bluuebunny/crossref_metadata_2025 huggingface dataset:
UCN-01-Induced Cell Cycle Arrest Requires the Transcriptional Induction of p21waf1/cip1 by Activation of Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase/Extracellular Signal-Regulated Kinase Pathway
Authors: Facchinetti Maria M. , De Siervi Adriana , Toskos Doreen , Senderowicz Adrian M.
Published: September 2005

The small molecule UCN-01 is a cyclin-dependent kinase (CDK) modulator shown to have antiproliferative effects against several in vitro and in vivo cancer models currently being tested in human clinical trials. Although UCN-01 may inhibit several serine-threonine kinases, the exact mechanism by which it promotes cell cycle arrest is still unclear. We have reported previously that UCN-01 promotes G1-S cell cycle arrest in a battery of head and neck squamous cancer cell lines. The arrest is accompanied by an increase in both p21waf1/cip1 and p27kip1 CDK inhibitors leading to loss in G1 CDK activity. In this report, we explore the role and the mechanism for the induction of these endogenous CDK inhibitors. We observed that p21 was required for the cell cycle effects of UCN-01, as HCT116 lacking p21 (HCT116 p21−/−) was refractory to the cell cycle effects of UCN-01. Moreover, UCN-01 promoted the accumulation of p21 at the mRNA level in the p53-deficient HaCaT cells without increase in the p21 mRNA half-life, suggesting that UCN-01 induced p21 at the transcriptional level. To study UCN-01 transcriptional activation of p21, we used several p21waf1/cip1 promoter-driven luciferase reporter plasmids and observed that UCN-01 activated the full-length p21waf1/cip1 promoter and a construct lacking p53 binding sites. The minimal promoter region required for UCN-01 (from −110 bp to the transcription start site) was the same minimal p21waf1/cip1 promoter region required for Ras enhancement of p21waf1/cip1 transcription. Neither protein kinase C nor PDK1/AKT pathways were relevant for the induction of p21 by UCN-01. In contrast, the activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase mitogen-activated protein kinase pathways was required for p21 induction as UCN-01 activated this pathway, and genetic or chemical MEK inhibitors blunted p21 accumulation. These results demonstrated for the first time that p21 is required for UCN-01 cell cycle arrest. Moreover, we showed that the accumulation of p21 is transcriptional via activation of the MEK pathway. This novel mechanism, by which UCN-01 exerts its antiproliferative effect, represents a promising strategy to be exploited in future clinical trials.
AIB1/SRC-3 Deficiency Affects Insulin-Like Growth Factor I Signaling Pathway and Suppresses
v
-Ha-
ras
-induced Breast Cancer Initiation and Progression in Mice
Authors: Kuang Shao-Qing , Liao Lan , Zhang Hao , Lee Adrian V. , O’Malley Bert W. , Xu Jianming
Published: September 2005

Although the amplified in breast cancer 1 (AIB1) coactivator is amplified and overexpressed in breast cancers, its role in mammary carcinogenesis remains unknown. We demonstrate that during mammary development and tumorigenesis, the elevation of AIB1 level and its nuclear localization correlate with normal and transformed mammary epithelial proliferation, whereas its lower expression and cytoplasmic localization correlate with mammary epithelial quiescence and differentiation. In this study, the role of AIB1 in breast tumor initiation, progression, and metastasis was studied by generating AIB1+/+, AIB1+/−, and AIB1−/− mice harboring the mouse mammary tumor virus/v-Ha-ras (ras) transgene that induces breast tumors. Breast tumor incidence was reduced dramatically in the intact AIB1−/−-ras virgin mice and inhibited completely in the ovariectomized AIB1−/−-ras mice. Breast tumor latency was delayed significantly in AIB1−/−-ras virgin mice with natural estrous cycles, multiparous mice with cyclically elevated reproductive hormones, and virgin mice bearing pituitary isografts with persistently elevated hormones. Although AIB1 deficiency significantly suppressed mammary tumorigenesis under all of the concentrations of ovarian hormones, it did not affect the promotional role of ovarian hormones on mammary tumorigenesis, suggesting that AIB1 and ovarian hormones contribute to mammary carcinogenesis through different pathways. AIB1 deficiency did not alter the expression of estrogen and progesterone-responsive genes in the mammary gland, but it caused partial resistance to the insulin-like growth factor I because of a significant reduction in the insulin receptor substrates. The impaired insulin-like growth factor I pathway in AIB1−/−-ras mammary epithelium and tumor cells was responsible in part for the suppression of mammary tumorigenesis and metastasis caused by inhibition of cell proliferation and migration. These results suggest that a more effective strategy to control breast cancer is to target AIB1-mediated and ovarian hormone-initiated pathways.
Syncytia Induction Enhances the Oncolytic Potential of Vesicular Stomatitis Virus in Virotherapy for Cancer
Authors: Ebert Oliver , Shinozaki Katsunori , Kournioti Chryssanthi , Park Man-Seong , García-Sastre Adolfo , Woo Savio L. C.
Published: September 2005

