bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of DNA Repair by a Herpes Simplex Virus Vector Enhances the Radiosensitivity of Human Glioblastoma Cells
Authors: Hadjipanayis Costas G. , DeLuca Neal A.
Published: September 2005

Expression of the herpes simplex virus (HSV) protein, ICP0, from the viral genome, rendered two radioresistant human glioblastoma multiforme cell lines more sensitive to the effects of ionizing radiation. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic survival assays, U87-MG and T98 cell survival was more greatly decreased as a function of ionizing radiation dose when ICP0 was preexpressed in cells compared with when ICP0 was not expressed. Consistent with previous results, we found that the catalytic subunit of DNA-dependent protein kinase was degraded as a function of ICP0 in both cell types. This most likely resulted in the inhibition of DNA repair as inferred by the persistence of γH2AX foci or DNA double-strand breaks. Enhanced apoptosis was also found to occur following irradiation of U87-MG cells preinfected with the ICP0-producing HSV-1 mutant, d106. Our results suggest that expression of ICP0 in human glioblastoma multiforme cells inhibits the repair of DNA double-strand breaks after ionizing radiation treatment, decreasing the survival of these cells in part by induction of apoptosis.
The Propeptide Mediates Formation of Stromal Stores of PROMIC-1: Role in Determining Prostate Cancer Outcome
Authors: Bauskin Asne R. , Brown David A. , Junankar Simon , Rasiah Krishan K. , Eggleton Sarah , Hunter Mark , Liu Tao , Smith Dave , Kuffner Tamara , Pankhurst Greg J. , Johnen Heiko , Russell Pamela J. , Barret Wade , Stricker Phillip D. , Grygiel John J. , Kench James G. , Henshall Susan M. , Sutherland Robert L. , Breit Samuel N.
Published: September 2005

The extracellular matrix (ECM) is a reservoir of cellular binding proteins and growth factors that are critical for normal cell behavior, and aberrations in the ECM invariably accompany malignancies such as prostate cancer. Carcinomas commonly overexpress macrophage inhibitory cytokine 1 (MIC-1), a proapoptotic and antitumorigenic transforming growth factor–β superfamily cytokine. Here we show that MIC-1 is often secreted in an unprocessed propeptide containing form. It is variably processed intracellularly, with unprocessed forms being secreted from several tumor lines, including prostate carcinoma lines, PC-3 and LNCaP. Once secreted, only unprocessed proMIC-1 binds ECM, demonstrating for the first time the occurrence of extracellular stores of MIC-1. The propeptide mediates this association via its COOH-terminal 89 amino acids. Xenograft models bearing tumors secreting various engineered forms of MIC-1 show that the propeptide regulates the balance between ECM stores and circulating serum levels of mature MIC-1 in vivo. The absence of propeptide results in ∼20-fold increase in serum MIC-1 levels. The significance of stromal MIC-1 stores was evaluated in prostate cancer tissue cores, which show major variation in stromal levels of MIC-1. Stromal MIC-1 levels are linked to prostate cancer outcome following radical prostatectomy, with decreasing stromal levels providing an important independent predictor of disease relapse. In low-grade localized prostate cancer (Gleason sum score ≤ 6), the level of MIC-1 stromal stores was the best predictor of future relapse when compared with all other clinicopathologic variables. The secretion and ECM association of unprocessed proMIC-1 is likely to play a central role in modulating local bioavailability of MIC-1 which can affect patient outcome in prostate cancer and other epithelial tumors.
ABL Oncogenes and Phosphoinositide 3-Kinase: Mechanism of Activation and Downstream Effectors
Authors: Kharas Michael G. , Fruman David A.
Published: September 2005

The BCR-ABL oncogene is responsible for most cases of chronic myelogenous leukemia and some acute lymphoblastic leukemias. The fusion protein encoded by BCR-ABL possesses an aberrantly regulated tyrosine kinase activity. Imatinib mesylate (Gleevec, STI-571) is an inhibitor of ABL tyrosine kinase activity that has been remarkably effective in slowing disease progression in patients with chronic phase chronic myelogenous leukemia, but the emergence of imatinib resistance underscores the need for additional therapies. Targeting signaling pathways activated by BCR-ABL is a promising approach for drug development. The study of signaling components downstream of BCR-ABL and the related murine oncogene v-Abl has revealed a complex web of signals that promote cell division and survival. Of these, activation of phosphoinositide 3-kinase (PI3K) has emerged as one of the essential signaling mechanisms in ABL leukemogenesis. This review describes molecular mechanisms by which PI3K is activated and the downstream PI3K effectors that propagate the signal to promote myeloid and lymphoid transformation. Of particular recent interest is the mammalian target of rapamycin, a PI3K-regulated kinase that regulates protein synthesis and contributes to leukemogenesis.
Specificity of the Methylation-Suppressed A Isoform of Candidate Tumor Suppressor RASSF1 for Microtubule Hyperstabilization Is Determined by Cell Death Inducer C19ORF5
Authors: Liu Leyuan , Vo Amy , McKeehan Wallace L.
Published: September 2005

