bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor-Specific Low Molecular Weight Forms of Cyclin E Induce Genomic Instability and Resistance to p21, p27, and Antiestrogens in Breast Cancer
Authors: Akli Said , Zheng Ping-Ju , Multani Asha S. , Wingate Hannah F. , Pathak Sen , Zhang Ning , Tucker Susan L. , Chang Sandy , Keyomarsi Khandan
Published: September 2005

The deregulated expression of cyclin E as measured by the overexpression of its low molecular weight (LMW) isoforms is a powerful predictor of poor outcome in patients with breast cancer. The mechanism by which these LMW forms give tumor cells a growth advantage is not known and is the subject of this article. In this article, we provide the pathobiological mechanisms of how these LMW forms are involved in disease progression. Specifically, we show that overexpression of the LMW forms of cyclin E but not the full-length form in MCF-7 results in (a) their hyperactivity because of increased affinity for cdk2 and resistance to inhibition by the cyclin-dependent kinase inhibitors p21 and p27, (b) resistance to the growth inhibiting effects of antiestrogens, and (c) chromosomal instability. Lastly, tumors from breast cancer patients overexpressing the LMW forms of cyclin E are polyploid in nature and are resistant to endocrine therapy. Collectively, the biochemical and functional differences between the full-length and the LMW isoforms of cyclin E provide a molecular mechanism for the poor clinical outcome observed in breast cancer patients harboring tumors expressing high levels of the LMW forms of cyclin E. These properties of the LMW forms cyclin E suggest that they are not just surrogate markers of poor outcome but bona fide mediators of aggressive disease and potential therapeutic targets for patients whose tumors overexpress these forms.
AEE788
Authors: Traxler Peter , Allegrini Peter R. , Brandt Ralf , Brueggen Josef , Cozens Robert , Fabbro Doriano , Grosios Konstantina , Lane Heidi A. , McSheehy Paul , Mestan Jürgen , Meyer Thomas , Tang Careen , Wartmann Markus , Wood Jeanette , Caravatti Giorgio
Published: September 2005

Aberrant epidermal growth factor receptor (EGFR) and ErbB2 expression are associated with advanced disease and poor patient prognosis in many tumor types (breast, lung, ovarian, prostate, glioma, gastric, and squamous carcinoma of head and neck). In addition, a constitutively active EGFR type III deletion mutant has been identified in non-small cell lung cancer, glioblastomas, and breast tumors. Hence, members of the EGFR family are viewed as promising therapeutic targets in the fight against cancer. In a similar vein, vascular endothelial growth factor (VEGF) receptor kinases are also promising targets in terms of an antiangiogenic treatment strategy. AEE788, obtained by optimization of the 7H-pyrrolo[2,3-d]pyrimidine lead scaffold, is a potent combined inhibitor of both epidermal growth factor (EGF) and VEGF receptor tyrosine kinase family members on the isolated enzyme level and in cellular systems. At the enzyme level, AEE788 inhibited EGFR and VEGF receptor tyrosine kinases in the nm range (IC50s: EGFR 2 nm, ErbB2 6 nm, KDR 77 nm, and Flt-1 59 nm). In cells, growth factor-induced EGFR and ErbB2 phosphorylation was also efficiently inhibited (IC50s: 11 and 220 nm, respectively). AEE788 demonstrated antiproliferative activity against a range of EGFR and ErbB2-overexpressing cell lines (including EGFRvIII-dependent lines) and inhibited the proliferation of epidermal growth factor- and VEGF-stimulated human umbilical vein endothelial cells. These properties, combined with a favorable pharmacokinetic profile, were associated with a potent antitumor activity in a number of animal models of cancer, including tumors that overexpress EGFR and or ErbB2. Oral administration of AEE788 to tumor-bearing mice resulted in high and persistent compound levels in tumor tissue. Moreover, AEE788 efficiently inhibited growth factor-induced EGFR and ErbB2 phosphorylation in tumors for >72 h, a phenomenon correlating with the antitumor efficacy of intermittent treatment schedules. Strikingly, AEE788 also inhibited VEGF-induced angiogenesis in a murine implant model. Antiangiogenic activity was also apparent by measurement of tumor vascular permeability and interstitial leakage space using dynamic contrast enhanced magnetic resonance imaging methodology. Taken together, these data indicate that AEE788 has potential as an anticancer agent targeting deregulated tumor cell proliferation as well as angiogenic parameters. Consequently, AEE788 is currently in Phase I clinical trials in oncology.
Glycine
N
-Methyltransferase Tumor Susceptibility Gene in the Benzo(
a
)pyrene-Detoxification Pathway
Authors: Chen Shih-Yin , Lin Jane-Ru Vivan , Darbha Ramalakshmi , Lin Pinpin , Liu Tsung-Yun , Chen Yi-Ming Arthur
Published: September 2005

