bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Gene Expression in Metastasizing Cells Shed from Kidney Tumors
Authors: Bockhorn Maximilian , Roberge Sylvie , Sousa Cristina , Jain Rakesh K. , Munn Lance L.
Published: September 2005

We developed a novel orthotopic mouse tumor model of renal cell carcinoma to collect and characterize cells spontaneously shed from SN12C (renal cell carcinoma) and SN12L1 (high metastatic variant of SN12C) tumors grown in kidneys of severe combined immunodeficient mice. Viability of the shed cell population was greater for SN12L1 tumors (25%) compared with SN12C tumors (11%, P < 0.05). Gene array analysis of 23 genes involved in metastasis showed that CD44, α3 integrin, and caveolin were down-regulated in the shed tumor cells compared with their primary counterparts, and blocking α3 integrin or CD44 function inhibited attachment and migration of both cell lines. These results suggest that cohesion of the cells within the primary tumor mediated by CD44 and α3 integrins hinders metastasis and that shedding is a passive process not necessarily mediated by cell migration in these tumors. Furthermore, resistance to apoptosis may enhance metastasis in the higher metastatic tumor.
Prolonged Culture of Vaccine-Primed Lymphocytes Results in Decreased Antitumor Killing and Change in Cytokine Secretion
Authors: Sussman Jeffrey J. , Parihar Robin , Winstead Karen , Finkelman Fred D.
Published: September 2005

Adoptive transfer of effector T cells has been used successfully to eliminate metastases in animal models. Because antitumor activity depends on the number of effector cells transferred, some human trials have used in vitro-repetitive activation and expansion techniques to increase cell number. We hypothesized that the prolonged culture period might contribute to the lack of human trial success by decreasing the potency of the effector T cells. Lymph nodes draining a progressively growing murine melanoma tumor transduced to secrete granulocyte/macrophage colony-stimulating factor were harvested and activated in vitro with anti-CD3 monoclonal antibody followed by expansion in IL-2 for a total of 5 days in culture. Some lymphocytes were reactivated and further expanded for a total of 9 days in culture. In vivo activity of the effector T cells was measured by the reduction in lung metastases and is shown to be dose dependent. The prolonged culture period resulted in nearly 3-fold more T cells but at least 8-fold less antitumor activity. This was accompanied by decreased secretion of the proinflammatory cytokine, IFN-γ, and increased secretion of the anti-inflammatory cytokine, IL-10. Thus, although increased cell number is important to maximize the effectiveness of adoptive immunotherapy, some culture conditions may actually be counterproductive in that decreases in cell potency can outweigh the benefits of increased cell numbers. The T-cell cytokine secretion pattern predicts decreased effector cell function and may explain the decreased antitumor effect.
In Vitroandin VivoCytotoxic Activities of Recombinant Immunotoxin 8H9(Fv)-PE38 against Breast Cancer, Osteosarcoma, and Neuroblastoma
Authors: Onda Masanori , Wang Qing-cheng , Guo Hong-fen , Cheung Nai-Kong V. , Pastan Ira
Published: September 2005

The 8H9 monoclonal antibody (MAb) is highly reactive with a cell surface glycoprotein expressed on human breast cancers, childhood sarcomas, and neuroblastomas but is not reactive with the cell surface of normal human tissues. This specific reactivity suggests that MAb 8H9 may be useful for targeted cancer therapy. To explore this possibility, we generated two recombinant immunotoxins (ITs) using the single-chain Fv (scFv) of MAb 8H9. Initially the 8H9(scFv) cDNA was fused to a DNA encoding a 38-kDa truncated form of Pseudomonas exotoxin (PE38) to generate the IT 8H9(scFv)-PE38. The fusion gene was expressed in Escherichia coli, and the IT was purified to near homogeneity from inclusion bodies. The purified IT showed specific cytotoxicity on nine different cancer cell lines derived from breast cancer, osteosarcoma, and neuroblastomas, known to react with MAb 8H9. The cytotoxic activity was inhibited by MAb 8H9, showing the cytotoxic activity is specific. The antitumor activity of 8H9(scFv)-PE38 was evaluated in severe combined immunodeficient mice bearing MCF-7 breast cancers or OHS-M1 osteosarcomas. The IT showed a specific dose-dependent antitumor activity at 0.075 and 0.15 mg/kg. Next, a more stable disulfide-linked IT, 8H9(dsFv)-PE38, was constructed. It was produced in high yield (16%) and showed cytotoxic and antitumor activities similar to those of 8H9(scFv)-PE38. 8H9(dsFv)-PE38 was given to two cynomolgus monkeys at doses of 0.1 and 0.2 mg/kg i.v. QOD × 3 and was well tolerated. This shows that a dose that causes significant tumor regressions in mice is well tolerated by monkeys. These results make 8H9(dsFv)-PE38 a candidate for further development as a therapeutic agent for breast cancers, osteosarcomas, and neuroblastomas.
Determination and Modeling of Kinetics of Cancer Cell Killing by Doxorubicin and Doxorubicin Encapsulated in Targeted Liposomes
Authors: Eliaz Rom E. , Nir Shlomo , Marty Cornelia , Szoka Francis C.
Published: September 2005

