bluuebunny/crossref_metadata_2025 huggingface dataset:
TEM8 Interacts with the Cleaved C5 Domain of Collagen α3(VI)
Authors: Nanda Akash , Carson-Walter Eleanor B. , Seaman Steven , Barber Thomas D. , Stampfl Jason , Singh Sujay , Vogelstein Bert , Kinzler Kenneth W. , St. Croix Brad
Published: September 2005

Tumor endothelial marker (TEM)8 was uncovered as a gene expressed predominantly in tumor endothelium, and its protein product was recently identified as the receptor for anthrax toxin. Here, we demonstrate that TEM8 protein is preferentially expressed in endothelial cells of neoplastic tissue. We used the extracellular domain of TEM8 to search for ligands and identified the α3 subunit of collagen VI as an interacting partner. The TEM8-interacting region on collagen α3(VI) was mapped to its COOH-terminal C5 domain. Remarkably, collagen α3(VI) is also preferentially expressed in tumor endothelium in a pattern concordant with that of TEM8. These results suggest that the TEM8/C5 interaction may play an important biological role in tumor angiogenesis.
Induction of Androgen Receptor-Dependent Apoptosis in Prostate Cancer Cells by the Retinoblastoma Protein
Authors: Wang Xintao , Deng Haiyun , Basu Indranil , Zhu Liang
Published: September 2005

Re-expression of a tumor suppressor in tumor cells that lack it is an effective way to study its functional activities. However, because tumor cells contain multiple mutations, tumor suppressor functions that are dependent on (an)other regulators are unlikely to be identified by its re-expression alone if the other regulators are also mutated. In this study, we show that re-expression of retinoblastoma (RB) together with the androgen receptor (AR) in RB- and AR-deficient prostate cancer DU-145 cells resulted in an apoptotic activity, acting through the mitochondria damage-initiated caspase activation pathway, which was not present when RB, or the AR, was re-expressed alone. The ability of RB + AR to induce mitochondria damage was dependent on the proapoptotic proteins Bax and Bak and could be blocked by the antiapoptotic protein Bcl-xL. Coexpressed AR did not detectably change RB’s regulation of E2F and cell cycle progression in culture. On the other hand, coexpressed RB could activate the transactivation activity of the AR in an androgen-depleted media. Although androgen induced greater AR transactivation activity in this condition, it did not induce apoptosis in the absence of coexpressed RB. Analysis of mutants of RB and the AR indicated that intact pocket function of RB and the transactivation activity of the AR were required for RB + AR-induced apoptosis. These results provide direct functional data for an AR-dependent apoptosis-inducing activity of RB and highlight the importance of cell type-specific regulators in obtaining a more complete understanding of RB.
Induction of Syndecan-1 Expression in Stromal Fibroblasts Promotes Proliferation of Human Breast Cancer Cells
Authors: Maeda Takashi , Alexander Caroline M. , Friedl Andreas
Published: September 2005

Infiltrating carcinomas characteristically elicit a reactive stromal response, and accumulating evidence indicates that tumor stroma fibroblasts reciprocally promote tumor development and growth. The cell surface heparan sulfate proteoglycan, syndecan-1 (Sdc1), is thought to function as a coreceptor for growth factor and extracellular matrix interactions, and Sdc1 expression is induced in reactive stromal cells in both mice and man. Mice with a targeted mutation in Sdc1 show reduced tumor development in response to oncogene expression and altered responses to other pathological stimuli that are associated with the induction of stromal Sdc1. Here, we test the hypothesis that Sdc1 is required for the growth-promoting activities of reactive stroma. We found that when highly invasive carcinoma cells (MDA-MB-231) were placed in contact with mouse embryonic fibroblasts (MEFs) in a coculture model, Sdc1 expression was induced. Sdc1 was not induced by less invasive or normal cell lines (T47D and NMuMG). Furthermore, the growth of MDA-MB-231 cells was enhanced by 42% when cocultured with Sdc1+/+ MEFs compared with Sdc1−/− MEFs. When T47D cells were cocultured with fibroblasts that expressed transfected Sdc1, these Sdc1-positive fibroblasts stimulated growth of the breast epithelial cells by 85% compared with untransfected controls. The growth-promoting effect was completely abolished when fibroblasts were transfected with mutant Sdc1 lacking heparan sulfate attachment sites. In conclusion, we have demonstrated that a growth-promoting loop exists between breast cancer cells and their stroma that depends on the activity of glycanated Sdc1.
Estrogen Receptor β Inhibits Human Breast Cancer Cell Proliferation and Tumor Formation by Causing a G2 Cell Cycle Arrest
Authors: Paruthiyil Sreenivasan , Parmar Hema , Kerekatte Vaishali , Cunha Gerald R. , Firestone Gary L. , Leitman Dale C.
Published: September 2005

Studies indicate that estrogen receptor (ER) α mediates breast cancer-promoting effects of estrogens. The role of ERβ in breast cancer is unknown. Elucidating the role of ERβ in the pathogenesis of breast cancer is important because many human breast tumors express both ERα and ERβ. We show that adenovirus-mediated expression of ERβ changes the phenotype of ERα-positive MCF-7 cells. Estradiol increases cell proliferation and causes tumor formation of MCF-7 cells expressing only ERα. In contrast, introducing ERβ into MCF-7 cells causes an inhibition of proliferation in vitro and prevents tumor formation in a mouse xenograft model in response to estradiol. ERβ inhibits proliferation by repressing c-myc, cyclin D1, and cyclin A gene transcription, and increasing the expression of p21Cip1 and p27Kip1, which leads to a G2 cell cycle arrest. These results demonstrate that ERα and ERβ produce opposite effects in MCF-7 cells on cell proliferation and tumor formation. Natural or synthetic ERβ-selective estrogens may lack breast cancer promoting properties exhibited by estrogens in hormone replacement regimens and may be useful for chemoprevention of breast cancer.
Radiation to Stromal Fibroblasts Increases Invasiveness of Pancreatic Cancer Cells through Tumor-Stromal Interactions
Authors: Ohuchida Kenoki , Mizumoto Kazuhiro , Murakami Mitsuhiko , Qian Li-Wu , Sato Norihiro , Nagai Eishi , Matsumoto Kunio , Nakamura Toshikazu , Tanaka Masao
Published: September 2005