Isoform-specific epigenetic silencing of RASSF1A (3p21.3) by promoter-specific CpG island hypermethylation occurs at high frequency in human tumors, whereas the closely related product of the same gene, RASSF1C, continues to be expressed. Both isoforms in isolation exhibit tumor suppressor properties and we show here similar cellular locations on mitochondria and microtubules, paclitaxel-like microtubule hyperstabilization, disruption of mitosis, and interaction with C19ORF5. We show both have identical but distinct sequence domains for microtubule association and hyperstabilization. C19ORF5 is a hyperstabilized microtubule-specific binding protein of which accumulation causes mitochondrial aggregation and cell death. We report herein that when A or C isoforms of RASSF1 are coexpressed with C19ORF5, the unique N-terminal sequence of RASSF1C prevents it from hyperstabilizing microtubules. This confers specificity on RASSF1A in microtubule hyperstabilization and accumulation of C19ORF5 on microtubules and could underlie a specific effect of hypermethylation-suppressed RASSF1A in tumor suppression.
Spatial Heterogeneity and Oxygen Dependence of Glucose Consumption in R3230Ac and Fibrosarcomas of the Fischer 344 Rat
Authors: Schroeder Thies , Yuan Hong , Viglianti Benjamin L. , Peltz Cathryn , Asopa Shubha , Vujaskovic Zeljko , Dewhirst Mark W.
Published: September 2005

To examine the oxygen-dependence of glucose consumption in solid tumors, we monitored gradients of glucose, lactate, and hypoxia in R3230Ac and FSA tumors growing in Fischer 344 rats. Bioluminescence imaging, detection of Hoechst 33342, and immunostaining of the hypoxia marker EF5 [2-8-N-(2,2,3,3,3-pentafluoropropyl)acetamide] were done in serial tumor slices. Glucose and lactate levels were also determined in liver and blood. Cells were further tested for glucose consumption and lactate production in vitro. In both tumor types, EF5 staining indicated similar maximum levels of hypoxia; the most intense staining occurred in perinecrotic regions. Glucose concentrations were highest in liver, declined from blood to tumor edge, further into vital tumor regions, and were lowest close to necrosis. Glucose was significantly lower in FSA than in R3230Ac tumors. Glucose concentrations in R3230Ac tumors were consistently higher in nonhypoxic than in hypoxic areas, with maximum values equal to systemic blood levels. Glucose in FSA tumors was close to zero, regardless of the presence or absence of hypoxia. Lactate did not differ significantly between the tumor types. FSA cells in culture showed a trend towards higher aerobic glucose consumption versus R3230Ac. Both cell lines increased their lactate production to similar levels under hypoxia. We conclude that both R3230Ac and FSA tumors retain the Pasteur effect, i.e., hypoxia triggers increased glycolysis. However, our results imply that increased aerobic glucose utilization leads to low glucose levels in FSA and a situation where supply limits uptake. This explains the repeatedly observed correlation between tumor blood flow and 18F-deoxyglucose uptake.
PGP9.5 Promoter Methylation Is an Independent Prognostic Factor for Esophageal Squamous Cell Carcinoma
Authors: Mandelker Diana L. , Yamashita Keishi , Tokumaru Yutaka , Mimori Koshi , Howard David L. , Tanaka Yoichi , Carvalho Andre Lopes , Jiang Wei-Wen , Park Hannah Lui , Kim Myoung Sook , Osada Motonobu , Mori Masaki , Sidransky David
Published: September 2005

PGP9.5/UCHL1 is a member of the carboxyl-terminal ubiquitin hydrolase family with a potential role in carcinogenesis. We previously identified PGP9.5 as a putative tumor-suppressor gene and methylation of the promoter as a cancer-specific event in primary cancer tissues. In this current study, we analyzed PGP9.5 methylation in 50 esophageal squamous cell carcinoma (ESCC) primary tumors with well characterized clinicopathologic variables including patient outcome. Two independent modalities for methylation analysis (TaqMan methylation-specific PCR and combined bisulfite restriction analysis) were used to analyze these samples. The two data sets were consistent with each other, as the 21 patients (42%) with highest methylation levels by TaqMan analysis all showed visible combined bisulfite restriction analysis bands on acrylamide gels. Using an optimized cutoff value by TaqMan quantitation, we found that patients with higher PGP9.5 methylation ratios in the primary tumor showed poorer 5-year survival rates than those without PGP9.5 methylation (P = 0.01). A significant correlation was also seen between PGP9.5 promoter methylation and the presence of regional lymph node metastases (P = 0.03). Multivariate analysis subsequently revealed that PGP9.5 methylation was an independent prognostic factor for ESCC survival (P = 0.03). These results suggest that PGP9.5 promoter methylation could be a clinically applicable marker for ESCC progression.
Noninvasive Magnetic Resonance Thermography of Recurrent Rectal Carcinoma in a 1.5 Tesla Hybrid System
Authors: Gellermann Johanna , Wlodarczyk Waldemar , Hildebrandt Bert , Ganter Hildegard , Nicolau Anett , Rau Beate , Tilly Wolfgang , Fähling Horst , Nadobny Jacek , Felix Roland , Wust Peter
Published: September 2005

To implement noninvasive thermometry, we installed a hybrid system consisting of a radiofrequency multiantenna applicator (SIGMA-Eye) for deep hyperthermia (BSD-2000/3D) integrated into the gantry of a 1.5 Tesla magnetic resonance (MR) tomograph Symphony. This system can record MR data during radiofrequency heating and is suitable for application and evaluation of methods for MR thermography.

