bluuebunny/crossref_metadata_2025 huggingface dataset:
Progression of Pregnancy-Dependent Mouse Mammary Tumors after Long Dormancy Periods. Involvement of Wnt Pathway Activation
Authors: Gattelli Albana , Cirio María Cecilia , Quaglino Ana , Schere-Levy Carolina , Martinez Natalia , Binaghi María , Meiss Roberto P. , Castilla Lucio H. , Kordon Edith C.
Published: September 2005

Mouse mammary tumor virus (LA) induces pregnancy-dependent mammary tumors that progress toward autonomy. Here we show that in virgin females, pregnancy-dependent tumor transplants are able to remain dormant for up to 300 days. During that period, these tumors synthesize DNA, express high levels of estrogen and progesterone receptors (ER+PR+) and are able to resume growth after hormone stimulation. Surprisingly, in a subsequent transplant generation, all these tumors are fully able to grow in virgin females, they express low levels of ER and PR (ER−PR−) and have a monoclonal origin; i.e., show all of the features we have described previously in pregnancy-independent tumors. Histologically, mouse mammary tumor virus (LA)-induced tumors are morphologically similar to genetically engineered mouse (GEM) mammary tumors that overexpress genes belonging to the Wnt pathway. Interestingly, in the virus-induced neoplasias, pregnancy-independent passages arising after a dormant phase usually display a lower level of glandular differentiation together with epithelial cell trans-differentiation, a specific feature associated to Wnt pathway activation. In addition, dormancy can lead to the specific selection of Int2/Fgf3 mutated and overexpressing cells. Therefore, our results indicate that during hormone-dependent tumor dormancy, relevant changes in cell population occur, allowing rapid progression after changes in the animal internal milieu.
Heat Shock Protein 27 Increases after Androgen Ablation and Plays a Cytoprotective Role in Hormone-Refractory Prostate Cancer
Authors: Rocchi Palma , So Alan , Kojima Satoko , Signaevsky Maxim , Beraldi Eliana , Fazli Ladan , Hurtado-coll Antonio , Yamanaka Kazuki , Gleave Martin
Published: September 2005

Heat shock protein 27 (Hsp27) is a chaperone implicated as an independent predictor of clinical outcome in prostate cancer. Our aim was to characterize changes in Hsp27 after androgen withdrawal and during androgen-independent progression in prostate xenografts and human prostate cancer to assess the functional significance of these changes using antisense inhibition of Hsp27. A tissue microarray was used to measure changes in Hsp27 protein expression in 232 specimens from hormone naive and posthormone-treated cancers. Hsp27 expression was low or absent in untreated human prostate cancers but increased beginning 4 weeks after androgen-ablation to become uniformly highly expressed in androgen-independent tumors. Androgen-independent human prostate cancer PC-3 cells express higher levels of Hsp27 mRNA in vitro and in vivo, compared with androgen-sensitive LNCaP cells. Phosphorothioate Hsp27 antisense oligonucleotides (ASOs) and small interference RNA potently inhibit Hsp27 expression, with increased caspase-3 cleavage and PC3 cell apoptosis and 87% decreased PC3 cell growth. Hsp27 ASO and small interference RNA also enhanced paclitaxel chemosensitivity in vitro, whereas in vivo, systemic administration of Hsp27 ASO in athymic mice decreased PC-3 tumor progression and also significantly enhanced paclitaxel chemosensitivity. These findings suggest that increased levels of Hsp27 after androgen withdrawal provide a cytoprotective role during development of androgen independence and that ASO-induced silencing can enhance apoptosis and delay tumor progression.
Expression of Toll-Like Receptor 4 on Dendritic Cells Is Significant for Anticancer Effect of Dendritic Cell-Based Immunotherapy in Combination with an Active Component of OK-432, a Streptococcal Preparation
Authors: Okamoto Masato , Furuichi Sachiko , Nishioka Yasuhiko , Oshikawa Tetsuya , Tano Tomoyuki , Ahmed Sharif Uddin , Takeda Kiyoshi , Akira Shizuo , Ryoma Yoshiki , Moriya Yoichiro , Saito Motoo , Sone Saburo , Sato Mitsunobu
Published: September 2005

A lipoteichoic acid-related molecule OK-PSA is an active component of OK-432, a Streptococcus-derived anticancer immunotherapeutic agent. In the present study, we first examined the effect of OK-PSA on the maturation of dendritic cells (DCs) in vitro by using the DCs derived from 5 healthy donors and 10 patients with head and neck cancer with or without expression of toll-like receptor 4 (TLR4) or MD-2 mRNA. OK-PSA treatment effectively increased the surface expression of MHC class II, CD80, CD83, and CD86. OK-PSA-stimulated DCs secreted the cytokines that can induce helper T-cell 1 (Th1)-type T-cell response, and stimulated allogeneic T cells to produce IFN-γ and to elicit an allogeneic antigen-specific cytotoxicity. These activities almost depended on expression of TLR4 and MD-2 genes. We next investigated the in vivo anticancer effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. C57BL/6 mice, which express wild-type TLR4, and C57BL/6-derived TLR4-knockout (TLR4−/−) mice were used. Although OK-PSA accelerated the antitumor effect of intratumoral DC administration in wild-type mice bearing syngeneic tumors, the antitumor effect of OK-PSA as well as of the combination therapy with DCs and OK-PSA was not significant in TLR4−/− mice. Interestingly, an administration of wild-type-mouse-derived DCs followed by OK-PSA exhibited a marked antitumor effect even in the TLR4−/− mice. These findings suggest that OK-PSA may be a potent adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anticancer activity even in a TLR4-deficient host when TLR4 is expressed only in DCs injected intratumorally.
HCMOGT-1 Is a Novel Fusion Partner to
PDGFRB
in Juvenile Myelomonocytic Leukemia with t(5;17)(q33;p11.2)
Authors: Morerio Cristina , Acquila Maura , Rosanda Cristina , Rapella Annamaria , Dufour Carlo , Locatelli Franco , Maserati Emanuela , Pasquali Francesco , Panarello Claudio
Published: September 2005

