bluuebunny/crossref_metadata_2025 huggingface dataset:
A Mouse Model of Human Breast Cancer Metastasis to Human Bone
Authors: Kuperwasser Charlotte , Dessain Scott , Bierbaum Benjamin E. , Garnet Dan , Sperandio Kara , Gauvin Gregory P. , Naber Stephen P. , Weinberg Robert A. , Rosenblatt Michael
Published: September 2005

Currently, an in vivo model of human breast cancer metastasizing from the orthotopic site to bone does not exist, making it difficult to study the many steps of skeletal metastasis. Moreover, models used to identify the mechanisms by which breast cancer metastasizes to bone are limited to intracardiac injection, which seeds the cancer cells directly into the circulation, thus bypassing the early steps in the metastatic process. Such models do not reflect the full process of metastasis occurring in patients. We have developed an animal model of breast cancer metastasis in which the breast cancer cells and the bone target of osteotropic metastasis are both of human origin. The engrafted human bone is functional, based on finding human IgG in the mouse bloodstream, human B cells in the mouse spleen, and normal bone histology. Furthermore, orthotopic injection of a specific human breast cancer cell line, SUM1315 (derived from a metastatic nodule in a patient), later resulted in both bone and lung metastases. In the case of bone, metastasis was to the human implant and not the mouse skeleton, indicating a species-specific osteotropism. This model replicates the events observed in patients with breast cancer skeletal metastases and serves as a useful and relevant model for studying the disease.
A Gene Expression Signature for Relapse of Primary Wilms Tumors
Authors: Li Wenliang , Kessler Patricia , Yeger Herman , Alami Jennifer , Reeve Anthony E. , Heathcott Rosemary , Skeen Jane , Williams Bryan R.G.
Published: September 2005

Anaplastic histology and metastasis are each associated with higher relapse and mortality rates in Wilms tumor patients. However, not all anaplastic tumors relapse and some nonanaplastic tumors relapse unexpectedly. To identify more accurate early prognostic indicators, we analyzed expression of 4,900 cancer-related genes in 26 primary Wilms tumors. This analysis revealed that expression of a set of four genes predicts future relapse of primary Wilms tumors with high accuracy, independent of anaplasia. Random permutation testing of this prognostic gene expression signature yielded P = 0.003. Real-time reverse transcription-PCR analysis of the four genes in an independent primary tumor set resulted in correct prediction of future relapse with an accuracy of 92%. One of the four genes in the prognostic signature, CCAAT/enhancer binding protein β (C/EBPB), is expressed at higher levels in both primary relapsing tumors and metastatic tumors than in primary nonrelapsing tumors. Short interfering RNA–mediated down-regulation of C/EBPB expression in WiT49, a cell line derived from a metastatic Wilms tumor, resulted in spontaneous apoptosis. These findings suggest that C/EBPB is a critical survival factor for Wilms tumor cells and that its expression contributes to the prognosis of Wilms tumor patients.
Lack of Telopeptides in Fibrillar Collagen I Promotes the Invasion of a Metastatic Breast Tumor Cell Line
Authors: Demou Zoe N. , Awad Michael , McKee Trevor , Perentes Jean Yannis , Wang Xiaoye , Munn Lance L. , Jain Rakesh K. , Boucher Yves
Published: September 2005

Defective fibrillar collagen polymerization in primary tumors has been correlated with increased metastasis. However, it is unclear how collagen organization influences tumor invasion. In this study, we show that collagen I polymerized without telopeptides (the flanking regions of collagen molecules) can differentially affect the three-dimensional migration of mammary carcinoma cells. MDA-MB-231 cells capable of proteolytic degradation and mesenchymal motion, invaded telopeptide-intact and telopeptide-free collagen gels to the same extent. In contrast, MDA-MB-435S cells, with typical features of amoeboid cells (poor collagenolytic activity, rounded cell morphology), were 5-fold more invasive in telopeptide-free than telopeptide-intact collagen. A fraction of the MDA-MB-435S cells that invaded telopeptide-intact or telopeptide-free collagen had a rounded morphology; however, in telopeptide-free collagen, a significant fraction of the cells switched from a rounded to elongated morphology (protrusion formation). The dynamic changes in cellular shape facilitated MDA-MB-435S locomotion through the narrow interfiber gaps, which were smaller than cell diameters. Based on the spherical morphology of MDA-MB-435S cells, we tested if the changes in cell shape and invasion were related to RhoA-ROCK activity; GTP-bound RhoA was measured in pull-down assays. RhoA activity was 1.8-fold higher for MDA-MB-435S cells seeded on telopeptide-free than telopeptide-intact collagen. Y27632 inhibition of ROCK, a Rho effector, significantly reduced the changes in cellular morphodynamics and the invasion of MDA-MB-435S cells but did not alter the invasion of MDA-MB-231 cells. Thus, the higher RhoA activity of MDA-MB-435S cells in telopeptide-free collagen enhances the changes in cellular morphodynamics associated with motility and invasion.
A 27-Amino-Acid Synthetic Peptide Corresponding to the NH2-Terminal Zinc-Binding Domain of Endostatin Is Responsible for Its Antitumor Activity
Authors: Tjin Tham Sjin Robert M. , Satchi-Fainaro Ronit , Birsner Amy E. , Ramanujam V.M. Sadagopa , Folkman Judah , Javaherian Kashi
Published: September 2005

