcinoma (RCC) is a heterogeneous disease that includes several histologically distinct subtypes. The most common RCC subtypes are clear cell, papillary, and chromophobe, and recent gene expression profiling studies suggest that classification of RCC based on transcriptional signatures could be beneficial. Traditionally, however, patterns of chromosomal alterations have been used to assist in the molecular classification of RCC. The purpose of this study was to determine whether it was possible to develop a classification model for the three major RCC subtypes that utilizes gene expression profiles as the bases for both molecular genetic and cytogenetic classification. Gene expression profiles were first used to build an expression-based RCC classifier. The RCC gene expression profiles were then examined for the presence of regional gene expression biases. Regional expression biases are genetic intervals that contain a disproportionate number of genes that are coordinately up- or down-regulated. The presence of a regional gene expression bias often indicates the presence of a chromosomal abnormality. In this study, we demonstrate an expression-based classifier can distinguish between the three most common RCC subtypes in 99% of cases (n = 73). We also demonstrate that detection of regional expression biases accurately identifies cytogenetic features common to RCC. Additionally, the in silico-derived cytogenetic profiles could be used to classify 81% of cases. Taken together, these data demonstrate that it is possible to construct a robust classification model for RCC using both transcriptional and cytogenetic features derived from a gene expression profile.
Differential Signaling Pathways Are Activated in the Epstein-Barr Virus-Associated Malignancies Nasopharyngeal Carcinoma and Hodgkin Lymphoma
Authors: Morrison Jennifer A. , Gulley Margaret L. , Pathmanathan Rajadurai , Raab-Traub Nancy
Published: September 2005

EBV is associated with the epithelial cancer, nasopharyngeal carcinoma (NPC), and the lymphoid malignancy, Hodgkin lymphoma (HL). The EBV latent membrane proteins 1 and 2A are expressed in these tumors. These proteins activate the phosphatidylinositol 3′-OH kinase (PI3K)/Akt pathway, which is commonly activated inappropriately in malignancy. In this study, the status of Akt activation and its targets, glycogen synthase kinase-3β (GSK-3β) and β-catenin, was investigated in NPC and HL clinical specimens. In the majority of HL and NPC specimens, Akt was activated, indicating an important role for this kinase in the development and/or progression of these tumors. Akt phosphorylates and inactivates GSK-3β, a negative regulator of the proto-oncoprotein β-catenin that is aberrantly activated in many cancers. GSK-3β was phosphorylated and inactivated with concomitant nuclear β-catenin accumulation in the majority of NPC specimens. The malignant cells of the majority of HL cases, however, did not have inactivated GSK-3β and lacked nuclear β-catenin expression. These data indicate that this signaling arm of PI3K/Akt is universal and important in NPC pathogenesis but is apparently not affected in HL. These findings point to a divergence in pathways activated by EBV in different cellular contexts.
Protein Kinase C θ (PKCθ) Expression and Constitutive Activation in Gastrointestinal Stromal Tumors (GISTs)
Authors: Duensing Anette , Joseph Nora E. , Medeiros Fabiola , Smith Felicity , Hornick Jason L. , Heinrich Michael C. , Corless Christopher L. , Demetri George D. , Fletcher Christopher D. M. , Fletcher Jonathan A.
Published: September 2005

KIT expression is a key diagnostic feature of gastrointestinal stromal tumors (GISTs), and virtually all of the GISTs express oncogenic forms of the KIT or PDGFRA receptor tyrosine kinase proteins, which serve as therapeutic targets of imatinib mesylate (Gleevec; Novartis, Basel, Switzerland). However, KIT expression can be low in PDGFRA-mutant GISTs, increasing the likelihood of misdiagnosis as other types of sarcoma. We report that the signaling intermediate protein kinase C θ (PKCθ) is a diagnostic marker in GISTs, including those that lack KIT expression and/or contain PDGFRA mutations. PKCθ is strongly activated in most GISTs and hence may serve, along with KIT/PDGFRA, as a novel therapeutic target.
Three Classes of Genes Mutated In Colorectal Cancers with Chromosomal Instability
Authors: Wang Zhenghe , Cummins Jordan M. , Shen Dong , Cahill Daniel P. , Jallepalli Prasad V. , Wang Tian-Li , Parsons D. Williams , Traverso Giovanni , Awad Mark , Silliman Natalie , Ptak Janine , Szabo Steve , Willson James K. V. , Markowitz Sanford D. , Goldberg Michael L. , Karess Roger , Kinzler Kenneth W. , Vogelstein Bert , Velculescu Victor E. , Lengauer Christoph
Published: September 2005