PDGFRB, a transmembrane tyrosine kinase receptor for plateletderived growth factor, is constitutively activated by gene fusion with different partners in myeloproliferative/myelodysplastic disorders with peculiar clinical characteristics. Six alternative partner genes have been described thus far. In this study, we report the molecular cloning of a novel translocation t(5;17)(q33;p11.2) in a case of juvenile myelomonocytic leukemia. The novel partner gene was identified as HCMOGT-1 using 5′-rapid amplification of cDNA ends; fluorescence in situ hybridization and reverse transcriptase-PCR analyses confirmed that the translocation resulted in PDGFRB/HCMOGT-1 fusion. We show that the breakpoint of PDGFRB occurred at the same site of all previously reported PDGFRB translocations.
Chemoprevention of Cancer of the Upper Respiratory Tract of the Syrian Golden Hamster by Aerosol Administration of Difluoromethylornithine and 5-Fluorouracil
Authors: Wattenberg Lee W. , Wiedmann Timothy S. , Estensen Richard D.
Published: September 2005

Research aimed at identifying effective chemopreventive compounds active against carcinogenesis of the upper respiratory tract (URT) has been largely unsuccessful. We are addressing this problem by efforts at agent identification and by using aerosol delivery. Two compounds, difluoromethylornithine (DFMO) and 5-fluorouracil (5-FU) were investigated. DFMO is an irreversible inhibitor of ornithine decarboxylase, an enzyme important in cell proliferation. It has been used widely by oral administration for chemoprevention. 5-FU is a pyrimidine analog used extensively as a chemotherapeutic agent. It is generally administered i.v. and can cause considerable toxicity. However, aerosol administration for therapy of lung cancer in humans has been reported to be without adverse effects (Tatsumura et al., Br J Cancer 1993;68:1146–9). The experimental model used herein entailed six intratracheal administrations of methylnitrosourea (MNU) to hamsters. Each of the test agents was started about 1 week after MNU and was continued for 29 weeks with DFMO. Infiltrating squamous cell carcinomas of the URT occurred in 92% of the controls and were reduced by 50% in animals receiving DFMO (P = 0.0001). The experiment with 5-FU was of shorter duration being terminated 20 weeks after MNU. Thirty percent of the controls had infiltrating carcinomas and were reduced by 60% in animals receiving 5-FU (P = 0.0274). Both compounds resulted in a significant increase in the percent of cancer-free animals. These two agents may have selected use in subjects at high risk of cancer of the URT.
Alternative Lengthening of Telomeres Is Characterized by High Rates of Telomeric Exchange
Authors: Londoño-Vallejo J. Arturo , Der-Sarkissian Héra , Cazes Lucien , Bacchetti Silvia , Reddel Roger R.
Published: September 2005

Telomere maintenance activity is a hallmark of cancer. In some telomerase-negative tumors, telomeres become lengthened by alternative lengthening of telomeres (ALT), a recombination-mediated DNA replication process in which telomeres use other telomeric DNA as a copy template. Using chromosome orientation fluorescence in situ hybridization, we found that postreplicative exchange events involving a telomere and another TTAGGG-repeat tract occur at remarkably high frequencies in ALT cells (range 28–280/100 metaphases) and rarely or never in non-ALT cells, including cell lines with very long telomeres. Like the ALT phenotype itself, the telomeric exchanges were not suppressed when telomerase was activated in ALT cells. These exchanges are telomere specific because there was no correlation with sister chromatid exchange rates at interstitial locations, and they were not observed in non-ALT Bloom syndrome cells with very high sister chromatid exchange rates.
Tumor-Microenvironment Interactions
Authors: Zipin Adi , Israeli-Amit Mira , Meshel Tsipi , Sagi-Assif Orit , Yron Ilana , Lifshitz Veronica , Bacharach Eran , Smorodinsky Nechama I. , Many Ariel , Czernilofsky Peter A. , Morton Donald L. , Witz Isaac P.
Published: September 2005

