bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Role for Mixed-Lineage Kinase-Like Mitogen-Activated Protein Triple Kinase α in Neoplastic Cell Transformation and Tumor Development
Authors: Cho Yong-Yeon , Bode Ann M. , Mizuno Hideya , Choi Bu Young , Choi Hong Seok , Dong Zigang
Published: September 2005

Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-α, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-α into MLTK-α-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-α fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-α-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-α. Very importantly, MLTK-α-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-α stably transfected cells. These results are the first to indicate that MLTK-α plays a key role in neoplastic cell transformation and cancer development.
Hyaluronan Facilitates Invasion of Colon Carcinoma Cells
in Vitro
via Interaction with CD44
Authors: Kim Hyeong-Rok , Wheeler Marie A. , Wilson Christopher M. , Iida Joji , Eng David , Simpson Melanie A. , McCarthy James B. , Bullard Kelli M.
Published: September 2005

Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in vitro. To assess HA functionality, HAS expression was inhibited in SW620 cells by transfection with antisense HAS constructs. Decreased HA secretion and retention in the transfectants were confirmed using competitive binding and particle exclusion assays. SW620 cells demonstrated greater invasion through Matrigel than did SW480 cells. Antisense transfection decreased Matrigel invasion by SW620 cells by >60%; addition of exogenous HA restored invasion. Because the cell-surface HA receptor CD44 has been implicated in cancer progression, HA-CD44 interaction was then inhibited by incubation with an anti-CD44 antibody. Anti-CD44 antibody impaired invasion into Matrigel by 95%. Taken together, these data suggest that pericellular HA is critical for colon carcinoma cell invasion and that this invasive capability is dependent on interaction with CD44.
Activation of Antioxidant Pathways in Ras-Mediated Oncogenic Transformation of Human Surface Ovarian Epithelial Cells Revealed by Functional Proteomics and Mass Spectrometry
Authors: Young Travis W. , Mei Fang C. , Yang Gong , Thompson-Lanza Jennifer A. , Liu Jinsong , Cheng Xiaodong
Published: September 2005

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic rasV12 allele. Of ∼2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.
The Selective Estrogen Receptor Modulator Arzoxifene and the Rexinoid LG100268 Cooperate to Promote Transforming Growth Factor β-Dependent Apoptosis in Breast Cancer
Authors: Rendi Mara H. , Suh Nanjoo , Lamph William W. , Krajewski Stan , Reed John C. , Heyman Richard A. , Berchuck Andrew , Liby Karen , Risingsong Renee , Royce Darlene B. , Williams Charlotte R. , Sporn Michael B.
Published: September 2005

We show that the selective estrogen receptor modulator arzoxifene (Arz) and the rexinoid LG100268 (268) synergize to promote apoptosis in a rat model of estrogen receptor-positive breast carcinoma and in estrogen receptor-positive human breast cancer cells in culture. We also show that it is not necessary to administer Arz and 268 continuously during tumor progression to prevent cancer in the rat model because dosing of these drugs in combination for relatively short periods, each followed by drug-free rests, is highly effective. This new approach to chemoprevention uses high doses of drugs that are too toxic for long-term administration. However, when given for short periods, the agents are nontoxic and still induce apoptosis in breast cancer cells. We also show that the ability of the two drugs to induce apoptosis is the combined result of induction of transforming growth factor β by Arz, together with inhibition of the prosurvival nuclear factor κB and phosphatidylinositol 3′ kinase signaling pathways by 268. The new protocol we have developed for chemoprevention allows the efficacious and safe administration of 268 and Arz, and these agents now should be considered for clinical use.
DNA Hypomethylation and Ovarian Cancer Biology
Authors: Widschwendter Martin , Jiang Guanchao , Woods Christian , Müller Hannes M. , Fiegl Heidi , Goebel Georg , Marth Christian , Müller-Holzner Elisabeth , Zeimet Alain G. , Laird Peter W. , Ehrlich Melanie
Published: September 2005

Hypomethylation of some portions of the genome and hypermethylation of others are very frequent in human cancer. The hypomethylation often involves satellite 2 (Sat2) DNA in the juxtacentromeric (centromere-adjacent) region of chromosome 1. In this study, we analyzed methylation in centromeric and juxtacentromeric satellite DNA in 115 ovarian cancers, 26 non-neoplastic ovarian specimens, and various normal somatic tissue standards. We found that hypomethylation of both types of satellite DNA in ovarian samples increased significantly from non-neoplastic toward cancer tissue. Furthermore, strong hypomethylation was significantly more prevalent in tumors of advanced stage or high grade. Importantly, extensive hypomethylation of Sat2 DNA in chromosome 1 was a highly significant marker of poor prognosis (relative risk for relapse, 4.1, and death, 9.4) and more informative than tumor grade or stage. Also, comparing methylation of satellite DNA and 15 5′ gene regions, which are often hypermethylated in cancer or implicated in ovarian carcinogenesis, we generally found no positive or negative association between methylation changes in satellite DNA and in the gene regions. However, hypermethylation at two loci, CDH13 (at 16q24) and RNR1 (at 13p12), was correlated strongly with lower levels of Sat2 hypomethylation. The CDH13/Sat2 epigenetic correlation was seen also in breast cancers. We conclude that satellite DNA hypomethylation is an important issue in ovarian carcinogenesis as demonstrated by: (a) an increase from non-neoplastic tissue toward ovarian cancer; (b) an increase within the ovarian cancer group toward advanced grade and stage; and (c) the finding that strong hypomethylation was an independent marker of poor prognosis.
Global Expression Profiling Identifies Signatures of Tumor Virulence in MMTV-PyMT-Transgenic Mice
Authors: Qiu Ting Hu , Chandramouli Gadisetti V. R. , Hunter Kent W. , Alkharouf Nawal W. , Green Jeffrey E. , Liu Edison T.
Published: September 2005