Glycine N-methyltransferase (GNMT) affects genetic stability by (a) regulating the ratio of S-adenosylmethionine to S-adenosylhomocystine and (b) binding to folate. Based on the identification of GNMT as a 4 S polyaromatic hydrocarbon-binding protein, we used liver cancer cell lines that expressed GNMT either transiently or stably in cDNA transfections to analyze the role of GNMT in the benzo(a)pyrene (BaP) detoxification pathway. Results from an indirect immunofluorescent antibody assay showed that GNMT was expressed in cell cytoplasm before BaP treatment and translocated to cell nuclei after BaP treatment. Compared with cells transfected with the vector plasmid, the number of BaP-7,8-diol 9,10-epoxide-DNA adducts that formed in GNMT-expressing cells was significantly reduced. Furthermore, the dose-dependent inhibition of BaP-7,8-diol 9,10-epoxide-DNA adduct formation by GNMT was observed in HepG2 cells infected with different multiplicities of infection of recombinant adenoviruses carrying GNMT cDNA. According to an aryl hydrocarbon hydroxylase enzyme activity assay, GNMT inhibited BaP-induced cytochrome P450 1A1 enzyme activity. Automated BaP docking using a Lamarckian genetic algorithm with GNMT X-ray crystallography revealed a BaP preference for the S-adenosylmethionine-binding domain of the dimeric form of GNMT, a novel finding of a cellular defense against potentially damaging exposures. In addition to GNMT, results from docking experiments showed that BaP binds readily with other DNA methyltransferases, including HhaI, HaeIII, PvuII methyltransferases and human DNA methyltransferase 2. We therefore hypothesized that BaP-DNA methyltransferase and BaP-GNMT interactions may contribute to carcinogenesis.
Role of the Retinoblastoma Pathway in Senescence Triggered by Repression of the Human Papillomavirus E7 Protein in Cervical Carcinoma Cells
Authors: Psyrri Amanda , DeFilippis Rosa Anna , Edwards Anne P. B. , Yates Kristin E. , Manuelidis Laertes , DiMaio Daniel
Published: September 2005

Repression of the endogenous human papillomavirus (HPV) type 18 E7 gene in HeLa cervical carcinoma cells by the bovine papillomavirus E2 transcription factor activates the retinoblastoma (Rb) pathway and induces cells to undergo senescence. To determine whether activation of the Rb pathway is responsible for senescence in response to HPV18 E7 repression, we tested the ability of wild-type and mutant E7 proteins to affect the activity of the Rb pathway and to modulate senescence in these cells. Enforced expression of the wild-type HPV16 E7 protein prevented Rb activation in response to E2 expression and impaired senescence. Importantly, there was an absolute correlation between the ability of mutant E7 proteins to inactivate the Rb pathway and to inhibit senescence in HeLa cells. Similar results were obtained in HT-3 cervical carcinoma cells. These results provide strong genetic evidence that activation of the Rb pathway is required for senescence in response to E7 repression. Hence, continuous neutralization of the Rb pathway by the E7 protein is required to maintain the proliferation of cervical carcinoma cells. Similarly, our results indicate that activation of the Rb pathway can prevent apoptosis induced by repression of the HPV18 E6 gene in HeLa cells.
Activation of Protein Kinase G Is Sufficient to Induce Apoptosis and Inhibit Cell Migration in Colon Cancer Cells
Authors: Deguchi Atsuko , Thompson W. Joseph , Weinstein I. Bernard
Published: September 2005

The activation of protein kinase G (PKG) by cGMP has become of considerable interest as a novel molecular mechanism for the induction of apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases and thereby increase cellular levels of cGMP appear to induce apoptosis via this mechanism. However, other effects of these compounds have not been excluded, and the precise mechanism by which PKG activation induces apoptosis has not been elucidated in detail. To directly examine the effects of PKG on cell growth and apoptosis, we generated a series of mutants of PKG Iα: PKG IαS65D, a constitutively activated point mutant; PKG IαΔ, a constitutively activated N-terminal truncated mutant; and PKG IαK390R, a dominant-negative point mutant. A similar series of mutants of PKG Iβ were also constructed (Deguchi et al., Mol. Cancer Ther., 1: 803–809, 2002). The present study demonstrates that when transiently expressed in SW480 colon cancer, the constitutively activated mutants of PKG Iβ, and to a lesser extent PKG Iα, inhibit colony formation and induce apoptosis. We were not able to obtain derivatives of SW480 cells that stably expressed these constitutively activated mutants, presumably because of toxicity. However, derivatives that stably overexpressed wild-type PKG Iβ displayed growth inhibition, whereas derivatives that stably expressed the dominant-negative mutant (KR) of PKG Iβ grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG Iβ was associated with decreased cellular levels of β-catenin and cyclin D1 and increased levels of p21CIP1. Reporter assays indicated that activation of PKG Iβ inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants of PKG Iβ inhibited cell migration. Taken together, these results indicate that activation of PKG Iβ is sufficient to inhibit growth and cell migration and induce apoptosis in human colon cancer cells and that these effects are associated with inhibition of the transcription of cyclin D1 and an increase in the expression of p21CIP1.
Serum Proteomic Profiles Suggest Celecoxib-Modulated Targets and Response Predictors
Authors: Xiao Zhen , Luke Brian T. , Izmirlian Grant , Umar Asad , Lynch Patrick M. , Phillips Robin K. S. , Patterson Sherri , Conrads Thomas P. , Veenstra Timothy D. , Greenwald Peter , Hawk Ernest T. , Ali Iqbal U.
Published: September 2005