Various mathematical approaches have been devised to relate the cytotoxic effect of drugs in cell culture to the drug concentration added to the cell culture medium. Such approaches can satisfactorily account for drug response when the drugs are free in solution, but the approach becomes problematic when the drug is delivered in a drug delivery system, such as a liposome. To address this problem, we have developed a simple model that assumes that the cytotoxic potency of a drug is a function of the intracellular drug level in a critical compartment. Upon exposure to drug, cell death commences after a lag time, and the cell kill rate is dependent on the amount of drug in the critical intracellular compartment. The computed number of cells in culture, at any time after exposure to the drug, takes into account the cell proliferation rate, the cell kill rate, the average intracellular drug concentration, and a lag time for cell killing. We have applied this model to compare the cytotoxic effect of doxorubicin (DOX), or DOX encapsulated in a liposome that is targeted to CD44 on B16F10 melanoma cells in culture. CD44 is the surface receptor that binds to hyaluronan and is overexpressed on various cancer cells, including B16F10. We have shown previously that the drug encapsulated in hyaluronan-targeted liposomes was more potent than was the free drug. The model required the determination of the cell-associated DOX after the cells were incubated with various concentrations of the free or the encapsulated drug for 3 h, and the quantification of cell number at various times after exposure to the drug. The uptake of encapsulated drug was greater than that of the free drug, and the ratio of cell association of encapsulated:free drug was 1.3 at 0.5 μg/ml and increased to 3.3 at 20 μg/ml DOX. The results demonstrate that the enhanced potency of the encapsulated drug could stem from its enhanced uptake. However, in certain cases, where larger amounts of the free drug were added, such that the intracellular amounts of drug exceeded those obtained from the encapsulated drug, the numbers of viable cells were still significantly smaller for the encapsulated drug. This finding demonstrates that for given amounts of intracellular DOX, the encapsulated form was more efficient in killing B16F10 cells than the free drug. The outcome was expressed in the kinetic model as a 5–6-fold larger rate constant of cell killing potency for the encapsulated drug versus the free drug. The model provides a quantitative framework for comparing the cytotoxic effect in cultured cells when applying the drug in the free form or in a delivery system.
Histone Deacetylase Is a Target of Valproic Acid-Mediated Cellular Differentiation
Authors: Gurvich Nadia , Tsygankova Oxana M. , Meinkoth Judy L. , Klein Peter S.
Published: September 2005

Valproic acid (VPA), a well-established therapy for seizures and bipolar disorder, has recently been shown to inhibit histone deacetylases (HDACs). Similar to more widely studied HDAC inhibitors, VPA can cause growth arrest and induce differentiation of transformed cells in culture. Whether this effect of VPA is through inhibition of HDACs or modulation of another target of VPA has not been tested. We have used a series of VPA analogs to establish a pharmacological profile for HDAC inhibition. We find that VPA and its analogs inhibit multiple HDACs from class I and class II (but not HDAC6 or HDAC10) with a characteristic order of potency in vitro. These analogs also induce hyperacetylation of core histones H3 and H4 in intact cells with an order of potency that parallels in vitro inhibition. VPA and VPA analogs induce differentiation in hematopoietic cell lines in a p21-dependent manner, and the order of potency for induction of differentiation parallels the potencies for inhibition in vitro, as well as for acetylation of histones associated with the p21 promoter, supporting the argument that differentiation caused by VPA is mediated through inhibition of HDACs. These findings provide additional evidence that VPA, a well-tolerated, orally administered drug with extensive clinical experience, may serve as an effective chemotherapeutic agent through targeting of HDACs.
Expression Profiling of t(12;22) Positive Clear Cell Sarcoma of Soft Tissue Cell Lines Reveals Characteristic Up-Regulation of Potential New Marker Genes Including
ERBB3
Authors: Schaefer Karl-Ludwig , Brachwitz Kristin , Wai Daniel H. , Braun Yvonne , Diallo Raihanatou , Korsching Eberhard , Eisenacher Martin , Voss Reinhard , van Valen Frans , Baer Claudia , Selle Barbara , Spahn Laura , Liao Shuen-Kuei , Lee Kevin A. W. , Hogendoorn Pancras C. W. , Reifenberger Guido , Gabbert Helmut E. , Poremba Christopher
Published: September 2005

Clear cell sarcoma of soft tissue (CCSST), also known as malignant melanoma of soft parts, represents a rare lesion of the musculoskeletal system usually affecting adolescents and young adults. CCSST is typified by a chromosomal t(12;22)(q13;q12) translocation resulting in a fusion between the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1), of which the activity in nontransformed cells is regulated by cyclic AMP. Our aim was to identify critical differentially expressed genes in CCSST tumor cells in comparison with other solid tumors affecting children and young adults to better understand signaling pathways regulating specific features of the development and progression of this tumor entity. We applied Affymetrix Human Genome U95Av2 oligonucleotide microarrays representing ∼12,000 genes to generate the expression profiles of the CCSST cell lines GG-62, DTC-1, KAO, MST2, MST3, and Su-CC-S1 in comparison with 8 neuroblastoma, 7 Ewing tumor, and 6 osteosarcoma cell lines. Subsequent hierarchical clustering of microarray data clearly separated all four of the tumor types from each other and identified differentially expressed transcripts, which are characteristically up-regulated in CCSST. Statistical analysis revealed a group of 331 probe sets, representing ∼300 significant (P < 0.001) differentially regulated genes, which clearly discriminated between the CCSST and other tumor samples. Besides genes that were already known to be highly expressed in CCSST, like S100A11 (S100 protein) or MITF (microphthalmia-associated transcription factor), this group shows an obvious portion of genes that are involved in cyclic AMP response or regulation, in pigmentation processes, or in neuronal structure and signaling. Comparison with other expression profile analyses on neuroectodermal childhood tumors confirms the high robustness of this strategy to characterize tumor entities based on their gene expression. We found the avian erythroblastic leukemia viral oncogene homologue 3 (ERBB3) to be one of the most dramatically up-regulated genes in CCSST. Quantitative real-time PCR and Northern blot analysis verified the mRNA abundance and confirmed the absence of the inhibitory transcript variant of this gene. The protein product of the member of the epidermal growth factor receptor family ERBB3 could be shown to be highly present in all of the CCSST cell lines investigated, as well as in 18 of 20 primary tumor biopsies. In conclusion, our data demonstrate new aspects of the phenotype and the biological behavior of CCSST and reveal ERBB3 to be a useful diagnostic marker.
The Tumor Suppressor p53 Down-Regulates Glucose Transporters
GLUT1
and
GLUT4
Gene Expression
Authors: Schwartzenberg-Bar-Yoseph Fabiana , Armoni Michal , Karnieli Eddy
Published: September 2005