FVB/N-Tg (MMTV-PyMT)634Mul-transgenic mice develop multifocal mammary tumors with a high incidence of pulmonary metastasis. We have demonstrated previously that mammary tumors derived from transgene-positive F1 progeny in particular inbred strains display altered latency, tumor growth rates, and metastatic rates when compared with the FVB/NJ homozygous parent. To identify genes with expression that might be critical in modifying the biological behavior of MMTV-PyMT tumors, we performed a detailed comparative analysis of expression profiles from mammary tumors arising in the parental FVB/NJ background and F1 progeny from crosses with I/LnJ, LP/J, MOLF/Ei, and NZB/B1NJ mice. Compared with normal mammary glands, gene expression profiles of tumors from all five strains exhibited up-regulation of genes involved in cell growth (e.g., Cks1 and CDC25C) and down-regulation of cell adhesion molecules, with many genes associated previously with human breast cancer such as STAT2, CD24 antigen, gelsolin, and lipocalin2. To identify genes with significant variation in expression between the five different genotypes, significance analysis of microarrays (SAM) and one-way ANOVA were used. Three definable groupings of tumors were identified: (a) tumors derived in the LP/J F1 and MOLF/Ei F1 strains in which tumor growth and dissemination are suppressed and latency prolonged; (b) the most aggressive tumors from the FVB/NJ parental strain and I/LnJ F1 genomic backgrounds; and (c) an intermediate virulence phenotype with tumors from NZB/B1NJ–F1 crosses. These array based assessments correlated well with a composite phenotype ranking using a “virulence” index. The gene expression signature that is associated with a high metastatic rate in the mouse contains the same 17 genes described recently as the signature gene set predictive of metastasis in human tumors (1) with 16 of the 17 genes exhibiting the same directional change in expression associated with human metastases. These results demonstrate that the genetic analysis of mouse models of tumorigenesis may be highly relevant to human cancer and that the metastatic phenotype of a tumor may be affected by the germline genetic configuration of the host.
Polymorphisms in DNA Double-Strand Break Repair Genes and Skin Cancer Risk
Authors: Han Jiali , Colditz Graham A. , Samson Leona D. , Hunter David J.
Published: September 2005

UV can cause a wide range of DNA lesions. UVA-induced oxidative DNA damage and blocked DNA replication by UVB-induced photoproducts can lead to double-strand breaks (DSBs). We selected 11 haplotype-tagging single nucleotide polymorphisms in three DSB repair genes XRCC2, XRCC3, and LigaseIV and evaluated their associations with skin cancer risk in a nested case-control study within the Nurses’ Health Study [219 melanoma, 286 squamous cell carcinoma (SCC), 300 basal cell carcinoma (BCC), and 873 controls]. We observed that the XRCC3 18085T (241Met) allele and its associated haplotype were significantly inversely associated with the risks of SCC and BCC, whereas the XRCC3 4552C allele along with its associated haplotype and the XRCC2 30833A allele were significantly associated with increased BCC risk. The LigaseIV 4044T and 4062T alleles were associated with decreased BCC risk; two of four haplotypes were significantly associated with altered BCC risk. A trend toward decreased risk of nonmelanoma skin cancer was found in those harboring a greater number of putative low risk alleles (P for trend, 0.05 for SCC, <0.0001 for BCC). The main effects of these genotypes were essentially null for melanoma risk. This study provides evidence to suggest the role of the DSB repair pathway in skin cancer development, especially for BCC.
A Fully Synthetic Therapeutic Vaccine Candidate Targeting Carcinoma-Associated Tn Carbohydrate Antigen Induces Tumor-Specific Antibodies in Nonhuman Primates
Authors: Lo-Man Richard , Vichier-Guerre Sophie , Perraut Ronald , Dériaud Edith , Huteau Valérie , BenMohamed Lbachir , Diop Ousmane M. , Livingston Philip O. , Bay Sylvie , Leclerc Claude
Published: September 2005

We recently developed an efficient strategy based on a fully synthetic dendrimeric carbohydrate display (multiple antigenic glycopeptide; MAG) to induce anticarbohydrate antibody responses for therapeutic vaccination against cancer. Here, we show the superior efficacy of the MAG strategy over the traditional keyhole limpet hemocyanin glycoconjugate to elicit an anticarbohydrate IgG response against the tumor-associated Tn antigen. We highlight the influence of the aglyconic carrier elements of such a tumor antigen for their recognition by the immune system. Finally, we additionally developed the MAG system by introducing promiscuous HLA-restricted T-helper epitopes and performed its immunological evaluation in nonhuman primates. MAG:Tn vaccines induced in all of the animals strong tumor-specific anti-Tn antibodies that can mediate antibody-dependent cell cytotoxicity against human tumor. Therefore, the preclinical evaluation of the MAG:Tn vaccine demonstrates that it represents a safe and highly promising immunotherapeutic molecularly defined tool for targeting breast, colon, and prostate cancers that express the carbohydrate Tn antigen.
Transcriptional Regulation of Signal Regulatory Protein α1 Inhibitory Receptors by Epidermal Growth Factor Receptor Signaling
Authors: Kapoor Gurpreet S. , Kapitonov Dmitri , O’Rourke Donald M.
Published: September 2005