Cyclooxygenase-2 is a valid target for cancer prevention and treatment. This has been shown in preclinical and clinical cancer prevention studies by using a cyclooxygenase-2 inhibitor, celecoxib. When used in a randomized cancer prevention clinical trial on patients with the inherited autosomal dominant condition, familial adenomatous polyposis, celecoxib proved efficacious. However, a remarkable heterogeneity in patients’ responses to the chemopreventive effects of celecoxib was observed. Proteomic profiling of sera from these patients identified several markers, the expression of which was specifically modulated after treatment with celecoxib. A decision tree algorithm identified classifiers for response to celecoxib with relatively high sensitivity but moderate to low specificity. In particular, a spectral feature at m/z 16,961.4 was identified as a strong discriminator between response and nonresponse to celecoxib at the highest dose.
Simultaneous Blockade of Platelet-Derived Growth Factor-Receptor and Epidermal Growth Factor-Receptor Signaling and Systemic Administration of Paclitaxel as Therapy for Human Prostate Cancer Metastasis in Bone of Nude Mice
Authors: Kim Sun Jin , Uehara Hisanori , Yazici Sertac , Langley Robert R. , He Junqin , Tsan Rachel , Fan Dominic , Killion Jerald J. , Fidler Isaiah J.
Published: September 2005

Once prostate cancer metastasizes to bone, conventional chemotherapy is largely ineffective. We hypothesized that inhibition of phosphorylation of the epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) expressed on tumor cells and tumor-associated endothelial cells, which is associated with tumor progression, in combination with paclitaxel would inhibit experimental prostate cancer bone metastasis and preserve bone structure. We tested this hypothesis in nude mice, using human PC-3MM2 prostate cancer cells. PC-3MM2 cells growing adjacent to bone tissue and endothelial cells within these lesions expressed phosphorylated EGF-R and PDGF-Rα and -β on their surfaces. The percentage of positive endothelial cells and the intensity of receptor expression directly correlated with proximity to bone tissue. Oral administration of PKI166 inhibited the phosphorylation of EGF-R but not PDGF-R, whereas oral administration of STI571 inhibited the phosphorylation of PDGF-R but not EGF-R. Combination therapy using oral PKI166 and STI571 with i.p. injections of paclitaxel induced a high level of apoptosis in tumor vascular endothelial cells and tumor cells in parallel with inhibition of tumor growth in the bone, preservation of bone structure, and reduction of lymph node metastasis. Collectively, these data demonstrate that blockade of phosphorylation of EGF-R and PDGF-R coupled with administration of paclitaxel significantly suppresses experimental human prostate cancer bone metastasis.
A Technical Triade for Proteomic Identification and Characterization of Cancer Biomarkers
Authors: Melle Christian , Ernst Günther , Schimmel Bettina , Bleul Annett , Koscielny Sven , Wiesner Andreas , Bogumil Ralf , Möller Ursula , Osterloh Dirk , Halbhuber Karl-Jürgen , von Eggeling Ferdinand
Published: September 2005

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 × 10−5 and 4.6 × 10−5) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.
Different Procarcinogenic Potentials of Lymphocyte Subsets in a Transgenic Mouse Model of Chronic Hepatitis B
Authors: Nakamoto Yasunari , Suda Takashi , Momoi Takashi , Kaneko Shuichi
Published: September 2005

The immune response to hepatitis viruses is believed to be involved in the development of chronic hepatitis; however, its pathogenetic potential has not been clearly defined. The current study, using a transgenic mouse model of chronic hepatitis B, was designed to determine the relative contributions of the immune cell subsets to the progression of liver disease that induces hepatocellular carcinogenesis. Hepatitis B virus transgenic mice were adoptively transferred with CD4+ and CD8+ T cell-enriched or -depleted and B cell-depleted splenocytes obtained from hepatitis B surface antigen-primed, syngeneic nontransgenic donors. The resultant liver disease, hepatocyte apoptosis, regeneration, and tumor development were assessed and compared with the manifestations in mice that had received unfractionated spleen cells. Transfer of CD8+-enriched splenocytes caused prolonged disease kinetics, and a marked increase in the extent of hepatocyte apoptosis and regeneration. In 12 of 14 mice the transfer resulted in multiple hepatocellular carcinomas (HCCs) comparable with the manifestations seen in the mice transferred with total splenocytes. In contrast, mice that had received CD4+-enriched cells demonstrated lower levels of liver disease and developed fewer incidences of HCC (4 of 17). The experiment also revealed that all of the groups of mice complicated with HCC developed comparable mean numbers and sizes of tumors. B-cell depletion had no effect on disease kinetics in this model. Taken together, these results demonstrate that the pathogenetic events induced by CD8+ T-cell subset are primarily responsible for the induction of chronic liver disease that increases tumor incidence, suggesting their potential in triggering the process of hepatocarcinogenesis.
Dose-Dependent Effects of Platelet-Derived Growth Factor-B on Glial Tumorigenesis
Authors: Shih Alan H. , Dai Chengkai , Hu Xiaoyi , Rosenblum Marc K. , Koutcher Jason A. , Holland Eric C.
Published: September 2005