The first recombinant endostatin that elicited strong antitumor activity was expressed in Escherichia coli and administered as a suspension. Under these conditions, the protein retained its full antiangiogenic activity. Lack of requirement for a folded structure prompted us to investigate antitumor properties of synthetic peptides corresponding to different regions of endostatin. Here, we show that the entire antitumor, antimigration, and antipermeability activities of endostatin are mimicked by a 27-amino-acid peptide corresponding to the NH2-terminal domain of endostatin. This peptide contains three histidines that are responsible for zinc binding. Mutations of the zinc-binding histidines abolished its antitumor and antimigration activities, but not antipermeability properties.
Macrophages Promote the Invasion of Breast Carcinoma Cells via a Colony-Stimulating Factor-1/Epidermal Growth Factor Paracrine Loop
Authors: Goswami Sumanta , Sahai Erik , Wyckoff Jeffrey B. , Cammer Michael , Cox Dianne , Pixley Fiona J. , Stanley E. Richard , Segall Jeffrey E. , Condeelis John S.
Published: September 2005

Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells. Colony stimulating factor 1 (CSF-1) produced by carcinoma cells promotes the expression of EGF by macrophages. In addition, EGF promotes the expression of CSF-1 by carcinoma cells thereby generating a positive feedback loop. Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion.
Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Alters Mitochondrial Membrane Lipids
Authors: Sandra Ferry , Degli Esposti Mauro , Ndebele Kenneth , Gona Philimon , Knight David , Rosenquist Magnus , Khosravi-Far Roya
Published: September 2005

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been shown to have selective antitumor activity. TRAIL induces ubiquitous pathways of cell death in which caspase activation is mediated either directly or via the release of apoptogenic factors from mitochondria; however, the precise components of the mitochondrial signaling pathway have not been well defined. Notably, mitochondria constitute an important target in overcoming resistance to TRAIL in many types of tumors. Bid is considered to be fundamental in engaging mitochondria during death receptor–mediated apoptosis, but this action is dependent on mitochondrial lipids. Here, we report that TRAIL signaling induces an alteration in mitochondrial membrane lipids, particularly cardiolipin. This occurs independently of caspase activation and primes mitochondrial membranes to the proapoptotic action of Bid. We unveil a link between TRAIL signaling and alteration of membrane lipid homeostasis that occurs in parallel to apical caspase activation but does not take over the mode of cell death because of the concurrent activation of caspase-8. In particular, TRAIL-induced alteration of mitochondrial lipids follows an imbalance in the cellular homeostasis of phosphatidylcholine, which results in an elevation in diacylglycerol (DAG). Elevated DAG in turn activates the δ isoform of phospholipid-dependent serine/threonine protein kinase C, which then accelerates the cleavage of caspase-8. We also show that preservation of phosphatidylcholine homeostasis by inhibition of lipid-degrading enzymes almost completely impedes the activation of pro-caspase-9 while scarcely changing the activation of caspase-8.
Bcl-2 Overexpression Enhances Tumor-Specific T-Cell Survival
Authors: Charo Jehad , Finkelstein Steven E. , Grewal Navrose , Restifo Nicholas P. , Robbins Paul F. , Rosenberg Steven A.
Published: September 2005

Although immunotherapy based on the adoptive transfer of tumor-specific T lymphocytes has been shown to result in dramatic clinical responses in some patients, the relatively low levels of engraftment and persistence of the adoptively transferred cells may limit these responses in many patients. In an attempt to develop strategies for prolonging the survival of adoptively transferred T cells, we have carried out studies in which T cells obtained from healthy donors as well as tumor-specific T cells were transduced with a retrovirus expressing the human Bcl-2 gene. Our results indicate that these transduced T cells overexpress Bcl-2, are resistant to death, and have a survival advantage following interleukin-2 withdrawal compared with control T cells transduced with a retrovirus expressing green fluorescent protein. Tumor-specific T cells overexpressing Bcl-2 maintained their ability to specifically recognize and respond to target cells. Furthermore, we show that adoptive immunotherapy of an established B16 tumor can be significantly enhanced by overexpressing Bcl-2 in melanoma-specific T-cell receptor transgenic T cells. Our data suggest that adoptive immunotherapy approaches to the treatment of cancer patients may be enhanced using Bcl-2-modified tumor-reactive T cells.
Mitotic Infidelity and Centrosome Duplication Errors in Cells Overexpressing Tripeptidyl-Peptidase II
Authors: Stavropoulou Vaia , Xie Jianjun , Henriksson Marie , Tomkinson Birgitta , Imreh Stefan , Masucci Maria G.
Published: September 2005

The oligopeptidase tripeptidyl-peptidase II (TPP II) is up-regulated Burkitt's lymphoma (BL) cells that overexpress the c-myc proto-oncogene and is required for their growth and survival. Here we show that overexpression of TPP II induces accelerated growth and resistance to apoptosis in human embryonic kidney 293 cells. This correlates with the appearance of multiple chromosomal aberrations, numerical and structural centrosome abnormalities, and multipolar cell divisions. Similar mitotic aberrations were also observed in a panel of BL lines and were suppressed, in parallel with TPP II down-regulation, upon reversion of BL-like characteristics in EBV-immortalized B lymphocytes carrying a tetracycline-regulated c-myc. Functional TPP II knockdown by small interfering RNA expression in BL cells caused the appearance of giant polynucleated cells that failed to complete cell division. Collectively, these data point to a role of TPP II in the regulation of centrosome homeostasis and mitotic fidelity suggesting that this enzyme may be a critical player in the induction and/or maintenance of genetic instability in malignant cells.
Up-regulation of Inhibitory Natural Killer Receptors CD94/NKG2A with Suppressed Intracellular Perforin Expression of Tumor-Infiltrating CD8+ T Lymphocytes in Human Cervical Carcinoma
Authors: Sheu Bor-Ching , Chiou Shin-Heng , Lin Ho-Hsiung , Chow Song-Nan , Huang Su-Cheng , Ho Hong-Nerng , Hsu Su-Ming
Published: September 2005