Vesicular stomatitis virus (VSV) selectively replicates in tumor but not in normal cells and is being developed as an oncolytic agent for cancer therapy. Here we report the construction of a recombinant VSV capable of inducing syncytia formation between tumor cells through membrane fusion at neutral pH, which led to enhanced oncolytic properties against multifocal hepatocellular carcinoma (HCC) in the livers of immunocompetent rats. Recombinant VSV vectors were constructed by insertion into their genome a transcription unit expressing a control or fusion protein derived from Newcastle disease virus. In vitro characterization of the recombinant fusogenic VSV vector on human and rat HCC cells showed extensive syncytia formation and significantly enhanced cytotoxic effects. In vivo, administration of fusogenic VSV into the hepatic artery of Buffalo rats bearing syngeneic multifocal HCC lesions in their livers resulted in syncytia formation exclusively within the tumors, and there was no collateral damage to the neighboring hepatic parenchyma. The fusogenic VSV also conferred a significant survival advantage over a nonfusogenic control virus in the treated animals (P = 0.0078, log-rank test). The results suggest that fusogenic VSV can be developed into an effective and safe therapeutic agent for cancer treatment in patients, including those with multifocal HCC in the liver.
Soluble Interleukin-13Rα2 Decoy Receptor Inhibits Hodgkin’s Lymphoma Growthin Vitroandin Vivo
Authors: Trieu Young , Wen Xiao-Yan , Skinnider Brian F. , Bray Mark R. , Li Zhihua , Claudio Jaime O. , Masih-Khan Esther , Zhu Yuan-Xiao , Trudel Suzanne , McCart J. Andrea , Mak Tak W. , Stewart A. Keith
Published: September 2005

Recent studies have demonstrated that the malignant Reed-Sternberg cells of Hodgkin’s lymphoma (HL) secrete and are responsive to interleukin (IL)-13. We hypothesized that overexpression of a soluble IL-13 decoy receptor (sIL-13Rα2) via adenoviral-mediated gene transfer would inhibit IL-13-induced Reed-Sternberg cell proliferation. Western blot and ELISA analysis verified expression of sIL-13Rα2 in cell lysates and supernatants of AdsIL-13Rα2-transduced COS-7 cells. Treatment of two IL-13-responsive HL-derived cell lines, HDLM-2 and l-1236, with AdsIL-13Rα2-conditioned medium, resulted in the inhibition of cell proliferation, and down-regulated the phosphorylation of signal transducer and activator of transcription 6 (STAT6), an important mediator of IL-13 signaling. i.v. delivery of AdsIL-13Rα2 in NOD/SCID mice with s.c. implanted HDLM-2 cells delayed tumor onset and growth while enhancing survival compared with control mice. Intratumoral administration of AdsIL-13Rα2 led to the regression or stabilization of established tumors and was associated with diminished STAT6 phosphorylation. Our data demonstrate that AdsIL-13Rα2 can suppress HL growth in vitro and in vivo.
The
NOD2
3020insC Mutation and the Risk of Colorectal Cancer
Authors: Kurzawski Grzegorz , Suchy Janina , Kładny Józef , Grabowska Ewa , Mierzejewski Marek , Jakubowska Anna , Dȩbniak Tadeusz , Cybulski Cezary , Kowalska Elsbieta , Szych Zbigniew , Domagała Wenancjusz , Scott Rodney J. , Lubiński Jan
Published: September 2005

Several predispositions to colorectal cancer have been identified, but little is known about genetic susceptibilities to disease in older persons. Colorectal cancer is a risk in Crohn’s disease and is believed to be associated with an inappropriate inflammatory response. Recently, the NOD2 gene has been associated with Crohn’s disease, which further strengthens the notion that the inflammatory response plays a crucial role in this disease. Several mutations have been identified in the NOD2 gene, which appear with significantly higher frequency in patients with the disease. One such mutation (3020insC) is believed to be clearly causative because it results in a prematurely truncated protein with a predicted reduction in functional efficiency. In this report, we have examined the frequency of the 3020insC mutation in a series of 856 individuals including 556 patients with colorectal cancer. The frequency of the 3020insC mutation in a consecutive series of 250 non-hereditary nonpolyposis colorectal cancer patients >50 years of age was significantly elevated compared with the control population (odds ratio, 2.23; P = 0.0046). The results indicate that NOD2 may be a predisposing factor to colorectal cancer characterized by an older average age of disease onset in persons who do not harbor any other genetic predisposition to disease.
Cytostatic and Cytotoxic Effects of Topotecan Decoded by a Novel Mathematical Simulation Approach
Authors: Lupi Monica , Matera Giada , Branduardi Davide , D’Incalci Maurizio , Ubezio Paolo
Published: September 2005

Topotecan (TPT) is a topoisomerase I inhibitor, and like the other drugs of this family, it is believed to act in a specific way on cells in S phase at the time of treatment. Exploiting a new method, coupling a particular experimental plan with computer simulation, a complete quantitative study of the time dependence and dose dependence of the activity of cell cycle controls has become feasible, and the overall scenario of events after treatment can be reconstructed in detail. We were able to demonstrate that the response of an ovarian cancer cell line to 1 h of treatment with TPT is not limited to inhibition of DNA synthesis, leading to cell death, but involves G1 and G2-M checkpoints. G1 and G2-M block, recycling, and death follow specific dose-dependent kinetics, lasting no less than 3 days after treatment. We also found that cells treated outside S phase contribute significantly to the overall activity. The utility of this analysis was demonstrated by reproducing more complex treatment schemes in which low TPT concentrations were applied for 1 h three times at 24-h intervals. In this case, the simulation clarified the origin of the auto-potentiation observed with repeated 0.2 μm treatments, in which the cytotoxicity, particularly against S-phase cells, was higher than the cytotoxicity in cells treated with 10 μm only once. We believe that this approach will help us to understand the complexity and heterogeneity of the response of a cell population to a drug challenge and could help us to establish the rationale for drug scheduling or drug combinations.
Ibuprofen Inhibits Survival of Bladder Cancer Cells by Induced Expression of the p75NTR Tumor Suppressor Protein
Authors: Khwaja Fatima , Allen Jeff , Lynch John , Andrews Peter , Djakiew Daniel
Published: September 2005