Tumorigenesis is associated with enhanced cellular glucose uptake and increased metabolism. Because the p53 tumor suppressor is mutated in a large number of cancers, we evaluated whether p53 regulates expression of the GLUT1 and GLUT4 glucose transporter genes. Transient cotransfection of osteosarcoma-derived SaOS-2 cells, rhabdomyosarcoma-derived RD cells, and C2C12 myotubes with GLUT1-P-Luc or GLUT4-P-Luc promoter-reporter constructs and wild-type p53 expression vectors dose dependently decreased both GLUT1 and GLUT4 promoter activity to approximately 50% of their basal levels. PG13-Luc activity, which was used as a positive control for functional p53 expression, was increased up to ∼250-fold by coexpression of wild-type p53. The inhibitory effect of wild-type p53 was greatly reduced or abolished when cells were transfected with p53 with mutations in amino acids 143, 248, or 273. A region spanning −66/+163 bp of the GLUT4 promoter was both necessary and sufficient to mediate the inhibitory effects of p53. Furthermore, in vitro translated p53 protein was found to bind directly to two sequences in that region. p53-DNA binding was completely abolished by excess unlabeled probe but not by nonspecific DNA and was super-shifted by the addition of an anti-p53 antibody. Taken together, our data strongly suggest that wild-type p53 represses GLUT1 and GLUT4 gene transcription in a tissue-specific manner. Mutations within the DNA-binding domain of p53, which are usually associated with malignancy, were found to impair the repressive effect of p53 on transcriptional activity of the GLUT1 and GLUT4 gene promoters, thereby resulting in increased glucose metabolism and cell energy supply. This, in turn, would be predicted to facilitate tumor growth.
Tumor Suppressor von Hippel-Lindau (VHL) Stabilization of Jade-1 Protein Occurs through Plant Homeodomains and Is VHL Mutation Dependent
Authors: Zhou Mina I. , Wang Hongmei , Foy Rebecca L. , Ross Jonathan J. , Cohen Herbert T.
Published: September 2005

The von Hippel-Lindau (VHL) gene is the major renal cancer gene in adults. The mechanism of renal tumor suppression by VHL protein is only partly elucidated. VHL loss increases expression of the hypoxia-inducible factor α transcription factors. However, clinical and biochemical data indicate that the hypoxia-inducible factors are necessary but not sufficient for renal tumorigenesis, which suggests other VHL effector pathways are involved. Jade-1 protein interacts strongly with VHL and is most highly expressed in renal proximal tubules, precursor cells of renal cancer. Short-lived Jade-1 protein contains plant homeodomain (PHD) and candidate PEST degradation motifs and is substantially stabilized by VHL. The effect of VHL on Jade-1 protein abundance and relative protein stability was further examined in immunoblots and metabolic labeling experiments using two time points. VHL-Jade-1 binding was tested in coimmunoprecipitations. In cotransfection studies with wild-type VHL, the Jade-1 PHD-extended PHD module, not the candidate PEST domain, was required for full VHL-mediated stabilization. This module is also found in leukemia transcription factors AF10 and AF17, as well as closely related Jade-like proteins, which suggests all might be VHL regulated. Intriguingly, naturally occurring truncations and mutations of VHL affected wild-type Jade-1 binding and stabilization. Although the VHL β domain was sufficient for Jade-1 binding, both the α and β domains were required for Jade-1 stabilization. Thus, truncating VHL mutations, which are severe and associated with renal cancer development, prevented Jade-1 stabilization. Moreover, well-controlled cotransfection and metabolic labeling experiments revealed that VHL missense mutations that cause VHL disease without renal cancer, such as Tyr98His and Tyr112His, stabilized Jade-1 fully. In contrast, like the VHL truncations, VHL missense mutations commonly associated with renal cancer, such as Leu118Pro or Arg167Trp, did not stabilize Jade-1 fully. Therefore, loss of Jade-1 stability may correlate with renal cancer risk. Endogenous Jade-1 in stable renal cancer lines also exhibited VHL mutation-dependent regulation. As in the cotransfections, VHL truncations did not increase endogenous Jade-1 abundance, whereas the VHL missense mutations tested partially increased Jade-1 expression. Additional studies with non-PHD proteins indicated that Jade-1 stabilization by VHL is highly specific. Fibronectin was not stabilized like Jade-1 by VHL, nor were candidate VHL interactors from a yeast screen. Thus, protein stabilization likely reflects the biological activity of largely intact VHL protein on the PHD-extended PHD module of Jade-1. Dysregulation of the VHL protein stabilization pathway or of Jade-1 itself may therefore contribute to VHL renal disease and renal cancer pathogenesis.
F16, a Mitochondriotoxic Compound, Triggers Apoptosis or Necrosis Depending on the Genetic Background of the Target Carcinoma Cell
Authors: Fantin Valeria R. , Leder Philip
Published: September 2005

Mutations that lead to the emergence of resistance to apoptosis are commonly observed among tumor cells. Some of the proteins affected are integral parts of the apoptotic cascade such as pro- and antiapoptotic members of the Bcl-2 family. F16 is a small molecule that accumulates in mitochondria of a variety of tumor cells and interferes with their physiological function. Because this interference ultimately triggers apoptosis in many affected cell lines, we examined the effect of antiapoptotic Bcl-2 overexpression on the response of cells to F16. Our results showed that high levels of Bcl-2 did not block the ability of F16 to induce cell death. However, unlike the apoptotic response that followed F16 treatment of cells with moderate Bcl-2 levels, cells resistant to a variety of apoptotic stimuli by virtue of Bcl-2 overexpression succumbed to F16 by necrosis. Thus, this dual ability of the mitochondriotoxic compound F16 to induce apoptosis and necrosis may represent an added advantage by expanding its spectrum of action toward genetically altered tumor cells incapable of apoptosis.
Loss of Thioredoxin-Binding Protein-2/Vitamin D3 Up-Regulated Protein 1 in Human T-Cell Leukemia Virus Type I-Dependent T-Cell Transformation
Authors: Nishinaka Yumiko , Nishiyama Akira , Masutani Hiroshi , Oka Shin-ichi , Ahsan Kaimul Md. , Nakayama Yukie , Ishii Yasuyuki , Nakamura Hajime , Maeda Michiyuki , Yodoi Junji
Published: September 2005