Although most colorectal cancers are chromosomally unstable, the basis for this instability has not been defined. To determine whether genes shown to cause chromosomal instability in model systems were mutated in colorectal cancers, we identified their human homologues and determined their sequence in a panel of colorectal cancers. We found 19 somatic mutations in five genes representing three distinct instability pathways. Seven mutations were found in MRE11, whose product is involved in double-strand break repair. Four mutations were found among hZw10, hZwilch/FLJ10036, and hRod/KNTC, whose products bind to one another in a complex that localizes to kinetochores and controls chromosome segregation. Eight mutations were found in Ding, a previously uncharacterized gene with sequence similarity to the Saccharomyces cerevisiae Pds1, whose product is essential for proper chromosome disjunction. This analysis buttresses the evidence that chromosomal instability has a genetic basis and provides clues to the mechanistic basis of instability in cancers.
Altered DNA Polymerase ι Expression in Breast Cancer Cells Leads to a Reduction in DNA Replication Fidelity and a Higher Rate of Mutagenesis
Authors: Yang Jin , Chen Zhiwen , Liu Yang , Hickey Robert J. , Malkas Linda H.
Published: September 2005

The recently discovered human enzyme DNA polymerase ι (pol ι) has been shown to have an exceptionally high error rate on artificial DNA templates. Although there is a considerable body of in vitro evidence for a role for pol ι in DNA lesion bypass, there is no in vivo evidence to confirm this action. We report here that pol ι expression is elevated in breast cancer cells and correlates with a significant decrease in DNA replication fidelity. We also demonstrate that UV treatment of breast cancer cells additionally increases pol ι expression with a peak occurring between 30 min and 2 h after cellular insult. This implies that the change in pol ι expression is an early event after UV-mediated DNA damage. That pol ι may play a role in the higher mutation frequencies observed in breast cancer cells was suggested when a reduction in mutation frequency was found after pol ι was immunodepleted from nuclear extracts of the cells. Analysis of the UV-induced mutation spectra revealed that >90% were point mutations. The analysis also demonstrated a decreased C→T nucleotide transition and an increased C→A transversion rate. Overall, our data strongly suggest that pol ι may be involved in the generation of both increased spontaneous and translesion mutations during DNA replication in breast cancer cells, thereby contributing to the accumulation of genetic damage.
TBX3
and Its Isoform
TBX3+2a
Are Functionally Distinctive in Inhibition of Senescence and Are Overexpressed in a Subset of Breast Cancer Cell Lines
Authors: Fan Weiwei , Huang Xu , Chen Chira , Gray Joe , Huang Taosheng
Published: September 2005

TBX3 is a transcription factor of the T-box gene family. Mutations of TBX3 cause ulnar-mammary syndrome (MIM 181450) in humans, an autosomal dominant disorder characterized by the absence or underdevelopment of the mammary glands and other congenital anomalies. It recently was found that TBX3 was able to immortalize mouse embryo fibroblast (MEF) cells. In addition, TBX2, a homologue of TBX3, is active in preventing senescence in rodent cells and was found to be amplified in some human breast cancers, suggesting TBX3 plays a role in breast cancer. This study examined the function of TBX3 and its isoform, TBX3 + 2a. TBX3 + 2a differs from TBX3 in the DNA binding domain with an extra 20 amino acids produced by alternative splicing. We first examined the tissue expression and alternative splicing patterns of these two isoforms. We found that TBX3 and TBX3 + 2a are widely expressed in humans and mice, and alternative splicing could be tissue specific and species specific. Overexpression of TBX3 is able to immortalize MEF cells, whereas TBX3 + 2a shows an acceleration of senescence, a functional difference that may be explained by the fact that these two isoforms may have different downstream targets. TBX3, but not TBX3 + 2a, is able to bind to the previously identified T-box binding site in a gel shift assay. A subset of human breast cancer cell lines overexpresses TBX3. Our results indicate that TBX3 and TBX3 + 2a are functionally distinctive in inhibition of senescence of MEF cells and may play a role in breast cancer.
Hydrogen Peroxide-Mediated Cytosolic Acidification Is a Signal for Mitochondrial Translocation of Bax during Drug-Induced Apoptosis of Tumor Cells
Authors: Ahmad Kashif A. , Iskandar Kartini B. , Hirpara Jayshree L. , Clement Marie-Veronique , Pervaiz Shazib
Published: September 2005