Inhibitory signals that govern the cytolytic functions of CD8+ T lymphocytes have been linked to the expression of natural killer cell receptors (NKRs) on CTLs. There is limited knowledge about the induction of inhibitory NKR (iNKR) expression in vivo. Up-regulation of iNKRs has been linked to the modulation of the virus- and/or tumor-specific immune responses in animal models. In the present study, we directly examined the expression of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human cervical cancer. We found that in human cervical cancer, the percentage expression of immunoglobulin-like NKR+CD8+ T lymphocytes were similar in gated CD8+-autologous TILs and peripheral blood mononuclear cells. On the contrary, cervical cancer–infiltrating CD8+ T lymphocytes expressed up-regulated C-type lectin NKRs CD94/NKG2A compared with either peripheral blood CD8+ T cells or normal cervix-infiltrating CD8+ T lymphocytes. Dual NKR coexpression analyses showed that CD94 and NKG2A were mainly expressed on CD56−CD161−CD8+ TILs within the cancer milieu. Immunohistochemical study showed that cervical cancer cells expressed abundant interleukin 15 (IL-15) and transforming growth factor-β (TGF-β). In kinetic coculture assay, cervical cancer cells can promote the expression of CD94/NKG2A on CD8+ T lymphocytes. The cancer-derived effects can be reversed by addition of rIL-15Rα/Fc and anti–TGF-β antibody. Functional analyses illustrated that intracellular perforin expression of CD8+ T cells was minimal upon up-regulation of CD94/NKG2A. Kinetic cytotoxicity assays showed that up-regulated expressions of CD94/NKG2A restrain CD8+ T lymphocyte cytotoxicity. Our study strongly indicated that cervical cancer cells could promote the expression of iNKRs via an IL-15– and possibly TGF-β–mediated mechanism and abrogate the antitumor cytotoxicity of TILs.
Chemoprevention of UV Light-Induced Skin Tumorigenesis by Inhibition of the Epidermal Growth Factor Receptor
Authors: El-Abaseri Taghrid B. , Fuhrman Jill , Trempus Carol , Shendrik Igor , Tennant Raymond W. , Hansen Laura A.
Published: September 2005

The epidermal growth factor receptor (EGFR) is activated in skin cells following UV irradiation, the primary cause of nonmelanoma skin cancer. The EGFR inhibitor AG1478 prevented the UV-induced activation of EGFR and of downstream signaling pathways through c-Jun NH2-terminal kinases, extracellular signal-regulated kinases, p38 kinase, and phosphatidylinositol 3-kinase in the skin. The extent to which the UV-induced activation of EGFR influences skin tumorigenesis was determined in genetically initiated v-rasHa transgenic Tg.AC mice, which have enhanced susceptibility to skin carcinogenesis. Topical treatment or i.p. injection of AG1478 before UV exposure blocked the UV-induced activation of EGFR in the skin and decreased skin tumorigenesis in Tg.AC mice. AG1478 treatment before each of several UV exposures decreased the number of papillomas arising and the growth of these tumors by ∼50% and 80%, respectively. Inhibition of EGFR suppressed proliferation, increased apoptotic cell death, and delayed the onset of epidermal hyperplasia following UV irradiation. Genetic ablation of Egfr similarly delayed epidermal hyperplasia in response to UV exposure. Thus, the UV-induced activation of EGFR promotes skin tumorigenesis by suppressing cell death, augmenting cell proliferation, and accelerating epidermal hyperplasia in response to UV. These results suggest that EGFR may be an appropriate target for the chemoprevention of UV-induced skin cancer.
Synergistic Antitumor Response of Interleukin 2 with Melphalan in Isolated Limb Perfusion in Soft Tissue Sarcoma–Bearing Rats
Authors: Hoving Saske , Brunstein Flavia , aan de Wiel-Ambagtsheer Gisela , van Tiel Sandra T. , de Boeck Gert , de Bruijn Ernst A. , Eggermont Alexander M.M. , ten Hagen Timo L.M.
Published: September 2005

