bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Akt/Protein Kinase B Signaling by Naltrindole in Small Cell Lung Cancer Cells
Authors: Chen Yulong L. , Law P. Y. , Loh Horace H.
Published: September 2005

The phosphatidylinositol 3-kinase-Akt/protein kinase B (PKB) survival signaling is very important for cancer cell survival and growth. Constitutively active phosphatidylinositol 3-kinase-Akt/PKB signaling in small cell lung cancer (SCLC) is a major factor for the survival of SCLC cells. Inhibitors of this signaling pathway would be potential antitumor agents, particularly for SCLC. Here we report that naltrindole, which has been used as a classic δ opioid antagonist, inhibited growth and induced apoptosis in the three characteristic SCLC cell lines, NCI-H69, NCI-H345, and NCI-H510. Naltrindole treatment reduced constitutive phosphorylation of Akt/PKB on serine 473 and threonine 308 in cells. We found that the levels of constitutive phosphorylation of Akt/PKB on serine 473 correlate with the sensitivity of the three cell lines to naltrindole treatment. Furthermore, naltrindole treatment not only reduced the phosphorylation of the Akt/PKB upstream kinase phosphoinositide-dependent kinase-1, but also its downstream effectors glycogen synthase kinase-3β and the Forkhead transcription factors AFX and FKHR. DNA array analysis of 205 apoptosis-related genes indicated that some Akt/PKB-dependent genes were either up- or down-regulated by naltrindole. Flow cytometric and microscopic analyses clearly showed that naltrindole induced apoptosis in SCLC cells. RNA interference experiments confirmed that naltrindole-induced cell death was associated with the Akt/PKB survival pathway. Together, these results show that naltrindole is a new inhibitor of the Akt/PKB signaling pathway, suggesting that naltrindole could be a potential lead for the development of a new type of inhibitors that target the constitutively active Akt/PKB signaling-dependent SCLC cells.
Analysis of the Drug Resistance Profile of Multidrug Resistance Protein 7 (ABCC10)
Authors: Hopper-Borge Elizabeth , Chen Zhe-Sheng , Shchaveleva Irina , Belinsky Martin G. , Kruh Gary D.
Published: September 2005

The multidrug resistance protein (MRP) family consists of nine members that can be categorized according to whether or not a third (NH2-terminal) membrane-spanning domain is present. Three (MRP1, MRP2, and MRP3) of the four members that have this structural feature are able to confer resistance to natural product anticancer agents. We previously established that MRP7, the remaining family member that has three membrane-spanning domains, possesses the cardinal biochemical activity of MRPs in that it is able to transport amphipathic anions such as 17β-estradiol 17-(β-d-glucuronide). However, the drug resistance profile of the pump has not been determined. In this study, the drug resistance capabilities of MRP7 are evaluated by analyzing the resistance profiles of two clones of HEK293 cells in which the pump was ectopically expressed. MRP7-transfected HEK293 cells exhibited the highest levels of resistance toward docetaxel (9–13-fold). In addition, lower levels of resistance were observed for paclitaxel (3-fold), vincristine (3-fold), and vinblastine (3–4-fold). Consistent with the operation of an ATP-dependent efflux pump, MRP7-transfected cells exhibited reduced accumulation of radiolabeled paclitaxel compared with HEK293 cells transfected with parental plasmid. These results indicate that MRP7, unlike other MRPs, is a resistance factor for taxanes.
NE-10 Neuroendocrine Cancer Promotes the LNCaP Xenograft Growth in Castrated Mice
Authors: Jin Ren Jie , Wang Yongqing , Masumori Naoya , Ishii Kenichiro , Tsukamoto Taiji , Shappell Scott B. , Hayward Simon W. , Kasper Susan , Matusik Robert J.
Published: September 2005

Increases in neuroendocrine (NE) cells and their secretory products are closely correlated with tumor progression and androgen-independent prostate cancer. However, the mechanisms by which NE cells influence prostate cancer growth and progression, especially after androgen ablation therapy, are poorly understood. To investigate the role of NE cells on prostate cancer growth, LNCaP xenograft tumors were implanted into nude mice. After the LNCaP tumors were established, the NE mouse prostate allograft (NE-10) was implanted on the opposite flank of these nude mice to test whether NE tumor-derived systemic factors can influence LNCaP growth. Mice bearing LNCaP tumors with or without NE allografts were castrated 2 weeks after NE tumor inoculation, and changes in LNCaP tumor growth rate and gene expression were investigated. After castration, LNCaP tumor growth decreased in mice bearing LNCaP tumors alone, and this was accompanied by a loss of nuclear androgen receptor (AR) localization. In contrast, in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continued to grow, had increased levels of nuclear AR, and secreted prostate-specific antigen. Therefore, in the absence of testicular androgens, NE secretions were sufficient to maintain LNCaP cell growth and androgen-regulated gene expression in vivo. Furthermore, in vitro experiments showed that NE secretions combined with low levels of androgens activated the AR, an effect that was blocked by the antiandrogen bicalutamide. Because an increase in AR level has been reported to be sufficient to account for hormone refractory prostate cancers, the NE cell population ability to increase AR level/activity can be another mechanism that allows prostate cancer to escape androgen ablation therapy.
Bone Marrow Stroma Influences Transforming Growth Factor-β Production in Breast Cancer Cells to Regulate c-myc Activation of the Preprotachykinin-I Gene in Breast Cancer Cells
Authors: Oh Hyun S. , Moharita Anabella , Potian Joseph G. , Whitehead Ian P. , Livingston Jason C. , Castro Tammy A. , Patel Prem S. , Rameshwar Pranela
Published: September 2005