Extravasation of tumor cells is a pivotal step in metastasis formation. This step is initiated by an interaction of extravasating tumor cells with endothelial cells. Among the molecules mediating tumor-endothelium interactions are selectins and their fucosylated ligands. In a previous study, we demonstrated that the fucose-generating FX enzyme regulates the expression of selectin ligands by B and T lymphocytes and by head and neck squamous cell carcinoma cells. It was also shown that the FX enzyme regulated important interaction parameters between these cancer cells and endothelial cells. The present study was aimed to determine whether the FX enzyme controls adhesive interactions between colorectal cancer cells and endothelial cells. The results clearly indicate that this is indeed the case. Overexpressing the FX enzyme by the transfer of FX cDNA to low FX-expressing colorectal cancer cells resulted in an increased adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. Down-regulating FX levels in colorectal cancer cells expressing high levels of endogenous FX by transfection with small-interfering RNA resulted in a down-regulated expression of the selectin ligand sialyl Lewis-a and a decrease in the adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. These transfection experiments also indicated that manipulating the levels of the FX enzyme affected global cellular fucosylation and altered the interaction of colorectal cancer cells with some extracellular matrix components such as fibronectin. We also found that highly metastatic colorectal cancer variants express higher levels of FX and of sialyl Lewis-a than low metastatic variants originating in the same tumors. These results lead us to hypothesize that the FX enzyme controls the capacity of colorectal cancer to extravasate and form metastasis. If this hypothesis will be confirmed the FX enzyme could become a target molecule for metastasis prevention.
A Transcriptional Profiling Study of CCAAT/Enhancer Binding Protein Targets Identifies Hepatocyte Nuclear Factor 3β as a Novel Tumor Suppressor in Lung Cancer
Authors: Halmos Balazs , Bassères Daniela S. , Monti Stefano , D‘Aló Francesco , Dayaram Tajhal , Ferenczi Katalin , Wouters Bas J. , Huettner Claudia S. , Golub Todd R. , Tenen Daniel G.
Published: September 2005

We showed previously that CCAAT/enhancer binding protein α (C/EBPα), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBPα target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3β (HNF3β), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBPα. We found down-regulation of HNF3β expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3β, as well as hypermethylation of the HNF3β promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3β expression. Conditional expression of HNF3β led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3β is a novel tumor suppressor in lung cancer. This is the first study to show genetic abnormalities of lung-specific differentiation pathways in the development of lung cancer.
DNA Microarrays Reveal Relationship of Ewing Family Tumors to Both Endothelial and Fetal Neural Crest-Derived Cells and Define Novel Targets
Authors: Staege Martin S. , Hutter Christoph , Neumann Ingo , Foja Sabine , Hattenhorst Uwe E. , Hansen Gesine , Afar Danny , Burdach Stefan E. G.
Published: September 2005

Ewing family tumors (EFTs) are small round blue cell tumors that show features of neuroectodermal differentiation. However, the histogenetic origin of EFTs is still a matter of debate. We used high-density DNA microarrays for the identification of EFT-specific gene expression profiles in comparison with normal tissues of diverse origin. We identified 37 genes that are up-regulated in EFTs compared with normal tissues and validated expression of these genes in EFTs by both conventional and quantitative reverse transcription-polymerase chain reaction. The expression pattern of EFT-associated genes in normal tissues indicated a high similarity between EFTs and fetal and neuronal as well as endothelial tissues and supports the concept that a primitive neural crest-derived progenitor at the transition to mesenchymal and endothelial differentiation is transformed in EFTs. EFT-associated genes could be used for molecular discrimination between EFTs and other small round blue cell tumors and clearly identified a cell line (SK-N-MC) that was initially established as neuroblastoma as being an EFT. Ectopic expression of the EFT-specific EWS-FLI1 fusion protein in human embryonic kidney (HEK293) cells was not sufficient to induce the complete EFT-specific gene expression signature, suggesting that the EFT-specific gene expression profile is not just a consequence of EWS-FLI1 expression but depends on the histogenetic background of the EFT stem cell.
Enhancement of the Efficacy of Chemotherapy for Lung Cancer by Simultaneous Suppression of Multidrug Resistance and Antiapoptotic Cellular Defense
Authors: Pakunlu Refika I. , Wang Yang , Tsao William , Pozharov Vitaly , Cook Thomas J. , Minko Tamara
Published: September 2005

The efficacy of chemotherapy of lung cancer is limited by the development of resistance in cancer cells during treatment. In most lung cancers, this resistance is associated with the overexpression of (a) multidrug resistance-associated protein (MRP) responsible for drug efflux from the cancer cells (pump resistance) and (b) BCL2 protein that activates antiapoptotic cellular defense (nonpump resistance). A novel liposomal proapoptotic anticancer drug delivery system was developed to enhance anticancer efficacy of the well-established drug doxorubicin (DOX). This multicomponent drug delivery system was tested on multidrug-sensitive and -resistant human small-cell lung cancer cells. The drug delivery system includes four components: (a) liposome as a carrier, (b) DOX as an inductor of apoptosis, (c) antisense oligonucleotides (ASOs) targeted to MRP1 mRNA as a suppressor of pump resistance, and (d) ASOs targeted to BCL2 mRNA as a suppressor of nonpump resistance. Intracellular internalization of ASOs and DOX; the influence of the proposed system on the expression of genes and proteins involved in the multidrug resistance, cytotoxicity, and apoptosis induction and antiapoptotic defense; and the activity of caspases were studied. It was found that the proposed liposomal delivery system successfully delivered ASOs and DOX to cell nuclei, inhibited MRP1 and BCL2 protein synthesis, and substantially increased the anticancer action of DOX by stimulating the caspase-dependent pathway of apoptosis in multidrug-resistant human lung cancer cells.
Excellent
In vivo
Bystander Activity of Fludarabine Phosphate against Human Glioma Xenografts that Express the
Escherichia coli
Purine Nucleoside Phosphorylase Gene
Authors: Hong Jeong S. , Waud William R. , Levasseur Dana N. , Townes Tim M. , Wen Hui , McPherson Sylvia A. , Moore Bryan A. , Bebok Zsuzsa , Allan Paula W. , Secrist John A. , Parker William B. , Sorscher Eric J.
Published: September 2005

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.
Mek1 Alters Epidermal Growth and Differentiation
Authors: Scholl Florence A. , Dumesic Phillip A. , Khavari Paul A.
Published: September 2005