Signal regulatory protein (SIRP) α1 is a membrane glycoprotein and a member of the SIRP receptor family. These transmembrane receptors have been shown to exert negative effects on signal transduction by receptor tyrosine kinases via immunoreceptor tyrosine-based inhibitory motifs in the carboxyl domain. Previous work has demonstrated that SIRPs negatively regulate many signaling pathways leading to reduction in tumor migration, survival, and cell transformation. Thus, modulation of SIRP expression levels or activity could be of great significance in the field of cancer therapy. The aim of the present study was to determine the factors that regulate levels of SIRPα1 in human glioblastoma cells that frequently overexpress the epidermal growth factor receptor (EGFR) because SIRPs have been shown to negatively regulate EGFR signaling. Northern blot analysis and immunoprecipitation assays showed variable expression levels of endogenous SIRPα transcripts in nine well-characterized glioblastoma cell lines. We examined SIRPα1 regulation in U87MG and U373MG cells in comparison with clonal derivatives that express a truncated form of erbB2, which negatively regulates EGFR signaling by inducing the formation of nonfunctional heterodimeric complexes. Mutant erbB2-expressing cells contained more SIRPα1 mRNA when compared with the parental cells in presence or absence of serum. Similarly, immunoprecipitation assays showed increased SIRPα1 protein levels in erbB-inhibited cells when compared with parental cells. Messenger RNA stability assays revealed that the increased mRNA levels in EGFR-inhibited cells were due to an induction of transcription. Consistent with this finding, expression of the erbB2 mutant receptor up-regulated SIRPα1 promoter activity in all cell lines tested. Interestingly, pharmacological inhibition of the kinase activities of EGFR, erbB2, and src and activation of mitogen-activated protein kinase, but not phosphatidylinositol 3′-kinase, significantly up-regulated SIRPα1 promoter activity. Based on these observations, we hypothesize that down-modulation of EGFR signaling leads to transcriptional up-regulation of the inhibitory SIRPα1 gene. These data may be important in the application of erbB-inhibitory strategies and for design of therapies for the treatment of glial tumors and other epithelial malignancies.
Assessing Tumor Angiogenesis
Authors: Rabascio Cristina , Muratori Elisabetta , Mancuso Patrizia , Calleri Angelica , Raia Valentina , Foutz Thomas , Cinieri Saverio , Veronesi Giulia , Pruneri Giancarlo , Lampertico Pietro , Iavarone Massimo , Martinelli Giovanni , Goldhirsch Aron , Bertolini Francesco
Published: September 2005

No markers are currently available to indicate the angiogenic profile of a specific malignant disease nor to predict response to antiangiogenic therapies. Nevertheless, many different antiangiogenic drugs are presently being tested in many clinical trials, with an obvious scarcity of useful endpoints for treatment outcome beside survival. By means of a quantitative reverse transcription-PCR approach, we measured VE-cadherin (VE-C), Tie-2, vascular endothelial growth factor receptor 2 and CD133 RNA in the blood of 14 healthy controls, 3 pregnant women, and 84 newly diagnosed (or relapsed) cancer patients. Circulating VE-C RNA was increased in pregnant women and cancer patients (P = 0.0002). VE-C RNA was particularly increased in patients affected by hematological malignancies and decreased to normal values in patients achieving complete remission. Conversely, circulating RNA levels of other endothelial or progenitor cell-specific markers Tie-2, vascular endothelial growth factor receptor 2, and CD133 were not significantly increased in either pregnant women or cancer patients. Comparison of various surrogate angiogenesis markers indicated a switch toward increased plasma vascular endothelial growth factor (VEGF) levels, viable circulating endothelial cells, and circulating VE-C RNA levels in patients affected by hematological malignancies. Taken together, our data indicate that the quantitative evaluation of circulating VE-C RNA is a specific and highly promising tool with which to investigate the angiogenic phenotype of cancer patients.
Phosphorylation of Akt (Ser473) is an Excellent Predictor of Poor Clinical Outcome in Prostate Cancer
Authors: Kreisberg Jeffrey I. , Malik Shazli N. , Prihoda Thomas J. , Bedolla Roble G. , Troyer Dean A. , Kreisberg Suzanne , Ghosh Paramita M.
Published: September 2005

We previously showed, by immunohistochemistry with phospho-specific antibodies, increased phosphorylation (activation) of Akt (Ser473) [phosphorylated Akt (pAkt)] in high-Gleason grade prostate cancer (Malik SN, et al., Clin Cancer Res 2002;8:1168–71). Elevation of pAkt was accompanied by decreased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) [phosphorylated ERK (pERK)], indicative of inactivation. In this report, we determined whether increased pAkt and decreased pERK predicted clinical outcome. Prostate-specific antigen (PSA) failure (detectable and rising PSA) versus PSA non-failure (undetectable PSA 5 years after prostatectomy) was used as a surrogate for clinical outcome. Prostate tumors from cases of PSA failure versus non-failure were stained for pAkt and pERK. A significant increase in mean pAkt staining (P < 0.001) in the PSA failures versus non-failures was seen based on the Wilcoxon signed ranks test [222.18 ± 33.9 (n = 37) versus 108.79 ± 104.57 (n = 16)]. Using the best-fitting multiple logistic regression equation, a 100-point increase in pAkt staining resulted in a 160% increase in the odds of being a PSA failure. There was decreased staining for pERK in PSA failures versus non-failures: a 100-point decrease resulted in an 80% increase in the odds of being a PSA failure. Each of these effects assumed the other biomarker was held constant. The area under the receiver-operating characteristic curve for these two biomarkers predicting PSA failure was 0.84, indicating excellent discrimination between PSA failure and non-failure cases. These data indicate that increased pAkt, alone or together with decreased pERK, is an important predictor of probability of PSA failure. However, pERK alone was not a significant predictor of PSA failure.
KAI1 COOH-Terminal Interacting Tetraspanin (KITENIN), a Member of the Tetraspanin Family, Interacts with KAI1, a Tumor Metastasis Suppressor, and Enhances Metastasis of Cancer
Authors: Lee Ji Hee , Park Sei Ryun , Chay Kee-Oh , Seo Young-Woo , Kook Hyun , Ahn Kyu Youn , Kim Young Jin , Kim Kyung Keun
Published: September 2005