Platelet-derived growth factor (PDGF) is expressed in many different tumors, but its precise roles in tumorigenesis remain to be fully defined. Here, we report on a mouse model that demonstrates dose-dependent effects of PDGF-B on glial tumorigenesis. By removing inhibitory regulatory elements in the PDGFB mRNA, we are able to substantially elevate its expression in tumor cells using a retroviral delivery system. This elevation in PDGF-B production results in tumors with shortened latency, increased cellularity, regions of necrosis, and general high-grade character. In addition, elevated PDGF-B in these tumors also mediates vascular smooth muscle cell recruitment that supports tumor angiogenesis. PDGF receptor (PDGFR) signaling appears to be required for the maintenance of these high-grade characteristics, because treatment of high-grade tumors with a small molecule inhibitor of PDGFR results in reversion to a lower grade tumor histology. Our data show that PDGFR signaling quantitatively regulates tumor grade and is required to sustain high-grade oligodendrogliomas.
Synthesis and Evaluation of Bombesin Derivatives on the Basis of Pan-Bombesin Peptides Labeled with Indium-111, Lutetium-177, and Yttrium-90 for Targeting Bombesin Receptor-Expressing Tumors
Authors: Zhang Hanwen , Chen Jianhua , Waldherr Christian , Hinni Karin , Waser Beatrice , Reubi Jean Claude , Maecke Helmut R.
Published: September 2005

Bombesin receptors are overexpressed on a variety of human tumors like prostate, breast, and lung cancer. The aim of this study was to develop radiolabeled (Indium-111, Lutetium-177, and Yttrium-90) bombesin analogues with affinity to the three bombesin receptor subtypes for targeted radiotherapy. The following structures were synthesized: diethylenetriaminepentaacetic acid-γ-aminobutyric acid-[d-Tyr6, β-Ala11, Thi13, Nle14] bombesin (6–14) (BZH1) and 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴ -tetraacetic acid-γ-aminobutyric acid-[d-Tyr6, β-Ala11, Thi13, Nle14] bombesin (6–14) (BZH2). [111In]-BZH1 and in particular [90Y]-BZH2 were shown to have high affinity to all three human bombesin receptor subtypes with binding affinities in the nanomolar range. In human serum metabolic cleavage was found between β-Ala11 and His12 with an approximate half-life of 2 hours. The metabolic breakdown was inhibited by EDTA and β-Ala11-His12 (carnosine) indicating that carnosinase is the active enzyme.

Both 111In-labeled peptides were shown to internalize into gastrin-releasing peptide-receptor–positive AR4–2J and PC-3 cells with similar high rates, which were independent of the radiometal. The biodistribution studies of [111In]-BZH1 and [111In]-BZH2 ([177Lu]-BZH2) in AR4–2J tumor-bearing rats showed specific and high uptake in gastrin-releasing peptide-receptor–positive organs and in the AR4–2J tumor. A fast clearance from blood and all of the nontarget organs except the kidneys was found. These radiopeptides were composed of the first pan-bombesin radioligands, which show great promise for the early diagnosis of tumors bearing not only gastrin-releasing peptide-receptors but also the other two bombesin receptor subtypes and may be of use in targeted radiotherapy of these tumors.
Urokinase-Type Plasminogen Activator System in Breast Cancer
Authors: Gelder Marion E. Meijer-van , Look Maxime P. , Peters Harry A. , Schmitt Manfred , Brünner Nils , Harbeck Nadia , Klijn Jan G. M. , Foekens John A.
Published: September 2005

The prognostic value of components of the urokinase-type plasminogen activator (uPA) system, its receptor uPAR (CD87), and plasminogen activator inhibitors PAI-1 and PAI-2 is well established. We studied the predictive value of these proteolytic factors by evaluating the association of their tumor expression level and the efficacy of tamoxifen therapy in patients with recurrent breast cancer. The antigen levels of the four factors were determined by ELISA in cytosols prepared from estrogen receptor-positive primary breast tumors of 691 hormone-naive breast cancer patients with recurrent disease and treated with tamoxifen as first-line systemic therapy. High tumor levels of uPA (P < 0.001), uPAR (P < 0.01), and PAI-1 (P = 0.01) were associated with a lower efficacy of tamoxifen therapy. In the multivariable analysis, uPA (P < 0.001) provided additional information independent of the traditional predictive factors to predict benefit from tamoxifen therapy. High levels of uPA, uPAR, and PAI-1 predicted a shorter progression-free survival (PFS) on tamoxifen in an analysis of the first 9 months of therapy. However in the analysis during the total follow-up period, high PAI-2 levels (P = 0.01) showed a longer response to tamoxifen. In conclusion, uPA, uPAR, and PAI-1, components of the urokinase system, are predictive for the efficacy of tamoxifen therapy in patients treated for recurrent breast cancer. Knowledge of their tumor expression levels might be helpful for future individualized therapy protocols, including possible new-targeted therapies based on the interference in the urokinase system.
EGF Gene Polymorphism and the Risk of Incident Primary Melanoma
Authors: Amend Kandace L. , Elder James T. , Tomsho Lynn P. , Bonner Joseph D. , Johnson Timothy M. , Schwartz Jennifer , Berwick Marianne , Gruber Stephen B.
Published: September 2005

