bluuebunny/crossref_metadata_2025 huggingface dataset:
Multidrug Transporter ABCG2 Prevents Tumor Cell Death Induced by the Epidermal Growth Factor Receptor Inhibitor Iressa (ZD1839, Gefitinib)
Authors: Elkind N. Barry , Szentpétery Zsófia , Apáti Ágota , Özvegy-Laczka Csilla , Várady György , Ujhelly Olga , Szabó Katalin , Homolya László , Váradi András , Buday László , Kéri György , Német Katalin , Sarkadi Balázs
Published: September 2005

Iressa (ZD1839, Gefitinib), used in clinics to treat non–small cell lung cancer patients, is a tyrosine kinase receptor inhibitor that leads to specific decoupling of epidermal growth factor receptor (EGFR) signaling. Recent data indicate that Iressa is especially effective in tumors with certain EGFR mutations; however, a subset of these tumors does not respond to Iressa. In addition, certain populations have an elevated risk of side effects during Iressa treatment. The human ABCG2 (BCRP/MXR/ABCP) transporter causes cancer drug resistance by actively extruding a variety of cytotoxic drugs, and it functions physiologically to protect our tissues from xenobiotics. Importantly, ABCG2 modifies absorption, distribution, and toxicity of several pharmacologic agents. Previously, we showed that ABCG2 displays a high-affinity interaction with several tyrosine kinase receptor inhibitors, including Iressa. Here, we show that the expression of ABCG2, but not its nonfunctional mutant, protects the EGFR signaling-dependent A431 tumor cells from death on exposure to Iressa. This protection is reversed by the ABCG2-specific inhibitor, Ko143. These data, reinforced with cell biology and biochemical experiments, strongly suggest that ABCG2 can actively pump Iressa. Therefore, variable expression and polymorphisms of ABCG2 may significantly modify the antitumor effect as well as the absorption and tissue distribution of Iressa.
Carboxyl-Terminal Src Kinase Homologous Kinase Negatively Regulates the Chemokine Receptor CXCR4 through YY1 and Impairs CXCR4/CXCL12 (SDF-1α)–Mediated Breast Cancer Cell Migration
Authors: Lee Byeong-Chel , Lee Tae-Hee , Zagozdzon Radoslaw , Avraham Shalom , Usheva Anny , Avraham Hava Karsenty
Published: September 2005

Using microarray gene analysis, we found that carboxyl-terminal Src kinase homologous kinase (CHK) regulated the expression of the chemokine receptor, CXCR4. Northern blot and fluorescence-activated cell-sorting analyses showed that CHK down-regulated CXCR4 mRNA and protein levels, respectively. Mutated CHK, which contains a mutation within the ATP binding site of CHK, failed to inhibit CXCR4 expression, thus suggesting that CHK kinase activity is involved in the regulation of CXCR4. Results from gel shift analysis indicated that CHK regulates CXCR4 transcriptional activity by altering YY1 binding to the CXCR4 promoter. Whereas CHK had no significant effects on the expression of YY1, c-Myc, Max, and other YY1-binding proteins, CHK was found to modulate the YY1/c-Myc association. Furthermore, CHK inhibited CXCR4-positive breast cancer cell migration. Taken together, these studies show a novel mechanism by which CHK down-regulates CXCR4 through the YY1 transcription factor, leading to decreased CXCR4-mediated breast cancer cell motility and migration.
Topotecan Blocks Hypoxia-Inducible Factor-1α and Vascular Endothelial Growth Factor Expression Induced by Insulin-Like Growth Factor-I in Neuroblastoma Cells
Authors: Beppu Kiichiro , Nakamura Katsuya , Linehan W. Marston , Rapisarda Annamaria , Thiele Carol J.
Published: September 2005

The extent of angiogenesis and/or vascular endothelial growth factor (VEGF) expression in neuroblastoma tumors correlates with metastases, N-myc amplification, and poor clinical outcome. Understanding the mechanisms regulating VEGF expression in neuroblastoma cells provides additional therapeutic options to control neuroblastoma tumor growth. VEGF mRNA is controlled by growth factors and hypoxia via the transcription factor hypoxia-inducible factor (HIF-1α). HIF-1α protein levels are regulated by the von Hippel Lindau tumor suppressor gene, VHL, which targets HIF-1α degradation. To determine whether the levels of VEGF in neuroblastomas are due to mutations in VHL, we evaluated genomic DNA from 15 neuroblastoma cell lines using PCR. We found no mutations in exons 1, 2, or 3 of the VHL gene. VEGF mRNA levels in neuroblastoma cells cultured in serum-free medium increased after 8 to 16 hours in serum, insulin-like growth factor–I (IGF–I), epidermal growth factor, or platelet-derived growth factor. Serum/IGF–I induced increases in HIF-1α protein that temporally paralleled increases in VEGF mRNA, whereas HIF-1β levels were unaffected. VEGF and HIF-1α levels were blocked by inhibitors of phosphatidylinositol 3-kinase and mammalian target of rapamycin. Furthermore, we confirmed that HIF-1α mediates ∼40% of the growth factor activity stimulating VEGF protein expression. Topotecan blocked the IGF-I-stimulated increase in HIF-1α but not HIF-1β, and this resulted in a decrease in VEGF in four neuroblastoma cell lines tested. These data indicate that growth factors in an autocrine or paracrine manner play a major role in regulating VEGF levels in neuroblastoma cells and that targeted therapies to phosphatidylinositol 3-kinase, mammalian target of rapamycin, and/or HIF-1α have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth.
Susceptibility to Aflatoxin B1-Induced Carcinogenesis Correlates with Tissue-Specific Differences in DNA Repair Activity in Mouse and in Rat
Authors: Bedard Leanne L. , Alessi Manlio , Davey Scott , Massey Thomas E.
Published: September 2005