We cloned recently an alternatively spliced variant of KAI1 mRNA that lacked exon 7 at the COOH-terminal region and showed differences in metastasis suppression when compared with the wild-type KAI1. These findings indicated that the COOH-terminal region of KAI1 is critical for its metastasis suppressor function. In this study, we isolated a cDNA clone of VANGL1, a member of the tetraspanin protein family, which interacted specifically with the COOH-terminal cytoplasmic domain of KAI1 in the yeast two-hybrid system. We renamed it KAI1 COOH-terminal interacting tetraspanin (KITENIN). We found that KITENIN-overexpressing CT-26 mouse colon cancer cells showed increased tumorigenicity and early hepatic metastasis in vivo, as well as increased invasiveness and adhesion to fibronectin in vitro compared with parental cells. Moreover, increased levels of KITENIN were observed in a human gastric tumor and its metastatic tissues, compared with the normal adjacent mucosa. Our results indicate that KITENIN promotes adhesion and invasion of cancer cells in vitro and in vivo, and suggest that KITENIN participates in the regulation of the tumor formation and metastasis by interacting with KAI1, a metastasis suppressor and antisense KITENIN strategy that can be used to inhibit metastasis in various cancers.
Hyperforin Inhibits Cancer Invasion and Metastasis
Authors: Donà Massimo , Dell’Aica Isabella , Pezzato Elga , Sartor Luigi , Calabrese Fiorella , Barbera Mila Della , Donella-Deana Arianna , Appendino Giovanni , Borsarini Anna , Caniato Rosy , Garbisa Spiridione
Published: September 2005

Hyperforin (Hyp), the major lipophilic constituent of St. John’s wort, was assayed as a stable dicyclohexylammonium salt (Hyp-DCHA) for cytotoxicity and inhibition of matrix proteinases, tumor invasion, and metastasis. Hyp-DCHA triggered apoptosis-associated cytotoxic effect in both murine (C-26, B16-LU8, and TRAMP-C1) and human (HT-1080 and SK-N-BE) tumor cells; its effect varied, with B16-LU8, HT-1080, and C-26 the most sensitive (IC50 = 5 to 8 μmol/L). At these concentrations, a marked and progressive decline of growth was observed in HT-1080 cells, whereas untransformed endothelial cells were only marginally affected. Hyp-DCHA inhibited in a dose-dependent and noncompetitive manner various proteinases instrumental to extracellular matrix degradation; the activity of leukocyte elastase was inhibited the most (IC50 = 3 μmol/L), followed by cathepsin G and urokinase-type plasminogen activator, whereas that of the matrix metalloproteinases (MMPs) 2 and 9 showed an IC50 > 100 μmol/L. Nevertheless, inhibition of extracellular signal-regulated kinase 1/2 constitutive activity and reduction of MMP-2 and MMP-9 secretion was triggered by 0.5 μmol/L Hyp-DCHA to various degrees in different cell lines, the most in C-26. Inhibition of C-26 and HT-1080 cell chemoinvasion (80 and 54%, respectively) through reconstituted basement membrane was observed at these doses. Finally, in mice that received i.v. injections of C-26 or B16-LU8 cells, daily i.p. administration of Hyp-DCHA—without reaching tumor-cytotoxic blood levels—remarkably reduced inflammatory infiltration, neovascularization, lung weight (−48%), and size of experimental metastases with C-26 (−38%) and number of lung metastases with B16-LU8 (−22%), with preservation of apparently healthy and active behavior. These observations qualify Hyp-DCHA as an interesting lead compound to prevent and contrast cancer spread and metastatic growth.
The Human Glutathione
S
-Transferase P1 Protein Is Phosphorylated and Its Metabolic Function Enhanced by the Ser/Thr Protein Kinases, cAMP-Dependent Protein Kinase and Protein Kinase C, in Glioblastoma Cells
Authors: Lo Hui-Wen , Antoun Gamil R. , Ali-Osman Francis
Published: September 2005