Radiotherapy represents a major treatment option for patients with pancreatic cancer, but recent evidence suggests that radiation can promote invasion and metastasis of cancer cells. Interactions between cancer cells and surrounding stromal cells may play an important role in aggressive tumor progression. In the present study, we investigated the invasive phenotype of pancreatic cancer cells in response to coculture with irradiated fibroblasts. Using in vitro invasion assay, we demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of pancreatic cancer cells and, surprisingly, the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. The hepatocyte growth factor (HGF) secretion from fibroblasts remained unchanged after irradiation, whereas exposure of pancreatic cancer cells to supernatant from irradiated fibroblasts resulted in increased phosphorylation of c-Met (HGF receptor) and mitogen-activated protein kinase activity, possibly or partially via increased expression of c-Met. We also demonstrated that scattering of pancreatic cancer cells was accelerated by the supernatant from irradiated fibroblasts. The enhanced invasiveness of pancreatic cancer cells induced by coculture with irradiated fibroblasts was completely blocked by NK4, a specific antagonist of HGF. These data suggest that invasive potential of certain pancreatic cancer cells is enhanced by soluble mediator(s) released from irradiated fibroblasts possibly through up-regulation of c-Met expression/phosphorylation and mitogen-activated protein kinase activity in pancreatic cancer cells. Our present findings further support the potential use of NK4 during radiotherapy for patients with pancreatic cancer.
Increased Immunogenicity of Colon Cancer Cells by Selective Depletion of Cytochromec
Authors: Schmitt Elise , Parcellier Arnaud , Ghiringhelli François , Casares Noelia , Gurbuxani Sandeep , Droin Nathalie , Hamai Ahmed , Pequignot Marie , Hammann Arlette , Moutet Monique , Fromentin Annie , Kroemer Guido , Solary Eric , Garrido Carmen
Published: September 2005

We and others have previously reported in an in vivo rat colon cancer cell model that cell death precedes and is necessary for the development of a specific antitumoral immune response. To sensitize colon cancer cells to death, we depleted cytochrome c by stable transfection with an antisense construct. Cytochrome c depletion sensitizes human and rat colon cancer cells to a nonapoptotic, nonautophagic death induced by various stimuli. This increased sensitization to a necrosis-like cell death may be related to a decrease in cellular ATP levels and an increase in reactive oxygen species production caused by cytochrome c depletion. In vivo, depletion of cytochrome c decreases the tumorigenicity of colon cancer cells in syngeneic rats without influencing their growth in immune-deficient animals. Furthermore, decreased expression of cytochrome c in tumor cells facilitates in vivo “necrotic” cell death and the induction of a specific immune response. These results delineate a novel strategy to sensitize colon cancer cells to chemotherapy and to increase their immunogenicity in immuno-competent hosts.
Herceptin-Geldanamycin Immunoconjugates
Authors: Mandler Raya , Kobayashi Hisataka , Hinson Ella R. , Brechbiel Martin W. , Waldmann Thomas A.
Published: September 2005

The efficacy of monoclonal antibodies (mAbs) as single agents in targeted cancer therapy has proven to be limited. Arming mAbs with a potent toxic drug could enhance their activity. Here we report that conjugating geldanamycin (GA) to the anti-HER2 mAb Herceptin improved the activity of Herceptin. The IC50s of the immunoconjugate H-GA were 10–200-fold lower than that of Herceptin in antiproliferative assays, depending on the cell line. The H-GA mode of action involved HER2 degradation, which was partially lactacystin sensitive and thus proteasome dependent. The linkage between GA and Herceptin remained stable in the circulation, as suggested by the pharmacokinetics of Herceptin and conjugated GA, which were almost identical and significantly different from that of free GA. Tumor uptake of Herceptin and H-GA were similar (52 ± 7 and 43 ± 7% of the initial injected dose per gram tissue, respectively; P = 0.077), indicating no apparent damage attributable to conjugation. Therapy experiments in xenograft-bearing mice consisted of weekly i.p. doses, 4 mg/kg for 4 months. H-GA showed a greater antitumor effect than Herceptin because it induced tumor regression in 69% of the recipients compared with 7% by Herceptin alone. Median survival time was 145 days as opposed to 78 days, and 31% of the recipients remained tumor free 2 months after therapy was terminated versus 0% in the Herceptin group. Enhancement of Herceptin activity could be of significant clinical value. In addition, the chemical linkage and the considerations in therapeutic regimen described here could be applied to other immunoconjugates for targeted therapy of a broad spectrum of cancers.
Transcriptional Activation of p21waf1/cip1 by Alkylphospholipids
Authors: De Siervi Adriana , Marinissen Maria , Diggs Jessica , Wang Xiao-Fan , Pages Gilles , Senderowicz Adrian
Published: September 2005