In 15 patients with preirradiated pelvic rectal recurrences, we acquired phase data sets (25 slices) every 10 to 15 minutes over the treatment time (60-90 minutes) using gradient echo sequences (echo time = 20 ms), transformed the phase differences to MR temperatures, and fused the color-coded MR-temperature distributions with anatomic T1-weighted MR data sets. We could generate one complete series of MR data sets per patient with satisfactory quality for further analysis. In fat, muscle, water bolus, prostate, bladder, and tumor, we delineated regions of interest (ROI), used the fat ROI for drift correction by transforming these regions to a phase shift zero, and evaluated the MR-temperature frequency distributions. Mean MR temperatures (TMR), maximum TMR, full width half maximum (FWHM), and other descriptors of tumors and normal tissues were noninvasively derived and their dependencies outlined. In 8 of 15 patients, direct temperature measurements in reference points were available. We correlated the tumor MR temperatures with direct measurements, clinical response, and tumor features (volume and location), and found reasonable trends and correlations. Therefore, the mean TMR of the tumor might be useful as a variable to evaluate the quality and effectivity of heat treatments, and consequently as optimization variable.

Feasibility of noninvasive MR thermography for regional hyperthermia has been shown and should be further investigated.
Tumor-Infiltrating Dendritic Cell Subsets of Progressive or Regressive Tumors Induce Suppressive or Protective Immune Responses
Authors: Liu Yongqing , Bi Xuguang , Xu Shulin , Xiang Jim
Published: September 2005

Tumor-infiltrating dendritic cells (TID) have an ambivalent role in regulation of tumor regression or growth. However, their precise natures and molecular mechanisms have not been elucidated. In this study, we studied TIDs recruited in progressive P815 and regressive P198 tumors of the same origin. Our data showed that P815 tumors contained CD4+8+ and CD4−8− TID815 subsets, whereas P198 tumors contained CD4+8+ and CD4+8− TID198 subsets. They similarly stimulate allogeneic T cell proliferation and have nitric oxide–mediated cytotoxicity to tumor cells with an exception of CD4−8− TID815 with less efficiency. The newly identified fourth CD4+8+ TID815 or TID198 subset and the CD4+8− TID198 all express high levels of IFN-γ and interleukin (IL)-6, whereas CD4−8− TID815 secrete a marked level of transforming growth factor-β. Vaccination of mice with P815 tumor lysate–pulsed CD4+8+ TID815 or TID198 and CD4+8− TID198 induced IFN-γ–secreting Th1 and effective CTL responses leading to protective immunity against P815 tumor, whereas CD4−8− TID815 stimulated IL-10–expressing Tr1 responses leading to immune suppression. Transfer of CD4+ Tr1 cells obtained from CD4−8− TID815-immunized wild-type, but not IL-10−/− mice, into CD4+8+ TID815 immunized mice abolished otherwise inevitable development of antitumor immunity. Taken together, our findings provide an important insight into immunologic alterations in progressive and regressive tumors and an implication for dendritic cell–based approaches in the design of cancer vaccines.
Peroxisome Proliferator-Activated Receptor δ and γ Agonists Differentially Alter Tumor Differentiation and Progression during Mammary Carcinogenesis
Authors: Yin Yuzhi , Russell Robert G. , Dettin Luis E. , Bai Renkui , Wei Zhi-Liang , Kozikowski Alan P. , Kopleovich Levy , Glazer Robert I.
Published: September 2005

Peroxisome proliferator-activated receptor (PPAR) represents a ligand-dependent nuclear receptor family that regulates multiple metabolic processes associated with fatty acid β-oxidation, glucose utilization, and cholesterol transport. These and other receptor-mediated actions pertain to their role in hypolipidemic and antidiabetic therapies and as potential targets for cancer chemopreventive agents. The present study evaluated the chemopreventive activity of two highly potent and selective PPARγ and PPARδ agonists in a progestin- and carcinogen-induced mouse mammary tumorigenesis model. Animals treated with the PPARγ agonist GW7845 exhibited a moderate delay in tumor formation. In contrast, animals treated with the PPARδ agonist GW501516 showed accelerated tumor formation. Significantly, tumors from GW7845-treated mice were predominantly ductal adenocarcinomas, whereas tumors from GW501516-treated animals were adenosquamous and squamous cell carcinomas. Gene expression analysis of tumors arising from GW7845- and GW501516-treated mice identified expression profiles that were distinct from each other and from untreated control tumors of the same histopathology. Only tumors from mice treated with the PPARγ agonist expressed estrogen receptor-α in luminal transit cells, suggesting increased ductal progenitor cell expansion. Tumors from mice treated with the PPARδ agonist exhibited increased PPARδ levels and activated 3-phosphoinositide–dependent protein kinase-1 (PDK1), which co-associated, suggesting a link between the known oncogenic activity of PDK1 in mammary epithelium and PPARδ activation. These results indicate that PPARδ and PPARγ agonists produce diverse, yet profound effects on mammary tumorigenesis that give rise to distinctive histopathologic patterns of tumor differentiation and tumor development.
Identification of the Ubiquitin-Proteasome Pathway in the Regulation of the Stability of Eukaryotic Elongation Factor-2 Kinase
Authors: Arora Sonia , Yang Jin-Ming , Hait William N.
Published: September 2005