We report here that the human glutathione S-transferase P1 (GSTP1) protein, involved in phase II metabolism of many carcinogens and anticancer agents and in the regulation of c-Jun NH2-terminal kinase-mediated cell signaling, undergoes phosphorylation by the Ser/Thr protein kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), resulting in a significant enhancement of its metabolic activity. GSTP1 phosphorylation by PKA was glutathione (GSH)-dependent, whereas phosphorylation by PKC did not require but was significantly enhanced by GSH. In the presence of GSH, the stoichiometry of phosphorylation was 0.4 ± 0.03 and 0.53 ± 0.02 mol incorporated phosphate per mole of dimeric GSTP1 protein. The GSTP1 protein was phosphorylated, in the presence of GSH, by eight different PKC isoforms (α, βΙ, βΙΙ, δ, ε, γ, η, and ζ), belonging to the three major PKC subclasses, albeit with various efficiencies. The catalytic efficiency, kcat/Km, of the phosphorylated GSTP1 was more than double that of the unphosphorylated protein. In MGR3 human glioblastoma cells, PKA and PKC activation resulted in a significant increase in the level of phosphorylation of the GSTP1 protein and was accompanied by a 2.1- and 2.7-fold increase, respectively, in specific GSTP1 activity in the cells. Peptide phosphorylation analyses and both phosphorylation and enzyme kinetic studies with GSTP1 proteins mutated at candidate amino acid residues established Ser-42 and Ser-184 as putative phospho-acceptor residues for both kinases in the GSTP1 protein. Together, these findings show PKA- and PKC-dependent phosphorylation as a significant post-translational mechanism of regulation of GSTP1 function. The GSH-dependence of the phosphorylation suggests that under high intracellular GSH conditions, such as is present in most drug-resistant tumors, the GSTP1 protein will exist in a hyper-phosphorylated and enzymatically more active state. In normal cells, the functional activation of the GSTP1 protein by PKA- and PKC-dependent phosphorylation could represent a potentially important mechanism of cellular protection, whereas in tumors, increased phase II metabolism of anticancer drugs by the more active phosphorylated GSTP1 protein could contribute to the drug resistance and therapeutic failure frequently associated with increased activities of these Ser/Thr kinases.
Antiangiogenic Properties of Fibstatin, an Extracellular FGF-2–Binding Polypeptide
Authors: Bossard Carine , Berghe Loic Van den , Laurell Henrik , Castano Caroline , Cerutti Martine , Prats Anne-Catherine , Prats Hervé
Published: September 2005

By using the two-hybrid system with basic fibroblast growth factor (FGF-2) as bait, we isolated and characterized fibstatin, an endogenous Mr 29,000 human basement membrane-derived inhibitor of angiogenesis and tumor growth. Fibstatin, a fragment containing the type III domains 12–14 of fibronectin, was produced as a recombinant protein and was shown to inhibit the proliferation, migration, and differentiation of endothelial cells in vitro. Antiangiogenic activity of fibstatin was confirmed in a Matrigel angiogenesis assay in vivo, and electrotransfer of the fibstatin gene into muscle tissue resulted in reduced B16F10 tumor growth. Taken together, these results suggest that fibstatin could act as a powerful molecule for antiangiogenic therapy.
Multiple Familial Trichoepithelioma Caused by Mutations in the Cylindromatosis Tumor Suppressor Gene
Authors: Salhi Aicha , Bornholdt Dorothea , Oeffner Frank , Malik Sajid , Heid Ernest , Happle Rudolf , Grzeschik Karl-Heinz
Published: September 2005

The recessive oncogene cylindromatosis (CYLD) mapping on 16q12-q13 is generally implicated in familial cylindromatosis, whereas a gene region for multiple familial trichoepithelioma has been assigned to 9p21. Markers from both chromosome intervals were subjected to linkage analysis in a large family with multiple hereditary trichoepithelioma (TE) from Algeria. Linkage to 9p21 was excluded, whereas CYLD remained as a candidate. Mutation analysis identified a single bp germ-line deletion expected to result in truncation or absence of the encoded protein, which segregated with the multiple TE phenotype. In individual tumors, loss of heterozygosity at 16q or a somatic point mutation in the CYLD gene was detected. Hence, mutations of the tumor suppressor gene CYLD at 16q12-q13 may give rise to familial TE indistinguishable from the phenotype assigned to 9p21.
Adhesion of Gastric Carcinoma Cells to Peritoneum Mediated by α3β1 Integrin (VLA-3)
Authors: Takatsuki Hironori , Komatsu Shinya , Sano Rikio , Takada Yoshikazu , Tsuji Tsutomu
Published: September 2005

The interaction between gastric carcinoma cells and the peritoneal lining is a key step in peritoneal dissemination. In this study, we examined the roles of the β1 family of integrin receptors in the adhesion of such cells to the peritoneum. The adhesion of several gastric carcinoma cell lines to peritonea excised from mice was inhibited most by an anti-α3 integrin antibody and to a lesser extent by an anti-α2 integrin antibody. In the peritoneal implantation of NUGC-4 human gastric carcinoma cells in athymic mice, treatment of the cells with anti-α2 or anti-α3 integrin antibody reduced the number of disseminated nodules; suppression by the anti-α3 integrin antibody was stronger than that by the anti-α2 integrin antibody. The cDNAs to human α2 and α3 integrins were introduced into K562 leukemic cells, which were positive for the integrin β1 subunit but negative for the α2 or α3 subunit. The α3 integrin-transfected cells adhered to excised peritoneum and to a monolayer of peritoneal mesothelial cells more firmly than did the α2 integrin-transfected cells or the mock transfectant. Reverse transcription-PCR was used to analyze the expression of laminin-5 and laminin-10/11, which have been reported to serve as high-affinity ligands for α3β1 integrin. mRNA for these laminin isoforms was found in mesothelial cells from the diaphragm and parietal peritoneum. These results strongly suggest that α3β1 integrin plays an essential role in mediating the initial attachment of cancer cells to the peritoneum, leading to the formation of peritoneal metastasis.
IRSp53/Eps8 Complex Is Important for Positive Regulation of Rac and Cancer Cell Motility/Invasiveness
Authors: Funato Yosuke , Terabayashi Takeshi , Suenaga Naoko , Seiki Motoharu , Takenawa Tadaomi , Miki Hiroaki
Published: September 2005