The highly homologous kinases, Mek1 and Mek2, act downstream of Ras and Raf to activate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In epidermis, Ras and Raf promote hyperplasia; however, they act on multiple Mek-independent effectors, and the extent to which Meks can mediate these effects is unknown. To address this, we expressed inducible Meks in transgenic murine and human epidermis. Both Mek1 and Mek2 triggered ERK phosphorylation. Only Mek1, however, recapitulated Ras/Raf effects in increasing proliferation and integrin expression while suppressing differentiation, which are impacts characteristic of epidermal neoplasia. Furthermore, a kinase-dead Mek1 mutant incapable of phosphorylating ERK proteins retained ability to mediate Mek1-driven epidermal proliferation. Mek1 is thus sufficient to promote the proliferative epithelial phenotype in a manner independent of intact kinase function.
Depletion of Methionine Aminopeptidase 2 Does Not Alter Cell Response to Fumagillin or Bengamides
Authors: Kim Sunkyu , LaMontagne Kenneth , Sabio Michael , Sharma Sushil , Versace Richard W. , Yusuff Naeem , Phillips Penny E.
Published: September 2005

Inhibition of endothelial cell growth by fumagillin has been assumed to be mediated by inhibition of the molecular target methionine aminopeptidase 2 (MetAp2). New data show that depletion of MetAp2 by siRNA does not inhibit endothelial cell growth. Moreover, MetAp2-depleted endothelial cells remain responsive to inhibition by either fumagillin or a newly identified MetAp2 enzyme inhibitor. These data suggest that MetAp2 function is not required for endothelial cell proliferation.
Lysophosphatidic Acid Stimulates Ovarian Cancer Cell Migration via a Ras-MEK Kinase 1 Pathway
Authors: Bian Dafang , Su Shibing , Mahanivong Chitladda , Cheng Robert K. , Han Qiwei , Pan Zhixing K. , Sun Peiqing , Huang Shuang
Published: September 2005

Lysophosphatidic acid (LPA) is present at high concentrations in ascites and plasma of ovarian cancer patients. Studies conducted in experimental models demonstrate that LPA promotes ovarian cancer invasion/metastasis by up-regulating protease expression, elevating protease activity, and enhancing angiogenic factor expression. In this study, we investigated the effect of LPA on ovarian cancer migration, an essential component of cancer cell invasion. LPA stimulates both chemotaxis and chemokinesis of ovarian cancer cells and LPA-stimulated cell migration is GI dependent. Moreover, constitutively active H-Ras enhances ovarian cancer cell migration, whereas dominant negative H-Ras blocks LPA-stimulated cell migration, suggesting that Ras works downstream of Gi to mediate LPA-stimulated cell migration. Interestingly, H-Ras mutants that specifically activate Raf-1, Ral-GDS, or phosphatidylinositol 3′-kinase are unable to significantly enhance ovarian cancer cell migration, suggesting that a Ras downstream effector distinct from Raf-1, Ral-GDS, and phosphatidylinositol 3′-kinase is responsible for LPA-stimulated cell migration. In this article, we demonstrate that LPA activates mitogen-activated protein kinase kinase 1 (MEKK1) in a Gi-Ras-dependent manner and that MEKK1 activity is essential for LPA-stimulated ovarian cancer cell migration. Inhibitors that block MEKK1 downstream pathways, including MEK1/2, MKK4/7, and nuclear factor-κB pathways, do not significantly alter LPA-stimulated cell migration. Instead, LPA induces the redistribution of focal adhesion kinase to focal contact regions of the cytoplasm membrane, and this event is abolished by pertussis toxin, dominant negative H-Ras, or dominant negative MEKK1. Our studies thus suggest that the Gi-Ras-MEKK1 signaling pathway mediates LPA-stimulated ovarian cancer cell migration by facilitating focal adhesion kinase redistribution to focal contacts.
Infectivity-Enhanced Cyclooxygenase-2-Based Conditionally Replicative Adenoviruses for Esophageal Adenocarcinoma Treatment
Authors: Davydova Julia , Le Long P. , Gavrikova Tatyana , Wang Minghui , Krasnykh Victor , Yamamoto Masato
Published: September 2005

The employment of conditionally replicative adenoviruses (CRAd) constitutes a promising alternative for cancer treatment; however, in the case of esophageal adenocarcinoma (EAC) the lack of an appropriate tumor-specific promoter and relative resistance to adenovirus infection have hampered the construction of CRAds with clinically applicable specificity and efficacy. By combining transcriptional targeting with infectivity enhancement for CRAds, we generated novel cyclooxygenase-2 (Cox-2) promoter-controlled replicative viral agents for the treatment of EAC. We used infectivity enhancement based on incorporation of an RGD-4C motif into the HI loop of the adenoviral (Ad) fiber knob domain as well as replacement of the Ad5 knob with the Ad3 knob. The Cox-2 promoter was highly active in EAC, whereas showing no significant activity in Cox-2-negative cell lines and primary cells isolated from normal mouse esophagus and stomach. Evaluation of infectivity-enhanced vectors revealed that the transduction and virus-cell binding ability of Ad5/Ad3-chimera were significantly more efficient than that of unmodified and Arg-Gly-Asp (RGD)-modified vectors. All of the Cox-2 CRAds demonstrated replication and subsequent oncolysis in EAC cells but not in Cox-2-negative cells in vitro, thus confirming the dependence of their replication on the Cox-2 promoter activity. Ad5/Ad3 CRAds exhibited significantly improved oncolysis and progeny production compared with unmodified and RGD-modified vectors without sacrificing tumor selectivity. Whereas unmodified and RGD-modified CRAds showed insignificant therapeutic effect in vivo, Ad5/Ad3 CRAds remarkably suppressed tumor growth of established xenografts in mice. Thus, our studies have demonstrated that Ad5/Ad3-chimeric Cox-2 promoter-driven CRAds are selective and potent agents for the treatment of EAC.
The Matrix Metalloproteinase Inhibitor Prinomastat Enhances Photodynamic Therapy Responsiveness in a Mouse Tumor Model
Authors: Ferrario Angela , Chantrain Christophe F. , von Tiehl Karl , Buckley Sue , Rucker Natalie , Shalinsky David R. , Shimada Hiroyuki , DeClerck Yves A. , Gomer Charles J.
Published: September 2005