Breast cancer cells (BCCs) have preference for the bone marrow (BM). This study used an in vitro coculture of BCCs and BM stroma to represent a model of early breast cancer metastasis to the BM. The overarching hypothesis states that once BCCs are in the BM, microenvironmental factors induce changes in the expression of genes for cytokines and preprotachykinin-I (PPT-I) in both BCCs and stromal cells. Consequently, the expression of both PPT-I and cytokines are altered to facilitate BCC integration within BM stroma. Cytokine and transcription factor arrays strongly suggested that transforming growth factor-β (TGF-β) and c-myc regulate the expression of PPT-I so as to facilitate BCC integration among stroma. Northern analyses and TGF-β bioassays showed that stromal cells and BCCs influence the level of PPT-I and TGF-β in each other. In cocultures, PPT-I and TGF-β expressions were significantly (P < 0.05) increased and decreased, respectively. TGF-β and PPT-I were undetectable in separate stromal cultures but were expressed as cocultures. Two consensus sequences for c-myc in the 5′ flanking region of the PPT-I gene were shown to be functional using gel shift and reporter gene assays. Mutagenesis of c-myc sites, neutralization studies with anti-TGF-β, and transient tranfections all showed that c-myc is required for TGF-β-mediated induction of PPT-I in BCCs. TGF-β was less efficient as a mediator of BCC integration within stroma for c-myc-BCCs. Because the model used in this study represents BCC integration within BM stroma, these studies suggest that TGF-β is important to the regulation of PPT-I in the early events of bone invasion by BCCs.
Thrombospondin-1 Associated with Tumor Microenvironment Contributes to Low-Dose Cyclophosphamide-Mediated Endothelial Cell Apoptosis and Tumor Growth Suppression
Authors: Hamano Yuki , Sugimoto Hikaru , Soubasakos Mary A. , Kieran Mark , Olsen Bjorn R. , Lawler Jack , Sudhakar Akulapalli , Kalluri Raghu
Published: September 2005

Low-dose cyclophosphamide (LDC) induces selective apoptosis of endothelial cells within the vascular bed of tumors. Here, we investigated a hypothesis that the effect of LDC is mediated by the pro-apoptotic action of endogenous inhibitors of angiogenesis. Tumors treated with LDC demonstrate similar expression of matrix metalloproteinases and also basement membrane-derived angiogenesis inhibitors when compared with wild-type tumors, whereas the expression of thrombospondin-1 (TSP-1) is significantly elevated in LDC-treated tumors. We used mice with an absence of type XVIII collagen (endostatin) or type IV collagen α3 chain (tumstatin) or TSP-1 to assess the contribution of these endogenous inhibitors of angiogenesis on LDC-mediated tumor suppression. Lack of TSP-1 in the host in addition to tumor cells leads to diminished capacity of LDC to suppress tumor growth, whereas the absence of endostatin and tumstatin did not alter the effect of LDC. LDC treatment predominantly induces selective expression of TSP-1 in tumor cells and peri-vascular cells and facilitates apoptosis of proliferating endothelial cells, with minimal direct effect on tumor cells and peri-vascular cells. These studies indicate that TSP-1 contributes to tumor growth suppression induced by LDC and suggest that tumors that express high basal level of TSP-1 may be more susceptible to tumor suppression by such a regimen. This study also makes a strong case for TSP-1 expression levels as a potential predictive marker for the successful use of LDC in cancer patients.
Genome-Wide Array-Based Comparative Genomic Hybridization Reveals Multiple Amplification Targets and Novel Homozygous Deletions in Pancreatic Carcinoma Cell Lines
Authors: Heidenblad Markus , Schoenmakers Eric F. P. M. , Jonson Tord , Gorunova Ludmila , Veltman Joris A. , van Kessel Ad Geurts , Höglund Mattias
Published: September 2005

Pancreatic carcinomas display highly complex chromosomal abnormalities, including many structural and numerical aberrations. There is ample evidence indicating that some of these abnormalities, such as recurrent amplifications and homozygous deletions, contribute to tumorigenesis by altering expression levels of critical oncogenes and tumor suppressor genes. To increase the understanding of gene copy number changes in pancreatic carcinomas and to identify key amplification/deletion targets, we applied genome-wide array-based comparative genomic hybridization to 31 pancreatic carcinoma cell lines. Two different microarrays were used, one containing 3,565 fluorescence in situ hybridization-verified bacterial artificial chromosome clones and one containing 25,468 cDNA clones representing 17,494 UniGene clusters. Overall, the analyses revealed a high genomic complexity, with several copy number changes detected in each case. Specifically, 60 amplicons at 32 different locations were identified, most frequently located within 8q (8 cases), 12p (7 cases), 7q (5 cases), 18q (5 cases), 19q (5 cases), 6p (4 cases), and 8p (4 cases). Amplifications of 8q and 12p were mainly clustered at 8q23–24 and 12p11–12, respectively, whereas amplifications on other chromosome arms were more dispersed. Furthermore, our analyses identified several novel homozygously deleted segments located to 9p24, 9p21, 9q32, 10p12, 10q22, 12q24, and 18q23. The individual complexity and aberration patterns varied substantially among cases, i.e., some cell lines were characterized mainly by high-level amplifications, whereas others showed primarily whole-arm imbalances and homozygous deletions. The described amplification and deletion targets are likely to contain genes important in pancreatic tumorigenesis.
c-Myc Interacts with Hypoxia to Induce Angiogenesis
In vivo
by a Vascular Endothelial Growth Factor-Dependent Mechanism
Authors: Knies-Bamforth Ulrike E. , Fox Stephen B. , Poulsom Richard , Evan Gerard I. , Harris Adrian L.
Published: September 2005