IRSp53 has been characterized as an adaptor protein that links Rho-family small GTPases, such as Rac, to reorganization of the actin cytoskeleton. Here, we search for other binding partners for the IRSp53 SH3 domain and identify Eps8 as the major binding protein in fibroblasts and various cancer cell lines. Eps8 has been shown to form a Rac-specific guanine nucleotide exchange factor complex with Abi-1 and Sos-1, which seems essential for ruffling formation induced by oncogenic Ras. We confirm the IRSp53/Eps8 complex formation in vivo and the direct association between Eps8 NH2-terminal proline-rich sequence and IRSp53 SH3 domain. This complex synergistically activates Rac by reinforcing the formation of the Eps8/Abi-1/Sos-1 Rac-guanine nucleotide exchange factor complex, which mediates positive regulation of Rac activity. In addition, IRSp53/Eps8 complex formation as determined by fluorescent resonance energy transfer analysis, occurs at the leading edge of motile cells, and the motility and invasiveness of HT1080 fibrosarcoma cells are suppressed by inhibiting complex formation. These findings implicate the importance of the IRSp53/Eps8 complex in Rac activation and metastatic behavior of the malignant tumor cells.
Array Comparative Genomic Hybridization Analysis of Colorectal Cancer Cell Lines and Primary Carcinomas
Authors: Douglas Eleanor J. , Fiegler Heike , Rowan Andrew , Halford Sarah , Bicknell David C. , Bodmer Walter , Tomlinson Ian P. M. , Carter Nigel P.
Published: September 2005

Array comparative genomic hybridization, with a genome-wide resolution of ∼1 Mb, has been used to investigate copy number changes in 48 colorectal cancer (CRC) cell lines and 37 primary CRCs. The samples were divided for analysis according to the type of genomic instability that they exhibit, microsatellite instability (MSI) or chromosomal instability (CIN). Consistent copy number changes were identified, including gain of chromosomes 20, 13, and 8q and smaller regions of amplification such as chromosome 17q11.2-q12. Loss of chromosome 18q was a recurrent finding along with deletion of discrete regions such as chromosome 4q34-q35. The overall pattern of copy number change was strikingly similar between cell lines and primary cancers with a few obvious exceptions such as loss of chromosome 6 and gain of chromosomes 15 and 12p in the former. A greater number of aberrations were detected in CIN+ than MSI+ samples as well as differences in the type and extent of change reported. For example, loss of chromosome 8p was a common event in CIN+ cell lines and cancers but was often found to be gained in MSI+ cancers. In addition, the target of amplification on chromosome 8q appeared to differ, with 8q24.21 amplified frequently in CIN+ samples but 8q24.3 amplification a common finding in MSI+ samples. A number of genes of interest are located within the frequently aberrated regions, which are likely to be of importance in the development and progression of CRC.
A Role for the RASSF1A Tumor Suppressor in the Regulation of Tubulin Polymerization and Genomic Stability
Authors: Vos Michele D , Martinez Alfredo , Elam Candice , Dallol Ashraf , Taylor Barbara J , Latif Farida , Clark Geoffrey J
Published: September 2005

The high frequency with which the novel tumor suppressor RASSF1A is inactivated by promoter methylation suggests that it plays a key role in the development of many primary human tumors. Yet the mechanism of RASSF1A action remains unknown. We now show that RASSF1A associates with microtubules and that this association is essential for RASSF1A to mediate its growth inhibitory effects. Overexpression of RASSF1A promotes the formation of stable microtubules, whereas a dominant-negative fragment of RASSF1A destabilizes microtubule networks. The RASSF1 protein is expressed as two main isoforms, 1A and 1C. The smaller 1C isoform also associates with microtubules but is less effective at stabilizing them. Because RASSF1A and RASSF1C localize to the mitotic spindle, we examined their effects upon genomic instability. RASSF1A and RASSF1C block activated Ras-induced genomic instability. However, a point mutant of RASSF1C, identified in human tumors, was severely defective for stabilizing tubulin and was unable to block the genomic destabilizing effects of Ras. Thus, we identify a role for RASSF1A/C in the control of microtubule polymerization and potentially in the maintenance of genomic stability.
Heritability and Linkage Analysis of Sensitivity to Cisplatin-Induced Cytotoxicity
Authors: Dolan M. Eileen , Newbold Karla G. , Nagasubramanian Ramamoorthy , Wu Xiaolin , Ratain Mark J. , Cook Edwin H. , Badner Judith A.
Published: September 2005