Absence of the proapoptotic protein Bax renders tumor cells resistant to drug-induced apoptosis. We have shown that hydrogen peroxide (H2O2)-mediated cytosolic acidification is an effector mechanism during drug-induced apoptosis of tumor cells. Here, we report that Bax is critical in determining the sensitivity of tumor cells to H2O2-induced apoptosis. More importantly, exposure of colorectal carcinoma (HCT116) and leukemia cells (HL60 and CEM) to H2O2 or its intracellular production during drug-induced apoptosis is a signal for mitochondrial translocation of Bax. Furthermore, we provide evidence that drug-induced H2O2-mediated Bax translocation in tumor cells is caspase independent but involves cytosolic acidification. Inhibiting cytosolic acidification prevents Bax translocation, and contrarily enforced acidification of the intracellular milieu results in mitochondrial recruitment of Bax, even in the absence of a trigger. These findings provide a novel mechanism for mitochondrial translocation of Bax and directly implicate H2O2-mediated cytosolic acidification in the recruitment of the mitochondrial pathway during drug-induced apoptosis of tumor cells.
Effects of Chronic Jet Lag on Tumor Progression in Mice
Authors: Filipski Elisabeth , Delaunay Franck , King Verdun M. , Wu Ming-Wei , Claustrat Bruno , Gréchez-Cassiau Aline , Guettier Catherine , Hastings Michael H. , Francis Lévi
Published: September 2005

Frequent transmeridian flights or predominant work at night can increase cancer risk. Altered circadian rhythms also predict for poor survival in cancer patients, whereas physical destruction of the suprachiasmatic nuclei (SCN), the hypothalamic circadian pacemaker, accelerates tumor growth in mice. Here we tested the effect of functional disruption of circadian system on tumor progression in a novel experimental model of chronic jet lag. B6D2F1 mice were synchronized with 12 hours of light and 12 hours of darkness or underwent repeat 8-hour advances of the light/dark cycle every 2 days before inoculation of Glasgow osteosarcoma. The 24-hour changes were assessed for plasma corticosterone, clock protein mPER1 expression in the SCN, and mRNA expression of clock genes mPer2 and mRev-erbα in liver and tumor. Time series were analyzed by spectral analysis and/or Cosinor. Differences were compared with analysis of variance (ANOVA). The 24-hour rest/activity cycle was ablated, and the rhythms of body temperature, serum corticosterone, and mPER1 protein expression in the SCN were markedly altered in jet-lagged mice as compared with controls (ANOVA, P < 0.001 for corticosterone and P = 0.01 for mPER1). Tumor grew faster in the jet-lagged animals as compared with controls (ANOVA, P < 0.001), whereas exposure to constant light or darkness had no effect (ANOVA, P = 0.66 and P = 0.8, respectively). The expression of mPer2 and mRev-erbα mRNAs in controls showed significant circadian rhythms in the liver (P = 0.006 and P = 0.003, respectively, Cosinor) and in the tumor (P = 0.04 and P < 0.001). Both rhythms were suppressed in the liver (P = 0.2 and P = 0.1, respectively, Cosinor) and in the tumor (P = 0.5) of jet-lagged mice. Altered environmental conditions can disrupt circadian clock molecular coordination in peripheral organs including tumors and play a significant role in malignant progression.
Inhibition of Melanoma Growth and Metastasis by ATF2-Derived Peptides
Authors: Bhoumik Anindita , Gangi Lisa , Ronai Ze’ev
Published: September 2005