Alkylphospholipids (ALKs) are a novel class of antitumor agents with an unknown mechanism of action. The first ALK tested in the clinic, miltefosine, has been approved recently in Europe for the local treatment of patients with cutaneous metastasis. Perifosine, the only available oral ALK, is being studied currently in human cancer clinical trials. We have shown previously that perifosine induces p21waf1/cip1 in a p53-independent fashion and that induction of p21waf1/cip1 is required for the perifosine-induced cell cycle arrest because cell lines lacking p21waf1/cip1 are refractory to perifosine. In this report, we investigated the mechanism by which perifosine induces p21waf1/cip1 protein expression. We observed that perifosine induces the accumulation of p21waf1/cip1 mRNA without affecting p21waf1/cip1 mRNA stability. Using several p21waf1/cip1 promoter-driven luciferase reporter plasmids, we observed that perifosine activates the 2.4-kb full-length p21waf1/cip1 promoter as well as a p21 promoter construct lacking p53-binding sites, suggesting that perifosine activates the p21waf1/cip1 promoter independent of p53. The minimal p21 promoter region required for perifosine-induced p21 promoter activation contains four consensus Sp1-binding sites. Mutations in each particular Sp1 site block perifosine-induced p21waf1/cip1 expression. Moreover, we showed that perifosine activates the mitogen-activated protein/extracellular signal-regulated kinase pathway, and this activation promotes the phosphorylation of Sp1 in known mitogen-activated protein kinase residues (threonine 453 and 739), thereby leading to increased Sp1 binding and enhanced p21waf1/cip1 transcription. These results represent a novel mechanism by which alkylphospholipids modulate transcription, and may contribute to the discovery of new signal transduction pathways crucial for normal and neoplastic cell cycle control.
Translational Regulation of X-Linked Inhibitor of Apoptosis Protein by Interleukin-6
Authors: Yamagiwa Yoko , Marienfeld Carla , Meng Fanyin , Holcik Martin , Patel Tushar
Published: September 2005

Interleukin-6 (IL-6) is a pleiotropic cytokine with diverse biological effects. IL-6 has been implicated in autocrine signaling pathways promoting tumor progression and chemoresistance in some human tumors. However, the mechanisms by which IL-6 modulates these responses are unknown. Aberrant apoptosis has been implicated as a fundamental mechanism of chemotherapeutic resistance. Thus, we investigated whether IL-6 alters the expression of apoptosis regulatory proteins as a mechanism of drug resistance. We provide evidence that IL-6 rapidly phosphorylates the translation initiation factor eukaryotic initiation factor-4E and triggers antiapoptotic responses in cholangiocarcinoma cells. Reduction of cellular eukaryotic initiation factor-4E by RNA interference decreases IL-6-induced effects on cytotoxic drug-induced caspase activation and apoptosis. Furthermore, IL-6 increases expression of the endogenous X-linked inhibitor of apoptosis protein expression by translation at an internal ribosome entry site. Our findings that IL-6 translationally regulates X-linked inhibitor of apoptosis protein expression reveal a novel mechanism by which IL-6 mediates tumor cell survival that may be targeted therapeutically to decrease tumor progression and chemoresistance.
The Inhibitor of Cyclin-Dependent Kinase 4a/Alternative Reading Frame (
INK4a/ARF
) Locus Encoded Proteins p16INK4a and p19ARF Repress Cyclin D1 Transcription through Distinct
cis
Elements
Authors: D’Amico Mark , Wu Kongming , Fu Maofu , Rao Mahadev , Albanese Chris , Russell Robert G. , Lian Hanzhou , Bregman David , White Michael A. , Pestell Richard G.
Published: September 2005

The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF (murine p19ARF). Invariant inactivation of either the p16INK4a-cyclin D/CDK-pRb pathway and/or p53-p14ARF pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16INK4a/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1−/− mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16INK4a expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16INK4a was observed in tissues of mice mutant for the Ink4a/Arf locus. p16INK4a and p19ARF inhibited DNA synthesis in MCF7 cells. p16INK4a repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16INK4a occurred independently of the p16INK4a-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19ARF repressed cyclin D1 through a novel distal cis-element at −1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.
Selective Activation of Ceruloplasmin Promoter in Ovarian Tumors
Authors: Lee Christine M. , Lo Hui-Wen , Shao Ru-Ping , Wang Shao-Chun , Xia Weiya , Gershenson David M. , Hung Mien-Chie
Published: September 2005

Gene therapy provides a novel treatment approach to cancer patients. Ideally, expression of therapeutic genes driven by cancer-specific promoters would only target tumors resulting in minimal toxicity to normal tissues. While there is a need of more effective and tolerable treatments for ovarian cancer patients, we aimed to identify gene promoters with high activity in ovarian tumors that can be potentially used in gene therapy to drive the expression of a therapeutic gene in tumors. To identify such promoters, a literature search was performed to reveal genes that are preferentially expressed in ovarian cancer compared with normal ovarian tissue. We found that the ceruloplasmin promoter drove up to 30-fold higher luciferase expression in ovarian cancer cells compared with immortalized normal cells. Furthermore, deletion studies revealed an activator protein-1 (AP-1) site in the ceruloplasmin promoter to be critical for optimal ceruloplasmin promoter activity. Ceruloplasmin promoter activity was significantly activated by 1-O-tetradecanoyl phorbol-13-acetate, a c-jun activator, and conversely suppressed by SP600125, a c-jun inhibitor. Consistently, the ceruloplasmin AP-1 site was specifically recognized by c-jun both in vitro and in vivo. Immunohistochemical analyses of human ovarian cancer specimens showed a direct correlation (r = 0.7, P = 0.007) between expression levels of c-jun and ceruloplasmin. In nude mice carrying SKOV3.ip1 xenografts, the ceruloplasmin promoter demonstrated significantly higher activities in tumors compared with normal organs. Together, these results suggest that the ceruloplasmin promoter activity is significantly enhanced in ovarian cancer and therefore may be exploited as a promising cancer-specific promoter in developing new gene therapy strategies for ovarian cancer.
Inhibition of Rat Mammary Gland Carcinogenesis by Simultaneous Targeting of Cyclooxygenase-2 and Peroxisome Proliferator-activated Receptor γ
Authors: Badawi Alaa F. , Eldeen Mazen B. , Liu Yingying , Ross Eric A. , Badr Mostafa Z.
Published: September 2005