Eukaryotic elongation factor-2 kinase (eEF-2 kinase) is a highly conserved calcium/calmodulin-dependent enzyme involved in the regulation of protein translation and cell proliferation. Rapid changes in the activity and abundance of eEF-2 kinase have been observed on growth stimulation, and increased enzyme activity is characteristic of malignant cell growth. Yet the mechanism for controlling the turnover of this kinase is unknown. The ubiquitin-proteasome pathway regulates the degradation of many cellular proteins, including transcription factors, cell cycle regulators, and signal transduction proteins. Therefore, we determined whether the ubiquitin-proteasome pathway regulates the turnover of eEF-2 kinase. We found that eEF-2 kinase was a relatively short-lived protein with a half-life of less than 6 hours. eEF-2 kinase was ubiquitinated in vivo as determined by coimmunoprecipitation and polyubiquitin affinity matrix. Incubation of purified eEF-2 kinase with a source of ubiquitination enzymes (rabbit reticulocyte lysate), purified ubiquitin, and ATP revealed the presence of increasing molecular weight species of ubiquitinated eEF-2 kinase. Treatment of cells with MG132, a proteasome inhibitor, inhibited eEF-2 kinase degradation and induced the accumulation of polyubiquitinated forms of the enzyme, resulting in an increase in its half-life. These results suggest involvement of the proteasome in the turnover of the ubiquitinated kinase. Because eEF-2 kinase is chaperoned by heat shock protein 90 (Hsp90), we next determined if disruption of the Hsp90-eEF-2 kinase complex promoted degradation of the kinase. Treatment of cells with geldanamycin, an Hsp90 inhibitor, enhanced ubiquitination of eEF-2 kinase and decreased the half-life of the kinase to less than 2 hours. These results indicate that cellular levels of eEF-2 kinase are maintained by a balance between association with Hsp90 and degradation by the ubiquitin-proteasome pathway. In conclusion, these data show that the turnover of eEF-2 kinase is regulated by the ubiquitin-proteasome pathway and, therefore, modulating the ubiquitination of eEF-2 kinase might control the abundance of this enzyme and have implications in the treatment of certain forms of cancer.
YB-1 Provokes Breast Cancer through the Induction of Chromosomal Instability That Emerges from Mitotic Failure and Centrosome Amplification
Authors: Bergmann Stephan , Royer-Pokora Brigitte , Fietze Ellen , Jürchott Karsten , Hildebrandt Barbara , Trost Detlef , Leenders Frauke , Claude Jenny-Chang , Theuring Franz , Bargou Ralf , Dietel Manfred , Royer Hans-Dieter
Published: September 2005

YB-1 protein levels are elevated in most human breast cancers, and high YB-1 levels have been correlated with drug resistance and poor clinical outcome. YB-1 is a stress-responsive, cell cycle–regulated transcription factor with additional functions in RNA metabolism and translation. In this study, we show in a novel transgenic mouse model that human hemagglutinin-tagged YB-1 provokes remarkably diverse breast carcinomas through the induction of genetic instability that emerges from mitotic failure and centrosome amplification. The increase of centrosome numbers proceeds during breast cancer development and explanted tumor cell cultures show the phenotype of ongoing numerical chromosomal instability. These data illustrate a mechanism that might contribute to human breast cancer development.
Loss of the Forkhead Transcription Factor FoxM1 Causes Centrosome Amplification and Mitotic Catastrophe
Authors: Wonsey Diane R. , Follettie Maximillian T.
Published: September 2005

Expression of the forkhead transcription factor FoxM1 correlates with proliferative status in a variety of normal and transformed cell types. Elevated expression of FoxM1 has been noted in both hepatocellular carcinoma and basal cell carcinoma. However, whether FoxM1 expression is essential for the viability of transformed cells is unknown. We report here that the expression of FoxM1 is significantly elevated in primary breast cancer. Microarray analysis shows that FoxM1 regulates genes that are essential for faithful chromosome segregation and mitosis, including Nek2, KIF20A, and CENP-A. Loss of FoxM1 expression generates mitotic spindle defects, delays cells in mitosis, and induces mitotic catastrophe. Time-lapse microscopy indicates that depletion of FoxM1 generates cells that enter mitosis but are unable to complete cell division, resulting in either mitotic catastrophe or endoreduplication. These findings indicate that FoxM1 depletion causes cell death due to mitotic catastrophe and that inhibiting FoxM1 represents a therapeutic strategy to target breast cancer.
Degradation of Human Exonuclease 1b upon DNA Synthesis Inhibition
Authors: El-Shemerly Mahmoud , Janscak Pavel , Hess Daniel , Jiricny Josef , Ferrari Stefano
Published: September 2005

In response to DNA damage, signaling pathways are triggered that either block the cell division cycle at defined transitions (G1-S and G2-M) or slow down progression through the S phase. Nucleases play important roles in DNA synthesis, recombination, repair, and apoptosis. In this study, we have examined the regulation of human exonuclease 1 (hEXO1b). The endogenous hEXO1b protein was only detected upon enrichment by immunoprecipitation. We found that hEXO1b was constantly expressed throughout the cell cycle. However, treatment of cells with agents that cause arrest of DNA replication led to rapid degradation of hEXO1b. This effect was fully reversed upon removal of the block. Analysis of synchronized cells showed that degradation of hEXO1b during the S phase was strictly dependent on DNA synthesis inhibition. DNA damage caused by UV-C radiation, ionizing radiation, cisplatin, or the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine did not affect hEXO1b stability. We show that hEXO1b was phosphorylated in response to inhibition of DNA synthesis and that phosphorylation coincided with rapid protein degradation through ubiquitin-proteasome pathways. Our data support the evidence that control of exonuclease 1 activity may be critical for the maintenance of stalled replication forks.
Perillyl Alcohol Inhibits a Calcium-Dependent Constitutive Nuclear Factor-κB Pathway
Authors: Berchtold Craig M. , Chen Kai-Shun , Miyamoto Shigeki , Gould Michael N.
Published: September 2005