The resistance of melanoma to apoptosis, as well as its growth and metastasis capabilities, can be overcome by expression of a peptide derived from amino acid (aa) 51 to 100 of ATF2. Here we show that expression of ATF2(51–100) in human melanoma cells reduced their growth in nude mice, which was additionally inhibited upon treatment with protein kinase inhibitors UCN-01 or SB203580. Injection of a fusion protein consisting of HIV-TAT and aa 51 to 100 of ATF2 into SW1 melanomas efficiently inhibits their growth and their metastasis up to complete regression. Additionally, expression of a 10aa peptide that corresponds to aa 51 to 60 of ATF2 sensitizes melanoma cells to spontaneous apoptosis, which coincides with activation of caspase 9 and poly(ADP-ribose) polymerase cleavage, and inhibit their growth in vivo. The 10aa peptide increases the association of c-Jun NH2-terminal kinase with c-Jun but not with ATF2, resulting in concomitant increase in TRE-mediated transcription. Our study points to mechanisms underlying the activities of the ATF2 peptide while highlighting its possible use in drug design.
Expression Profiling Reveals Novel Pathways in the Transformation of Melanocytes to Melanomas
Authors: Hoek Keith , Rimm David L. , Williams Kenneth R. , Zhao Hongyu , Ariyan Stephan , Lin Aiping , Kluger Harriet M. , Berger Aaron J. , Cheng Elaine , Trombetta E. Sergio , Wu Terence , Niinobe Michio , Yoshikawa Kazuaki , Hannigan Gregory E. , Halaban Ruth
Published: September 2005

Affymetrix and spotted oligonucleotide microarrays were used to assess global differential gene expression comparing normal human melanocytes with six independent melanoma cell strains from advanced lesions. The data, validated at the protein level for selected genes, confirmed the overexpression in melanoma cells relative to normal melanocytes of several genes in the growth factor/receptor family that confer growth advantage and metastasis. In addition, novel pathways and patterns of associated expression in melanoma cells not reported before emerged, including the following: (a) activation of the NOTCH pathway; (b) increased Twist expression and altered expression of additional transcriptional regulators implicated in embryonic development and epidermal/mesenchymal transition; (c) coordinated activation of cancer/testis antigens; (d) coordinated down-regulation of several immune modulation genes, in particular in the IFN pathways; (e) down-regulation of several genes implicated in membrane trafficking events; and (f) down-regulation of growth suppressors, such as the Prader-Willi gene NECDIN, whose function was confirmed by overexpression of ectopic Flag-necdin. Validation of differential expression using melanoma tissue microarrays showed that reduced ubiquitin COOH-terminal esterase L1 in primary melanoma is associated with worse outcome and that increased expression of the basic helix-loop-helix protein Twist is associated with worse outcome. Some differentially expressed genes reside on chromosomal regions displaying common loss or gain in melanomas or are known to be regulated by CpG promoter methylation. These results provide a comprehensive view of changes in advanced melanoma relative to normal melanocytes and reveal new targets that can be used in assessing prognosis, staging, and therapy of melanoma patients.
Three Biomarkers Identified from Serum Proteomic Analysis for the Detection of Early Stage Ovarian Cancer
Authors: Zhang Zhen , Bast Robert C. , Yu Yinhua , Li Jinong , Sokoll Lori J. , Rai Alex J. , Rosenzweig Jason M. , Cameron Bonnie , Wang Young Y. , Meng Xiao-Ying , Berchuck Andrew , van Haaften-Day Carolien , Hacker Neville F. , de Bruijn Henk W. A. , van der Zee Ate G. J. , Jacobs Ian J. , Fung Eric T. , Chan Daniel W.
Published: September 2005