Little is known about the genetic determinants explaining variation in sensitivity to chemotherapeutic cytotoxicity. We characterized the degree of cisplatin sensitivity, using lymphoblastoid cell lines derived from 10 Centre d’Etude du Polymorphisme Humain pedigrees. We estimated the heritability for susceptibility to cisplatin-induced cytotoxicity to be ∼0.47; therefore, sensitivity to the cytotoxic effects of cisplatin is under appreciable genetic influence. Linkage analysis was performed, and the strongest signal (lod score, 2.16; empirical P = 0.0005) was found on chromosome 1 at 44 cM. Susceptibility to cisplatin-induced cytotoxicity is likely due to multiple loci, with low locus-specific heritability contributing to the trait. These data show the power of using large pedigrees that have been extensively genotyped for evaluating the genetic contribution to sensitivity to cell growth inhibition by anticancer agents.
A Novel Founder CHEK2 Mutation is Associated with Increased Prostate Cancer Risk
Authors: Cybulski Cezary , Huzarski Tomasz , Górski Bohdan , Masojć Bartłomiej , Mierzejewski Marek , Dębniak Tadeusz , Gliniewicz Bartłomiej , Matyjasik Joanna , Złowocka Elżbieta , Kurzawski Grzegorz , Sikorski Andrzej , Posmyk Michał , Szwiec Marek , Czajka Ryszard , Narod Steven A. , Lubiński Jan
Published: September 2005

Variants in the CHEK2 have been found to be associated with prostate cancer risk in the United States and Finland. We sequenced CHEK2 gene in 140 Polish patients with prostate cancer and then genotyped the three detected variants in a larger series of prostate cancer cases and controls. CHEK2 truncating mutations (IVS2 + 1G>A or 1100delC) were identified in 9 of 1921 controls (0.5%) and in 11 of 690 (1.6%) unselected patients with prostate cancer [odds ratio (OR) = 3.4; P = 0.004]. These mutations were found in 4 of 98 familial prostate cases (OR = 9.0; P = 0.0002). The missense variant I157T was also more frequent in men with prostate cancer (7.8%) than in controls (4.8%), but the relative risk was more modest (OR = 1.7; P = 0.03). I157T was identified in 16% of men with familial prostate cancer (OR = 3.8; P = 0.00002). Loss of the wild-type CHEK2 allele was not observed in any of prostate cancers from five men who carried CHEK2-truncating mutations. Our results provide evidence that the two truncating mutations of CHEK2 confer a moderate risk of prostate cancer in Polish men and that the missense change appears to confer a modest risk.
Oncogenic H-Ras Up-Regulates Expression of ERCC1 to Protect Cells from Platinum-Based Anticancer Agents
Authors: Youn Cha-Kyung , Kim Mi-Hwa , Cho Hyun-Ju , Kim Hong-Beum , Chang In-Youb , Chung Myung-Hee , You Ho Jin
Published: September 2005

Tumors frequently contain mutations in the ras genes, resulting in the constitutive activation of the Ras-activated signaling pathway. The activation of Ras is involved not only in tumor progression but also in the development of resistance of the tumor cells to platinum-based chemotherapeutic agents. To investigate the potential mechanisms underlying this resistance, we analyzed the effect of activated H-Ras on the expression of the nucleotide excision repair genes. Here we identified ERCC1, which is one of the key enzymes involved in nucleotide excision repair, as being markedly up-regulated by the activated H-Ras. From promoter analysis of ERCC1, an increase in the Ap1 transcriptional activity as a result of the expression of the oncogenic H-Ras was found to be crucial for this induction. In addition, ERCC1 small interfering RNA expression was shown to reduce the oncogenic H-Ras-mediated increase in the DNA repair activity as well as to suppress the oncogenic H-Ras-mediated resistance of the cells to platinum-containing chemotherapeutic agents. These results suggest that the oncogenic H-Ras-induced ERCC1, which activates the DNA repair capacity, may be involved in the protection of the cells against platinum-based anticancer agents.
p53 Heterozygosity Results in an Increased 2-Acetylaminofluorene-Induced Urinary Bladder but not Liver Tumor Response in DNA Repair-Deficient
Xpa
Mice
Authors: Hoogervorst Esther M. , van Oostrom Conny Th. M. , Beems Rudolf B. , van Benthem Jan , Gielis Siska , Vermeulen Jolanda P. , Wester Piet W. , Vos Joseph G. , de Vries Annemieke , van Steeg Harry
Published: September 2005

Both nucleotide excision repair (NER) and the p53 tumor suppressor protein play crucial roles in the prevention of cells becoming cancerous. This is clearly demonstrated by the fact that NER-deficient xeroderma pigmentosum patients and Li-Fraumeni patients who carry a germ-line p53 mutation are highly tumor prone. The NER-deficient Xpa and the p53+/− mouse models clearly mimic their human counterparts, because they are both tumor prone as well. The aim of the study presented here was to analyze the relative contribution of these two pathways in tumor suppression and to analyze a possible link between NER and p53 activation in vivo. For this, we exposed Xpa, p53+/−, and Xpa/p53+/− mice to 2-acetylaminofluorene (2-AAF). We show that 2-AAF-induced urinary bladder tumor suppression is dependent on p53 status, because p53+/− mice were highly tumor prone. Xpa/p53+/− mice were even more tumor prone, whereas no increased tumor response was found in Xpa mice. Short-term assays revealed a decreased apoptotic response in Xpa/p53+/− mice, pointing in vivo toward a link between NER and p53-mediated apoptosis. In contrast, liver tumor response was primarily dependent on appropriate DNA repair, because Xpa-deficient mice were liver tumor prone. p53 heterozygosity had no influence on liver tumor incidences, in line with the results obtained from the short-term 2-AAF studies revealing no altered cellular response in p53+/− or Xpa/p53+/− mice. Interestingly, however, mice completely deficient in both NER and p53 (Xpa/p53−/− mice) showed a dramatic increase of hepatocellular proliferation accompanied by lacZ reporter gene mutations.
Accumulation of the Oxidative Base Lesion 8-Hydroxyguanine in DNA of Tumor-Prone Mice Defective in Both the
Myh
and
Ogg1
DNA Glycosylases
Authors: Russo Maria Teresa , De Luca Gabriele , Degan Paolo , Parlanti Eleonora , Dogliotti Eugenia , Barnes Deborah E. , Lindahl Tomas , Yang Hanjing , Miller Jeffrey H. , Bignami Margherita
Published: September 2005