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B1 (AFB1)–induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB1 to alter repair of AFB1-DNA damage was determined. Plasmid DNA containing AFB1-N7-guanine or AFB1-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB1-N7-guanine and AFB1-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and ∼6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB1-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB1 i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB1-N7-guanine damage was ∼3.5-fold higher in liver of AFB1-treated mice relative to control. This is the first study to show that in vivo treatment with AFB1 can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB1, and selective induction of repair in liver contribute to the susceptibility of mice to AFB1-induced lung tumorigenesis relative to hepatocarcinogenesis.
PTEN and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation by Glioblastoma
Authors: Rong Yuan , Post Dawn E. , Pieper Russell O. , Durden Donald L. , Van Meir Erwin G. , Brat Daniel J.
Published: September 2005

We have previously proposed that intravascular thrombosis and subsequent vasoocclusion contribute to the development of pseudopalisading necrosis, a pathologic hallmark that distinguishes glioblastoma (WHO grade 4) from lower grade astrocytomas. To better understand the potential prothrombotic mechanisms underlying the formation of these structures that drive tumor angiogenesis, we investigated tissue factor (TF), a potent procoagulant protein known to be overexpressed in astrocytomas. We hypothesized that PTEN loss and tumor hypoxia, which characterize glioblastoma but not lower grade astrocytomas, could up-regulate TF expression and cause intravascular thrombotic occlusion. We examined the effect of PTEN restoration and hypoxia on TF expression and plasma coagulation using a human glioma cell line containing an inducible wt-PTEN cDNA. Cell exposure to hypoxia (1% O2) markedly increased TF expression, whereas restoration of wt-PTEN caused decreased cellular TF. The latter effect was at least partially dependent on PTEN's protein phosphatase activity. Hypoxic cells accelerated plasma clotting in tilt tube assays and this effect was prevented by both inhibitory antibodies to TF and plasma lacking factor VII, implicating TF-dependent mechanisms. To further examine the genetic events leading to TF up-regulation during progression of astrocytomas, we investigated its expression in a series of human astrocytes sequentially infected with E6/E7/human telomerase, Ras, and Akt. Cells transformed with Akt showed the greatest incremental increase in hypoxia-induced TF expression and secretion. Together, our results show that PTEN loss and hypoxia up-regulate TF expression and promote plasma clotting by glioma cells, suggesting that these mechanisms may underlie intravascular thrombosis and pseudopalisading necrosis in glioblastoma.
Apoptosis Factor EI24/PIG8 Is a Novel Endoplasmic Reticulum–Localized Bcl-2–Binding Protein which Is Associated with Suppression of Breast Cancer Invasiveness
Authors: Zhao Xiansi , Ayer Robert E. , Davis Shannon L. , Ames Sarah J. , Florence Brian , Torchinsky Cyrus , Liou James S. , Shen Ling , Spanjaard Remco A.
Published: September 2005

p53 is a critical tumor suppressor which removes cells with DNA damage by regulating expression and activity of a select group of p53-induced genes (PIG) that subsequently induce apoptosis. PIG8 was also identified as a gene induced by etoposide and named etoposide-induced gene 24 (EI24). Later experiments established EI24/PIG8 as a proapoptotic factor and suggested that it may function as a tumor suppressor. Indeed, EI24/PIG8 is relatively highly mutated in aggressive breast cancers and is located in a region which expresses frequent loss of heterozygosity. However, despite these important observations, the activity and role of EI24/PIG8 remain largely unknown. We used (immmuno)fluorescence microscopy and subcellular fractionation techniques to show that EI24/PIG8 is localized in the endoplasmic reticulum (ER). Pull-down experiments showed that it specifically binds with Bcl-2, a death regulator known to reside in mitochondria, ER, and the nuclear envelope. EI24/PIG8-Bcl-2 binding was corroborated by coimmunoprecipitation and other in vitro and in vivo protein-protein binding assays. Further analysis showed that EI24/PIG8 uses its N-terminal region to bind the BH3 domain in Bcl-2. Finally, we used immunohistochemical techniques to analyze expression of EI24/PIG8 in breast cancer tissue progression arrays and showed that loss of EI24/PIG8 is associated with tumor invasiveness but not with the development of the primary tumor. These results suggest that EI24/PIG8 is a novel, ER-localized Bcl-2–binding protein which may contribute to apoptosis by modulating the activity and/or function of Bcl-2 in this organelle. EI24/PIG8 may serve to prevent tumor spreading, consistent with its suspected role as a tumor suppressor.
Inactivation of the Invasion Inhibitory Gene IIp45 by Alternative Splicing in Gliomas
Authors: Song Sonya W. , Fuller Gregory N. , Zheng Hong , Zhang Wei
Published: September 2005