Early detection remains the most promising approach to improve long-term survival of patients with ovarian cancer. In a five-center case-control study, serum proteomic expressions were analyzed on 153 patients with invasive epithelial ovarian cancer, 42 with other ovarian cancers, 166 with benign pelvic masses, and 142 healthy women. Data from patients with early stage ovarian cancer and healthy women at two centers were analyzed independently and the results cross-validated to discover potential biomarkers. The results were validated using the samples from two of the remaining centers. After protein identification, biomarkers for which an immunoassay was available were tested on samples from the fifth center, which included 41 healthy women, 41 patients with ovarian cancer, and 20 each with breast, colon, and prostate cancers. Three biomarkers were identified as follows: (a) apolipoprotein A1 (down-regulated in cancer); (b) a truncated form of transthyretin (down-regulated); and (c) a cleavage fragment of inter-α-trypsin inhibitor heavy chain H4 (up-regulated). In independent validation to detect early stage invasive epithelial ovarian cancer from healthy controls, the sensitivity of a multivariate model combining the three biomarkers and CA125 [74% (95% CI, 52–90%)] was higher than that of CA125 alone [65% (95% CI, 43–84%)] at a matched specificity of 97% (95% CI, 89–100%). When compared at a fixed sensitivity of 83% (95% CI, 61–95%), the specificity of the model [94% (95% CI, 85–98%)] was significantly better than that of CA125 alone [52% (95% CI, 39–65%)]. These biomarkers demonstrated the potential to improve the detection of early stage ovarian cancer.
Genomic and Expression Profiling of Chromosome 17 in Breast Cancer Reveals Complex Patterns of Alterations and Novel Candidate Genes
Authors: Orsetti Béatrice , Nugoli Mélanie , Cervera Nathalie , Lasorsa Laurence , Chuchana Paul , Ursule Lisa , Nguyen Catherine , Redon Richard , du Manoir Stanislas , Rodriguez Carmen , Theillet Charles
Published: September 2005

Chromosome 17 is severely rearranged in breast cancer. Whereas the short arm undergoes frequent losses, the long arm harbors complex combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic array-comparative genomic hybridization and of associated RNA expression changes by cDNA arrays. We built a genomic array covering the entire chromosome at an average density of 1 clone per 0.5 Mb, and patterns of gains and losses were characterized in 30 breast cancer cell lines and 22 primary tumors. Genomic profiles indicated severe rearrangements. Compiling data from all samples, we subdivided chromosome 17 into 13 consensus segments: 4 regions showing mainly losses, 6 regions showing mainly gains, and 3 regions showing either gains or losses. Within these segments, smallest regions of overlap were defined (17 for gains and 16 for losses). Expression profiles were analyzed by means of cDNA arrays comprising 358 known genes at 17q. Comparison of expression changes with quantitative anomalies revealed that about half of the genes were consistently affected by copy number changes. We identified 85 genes overexpressed when gained (39 of which mapped within the smallest regions of overlap), 67 genes underexpressed when lost (32 of which mapped to minimal intervals of losses), and, interestingly, 32 genes showing reduced expression when gained. Candidate genes identified in this study belong to very diverse functional groups, and a number of them are novel candidates.
High-Dose Granulocyte-Macrophage Colony-Stimulating Factor-Producing Vaccines Impair the Immune Response through the Recruitment of Myeloid Suppressor Cells
Authors: Serafini Paolo , Carbley Rebecca , Noonan Kimberly A. , Tan Gladys , Bronte Vincenzo , Borrello Ivan
Published: September 2005

Tumor vaccines have shown promise in early clinical trials. Among them, tumor cells genetically engineered to secrete biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF) can generate a systemic antitumor immune response. Although the minimal required GM-CSF dose produced by modified tumor cells to achieve a measurable antitumor effect is well known, no data examined whether an upper therapeutic limit may exist for this vaccination strategy. Because recent data demonstrate an immunosuppressive effect of GM-CSF produced by growing tumors, we thus sought to determine whether high GM-CSF doses administered in a vaccine formulation could impair antitumor immunity. Using a vaccine strategy involving a GM-CSF-producing bystander cell line (B78H1-GM) admixed with autologous tumor, we assessed the impact of varying doses of GM-CSF while maintaining a constant antigen dose. Our results defined a threshold above which a GM-CSF-based vaccine not only lost its efficacy, but more importantly for its clinical implications resulted in substantial immunosuppression in vivo. Above this threshold, GM-CSF induced Gr1+/CD11b+ myeloid suppressor cells that substantially impaired antigen-specific T-cell responses and adversely affected antitumor immune responses in vivo. The dual effects of GM-CSF are mediated by the systemic and not local concentration of this cytokine. Myeloid suppressor cell-induced immunosuppression is mediated by nitric oxide production via inducible nitric oxide synthase (iNOS) because the specific iNOS inhibitor, l-NMMA, restored antigen-specific T-cell responsiveness in vitro. Taken together, our data demonstrated the negative impact of supra-therapeutic vaccine doses of GM-CSF and underscored the importance of identifying these critical variables in an effort to increase the therapeutic efficacy of tumor vaccines.
Casein Kinase I Attenuates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis by Regulating the Recruitment of Fas-Associated Death Domain and Procaspase-8 to the Death-Inducing Signaling Complex
Authors: Izeradjene Kamel , Douglas Leslie , Delaney Addison B. , Houghton Janet A.
Published: September 2005