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to reduce inflammation and as analgesics by inhibition of cyclooxygenase-2. At higher concentrations, some NSAIDs inhibit proliferation and induce apoptosis of cancer cells. Although several molecular mechanisms have been postulated to explain the anticancer effects of NSAIDs, they do not involve merely the inhibition of cyclooxygenase-2, and a more proximate initiator molecule may be regulated by NSAIDs to inhibit growth. The p75 neurotrophin receptor (p75NTR) is a proximate cell membrane receptor glycoprotein that has been identified as a tumor and metastasis suppressor. We observed that NSAID treatment of cell lines from bladder and other organs induced expression of the p75NTR protein. Of the different types of NSAIDs examined, ibuprofen was more efficacious than aspirin and acetaminophen and comparable with (R)-flurbiprofen and indomethacin in induction of p75NTR protein expression. This rank order NSAID induction of the p75NTR protein correlated with the ability of these NSAIDs to reduce cancer cell survival. To examine a mechanistic relationship between ibuprofen induction of p75NTR protein and inhibition of survival, bladder cancer cells were transfected with ponasterone A-inducible vectors that expressed a death domain-deleted (ΔDD) or intracellular domain-deleted (ΔICD) p75NTR product that acts as a dominant negative antagonist of the intact p75NTR protein. Expression of ΔDD and ΔICD rescued cells from ibuprofen inhibition of growth. These observations suggest that p75NTR is an important upstream modulator of the anticancer effects of NSAIDs and that ibuprofen induction of the p75NTR protein establishes an alternate mechanism by which ibuprofen may exert an anticancer effect.
Expression of a Novel Human Gene,
Human Wings Apart-Like (hWAPL)
, Is Associated with Cervical Carcinogenesis and Tumor Progression
Authors: Oikawa Kosuke , Ohbayashi Tetsuya , Kiyono Tohru , Nishi Hirotaka , Isaka Keiichi , Umezawa Akihiro , Kuroda Masahiko , Mukai Kiyoshi
Published: September 2005

In Drosophila melanogaster, the wings apart-like (wapl) gene encodes a protein that regulates heterochromatin structure. Here, we characterize a novel human homologue of wapl (termed human WAPL; hWAPL). The hWAPL mRNA was predominantly expressed in uterine cervical cancer, with weak expression in all other normal and tumor tissues examined. hWAPL expression in benign epithelia was confined to the basal cell layers, whereas in dysplasias it increasingly appeared in more superficial cell layers and showed a significant correlation with severity of dysplasia. Diffuse hWAPL expression was found in all invasive squamous cell carcinomas examined. In addition, NIH3T3 cells overexpressing hWAPL developed into tumors on injection into nude mice. Furthermore, repression of hWAPL expression by RNA interference induced cell death in SiHa cells. These results demonstrate that hWAPL is associated with cell growth, and the hWAPL expression may play a significant role in cervical carcinogenesis and tumor progression.
Molecular Causes of the Aberrant Choline Phospholipid Metabolism in Breast Cancer
Authors: Glunde Kristine , Jie Chunfa , Bhujwalla Zaver M.
Published: September 2005

Proton magnetic resonance spectroscopy (1H MRS) consistently detects significant differences in choline phospholipid metabolites of malignant versus benign breast lesions. It is critically important to understand the molecular causes underlying these metabolic differences, because this may identify novel targets for attack in cancer cells. In this study, differences in choline membrane metabolism were characterized in breast cancer cells and normal human mammary epithelial cells (HMECs) labeled with [1,2-13C]choline, using 1H and 13C magnetic resonance spectroscopy. Metabolic fluxes between membrane and water-soluble pool of choline-containing metabolites were assessed by exposing cells to [1,2-13C]choline for long and short periods of time to distinguish between catabolic and anabolic pathways in choline metabolism. Gene expression analysis using microarrays was performed to understand the molecular mechanisms underlying these changes. Breast cancer cells exhibited increased phosphocholine (PC; P < 0.001), total choline-containing metabolites (P < 0.01), and significantly decreased glycerophosphocholine (P < 0.05) compared with normal HMECs. Decreased 13C-enrichment was detected in choline (P < 0.001) and phosphocholine (P < 0.05, P < 0.001) of breast cancer cells compared with HMECs, indicating a higher metabolic flux from membrane phosphatidylcholine to choline and phosphocholine in breast cancer cells. Choline kinase and phospholipase C were significantly overexpressed, and lysophospholipase 1, phospholipase A2, and phospholipase D were significantly underexpressed, in breast cancer cells compared with HMECs. The magnetic resonance spectroscopy data indicated that elevated phosphocholine in breast cancer cells was primarily attributable to increased choline kinase activity and increased catabolism mediated by increased phospholipase C activity. These observations were consistent with the overexpression of choline kinase and phospholipase C detected in the microarray analyses.
Deficiencies in Mouse
Myh
and
Ogg1
Result in Tumor Predisposition and G to T Mutations in Codon 12 of the
K-Ras
Oncogene in Lung Tumors
Authors: Xie Yali , Yang Hanjing , Cunanan Cristina , Okamoto Kimberly , Shibata Darryl , Pan Janet , Barnes Deborah E. , Lindahl Tomas , McIlhatton Michael , Fishel Richard , Miller Jeffrey H.
Published: September 2005