The invasion inhibitory protein 45 (IIp45) we recently identified was underexpressed in glioblastoma multiforme, the most malignant form of glioma. The IIp45 gene is located at chromosome 1p36 where frequent deletions have been reported in various types of tumors, including gliomas, raising the possibility that IIp45 may be a classic tumor suppressor gene that can be inactivated by frequent point mutations. To test this hypothesis, we sequenced the IIp45 gene in 59 diffuse glioma samples of different grades and histologic subtypes and identified a possible point mutation or a rare polymorphism in only one sample (1.7%), suggesting that IIp45 is not a classic tumor suppressor gene such as p53. Instead, reverse transcription-PCR and subsequent sequencing results revealed a tumor-specific IIp45 spliced isoform (IIp45S) in 20 of 59 (34%) gliomas examined, particularly in glioblastoma multiformes, including native tissue samples (15 of 25; 60%) and cell lines (5 of 5; 100%). The alternative splicing event is independent of 1p36 deletion, which is not common in glioblastoma multiforme. The IIp45S transcript was not detected in any of 18 normal organs, including fetal and adult brain. We determined that the IIp45S isoform results from exclusion of IIp45 exon 7 and encodes a variant protein that carries a COOH terminus different from that of IIp45 due to a frame-shift mutation. IIp45S protein was undetectable in glioma tissues, although IIp45S mRNA was prevalent. We found that IIp45S, once translated, is rapidly degraded by an ubiquitin-proteasome mechanism. Thus, the IIp45 gene is inactivated by a tumor-specific alternative splicing that generates an aberrant and unstable IIp45 isoform in infiltrative gliomas.
Cannabinoid Receptor as a Novel Target for the Treatment of Prostate Cancer
Authors: Sarfaraz Sami , Afaq Farrukh , Adhami Vaqar M. , Mukhtar Hasan
Published: September 2005

Cannabinoids, the active components of Cannabis sativa Linnaeus (marijuana) and their derivatives have received renewed interest in recent years due to their diverse pharmacologic activities such as cell growth inhibition, anti-inflammatory effects and tumor regression. Here we show that expression levels of both cannabinoid receptors, CB1 and CB2, are significantly higher in CA-human papillomavirus-10 (virally transformed cells derived from adenocarcinoma of human prostate tissue), and other human prostate cells LNCaP, DUI45, PC3, and CWR22Rν1 than in human prostate epithelial and PZ-HPV-7 (virally transformed cells derived from normal human prostate tissue) cells. WIN-55,212-2 (mixed CB1/CB2 agonist) treatment with androgen-responsive LNCaP cells resulted in a dose- (1-10 μmol/L) and time-dependent (24-48 hours) inhibition of cell growth, blocking of CB1 and CB2 receptors by their antagonists SR141716 (CB1) and SR144528 (CB2) significantly prevented this effect. Extending this observation, we found that WIN-55,212-2 treatment with LNCaP resulted in a dose- (1-10 μmol/L) and time-dependent (24-72 hours) induction of apoptosis (a), decrease in protein and mRNA expression of androgen receptor (b), decrease in intracellular protein and mRNA expression of prostate-specific antigen (c), decrease in secreted prostate-specific antigen levels (d), and decrease in protein expression of proliferation cell nuclear antigen and vascular endothelial growth factor (e). Our results suggest that WIN-55,212-2 or other non–habit-forming cannabinoid receptor agonists could be developed as novel therapeutic agents for the treatment of prostate cancer.
Cytoplasmic Phospholipase A2 Deletion Enhances Colon Tumorigenesis
Authors: Ilsley Jillian N.M. , Nakanishi Masako , Flynn Christopher , Belinsky Glenn S. , De Guise Sylvain , Adib John N. , Dobrowsky Rick T. , Bonventre Joseph V. , Rosenberg Daniel W.
Published: September 2005

Cellular pools of free arachidonic acid are tightly controlled through enzymatic release of the fatty acid and subsequent utilization by downstream enzymes including the cyclooxygenases. Arachidonic acid cleavage from membrane phospholipids is accomplished by the actions of phospholipase A2 (PLA2). Upon release, free arachidonic acid provides substrate for the synthesis of eicosanoids. However, under certain conditions, arachidonic acid may participate in ceramide-mediated apoptosis. Disruption of arachidonic acid homeostasis can shift the balance of cell turnover in favor of tumorigenesis, via overproduction of tumor-promoting eicosanoids or alternatively by limiting proapoptotic signals. In the following study, we evaluated the influence of genetic deletion of a key intracellular phospholipase, cytoplasmic PLA2 (cPLA2), on azoxymethane-induced colon tumorigenesis. Heterozygous and null mice, upon treatment with the organotropic colon carcinogen, azoxymethane, developed a significant (P < 0.05) increase in colon tumor multiplicity (7.2-fold and 5.5-fold, respectively) relative to their wild-type littermates. This enhanced tumor sensitivity may be explained, in part, by the attenuated levels of apoptosis observed by terminal deoxynucleotidyl transferase–mediated nick end labeling staining within the colonic epithelium of heterozygous and null mice (≈50% of wild type). The lower frequency of apoptotic cells corresponded with reduced ceramide levels (69% and 46% of wild-type littermates, respectively). Remarkably, increased tumorigenesis resulting from cPLA2 deletion occurred despite a significant reduction in prostaglandin E2 production, even in cyclooxygenase-2–overexpressing tumors. These data contribute new information that supports a fundamental role of cPLA2 in the control of arachidonic acid homeostasis and cell turnover. Our findings indicate that the proapoptotic role of cPLA2 in the colon may supercede its contribution to eicosanoid production in tumor development.
Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP-Dependent Protein Kinase A Signaling Pathway
Authors: Huang Wen-Chin , Xie Zhihui , Konaka Hiroyuki , Sodek Jaro , Zhau Haiyen E. , Chung Leland W.K.
Published: September 2005