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor–mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the death-inducing signaling complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the death-inducing signaling complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKIα isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKIα plays an antiapoptotic role through the generation of phosphorylated sites at the level of the death-inducing signaling complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.
Deficiency of NRH:Quinone Oxidoreductase 2 Increases Susceptibility to 7,12-Dimethylbenz(
a
)anthracene and Benzo(
a
)pyrene-Induced Skin Carcinogenesis
Authors: Iskander Karim , Paquet Marilene , Brayton Cory , Jaiswal Anil K.
Published: September 2005

NRH:Quinone oxidoreductase 2 (NQO2) is an enzyme that catalyzes the reductive metabolism of quinones. C57BL/6 NQO2−/− mice lacking NQO2 gene expression were generated in our laboratory. The dorsal skin of NQO2-deficient mice was exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene alone (complete carcinogen) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (initiation/promotion model) to determine the in vivo role of NQO2 in chemical carcinogenesis. The NQO2−/− mice showed significantly increased tumor frequency with DMBA + TPA when compared with their wild-type littermates. The benzo(a)pyrene + TPA also showed increase in tumor incidence in NQO2−/− mice but to a less extent than DMBA. DMBA alone resulted in low frequency of tumor development with no difference in susceptibility between wild-type and NQO2−/− mice. Benzo(a)pyrene alone failed to induce tumors in either wild-type or NQO2−/− mice. Histologic analysis of the NQO2−/− mice tumors demonstrated proliferative activity. The treatment of NQO2−/− mice skin with benzo(a)pyrene failed to significantly increase tumor suppressor protein p53 and p53-regulated growth-related protein p21 and proapoptotic protein Bax as observed in case of wild-type mice. These results demonstrate that NQO2 protects against DMBA- and benzo(a)pyrene-induced skin carcinogenesis and suggest that NQO2 protection might be against tumor promotion. The results also suggest that lack of induction of p53, p21, and Bax proteins might contribute to increased sensitivity of NQO2−/− mice skin to benzo(a)pyrene carcinogenicity.
Peloruside A Does Not Bind to the Taxoid Site on β-Tubulin and Retains Its Activity in Multidrug-Resistant Cell Lines
Authors: Gaitanos Thomas N. , Buey Rubén M. , Díaz J. Fernando , Northcote Peter T. , Teesdale-Spittle Paul , Andreu José M. , Miller John H.
Published: September 2005

Peloruside A (peloruside), a microtubule-stabilizing agent from a marine sponge, is less susceptible than paclitaxel to multidrug resistance arising from overexpression of the P-glycoprotein efflux pump and is not affected by mutations that affect the taxoid binding site of β-tubulin. In vitro studies with purified tubulin indicate that peloruside directly induces tubulin polymerization in the absence of microtubule-associated proteins. Competition for binding between peloruside, paclitaxel, and laulimalide revealed that peloruside binds to a different site on tubulin to paclitaxel. Moreover, laulimalide was able to displace peloruside, indicating that peloruside and laulimalide may compete for the same or overlapping binding sites. It was concluded that peloruside and laulimalide have binding properties that are distinct from other microtubule-stabilizing compounds currently under investigation.
Heparin-Binding EGF-Like Growth Factor Is a Promising Target for Ovarian Cancer Therapy
Authors: Miyamoto Shingo , Hirata Michinari , Yamazaki Ayano , Kageyama Takuya , Hasuwa Hidetoshi , Mizushima Hiroto , Tanaka Yoshihiro , Yagi Hiroshi , Sonoda Kenzo , Kai Masahiro , Kanoh Hideo , Nakano Hitoo , Mekada Eisuke
Published: September 2005