Oxidative DNA damage is unavoidably and continuously generated by oxidant byproducts of normal cellular metabolism. The DNA damage repair genes, mutY and mutM, prevent G to T mutations caused by reactive oxygen species in Escherichia coli, but it has remained debatable whether deficiencies in their mammalian counterparts, Myh and Ogg1, are directly involved in tumorigenesis. Here, we demonstrate that deficiencies in Myh and Ogg1 predispose 65.7% of mice to tumors, predominantly lung and ovarian tumors, and lymphomas. Remarkably, subsequent analyses identified G to T mutations in 75% of the lung tumors at an activating hot spot, codon 12, of the K-ras oncogene, but none in their adjacent normal tissues. Moreover, malignant lung tumors were increased with combined heterozygosity of Msh2, a mismatch repair gene involved in oxidative DNA damage repair as well. Thus, oxidative DNA damage appears to play a causal role in tumorigenesis, and codon 12 of K-ras is likely to be an important downstream target in lung tumorigenesis. The multiple oxidative repair genes are required to prevent mutagenesis and tumor formation. The mice described here provide a valuable model for studying the mechanisms of oxidative DNA damage in tumorigenesis and investigating preventive or therapeutic approaches.
The Tumor Microenvironment Controls Primary Effusion Lymphoma Growthin Vivo
Authors: Staudt Michelle R. , Kanan Yogita , Jeong Joseph H. , Papin James F. , Hines-Boykin Rebecca , Dittmer Dirk P.
Published: September 2005

Certain lymphomas in AIDS patients, such as primary effusion lymphoma (PEL), are closely associated with the lymphotropic γ herpes virus Kaposi’s sarcoma-associated herpes virus (KSHV), also called human herpesvirus 8. The virus is thought to be essential for tumorigenesis, yet systems to investigate PEL in vivo are rare. Here we describe PEL tumorigenesis in a new xenograft model. Embedded in Matrigel, PEL cells formed rapid, well-organized, and angiogenic tumors after s.c. implantation of C.B.17 SCID mice. Without Matrigel we did not observe comparable tumors, which implies that extracellular support and/or signaling aids PEL. All of the tumors maintained the KSHV genome, and the KSHV latent protein LANA/orf73 was uniformly expressed. However, the expression profile for key lytic mRNAs, as well as LANA-2/vIRF3, differed between tissue culture and sites of implantation. We did not observe a net effect of ganciclovir on PEL growth in culture or as xenograft. These findings underscore the importance of the microenvironment for PEL tumorigenesis and simplify the preclinical evaluation of potential anticancer agents.
The HER-2-Targeting Antibodies Trastuzumab and Pertuzumab Synergistically Inhibit the Survival of Breast Cancer Cells
Authors: Nahta Rita , Hung Mien-Chie , Esteva Francisco J.
Published: September 2005

Trastuzumab (herceptin) and pertuzumab (Omnitarg, 2C4) are recombinant humanized monoclonal antibodies that target different extracellular regions of the HER-2 tyrosine kinase receptor. We explored combination effects of these agents in the HER-2-overexpressing BT474 breast cancer cell line. Trastuzumab and 2C4 synergistically inhibited the survival of BT474 cells, in part, because of increased apoptosis. Trastuzumab increased 2C4-mediated disruption of HER-2 dimerization with the epidermal growth factor receptor and HER-3. Combination drug treatment reduced levels of total and phosphorylated HER-2 protein and blocked receptor signaling through Akt but did not affect mitogen-activated protein kinase. These results suggest that combining HER-2-targeting agents may be a more effective therapeutic strategy in breast cancer rather than treating with a single HER-2 monoclonal antibody.
Reduction in Raf Kinase Inhibitor Protein Expression Is Associated with Increased Ras-Extracellular Signal-Regulated Kinase Signaling in Melanoma Cell Lines
Authors: Schuierer Marion M. , Bataille Frauke , Hagan Suzanne , Kolch Walter , Bosserhoff Anja-Katrin
Published: September 2005

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.
B-Raf Is Dispensable for K-Ras-Mediated Oncogenesis in Human Cancer Cells
Authors: Kim Jung-Sik , Lee Carolyn , Foxworth Aaron , Waldman Todd
Published: September 2005