The cell death induced by the monoterpene anticancer agent perillyl alcohol correlates to the increased expression of certain proapoptotic genes known to influence cell survival. Whereas sequence-specific DNA-binding factors dictate the expression patterns of genes through transcriptional regulation, those transcriptional factors influencing constitutive cell survival with perillyl alcohol treatment are not well studied. Here, we investigated whether the monoterpenes can regulate the activity of nuclear factor-κB (NF-κB), a calcium-dependent transcription factor necessary for survival in the WEHI-231 B-lymphoma cells. Unique among the monoterpenes, perillyl alcohol short-term treatment induced a persistent decrease of calcium levels, whereas other various monoterpenes caused transient reductions in calcium levels. Perillyl alcohol treatment also rapidly elicited reductions of NF-κB DNA-binding activity and target gene induction, which was associated with an increase in apoptosis in these B-lymphoma cells. This apoptosis was directly due to NF-κB because its prior activation abolished the cell killing effects of perillyl alcohol treatment. Our findings suggest that perillyl alcohol can inhibit NF-κB function to modulate gene expression patterns and cell survival of certain B-lymphoma cells. The effects of perillyl alcohol were not limited to these B-lymphoma cells but were also observed in MDA-MB 468 cells, an estrogen receptor–negative breast cancer cell line. These results identify a calcium-dependent NF-κB pathway as a molecular target of perillyl alcohol activity in different cancer cell types.
The Role of Growth Factor Receptor Pathways in Human Breast Cancer Cells Adapted to Long-term Estrogen Deprivation
Authors: Sabnis Gauri J. , Jelovac Danijela , Long Brian , Brodie Angela
Published: September 2005

To study the long-term effects of estrogen deprivation on breast cancer, MCF-7Ca human estrogen receptor–positive breast cancer cells stably transfected with human aromatase gene were cultured in the steroid-depleted medium for 6 to 8 months until they had acquired the ability to grow. Proliferation of these cells (UMB-1Ca) was accompanied by increased expression of human epidermal growth factor receptor 2, increased activation of AKT through phosphorylation at Ser473 and Thr308, and increased invasion compared with parental MCF-7Ca cells. Estrogen receptor expression was also increased 5-fold. Although growth was inhibited by the antiestrogen fulvestrant, the IC50 was 100-fold higher than for parental MCF-7Ca cells. Aromatase inhibitor letrozole also inhibited growth at 10,000-fold higher concentration than required for MCF-7Ca cells, whereas anastrozole, exemestane, formestane, and tamoxifen were ineffective at 100 nmol/L. Growth of UMB-1Ca cells was inhibited by phosphatidylinositol 3-kinase inhibitor wortmannin (IC50 ∼25 nmol/L) and epidermal growth factor receptor kinase inhibitor gefitinib (ZD 1839; IC50 ∼10 μmol/L) whereas parental MCF-7Ca cells were insensitive to these agents. Concomitant treatment of UMB-1Ca cells with the signal transduction inhibitors and anastrozole and tamoxifen restored their growth inhibitory effects. These studies show that estrogen deprivation results in up-regulation of growth factor signaling pathways, which leads to a more aggressive and hormone refractory phenotype. Cross-talk between ER and growth factor signaling was evident as inhibition of these pathways could restore estrogen responsiveness to these cells.
Motexafin Gadolinium Disrupts Zinc Metabolism in Human Cancer Cell Lines
Authors: Magda Darren , Lecane Philip , Miller Richard A. , Lepp Cheryl , Miles Dale , Mesfin Mimi , Biaglow John E. , Ho Vincent V. , Chawannakul Danny , Nagpal Shailender , Karaman Mazen W. , Hacia Joseph G.
Published: September 2005

To gain a better understanding of the mechanism of action of the metal cation–containing chemotherapeutic drug motexafin gadolinium (MGd), gene expression profiling analyses were conducted on plateau phase human lung cancer (A549) cell cultures treated with MGd. Drug treatment elicited a highly specific response that manifested in elevated levels of metallothionein isoform and zinc transporter 1 (ZnT1) transcripts. A549 cultures incubated with MGd in the presence of exogenous zinc acetate displayed synergistic increases in the levels of intracellular free zinc, metallothionein transcripts, inhibition of thioredoxin reductase activity, and cell death. Similar effects were observed in PC3 prostate cancer and Ramos B-cell lymphoma cell lines. Intracellular free zinc levels increased in response to treatment with MGd in the absence of exogenous zinc, indicating that MGd can mobilize bound intracellular zinc. These findings lead us to suggest that an important component of the anticancer activity of MGd is related to its ability to disrupt zinc metabolism and alter cellular availability of zinc. This class of compounds may provide insight into the development of novel cancer drugs targeting control of intracellular free zinc and the roles that zinc and other metal cations play in biochemical pathways relevant to cancer.
Alteration of SMRT Tumor Suppressor Function in Transformed Non-Hodgkin Lymphomas
Authors: Song Lynda , Zlobin Andrei , Ghoshal Pushpankur , Zhang Qing , Houde Christiane , Weijzen Sanne , Jiang Qun , Nacheva Elizabeth , Yagan Danny , Davis Eric , Galiegue-Zouitina Sylvie , Catovsky Daniel , Grogan Thomas , Fisher Richard I. , Miele Lucio , Coignet Lionel J.
Published: September 2005