Osteocalcin and bone sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)–responsive elements (CRE) as the critical determinants for conditioned medium–mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium–mediated stimulation of human osteocalcin and bone sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background–dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.
Manganese Superoxide Dismutase Polymorphism, Prediagnostic Antioxidant Status, and Risk of Clinical Significant Prostate Cancer
Authors: Li Haojie , Kantoff Philip W. , Giovannucci Edward , Leitzmann Michael F. , Gaziano J. Michael , Stampfer Meir J. , Ma Jing
Published: September 2005

Oxidative stress may enhance prostatic carcinogenesis. A polymorphism [valine (V) → alanine (A)] of manganese superoxide dismutase (MnSOD), the primary antioxidant enzyme in mitochondria, has been recently associated with prostate cancer. We examined the relationship between prostate cancer and the MnSOD polymorphism and its interactions with baseline plasma antioxidant levels (selenium, lycopene, and α-tocopherol) and β-carotene treatment among 567 cases and 764 controls nested in the prospective Physicians' Health Study. We found little overall association between MnSOD polymorphism and prostate cancer risk; however, this polymorphism significantly modified risk of prostate cancer associated with prediagnostic plasma antioxidants (Pinteraction ≥ 0.05). Among men with the AA genotype, high selenium level (4th versus 1st quartile) was associated with a relative risk (RR) of 0.3 [95% confidence interval (CI), 0.2-0.7] for total prostate cancer; for clinically aggressive prostate cancer, the RR was 0.2 (95% CI, 0.1-0.5). In contrast, among men with the VV/VA genotype, the RRs were 0.6 (0.4-1.0) and 0.7 (0.4-1.2) for total and clinically aggressive prostate cancer. These patterns were similar for lycopene and α-tocopherol and were particularly strong when these antioxidants and selenium were combined; men with the AA genotype had a 10-fold gradient in risk for aggressive prostate cancer across quartiles of antioxidant status. Men with AA genotype who were randomly assigned to β-carotene treatment (versus placebo) had a RR of 0.6 (95% CI, 0.2-0.9; Pinteraction = 0.03) for fatal prostate cancer, but no significant association was observed in men with the VV/VA genotype. Both endogenous and exogenous antioxidants play an important and interdependent role in preventing clinically significant prostate cancer.
Association of CYP1A1 Polymorphisms with Differential Metabolic Activation of 17β-Estradiol and Estrone
Authors: Kisselev Pyotr , Schunck Wolf-Hagen , Roots Ivar , Schwarz Dieter
Published: September 2005

Several epidemiologic studies associate certain CYP1A1 genotypes, alone or in combination, with an increased risk of estrogen-related cancers. To answer the question of whether genotype-dependent activation of estrogens by CYP1A1 could be the underlying mechanism, we studied the hydroxylation activity of the most common allelic variants of human CYP1A1 towards both endogenously occurring estrogens, 17β-estradiol (E2) and estrone (E1). We expressed and purified CYP1A1.1 (wild-type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn) and did enzymatic assays of NADPH-dependent estrogen hydroxylation in reconstituted CYP1A1 systems. All CYP1A1 variants catalyzed the formation of 2-, 4-, 6α-, and 15α-hydroxylated estrogen metabolites from E2 and E1, yet with varying catalytic efficiency and distinct regiospecificity. Whereas the variant CYP1A1.2 (Ile462Val) had a significant higher catalytic activity for all hydroxylation sites and both substrates, it was most pronounced for 2-hydroxylation. Catalytic efficiencies for the formation of the major metabolites, 2-OH-E2 and 2-OH-E1, by CYP1A1.2 were 5.7- and 12-fold higher, respectively, compared with the wild-type enzyme. The catalytic efficiencies for hydroxylations catalyzed by CYP1A1.4 were roughly comparable with those of the wild-type enzyme. Enzyme kinetics showed that the superior activity of CYP1A1.2 (Ile462Val) is mainly caused by a higher Vmax, whereas Km values of all variants were similar. The data suggest that risk of estrogen-induced cancers and cardiovascular diseases might be—at least partially—determined by the CYP1A1 genotype.
X-Linked Dominant Growth Suppression of Transplanted Tumors in C57BL/6J-scidMice
Authors: Wood Mark , Udagawa Taturo , Hida Yasuhiro , D'Amato Robert J.
Published: September 2005