The proto-oncogene c-myc is involved in the regulation of cell proliferation, differentiation, and apoptosis. In this study, we used an inducible transgenic mouse model in which c-Myc was targeted to the epidermis and, after activation, gave rise to hyperplastic and dysplastic skin lesions and to dermal angiogenesis, involving both vascular endothelial growth factor (VEGF) receptor-1 and VEGF receptor-2. After c-Myc activation, VEGF mRNA was expressed in postmitotic keratinocytes where it colocalized with transgene expression and areas of tissue hypoxia, suggesting a role of hypoxia in VEGF induction. In vitro, c-Myc activation alone was able to induce VEGF protein release and in conjunction with hypoxia, c-Myc activation further increased VEGF protein. Blocking VEGF signaling in vivo significantly reduced dermal angiogenesis, demonstrating the importance of VEGF as a mediating factor for the c-Myc–induced angiogenic phenotype.
Ammonium Trichloro(dioxoethylene-o,o′)tellurate (AS101) Sensitizes Tumors to Chemotherapy by Inhibiting the Tumor Interleukin 10 Autocrine Loop
Authors: Sredni Benjamin , Weil Merav , Khomenok Gennadi , Lebenthal Ilana , Teitz Seagal , Mardor Yael , Ram Zvi , Orenstein Arie , Kershenovich Amir , Michowiz Shalom , Cohen Yan I. , Rappaport Zvi H. , Freidkin Ilya , Albeck Michael , Longo Dan L. , Kalechman Yona
Published: September 2005

Cancer cells of different solid and hematopoietic tumors express growth factors in respective stages of tumor progression, which by autocrine and paracrine effects enable them to grow autonomously. Here we show that the murine B16 melanoma cell line and two human primary cultures of stomach adenocarcinoma and glioblastoma multiforme (GBM) constitutively secrete interleukin (IL)-10 in an autocrine/paracrine manner. This cytokine is essential for tumor cell proliferation because its neutralization decreases clonogenicity of malignant cells, whereas addition of recombinant IL-10 increases cell proliferation. The immunomodulator ammonium trichloro(dioxoethylene-o,o′)tellurate (AS101) decreased cell proliferation by inhibiting IL-10. This activity was abrogated by exogenous addition of recombinant IL-10. IL-10 inhibition by AS101 results in dephosphorylation of Stat3, followed by reduced expression of Bcl-2. Moreover, these activities of AS101 are associated with sensitization of tumor cells to chemotherapeutic drugs, resulting in their increased apoptosis. More importantly, AS101 sensitizes the human aggressive GBM tumor to paclitaxel both in vitro and in vivo by virtue of IL-10 inhibition. AS101 sensitizes GBM cells to paclitaxel at concentrations that do not affect tumor cells. This sensitization can also be obtained by transfection of GBM cells with IL-10 antisense oligonucleotides. Sensitization of GBM tumors to paclitaxel (Taxol) in vivo was obtained by either AS101 or by implantation of antisense IL-10-transfected cells. The results indicate that the IL-10 autocrine/paracrine loop plays an important role in the resistance of certain tumors to chemotherapeutic drugs. Therefore, anti-IL-10 treatment modalities with compounds such as AS101, combined with chemotherapy, may be effective in the treatment of certain malignancies.
Small Molecule Antagonists of the σ-1 Receptor Cause Selective Release of the Death Program in Tumor and Self-Reliant Cells and Inhibit Tumor Growth
in Vitro
and
in Vivo
Authors: Spruce Barbara A. , Campbell Lorna A. , McTavish Niall , Cooper Michelle A. , Appleyard M. Virginia L. , O’Neill Mary , Howie Jacqueline , Samson Jayne , Watt Stephen , Murray Karen , McLean Doris , Leslie Nick R. , Safrany Stephen T. , Ferguson Michelle J. , Peters John A. , Prescott Alan R. , Box Gary , Hayes Angela , Nutley Bernard , Raynaud Florence , Downes C. Peter , Lambert Jeremy J. , Thompson Alastair M. , Eccles Suzanne
Published: September 2005

The acquisition of resistance to apoptosis, the cell’s intrinsic suicide program, is essential for cancers to arise and progress and is a major reason behind treatment failures. We show in this article that small molecule antagonists of the σ-1 receptor inhibit tumor cell survival to reveal caspase-dependent apoptosis. σ antagonist-mediated caspase activation and cell death are substantially attenuated by the prototypic σ-1 agonists (+)-SKF10,047 and (+)-pentazocine. Although several normal cell types such as fibroblasts, epithelial cells, and even σ receptor-rich neurons are resistant to the apoptotic effects of σ antagonists, cells that can promote autocrine survival such as lens epithelial and microvascular endothelial cells are as susceptible as tumor cells. Cellular susceptibility appears to correlate with differences in σ receptor coupling rather than levels of expression. In susceptible cells only, σ antagonists evoke a rapid rise in cytosolic calcium that is inhibited by σ-1 agonists. In at least some tumor cells, σ antagonists cause calcium-dependent activation of phospholipase C and concomitant calcium-independent inhibition of phosphatidylinositol 3′-kinase pathway signaling. Systemic administration of σ antagonists significantly inhibits the growth of evolving and established hormone-sensitive and hormone-insensitive mammary carcinoma xenografts, orthotopic prostate tumors, and p53-null lung carcinoma xenografts in immunocompromised mice in the absence of side effects. Release of a σ receptor-mediated brake on apoptosis may offer a new approach to cancer treatment.
Seprase Promotes Rapid Tumor Growth and Increased Microvessel Density in a Mouse Model of Human Breast Cancer
Authors: Huang Yan , Wang Sophie , Kelly Thomas
Published: September 2005