We examined the effect of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, and N-(9-fluorenyl-methyloxycarbonyl)-l-leucine (F-l-Leu), a peroxisome proliferator-activated receptor γ (PPARγ) agonist, separately and combined, on the development of methylnitrosourea (MNU)-induced rat mammary gland carcinogenesis. Celecoxib and F-l-Leu significantly reduced tumor incidence and multiplicity (P < 0.05). Combining both agents exerted higher (synergistic) cancer inhibition than separate treatments (P < 0.05). The effects of the test drugs on COX-2 and PPARγ expression and on the synthesis of prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) were examined in rat mammary normal (MNU-untreated), uninvolved, and tumor (MNU-treated) tissues. Celecoxib and F-l-Leu, separately, inhibited COX-2 and up-regulated PPARγ expression. These effects were paralleled by inhibition of PGE2 synthesis and up-regulation of 15d-PGJ2. Combined treatment resulted in higher alterations in COX-2 and PPARγ transcripts and PG synthesis compared with separate administrations. The effect of the test agents on Bcl2, BAX, and protein kinase Cα expression levels were examined in the rat mammary gland and the pro-(BAX:Bcl2) and anti-[PKCα*(Bcl2/BAX)] apoptotic ratios were evaluated. Each drug increased the proapoptotic ratio by 2- to 7-fold and reduced the antiapoptotic ratio by 2- to >8-fold in all tissues. Combined treatment, however, resulted in >9- to 14-fold up-regulation in the proapoptotic processes and 15- to >30-fold down-regulation in the antiapoptotic ones. Analyses were also carried out on the drug-induced modulation of cell cycle regulators and proliferation markers (cyclin-dependent kinase 1 and proliferating cell nuclear antigen). F-l-Leu and celecoxib each reduced the cyclin-dependent kinase 1 and proliferating cell nuclear antigen expression in the tumor. Higher down-regulation was attained in all tissues by combined treatment where cyclin-dependent kinase 1 and proliferating cell nuclear antigen almost retained the expression levels observed in the normal glands. In conclusion, simultaneous targeting of COX-2 and PPARγ may inhibit mammary cancer development more effectively than targeting each molecule alone. COX-2 inhibitors and PPARγ agonists coordinately mediate their anticancer effect via both COX-dependent (inhibition of COX-2, activation of PPARγ, and modulation PG synthesis) and COX-independent (induction of proapoptotic factors and inhibition of cell proliferation) pathways.
Normal Cells Control the Growth of Neighboring Transformed Cells Independent of Gap Junctional Communication and Src Activity
Authors: Alexander David B. , Ichikawa Hitoshi , Bechberger John F. , Valiunas Virginijus , Ohki Misao , Naus Christian C. G. , Kunimoto Takehiko , Tsuda Hiroyuki , Miller W. Todd , Goldberg Gary S.
Published: September 2005

The growth of many types of cancer cells can be controlled by surrounding normal cells. However, mechanisms underlying this phenomenon have not been defined. We used a layered culture system to investigate how nontransformed cells suppress the growth of neighboring transformed cells. Direct physical contact between transformed and nontransformed cells was required for growth suppression of transformed cells in this system; communication by diffusible factors was not sufficient. However, significant gap junctional communication was not required, indicating that other intercellular junctions mediated this growth regulatory response. We also report that the Src kinase activity in transformed cells was not directly inhibited by contact with nontransformed cells. Instead, nontransformed cells increased the expression of serum deprivation-response protein and the transcription factor four and a half LIM domain 1 in tumor cells. In addition, these results suggest mechanisms by which normal cells may block Wnt signaling, inhibit insulin-like growth factor activity, and promote host recognition of neighboring tumor cells.
Control of Radiosensitivity of F9 Mouse Teratocarcinoma Cells by Regulation of Histone
H2AX
Gene Expression using a Tetracycline Turn-Off System
Authors: Yoshida Kayo , Morita Takashi
Published: September 2005

The mouse histone H2AX has unique COOH-terminal serine residues that are phosphorylated in response to double-strand DNA breaks introduced by ionizing radiation. This suggests that H2AX acts to maintain genomic stability. We constructed a tetracycline (tet)-directed turn-off vector and integrated it into F9 mouse teratocarcinoma cells by homologous recombination. In homozygously recombined cells, expression of the histone H2AX gene was repressed to 0.02% of the expression observed in wild-type cells by the addition of doxycycline, an analog of tet. Sensitivity of cells with repressed H2AX expression to X-irradiation was increased 1.95×, indicating that DNA repair was impaired by repression of H2AX. When we s.c. injected tet-regulated F9 cells into the flanks of mice, tumor growth was slightly suppressed by X-irradiation in H2AX-repressed tumors, whereas without X-irradiation, tumor growth did not differ by H2AX status. Thus, H2AX might be a potential molecular target for sensitizing cancer cells to radiotherapy to minimize required irradiation doses.
Loss of Heterozygosity and Its Correlation with Expression Profiles in Subclasses of Invasive Breast Cancers
Authors: Wang Zhigang C. , Lin Ming , Wei Lee-Jen , Li Cheng , Miron Alexander , Lodeiro Gabriella , Harris Lyndsay , Ramaswamy Sridhar , Tanenbaum David M. , Meyerson Matthew , Iglehart James D. , Richardson Andrea
Published: September 2005