Indolent non-Hodgkin lymphomas are characterized by a prolonged phase that is typically followed by a clinical progression associated with an accelerated clinical course and short survival time. Previous studies have not identified a consistent cytogenetic or molecular abnormality associated with transformation. The development of a transformed phenotype, evolving from the original low-grade component, most likely depends on multiple genetic events, including the activation of synergistic dominant oncogenes and a loss of tumor suppressor gene functions. Complex karyotypes and relatively bad chromosome morphology are typical of transformed non-Hodgkin lymphomas, rendering complete cytogenetic analysis difficult. Here, we report the use of transformed non-Hodgkin lymphoma cell lines and primary samples to identify the involvement of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) gene that maps at chromosome 12q24 in transformed non-Hodgkin lymphomas. We also show that down-regulation of SMRT in the immortalized “Weinberg's model” cell lines induces transformation of the cells. Assessment of cDNA array profiles should further help us to design a working model for SMRT involvement in non-Hodgkin lymphoma transformation as a novel, nonclassical tumor suppressor.
Human Anti-CD40 Antagonist Antibody Triggers Significant Antitumor Activity against Human Multiple Myeloma
Authors: Tai Yu-Tzu , Li Xianfeng , Tong Xia , Santos Daniel , Otsuki Takemi , Catley Laurence , Tournilhac Olivier , Podar Klaus , Hideshima Teru , Schlossman Robert , Richardson Paul , Munshi Nikhil C. , Luqman Mohammad , Anderson Kenneth C.
Published: September 2005

Monoclonal antibodies (mAb) directed against lineage-specific B-cell antigens have provided clinical benefit for patients with hematologic malignancies, but to date no antibody-mediated immunotherapy is available for multiple myeloma. In the present study, we assessed the efficacy of a fully human anti-CD40 mAb CHIR-12.12 against human multiple myeloma cells. CHIR-12.12, generated in XenoMouse mice, binds to CD138-expressing multiple myeloma lines and freshly purified CD138-expressing cells from >80% multiple myeloma patients, as assessed by flow cytometry. Importantly, CHIR-12.12 abrogates CD40L-induced growth and survival of CD40-expressing patient multiple myeloma cells in the presence or absence of bone marrow stromal cells (BMSC), without altering constitutive multiple myeloma cell proliferation. Immunoblotting analysis specifically showed that PI3-K/AKT, nuclear factor-κB (NF-κB), and extracellular signal-regulated kinase activation induced by CD40L (5 μg/mL) was inhibited by CHIR-12.12 (5 μg/mL). Because CD40 activation induces multiple myeloma cell adhesion to both fibronectin and BMSCs, we next determined whether CHIR-12.12 inhibits this process. CHIR-12.12 decreased CD40L-induced multiple myeloma cell adhesion to fibronectin and BMSCs, whereas control human IgG1 did not. Adhesion of multiple myeloma cells to BMSCs induces interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion, and treatment of multiple myeloma cells with CD40L further enhanced adhesion-induced cytokine secretion; conversely, CHIR-12.12 blocks CD40L-enhanced IL-6 and VEGF secretion in cocultures of multiple myeloma cells with BMSCs. Finally, CHIR-12.12 triggered lysis of multiple myeloma cells via antibody-dependent cellular cytotoxicity (ADCC) but did not induce ADCC against CD40-negative multiple myeloma cells, confirming specificity against CD40-expressing multiple myeloma cells. These results provide the preclinical rationale for clinical trials of CHIR-12.12 to improve patient outcome in multiple myeloma.
Cathepsin D Is a Potential Serum Marker for Poor Prognosis in Glioma Patients
Authors: Fukuda Mieko E. , Iwadate Yasuo , Machida Toshio , Hiwasa Takaki , Nimura Yoshinori , Nagai Yuichiro , Takiguchi Masaki , Tanzawa Hideki , Yamaura Akira , Seki Naohiko
Published: September 2005

Cathepsin D is an aspartyl protease involved in protein catabolism and tissue remodeling which can be secreted from cancer cells. To identify a potential serum marker for gliomas, we investigated the gene expression levels of cathepsin D in 87 tissue samples and measured the protein concentrations in sera of glioma patients. The tissue samples consisted of 43 glioblastomas, 13 anaplastic astrocytomas, 22 astrocytomas, and 9 normal brain tissues. The results of real-time quantitative reverse transcription-PCR analysis showed that cathepsin D transcript levels became significantly higher as the glioma grade advanced (P = 0.0466, glioblastoma and anaplastic astrocytoma; P = 0.0008, glioblastoma and astrocytoma; P = 0.0271, glioblastoma and normal brain tissue; unpaired t test). Immunohistochemical analysis with anti-cathepsin D antibody revealed dense and spotty staining in the tumor cells with high transcript levels. The low expression of cathepsin D significantly correlated with long survival of the glioma patients. Furthermore, the glioblastoma patients with high gene expression of cathepsin D lived significantly shorter than those with low expression (P = 0.0104, Cox-Mantel log-rank test) and frequently had leptomeningeal dissemination (P = 0.0016, χ2 test). The multivariate analysis confirmed that the cathepsin D expression level was an independent predictor for short survival (P = 0.0102, Cox proportional hazard regression model). Measurement of the serum cathepsin D concentrations by ELISA showed a significant increase in the patients with high-grade gliomas as compared with the low-grade tumors (P = 0.0081, χ2 test). These results collectively suggest that cathepsin D could be a potential serum marker for the prediction of aggressive nature of human gliomas.
Cooperation between Cdk4 and p27kip1 in Tumor Development: A Preclinical Model to Evaluate Cell Cycle Inhibitors with Therapeutic Activity
Authors: Sotillo Rocío , Renner Oliver , Dubus Pierre , Ruiz-Cabello Jesús , Martín-Caballero Juan , Barbacid Mariano , Carnero Amancio , Malumbres Marcos
Published: September 2005