Seprase is a cell surface serine protease that is expressed to high levels by invading human breast carcinoma cells. To investigate the role of seprase in breast cancer, MDA MB-231 human mammary adenocarcinoma cells were engineered to express active seprase to high levels. All cells grow rapidly in cell culture. But differences are discovered when the cells are tested for tumorigenicity, growth, and microvessel density by implantation into the mammary fat pads of female severe combined immunodeficient mice. Control transfectants that do not express seprase grow slowly whereas cells that express seprase to high levels form fast-growing tumors that are highly vascular. Microvessel density is elevated in tumors of two different lines of seprase transfectants to 146 ± 67.4 and 144 ± 33.42 vessels/mm2 as compared with 50.5 ± 12.9 vessels/mm2 for tumors of control-transfected cells that do not express seprase. Seprase-expressing cells are better able to attract blood vessels and exhibit rapid tumor growth.
Acute Hypoxia Enhances Spontaneous Lymph Node Metastasis in an Orthotopic Murine Model of Human Cervical Carcinoma
Authors: Cairns Rob A. , Hill Richard P.
Published: September 2005

An orthotopic mouse model of cervical carcinoma has been used to investigate the relationship between acute (cyclic) hypoxia and spontaneous lymph node metastasis in vivo. The human cervical carcinoma cell line ME-180 was stably transfected to express the fluorescent protein DsRed2, which allowed the in vivo optical monitoring of tumor growth and metastasis by fluorescent microscopy. The surgically implanted primary tumors metastasize initially to local lymph nodes and later to lung, a pattern consistent with the clinical course of the disease. The effect of acute hypoxia on the growth and spread of these tumors was examined by exposing tumor-bearing mice to treatment consisting of exposure to 12 cycles of 10 min 7% O2 followed by 10 min air (total 4 h) daily during tumor growth. After 21 days, the tumors were excised, lymph node and lung metastases were quantified, and the hypoxic fraction and relative vascular area of the primary tumors were assessed by immunohistochemical staining for the hypoxic marker drug EF5 [2-(2-nitro-1H-imidazole-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] and the vascular marker CD31, respectively. In untreated mice, the primary tumor size was directly correlated with lymph node metastatic burden. The acute hypoxia treatment resulted in a significant decrease in the size of the primary tumors at the time of excision. However, the mice in the acute hypoxia group had an increased number of positive lymph nodes (2–4) as compared with control mice (1–3). Lung metastasis was not affected. The acute hypoxia treatment also decreased the relative vascular area in the primary tumors but did not affect the hypoxic fraction. These results suggest that fluctuating oxygenation in cervical carcinoma tumors may reduce tumor growth rate, but it may also enhance the ability of tumor cells to metastasize to local lymph nodes.
Correlation between
UDP-Glucuronosyltransferase
Genotypes and 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Glucuronidation Phenotype in Human Liver Microsomes
Authors: Wiener Doris , Fang Jia-Long , Dossett Nicole , Lazarus Philip
Published: September 2005

The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke, and glucuronidation of its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), is an important mechanism for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone detoxification. Substantial interindividual variability in urinary NNAL glucuronide formation has been observed in smokers and tobacco chewers. To determine whether genetic variations may play a role in this interindividual variability, NNAL-glucuronidating activities were analyzed in 78 human liver microsomal specimens and compared with the prevalence of missense polymorphisms in the two major NNAL-glucuronidating enzymes UGT1A4 and UGT2B7. In vitro assays using liver microsomal specimens from individual subjects demonstrated a 70- and 50-fold variability in NNAL-N-Gluc and NNAL-O-Gluc formation, respectively, and a 20-fold variability in the ratio of NNAL-N-Gluc:NNAL-O-Gluc formation. Microsomes from subjects with a homozygous polymorphic UGT1A424Thr/UGT1A424Thr genotype exhibited a significantly higher (P < 0.05) level of NNAL-N-Gluc activity compared with microsomes from subjects with the wild-type UGT1A424Pro/UGT1A424Pro genotype, and a significantly higher (P < 0.05) number of subjects with liver microsomes having high NNAL-N-Gluc formation activity contained the UGT1A424Thr/UGT1A424Thr genotype. Microsomes from subjects with the homozygous polymorphic UGT2B7268Tyr/UGT2B7268Tyr genotype exhibited a significantly lower level (P < 0.025) of NNAL-O-Gluc activity when compared with microsomes from subjects with the wild-type UGT2B7268His/UGT2B7268His genotype, and a significantly (P < 0.05) higher number of subjects with liver microsomes having low NNAL-O-Gluc formation activity contained the UGT2B7268Tyr/UGT2B7268Tyr genotype. These data suggest that the UGT1A4 codon 24 and UGT2B7 codon 268 polymorphisms may be associated with altered rates glucuronidation and detoxification of NNAL in vivo.
In Vivo
Imaging of
β-Galactosidase
Activity Using Far Red Fluorescent Switch
Authors: Tung Ching-Hsuan , Zeng Qing , Shah Khalid , Kim Dong-Eog , Schellingerhout Dawid , Weissleder Ralph
Published: September 2005