The cytokine interleukin 2 (IL-2) is a mediator of immune cell activation with some antitumor activity, mainly in renal cell cancer and melanoma. We have previously shown that tumor necrosis factor (TNF)-α has strong synergistic antitumor activity in combination with chemotherapeutics in the isolated limb perfusion (ILP) setting based on a TNF-mediated enhanced tumor-selective uptake of the chemotherapeutic drug followed by a selective destruction of the tumor vasculature. IL-2 can cause vascular leakage and edema and for this reason we examined the antitumor activity of a combined treatment with IL-2 and melphalan in our well-established ILP in soft tissue sarcoma–bearing rats (BN175). ILP with either IL-2 or melphalan alone has no antitumor effect, but the combination of IL-2 and melphalan resulted in a strong synergistic tumor response, without any local or systemic toxicity. IL-2 enhanced significantly melphalan uptake in tumor tissue. No signs of significant vascular damage were detected to account for this observation, although the tumor sections of the IL-2– and IL-2 plus melphalan–treated animals revealed scattered extravasation of erythrocytes compared with the untreated animals. Clear differences were seen in the localization of ED-1 cells, with an even distribution in the sham, IL-2 and melphalan treatments, whereas in the IL-2 plus melphalan–treated tumors clustered ED-1 cells were found. Additionally, increased levels of TNF mRNA were found in tumors treated with IL-2 and IL-2 plus melphalan. These observations indicate a potentially important role for macrophages in the IL-2–based perfusion. The results in our study indicate that the novel combination of IL-2 and melphalan in ILP has synergistic antitumor activity and may be an alternative for ILP with TNF and melphalan.
Sensitivity of Undifferentiated, High-TCR Density CD8+ Cells to Methylene Groups Appended to Tumor Antigen Determines Their Differentiation or Death
Authors: Kawano Kouichiro , Efferson Clay L. , Peoples George E. , Carter Darrick , Tsuda Naotake , Murray James L. , Ioannides Constantin G.
Published: September 2005

CD8+ cells expressing high numbers of TCR per cell (TCRhi) are considered important mediators of antitumor effects. To understand the relationship between TCR density and antigen affinity for TCR in the outcome of stimulation with antigen and differentiation of CTL recognizing tumor antigen, we analyzed perforin induction in ovarian tumor-associated lymphocytes in response to the smallest possible changes in the atomic forces of interaction between antigen and TCR. Stimulating undifferentiated, apoptosis-resistant CD8+ cells expressing high levels of E75-TCR (TCRhi) with variants of the CTL epitope E75, HER-2 (369-377), induced their stepwise differentiation, first to IFN-γ+ Perf− and to TCRhi IFN-γ+ Perf+ cells. Blocking caspase-9 activation at antigen stimulation also enhanced the generation of TCRhi Perfhi cells, demonstrating that TCR density dictated the pathway of death activated by stimulation with the same agonist. Expansion and differentiation of TCRhi Perf+ CTL required an agonist of optimal CH2 side chain length, which in this study was equal to two CH2 groups appended to E75 at the Gly4 position. Side chains one CH2 shorter or longer than optimal were either less stimulatory or induced death of TCRhi Perf+ cells. Differentiation of TCRhi CD8+ cells can be finely tuned by synthetic amino acids in the peptide, whose side chains induce small increments in the affinity of the antigen for TCR below the affinity which induce apoptosis.
The G Protein–Coupled Receptor S1P2 Regulates Rho/Rho Kinase Pathway to Inhibit Tumor Cell Migration
Authors: Lepley Denise , Paik Ji-Hye , Hla Timothy , Ferrer Fernando
Published: September 2005

Sphingosine 1-phosphate (S1P) is a lysophospholipid that exerts a variety of responses in cells such as proliferation, migration, and survival. These effects are mediated by G protein–coupled receptors on the cell surface (S1P1-5), which activate downstream signaling intermediates such as Rac and Rho GTPases. Mechanisms of S1P action in human glioblastoma cells are not well defined. S1P receptors (1–5) and S1P-metabolizing enzymes were expressed in three human glioblastoma cell lines. S1P had a profound and differential effect on glioblastoma cell migration. U87 cells treated with S1P showed a significant increase in migration, whereas U118 and U138 cell lines were strongly inhibited. S1P-mediated inhibition correlated with S1P2 receptor expression. FTY720-P, an S1P analogue that binds all S1P receptors except S1P2, did not inhibit glioblastoma cell migration. Overexpression of S1P2 further suppressed migration, and blockage of S1P2 mRNA expression by small interfering RNA reversed the inhibitory effect. Contrary to previous reports showing bimodal regulation of Rac activity and migration by S1P2 receptor stimulation, both Rac1 and RhoA GTPases were activated by S1P treatment in native cells and cells overexpressing S1P2. Treatment of U118 cells with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 restored migration suggesting that ROCK-dependent mechanisms are important. Actin staining of S1P stimulated U118 cells overexpressing β-galactosidase resulted in pronounced stress fiber formation that was exacerbated by S1P2 overexpression, partially blocked by S1P1, or totally abolished by pretreatment with Y-27632. These data provide evidence of a novel mechanism of S1P inhibition of tumor cell migration via Rho kinase–dependent pathway.
Valproic Acid Alters Chromatin Structure by Regulation of Chromatin Modulation Proteins
Authors: Marchion Douglas C. , Bicaku Elona , Daud Adil I. , Sullivan Daniel M. , Munster Pamela N.
Published: September 2005

Histone acetylation and deacetylation are crucial in the regulation of gene expression. Dynamic changes in gene expression may affect chromatin structure and, consequently, the interaction of chromatin with regulatory factors. In this study, the effects of the antiseizure drug valproic acid (VPA) on the expression of genes that regulate the structure of chromatin and the access of macromolecules to the DNA were investigated. Exposure of breast cancer cells to VPA resulted in rapid dose-dependent hyperacetylation of the histones H3 and H4. VPA further induced a depletion of several members of the structural maintenance of chromatin (SMC) proteins, SMC-associated proteins, DNA methyltransferase, and heterochromatin proteins. Down-regulation of these proteins was associated with chromatin decondensation. The observed alterations of chromatin structure correlated with enhanced sensitivity of DNA to nucleases and increased interaction of DNA with intercalating agents. VPA-induced chromatin decondensation led to a sequence-specific potentiation of DNA-damaging agents in cell culture and xenograft models. Modulation of heterochromatin maintenance proteins was not a direct, but a downstream, effect of histone acetylation. The effects on the chromatin structure were reversible upon drug withdrawal, but obligatory for the potentiation of DNA-damaging agents. In addition to their antitumor activity as single agents, the chromatin decondensation induced by histone deacetylase inhibitors may enhance the efficacy of cytotoxic agents that act by targeting DNA. The proposed mechanism of action suggests an effect of drug sequencing on the antitumor activity of these drugs.
RNA Silencing of Checkpoint Regulators Sensitizesp53-Defective Prostate Cancer Cells to Chemotherapy while Sparing Normal Cells
Authors: Mukhopadhyay Utpal K. , Senderowicz Adrian M. , Ferbeyre Gerardo
Published: September 2005