Gene expression array profiles identify subclasses of breast cancers with different clinical outcomes and different molecular features. The present study attempted to correlate genomic alterations (loss of heterozygosity; LOH) with subclasses of breast cancers having distinct gene expression signatures. Hierarchical clustering of expression array data from 89 invasive breast cancers identified four major expression subclasses. Thirty-four of these cases representative of the four subclasses were microdissected and allelotyped using genome-wide single nucleotide polymorphism detection arrays (Affymetrix, Inc.). LOH was determined by comparing tumor and normal single nucleotide polymorphism allelotypes. A newly developed statistical tool was used to determine the chromosomal regions of frequent LOH. We found that breast cancers were highly heterogeneous, with the proportion of LOH ranging widely from 0.3% to >60% of heterozygous markers. The most common sites of LOH were on 17p, 17q, 16q, 11q, and 14q, sites reported in previous LOH studies. Signature LOH events were discovered in certain expression subclasses. Unique regions of LOH on 5q and 4p marked a subclass of breast cancers with “basal-like” expression profiles, distinct from other subclasses. LOH on 1p and 16q occurred preferentially in a subclass of estrogen receptor-positive breast cancers. Finding unique LOH patterns in different groups of breast cancer, in part defined by expression signatures, adds confidence to newer schemes of molecular classification. Furthermore, exclusive association between biological subclasses and restricted LOH events provides rationale to search for targeted genes.
Effector Cell-Derived Lymphotoxin α and Fas Ligand, but not Perforin, Promote Tc1 and Tc2 Effector Cell-Mediated Tumor Therapy in Established Pulmonary Metastases
Authors: Dobrzanski Mark J. , Reome Joyce B. , Hollenbaugh Joseph A. , Hylind James C. , Dutton Richard W.
Published: September 2005

Cytolytic CD8+ effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8+ T cells (Tc1) secrete IFN-γ, whereas type 2 CD8+ T cells (Tc2) secrete interleukin (IL)-4 and IL-5. Although both effector cell subpopulations display Fas ligand (FasL) and tumor necrosis factor (TNF), tumor lysis is predominantly perforin dependent in vitro. Using an ovalbumin-transfected B16 lung metastasis model, we show that heightened numbers of adoptively transferred ovalbumin-specific Tc1 and Tc2 cells accumulated at the tumor site by day 2 after therapy and induced tumor regression that enhanced survival in mice with pulmonary metastases. Transfer of either TNF-α- or perforin-deficient Tc1 or Tc2 effector cells generated from specified gene-deficient mice showed no differences in therapeutic efficiency when compared with corresponding wild-type cells. In contrast, both Tc1 and Tc2 cells, derived from either FasL or TNF-α/lymphotoxin (LT) α double knockout mice, showed that therapeutic effects were dependent, in part, on effector cell-derived FasL or LTα. Six days after effector cell therapy, elevated levels of activated endogenous CD8/CD44High and CD4/CD44High T cells localized and persisted at sites of tumor growth, whereas donor cell numbers concomitantly decreased. Both Tc1 and Tc2 effector cell subpopulations induced endogenous antitumor responses that were dependent, in part, on recipient-derived IFN-γ and TNF-α. However, neither effector cell-mediated therapy was dependent on recipient-derived perforin, IL-4, IL-5, or nitric oxide. Collectively, tumor antigen-specific Tc1 and Tc2 effector cell-mediated therapy is initially dependent, in part, on effector cell-derived FasL or LTα that may subsequently potentiate endogenous recipient-derived type 1 antitumor responses dependent on TNF-α and IFN-γ.
Effects of Dexamethasone or Celecoxib on Biliary Toxicity after Hepatic Arterial Infusion of 5-Fluorodeoxyuridine in a Canine Model
Authors: Ensminger William , Knol James , DeRemer Susan , Wilkinson Erby , Walker Suzette , Williams David , Maybaum Jonathan
Published: September 2005

Previous work has shown that in humans the dose-limiting toxicity for fluorodeoxyuridine [2-fluoro-5′-deoxyuridine (FdUrd)] when administered by hepatic arterial infusion is biliary sclerosis. The current study was undertaken to attempt to modify this toxicity in a canine model that has been demonstrated to closely mimic the clinical situation. Unlike previous studies using this model, in which animals were sacrificed after extensive fibrosis had already occurred, the current experiments were designed so that observations of pathology were made at an earlier time, when the initial inflammatory injury underlying the fibrotic process was still taking place. Implantable pumps were used to deliver FdUrd into the hepatic artery of animals at a rate of 0.3 mg/kg/day in the presence or absence of 10 mg/week dexamethasone or 100 mg/day of celecoxib for 35 days, at which time the animals were beginning to show signs of toxicity. After evaluation for radiological evidence of biliary obstruction, the animals were sacrificed and portions of their livers were processed for examination of microscopic pathology and 2-bromo-5′deoxyuridine labeling index. Dexamethasone treatment protected the animals from biliary sclerosis determined radiologically, further validating this model as being representative of the response in humans. Similarly the Cox-2 inhibitor, celecoxib, appeared to provide protection against radiological changes of biliary stricture, although possibly to a lesser degree than the resultant from dexamethasone. In addition, FdUrd treatment caused elevation of the DNA 2-bromo-5′deoxyuridine labeling index above control levels in biliary epithelial cells. Dexamethasone and celecoxib each significantly attenuated the FdUrd-induced elevation of DNA labeling index in biliary epithelium. These findings demonstrate the usefulness of this canine model for studying the mechanisms of drug-induced biliary sclerosis and reinforce the hypothesis that blocking inflammation may retard the progression of injury that eventually leads to fibrosis. This study suggests that clinical testing of celecoxib as a preventive for hepatic arterial-FdUrd induced biliary damage could prove valuable.
RTVP-1, a Tumor Suppressor Inactivated by Methylation in Prostate Cancer
Authors: Ren Chengzhen , Li Likun , Yang Guang , Timme Terry L. , Goltsov Alexei , Ren Chenghui , Ji Xiaorong , Addai Josephine , Luo Hongbin , Ittmann Michael M. , Thompson Timothy C.
Published: September 2005