Oncogenic mutations in B-Raf and Kirsten-Ras (K-Ras) are mutually exclusive during human cancer pathogenesis. In an effort to study the biological basis of this epistasis, gene targeting was used to create isogenic sets of human cancer cells differing only in presence or absence of endogenous oncogenic K-Ras or wild-type B-Raf. Whereas cells lacking the K-Ras oncogene were unable to efficiently form xenograft tumors, isogenic cells retaining activated K-Ras but deleted for B-Raf remained highly tumorigenic. Deletion of oncogenic K-Ras failed to reduce the activation state of B-Raf or ERK1/2, despite the requirement of oncogenic K-Ras for tumorigenesis. Genechip analysis revealed numerous genes in which the regulation by oncogenic K-Ras did not require B-Raf. These studies suggest that despite the mutual exclusivity of K-Ras and B-Raf mutations in human cancer and the well-described role for Raf proteins as Ras effectors, B-Raf is dispensable for K-Ras-mediated oncogenesis in a human cancer cell line. Additional studies are required to demonstrate the generalizability of these unexpected findings.
Functional Characterization of the Candidate Tumor Suppressor Gene
NPRL2
/
G21
Located in 3p21.3C
Authors: Li Jingfeng , Wang Fuli , Haraldson Klas , Protopopov Alexey , Duh Fuh-Mei , Geil Laura , Kuzmin Igor , Minna John D. , Stanbridge Eric , Braga Eleonora , Kashuba Vladimir I. , Klein George , Lerman Michael I. , Zabarovsky Eugene R.
Published: September 2005

Initial analysis identified the NPRL2/G21 gene located in 3p21.3C, the lung cancer region, as a strong candidate tumor suppressor gene. Here we provide additional evidence of the tumor suppressor function of NPRL2/G21. The gene has highly conserved homologs/orthologs ranging from yeast to humans. The yeast ortholog, NPR2, shows three highly conserved regions with 32 to 36% identity over the whole length. By sequence analysis, the main product of NPRL2/G21 encodes a soluble protein that has a bipartite nuclear localization signal, a protein-binding domain, similarity to the MutS core domain, and a newly identified nitrogen permease regulator 2 domain with unknown function. The gene is highly expressed in many tissues.

We report inactivating mutations in a variety of tumors and cancer cell lines, growth suppression of tumor cells with tet-controlled NPRL2/G21 transgenes on plastic Petri dishes, and suppression of tumor formation in SCID mice. Screening of 7 renal, 5 lung, and 7 cervical carcinoma cell lines showed homozygous deletions in the 3′ end of NPRL2 in 2 renal, 3 lung, and 1 cervical (HeLa) cell line. Deletions in the 3′ part of NPRL2 could result in improper splicing, leading to the loss of the 1.8 kb functional NPRL2 mRNA. We speculate that the NPRL2/G21 nuclear protein may be involved in mismatch repair, cell cycle checkpoint signaling, and activation of apoptotic pathway(s). The yeast NPR2 was shown to be a target of cisplatin, suggesting that the human NPRL2/G21 may play a similar role. At least two homozygous deletions of NPRL2/G21 were detected in 6 tumor biopsies from various locations and with microsatellite instability. This study, together with previously obtained results, indicates that NPRL2 is a multiple tumor suppressor gene.
BN80927
Authors: Demarquay Danièle , Huchet Marion , Coulomb Helène , Lesueur-Ginot Laurence , Lavergne Olivier , Camara José , Kasprzyk Philip G. , Prévost Grégoire , Bigg Dennis C. H.
Published: September 2005

BN80927 belongs to a novel family of camptothecin analogs, the homocamptothecins, developed on the concept of topoisomerase I (Topo I) inhibition and characterized by a stable seven-membered β-hydroxylactone ring. Preclinical data reported here show that BN80927 retains Topo I poisoning activity in cell-free assay (DNA relaxation) as well as in living cells, in which in vivo complexes of topoisomerase experiments and quantification of DNA-protein-complexes stabilization, have confirmed the higher potency of BN80927 as compared with the Topo I inhibitor SN38. In addition, BN80927 inhibits Topo II-mediated DNA relaxation in vitro but without cleavable-complex stabilization, thus indicating catalytic inhibition. Moreover, a Topo I-altered cell line (KBSTP2), resistant to SN38, remains sensitive to BN80927, suggesting that a part of the antiproliferative effects of BN80927 are mediated by a Topo I-independent pathway. This hypothesis is also supported by in vitro data showing an antiproliferative activity of BN80927 on a model of resistance related to the noncycling state of cells (G0-G1 synchronized). In cell growth assays, BN80927 is a very potent antiproliferative agent as shown by IC50 values consistently lower than those of SN38 in tumor cell lines as well as in their related drug-resistant lines. BN80927 shows high efficiency in vivo in tumor xenograft studies using human androgen-independent prostate tumors PC3 and DU145. Altogether, these data strongly support the clinical development of BN80927.
High-Throughput Retroviral Tagging for Identification of Genes Involved in Initiation and Progression of Mouse Splenic Marginal Zone Lymphomas
Authors: Shin Min Sun , Fredrickson Torgny N. , Hartley Janet W. , Suzuki Takeshi , Agaki Keiko , Morse Herbert C.
Published: September 2005