p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G2 checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.
Mouse Mammary Tumor Virus Infects Human Cells
Authors: Indik Stanislav , Günzburg Walter H. , Salmons Brian , Rouault Francoise
Published: September 2005

Mouse mammary tumor virus (MMTV) has long been speculated to be involved in human breast cancer and more recently in human primary biliary cirrhosis. Despite complete proviral sequences markedly homologous to MMTV being identified in human breast cancer tissue, no convincing evidence has been presented to date that MMTV can infect human cells. Using both wild-type and a genetically marked virus (MMTV-EGFP), we show here the successful infection of a number of different human cells by MMTV. Furthermore, infection of human cells is shown to be almost as efficient as the infection of murine mammary epithelial cells. Sequencing of PCR products from integrated proviruses reveals that reverse transcription and integration of the viral genome has occurred as expected. Furthermore, sequencing of two independent MMTV proviral integration sites reveal them to be present only in the human and not in the mouse genome. Infection requires an intact MMTV envelope protein and is blocked either by heat inactivation of the virus or by specific neutralizing anti-MMTV serum, ruling out a nonspecific mechanism of viral transfer. Thus, MMTV can infect human cells and this finding provides a possible explanation for the detection by others of MMTV sequences in human breast cancer patients.
Regulation of p38 Phosphorylation and Topoisomerase IIα Expression in the B-Cell Lymphoma Line Jiyoye by CD26/Dipeptidyl Peptidase IV Is Associated with EnhancedIn vitroandIn vivoSensitivity to Doxorubicin
Authors: Yamochi Toshiko , Yamochi Tadanori , Aytac Ugur , Sato Tsutomu , Sato Kazuya , Ohnuma Kei , McKee Kathryn S. , Morimoto Chikao , Dang Nam H.
Published: September 2005

CD26 is a Mr 110,000 surface-bound glycoprotein with diverse functional properties, including having a key role in normal T-cell physiology and the development of certain cancers. In this article, we show that surface expression of CD26, especially its intrinsic dipeptidyl peptidase IV (DPPIV) enzyme activity, results in enhanced topoisomerase IIα level in the B-cell line Jiyoye and subsequent in vitro sensitivity to doxorubicin-induced apoptosis. In addition, we show that expression of CD26/DPPIV is associated with increased phosphorylation of p38 and its upstream regulators mitogen-activated protein kinase kinase 3/6 and apoptosis signal-regulating kinase 1 and that p38 signaling pathway plays a role in the regulation of topoisomerase IIα expression. Besides demonstrating that CD26 effect on topoisomerase IIα and doxorubicin sensitivity is applicable to cell lines of both B-cell and T-cell lineages, the potential clinical implication of our work lies with the fact that we now show for the first time that our in vitro results can be extended to a severe combined immunodeficient mouse model. Our findings that CD26 expression can be an in vivo marker of tumor sensitivity to doxorubicin treatment may lead to future treatment strategies targeting CD26/DPPIV for selected human cancers in the clinical setting. Our article thus characterizes the biochemical linkage among CD26, p38, and topoisomerase IIα while providing evidence that CD26-associated topoisomerase IIα expression results in greater in vitro and in vivo tumor sensitivity to the antineoplastic agent doxorubicin.
Akt Activation Suppresses Chk2-Mediated, Methylating Agent–Induced G2 Arrest and Protects from Temozolomide-Induced Mitotic Catastrophe and Cellular Senescence
Authors: Hirose Yuchi , Katayama Makoto , Mirzoeva Olga K. , Berger Mitchel S. , Pieper Russell O.
Published: September 2005

Pharmacologic inhibition of the DNA signal transducers Chk1 and p38 blocks G2 arrest and sensitizes glioblastoma cells to chemotherapeutic methylating agent–induced cytotoxicity. Because Akt pathway activation has been suggested to also block G2 arrest induced by DNA-damaging agents and because glioma cells frequently have high levels of Akt activation, we examined the contribution of the Akt pathway to methylating agent–induced G2 arrest and toxicity. U87MG human glioma cells containing an inducible Akt expression construct were incubated with inducing agent or vehicle, after which the cells were exposed to temozolomide and assayed for activation of the components of the G2 arrest pathway and survival. Temozolomide-treated control cells activated the DNA damage signal transducers Chk1, Chk2, and p38, leading to Cdc25C and Cdc2 inactivation, prolonged G2 arrest, and loss of clonagenicity by a combination of senescence and mitotic catastrophe. Temozolomide-treated cells induced to overexpress Akt, however, exhibited significantly less drug-induced Cdc25C/Cdc2 inactivation and less G2 arrest. Akt-mediated suppression of G2 arrest was associated not with alterations in Chk1 or p38 activation but rather with suppression of Chk2 activation and reduced recruitment of Chk2 to sites of damage in chromatin. Unlike bypass of the G2 checkpoint induced by pharmacologic inhibitors of Chk1 or p38, however, Akt-induced bypass of G2 arrest suppressed, rather than enhanced, temozolomide-induced senescence and mitotic catastrophe. These results show that whereas Akt activation suppresses temozolomide-induced Chk2 activation and G2 arrest, the overriding effect is protection from temozolomide-induced cytotoxicity. The Akt pathway therefore represents a new target for the sensitization of gliomas to chemotherapeutic methylating agents such as temozolomide.
Altered ErbB Receptor Signaling and Gene Expression in Cisplatin-Resistant Ovarian Cancer
Authors: Macleod Kenneth , Mullen Peter , Sewell Jane , Rabiasz Genevieve , Lawrie Sandra , Miller Eric , Smyth John F. , Langdon Simon P.
Published: September 2005