Ovarian cancer is the most frequent cause of cancer death among all gynecologic cancers. We demonstrate here that lysophosphatidic acid (LPA)-induced ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) is a critical to tumor formation in ovarian cancer. We found that among the epidermal growth factor receptor (EGFR) family of growth factors, HB-EGF gene expression in cancerous tissues and HB-EGF protein levels in patients’ ascites fluid were significantly elevated. The human ovarian cancer cell lines SKOV3 and RMG-1 form tumors in nude mice. Tumor formation of these cells was enhanced by exogenous expression of pro-HB-EGF and completely blocked by pro-HB-EGF gene RNA interference or by CRM197, a specific HB-EGF inhibitor. Transfection with mutant forms of HB-EGF indicated that the release of soluble HB-EGF is essential for tumor formation. LPA, which is constitutively produced by ovarian cancer cells, induced HB-EGF ectodomain shedding in SKOV3 and RMG-1 cells, resulting in the transactivation of EGFR and the downstream kinase extracellular signal-regulated kinase/mitogen-activated protein kinase. LPA-induced transactivation was abrogated by HB-EGF gene RNA interference or by CRM197. Introduction of lipid phosphate phosphohydrolase, which hydrolyzes LPA, decreased the constitutive shedding of HB-EGF, EGFR transactivation, and the tumorigenic potential of SKOV3 and RMG-1 cells. These results indicate that HB-EGF is the primary member of the EGFR family of growth factors expressed in ovarian cancer and that LPA-induced ectodomain shedding of this growth factor is a critical step in tumor formation, making HB-EGF a novel therapeutic target for ovarian cancer.
Cystatin M
Authors: Zhang Jun , Shridhar Ravi , Dai Qun , Song Jin , Barlow Shayne C. , Yin Lijia , Sloane Bonnie F. , Miller Fred R. , Meschonat Carol , Li Benjamin D. L. , Abreo Fleurette , Keppler Daniel
Published: September 2005

The contribution of pericellular proteolysis to tumor progression is well documented. To better understand protease biology and facilitate clinical translation, specific proteolytic systems need to be better defined. In particular, the precise role of endogenous protease inhibitors still needs to be deciphered. We reported previously that cystatin M, a potent endogenous inhibitor of lysosomal cysteine proteases, significantly suppressed in vitro cell proliferation, migration, and Matrigel invasion. Here, we show that scid mice orthotopically implanted with breast cancer cells expressing cystatin M show significantly delayed primary tumor growth and lower metastatic burden in the lungs and liver when compared with mice implanted with mock controls. The incidence of metastasis, however, appeared to be unaltered between the cystatin M group and the control group. Experimental metastasis assays suggest that cystatin M suppressed tumor cell proliferation at the secondary site. By using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we found consistent expression of cystatin M in normal human breast epithelial cells, whereas expression was decreased by 86% in invasive ductal carcinoma (IDC) cells of stage I to IV patients. Complete loss of expression of cystatin M was observed in two of three IDCs from stage IV patients. Immunohistochemical studies confirmed that expression of cystatin M in IDCs was partially or completely lost. We propose cystatin M as a novel candidate tumor suppressor gene for breast cancer.
Human Papillomavirus Type 16-Positive Cervical Cancer Is Associated with Impaired CD4+ T-Cell Immunity against Early Antigens E2 and E6
Authors: de Jong Annemieke , van Poelgeest Mariëtte I. E. , van der Hulst Jeanette M. , Drijfhout Jan Wouter , Fleuren Gert Jan , Melief Cornelis J. M. , Kenter Gemma , Offringa Rienk , van der Burg Sjoerd H.
Published: September 2005

Cervical cancer is the possible outcome of genital infection with high-risk human papillomavirus (HPV) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Fortunately, the majority of genital HPV infections are cleared before the development of (pre)malignant lesions. Analysis of CD4+ T-helper (Th) immunity against the E2, E6, and E7 antigens of HPV16 in healthy women revealed strong proliferative E2- and E6-specific responses associated with prominent IFN-γ and interleukin 5 secretion. This indicates that the naturally arising virus-induced immune response displays a mixed Th1/Th2 cytokine profile. Of all HPV16+ cervical cancer patients, approximately half failed to mount a detectable immune response against the HPV16-derived peptides. The other half of the patients showed impaired HPV16-specific proliferative responses, which generally lacked both IFN-γ and interleukin 5. This indicates that the HPV16-specific CD4+ T-cell response in cervical cancer patients is either absent or severely impaired, despite a relatively good immune status of the patients, as indicated by intact responses against recall antigens. It is highly conceivable that proper CD4+ T-cell help is important for launching an effective immune attack against HPV because infection of cervical epithelia by this virus is, at least initially, not accompanied by gross disturbance of this tissue and/or strong proinflammatory stimuli. Therefore, our observations concerning the lack of functional HPV16-specific CD4+ T-cell immunity in patients with cervical cancer offer a possible explanation for the development of this disease.
Nuclear Factor-κB is an Important Modulator of the Altered Gene Expression Profile and Malignant Phenotype in Squamous Cell Carcinoma
Authors: Loercher Amy , Lee Tin Lap , Ricker Justin L. , Howard April , Geoghegen Joel , Chen Zhong , Sunwoo John B. , Sitcheran Raquel , Chuang Eric Y. , Mitchell James B. , Baldwin Albert S. , Van Waes Carter
Published: September 2005