Human B-cell lymphomas are frequently associated with specific genetic changes caused by chromosomal translocations that activate proto-oncogenes. For lymphomas of mice expressing murine leukemia virus, mutagenic proviral insertions are thought to play a similar role. Here we report studies designed to determine whether specific retroviral integration sites might be associated with a specific subset of mouse B-cell lymphomas and if the genes associated with these sites are regularly altered in expression. We studied splenic marginal zone lymphomas (MZL) of NFS.V+ mice that are unusual in exhibiting frequent progression from low to high grade, potentially allowing assignment of cancer genes to processes of initiation and progression. We used inverse PCR to clone and analyze 212 retroviral integration sites from 43 MZL at different stages of progression. Sixty-two marked common integration sites and included 31 that had been marked previously. Among the new common integration sites, seven were unique to MZL. Using microarrays and real-time quantitative PCR analysis, we defined differential patterns of gene expression in association with disease progression for Gfi1, Sox4, Brca2, Snf1lk, Nfkb1, Pou2af1, Prdm1, Stat6, and Blnk. Heightened expression of Gfi1 distinguishes MZL from other lymphoma types. The combined use of proviral tagging and analyses of gene expression thus provides a powerful approach to understanding of genes that collaborate in tumorigenesis.
Cripto
Authors: Xing Pei Xiang , Hu Xiu Feng , Pietersz Geoffrey A. , Hosick Howard L. , McKenzie Ian F. C.
Published: September 2005

Cripto, a member of the epidermal growth factor-Cripto-FRL-Criptic (EGF-CFC) family, has been described recently as a potential target for immunotherapy (Adkins et al., J Clin Invest 2003;112:575–87). We have produced rat monoclonal antibodies (mAbs) to a Cripto 17-mer peptide, corresponding to the “EGF-like” motif of Cripto. The mAbs react with most cancers of the breast, colon, lung, stomach, and pancreas but do not react or react weakly with normal tissues. The mAbs inhibit cancer cell growth in vitro, and this effect was greater with cytotoxic drugs such as 5-fluorouracil, epirubicin, and cisplatin. The anti-Cripto mAbs prevent tumor development in vivo and inhibit the growth of established tumors of LS174T colon xenografts in Scid mice. The growth inhibitory effects with these mAbs may be greater than those described elsewhere, possibly because of IgM giving more effective cross-linking or binding to a different epitope (EGF-like region versus CFC region). The mechanism of inhibitory effects of the Cripto mAbs includes both cancer cell apoptosis, activation of c-Jun-NH2-terminal kinase and p38 kinase signaling pathways and blocking of Akt phosphorylation. Thus, Cripto is a unique target, and mAbs to Cripto could be of therapeutic value for human cancers.
Efflux Kinetics and Intracellular Distribution of Daunorubicin Are Not Affected by Major Vault Protein/Lung Resistance-Related Protein (Vault) Expression
Authors: van Zon Arend , Mossink Marieke H. , Schoester Martijn , Scheper Rik J. , Sonneveld Pieter , Wiemer Erik A. C.
Published: September 2005

Vaults may contribute to multidrug resistance by transporting drugs away from their subcellular targets. To study the involvement of vaults in the extrusion of anthracyclines from the nucleus, we investigated the handling of daunorubicin by drug-sensitive and drug-resistant non-small lung cancer cells, including a green fluorescent protein (GFP)-tagged major vault protein (MVP)-overexpressing transfectant (SW1573/MVP-GFP). Cells were exposed to 1 μm daunorubicin for 60 min, after which the cells were allowed to efflux the accumulated drug. No significant differences in daunorubicin efflux kinetics were observed between the sensitive SW1573 and SW1573/MVP-GFP transfectant, whereas the drug-resistant SW1573/2R120 cells clearly demonstrated an increased efflux rate. It was noted that the redistribution of daunorubicin from the nucleus into distinct vesicular structures in the cytoplasm was not accompanied by changes in the intracellular localization of vaults. Similar experiments were performed using mouse embryonic fibroblasts derived from wild-type and MVP knockout mice, which were previously shown to be devoid of vault particles. Both cell lines showed comparable drug efflux rates, and the intracellular distribution of daunorubicin in time was identical. Reintroduction of a human MVP tagged with GFP in the MVP−/− cells results in the formation of vault particles but did not give rise an altered daunorubicin handling compared with MVP−/− cells expressing GFP. Our results indicate that vaults are not directly involved in the sequestration of anthracyclines in vesicles nor in their efflux from the nucleus.
Overexpression of Histone Deacetylase 1 Confers Resistance to Sodium Butyrate–Mediated Apoptosis in Melanoma Cells through a p53-Mediated Pathway
Authors: Bandyopadhyay Debdutta , Mishra Anupam , Medrano Estela E.
Published: September 2005

Melanoma cells typically express wild-type p53, yet they are notoriously resistant to DNA-damaging agents. Here, we show that sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, induced apoptosis in human melanoma cells in a dose- and time-dependent manner. Apoptosis was associated with HDAC1-dependent induction of Bax and acetylation of p53. Down-regulation of HDAC1 by an antisense vector sensitized the cells to NaB-induced apoptosis, whereas its overexpression conferred resistance to this agent. Increased HDAC1 levels and activity impaired NaB-mediated activation of Bax promoter and Bax protein levels. Finally, using p53-null melanoma cell line and RNA interference in cells expressing wild-type p53 protein, we show that Bax induction and NaB-mediated apoptosis is p53 dependent.
Proteasome-Mediated Destruction of the Cyclin A/Cyclin-Dependent Kinase 2 Complex Suppresses Tumor Cell Growth
in Vitro
and
in Vivo
Authors: Chen Wei , Lee Jeongwu , Cho Steve Y. , Fine Howard A.
Published: September 2005