Human T-cell leukemia virus type I (HTLV-I) is the causative agent of adult T-cell leukemia (ATL). However, the low incidence of ATL among HTLV-I-infected carriers, together with a long latent period, suggests that multiple host-viral events are involved in the progression of HTLV-I-dependent transformation and subsequent development of ATL. Human thioredoxin (TRX) is a redox active protein highly expressed in HTLV-I-transformed cell lines, whereas the TRX-binding protein-2/vitamin D3 up-regulated protein 1 (TBP-2/VDUP1) was recently identified as a negative regulator of TRX. We report here that expression of TBP-2 is lost in HTLV-I-positive, interleukin-2-independent T-cell lines but maintained in HTLV-I-positive, interleukin-2-dependent T-cell lines, as well as HTLV-I-negative T-cell lines. Ectopic overexpression of TBP-2 in HTLV-I-positive T cells resulted in growth suppression. In the TBP-2-overexpressing cells, a G1 arrest was observed in association with an increase of p16 expression and reduction of retinoblastoma phosphorylation. The results suggest that TBP-2 plays a crucial role in the growth regulation of T cells and that the loss of TBP-2 expression in HTLV-I-infected T cells is one of the key events involved in the multistep progression of ATL leukemogenesis.
Tumor-dependent Kinetics of Partial Pressure of Oxygen Fluctuations during Air and Oxygen Breathing
Authors: Cárdenas-Navia L. Isabel , Yu Daohai , Braun Rod D. , Brizel David M. , Secomb Timothy W. , Dewhirst Mark W.
Published: September 2005

The primary purpose of this study was to examine the kinetics of partial pressure of oxygen (pO2) fluctuations in fibrosarcoma (FSA) and 9L tumors under air and O2 breathing conditions. The overall hypothesis was that key factors relating to oxygen tension fluctuations would vary between the two tumor types and as a function of the oxygen content of the breathing gas. To assist in the interpretation of the temporal data, spatial pO2 distributions were measured in 10 FSA and 8 9L tumors transplanted into the subcutis of the hind leg of Nembutal-anesthetized (50 mg/kg) Fischer 344 rats. Recessed-tip oxygen microelectrodes were inserted into the tumor, and linear pO2 measurements were recorded in 50-μm steps along a 3-mm path, and blood pressure was simultaneously measured via femoral arterial access. Additionally, pO2 was measured at a single location for 90 to 120 minutes in FSA (n = 11) or 9L tumors (n = 12). Rats were switched from air to 100% O2 breathing after 45 minutes. Temporal pO2 records were evaluated for their potential radiobiological significance by assessing the number of times they crossed a 10-mm-Hg threshold. In addition, the data were subjected to Fourier analysis for air and O2 breathing.

FSA and 9L tumors had spatial median pO2 measurements of 4 and 1 mm Hg, respectively. 9L had more low pO2 measurements ≤2.5 mm Hg than did FSA, whereas between 2.5 and 10 mm Hg this pattern was reversed. Pimonidazole staining patterns in FSA and 9L tumors supported these results. Temporal pO2 instability was observed in all experiments during air and O2 breathing. Threshold analyses indicated that the 10 mm Hg threshold was crossed 2 to 5 times per hour, independent of tumor type. However, the magnitude of 9L pO2 fluctuations was approximately eight times greater than FSA fluctuations, as assessed with Fourier transform analysis (Wilcoxon, P < 0.005). O2 breathing significantly increased median pO2 in FSA from 3 to 8 mm Hg (P < 0.005) and caused a significant increase in frequency and magnitude of pO2 fluctuations. One hundred percent O2 breathing had no effect on 9L tumor pO2, and it decreased the magnitude of pO2 fluctuations with borderline significance.

These results show that these two tumors differ significantly with respect to spatial and temporal oxygenation conditions under air and O2 breathing. Fluctuations of pO2 of the type reported herein are predicted to significantly affect radiotherapy response and could be a source for genetic instability, increased angiogenesis, and metastases.
Overexpression of Activating Transcription Factor-2 Is Required for Tumor Growth and Progression in Mouse Skin Tumors
Authors: Papassava Paraskevi , Gorgoulis Vassilis G. , Papaevangeliou Dimitra , Vlahopoulos Spiros , van Dam Hans , Zoumpourlis Vassilis
Published: September 2005