We previously identified and characterized a novel p53-regulated gene in mouse prostate cancer cells that was homologous to a human gene that had been identified in brain cancers and termed RTVP-1 or GLIPR. In this report, we document that the human RTVP-1 gene is also regulated by p53 and induces apoptosis in human prostate cancer cell lines. We show that the expression of the human RTVP-1 gene is down-regulated in human prostate cancer specimens compared with normal human prostate tissue at the mRNA and protein levels. We further document epigenetic changes consistent with RTVP-1 being a tumor suppressor in human prostate cancer.
Selective Usage of D-Type Cyclins by Ewing’s Tumors and Rhabdomyosarcomas
Authors: Zhang Jingsong , Hu Siwen , Schofield Deborah E. , Sorensen Poul H. B. , Triche Timothy J.
Published: September 2005

The genetic mechanisms that control proliferation of childhood musculoskeletal malignancies, notably Ewing’s tumor (ET) and rhabdomyosarcoma (RMS), remain largely unknown. Most human cancers appear to overexpress at least one of the G1 cyclins (cyclins D1, D2, D3, E1, and E2) to bypass normal regulation of cell cycle G1 progression. We compared the gene expression profiles of 7 ET and 13 RMS primary tumor samples and found overexpression of cyclin D1 in all 7 ET samples. In contrast, RMS samples expressed higher levels of cyclin D2, cyclin D3, and cyclin E1. This was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot. The relative roles of RAS-extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3′-kinase (PI3K)-AKT pathways in the regulation of D-type cyclin expression in these tumors were then assessed. Inhibition of either pathway reduced expression of cyclins D1, D2, and D3 in RMS lines, whereas only PI3K inhibitors blocked cyclin D1, D2, and D3 expression in ET lines. Furthermore, PI3K-AKT appeared to regulate D-type cyclin transcription in RMS lines through FKHR and FKHRL1. Finally, the role of the ET-associated EWS-FLI1 fusion gene in regulating D cyclin expression was studied. Inhibition of EWS-FLI1 expression in the TC71 ET line decreased cyclin D1 levels but increased cyclin D3 levels. In contrast, induction of EWS-FLI1 expression in the RD RMS cell line increased cyclin D1 expression but decreased cyclin D3 expression. Our results demonstrate distinct regulation of D-type cyclins in ET and RMS and indicate that EWS-FLI1 can modulate the expression of D-type cyclins independent of cellular backgrounds.
Inhibition of Metastasis of Tumor Cells Overexpressing Thymidine Phosphorylase by 2-Deoxy-l-Ribose
Authors: Nakajima Yuichi , Gotanda Takenari , Uchimiya Hiroshi , Furukawa Tatsuhiko , Haraguchi Misako , Ikeda Ryuji , Sumizawa Tomoyuki , Yoshida Hiroki , Akiyama Shin-ichi
Published: September 2005

Thymidine phosphorylase (TP) catalyzes the reversible conversion of thymidine to thymine, thereby generating 2-deoxy-d-ribose-1-phosphate, which upon dephosphorylation forms 2-deoxy-d-ribose (d-dRib), a degradation product of thymidine. We have previously shown that d-dRib promotes angiogenesis and chemotaxis of endothelial cells and also confers resistance to hypoxia-induced apoptosis in some cancer cell lines. 2-Deoxy-l-ribose (l-dRib), a stereoisomer of d-dRib, can inhibit d-dRib anti-apoptotic effects and suppressed the growth of KB cells overexpressing TP (KB/TP cells) transplanted into nude mice. In this study, we examined the ability of l-dRib to suppress metastasis of KB/TP cells using two different models of metastasis. The antimetastatic effect of l-dRib was first investigated in a liver-metastasis model in nude mice inoculated with KB/TP cells. Oral administration of l-dRib for 28 days at a dose of 20 mg/kg/day significantly reduced the number of metastatic nodules in the liver and suppressed angiogenesis and enhanced apoptosis in KB/TP metastatic nodules. Next, we compared the ability of l-dRib and tegafur alone or in combination to decrease the number of metastatic nodules in organs in the abdominal cavity in nude mice receiving s.c. of KB/TP cells into their backs. l-dRib (20 mg/kg/day) was significantly (P < 0.05) more efficient than tegafur (100 mg/kg/day) in decreasing the number of metastatic nodules in organs in the abdominal cavity. By in vitro invasion assay, l-dRib also reduced the number of invading KB/TP cells. l-dRib anti-invasive activity may be mediated by its ability to suppress the enhancing effect of TP and d-dRib on both mRNA and protein expression of vascular endothelial growth factor and interleukin-8 in cultured KB cells. These findings suggest that l-dRib may be useful in a clinical setting for the suppression of metastasis of tumor cells expressing TP.
Moderate Hypermutability of a Transgenic
lacZ
Reporter Gene in
Myc
-Dependent Inflammation-Induced Plasma Cell Tumors in Mice
Authors: Felix Klaus , Polack Axel , Pretsch Walter , Jackson Sharon H. , Feigenbaum Lionel , Bornkamm Georg-Wilhelm , Janz Siegfried
Published: September 2005