Cyclin-dependent kinases (cdks) represent potentially promising molecular targets for cancer therapeutic strategies. To evaluate the antitumor activity of selective cyclin/cdk inhibition, we constructed a chimeric protein composed of a F-box protein (TrCP) fused to a peptide comprising the cyclin/cdk2 binding motif in p21-like cdk inhibitors (TrCP-LFG). We now demonstrate that endogenous cyclin A and its binding substrate, cdk2, can be tethered to β-TrCP, ubiquitinated, and effectively degraded. Degradation of cdk2 and cyclin A together, but not cdk2 alone, results in massive tumor cell apoptosis in vitro and in vivo in a proteasome-dependent manner with no toxicity to normal tissue. These data demonstrate that cyclin A and/or the cyclin A/cdk2 complex is a promising anticancer target with a high therapeutic index.
The Superoxide-Generating Oxidase Nox1 Is Functionally Required for Ras Oncogene Transformation
Authors: Mitsushita Junji , Lambeth J. David , Kamata Tohru
Published: September 2005

The activated Ras oncogene can transform various mammalian cells and has been implicated in development of a high population of malignant human tumors. Recent studies suggest that generation of reactive oxygen species such as superoxide and H2O2 is involved in cell transformation by the activated Ras. However, the nature of an oxidase participating in Ras-transformation is presently unknown. Here, we report that Ras oncogene up-regulates the expression of Nox1, a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase, via the mitogen-activated protein kinase kinase-mitogen-activated protein kinase pathway, and that small interfering RNAs designed to target Nox1 mRNA effectively blocks the Ras transformed phenotypes including anchorage-independent growth, morphological changes, and production of tumors in athymic mice. Therefore, we propose that increased reactive oxygen species generation by Ras-induced Nox1 is required for oncogenic Ras transformation.
Transactivation of the PPAR-Responsive Enhancer Module in Chemopreventive Glutathione
S
-Transferase Gene by the Peroxisome Proliferator-Activated Receptor-γ and Retinoid X Receptor Heterodimer
Authors: Park Eun Young , Cho Il Je , Kim Sang Geon
Published: September 2005

Cancer chemopreventive agents transcriptionally induce glutathione S-transferase (GST), which can protect cells from chemical-induced carcinogenesis. Activation of either NF-E2-related factor-2 (Nrf2) or the CCAAT/enhancer binding protein-β (C/EBPβ) contributes to GST induction. Peroxisome proliferator-activated receptor-γ (PPARγ) and the retinoic acid X receptor (RXR) play roles in regulating cell differentiation and chemoprevention. This study examined GSTA2 gene induction by the PPARγ activator and 9-cis-retinoic acid (RA), a RXR ligand, and investigated the molecular basis of PPAR-RXR-mediated GSTA2 induction in the H4IIE hepatocytes. Either 15-deoxy-δ (12, 14)-prostaglandin J2 (PGJ2) or RA induced GSTA2 with Nrf2 and C/EBPβ activation. When compared with PGJ2 or RA alone, PGJ2 + RA enhanced GSTA2 induction, with increases in Nrf2 and C/EBPβ activation. PGJ2 + RA increased the luciferase reporter gene activity in the cells transfected with the −1.65-kb flanking region of the GSTA2 gene. Thiazolidinedione PPARγ agonists, troglitazone, rosiglitazone, and pioglitazone, in combination with RA, potentiated GSTA2 induction, confirming that the activation of the PPARγ and RXR heterodimer contributed to GSTA2 expression. Deletion of the antioxidant response element- or C/EBP-binding sites or the overexpression of dominant-negative mutant of C/EBP abolished the reporter gene expression. PGJ2 + RA increased the binding of the PPARγ − RXR heterodimer to the putative PPAR-response elements (PPREs) in the GSTA2 promoter. Specific mutations of these multiple PPRE sites resulted in the complete loss of its responsiveness to PGJ2 + RA, which suggests that these binding sites function as a PPRE-responsive enhancer module (PPREM). Transactivation of PPREM by the PPARγ − RXR heterodimer was verified by the effective GSTA2 induction in the cells treated with PGJ2 + RA after transfecting them with the plasmids encoding PPARγ1 and RXRα. In conclusion, the PPARγ − RXR heterodimer promotes GSTA2 induction by activating PPREM in the GSTA2 gene, as well as inducing Nrf2 and C/EBPβ activation.
Genotoxicity of Therapeutic Intervention in Children with Acute Lymphocytic Leukemia
Authors: Rice Sederick C. , Vacek Pamela , Homans Alan H. , Messier Terri , Rivers Jami , Kendall Heather , Finette Barry A.
Published: September 2005