Activating transcription factor (ATF)-2 is a member of the ATF/cyclic AMP-responsive element binding protein family of transcription factors. It has been shown, in vitro, to possess growth factor-independent proliferation and transformation capacity. The information concerning the involvement of ATF-2 in carcinogenesis is rather limited. In a previous report, we showed a progressive increase in the levels of various activator protein (AP)-1 components, including phosphorylated ATF-2, in a series of mouse skin cell lines that represented developmental stages of the mouse skin carcinogenesis system. In the present study, we examined in detail the role of ATF-2 in the development of mouse skin spindle cells A5 and CarB, which correspond to the late and most aggressive stage of the mouse skin carcinogenesis model. To address this issue, we overexpressed a dominant negative form of ATF-2 in the A5 and CarB cell lines and examined their behavior in vitro and in vivo at the molecular and cellular level. The stable transfectants expressed decreased levels of phosphorylated ATF-2 and c-Jun. Subsequently, we observed that dominant negative ATF-2 affected the composition and reduced the activity of AP-1. The above biochemical changes were followed, both in vitro and in vivo in BALB/c severe combined immunodeficient mice, by suppression of the aggressive characteristics of the A5 and CarB mouse skin spindle cells. We attributed this behavior to the significant down-regulation of cyclin D1, cyclin A, and ATF-3, known AP-1 targets implicated in cell cycle control and promotion. In conclusion, our findings underscore a key regulatory role of ATF-2 in tumor growth and progression of mouse skin tumors.
Genomic and Expression Analysis of the 8p11–12 Amplicon in Human Breast Cancer Cell Lines
Authors: Ray Michael E. , Yang Zeng Quan , Albertson Donna , Kleer Celina G. , Washburn Joseph G. , Macoska Jill A. , Ethier Stephen P.
Published: September 2005

Gene amplification is an important mechanism of oncogene activation in breast and other cancers. Characterization of amplified regions of the genome in breast cancer has led to the identification of important oncogenes including erbB-2/HER-2, C-MYC, and fibroblast growth factor receptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10–15% of human breast cancers. The putative oncogene FGFR1 localizes to this region; however, we show evidence that FGFR inhibition fails to slow growth of three breast cancer cell lines with 8p11-p12 amplification. We present a detailed analysis of this amplicon in three human breast cancer cell lines using comparative genomic hybridization, traditional Southern and Northern analysis, and chromosome 8 cDNA microarray expression profiling. This study has identified new candidate oncogenes within the 8p11-p12 region, supporting the hypothesis that genes other than FGFR1 may contribute to oncogenesis in breast cancers with proximal 8p amplification.
EphB2 as a Therapeutic Antibody Drug Target for the Treatment of Colorectal Cancer
Authors: Mao Weiguang , Luis Elizabeth , Ross Sarajane , Silva Johnny , Tan Christine , Crowley Craig , Chui Clarissa , Franz Gretchen , Senter Peter , Koeppen Hartmut , Polakis Paul
Published: September 2005

Analysis of human colorectal cancer specimens revealed overexpression of the EphB2 receptor tyrosine kinase. Monoclonal antibodies (MAbs) to extracellular sequence of EphB2 were raised and tested for activity against colorectal cancer cells. One of the MAbs, 2H9, effectively blocked the interaction of ephB2 with ephrin ligands and inhibited the resulting autophosphorylation of the receptor. However, this antibody did not affect the proliferation of cancer cells expressing ephB2. Immunocytochemical analysis revealed rapid internalization of the MAb 2H9 on binding ephB2, suggesting that target-dependent cell killing could be achieved with an antibody-drug conjugate. When MAb 2H9 was conjugated to monomethylauristatin E through a cathepsin B-cleavable linker, it specifically killed ephB2-expressing cancer cells in vitro and in vivo. Our results suggest that ephB2 is an attractive target for immunoconjugate cancer therapy.
Cyclooxygenase-2 Promotes Human Cholangiocarcinoma Growth
Authors: Han Chang , Leng Jing , Demetris A. Jake , Wu Tong
Published: September 2005

The expression of cyclooxygenase-2 (COX-2) is increased in human cholangiocarcinoma. However, the biologic function and molecular mechanisms of COX-2 in the control of cholangiocarcinoma cell growth have not been well established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth of human intrahepatic cholangiocarcinoma cells. Overexpression of COX-2 or treatment with prostaglandin E2 (PGE2) enhanced human cholangiocarcinoma cell growth, whereas antisense depletion of COX-2 in these cells decreased PGE2 production and inhibited growth. These findings demonstrate a direct role of COX-2-mediated PGE2 in the growth regulation of human cholangiocarcinoma cells. Furthermore, the COX-2 inhibitor celecoxib induced a dose-dependent inhibition of cell growth, cell cycle arrest at the G1-S checkpoint, and induction of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1. However, the high concentration of celecoxib (50 μm) required for inhibition of growth, the incomplete protection of celecoxib-induced inhibition of cell growth by PGE2 or COX-2 overexpression, and the fact that overexpression or antisense depletion of COX-2 failed to alter the level of p21waf1/cip1 and p27kip1 indicate the existence of a COX-2-independent mechanism in celecoxib-induced inhibition of cholangiocarcinoma cell growth.
Antisense Targeting Protein Kinase C α and β1 Inhibits Gastric Carcinogenesis
Authors: Jiang Xiao-Hua , Tu Shui-Ping , Cui Jian-Tao , Lin Marie C. M. , Xia Harry H. X. , Wong Wai Man , Chan Annie On-On , Yuen Man Fung , Jiang Shi-Hu , Lam Shiu-Kum , Kung Hsiang-Fu , Soh Jae Won , Weinstein I. Bernard , Wong Benjamin Chun-Yu
Published: September 2005

Protein kinase C (PKC) family, which functions through serine/threonine kinase activity, is involved in signal transduction pathways necessary for cell proliferation, differentiation, and apoptosis. Its critical role in neoplastic transformation and tumor invasion renders PKC a potential target for anticancer therapy. In this study, we investigated the effect of targeting individual PKCs on gastric carcinogenesis. We established gastric cancer cell lines stably expressing antisense PKCα, PKCβ1, and PKCβ2 cDNA. These stable transfectants were characterized by cell morphology, cell growth, apoptosis, and tumorigenicity in vitro and in vivo. PKCα-AS and PKCβ1-AS transfectants showed a different morphology with flattened, long processes and decreased nuclear:cytoplasmic ratio compared with the control cells. Cell growth was markedly inhibited in PKCα-AS and PKCβ1-AS transfectants. PKCα-AS and PKCβ1-AS cells were more responsive to mitomycin C- or 5-fluorouracil-induced apoptosis. However, antisense targeting of PKCβ2 did not have any significant effect on cell morphology, cell growth, or apoptosis. Furthermore, antisense inhibition of PKCα and PKCβ1 markedly suppressed colony-forming efficiency in soft agar and in nude mice xenografts. Inhibition of PKCα or PKCβ1 significantly suppressed transcriptional and DNA binding activity of activator protein in gastric cancer cells, suggesting that PKCα or PKCβ1 exerts their effects on cell growth through regulation of activator protein activity. These data provide evidence that targeting PKCα and PKCβ1 by antisense method is a promising therapy for gastric cancer.
Expression of Chicken Ovalbumin Upstream Promoter-Transcription Factor II Enhances Invasiveness of Human Lung Carcinoma Cells
Authors: Navab Roya , Gonzalez-Santos Juana Maria , Johnston Michael R. , Liu Jiang , Brodt Pnina , Tsao Ming-Sound , Hu Jim
Published: September 2005