The OGG1 and MYH DNA glycosylases prevent the accumulation of DNA 8-hydroxyguanine. In Myh−/− mice, there was no time-dependent accumulation of DNA 8-hydroxyguanine in brain, small intestine, lung, spleen, or kidney. Liver was an exception to this general pattern. Inactivation of both MYH and OGG1 caused an age-associated accumulation of DNA 8-hydroxyguanine in lung and small intestine. The effects of abrogated OGG1 and MYH on hepatic DNA 8-hydroxyguanine levels were additive. Because there is an increased incidence of lung and small intestine cancer in Myh−/−/Ogg1−/− mice, these findings support a causal role for unrepaired oxidized DNA bases in cancer development.
Suppression of Colorectal Cancer Liver Metastasis and Extension of Survival by Expression of Apolipoprotein(a) Kringles
Authors: Yu Hyun-Kyung , Kim Jang-Seong , Lee Ho-Jeong , Ahn Jin-Hyung , Lee Suk-Keun , Hong Soon-Won , Yoon Yeup
Published: September 2005

The formation of hepatic metastases in colorectal cancer is the main cause of patient death. Current therapies directed at hepatic metastasis of colorectal cancer have had minimal impact on outcome. Therefore, alternative treatment strategies for liver metastasis require development. The present study was performed to evaluate the application of cDNA of LK68 encoding apolipoprotein(a) kringles IV-9, IV-10, and V as possible candidates for gene therapy treatment of this life-threatening disease. The murine colorectal cancer cell line CT26 was transduced ex vivo with LK68 cDNA via retroviral gene transfer, and an experimental model of hepatic metastasis was established by injecting LK68-expressing and control cells into the spleens of BALB/c mice. Expression of LK68 did not affect the growth characteristics and viability of transduced CT26 cells in vitro. LK68 produced from CT26 cells substantially inhibited the migration of endothelial cells in vitro. In vivo, substantial suppression of liver metastasis and prolonged survival were observed in mice bearing LK68-expressing CT26 cells, compared with controls. LK68-expressing liver metastases were restricted to smaller sizes and displayed decreased microvessel density and increased tumor cell apoptosis. Our data collectively indicate that LK68 suppresses angiogenesis-dependent progression of prevascular micrometastases to macroscopic tumors and their growth, which are clinically accessible and biologically relevant therapeutic targets. We propose that antiangiogenic gene therapy with LK68 is a promising strategy for the treatment of colorectal cancer liver metastasis.
A Novel Role for Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6 as a Determinant of Gemcitabine Chemoresistance in Pancreatic Adenocarcinoma Cells
Authors: Duxbury Mark S. , Ito Hiromichi , Benoit Eric , Waseem Talat , Ashley Stanley W. , Whang Edward E.
Published: September 2005

Most patients with pancreatic adenocarcinoma present with surgically incurable disease. Gemcitabine, the principal agent used to treat such patients, has little impact on outcome. Overexpression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6, a feature of this malignancy, is associated with resistance to anoikis and increased metastasis. The purpose of this study was to determine the role of CEACAM6 in cellular chemoresistance to gemcitabine. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express very low levels of the protein. Suppression of CEACAM6 expression was achieved in BxPC3 cells, which inherently overexpress CEACAM6, by stable transfection of a CEACAM6 small interfering RNA-generating vector. The effects of modulating CEACAM6 expression on gemcitabine-induced cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay, flow cytometric apoptosis quantification, caspase profiling, and Western analysis of cytoplasmic cytochrome c release. The roles of Akt and c-Src kinases as downstream targets of CEACAM6 signaling were examined. Stable overexpression of CEACAM6 in Capan2 increased gemcitabine chemoresistance, whereas CEACAM6 gene silencing in BxPC3 markedly increased the sensitivity of these cells to gemcitabine. Differential expression of CEACAM6 modulates Akt activity in a c-Src-dependent manner, and CEACAM6 overexpression appears to protect cells from cytochrome c-induced caspase 3 activation and apoptosis.
Noninvasive Imaging of the Transcriptional Activities of Human Telomerase Promoter Fragments in Mice
Authors: Groot-Wassink Thomas , Aboagye Eric O. , Wang Yaohe , Lemoine Nicholas R. , Keith W. Nicol , Vassaux Georges
Published: September 2005

We have assessed the feasibility of positron emission tomography (PET) and ex vivo γ-counting to measure the pattern of expression of telomerase promoter fragments in vivo. Promoter fragments from either the RNA [human telomerase RNA (hTR)] or the catalytic components [human telomerase reverse transcriptase (hTERT)] of the telomerase genes were used to drive the expression of the sodium iodide symporter PET reporter gene in recombinant adenoviruses. Both promoter fragments provided cancer-selective expression that could be visualized and quantitated by PET. The transcriptional activity of the hTR promoter was found to be consistently stronger than that of the hTERT promoter. Both promoters appear therefore to be good candidates for safe use in gene therapy, and PET imaging can be used to assess the selectivity of promoters in vivo. Given that this methodology is directly scalable to humans, imaging gene expression using the sodium iodide symporter PET reporter gene could be applied to measure telomerase promoter activity in humans.