Photodynamic therapy (PDT) clinical results are promising; however, tumor recurrences can occur and, therefore, methods for improving treatment efficacy are needed. PDT elicits direct tumor cell death and microvascular injury as well as expression of angiogenic, inflammatory, and prosurvival molecules. Preclinical studies combining antiangiogenic drugs or cyclooxygenase-2 inhibitors with PDT show improved treatment responsiveness (A. Ferrario et al., Cancer Res 2000;60:4066–9; A. Ferrario et al., Cancer Res 2002;62:3956–61). In the present study, we evaluated the role of Photofrin-mediated PDT in eliciting expression of matrix metalloproteinases (MMPs) and modulators of MMP activity. We also examined the efficacy of a synthetic MMP inhibitor, Prinomastat, to enhance tumoricidal activity after PDT, using a mouse mammary tumor model. Immunoblot analysis of extracts from PDT-treated tumors demonstrated strong expression of MMPs and extracellular MMP inducer along with a concomitant decrease in expression of tissue inhibitor of metalloproteinase-1. Gelatin zymography and enzyme activity assays performed on protein extracts from treated tumors confirmed the induction of both latent and enzymatically active forms of MMP-9. Immunohistochemical analysis indicated that infiltrating inflammatory cells and endothelial cells were primary sources of MMP-9 expression after PDT, whereas negligible expression was observed in tumor cells. Administration of Prinomastat significantly improved PDT-mediated tumor response (P = 0.02) without affecting normal skin photosensitization. Our results indicate that PDT induces MMPs and that the adjunctive use of an MMP inhibitor can improve PDT tumor responsiveness.
External Beam Radiation of Tumors Alters Phenotype of Tumor Cells to Render Them Susceptible to Vaccine-Mediated T-Cell Killing
Authors: Chakraborty Mala , Abrams Scott I. , Coleman C. Norman , Camphausen Kevin , Schlom Jeffrey , Hodge James W.
Published: September 2005

Local radiation is an established therapy for human tumors. Radiation also has been shown to alter the phenotype of target tissue, including gene products that may make tumor cells more susceptible to T-cell-mediated immune attack. We demonstrate a biological synergy between local radiation of tumor and active vaccine therapy. The model used consisted of mice transgenic for human carcinoembryonic antigen (CEA) and a murine carcinoma cell line transfected with CEA. The vaccine regimen consisted of a prime and boost strategy using vaccinia and avipox recombinants expressing CEA and three T-cell costimulatory molecules. One dose of 8-Gy radiation to tumor induced up-regulation of the death receptor Fas in situ for up to 11 days. However, neither radiation at this dose nor vaccine therapy was capable of inhibiting growth of 8-day established tumor. When vaccine therapy and local radiation of tumor were used in combination, dramatic and significant cures were achieved. This was mediated by the engagement of the Fas/Fas ligand pathway because Ag-bearing tumor cells expressing dominant-negative Fas were not susceptible to this combination therapy. Following the combination of vaccine and local radiation, tumors demonstrated a massive infiltration of T cells not seen with either modality alone. Mice cured of tumors demonstrated CD4+ and CD8+ T-cell responses specific for CEA but also revealed the induction of high levels of T-cell responses to two other antigens (gp70 and p53) overexpressed in tumor, indicating the presence of a consequential antigen cascade. Thus, these studies demonstrate a new paradigm for the use of local tumor irradiation in combination with active specific vaccine therapy to elicit durable antitumor responses of established tumors.
An Autoantibody-Mediated Immune Response to Calreticulin Isoforms in Pancreatic Cancer
Authors: Hong Su-Hyung , Misek David E. , Wang Hong , Puravs Eric , Giordano Thomas J. , Greenson Joel K. , Brenner Dean E. , Simeone Diane M. , Logsdon Craig D. , Hanash Samir M.
Published: September 2005

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Aliquots of solubilized proteins from a pancreatic cancer cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which sera of individual patients were tested for primary antibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis, 33 patients with other cancers, and 15 healthy subjects were analyzed. Autoantibodies were detected against either one or two calreticulin isoforms identified by mass spectrometry in sera from 21 of 36 patients with pancreatic cancer. One of 18 chronic pancreatitis patients and 1 of 15 healthy controls demonstrated autoantibodies to calreticulin isoform 1; none demonstrated autoantibodies to isoform 2. None of the sera from patients with colon cancer exhibited reactivity against either of these two proteins. One of 14 sera from lung adenocarcinoma patients demonstrated autoantibodies to calreticulin isoform 1; 2 of 14 demonstrated autoantibodies to isoform 2. Immunohistochemical analysis of calreticulin in pancreatic/ampullary tumor tissue arrays using an isoform nonspecific antibody revealed diffuse and consistent cytoplasmic staining in the neoplastic epithelial cells of the pancreatic and ampullary adenocarcinomas. The detection of autoantibodies to calreticulin isoforms may have utility for the early diagnosis of pancreatic cancer.
Phytoestrogens/Flavonoids Reverse Breast Cancer Resistance Protein/ABCG2-Mediated Multidrug Resistance
Authors: Imai Yasuo , Tsukahara Satomi , Asada Sakiyo , Sugimoto Yoshikazu
Published: September 2005