Overexpression of the epidermal growth factor (EGF) pathway has been implicated in melanoma pathogenesis, and a recent case-control study identified a single nucleotide polymorphism (G to A) in the EGF gene where the G allele was associated with increased EGF expression and an increased risk of melanoma. To further evaluate this association, we conducted a case-control analysis from the Genes, Environment, and Melanoma study at the University of Michigan site using two different study designs. Incident cases of histopathologically confirmed first primary melanoma that were diagnosed between January 1, 2000 and December 31, 2000 from the University of Michigan Melanoma Clinic (n = 330) were compared with the following two different sources of nonmelanoma controls: spouse/friend controls (n = 84) and healthy volunteer controls from a case-control study of psoriasis (n = 148). Using a second analytic design, comparisons between multiple primary melanoma cases (n = 62) and single primary melanoma cases (n = 330) were also evaluated to estimate odds ratios (ORs). Genotyping for the single nucleotide substitution (G to A) at position 61 in the 5′ untranslated region of the EGF gene was performed from genomic DNA, and epidemiological risk factors were assessed through a telephone interview. When EGF genotypes were compared between incident primary melanoma cases and the nonmelanoma controls, the risk associated with the homozygous G/G genotype was not statistically significantly associated with an increased risk for incident primary melanoma compared with the homozygous A/A genotype [OR, 1.09; 95% confidence interval (CI); 0.65–1.85]. No strong associations with EGF G/G genotype were observed in comparisons of multiple primary and single primary melanoma cases (OR, 0.66; 95% CI; 0.25–1.73). Case subjects with tumors ≥3.5 mm compared with those <3.5 mm were not significantly associated with the G/G genotype (OR, 0.54; 95% CI; 0.12–2.35). Our data do not support a significant association between melanoma and the EGF 61*G allele or the homozygous G/G genotype. The EGF polymorphism is not a reproducible risk factor for melanoma or thick melanoma in our data. The two analytic approaches used in the study provide evidence against a strong association between EGF 61*G and melanoma and demonstrate the potential utility of case-case designs for evaluating the role of single nucleotide polymorphisms and cancer. Additional independent studies will be required to elucidate relationships between genetic variation in the EGF gene and risk of melanoma.
Activation of the Platelet-Derived Growth Factor-Receptor Enhances Survival of Murine Bone Endothelial Cells
Authors: Langley Robert R. , Fan Dominic , Tsan Rachel Z. , Rebhun Robert , He Junqin , Kim Sun-Jin , Fidler Isaiah J.
Published: September 2005

The activation of the microvascular endothelial cell platelet-derived growth factor (PDGF) receptor (PDGF-R) by PDGF has been implicated in neoplastic angiogenesis. Here, we established cultures of murine bone microvascular endothelial cells and examined their response to stimulation with PDGF BB ligand and to blockade of PDGF-R signaling with the tyrosine kinase inhibitor STI571 (Gleevec). The addition of STI571 to cultures of bone endothelial cells blocked PDGF BB-induced phosphorylation in a dose-dependent manner and completely abrogated the activation of downstream targets Akt and ERK1/2. Coadministration of STI571 and Taxol also induced the activation of procaspase-3 and significant apoptosis. These data suggest that phosphorylation of PDGF-R stimulates survival pathways in bone endothelial cells and that by selectively inhibiting PDGF-R signaling with STI571, the cells are rendered sensitive to Taxol treatment. The therapeutic combination of STI571 and Taxol may be a powerful tool for targeting tumor-associated endothelial cells in the skeletal compartment.
Combination of a Poxvirus-Based Vaccine with a Cyclooxygenase-2 Inhibitor (Celecoxib) Elicits Antitumor Immunity and Long-Term Survival in CEA.Tg/MIN Mice
Authors: Zeytin Hasan E. , Patel Arti C. , Rogers Connie J. , Canter Daniel , Hursting Stephen D. , Schlom Jeffrey , Greiner John W.
Published: September 2005