We reported previously that transcription factor nuclear factor (NF)-κB is constitutively activated in human and murine squamous cell carcinomas (SCCs). The role of NF-κB in the cumulative changes in gene expression with transformation and progression of the murine SCC Pam 212 and after switching off NF-κB by a dominant negative inhibitor κB mutant (IκBαM) was explored by profiling with a 15,000-element cDNA micoarrray. Remarkably, NF-κB modulated the expression of >60% of the 308 genes differentially expressed between normal keratinocytes and metastatic SCCs. NF-κB directly or indirectly modulated expression of programs of genes functionally linked to proliferation, apoptosis, adhesion, and angiogenesis. Among these, changes in expression of cyclin D1, inhibitor of apoptosis-1, mutant Trp53, and β-catenin detected with modulation of NF-κB by microarray were confirmed by Western and Northern blot. NF-κB DNA binding motifs were detected in the promoter of ∼63% of genes showing increased expression and 33% of the genes showing decreased expression. The ACTACAG motif implicated in the NF-κB-dependent down-regulation of mRNA expression of MyoD and Sox9 was detected in the coding portion of about 15% of genes showing increased or decreased expression. Inactivation of NF-κB inhibited malignant phenotypic features including proliferation, cell survival, migration, angiogenesis, and tumorigenesis. These results provide evidence that NF-κB is an important modulator of gene expression programs that contribute to the malignant phenotype of SCC.
Blocking the Raf/MEK/ERK Pathway Sensitizes Acute Myelogenous Leukemia Cells to Lovastatin-Induced Apoptosis
Authors: Wu Jianghong , Wong W. Wei-Lynn , Khosravi Fereshteh , Minden Mark D. , Penn Linda Z.
Published: September 2005

The statin family of drugs are well-established inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and are used clinically in the control of hypercholesterolemia. Recent evidence, from ourselves and others, shows that statins can also trigger tumor-specific apoptosis by blocking protein geranylgeranylation. We and others have proposed that statins disrupt localization and function of geranylgeranylated proteins responsible for activating signal transduction pathways essential for the growth and/or survival of transformed cells. To explore this further, we have investigated whether the mitogen-activated protein kinase (MAPK) signaling cascades play a role in regulating statin-induced apoptosis. Cells derived from acute myelogenous leukemia (AML) are used as our model system. We show that p38 and c-Jun NH2-terminal kinase/stress-activated kinase MAPK pathways are not altered during lovastatin-induced apoptosis. By contrast, exposure of primary and established AML cells to statins results in significant disruption of basal extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Addition of geranylgeranyl PPi reverses statin-induced loss of ERK1/2 phosphorylation and apoptosis. By establishing and evaluating the inducible Raf-1:ER system in AML cells, we show that constitutive activation of the Raf/MAPK kinase (MEK)/ERK pathway significantly represses but does not completely block lovastatin-induced apoptosis. Our results strongly suggest statins trigger apoptosis by regulating several signaling pathways, including the Raf/MEK/ERK pathway. Indeed, down-regulation of the Raf/MEK/ERK pathway potentiates statin-induced apoptosis because exposure to the MEK1 inhibitor PD98059 sensitizes AML cells to low, physiologically achievable concentrations of lovastatin. Our study suggests that lovastatin, alone or in combination with a MEK1 inhibitor, may represent a new and immediately available therapeutic approach to combat tumors with activated ERK1/2, such as AML.