Breast cancer resistance protein (BCRP), also called ABCG2, confers resistance to anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan. We found previously that sulfated estrogens are physiologic substrates of BCRP. Flavonoids with weak estrogenic activities are called phytoestrogens. In this study, we show that phytoestrogens/flavonoids, such as genistein, naringenin, acacetin, and kaempferol, potentiated the cytotoxicity of SN-38 and mitoxantrone in BCRP-transduced K562 (K562/BCRP) cells. Some glycosylated flavonoids, such as naringenin-7-glucoside, also effectively inhibited BCRP. These flavonoids showed marginal effect on the drug sensitivity of K562 cells. Genistein and naringenin reversed neither P-glycoprotein-mediated vincristine resistance nor multidrug resistance-related protein 1-mediated VP-16 resistance. Genistein and naringenin increased cellular accumulation of topotecan in K562/BCRP cells. K562/BCRP cells also accumulated less [3H]genistein than K562 cells. [3H]genistein transport in the basal-to-apical direction was greater in BCRP-transduced LLC-PK1 (LLC/BCRP) cells, which express exogenous BCRP in the apical membrane, than in parental cells. Fumitremorgin C abolished the increased transport of [3H]genistein in LLC/BCRP cells compared with parental cells. TLC analysis revealed that genistein was transported in its native form but not in its metabolized form. These results suggest that genistein is among the natural substrates of BCRP and competitively inhibits BCRP-mediated drug efflux. The results have two important clinical implications: (a) flavonoids and glycosylated flavonoids may be useful in overcoming BCRP-mediated drug resistance in tumor cells; and (b) coadministration of flavonoids with BCRP-substrate antitumor agents may alter the pharmacokinetics and consequently increase the toxicity of specific antitumor agents in cancer patients.
Gefitinib Enhances the Antitumor Activity and Oral Bioavailability of Irinotecan in Mice
Authors: Stewart Clinton F. , Leggas Markos , Schuetz John D. , Panetta John C. , Cheshire Pamela J. , Peterson Jennifer , Daw Najat , Jenkins Jesse J. , Gilbertson Richard , Germain Glen S. , Harwood Franklin C. , Houghton Peter J.
Published: September 2005

As a single agent the ERBB1 inhibitor, gefitinib (Iressa; ZD1839) showed minimal activity against a panel of 10 pediatric tumor xenografts that do not express the ERBB1 receptor. However, combined with irinotecan (CPT-11), significantly greater than additive activity was observed in four of eight models (P < 0.05), and the combination showed enhanced activity against three additional tumor lines. Breast cancer resistance protein (ABCG2), a transporter that confers resistance to SN-38 (the active metabolite of irinotecan), was readily detected in six of nine xenograft models examined by immunohistochemistry. In vitro gefitinib potently reversed resistance to SN-38 only in a cell line that overexpressed functional ABCG2. However, overexpression of ABCG2 did not decrease accumulation nor increase the rate of efflux of [14C]gefitinib. On the basis of these results and the distribution of Abcg2 in mouse tissues, we assessed the ability of gefitinib to modulate irinotecan pharmacokinetics. Oral gefitinib coadministration resulted in no change in clearance of intravenously administered irinotecan. However, gefitinib treatment dramatically increased the oral bioavailability of irinotecan after simultaneous oral administration. It is concluded that gefitinib may modulate SN-38 activity at the cellular level to reverse tumor resistance mediated by ABCG2 through inhibiting drug efflux and may be used potentially in humans to modulate the oral bioavailability of a poorly absorbed camptothecin such as irinotecan.
Caveolin-1 Is Required for Vascular Endothelial Growth Factor-Triggered Multiple Myeloma Cell Migration and Is Targeted by Bortezomib
Authors: Podar Klaus , Shringarpure Reshma , Tai Yu-Tzu , Simoncini Melissa , Sattler Martin , Ishitsuka Kenji , Richardson Paul G. , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C.
Published: September 2005

We recently demonstrated that caveolae, vesicular flask-shaped invaginations of the plasma membrane, represent novel therapeutic targets in multiple myeloma. In the present study, we demonstrate that vascular endothelial growth factor (VEGF) triggers Src-dependent phosphorylation of caveolin-1, which is required for p130Cas phosphorylation and multiple myeloma cell migration. Conversely, depletion of caveolin-1 by antisense methodology abrogates p130Cas phosphorylation and VEGF-triggered multiple myeloma cell migration. The proteasome inhibitor bortezomib both inhibited VEGF-triggered caveolin-1 phosphorylation and markedly decreased caveolin-1 expression. Consequently, bortezomib inhibited VEGF-induced multiple myeloma cell migration. Bortezomib also decreased VEGF secretion in the bone marrow microenvironment and inhibited VEGF-triggered tyrosine phosphorylation of caveolin-1, migration, and survival in human umbilical vascular endothelial cells. Taken together, these studies demonstrate the requirement of caveolae for VEGF-triggered multiple myeloma cell migration and identify caveolin-1 in multiple myeloma cells and human umbilical vascular endothelial cells as a molecular target of bortezomib.
DNA Polymerase β Interacts with TRF2 and Induces Telomere Dysfunction in a Murine Mammary Cell Line
Authors: Fotiadou Poppy , Henegariu Octavian , Sweasy Joann B.
Published: September 2005