The present study was designed to determine whether: (a) chronic administration of dietary celecoxib (Celebrex), a potent nonsteroidal anti-inflammatory drug, which targets the cyclooxygenase-2 (COX-2) enzyme, negatively impacts host immunity; and (b) celecoxib can be coupled with a poxvirus-based vaccine to impact tumor burden in a murine tumor model of spontaneous adenomatous polyposis coli. Naive mice fed the celecoxib-supplemented diets developed eosinophilia with lowered plasma prostaglandin E2 levels and reduced COX-2 mRNA expression levels in their splenic T cells. Responses of splenic T, B, and natural killer cells to broad-based and antigen-specific stimuli were, for the most part, unchanged in those mice as well as COX-2 knockout mice; exceptions included: (a) reduced IFN-γ production by concanavalin A- or antigen-stimulated T cells; and (b) heightened lipopolysaccharide response of naive B cells from mice fed a diet supplemented with 1000 ppm of celecoxib. When transgenic mice that express the human carcinoembryonic antigen (CEA) gene (CEA transgenic) were bred with mice bearing a mutation in the ApcΔ850 gene (multiple intestinal neoplasia mice), the progeny (CEA transgenic/multiple intestinal neoplasia) spontaneously develop multiple intestinal neoplasms that overexpress CEA and COX-2. Beginning at 30 days of age, the administration of a diversified prime/boost recombinant CEA-poxvirus-based vaccine regimen or celecoxib (1000 ppm)-supplemented diet reduced the number of intestinal neoplasms by 54% and 65%, respectively. Combining the CEA-based vaccine with the celecoxib-supplemented diet reduced tumor burden by 95% and significantly improved overall long-term survival. Both tumor reduction and improved overall survival were achieved without any evidence of autoimmunity directed at CEA-expressing or other normal tissues. Celecoxib is prescribed for the treatment of familial adenomatous polyposis in humans, and the CEA-based vaccines have been well tolerated and capable of eliciting anti-CEA host immune responses in early clinical studies. The results suggest that the administration of a recombinant poxvirus-based vaccine is compatible with celecoxib, and this combined chemoimmuno-based approach might lead to an additive therapeutic antitumor benefit not only in patients diagnosed with familial adenomatous polyposis but, perhaps, in other preventive settings in which COX-2 overexpression is associated with progression from premalignancy to neoplasia.
An Oncolytic Adenovirus Vector Combining Enhanced Cell-to-Cell Spreading, Mediated by the ADP Cytolytic Protein, with Selective Replication in Cancer Cells with Deregulated
Wnt
Signaling
Authors: Toth Karoly , Djeha Hakim , Ying Baoling , Tollefson Ann E. , Kuppuswamy Mohan , Doronin Konstantin , Krajcsi Peter , Lipinski Kai , Wrighton Christopher J. , Wold William S. M.
Published: September 2005

We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3–11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear β-catenin.
Membrane Transporters and Channels
Authors: Huang Ying , Anderle Pascale , Bussey Kimberly J. , Barbacioru Catalin , Shankavaram Uma , Dai Zunyan , Reinhold William C. , Papp Audrey , Weinstein John N. , Sadée Wolfgang
Published: September 2005

Membrane transporters and channels (collectively the transportome) govern cellular influx and efflux of ions, nutrients, and drugs. We used oligonucleotide arrays to analyze gene expression of the transportome in 60 human cancer cell lines used by the National Cancer Institute for drug screening. Correlating gene expression with the potencies of 119 standard anticancer drugs identified known drug-transporter interactions and suggested novel ones. Folate, nucleoside, and amino acid transporters positively correlated with chemosensitivity to their respective drug substrates. We validated the positive correlation between SLC29A1 (nucleoside transporter ENT1) expression and potency of nucleoside analogues, azacytidine and inosine-glycodialdehyde. Application of an inhibitor of SLC29A1, nitrobenzylmercaptopurine ribonucleoside, significantly reduced the potency of these two drugs, indicating that SLC29A1 plays a role in cellular uptake. Three ABC efflux transporters (ABCB1, ABCC3, and ABCB5) showed significant negative correlations with multiple drugs, suggesting a mechanism of drug resistance. ABCB1 expression correlated negatively with potencies of 19 known ABCB1 substrates and with Baker’s antifol and geldanamycin. Use of RNA interference reduced ABCB1 mRNA levels and concomitantly increased sensitivity to these two drugs, as expected for ABCB1 substrates. Similarly, specific silencing of ABCB5 by small interfering RNA increased sensitivity to several drugs in melanoma cells, implicating ABCB5 as a novel chemoresistance factor. Ion exchangers, ion channels, and subunits of proton and sodium pumps variably correlated with drug potency. This study identifies numerous potential drug-transporter relationships and supports a prominent role for membrane transport in determining chemosensitivity. Measurement of transporter gene expression may prove useful in predicting anticancer drug response.
Regulation of p27Kip1 Protein Levels Contributes to Mitogenic Effects of the RET/PTC Kinase in Thyroid Carcinoma Cells
Authors: Vitagliano Donata , Carlomagno Francesca , Motti Maria Letizia , Viglietto Giuseppe , Nikiforov Yuri E. , Nikiforova Marina N. , Hershman Jerome M. , Ryan Anderson J. , Fusco Alfredo , Melillo Rosa Marina , Santoro Massimo
Published: September 2005