The majority of ovarian cancer patients are treated with platinum-based chemotherapy, but the emergence of resistance to such chemotherapy severely limits its overall effectiveness. We have shown that development of resistance to this treatment can modify cell signaling responses in a model system wherein cisplatin treatment has altered cell responsiveness to ligands of the erbB receptor family. A cisplatin-resistant ovarian carcinoma cell line PE01CDDP was derived from the parent PE01 line by exposure to increasing concentrations of cisplatin, eventually obtaining a 20-fold level of resistance. Whereas PE01 cells were growth stimulated by the erbB receptor-activating ligands, such as transforming growth factor-α (TGFα), NRG1α, and NRG1β, the PE01CDDP line was growth inhibited by TGFα and NRG1β but unaffected by NRG1α. TGFα increased apoptosis in PE01CDDP cells but decreased apoptosis in PE01 cells. Differences in extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling were also found, which may be implicated in the altered cell response to ligands. Microarray analysis revealed 51 genes whose mRNA increased by at least 2-fold in PE01CDDP cells relative to PE01 (including FRA1, ETV4, MCM2, AXL, MT3, TRAP1, and FANCG), whereas 36 genes (including IGFBP3, TRAM1, and KRT4 and KRT19) decreased by a similar amount. Differential display reverse transcriptase-PCR identified altered mRNA expression for TCP1, SLP1, proliferating cell nuclear antigen, and ZXDA. Small interfering RNA inhibition of FRA1, TCP1, and MCM2 expression was associated with reduced growth and FRA1 inhibition with enhanced cisplatin sensitivity. Altered expression of these genes by cytotoxic exposure may provide survival advantages to cells including deregulation of signaling pathways, which may be critical in the development of drug resistance.
Visualizing Chromosomes as Transcriptome Correlation Maps: Evidence of Chromosomal Domains Containing Co-expressed Genes—A Study of 130 Invasive Ductal Breast Carcinomas
Authors: Reyal Fabien , Stransky Nicolas , Bernard-Pierrot Isabelle , Vincent-Salomon Anne , de Rycke Yann , Elvin Paul , Cassidy Andrew , Graham Alexander , Spraggon Carolyn , Désille Yoann , Fourquet Alain , Nos Claude , Pouillart Pierre , Magdelénat Henri , Stoppa-Lyonnet Dominique , Couturier Jérôme , Sigal-Zafrani Brigitte , Asselain Bernard , Sastre-Garau Xavier , Delattre Olivier , Thiery Jean Paul , Radvanyi François
Published: September 2005

Completion of the working draft of the human genome has made it possible to analyze the expression of genes according to their position on the chromosomes. Here, we used a transcriptome data analysis approach involving for each gene the calculation of the correlation between its expression profile and those of its neighbors. We used the U133 Affymetrix transcriptome data set for a series of 130 invasive ductal breast carcinomas to construct chromosomal maps of gene expression correlation (transcriptome correlation map). This highlighted nonrandom clusters of genes along the genome with correlated expression in tumors. Some of the gene clusters identified by this method probably arose because of genetic alterations, as most of the chromosomes with the highest percentage of correlated genes (1q, 8p, 8q, 16p, 16q, 17q, and 20q) were also the most frequent sites of genomic alterations in breast cancer. Our analysis showed that several known breast tumor amplicons (at 8p11-p12, 11q13, and 17q12) are located within clusters of genes with correlated expression. Using hierarchical clustering on samples and a Treeview representation of whole chromosome arms, we observed a higher-order organization of correlated genes, sometimes involving very large chromosomal domains that could extend to a whole chromosome arm. Transcription correlation maps are a new way of visualizing transcriptome data. They will help to identify new genes involved in tumor progression and new mechanisms of gene regulation in tumors.
Farnesyltransferase Inhibitors Induce DNA Damage via Reactive Oxygen Species in Human Cancer Cells
Authors: Pan Jingxuan , She Miaorong , Xu Zhi-Xiang , Sun Lily , Yeung Sai-Ching Jim
Published: September 2005