β-Galactosidase (β-gal) has been widely used as a transgene reporter enzyme, and several substrates are available for its in vitro detection. The ability to image β-gal expression in living animals would further extend the use of this reporter. Here we show that DDAOG, a conjugate of β-galactoside and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), is not only a chromogenic β-gal substrate but that the cleavage product has far-red fluorescence properties detectable by imaging. Importantly, the cleavage substrate shows a 50-nm red shift, enabling its specific detection in a background of intact probe, a highly desirable feature for in vivo imaging. Specifically, we show that β-gal-expressing 9L gliomas are readily detectable by red fluorescence imaging in comparison with the native 9L gliomas. We furthermore show that herpes simplex virus amplicon-mediated LacZ gene transfer into tumors can be transiently and thus serially visualized over time. The results indicated that in vivo real-time detection of β-gal activity is possible by fluorescence imaging technology.
Vaccination-Induced Autoimmune Vitiligo Is a Consequence of Secondary Trauma to the Skin
Authors: Lane Cecilia , Leitch Jaina , Tan Xiaohua , Hadjati Jamishid , Bramson Jonathan L. , Wan Yonghong
Published: September 2005

A major concern for cancer vaccines targeting self-tumor antigens is the risk of autoimmune sequelae. Although antitumor immunity correlates with autoimmune disease in some preclinical models, the mechanism(s) linking antitumor immunity and subsequent autoimmune pathology remain(s) to be determined. In the current study, we demonstrated that intradermal (i.d.) immunization with a recombinant adenovirus (Ad) expressing the murine melanoma antigen tyrosinase-related protein 2 (AdmTrp-2) results in a moderate level of tumor protection against the B16F10 murine melanoma without any vitiligo. Similar immunization with an Ad encoding human Trp-2 (AdhTrp-2) resulted in 50-fold greater protective immunity and produced vitiligo in all of the mice, suggesting that the development of autoimmunity may reflect the potency of the vaccine. Interestingly, delivery of AdhTrp-2 by i.m. injection generated protective immunity comparable with that seen in mice that received the vaccine by the i.d. route, but none of the recipients in the i.m. group developed vitiligo. The cellular and humoral responses in the i.m. immunized mice were greater than in the i.d. group; therefore, the lack of vitiligo was not caused by reduced efficacy of the vaccine. These results led us to hypothesize that vaccine-induced vitiligo was associated with local inflammatory responses. Mice immunized i.m. with AdhTrp-2 did develop vitiligo when they subsequently were injected i.d. with either a control Ad vector or complete Freund’s adjuvant, suggesting that vitiligo is initiated by some form of trauma within the skin. Our data demonstrated that autoimmune pathology is not an unavoidable outcome of effective cancer vaccines directed against self-tumor antigens.
In Vivo
Targeting of Underglycosylated MUC-1 Tumor Antigen Using a Multimodal Imaging Probe
Authors: Moore Anna , Medarova Zdravka , Potthast Andreas , Dai Guangping
Published: September 2005

One of the most difficult challenges of oncology is to improve methods for early tumor detection, which is crucial for the success of cancer therapy and greatly improves the survival rate. Underglycosylated mucin-1 antigen (uMUC-1) is one of the early hallmarks of tumorigenesis and is overexpressed and underglycosylated on almost all human epithelial cell adenocarcinomas as well as in nonepithelial cancer cell lines, as well as in hematological malignancies such as multiple myeloma, and some B-cell non-Hodgkin lymphomas. In this study, we designed, synthesized, and tested a novel multimodal imaging probe specifically recognizing in vivo uMUC-1 antigen in an animal model of human cancer. Furthermore, in vivo magnetic resonance- and near-infrared-imaging experiments on tumor-bearing animals showed specific accumulation of the probe in uMUC-1-positive tumors and virtually no signal in control tumors. We expect that this probe has a potential to greatly aid in screening prospective patients for early cancer detection and in monitoring the efficacy of drug therapy.
Microarray and Serial Analysis of Gene Expression Analyses Identify Known and Novel Transcripts Overexpressed in Hematopoietic Stem Cells
Authors: Georgantas Robert W. , Tanadve Vivek , Malehorn Matthew , Heimfeld Shelly , Chen Chen , Carr Laura , Martinez-Murillo Francisco , Riggins Greg , Kowalski Jeanne , Civin Curt I.
Published: September 2005

The human CD34+/CD38−/Lin− cell subset, comprising ∼1–10% of the CD34+ cell population, contains few of the less primitive hematopoietic (lineage-committed) progenitor cells (HPCs) but most of the primitive in vivo engrafting (lympho-)hematopoietic stem cells (HSCs). We analyzed gene expression in CD34+/CD38−/Lin− cell populations isolated from normal human adult donor bone marrow, neonatal placental/umbilical cord blood, and mobilized adult donor peripheral blood stem-progenitor cells. As measured by Affymetrix microarrays, 4746 genes were expressed in CD34+/CD38−/Lin− cells from all three tissues. We also determined the transcriptomes of the stem cell-depleted, HPC-enriched CD34+/[CD38/Lin]++ cell population from each tissue. Comparison of CD34+/CD38−/Lin− (HSC-enriched) versus CD34+/[CD38/Lin]++ (HPC-enriched, HSC-depleted) cells from each tissue yielded 81 genes overrepresented and 90 genes underrepresented, common to all three of the CD34+/CD38−/Lin− cell populations. These transcripts, which are selectively expressed in HSCs from all three tissues, include a number of known genes (e.g., transcription factors, receptors, and signaling molecules) that might play roles in key functions (e.g., survival, self-renewal, differentiation, and/or migration/adhesion) of human HSCs. Many genes/transcripts of unknown function were also detected by microarray analysis. Serial analysis of gene expression of the bone marrow HSC and HPC populations confirmed expression of most of the overrepresented transcripts for which reliable serial analysis of gene expression tags were detected and additionally suggested that current microarrays do not detect as many as 30% of the transcripts expressed in HSCs, including a number of previously unknown transcripts. This work is a step toward full definition of the transcriptome of normal human HSCs and may identify new genes involved in leukemogenesis and cancer stem cells.
Selectively Advantageous Mutations and Hitchhikers in Neoplasms
Authors: Maley Carlo C. , Galipeau Patricia C. , Li Xiaohong , Sanchez Carissa A. , Paulson Thomas G. , Reid Brian J.
Published: September 2005