Tumor susceptibility, angiogenesis, and immune response differ between mouse strains. We, therefore, examined the growth rates of tumor xenografts in three genetically isolated strains of severe combined immunodeficient mice (C.B-17, C57BL/6J, and C3H). Tumors grew at significantly reduced rates in the C57BL/6J-scid strain. Engrafting bone marrow from the C57BL/6J-scid strain onto C.B-17-scid mice did not transfer the slow-growing tumor phenotype to the recipient mice; this counters the supposition that the slow-growing tumor phenotype is caused by a greater immune response to the xenograft in the C57BL/6J-scid strain. To establish the inheritance pattern of the slow-growing tumor phenotype, we reciprocally crossed C.B-17-scid mice and C57BL/6J-scid mice. Tumor growth was suppressed in all of the F1 progeny except the male mice derived from the cross between C.B-17-scid female and C57BL/6J-scid male mice. The F1 male mice that received the X chromosome from the C.B-17 strain displayed a fast-growing tumor phenotype. These results confirm that there are significant strain differences in capacity to support the growth of tumor xenografts. In addition, these results reveal the existence of a dominant allele involved in host suppression of tumor growth on the X chromosome of C57BL/6J mice.
Monoclonal Anti-idiotype Antibody 6G6.C4 Fused to GM-CSF Is Capable of Breaking Tolerance to Carcinoembryonic Antigen (CEA) in CEA–Transgenic Mice
Authors: Schwegler Christian , Dorn-Beineke Alexandra , Nittka Stefanie , Stocking Carol , Neumaier Michael
Published: September 2005

Internal image anti-idiotypic antibodies are capable of mimicking tumor-associated antigens and thus may serve as surrogate for vaccination strategies in cancer patients. The monoclonal antibody (mAb) 6G6.C4 mimics an epitope specific for the human carcinoembryonic antigen (CEA) and generates a CEA-specific response (Ab3) in various experimental animals. In humans, however, 6G6.C4 only yields a very limited humoral anti-CEA reaction presumably due to tolerance against the CEA autoantigen. In this study, we investigated the CEA-specific Ab3 response in mice transgenic for the human CEA and tested whether the antigen tolerance could be overcome by fusing a recombinant single-chain variable fragment of 6G6.C4 (scFv6G6.C4) to the murine granulocyte macrophage colony-stimulating factor (GM-CSF).

Like mAb 6G6.C4, the fusion protein (scFv6G6.C4/GM-CSF) retained binding to the CEA-specific idiotype mAb T84.66. Also, scFv6G6.C4/GM-CSF was biologically active as measured by proliferation of the GM-CSF-dependent murine FDC-P1 cells in vitro. After immunization with the scFv6G6.C4/GM-CSF fusion protein, CEA-transgenic animals showed significantly enhanced Ab3 antibody responses to scFv6G6.C4 (P = 0.005) and to CEA (P = 0.012) compared with the scFV6G6.C4 alone. Sera from mice immunized with the fusion protein specifically recognized CEA in Western blot analyses with no cross-reaction to CEA-related antigens. Finally, the Ab3 antisera detected single CEA-expressing tumor cells in suspension as shown by flow cytometry. Taken together, these data show in a model antigenically related to the human system that vaccination with scFv6G6.C4/GM-CSF improves vaccination against an endogenous tumor-associated antigen resulting in a highly specific humoral Ab3 response in vivo that is capable of bind single circulating CEA-positive tumor cells.
Glycogen Synthase Kinase-3β Participates in Nuclear Factor κB–Mediated Gene Transcription and Cell Survival in Pancreatic Cancer Cells
Authors: Ougolkov Andrei V. , Fernandez-Zapico Martin E. , Savoy Doris N. , Urrutia Raul A. , Billadeau Daniel D.
Published: September 2005

Recent studies using glycogen synthase kinase-3β (GSK-3β)–deficient mouse embryonic fibroblasts suggest that GSK-3β positively regulates nuclear factor κB (NFκB)–mediated gene transcription. Because NFκB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3β in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3β and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3β by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3β influences NFκB-mediated gene transcription at a point distal to the Iκ kinase complex, as only ectopic expression of the NFκB subunits p65/p50, but not an Iκ kinase β constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3β inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3β in cancer cell survival and proliferation and suggest GSK-3β as a potential therapeutic target in the treatment of pancreatic cancer.
Lyn Kinase Activity Is the Predominant Cellular Src Kinase Activity in Glioblastoma Tumor Cells
Authors: Stettner Michelle R. , Wang Wenquan , Nabors L. Burton , Bharara Suman , Flynn Daniel C. , Grammer J. Robert , Gillespie G. Yancey , Gladson Candece L.
Published: September 2005

Cellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. As we have found that Lyn, but not Fyn, activity promotes migration of glioblastoma cells in response to the cooperative signal generated by platelet-derived growth factor receptor β and integrin αvβ3, we compared the activity and expression of Lyn and Fyn in glioblastoma (grade IV) tumor biopsy samples with that in anaplastic astrocytoma (grade III) tumors, nonneoplastic brain, and normal autopsy brain samples. Lyn kinase activity was significantly elevated in glioblastoma tumor samples. Notably, the Lyn kinase activity accounted for >90% of pan-Src kinase activity in glioblastoma samples but only ≈30% of pan-Src kinase activity in the other groups. The levels of phosphorylation of the autophosphorylation site were consistent with significantly higher Lyn activity in glioblastoma tumor tissue than nonneoplastic brain. Although the normalized levels of Lyn protein and the relative levels of Lyn message were significantly higher in glioblastoma samples than nonneoplastic brain, the normalized levels of Lyn protein did not correlate with Lyn activity in the glioblastoma samples. There was no significant difference in the normalized levels of c-Src and Fyn protein and message in the glioblastoma and nonneoplastic brain. Immunostaining revealed that Lyn is located primarily in the glioblastoma cells in the tumor biopsies. These data indicate that Lyn kinase activity is significantly elevated in glioblastoma tumors and suggest that it is the Lyn activity that promotes the malignant phenotype in these tumors.
Molecular Staging of Cervical Lymph Nodes in Squamous Cell Carcinoma of the Head and Neck
Authors: Ferris Robert L. , Xi Liqiang , Raja Siva , Hunt Jennifer L. , Wang Jun , Gooding William E. , Kelly Lori , Ching Jesus , Luketich James D. , Godfrey Tony E.
Published: September 2005