Mutator phenotypes, a common and largely unexplained attribute of human cancer, might be better understood in mouse tumors containing reporter genes for accurate mutation enumeration and analysis. Previous work on peritoneal plasmacytomas (PCTs) in mice suggested that PCTs have a mutator phenotype caused by Myc-deregulating chromosomal translocations and/or phagocyte-induced mutagenesis due to chronic inflammation. To investigate this hypothesis, we generated PCTs that harbored the transgenic shuttle vector, pUR288, with a lacZ reporter gene for the assessment of mutations in vivo. PCTs exhibited a 5.5 times higher mutant frequency in lacZ (40.3 ± 5.1 × 10−5) than in normal B cells (7.36 ± 0.77 × 10−5), demonstrating that the tumors exhibit the phenotype of increased mutability. Studies on lacZ mutant frequency in serially transplanted PCTs and phagocyte-induced lacZ mutations in B cells in vitro indicated that mutant levels in tumors are not determined by exogenous damage inflicted by inflammatory cells. In vitro studies with a newly developed transgenic model of inducible Myc expression (Tet-off/MYC) showed that deregulated Myc sensitizes B cells to chemically induced mutations, but does not cause, on its own, mutations in lacZ. These findings suggested that the hypermutability of PCT is governed mainly by intrinsic features of tumor cells, not by deregulated Myc or chronic inflammation.
Proceedings of the Oxygen Homeostasis/Hypoxia Meeting
Authors: Kaufman Bennett , Scharf Orit , Arbeit Jeffrey , Ashcroft Margaret , Brown J. Martin , Bruick Richard K. , Chapman J. Donald , Evans Sydney M. , Giaccia Amato J. , Harris Adrian L. , Huang Eric , Johnson Randall , Kaelin William , Koch Cameron J. , Maxwell Patrick , Mitchell James , Neckers Len , Powis Garth , Rajendran Joseph , Semenza Gregg L. , Simons Jonathan , Storkebaum Erik , Welch Michael J. , Whitelaw Murray , Melillo Giovanni , Ivy S. Percy
Published: September 2005

The first Oxygen Homeostasis/Hypoxia Meeting was held on February 12, 2003, at the Sheraton National Hotel, Washington, D.C. The meeting was hosted by Drs. S. Percy Ivy and Giovanni Melillo of the National Cancer Institute, NIH. The purpose of the meeting was to stimulate collaborations among the participants who are engaged in different areas of hypoxia research and application, including basic research on hypoxia, and its induction and consequences; the development of drugs targeting hypoxia and factors involved in pathways leading to (or controlled by) hypoxia; and the development and application of hypoxia imaging techniques and reagents.
HOXB13 Homeodomain Protein Suppresses the Growth of Prostate Cancer Cells by the Negative Regulation of T-Cell Factor 4
Authors: Jung Chaeyong , Kim Ran-Sook , Lee Sang-Jin , Wang Chihuei , Jeng Meei-Huey
Published: September 2005

In prostate gland, HOXB13 is highly expressed from the embryonic stages to adulthood. However, the function of HOXB13 in normal cell growth and tumorigenesis is not yet known. We investigated the role of HOXB13 and mechanism by which it functions in HOXB13-negative cells. Expression of HOXB13 was forced in HOXB13-negative PC3 prostate cancer cells using a liposome-mediated gene transfer approach. Compared with the control clones, HOXB13-expressing PC3 cells exhibited significant inhibition of in vitro and in vivo cell growth with G1 cell cycle arrest mediated by the suppression of cyclin D1 expression. Because cyclin D1 is mainly regulated by β-catenin/T-cell factor (TCF), TCF-4 response element was used in a reporter gene transcription assay, demonstrating that HOXB13 significantly inhibits TCF-4-mediated transcriptional activity in both prostate and nonprostate cells. This inhibition occurred in a dose-responsive manner and was specific to TCF-4 response element. Western blot analysis demonstrated that HOXB13 down-regulates the expression of TCF-4 and its responsive genes, c-myc and cyclin D1. HOXB13 also suppressed the activity of natural c-myc promoter. This study suggests that HOXB13, a transcription factor, functions as a cell growth suppressor by negatively regulating the expression of TCF-4, which eventually provides negative signals for cell proliferation. This observation will provide valuable insight into the molecular basis of prostate tumorigenesis.
Identification of the E1A-Regulated Transcription Factor p120E4F as an Interacting Partner of the RASSF1A Candidate Tumor Suppressor Gene
Authors: Fenton Sarah L. , Dallol Ashraf , Agathanggelou Angelo , Hesson Luke , Ahmed-Choudhury Jalal , Baksh Shairaz , Sardet Claude , Dammann Reinhard , Minna John D. , Downward Julian , Maher Eamonn R. , Latif Farida
Published: September 2005

Epigenetic inactivation of the candidate tumor suppressor gene RASSF1A is a frequent and critical event in the pathogenesis of many human cancers. The RASSF1A protein contains a Ras association domain, suggesting a role in Ras-like signaling pathways, and has also been implicated in cell cycle progression. However, the preliminary data suggests that the RASSF1A gene product is likely to have multiple functions. To identify novel RASSF1A functions, we have sought to identify interacting proteins by yeast two-hybrid analysis in a human brain cDNA library. We identified the E1A-regulated transcription factor p120E4F as a RASSF1A interacting partner in yeast and mammalian cells, and demonstrated that RASSF1A protein and p120E4F form a complex in vivo. The interaction between RASSF1A and p120E4F was confirmed by both in vitro and in vivo pull downs and coimmunoprecipitation assays. In addition, specific inactivation of RASSF1A by short interfering RNA disrupts binding of RASSF1A to p120E4F in coimmunoprecipitation assays. In addition, we demonstrated enhanced G1 cell cycle arrest and S phase inhibition by propidium iodide staining of p120E4F in the presence of RASSF1A. As p120E4F has been reported previously to interact with p14ARF, retinoblastoma, and p53, these findings provide an important link between the function of RASSF1A and other major human tumor suppressor genes.
Cotreatment with Histone Deacetylase Inhibitor LAQ824 Enhances Apo-2L/Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Induced Death Inducing Signaling Complex Activity and Apoptosis of Human Acute Leukemia Cells
Authors: Guo Fei , Sigua Celia , Tao Jianguo , Bali Purva , George Prince , Li Yunqing , Wittmann Sylvie , Moscinski Lynn , Atadja Peter , Bhalla Kapil
Published: September 2005