DNA polymerase β (Polβ) is a DNA repair protein that functions in base excision repair and meiosis. The enzyme has deoxyribose phosphate lyase and polymerase activity, but it is error prone because it bears no proofreading activity. Errors in DNA repair can lead to the accumulation of mutations and consequently to tumorigenesis. Polβ expression has been found to be higher in tumors, and deregulation of its expression has been found to induce chromosomal instability, a hallmark of tumorigenesis, but the underlying mechanisms are unclear. In the present study, we have investigated whether ectopic expression of Polβ influences the stability of chromosomes in a murine mammary cell line. The results demonstrate a telomere dysfunction phenotype: an increased rate of telomere loss and chromosome fusion, suggesting that ectopic expression of Polβ leads to telomere dysfunction. In addition, Polβ interacts with TRF2, a telomeric DNA binding protein. Colocalization of the two proteins occurs at nontelomeric sites and appears to be influenced by the change in the status of the telomeric complex.
In Vitroandin VivoActivity of the Maytansinoid Immunoconjugate huN901-N2′-Deacetyl-N2′-(3-Mercapto-1-Oxopropyl)-Maytansine against CD56+ Multiple Myeloma Cells
Authors: Tassone Pierfrancesco , Gozzini Antonella , Goldmacher Victor , Shammas Masood A. , Whiteman Kathleen R. , Carrasco Daniel R. , Li Cheng , Allam Charles K. , Venuta Salvatore , Anderson Kenneth C. , Munshi Nikhil C.
Published: September 2005

HuN901 is a humanized monoclonal antibody that binds with high affinity to CD56, the neuronal cell adhesion molecule. HuN901 conjugated with the maytansinoid N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), a potent antimicrotubular cytotoxic agent, may provide targeted delivery of the drug to CD56 expressing tumors. Based on gene expression profiles of primary multiple myeloma (MM) cells showing expression of CD56 in 10 out of 15 patients (66.6%) and flow cytometric profiles of MM (CD38brightCD45lo) cells showing CD56 expression in 22 out of 28 patients (79%), we assessed the efficacy of huN901-DM1 for the treatment of MM. We first examined the in vitro cytotoxicity and specificity of huN901-DM1 on a panel of CD56+ and CD56− MM cell lines, as well as a CD56− Waldenstrom’s macroglobulinemia cell line. HuN901-DM1 treatment selectively decreased survival of CD56+ MM cell lines and depleted CD56+ MM cells from mixed cultures with a CD56− cell line or adherent bone marrow stromal cells. In vivo antitumor activity of huN901-DM1 was then studied in a tumor xenograft model using a CD56+ OPM2 human MM cell line in SCID mice. We observed inhibition of serum paraprotein secretion, inhibition of tumor growth, and increase in survival of mice treated with huN901-DM1. Our data therefore demonstrate that huN901-DM1 has significant in vitro and in vivo antimyeloma activity at doses that are well tolerated in a murine model. Taken together, these data provide the framework for clinical trials of this agent to improve patient outcome in MM.
Verapamil and Its Derivative Trigger Apoptosis through Glutathione Extrusion by Multidrug Resistance Protein MRP1
Authors: Trompier Doriane , Chang Xiu-Bao , Barattin Régis , d’Hardemare Amaury du Moulinet , Di Pietro Attilio , Baubichon-Cortay Hélène
Published: September 2005

This study demonstrates that verapamil and a newly synthesized verapamil derivative, NMeOHI2, behave as apoptogens in multidrug resistance protein 1 (MRP1)-expressing cells. When treated with either verapamil or NMeOHI2, surprisingly, baby hamster kidney-21 (BHK) cells transfected with human MRP1 were killed. Because parental BHK cells were not, as well as cells expressing an inactive (K1333L) MRP1 mutant, this indicated that cell death involved functional MRP1 transporter. Cell death was identified as apoptosis by using annexin V-fluorescein labeling and was no longer observed in the presence of the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F (Z-VAD-FMK). In vitro, both verapamil and its derivative inhibited leukotriene C4 transport by MRP1-enriched membrane vesicles in a competitive manner, with a Ki of 48.6 μm for verapamil and 5.5 μm for NMeOHI2, and stimulated reduced glutathione (GSH) transport 3-fold and 9-fold, respectively. Treatment of MRP1-expressing cells with either verapamil or the derivative quickly depleted intracellular GSH content with a strong decrease occurring in the first hour of treatment, which preceded cell death beginning at 8–16 h. Furthermore, addition of GSH to the media efficiently prevented cell death. Therefore, verapamil and its derivative trigger apoptosis through stimulation of GSH extrusion mediated by MRP1. This new information on the mechanism of induced apoptosis of MDR cells may represent a novel approach in the selective treatment of MRP1-positive tumors.
Restoration of Bone Morphogenetic Protein Receptor Type II Expression Leads to a Decreased Rate of Tumor Growth in Bladder Transitional Cell Carcinoma Cell Line TSU-Pr1
Authors: Kim Isaac Yi , Lee Dong-Hyeon , Lee Dug Keun , Kim Wun Jae , Kim Moses M. , Morton Ronald A. , Lerner Seth P. , Kim Seong Jin
Published: September 2005