Neoplastic progression is an evolutionary process characterized by genomic instability and waves of clonal expansions carrying genetic and epigenetic lesions to fixation (100% of the cell population). However, an evolutionarily neutral lesion may also reach fixation if it spreads as a hitchhiker on a selective sweep. We sought to distinguish advantageous lesions from hitchhikers in the premalignant condition Barrett’s esophagus. Patients (211) had biopsies taken at 2-cm intervals in their Barrett’s segments. Purified epithelial cells were assayed for loss of heterozygosity and microsatellite shifts on chromosomes 9 and 17, sequence mutations in CDKN2A/MTS1/INK4a (p16) and TP53 (p53), and methylation of the p16 promoter. We measured the expanse of a lesion in a Barrett’s segment as the proportion of proliferating cells that carried a lesion in that locus. We then selected the lesion having expanses >90% in the greatest number of patients as our first putative advantageous lesion. We filtered out hitchhikers by removing all expanses of other lesions that did not occur independent of the advantageous lesion. The entire process was repeated on the remaining expanses to identify additional advantageous lesions. p16 loss of heterozygosity, promoter methylation, and sequence mutations have strong, independent, advantageous effects on Barrett’s cells early in progression. Second lesions in p16 and p53 are associated with later selective sweeps. Virtually all of the other lesion expansions, including microsatellite shifts, could be explained as hitchhikers on p16 lesion clonal expansions. These techniques can be applied to any neoplasm.
Transforming Growth Factor β Receptor Type II Inactivation Promotes the Establishment and Progression of Colon Cancer
Authors: Biswas Swati , Chytil Anna , Washington Kay , Romero-Gallo Judith , Gorska Agnieszka E. , Wirth Pamela S. , Gautam Shiva , Moses Harold L. , Grady William M.
Published: September 2005

Deregulation of members of the transforming growth factor (TGF)-β signaling pathway occurs often in colon cancers and is believed to affect the formation of primary colon cancer. Mutational inactivation of TGFBR2 is the most common genetic event affecting the TGF-β signaling pathway and occurs in ∼20–30% of all colon cancers. By mating Fabpl4xat-132 Cre mice with Tgfbr2flx/flx mice, we have generated a mouse model that is null for Tgfbr2 in the colonic epithelium, and in this model system, we have assessed the effect of loss of TGF-β signaling in vivo on colon cancer formation induced by azoxymethane (AOM). We have observed a significant increase in the number of AOM-induced adenomas and adenocarcinomas in the Fabpl4xat-132 Cre Tgfbr2flx/flx mice compared with Tgfbr2flx/flx mice, which have intact TGF-β receptor type II (TGFBR2) in the colon epithelium, and we have found increased proliferation in the neoplasms occurring in the Fabpl4xat-132 Cre Tgfbr2flx/flx mice. These results implicate the loss of TGF-β-mediated growth inhibition as one of the in vivo mechanisms through which TGFBR2 inactivation contributes to colon cancer formation. Thus, we have demonstrated that loss of TGFBR2 in colon epithelial cells promotes the establishment and progression of AOM-induced colon neoplasms, providing evidence from an in vivo model system that TGFBR2 is a tumor suppressor gene in the colon.
Alteration of Gene Expression in Normal-Appearing Colon Mucosa of
APC

min
Mice and Human Cancer Patients
Authors: Chen Ling-Chun , Hao Chun-Yi , Chiu Yanek S. Y. , Wong Patrick , Melnick Jane S. , Brotman Martin , Moretto John , Mendes Fredrick , Smith Andrew P. , Bennington James L. , Moore Dan , Lee Nancy M.
Published: September 2005

The expression of many genes is altered in colon cancer, but the roles of these genes in carcinogenesis are unclear. Using real-time quantitative PCR, we demonstrated that several genes previously implicated in human colon cancer undergo altered expression in the APCmin mouse adenomatous polyp, a precursor of cancer, as well as in normal-appearing surrounding mucosa. The five genes that were most highly up-regulated in mouse polyp were also significantly up-regulated in polyp-free colon mucosa. Similar changes occurred in morphologically normal mucosa of surgical sections taken from human cancer patients, frequently extending to the margins. Thus, morphologically normal colon mucosa in APCmin mice and in human cancer patients is not metabolically normal. Altered gene expression in this tissue does not appear to result from a field effect because there was no correlation between extent of altered regulation and distance from polyp or tumor. Our data suggest that alterations of expression levels of these genes may be an early event in carcinogenesis and a marker of risk for the development of colon cancer.
Evidence of Antiangiogenic and Antimetastatic Activities of the Recombinant Disintegrin Domain of Metargidin
Authors: Trochon-Joseph Véronique , Martel-Renoir Dominique , Mir Lluis M. , Thomaïdis Annick , Opolon Paule , Connault Elisabeth , Li Hong , Grenet Christophe , Fauvel-Lafève Françoise , Soria Jeannette , Legrand Chantal , Soria Claudine , Perricaudet Michel , Lu He
Published: September 2005