Deregulation of the G1-S transition of the cell cycle is a common feature of human cancer. Tumor-associated alterations in this process frequently affect cyclin-dependent kinases (Cdk), their regulators (cyclins, INK4 inhibitors, or p27Kip1), and their substrates (retinoblastoma protein). Although these proteins are generally thought to act in a linear pathway, mutations in different components frequently cooperate in tumor development. Using gene-targeted mouse models, we report in this article that Cdk4 resistance to INK4 inhibitors, due to the Cdk4 R24C mutation, strongly cooperates with p27Kip1 deficiency in tumor development. No such cooperation is observed between Cdk4 R24C and p18INK4c absence, suggesting that the only function of p18INK4c is inhibiting Cdk4 in this model. Cdk4R/R knock in mice, which express the Cdk4 R24C mutant protein, develop pituitary tumors with complete penetrance and short latency in a p27Kip1−/− or p27Kip1+/− background. We have investigated whether this tumor model could be useful to assess the therapeutic activity of cell cycle inhibitors. We show here that exposure to flavopiridol, a wide-spectrum Cdk inhibitor, significantly delays tumor progression and leads to tumor-free survival in a significant percentage of treated mice. These data suggest that genetically engineered tumor models involving key cell cycle regulators are a valuable tool to evaluate drugs with potential therapeutic benefit in human cancer.
Liver-Specific pRB Loss Results in Ectopic Cell Cycle Entry and Aberrant Ploidy
Authors: Mayhew Christopher N. , Bosco Emily E. , Fox Sejal R. , Okaya Tomohisa , Tarapore Pheruza , Schwemberger Sandy J. , Babcock George F. , Lentsch Alex B. , Fukasawa Kenji , Knudsen Erik S.
Published: September 2005

The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during post-natal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals.
A Genomic Map of p53 Binding Sites Identifies Novel p53 Targets Involved in an Apoptotic Network
Authors: Miled Chaouki , Pontoglio Marco , Garbay Serge , Yaniv Moshe , Weitzman Jonathan B.
Published: September 2005

The transcriptional activity of the p53 protein is central to its role in tumor suppression. Identification of the complete repertoire of p53-regulated genes is critical for dissecting the complexity of the p53 network. Although several different approaches have been used to characterize the p53 genetic program, we still lack a comprehensive molecular understanding of how p53 prevents cancer. Using a computational approach, we generated a genome-wide map of p53 binding sites (p53BS) to identify novel p53 target genes. We show that the presence of nearby p53BS can identify new proapoptotic members of the Bcl2 family. We show that p53 binds to p53BS identified in the BCL-G/BCL2L14 gene and that induction of this gene contributes to p53-mediated apoptosis. We found that p53 activates the COL18A1 gene encoding the precursor for the antiangiogenic factor endostatin. We also show that p53 up-regulates the MAP4K4 gene and activates the c-Jun NH2-terminal kinase (JNK) pathway to drive apoptosis. Thus, unbiased mapping of the genomic landscape of p53BS provides a systematic and complementary approach to identify novel factors and connections in the p53 genetic network. Our study illustrates how systematic genomic approaches can identify binding sites that are functionally relevant for a p53 transcriptional program. The genetic link among p53, antiangiogenic factors, and the JNK signaling pathway adds new dimensions to understanding p53 function in highly connected genetic networks.
Glypican-3 Promotes the Growth of Hepatocellular Carcinoma by Stimulating Canonical Wnt Signaling
Authors: Capurro Mariana I. , Xiang Yun-Yan , Lobe Corrinne , Filmus Jorge
Published: September 2005

Glypican-3 (GPC3) is a heparan sulfate proteoglycan that is bound to the cell membrane by a glycosyl-phosphatidylinositol anchor. GPC3 is expressed by most hepatocellular carcinomas but not by normal hepatocytes and benign liver lesions. We report here that GPC3 stimulates the in vitro and in vivo growth of hepatocellular carcinoma cells by increasing autocrine/paracrine canonical Wnt signaling. Coimmunoprecipitation experiments showed that GPC3 is able to form complexes with Wnts, and cell-binding assays indicated that GPC3-expressing cells have an increased capacity to bind Wnt. Collectively, these results suggest that GPC3 stimulates Wnt activity by facilitating the interaction of this polypeptide with its signaling receptors. Surprisingly, in contrast to the current model that proposes that Wnt-glypican binding is mediated by the heparan sulfate chains, we found that the nonglycanated GPC3 core protein can form complexes with Wnts. Furthermore, we showed that the glycosaminoglycan chains are not required for the stimulatory effect on Wnt signaling and hepatocellular carcinoma growth.
A Germline DNA Polymorphism Enhances Alternative Splicing of the KLF6 Tumor Suppressor Gene and Is Associated with Increased Prostate Cancer Risk
Authors: Narla Goutham , DiFeo Analisa , Reeves Helen L. , Schaid Daniel J. , Hirshfeld Jennifer , Hod Eldad , Katz Amanda , Isaacs William B. , Hebbring Scott , Komiya Akira , McDonnell Shannon K. , Wiley Kathleen E. , Jacobsen Steven J. , Isaacs Sarah D. , Walsh Patrick C. , Zheng S. Lilly , Chang Bao-Li , Friedrichsen Danielle M. , Stanford Janet L. , Ostrander Elaine A. , Chinnaiyan Arul M. , Rubin Mark A. , Xu Jianfeng , Thibodeau Stephen N. , Friedman Scott L. , Martignetti John A.
Published: September 2005