Clinical staging of cervical lymph nodes from patients with squamous cell carcinoma of the head and neck (SCCHN) has only 50% accuracy compared with definitive pathologic assessment. Consequently, both clinically positive and clinically negative patients frequently undergo neck dissections that may not be necessary. To address this potential overtreatment, sentinel lymph node (SLN) biopsy is currently being evaluated to provide better staging of the neck. However, to fully realize the potential improvement in patient care afforded by the SLN procedure, a rapid and accurate SLN analysis is necessary. We used quantitative reverse transcription–PCR (QRT-PCR) to screen 40 potential markers for their ability to detect SCCHN metastases to cervical lymph nodes. Seven markers were identified with good characteristics for identifying metastatic disease, and these were validated using a set of 26 primary tumors, 19 histologically positive lymph nodes, and 21 benign nodes from patients without cancer. Four markers discriminated between positive and benign nodes with accuracy >97% but only one marker, pemphigus vulgaris antigen (PVA), discriminated with 100% accuracy in both the observed data and a statistical bootstrap analysis. A rapid QRT-PCR assay for PVA was then developed and incorporated into a prototype instrument capable of performing fully automated RNA isolation and QRT-PCR. The automated analysis with PVA provided perfect discrimination between histologically positive and benign lymph nodes and correctly identified two lymph nodes with micrometastatic tumor deposits. These assays were completed (from tissue to result) in ∼30 minutes, thus demonstrating the feasibility of intraoperative staging of SCCHN SLNs by QRT-PCR.
Selective Identification of Secreted and Transmembrane Breast Cancer Markers using Escherichia coli Ampicillin Secretion Trap
Authors: Ferguson Deborah A. , Muenster Matthew R. , Zang Qun , Spencer Jeffrey A. , Schageman Jeoffrey J. , Lian Yun , Garner Harold R. , Gaynor Richard B. , Huff J. Warren , Pertsemlidis Alexander , Ashfaq Raheela , Schorge John , Becerra Carlos , Williams Noelle S. , Graff Jonathan M.
Published: September 2005

Secreted and cell surface proteins play important roles in cancer and are potential drug targets and tumor markers. Here, we describe a large-scale analysis of the genes encoding secreted and cell surface proteins in breast cancer. To identify these genes, we developed a novel signal sequence trap method called Escherichia coli ampicillin secretion trap (CAST). For CAST, we constructed a plasmid in which the signal sequence of β-lactamase was deleted such that it does not confer ampicillin resistance. Eukaryotic cDNA libraries cloned into pCAST produced tens of thousands of ampicillin-resistant clones, 80% of which contained cDNA fragments encoding secreted and membrane spanning proteins. We identified 2,708 unique sequences from cDNA libraries made from surgical breast cancer specimens. We analyzed the expression of 1,287 of the 2,708 genes and found that 166 were overexpressed in breast cancers relative to normal breast tissues. Eighty-five percent of these genes had not been previously identified as markers of breast cancer. Twenty-three of the 166 genes (14%) were relatively tissue restricted, suggesting use as cancer-specific targets. We also identified several new markers of ovarian cancer. Our results indicate that CAST is a robust, rapid, and low cost method to identify cell surface and secreted proteins and is applicable to a variety of relevant biological questions.
Cytoplasmic HuR Expression Is a Prognostic Factor in Invasive Ductal Breast Carcinoma
Authors: Heinonen Mira , Bono Petri , Narko Kirsi , Chang Sung-Hee , Lundin Johan , Joensuu Heikki , Furneaux Henry , Hla Timothy , Haglund Caj , Ristimäki Ari
Published: September 2005

HuR is a ubiquitously expressed mRNA-binding protein. Intracellular localization of HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm. In the cytoplasm it can stabilize certain transcripts. Because nucleocytoplasmic translocation of HuR is necessary for its activity, it was hypothesized that cytoplasmic HuR expression in cancer cells could be a prognostic marker. To test the significance of HuR in carcinogenesis of the breast, we have investigated HuR expression in a mouse mammary gland tumor model and from 133 invasive ductal breast carcinoma specimens. HuR expression was elevated in the cyclooxygenase-2 transgene–induced mouse mammary tumors, and its expression was predominantly cytoplasmic in the tumor cells. In the human carcinoma samples, high cytoplasmic immunoreactivity for HuR was found in 29% (38 of 133) of the cases. Cytoplasmic HuR expression associated with high grade (P = 0.0050) and tumor size over 2 cm (P = 0.0082). Five-year distant disease-free survival rate was 42% [95% confidence interval (95% CI), 26-58] in cytoplasm-high category and 84% (95% CI, 76-91) in cytoplasm-negative or -low category (P < 0.0001), and high cytoplasmic expression of HuR was an independent prognostic factor in a Cox multivariate model (relative risk 2.07; 95% CI, 1.05-4.07). Moreover, high cytoplasmic HuR immunopositivity was significantly associated with poor outcome in the subgroup of node-negative breast cancer in a univariate analysis (P < 0.0007). Our results show that high cytoplasmic HuR expression is associated with a poor histologic differentiation, large tumor size, and poor survival in ductal breast carcinoma. Thus, HuR is the first mRNA stability protein of which expression associates with poor outcome in breast cancer.
Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells
Authors: Gastpar Robert , Gehrmann Mathias , Bausero Maria A. , Asea Alexzander , Gross Catharina , Schroeder Josef A. , Multhoff Gabriele
Published: September 2005