We show that treatment of a panel of thyroid carcinoma cell lines naturally harboring the RET/PTC1 oncogene, with the RET kinase inhibitors PP1 and ZD6474, results in reversible G1 arrest. This is accompanied by interruption of Shc and mitogen-activated protein kinase (MAPK) phosphorylation, reduced levels of G1 cyclins, and increased levels of the cyclin-dependent kinase inhibitor p27Kip1 because of a reduced protein turnover. MAP/extracellular signal-regulated kinase 1/2 inhibition by U0126 caused G1 cyclins down-regulation and p27Kip1 up-regulation as well. Forced expression of RET/PTC in normal thyroid follicular cells caused a MAPK- and proteasome-dependent down-regulation of p27Kip1. Reduction of p27Kip1 protein levels by antisense oligonucleotides abrogated the G1 arrest induced by RET/PTC blockade. Therefore, in thyroid cancer, RET/PTC-mediated MAPK activation contributes to p27Kip1 deregulation. This pathway is implicated in cell cycle progression and in response to small molecule kinase inhibitors.
Efficient Cancer Therapy with a Nanobody-Based Conjugate
Authors: Cortez-Retamozo Virna , Backmann Natalija , Senter Peter D. , Wernery Ullrich , De Baetselier Patrick , Muyldermans Serge , Revets Hilde
Published: September 2005

Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae β-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody–enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.
Microarray Analyses Uncover UBE1L as a Candidate Target Gene for Lung Cancer Chemoprevention
Authors: Pitha-Rowe Ian , Petty W. Jeffrey , Feng Qing , Koza-Taylor Petra H. , DiMattia Debra A. , Pinder Lynn , Dragnev Konstantin H. , Memoli Natalie , Memoli Vincent , Turi Tom , Beebe Jean , Kitareewan Sutisak , Dmitrovsky Ethan
Published: September 2005

Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2BNNK), and RA-chemoprevented (BEAS-2BNNK/RA) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.
Different Roles for Caveolin-1 in the Development of Non-Small Cell Lung Cancer
versus
Small Cell Lung Cancer
Authors: Sunaga Noriaki , Miyajima Kuniharu , Suzuki Makoto , Sato Mitsuo , White Michael A. , Ramirez Ruben D. , Shay Jerry W. , Gazdar Adi F. , Minna John D.
Published: September 2005

Caveolin-1 (CAV1), an essential structural constituent of caveolae that plays an important role in cellular processes such as transport and signaling, has been implicated in the development of human cancers. However, it is unclear whether CAV1 is acting like an oncogene or tumor suppressor gene. We found that CAV1 expression was reduced or absent in 95% of small cell lung cancers (SCLCs; n = 21 lines), whereas it was retained in 76% of non-small cell lung cancers (NSCLCs; n = 25 lines) compared with normal human lung epithelial cultures, where it was abundantly expressed. CAV1 expression was tightly linked to the ability to grow attached to the plastic cell culture surface, whereas CAV1-nonexpressing lung cancers of both SCLC and NSCLC type grew as suspension cultures. In addition, attached lung cancer cultures expressed phosphorylated focal adhesion kinase, whereas suspension cultures did not. Lack of CAV1 expression was tightly associated with CAV1 promoter methylation (P < 0.0001) such that CAV1 methylation was found in 93% of SCLCs (n = 15) and 9% of NSCLCs (n = 11), whereas 5-aza-2′deoxycytidine treatment restored CAV1 expression in SCLCs. Exogenous CAV1 expression in SCLCs significantly inhibited soft-agar colony formation but did not lead to attachment. By contrast, CAV1 knockdown in NSCLCs mediated by small interfering RNA against CAV1 led to inhibition of cellular proliferation and soft-agar and liquid colony formation. Importantly, CAV1 knockdown led to reduced phospho-focal adhesion kinase and RalA, but not RalB, levels in NSCLC cells. These results suggest different roles for CAV1 in SCLC, where CAV1 acts like a tumor suppressor gene, and NSCLC, where it appears required for survival and growth.
A Specific Antagonist of the p110δ Catalytic Component of Phosphatidylinositol 3′-Kinase, IC486068, Enhances Radiation-Induced Tumor Vascular Destruction
Authors: Geng Ling , Tan Jiahuai , Himmelfarb Eric , Schueneman Aaron , Niermann Ken , Fu Allie , Cuneo Kyle , Kesicki Edward A. , Treiberg Jennifer , Hayflick Joel S. , Hallahan Dennis E.
Published: September 2005