Present studies demonstrate that treatment with the histone deacetylases inhibitor LAQ824, a cinnamic acid hydroxamate, increased the acetylation of histones H3 and H4, as well as induced p21WAF1 in the human T-cell acute leukemia Jurkat, B lymphoblast SKW 6.4, and acute myelogenous leukemia HL-60 cells. This was associated with increased accumulation of the cells in the G1 phase of the cell cycle, as well as accompanied by the processing and activity of caspase-9 and -3, and apoptosis. Exposure to LAQ824 increased the mRNA and protein expressions of the death receptors DR5 and/or DR4, but reduced the mRNA and protein levels of cellular FLICE-inhibitory protein (c-FLIP). As compared with treatment with Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or LAQ824 alone, pretreatment with LAQ824 increased the assembly of Fas-associated death domain and caspase-8, but not of c-FLIP, into the Apo-2L/TRAIL-induced death-inducing signaling complex. This increased the processing of caspase-8 and Bcl-2 interacting domain (BID), augmented cytosolic accumulation of the prodeath molecules cytochrome-c, Smac and Omi, as well as led to increased activity of caspase-3 and apoptosis. Treatment with LAQ824 also down-regulated the levels of Bcl-2, Bcl-xL, XIAP, and survivin. Partial inhibition of apoptosis due to LAQ824 or Apo-2L/TRAIL exerted by Bcl-2 overexpression was reversed by cotreatment with LAQ824 and Apo-2L/TRAIL. Significantly, cotreatment with LAQ824 increased Apo-2L/TRAIL-induced apoptosis of primary acute myelogenous leukemia blast samples isolated from 10 patients with acute myelogenous leukemia. Taken together, these findings indicate that LAQ824 may have promising activity in augmenting Apo-2L/TRAIL-induced death-inducing signaling complex and apoptosis of human acute leukemia cells.
Activation of Tumor-specific CD4+ T Lymphocytes by Major Histocompatibility Complex Class II Tumor Cell Vaccines
Authors: Dissanayake Samudra K. , Thompson James A. , Bosch Jacobus J. , Clements Virginia K. , Chen Peter W. , Ksander Bruce R. , Ostrand-Rosenberg Suzanne
Published: September 2005

Mouse tumor cells transfected with syngeneic MHC class II and costimulatory molecule genes are therapeutic vaccines in mice, provided they do not coexpress the class II-associated invariant chain (Ii). We demonstrated previously that the vaccine cells present tumor peptides via the endogenous antigen presentation pathway to activate CD4+ and CD8+ T cells. Because of their efficacy in mice, we are translating this vaccine strategy for clinical use. To obtain MHC class II+CD80+Ii− human tumor cells, we developed retroviruses encoding HLA-DR and CD80. The HLA-DR virus encodes the DRα and DRβ0101 chains using an internal ribosomal entry site to coordinate expression. SUM159PT mammary carcinoma and Mel 202 ocular melanoma cells transduced with the retroviruses DRB1/CD80 express high levels of DRB0101 and CD80 on the cell surface in the absence of Ii. Irradiated SUM159PT/DR1/CD80 vaccines stimulate proliferation of non-HLA-DRB0101 peripheral blood mononuclear cells and present an exogenous DR1-restricted tetanus toxoid (TT) peptide, indicating that the transduced DRB0101 is functional. SUM159PT/DR1/CD80 vaccines were further transduced with a retrovirus encoding the TT fragment C gene, as a model tumor antigen. These cells stimulate IFN-γ release from TT-primed human DRB0101 peripheral blood mononuclear cells, demonstrating their ability to present “endogenous” tumor antigen. Depletion and antibody blocking experiments confirm that MHC class II-restricted, endogenously synthesized epitopes are presented to CD4+ T cells. Therefore, the MHC class II vaccines are efficient antigen-presenting cells that activate tumor-specific MHC class II-restricted, CD4+ T lymphocytes, and they are a novel and potential immunotherapeutic for metastatic cancers.
Inhibition of
MUC4
Expression Suppresses Pancreatic Tumor Cell Growth and Metastasis
Authors: Singh Ajay P. , Moniaux Nicolas , Chauhan Subhash C. , Meza Jane L. , Batra Surinder K.
Published: September 2005

The MUC4 mucin is a high molecular weight membrane-bound glycoprotein. It is aberrantly expressed in pancreatic tumors and tumor cell lines with no detectable expression in the normal pancreas. A progressive increase of MUC4 expression has also been observed in pancreatic intraepithelial neoplasia, suggesting its association with disease development. Here, we investigated the consequences of silencing MUC4 expression in an aggressive and highly metastatic pancreatic tumor cell line CD18/HPAF that expresses high levels of MUC4. The expression of MUC4 was down-regulated by the stable integration of a plasmid-construct expressing antisense-MUC4 RNA. A decrease in MUC4 expression, confirmed by Western blot and immunofluorescence analyses, resulted in diminished growth and clonogenic ability of antisense-MUC4-transfected (EIAS19) cells compared with parental, empty vector (ZEO) and sense transfected (ES6) control cells. In addition, EIAS19 cells displayed a significant decrease in tumor growth and metastatic properties when transplanted orthotopically into the immunodeficient mice. In vitro biological assays for motility, adhesion, and aggregation demonstrated a 3-fold decrease in motility of EIAS19 cells compared with control cells, whereas these cells adhered more and showed an increase in cellular aggregation. Interestingly, MUC4 down-regulation also correlated with the reduced expression of its putative interacting partner, HER2/neu, in antisense-MUC4-transfected cells. In conclusion, the present work demonstrates, for the first time, a direct association of the MUC4 mucin with the metastatic pancreatic cancer phenotype and provides experimental evidence for a functional role of MUC4 in altered growth and behavioral properties of the tumor cell.