Farnesyltransferase inhibitors (FTIs) possess antitumor activity. Based on recent findings, we hypothesized that FTIs induce reactive oxygen species (ROS) that damage DNA, leading to DNA damage responses. To test this hypothesis, we investigated the effects of FTIs on the generation of ROS, DNA double-strand breaks (DSB), DNA damage responses, and RhoB, and the effects of quenching ROS on these FTI effects. We evaluated four FTIs in human cancer cell lines of different tissue origins. We found that FTIs induced ROS and DSBs. Suppressing expression of the β-subunit of farnesyltransferase with siRNA did not induce ROS, but slightly attenuated the ROS induced by FTIs. N-acetyl-l-cysteine (NAC), but not caspase inhibitors, blocked FTI-induced DSBs, suggesting that the DSBs were caused by ROS and did not result from apoptosis. The DSBs led to DNA damage responses. H2AX became phosphorylated and formed nuclear foci. The DNA-damage-sensing molecules involved were probably ataxia-telangiectasia mutated protein (ATM) and DNA-dependent protein kinase (DNA-PK) but not ATM- and Rad3-related protein (ATR). Key components of the homologous recombination and nonhomologous end joining repair pathways (DNA-PK, BRCA1, and NBS1) underwent phosphorylation and formed nuclear foci. RhoB, a mediator of the antineoplastic effect of FTIs and a protein inducible by DNA damage, was increased by FTIs. This increase was blocked by NAC. We concluded that FTIs induced oxidative DNA damage by inducing ROS and initiated DNA damage responses, including RhoB induction, and there was a complex relationship among FTIs, farnesyltransferase, ROS, and RhoB. Our data also imply that inhibitors of DNA repair may accentuate the clinical efficacy of FTIs.
Additive Antitumor Effect of Aromatase Inhibitor Letrozole and Antiestrogen Fulvestrant in a Postmenopausal Breast Cancer Model
Authors: Jelovac Danijela , Macedo Luciana , Goloubeva Olga G. , Handratta Venkatesh , Brodie Angela M.H.
Published: September 2005

Blocking estrogen receptors with antiestrogens and blocking estrogen synthesis with aromatase inhibitors are two strategies currently being used for reducing the effect of estrogen in postmenopausal estrogen receptor–positive breast cancer patients. To optimize these treatment strategies, we have investigated whether tumor progression can be delayed by combining the pure antiestrogen fulvestrant with the nonsteroidal aromatase inhibitor letrozole. These studies were done in ovariectomized, athymic mice bearing tumors of estrogen receptor–positive human breast cancer cells stably transfected with the aromatase gene (MCF-7Ca). Groups of mice with equivalent tumor volumes were injected s.c. daily with vehicle (control; n = 6), fulvestrant (1 mg/d; n = 7), letrozole (10 μg/d; n = 18), or letrozole (10 μg/d) plus fulvestrant (1 mg/d; n = 5). All treatments were effective in suppressing tumor growth compared with controls (P < 0.001). Tumor volumes of the fulvestrant-treated group had doubled in 10 weeks. After 19 weeks of letrozole (10 μg/d) treatment when tumors had nearly doubled in volume, mice (n = 18) were assigned to second-line therapy with letrozole (100 μg/d; n = 6), tamoxifen (100 μg/d; n = 6), or remained on letrozole treatment (10 μg/d; n = 6). However, tumors continued to increase in volume in these groups. Tumors of animals treated with the combination of letrozole plus Faslodex regressed over 29 weeks of treatment by 45%. Thus, the combination of letrozole plus fulvestrant was more effective in suppressing tumor growth than either letrozole or fulvestrant alone or sequential therapies with tamoxifen or a higher dose of letrozole (100 μg/d).
19-Nor-1,25(OH)2D2 (a Novel, Noncalcemic Vitamin D Analogue), Combined with Arsenic Trioxide, Has Potent Antitumor Activity against Myeloid Leukemia
Authors: Kumagai Takashi , Shih Lee-Yung , Hughes Susan V. , Desmond Julian C. , O'Kelly James , Hewison Martin , Koeffler H. Phillip
Published: September 2005

Recently, we reported that a novel, noncalcemic vitamin D analogue (19-nor-1,25(OH)2D2; paricalcitol) had anticancer activity. In this study, we explored if paricalcitol enhanced anticancer effects of other clinically useful drugs in vitro against a large variety of cancer cells. Paricalcitol, when combined with As2O3, showed a markedly enhanced antiproliferative effect against acute myeloid leukemia (AML) cells. This combination induced monocytic differentiation of NB-4 acute promyelocytic leukemia (APL) cells and HL-60 AML cells and caused both to undergo apoptosis associated with down-regulation of Bcl-2 and Bcl-xL. Paricalcitol induced monocytic differentiation of U937 AML cells, which was partially blocked by inducing expression of APL-related PML-retinoic acid receptor α (RARα) chimeric protein in the U937 cells containing a Zn2+-inducible expression vector coding for this fusion protein (PR9 cells). Exposure to As2O3 decreased levels of PML-RARα in PR9 cells, and the combination of paricalcitol and As2O3 enhanced their monocytic differentiation in parallel with the As2O3-mediated decrease of PML-RARα. Furthermore, As2O3 increased the transcriptional activity of paricalcitol probably by increasing intracellular levels of paricalcitol by decreasing the function of the mitochondrial enzyme 25-hydroxyvitamin D3-24-hydroxylase, which functions to metabolize the active vitamin D in cells. In summary, the combination of paricalcitol and As2O3 potently decreased growth and induced differentiation and apoptosis of AML cells. This probably occurred by As2O3 decreasing levels of both the repressive PML-RARα fusion protein and the vitamin D metabolizing protein, 25-hydroxyvitamin D3-24-hydroxylase, resulting in increased activity of paricalcitol. The combination of both of these Food and Drug Administration–approved drugs should be considered for treatment of all-trans retinoic acid–resistant APL patients as well as those with other types of AML.
Genomic Organization of Amplified MYC Genes Suggests Distinct Mechanisms of Amplification in Tumorigenesis
Authors: Herrick John , Conti Chiara , Teissier Sebastien , Thierry Francoise , Couturier Jérôme , Sastre-Garau Xavier , Favre Michel , Orth Gérard , Bensimon Aaron
Published: September 2005