The phosphatidylinositol 3′-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival and function during oxidative stress. The role of the PI3k/Akt signaling pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was rapidly phosphorylated in response to radiation in primary culture endothelial cells (human umbilical vascular endothelial cells) in the absence of serum or growth factors. PI3k consists of p85 and p110 subunits, which play a central upstream role in Akt activation in response to exogenous stimuli. The δ isoform of the p110 subunit is expressed in endothelial cells. We studied the effects of the p110δ specific inhibitor IC486068, which abrogated radiation-induced phosphorylation of Akt. IC486068 enhanced radiation-induced apoptosis in endothelial cells and reduced cell migration and tubule formation of endothelial cells in Matrigel following irradiation. In vivo tumor growth delay was studied in mice with Lewis lung carcinoma and GL261 hind limb tumors. Mice were treated with daily i.p. injections (25 mg/kg) of IC486068 during 6 days of radiation treatment (18 Gy). Combined treatment with IC486068 and radiation significantly reduced tumor volume as compared with either treatment alone. Reduction in vasculature was confirmed using the dorsal skinfold vascular window model. The vascular length density was measured by use of the tumor vascular window model and showed IC486068 significantly enhanced radiation-induced destruction of tumor vasculature as compared with either treatment alone. IC486068 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. These findings suggest that p110δ is a therapeutic target to enhance radiation-induced tumor control.
Thioredoxin Reductase as a Potential Molecular Target for Anticancer Agents That Induce Oxidative Stress
Authors: Smart DeeDee K. , Ortiz Karen L. , Mattson David , Bradbury C. Matthew , Bisht Kheem S. , Sieck Leah K. , Brechbiel Martin W. , Gius David
Published: September 2005

Redox-sensitive signaling factors regulate multiple cellular processes, including proliferation, cell cycle, and prosurvival signaling cascades, suggesting their potential as molecular targets for anticancer agents. It is logical to set constraints that a molecular target should meet at least one of the following criteria: (1) inhibition of prosurvival signaling pathways; (2) inhibition of cell cycle progression; or (3) enhancement of the cytotoxic effects of anticancer agents. Therefore, we hypothesized that thioredoxin reductase 1 (TR), a component of several redox-regulated pathways, might represent a potential molecular target candidate in response to agents that induce oxidative stress. To address this issue, permanent cell lines overexpressing either the wild-type (pCXN2-myc-TR-wt) or a Cys-Ser mutant (pCXN2-myc-mTR) TR gene were used, as were parental HeLa cells treated with 1-methyl-1-propyl-2-imidazolyl disulfide (IV-2), a pharmacologic inhibitor of TR. Cells were exposed to the oxidative stressors, H2O2 and ionizing radiation (IR), and analyzed for changes in signal transduction, cell cycle, and cytotoxicity. Analysis of HeLa cells overexpressing the pCXN2-myc-TR-wt gene showed increased basal activity of nuclear factor κB (NFκB) and activator protein (AP-1), whereas HeLa cells expressing a pCXN2-myc-mTR gene and HeLa cells treated with IV-2 were unable to induce NFκB or AP-1 activity following H2O2 or IR exposure. Fluorescence-activated cell sorting analysis showed a marked accumulation of pCXN2-myc-mTR cells in the late G1 phase, whereas pCXN2-myc-TR-wt cells showed a decreased G1 subpopulation. Chemical inhibition of TR with IV-2 also completely inhibited cellular proliferation at concentrations between 10 and 25 μmol/L, resulting in a G1 phase cell cycle arrest consistent with the results from cells expressing the pCXN2-myc-mTR gene. Following exposure to H2O2 and IR, pCXN2-myc-mTR– and IV-2–treated cells were significantly more sensitive to oxidative stress-induced cytotoxicity as measured by clonogenic survival assays. Finally, IV-2–treated cells showed increased tumor cell death when treated with H2O2 and IR. These results identify TR as a potential target to enhance the cytotoxic effects of agents that induce oxidative stress, including IR.
A Novel System for Simultaneousin VivoTracking and Biological Assessment of Leukemia Cells andex VivoGenerated Leukemia-Reactive Cytotoxic T Cells
Authors: Sauer Martin G. , Ericson Marna E. , Weigel Brenda J. , Herron Michael J. , Panoskaltsis-Mortari Angela , Kren Betsy T. , Levine Bruce L. , Serody Jon S. , June Carl H. , Taylor Patricia A. , Blazar Bruce R.
Published: September 2005

To determine the mechanisms by which adoptive immunotherapy could reduce lethality to acute myelogenous leukemia (AML), a novel technique was developed to track both leukemic blasts and adoptively transferred cytotoxic T cells (CTLs) independently and simultaneously in mice. To follow the fate of ex vivo generated anti-AML-reactive CTLs, splenocytes obtained from enhanced green fluorescent protein transgenic mice were cocultured with AML lysate-pulsed dendritic cells, which subsequently were expanded by exposure to anti-CD3/CD28 monoclonal antibody-coated magnetic microspheres. To track AML cells, stable transfectants of C1498 expressing DsRed2, a red fluorescent protein, were generated. Three factors related to CTLs correlated with disease-free survival: (a) CTL l-selectin expression. l-Selectin high fractions resulted in 70% disease-free survival, whereas l-selectin low-expressing CTLs resulted in only 30% disease-free survival. (b) Duration of ex vivo expansion (9 versus 16 days). Short-term expanded CTLs could be found at high frequency in lymphoid organs for longer than 4 weeks after transfer, whereas long-term expanded CTLs were cleared from the system after 2 weeks. Duration of expansion correlated inversely with l-selectin expression. (c) CTL dose. A higher dose (40 versus 5 × 106) resulted in superior disease-free survival. This survival advantage was achieved with short-term expanded CTLs only. The site of treatment failure was mainly the central nervous system where no CTLs could be identified at AML sites.