Prostate cancer is a leading and increasingly prevalent cause of cancer death in men. Whereas family history of disease is one of the strongest prostate cancer risk factors and suggests a hereditary component, the predisposing genetic factors remain unknown. We first showed that KLF6 is a tumor suppressor somatically inactivated in prostate cancer and since then, its functional loss has been further established in prostate cancer cell lines and other human cancers. Wild-type KLF6, but not patient-derived mutants, suppresses cell growth through p53-independent transactivation of p21. Here we show that a germline KLF6 single nucleotide polymorphism, confirmed in a tri-institutional study of 3,411 men, is significantly associated with an increased relative risk of prostate cancer in men, regardless of family history of disease. This prostate cancer–associated allele generates a novel functional SRp40 DNA binding site and increases transcription of three alternatively spliced KLF6 isoforms. The KLF6 variant proteins KLF6-SV1 and KLF6-SV2 are mislocalized to the cytoplasm, antagonize wtKLF6 function, leading to decreased p21 expression and increased cell growth, and are up-regulated in tumor versus normal prostatic tissue. Thus, these results are the first to identify a novel mechanism of self-encoded tumor suppressor gene inactivation and link a relatively common single nucleotide polymorphism to both regulation of alternative splicing and an increased risk in a major human cancer.
Natural Killer Cell–Mediated Lysis of Hepatoma Cells via Specific Induction of NKG2D Ligands by the Histone Deacetylase Inhibitor Sodium Valproate
Authors: Armeanu Sorin , Bitzer Michael , Lauer Ulrich M. , Venturelli Sascha , Pathil Anita , Krusch Matthias , Kaiser Stephan , Jobst Jürgen , Smirnow Irina , Wagner Annika , Steinle Alexander , Salih Helmut R.
Published: September 2005

Natural killer (NK) cells as components of the innate immunity substantially contribute to antitumor immune responses. However, the tumor-associated ligands engaging activating NK cell receptors are largely unknown. An exception are the MHC class I chain-related molecules MICA and MICB and the UL16-binding proteins (ULBP) which bind to the activating immunoreceptor NKG2D expressed on cytotoxic lymphocytes. A therapeutic induction of NKG2D ligands that primes cancer cells for NK cell lysis has not yet been achieved. By microarray studies, we found evidence that treatment of human hepatocellular carcinoma cells with the histone deacetylase inhibitor (HDAC-I) sodium valproate (VPA) mediates recognition of cancer cells by cytotoxic lymphocytes via NKG2D. VPA induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression. No significant changes in the expression of the NKG2D ligands ULBP1-3 were observed. The induction of MIC molecules increased lysis of hepatocellular carcinoma cells by NK cells which was abolished by addition of a blocking NKG2D antibody. Importantly, in primary human hepatocytes, VPA treatment did not induce MIC protein expression. Taken together, our data show that the HDAC-I VPA mediates specific priming of malignant cells for innate immune effector mechanisms. These results suggest the clinical evaluation of HDAC-I in solid tumors such as hepatocellular carcinoma, especially in combination with immunotherapy approaches employing adoptive NK cell transfer.
Blocking Platelet-Derived Growth Factor-D/Platelet-Derived Growth Factor Receptor β Signaling Inhibits Human Renal Cell Carcinoma Progression in an Orthotopic Mouse Model
Authors: Xu Lei , Tong Ricky , Cochran David M. , Jain Rakesh K.
Published: September 2005

Renal cell carcinoma is a highly malignant and often fatal disease of the kidney. It is difficult to treat, often because metastases are common at the time of presentation. Platelet-derived growth factor-D (PDGF-D) is a newly discovered member of the PDGF family; its function in tumor progression is largely unknown. Here, we examined the expression level of PDGF-D in human renal cell carcinoma by immunohistochemical staining using tissue arrays. We showed that human renal cell carcinoma expresses high levels of PDGF-D protein. The human renal cell carcinoma cell line SN12-C was stably transfected with pdgf-d cDNA. Overexpression of PDGF-D in SN12-C cells promoted tumor growth, angiogenesis, and metastasis of human renal cell carcinoma in an orthotopic severe combined immunodeficient (SCID) mouse model. PDGF-D overproduction in SN12-C cells increased the proliferation and migration of mural cells in vitro and improved perivascular cell coverage in vivo. Overexpression of PDGF-D led to increased expression of angiopoietin-1 and matrix metalloproteinase-9 in tumor tissues. ShRNAi and Gleevec were used to block PDGF-D expression and PDGF receptor β (PDGFRβ) signaling. Inhibition of PDGF-D expression by short hairpin RNA interference (shRNAi) and blockage of PDGFRβ signaling by Gleevec inhibited the growth and lung metastasis of SN12-C cells grown orthotopically in SCID mice. Thus, PDGF-D is a potential candidate for controlling the progression of metastatic renal cell carcinoma. This opens up an avenue of investigation into novel therapeutic strategies for the treatment of renal cell carcinoma, including the use of recently developed tyrosine kinase inhibitors, such as Gleevec, which inhibit PDGF activity through inhibition of its receptor tyrosine kinase.