Integration of the human papillomavirus (HPV) genome into the host genome is associated with the disruption of the HPV E2 gene and with amplification and rearrangement of the viral and flanking cellular sequences. Molecular characterization of the genomic structures of coamplified HPV sequences and oncogenes provides essential information concerning the mechanisms of amplification and their roles in carcinogenesis. Using fluorescent hybridization on stretched DNA molecules in two cervical cancer–derived cell lines, we have elucidated the genomic structures of amplified regions containing HPV/myc genes over several hundreds of kilobases. Direct visualization of hybridization signals on individual DNA molecules suggests that overreplication and breakage-fusion-bridge–type mechanisms are involved in the genomic instability associated with HPV cervical cancers. Further analysis from two other genital cancer–derived cell lines reveals a recurrent motif of amplification, probably generated by a common mechanism involving overreplication upon viral integration. Interestingly, different amplification patterns seem to be correlated with the disease outcome, thus providing new insights into HPV-related cancer development and tumor progression.
HYAL1 Hyaluronidase: A Molecular Determinant of Bladder Tumor Growth and Invasion
Authors: Lokeshwar Vinata B. , Cerwinka Wolfgang H. , Lokeshwar Bal L.
Published: September 2005

Hyaluronic acid and HYAL1-type hyaluronidase show high accuracy in detecting bladder cancer and evaluating its grade, respectively. Hyaluronic acid promotes tumor progression; however, the functions of hyaluronidase in cancer are largely unknown. In this study, we stably transfected HT1376 bladder cancer cells with HYAL1-sense (HYAL1-S), HYAL1-antisense (HYAL1-AS), or vector cDNA constructs. Whereas HYAL1-S transfectants produced 3-fold more HYAL1 than vector transfectants, HYAL1-AS transfectants showed ∼90% reduction in HYAL1 production. HYAL1-AS transfectants grew four times slower than vector and HYAL1-S transfectants and were blocked in the G2-M phase of the cell cycle. The expression of cdc25c and cyclin B1 and cdc2/p34-associated H1 histone kinase activity also decreased in HYAL1-AS transfectants. HYAL1-S transfectants were 30% to 44% more invasive, and HYAL1-AS transfectants were ∼50% less invasive than the vector transfectants in vitro. In xenografts, there was a 4- to 5-fold delay in the generation of palpable HYAL1-AS tumors, and the weight of HYAL1-AS tumors was 9- to 17-fold less than vector and HYAL1-S tumors, respectively (P < 0.001). Whereas HYAL1-S and vector tumors infiltrated skeletal muscle and blood vessels, HYAL1-AS tumors resembled benign neoplasia. HYAL1-S and vector tumors expressed significantly higher amounts of HYAL1 (in tumor cells) and hyaluronic acid (in tumor-associated stroma) than HYAL1-AS tumors. Microvessel density in HYAL1-S tumors was 3.8- and 9.5-fold higher than that in vector and HYAL1-AS tumors, respectively. These results show that HYAL1 expression in bladder cancer cells regulates tumor growth and progression and therefore serves as a marker for high-grade bladder cancer.
Human Carcinoma Cell Growth and Invasiveness Is Impaired by the Propeptide of the Ubiquitous Proprotein Convertase Furin
Authors: López de Cicco Ricardo , Bassi Daniel E. , Zucker Stanley , Seidah Nabil G. , Klein-Szanto Andrés J.P.
Published: September 2005

Furin, a potent proprotein convertase involved in activation of several cancer-related substrates, is synthesized as an inactive zymogen, thus minimizing the occurrence of premature enzymatic activity that would lead to inappropriate protein activation or degradation. This natural inhibitory mechanism is based on the presence of an inactivating prosegment at the NH2 terminal of the zymogen. After initial autocatalytic cleavage, the prosegment remains tightly associated with the convertase until it reaches the trans-Golgi network where the dissociation of the prosegment and activation of furin occurs. We hypothesized that the inhibitory properties of the preprosegment of furin (ppFur) could be beneficial if ectopically expressed in tumor cells. Transfection of four human head and neck squamous cell carcinoma cell lines with the complete ppFur cDNA sequence (pIRES-EGFP-ppFur) or with the empty expression vector (pIRES-EGFP) was done. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, anchorage-independent growth in soft agar, and proliferation assays, as well as by investigating impairment of furin substrates processing. Following transfection of ppFur, a significant reduction in cell proliferation, tumorigenicity, and invasiveness was observed in vitro and in vivo. These biological changes are directly related to the inhibition of furin-mediated activation of crucial cancer-related substrates, such as membrane type 1 matrix metalloproteinase, transforming growth factor-β, insulin-like growth factor-1 receptor, and vascular endothelial growth factor-C. PpFur expression in head and neck squamous cell carcinoma cell lines showed a mechanistic link between furin inhibition, decreased substrate processing, cell proliferation, and invasive ability. These findings suggest that furin inhibition is a feasible approach to ameliorate and even abolish the malignant phenotype of various malignancies.