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX− derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.
Blockade of the Stromal Cell–Derived Factor-1/CXCR4 Axis Attenuates In vivo Tumor Growth by Inhibiting Angiogenesis in a Vascular Endothelial Growth Factor–Independent Manner
Authors: Guleng Bayasi , Tateishi Keisuke , Ohta Miki , Kanai Fumihiko , Jazag Amarsanaa , Ijichi Hideaki , Tanaka Yasuo , Washida Miwa , Morikane Keita , Fukushima Yasushi , Yamori Takao , Tsuruo Takashi , Kawabe Takao , Miyagishi Makoto , Taira Kazunari , Sata Masataka , Omata Masao
Published: September 2005

The interaction between the chemokine receptor CXCR4 and its specific ligand, stromal cell–derived factor-1 (SDF-1/CXCL12), mediates several cellular functions. In cancer, SDF-1-positive or CXCR4-positive cells of various lineages are detected within tumor tissues. Recent intensive research has indicated the possibility that blocking CXCR4 could reduce the metastatic potential of cancer cells. Here, we show that the inhibition of the SDF-1/CXCR4 axis decreases the growth of s.c. gastrointestinal tumors through the suppression of tumor neoangiogenesis. The neutralization of CXCR4 suppressed the growth in vivo of tumors derived from mouse Colon38 and PancO2 cells, whereas it did not affect the growth of Colon38 and PancO2 cells in vitro. This attenuation of tumor growth was found to be independent of the expression of CXCR4 by the cancer cells themselves, because CXCR4 knocked-down Colon38 cells grew similarly to control cells. Furthermore, CD31-positive tumor capillaries were reduced to 45% (P < 0.001) and intratumor blood flows were decreased to 65% (P < 0.01) by blockade of CXCR4. The vascular endothelial growth factor (VEGF) concentration in the tumors was not affected by the neutralization of CXCR4. Taken together with the detection of CXCR4-positive endothelial cells in the tumor tissues, the findings suggest that the antiangiogenic effects of the blockade of CXCR4 are related to a reduction of the establishment of tumor endothelium independently of VEGF inhibition. Our data indicate that the SDF-1/CXCR4 pathway might be a general target for anticancer strategies and that blocking this system could be cooperatively effective in combination with other antiangiogenic therapies, such as blockade of VEGF.
Estrogen Enhances whereas Tamoxifen Retards Development of Tsc Mouse Liver Hemangioma: A Tumor Related to Renal Angiomyolipoma and Pulmonary Lymphangioleiomyomatosis
Authors: El-Hashemite Nisreen , Walker Victoria , Kwiatkowski David J.
Published: September 2005

Pulmonary lymphangioleiomyomatosis and abdominal angiomyolipoma are related lesions for which there is no authentic animal model. Both of these proliferative lesions occur in sporadic patients, and at much higher frequency in patients with tuberous sclerosis, which is due to mutations in the TSC1 and TSC2 genes. Tsc1+/− and Tsc2+/− mice frequently develop liver hemangioma. We found that the Tsc mouse liver hemangioma are composed predominantly of endothelial cells but with a smooth muscle component, and express HMB45 antigen, estrogen receptor, and progesterone receptor, similar to lymphangioleiomyomatosis and angiomyolipoma. Estrogen treatment significantly accelerated the development of liver hemangioma in Tsc1+/− female mice, with 91% having liver hemangioma and 55% having severe lesions by 7 months of age. Similarly, an increased frequency and severity of liver hemangiomas was seen in Tsc1+/− males treated with estrogen. In contrast, tamoxifen treatment for 9 months significantly reduced the frequency and severity of hemangiomas in Tsc1+/− female mice. In addition, estrogen treatment significantly increased serum vascular endothelial growth factor levels in Tsc1+/− mice, whereas tamoxifen reduced vascular endothelial growth factor levels. These mouse model observations indicate the importance of estrogen signaling in vivo for the growth of tuberous sclerosis lesions, suggesting the possible benefits of tamoxifen therapy for the treatment of angiomyolipoma and lymphangioleiomyomatosis.
Oncogenic H-Ras Up-regulates Expression of Ku80 to Protect Cells from γ-Ray Irradiation in NIH3T3 Cells
Authors: Chang In-Youb , Youn Cha-Kyung , Kim Hong-Beum , Kim Mi-Hwa , Cho Hyun-Ju , Yoon Young , Lee Yun-Sil , Chung Myung-Hee , You Ho Jin
Published: September 2005