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) plays an essential role in angiogenesis and development. It is differentially expressed in tumor cell lines, but its role in carcinogenesis is largely unknown. We demonstrate here that noninvasive human lung cancer cells become invasive when COUP-TFII was expressed. The expression of extracellular matrix degrading proteinases, such as matrix metalloproteinase 2 and urokinase-type plasminogen activator, was up-regulated in these cells. This finding was confirmed by transduction of different human lung cancer cell lines with COUP-TFII protein and also by using antisense expression. We observed disorganization of actin filaments and focal adhesion kinase phosphorylation in COUP-TFII-transfected human lung cancer cells in addition to the increase in extracellular metalloproteinase activity. These results suggest that COUP-TFII may be considered as a new target for anticancer therapies.
Small Interfering Double-Stranded RNAs as Therapeutic Molecules to Restore Chemosensitivity to Thymidylate Synthase Inhibitor Compounds
Authors: Schmitz John C. , Chen Tian-min , Chu Edward
Published: September 2005

RNA interference is a post-transcriptional mechanism by which double-stranded RNA specifically silence expression of a corresponding gene. Small interfering double-stranded RNA (siRNA) of 21–23 nucleotides can induce the process of RNA interference. Studies from our laboratory have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this process gives rise to increased synthesis of TS and leads to the development of cellular drug resistance to TS-targeted compounds. As a strategy to inhibit TS expression at the mRNA level, siRNAs were designed to target nucleotides 1058–1077 on human TS mRNA. Transfection of TS1058 siRNA into human colon cancer RKO cells resulted in a dose-dependent inhibition of TS expression with an IC50 value of 10 pm but had no effect on the expression of α-tubulin or topoisomerase I. Inhibition of TS expression by TS1058 was maximal at 48 h and remained suppressed for up to 5 days. Pretreatment of RKO cells with TS1058 siRNA suppressed TS protein induction following exposure to raltitrexed. In addition, TS1058 restored chemosensitivity of the resistant RKO-HTStet cell line to various TS inhibitor compounds. On treatment with TS1058, IC50 values for raltitrexed, 1843U89, and 5-fluoro-2′-deoxyuridine decreased by ∼15–16-fold. These studies suggest that TS-targeted siRNAs are effective inhibitors of TS expression and may have therapeutic potential by themselves or as chemosensitizers in combination with TS inhibitor compounds.
Cyclooxygenase-2 Induces EP1- and HER-2/Neu-Dependent Vascular Endothelial Growth Factor-C Up-Regulation
Authors: Su Jen-Liang , Shih Jin-Yuan , Yen Men-Luh , Jeng Yung-Ming , Chang Cheng-Chi , Hsieh Chang-Yao , Wei Lin-Hung , Yang Pan-Chyr , Kuo Min-Liang
Published: September 2005

Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the progression of human lung adenocarcinoma. However, the mechanism underlying COX-2’s effect on tumor progression remains largely unknown. Lymphangiogenesis, the formation of new lymphatic vessels, has recently received considerable attention and become a new frontier of tumor metastasis research. Here, we study the interaction between COX-2 and the lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, in human lung cancer cells and their implication in patient outcomes. We developed an isopropyl-β-d-thiogalactopyranoside-inducible COX-2 gene expression system in human lung adenocarcinoma CL1.0 cells. We found that VEGF-C gene expression but not VEGF-D was significantly elevated in cells overexpressing COX-2. COX-2-mediated VEGF-C up-regulation was commonly observed in a broad array of non-small cell lung cancer cell lines. The use of pharmacological inhibitors or activators and genetic inhibition by EP receptor-antisense oligonucleotides revealed that prostaglandin EP1 receptor but not other prostaglandin receptors is involved in COX-2-mediated VEGF-C up-regulation. At the mechanistic level, we found that COX-2 expression or prostaglandin E2 (PGE2) treatment could activate the HER-2/Neu tyrosine kinase receptor through the EP1 receptor-dependent pathway and that this activation was essential for VEGF-C induction. The transactivation of HER-2/Neu by PGE2 was inhibited by way of blocking the Src kinase signaling using the specific Src family inhibitor, PP1, or transfection with the mutant dominant negative src plasmid. Src kinase was involved in not only the HER-2/Neu transactivation but also the following VEGF-C up-regulation by PGE2 treatment. In addition, immunohistochemical staining of 59 lung adenocarcinoma specimens showed that COX-2 level was highly correlated with VEGF-C, lymphatic vessels density, and other clinicopathological parameters. Taken together, our results provided evidence that COX-2 up-regulated VEGF-C and promotes lymphangiogenesis in human lung adenocarcinoma via the EP1/Src/HER-2/Neu signaling pathway.
CD30 Is Involved in Inhibition of T-Cell Proliferation by Hodgkin’s Reed-Sternberg Cells
Authors: Su Che-Chun , Chiu Hsiu-Hui , Chang Chia-Che , Chen Jui-Chieh , Hsu Su-Ming
Published: September 2005