Metargidin, a transmembrane protein of the adamalysin family, and integrins, e.g., α5β1 and αv, are preferentially expressed on endothelial cells on angiogenesis. Furthermore, metargidin interacts with these integrins via its disintegrin domain. In this study, recombinant human disintegrin domain (RDD) was produced in Escherichia coli by subcloning its cDNA into the pGEX-2T vector, and the effect of purified RDD on different steps of angiogenesis was evaluated. At concentrations of 2–10 μg/ml, RDD exhibited inhibitory activities in a variety of in vitro functional assays, including endothelial cell proliferation and adhesion on the integrin substrates fibronectin, vitronectin, and fibrinogen. RDD (10 μg/ml) totally abrogated endothelial cell migration and blocked most capillary formation in a three-dimensional fibrin gel. To test RDD efficacy in vivo, the RDD gene inserted into pBi vector containing a tetracycline-inducible promoter was electrotransferred into nude mouse muscle. RDD was successfully synthesized by muscle cells in vivo as shown by immunolabeling and Western blotting. In addition, 78% less MDA-MB-231 tumor growth, associated with strong inhibition of tumor angiogenesis, was observed in athymic mice bearing electrotransferred RDD. Moreover, in the presence of RDD, 74% fewer B16F10 melanoma lung metastases were found in C57BL/6 mice. Taken together, these results identified this RDD as a potent intrinsic inhibitor of angiogenesis, tumor growth, and metastasis, making it a promising tool for use in anticancer treatment.
Pheophorbide a Is a Specific Probe for ABCG2 Function and Inhibition
Authors: Robey Robert W. , Steadman Kenneth , Polgar Orsolya , Morisaki Kuniaki , Blayney Margaret , Mistry Prakash , Bates Susan E.
Published: September 2005

Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2−/− knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649–15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumitremorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 μm of the cyclin-dependent kinase inhibitor UCN-01 or 1 μm of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected HEK-293 cells treated with 1 μm tariquidar, and ABCG2-transfected cells were 6–7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples.
Presence of Simian Virus 40 DNA Sequences in Human Lymphoid and Hematopoietic Malignancies and Their Relationship to Aberrant Promoter Methylation of Multiple Genes
Authors: Shivapurkar Narayan , Takahashi Takao , Reddy Jyotsna , Zheng Yingye , Stastny Victor , Collins Robert , Toyooka Shinichi , Suzuki Makato , Parikh Gunjan , Asplund Sheryl , Kroft Steven H. , Timmons Charles , McKenna Robert W. , Feng Ziding , Gazdar Adi F.
Published: September 2005

The simian polyoma virus SV40 has been detected in specific human tumors including non-Hodgkin’s lymphomas, although a causative role for the virus has not been convincingly demonstrated. Aberrant methylation of CpG islands in promoter regions is a frequent method of silencing tumor suppressor genes (TSGs) in cancers and may be induced by oncogenic viruses. We investigated the relationship between the presence of SV40 or EBV DNA sequences and the methylation profiles for 10 TSGs in 90 cases of non-Hodgkin’s lymphomas/leukemias and 56 control tissues. SV40 sequences were present in 33/90 (37%) non-Hodgkin’s lymphomas/leukemias, and EBV was present in 11/42 (26%) of non-Hodgkin’s lymphomas. We found a highly significant correlation between the presence of SV40 and methylation of seven genes (P values, 0.006 to <0.0001). In lymphomas, there was no relationship between EBV and methylation. Oncogenic viruses and methylation were rarely present in control tissues. We investigated methylation of the same 10 TSGs in peripheral blood mononuclear cells (PBMC) from a healthy volunteer infected with EBV or EBV and SV40. Promoter methylation of CDH1 and CDH13 were noted in dual SV40- and EBV-infected PBMC, and these two genes were also highly significantly correlated to the presence of SV40 sequences in tumors. SV40 infection also resulted in appearance of the lymphoma/leukemia-specific marker, methylated SHP1. Methylation was completely absent in uninfected and EBV-infected PBMC. Our results demonstrate that the presence of SV40 in hematological malignancies is associated with promoter methylation of TSGs and that in all probability, the virus plays a role in tumor pathogenesis.
PrLZ,
a Novel Prostate-Specific and Androgen-Responsive Gene of the TPD52 Family, Amplified in Chromosome 8q21.1 and Overexpressed in Human Prostate Cancer
Authors: Wang Ruoxiang , Xu Jianchun , Saramäki Outi , Visakorpi Tapio , Sutherland William M. , Zhou Jianguang , Sen Buer , Lim So Dug , Mabjeesh Nicola , Amin Mahul , Dong Jin-Tang , Petros John A. , Nelson Peter S. , Marshall Fray F. , Zhau Haiyen E. , Chung Leland W. K.
Published: September 2005