The Ras activation contributes to radioresistance, but the mechanism is unclear. This article shows that the expression of the dominant-positive H-Ras increased the Ku80 level, which is one of the key enzymes involved in repairing dsDNA breaks (DSB). After exposing the cells to ionizing radiation and analyzing them using an electrophoretic mobility shift assay and pulsed-field gel electrophoresis, it was found that activated H-Ras expression in NIH3T3 cells increases the DNA-binding activity of Ku80 and increases the DSB repair activity. Ku80 small interfering RNA expression was shown to reduce the oncogenic H-Ras-mediated increase in the DSBs and suppress the oncogenic H-Ras-mediated resistance of the cells to γ-ray irradiation, whereas Ku80 overexpression in the NIH3T3 cells significantly increased the radioresistance. These results suggest that the Ku80 expression induced by oncogenic H-Ras seems to play an important role in protecting cells against γ-ray irradiation.
5-Lipoxygenase, a Marker for Early Pancreatic Intraepithelial Neoplastic Lesions
Authors: Hennig Rene , Grippo Paul , Ding Xian-Zhong , Rao Sambasiva M. , Buchler Markus W. , Friess Helmut , Talamonti Mark S. , Bell Richard H. , Adrian Thomas E.
Published: September 2005

Pancreatic cancer has an abysmal prognosis because of late diagnosis. Therefore, it is important to identify risk factors if we are to be able to prevent and detect this cancer in an early, noninvasive stage. Pancreatic intraepithelial neoplasias (PanIN) are the precursor lesions which could be an ideal target for chemoprevention. This study shows up-regulation of 5-lipoxygenase (5-LOX) in all grades of human PanINs and early lesions of pancreatic cancer in two different animal models (EL-Kras mice and N-nitrosobis(2-oxopropyl)amine–treated hamsters) by immunohistochemistry. The results were consistent in all tissues examined, including seven chronic pancreatitis patients, four pancreatic cancer patients, one multiorgan donor, nine EL-Kras mice, and three N-nitrosobis(2-oxopropyl)amine–treated hamsters, all with PanINs. Overexpression of 5-LOX in NIH3T3 cells resulted in greater sensitivity of these cells to the growth inhibitory effects of the 5-LOX inhibitor Rev5901. These findings provide evidence that 5-LOX plays a key role in the development of pancreatic cancer. Furthermore, the lipoxygenase pathway may be a target for the prevention of this devastating disease.
Role of Hepatic Cytochrome P450s in the Pharmacokinetics and Toxicity of Cyclophosphamide: Studies with the Hepatic Cytochrome P450 Reductase Null Mouse
Authors: Pass Georgia J. , Carrie Dianne , Boylan Michael , Lorimore Sally , Wright Eric , Houston Brian , Henderson Colin J. , Wolf C. Roland
Published: September 2005

Cyclophosphamide (CPA) is an anticancer prodrug that is dependent on cytochrome P450 (CYP) metabolism for its therapeutic effectiveness. In spite of the use of CPA in the clinic for over 50 years, little is known about the relationship between its toxicokinetics and therapeutic response. We have employed a powerful new model, the Hepatic Cytochrome P450 Reductase Null (HRN) mouse, which has almost no hepatic cytochrome P450 activity, to study the toxicokinetics of CPA and to establish in vivo the role of hepatic P450 metabolism in its pharmacokinetics. In HRN mice the in vitro metabolism and intrinsic clearance of CPA was over 6-fold lower than in wild-type animals. This change in CPA metabolism was also reflected in vivo, with a profound difference in the pharmacokinetics of both CPA and its metabolites. At a CPA dose of 100 mg/kg, the Cmax, plasma area under the curve (AUC) and half-life were increased by 2.6-, 6.2-, and 3.2-fold, respectively, in the HRN mice. Similar changes were also observed at a dose of 300 mg/kg. These data confirm that hepatic metabolism is the major route of CPA elimination and disposition. The primary metabolites of CPA, 4-hydroxycyclophosphamide (4-OH-CPA) and 3-dechloroethylcyclophosphamide, were still formed, but at altered rates in the HRN mice. At 100 mg/kg the t1/2 for 4-OH-CPA was increased 1.8-fold, the Cmax reduced 1.7-fold, and the AUC remained unchanged. This latter finding shows that P450-mediated oxidative metabolism is essential for the clearance of this compound. Toxicokinetic analysis of CPA-induced myelosuppression and granulocytopenia showed that at high doses (≥100 mg/kg) there was no difference in myelotoxicity between the wild-type and HRN mice. However, at lower doses (≤70 mg/kg) a significant difference was observed, with little toxicity seen in HRN mice but at least a 45% reduction in the bone marrow granulocyte population in wild-type mice. Meta-analysis of the toxicity experiments showed the myelotoxicity of CPA was found to be closely correlated with the Cmax of 4-OH-CPA (r2 = 0.80, P = 0.002). As the therapeutic effectiveness of CPA has been linked to the AUC for 4-OH-CPA, the finding that 4-OH-CPA Cmax may determine its level of myelotoxicity indicates that the therapeutic index could be altered by changing the method of CPA administration. Furthermore, monitoring 4-OH-CPA Cmax may identify individuals at most risk of CPA side effects.