Bone morphogenetic proteins (BMPs), potential regulators of cellular growth and metastasis that signal through an interaction with plasma membrane receptors, have been suggested to be important regulators of malignant cells. The present study was carried out to evaluate the potential role of BMP receptor (BMP-R) types IA, IB, and II in bladder transitional cell carcinoma (TCC) cells. Initially, we investigated the expression of these BMP-Rs in 30 archival tissues of human bladder TCC using immunohistochemistry; 10 benign bladder specimens were used for comparison. The results demonstrated that the expression of BMP-Rs is localized preferentially to the transitional epithelium and that there was a significant association between loss of BMP-RII expression and tumor grade. To find a cell line that can serve as a model system for clinical observation, we subsequently examined sensitivity to BMP-4 and expression of BMP-RII, BMP-RIA, and BMP-RIB in three human bladder cancer cell lines, TCC-Sup, RT4, and TSU-Pr1. Of the three cell lines, TSU-Pr1 exhibited a decreased level of BMP-RII expression and was resistant to the growth-inhibitory effect of BMP-4. Overexpression of BMP-RII in TSU-Pr1 cells not only restored BMP-4 responsiveness but also significantly decreased tumorigenicity in vivo. Taken together, these results demonstrate that human bladder TCC tissues have a frequent loss of BMP-RII expression and that overexpression of BMP-RII leads to restoration of BMP signaling and decreased tumor growth in the human bladder TCC cell line TSU-Pr1.
Krüppel-Like Factor 6 Is Frequently Down-Regulated and Induces Apoptosis in Non-Small Cell Lung Cancer Cells
Authors: Ito Genshi , Uchiyama Mika , Kondo Masashi , Mori Shoichi , Usami Noriyasu , Maeda Osamu , Kawabe Tsutomu , Hasegawa Yoshinori , Shimokata Kaoru , Sekido Yoshitaka
Published: September 2005

Krüppel-like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcriptional factor, which has been suggested to be a candidate tumor suppressor gene in prostate cancer and astrocytic glioma. Because KLF6 is located at chromosome 10p15, where non-small cell lung cancers (NSCLCs) also exhibit frequent allelic loss, we hypothesized that the inactivation of KLF6 is also involved in the development of NSCLC. To determine this, we performed mutational analysis for 105 NSCLCs, including 9 cell lines and 96 primary tumors, and Northern blot analysis for 74 NSCLCs, including the 9 cell lines and 65 primary tumors. Although somatic mutations were not detected in the coding sequence of KLF6, expression of KLF6 mRNA was down-regulated in the 9 cell lines and in 55 (85%) of the 65 primary tumors compared with normal lung tissue. Treatment of two cell lines expressing KLF6 at low levels with 5-azacytidine did not induce KLF6 expression, suggesting that KLF6 down-regulation is not due to promoter hypermethylation. We also performed loss of heterozygosity (LOH) analysis using the laser capture microdissection technique, and found that 21 of 62 (34%) informative samples had LOH in the KLF6 gene locus. Comparing the LOH status with mRNA expression of KLF6, we found that 14 of the 14 (100%) samples with LOH showed KLF6 down-regulation, and that even 23 of 31 (74%) samples without LOH also showed this down-regulation. We also studied the expression of the WAF1 gene, a possible downstream gene of KLF6, and detected simultaneous down-regulation of WAF1 and KLF6 mRNA in 6 of 9 (67%) cell lines and 48 of the 55 (87%) primary tumors, although there was not a significant association between loss of KLF6 and WAF1 expression. Furthermore, colony formation assay of two NSCLC cell lines (NCI-H1299 and NCI-H2009) induced a markedly reduced colony formation by KLF6 transfection, and Annexin V staining and terminal deoxynucleotidyl transferase-mediated nick end labeling assays revealed that KLF6 induced apoptosis. Our present studies demonstrated that KLF6 is frequently down-regulated in NSCLC and suppresses tumor growth via induction of apoptosis in NSCLC, which may suggest that KLF6 is a tumor suppressor for NSCLC.
Synergistic Tumor Suppression by Coexpression of FHIT and p53 Coincides with FHIT-Mediated MDM2 Inactivation and p53 Stabilization in Human Non-Small Cell Lung Cancer Cells
Authors: Nishizaki Masahiko , Sasaki Ji-ichiro , Fang Bingliang , Atkinson Edward. N. , Minna John D. , Roth Jack A. , Ji Lin
Published: September 2005

Aberrations of the tumor suppressor genes FHIT and p53 are frequently associated with a wide range of human cancers, including lung cancer. We studied the combined effects of FHIT and p53 proteins on tumor cell proliferation and apoptosis in human non-small cell lung carcinoma (NSCLC) cells in vitro and on tumor growth in animal models by adenoviral vector-mediated cotransfer of wild-type FHIT and p53 genes. We found that the coexpression of FHIT and p53 synergistically inhibited tumor cell proliferation in NSCLC cells in vitro and suppressed the growth of human tumor xenografts in nude mice. Furthermore, we found that this synergistic inhibition of tumor cell growth corresponded with the FHIT-mediated inactivation of MDM2, which thereby blocked the association of MDM2 with p53, thus stabilizing the p53 protein. Our results therefore reveal a novel molecular mechanism consisting of FHIT-mediated tumor suppression and the interaction of FHIT with other cellular components in the pathways regulating p53 activity. These findings show that combination treatment with synergistic tumor-suppressing gene therapy such as Ad-FHIT and Ad-p53 may be an effective therapeutic strategy for NSCLC and other cancers.