CD30 is expressed on Hodgkin’s Reed-Sternberg (H-RS) cells, the tumor cells in Hodgkin’s disease. Increased levels of serum CD30 are observed in Hodgkin’s disease patients and are a good marker for predicting a poor prognosis and a poor response to therapy. In this study, we addressed the effect of CD30 on T cells. We showed that CD30, either as a membranous protein on H-RS cells and Chinese hamster ovary cells or as a plate-bound chimeric protein, inhibited T-cell proliferation. Anti-CD3-stimulated T cells in the presence of CD30 failed to increase tritium uptake and failed to express CD25 and CD26 and to produce interleukin 2. The inhibition of T-cell proliferation was, however, reversed with addition of exogenous interleukin 2 or pretreatment of H-RS cells with anti-CD30. Inability of T cells to express CD25 and CD26 in cocultures with H-RS cells or a plate-bound CD30 chimeric protein is in accordance with the results of immunohistochemistry on disease-involved tissues. We conclude that H-RS cells are able to inhibit the proliferation and activation of T cells through CD30-related interaction. The outcome of CD30-related interaction is an ineffective antitumor immunity, which is clearly in favor of the growth and survival of the tumor cells.
Increase of Nuclear Ceramide through Caspase-3-Dependent Regulation of the “Sphingomyelin Cycle” in Fas-Induced Apoptosis
Authors: Watanabe Mitsumasa , Kitano Toshiyuki , Kondo Tadakazu , Yabu Takeshi , Taguchi Yoshimitsu , Tashima Masaro , Umehara Hisanori , Domae Naochika , Uchiyama Takashi , Okazaki Toshiro
Published: September 2005

Regardless of the existence of ceramide-related molecules, such as sphingomyelin (SM), neutral sphingomyelinase (nSMase), and SM synthase, in the nucleus, the regulation of ceramide in the nucleus is poorly understood in stress-induced apoptosis. In Fas-induced Jurkat T-cell apoptosis, we found a time- and dose-dependent increase of ceramide content in the nuclear and microsomal fractions. Fas-induced increase of ceramide content in the nucleus also was detected by confocal microscopy using anticeramide antibody. Activation of nSMase and inhibition of SM synthase were evident in the nuclear fraction after Fas cross-linking, whereas nSMase was activated, but SM synthase was not affected, in the microsomal fraction. Pretreatment with d-609, a putative SM synthase inhibitor, enhanced Fas-induced increase of ceramide in the nucleus and induction of apoptosis along with increase of Fas-induced inhibition of nuclear SM synthase. Fas-induced activation of caspase-3 was detected in the nuclear fraction and in whole cell lysate. A caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-chloromethyl ketone, blocked not only Fas-induced increases of apoptosis and ceramide content but also Fas-induced activation of nSMase and inhibition of SM synthase in the nuclear fraction. Taken together, it is suggested that the nucleus is a site for ceramide increase and caspase-3 activation in Fas-induced Jurkat T-cell apoptosis and that caspase-3-dependent regulation of the “SM cycle” consisting of nSMase and SM synthase plays a role in Fas-induced ceramide increase in the nucleus.
Graft-versus-Tax Response in Adult T-Cell Leukemia Patients after Hematopoietic Stem Cell Transplantation
Authors: Harashima Nanae , Kurihara Kiyoshi , Utsunomiya Atae , Tanosaki Ryuji , Hanabuchi Shino , Masuda Masato , Ohashi Takashi , Fukui Fumiyo , Hasegawa Atsuhiko , Masuda Takao , Takaue Yoichi , Okamura Jun , Kannagi Mari
Published: September 2005

Adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) is characterized by poor prognosis after chemotherapy. Recent clinical trials have indicated, however, that allogeneic but not autologous hematopoietic stem cell transplantation (HSCT) for ATL can yield better clinical outcomes. In the present study, we investigated cellular immune responses of ATL patients who obtained complete remission after nonmyeloablative allogeneic peripheral blood HSCT from HLA-identical sibling donors. In the culture of peripheral blood mononuclear cells (PBMCs) from a post-HSCT but not pre-HSCT ATL patient, CD8+ CTLs proliferated vigorously in response to stimulation with autologous HTLV-I-infected T cells that had been established before HSCT in vitro. These CTLs contained a large number of monospecific CTL population directed to a HLA-A2-restricted HTLV-I Tax 11-19 epitope. The frequency of Tax 11-19-specific CD8+ CTLs in this patient markedly increased also in vivo after HSCT, as determined by staining with HLA-A2/Tax 11-19 tetramers. Similar clonal expansion of HTLV-I Tax-specific CTLs exclusively directed to a HLA-A24-restricted Tax 301-309 epitope was observed in the PBMCs from another ATL patient after HSCT from a HTLV-I-negative donor. Among four post-HSCT ATL patients tested, HTLV-I-specific CTLs were induced in the PBMC culture from three patients but not from the remaining one who had later recurrence of ATL. These observations suggested that reconstituted immunity against antigen presentation in ATL patients after HSCT resulted in strong and selective graft-versus-HTLV-I response, which might contribute to graft-versus-leukemia effects.
Genome-Wide Profiling of Papillary Thyroid Cancer Identifies MUC1 as an Independent Prognostic Marker
Authors: Wreesmann Volkert B. , Sieczka Elizabeth M. , Socci Nicholas D. , Hezel Michael , Belbin Thomas J. , Childs Geoffrey , Patel Snehal G. , Patel Kepal N. , Tallini Giovanni , Prystowsky Michael , Shaha Ashok R. , Kraus Dennis , Shah Jatin P. , Rao Pulivarthi H. , Ghossein Ronald , Singh Bhuvanesh
Published: September 2005