We report a previously unrecognized prostate-specific protein, PrLZ (prostate leucine zipper), a new member of the Tumor Protein D52 (TPD52) family. The gene for PrLZ was localized at chromosome 8q21.1, a locus most frequently amplified in human prostate cancer. Multiple tissue analyses demonstrated PrLZ predominantly in the prostate gland. Although its expression was enhanced by androgens in androgen receptor-expressing cells, PrLZ was detected in all of the human prostate cancer cell lines, regardless of androgen receptor status. Monoclonal anti-PrLZ antibodies were produced and intense immunohistochemical staining of PrLZ was observed in prostate epithelial cells in intraepithelial neoplasia and prostate cancer, whereas lower-level staining was detected in normal and benign epithelial components of the prostate gland. As the only prostate-specific gene identified in the most frequently amplified genomic region in prostate cancer, PrLZ may be the link between chromosome 8q amplification and malignant transformation of the prostate epithelia.
Imatinib Mesylate Is a Potent Inhibitor of the ABCG2 (BCRP) Transporter and Reverses Resistance to Topotecan and SN-38
in Vitro
Authors: Houghton Peter J. , Germain Glen S. , Harwood Franklin C. , Schuetz John D. , Stewart Clinton F. , Buchdunger Elisabeth , Traxler Peter
Published: September 2005

Imatinib mesylate (Gleevec, STI571) is a kinase inhibitor selective for Bcr-Abl, activated c-Kit kinases, and platelet-derived growth factor receptor tyrosine kinase. Imatinib mesylate, similar to many other tyrosine kinase inhibitors (TKIs), such as members of the 4-anilinoquinazoline class, competes for ATP binding. Previously, 4-anilinoquinazoline TKIs have been shown to inhibit the function of the breast cancer resistance-associated drug transporter (ABCG2), reversing resistance to camptothecin derivatives topotecan and SN-38. However, the potential to inhibit ABCG2 for the 2-phenylamino-pyrimidine class of TKIs, exemplified by imatinib mesylate, has not been examined. Here, we show that imatinib mesylate potently reverses ABCG2-mediated resistance to topotecan and SN-38 and significantly increases accumulation of topotecan only in cells expressing functional ABCG2. However, overexpression of ABCG2 does not confer resistance to imatinib mesylate. Furthermore, accumulation and efflux of [14C]imatinib mesylate are unaltered between ABCG2-expressing and non-ABCG2-expressing cells or by ATP depletion. These results suggest that imatinib mesylate inhibits the function of ABCG2 but is not a substrate for this transporter.
Reactive Oxygen Species (ROS), Troublemakers between Nuclear Factor-κB (NF-κB) and c-Jun NH2-terminal Kinase (JNK)
Authors: Zhang Yadong , Chen Fei
Published: September 2005

Nuclear factor-κB (NF-κB) and c-Jun NH2-terminal kinase (JNK) are activated simultaneously under a variety of stress conditions. They also share several common signaling pathways for their activation in response to cytokines or growth factors. Recent studies, however, demonstrated a new form of interplay between these two allies. Inhibition of NF-κB by ikkβ or rela gene deficiency sensitizes stress responses through enhanced or prolonged activation of JNK. Conversely, sustained activation of NF-κB inhibits cytokine-induced JNK activation. The mechanisms of how NF-κB and JNK become rivals for each other are under extensive debate.
Classification of Proliferative Pulmonary Lesions of the Mouse
Authors: Nikitin Alexander Yu. , Alcaraz Ana , Anver Miriam R. , Bronson Roderick T. , Cardiff Robert D. , Dixon Darlene , Fraire Armando E. , Gabrielson Edward W. , Gunning William T. , Haines Diana C. , Kaufman Matthew H. , Linnoila R. Ilona , Maronpot Robert R. , Rabson Alan S. , Reddick Robert L. , Rehm Sabine , Rozengurt Nora , Schuller Hildegard M. , Shmidt Elena N. , Travis William D. , Ward Jerrold M. , Jacks Tyler
Published: September 2005

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.
A Serial Analysis of Gene Expression (SAGE) Database Analysis of Chemosensitivity
Authors: Stein Wilfred D , Litman Thomas , Fojo Tito , Bates Susan E
Published: September 2005

Drug sensitivity and resistance has been most extensively studied in cell lines carried in tissue culture. Furthermore, cell lines have been widely used in testing new anticancer agents, despite the widely recognized observation that cell lines are more sensitive to cytotoxic drugs than are their corresponding solid tumors. We used the Serial Analysis of Gene Expression (SAGE) database to identify differences between solid tumors and cell lines, hoping to detect genes that could potentially explain differences in drug sensitivity. SAGE libraries were available for both solid tumors and cell lines from breast, colon, ovarian, pancreatic, and prostate carcinomas and from gliomas and medulloblastomas. Sixty-two genes were identified as overexpressed in tumors. The immune response and complement pathways were the significant common themes, with extracellular matrix (ECM) proteins third. For the 61 genes overexpressed in cell lines, protein synthesis was the dominant theme. We next used the SAGE database to identify genetic differences between tumor types that convey a broad range of survival to the patients that bear them as distant metastases. SAGE gene expression data were correlated with 5-year survivals documented in the SEER (Surveillance, Epidemiology and End-Results) database for patients diagnosed with “distant” or metastatic cancers. These are unlikely to be amenable to surgical resection; therefore, survival here reflects, to some extent, sensitivity to systemic therapy, i.e., chemotherapy. Using survival data as a surrogate of chemotherapy sensitivity, a spectrum can be generated, with testicular cancer at one end and pancreatic cancer at the other. Favorable 5-year survival, despite a distant presentation, correlates with expression of protein synthesis genes. Poor 5-year survival correlates with expression of cell adhesion, cytoskeletal, and ECM genes, a pattern similar to that found to distinguish solid tumors from the more cytotoxin-sensitive cancer cell lines. One interpretation is that resistance to chemotherapy may arise, in part, from the adherent, relatively inert condition (i.e., low in protein synthesis potential) of refractory cancers. Thus, attachment or ECM genes could be targets for anticancer therapy.