Clinicopathological variables used at present for prognostication and treatment selection for papillary thyroid carcinomas (PTCs) do not uniformly predict tumor behavior, necessitating identification of novel prognostic markers. Complicating the assessment is the long natural history of PTC and our rudimentary knowledge of its genetic composition. In this study we took advantage of differences in clinical behavior of two distinct variants of PTC, the aggressive tall-cell variant (TCV) and indolent conventional PTC (cPTC), to identify molecular prognosticators of outcome using complementary genome wide analyses. Comparative genome hybridization (CGH) and cDNA microarray (17,840 genes) analyses were used to detect changes in DNA copy number and gene expression in pathological cPTC and TCV. The findings from CGH and cDNA microarray analyses were correlated and validated by real-time PCR and immunohistochemical analyses on a series of 100 cases of cPTC and TCV. Genes identified by this approach were evaluated as prognostic markers in cPTC by immunohistochemistry on tissue arrays. CGH identified significant differences in the presence (76 versus 27%; P = 0.001) and type of DNA copy number aberrations in TCV compared with cPTC. Recurrent gains of 1p34–36, 1q21, 6p21–22, 9q34, 11q13, 17q25, 19, and 22 and losses of 2q21–31, 4, 5p14-q21, 6q11–22, 8q11–22, 9q11–32, and 13q21–31 were unique to TCV. Hierarchical clustering of gene expression profiles revealed significant overlap between TCV and cPTC, but further analysis identified 82 dysregulated genes differentially expressed among the PTC variants. Of these, MUC1 was of particular interest because amplification of 1q by CGH correlated with MUC1 amplification by real-time PCR analysis and protein overexpression by immunohistochemistry in TCV (P = 0.005). Multivariate analysis revealed a significant association between MUC1 overexpression and treatment outcome, independent of histopathological categorization (P = 0.03). Analysis of a validation series containing a matched group of aggressive and indolent cPTCs confirmed the association between MUC1 overexpression and survival (relative risk, 2.3; 95% confidence interval, 1.1–5.5; P = 0.03). Our data suggest that MUC1 dysregulation is associated with aggressive behavior of PTC and may serve as a prognostic marker and potential therapeutic target in this disease.
Enhancement of DNA Vaccine Potency by Coadministration of a Tumor Antigen Gene and DNA Encoding Serine Protease Inhibitor-6
Authors: Kim Tae Woo , Hung Chien-Fu , Boyd David A. K. , He Liangmei , Lin Cheng-Tao , Kaiserman Dion , Bird Phillip I. , Wu T-C.
Published: September 2005

Serine protease inhibitor 6 (SPI-6), also called Serpinb9, inhibits granzyme B and thus may provide a method for delaying apoptotic cell death in dendritic cells. We have previously enhanced DNA vaccine potency by targeting antigen to MHC antigen presentation pathways, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, domain II of Pseudomonas aeruginosa exotoxin A, or the sorting signal of the lysosome-associated membrane protein type 1. In this study, we explored intradermal coadministration of DNA encoding SPI-6 with DNA constructs encoding human papillomavirus type 16 E7 linked to these intracellular targeting molecules for its ability to generate E7-specific CD8+ T-cell immune responses and E7-specific antitumor effects. This combination of strategies resulted in significantly increased E7-specific CD8+ T-cell and CD4+ Th1-cell responses, enhanced tumor treatment ability, and stronger tumor protection when compared with vaccination without SPI-6. Among these targeting strategies tested, mice vaccinated with Sig/E7/lysosome-associated membrane protein type 1 mixed with SPI-6 showed the greatest fold increase in E7-specific CD8+ T cells (∼5-fold). Vaccination with a nonfunctional mutant of SPI-6 did not result in immune enhancement, indicating that enhancement was dependent on the antiapoptotic function of SPI-6. Our results suggest that DNA vaccines combining strategies that enhance MHC class I and II antigen processing with SPI-6 have potential clinical implications for control of viral infection and neoplasia.
Ceramide Reduction and Transcriptional Up-Regulation of Glucosylceramide Synthase through Doxorubicin-Activated Sp1 in Drug-Resistant HL-60/ADR Cells
Authors: Uchida Yoshikazu , Itoh Mitsuru , Taguchi Yoshimitsu , Yamaoka Syohei , Umehara Hisanori , Ichikawa Shin-ichi , Hirabayashi Yoshio , Holleran Walter M. , Okazaki Toshiro
Published: September 2005

Treatment with doxorubicin (DOX) induced apoptosis with an increase of ceramide content in drug-sensitive HL-60 cells, but not in drug-resistant HL-60/ADR cells. In HL-60/ADR cells (but not in HL-60 cells), the levels of mRNA, protein, and activity in glucosylceramide synthase (GCS), which converts ceramide to glucosylceramide, were up-regulated in response to DOX. Thus, abrogation of apoptosis in HL-60/ADR cells might be involved in ceramide reduction through DOX-induced up-regulation of GCS function. Because we reported that a GC-rich/Sp1 promoter binding region was of importance in the regulation of GCS expression, the role of Sp1 in DOX-induced up-regulation of GCS and apoptosis was investigated. DOX induced Sp1 activation in HL-60/ADR cells, as assessed by Sp1 gel shift and promoter-luciferase reporter assays, whereas transfection of double-stranded oligodeoxynucleotides (ODNs) containing a GC-rich/Sp1 region (Sp1 decoy ODNs) inhibited DOX-induced Sp1 activation. In addition, DOX-increased mRNA and enzyme activity in GCS were inhibited by Sp1 decoy, in conjunction with corresponding elevations of ceramide content. Moreover, DOX-induced apoptotic cell death was significantly increased in Sp1 decoy ODN-transfected HL-60/ADR cells over mock-transfected HL-60/ADR cells. Together, the results suggest that transcriptional up-regulation of GCS through DOX-induced activation of Sp1 is one potential mechanism to regulate ceramide increase and apoptosis in HL-60/ADR cells.