The survival rates of children treated for cancer have dramatically increased after the development of standardized multiple-modality treatment protocols. As a result, there is a rapidly growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapeutic intervention is unknown. To study the genotoxic effects of antineoplastic treatment in children, we performed a comparative analysis of the changes in the frequency of somatic mutations (Mfs) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT)-reporter gene in children treated for acute lymphocytic leukemia (ALL). We measured HPRT Mfs from 130 peripheral blood samples from 45 children with ALL (13, low risk; 22, standard risk; and 10, high risk) from the time of diagnosis, as well as during and after the completion of therapy. We observed a significant increase in mean HPRT Mfs during each phase of therapy (diagnosis, 1.4 × 10−6; consolidation, 52.1 × 10−6; maintenance, 93.2 × 10−6; and off-therapy, 271.7 × 10−6) that were independent of the risk group treatment protocol used. This 200-fold increase in mean somatic Mf remained elevated years after the completion of therapy. We did not observe a significant difference in the genotoxicity of each risk group treatment modality despite differences in the compositional and clinical toxicity associated with these treatment protocols. These findings suggest that combination chemotherapy used to treat children with ALL is quite genotoxic, resulting in an increased somatic mutational load that may result in an elevated risk for the development of multi-factorial diseases, in particular second malignancies.
In Situ
Tumor Ablation Creates an Antigen Source for the Generation of Antitumor Immunity
Authors: den Brok Martijn H. M. G. M. , Sutmuller Roger P. M. , van der Voort Robbert , Bennink Erik J. , Figdor Carl G. , Ruers Theo J. M. , Adema Gosse J.
Published: September 2005

Tumor-destructing techniques, like radiofrequency ablation (RFA), allow eradication of large tumors. Potentially, in situ tumor destruction also can provide the immune system with an antigen source for the induction of antitumor immunity. Antigen-presenting cells could take up antigens in the periphery after which they induce specific immune responses. Recent data show that especially antigen-presenting dendritic cells are crucial for the induction of potent immune responses. However, virtually nothing is known regarding the induction of immune responses after in situ tumor destruction in mice or humans. We used the well-defined murine B16-OVA melanoma cell line to develop a novel tumor model to explore: (a) the immunologic consequences of in situ tumor destruction; and (b) the efficacy of a combination approach of tumor destruction and immunostimulation. Applying this model system we demonstrate that following RFA, a weak but detectable immune response develops, directed against OVA, but also against a broader range of B16 antigens. Adoptive transfer experiments further indicate that antitumor reactivity can be transferred to naïve mice by splenocytes. To augment the response observed, we administered a blocking monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 at the time of tumor destruction. Interestingly, this strongly enhanced antitumor immunity, resulting in long-lasting tumor protection. These results illustrate that in situ tumor destruction can provide a useful antigen source for the induction of antitumor immunity, provided that additional immunostimulatory signals are coadministered.
Selective Preservation of Pemetrexed Pharmacological Activity in HeLa Cells Lacking the Reduced Folate Carrier
Authors: Zhao Rongbao , Hanscom Marie , Chattopadhyay Shrikanta , Goldman I. David
Published: September 2005

A methotrexate (MTX)-resistant HeLa subline (R5), developed in this laboratory, with impaired transport due to a genomic deletion of the reduced folate carrier (RFC) was only 2-fold resistant to pemetrexed (PMX), but 200- and 400-fold resistant to raltitrexed (ZD1694) and Nα-(-4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-1-ornithine (PT523), respectively, compared with parental HeLa cells when grown with 2 μm folic acid. When folic acid was replaced with the more physiological 25 nm 5-formyltetrahydrofolate, R5 cells were 2-fold collaterally sensitive to PMX but still 40- and 200-fold resistant to ZD1694 and PT523, respectively. Sensitivity to PT523 and PMX could be completely restored, and sensitivity to ZD1694 nearly restored, by transfection of RFC cDNA into R5 cells, indicating that the defect in drug transport was the only, or major, factor in resistance. The preserved PMX activity in R5 cells could not be related to the very low expression of folate receptors. Rather, retained PMX activity in R5 cells was associated with residual transport by another process that exhibits good affinity for PMX (Kt = 12 μm) with much lower affinities for ZD1694, MTX, and PT523 (Kis of ∼ 90, 100, and 250 μm, respectively). PMX transported by this route was rapidly converted to higher polyglutamates and, when grown with 25 nm 5-formyl-tetrahydrofolate, the rate of formation of these derivatives and their net accumulation in R5 cells was comparable to that of wild-type cells. These data suggest that selective preservation of PMX pharmacological activity in RFC-null R5 cells is due, in part, to partial preservation of transport by secondary process with a higher affinity for PMX than the other antifolates evaluated.
Microarray-Based Screening for Molecular Markers in Medulloblastoma Revealed
STK15
as Independent Predictor for Survival
Authors: Neben Kai , Korshunov Andrey , Benner Axel , Wrobel Gunnar , Hahn Meinhard , Kokocinski Felix , Golanov Andrey , Joos Stefan , Lichter Peter
Published: September 2005

Medulloblastoma, a primitive neuroectodermal tumor of the cerebellum, is one of the most common central nervous system malignancies of childhood. Despite aggressive multimodal therapy, including surgery, irradiation, and chemotherapy, 5-year survival rates have only approached 50–60%. To identify potential candidate genes that predict for overall survival (OS), we performed a gene expression profiling analysis in 35 newly diagnosed medulloblastoma neoplasms. Subsequently, the nine most promising candidate genes were analyzed by immunohistochemistry and fluorescence in situ hybridization on tumor tissue microarrays representing a series of 180 tumors. We found 54 genes in which expression levels predicted for unfavorable survival in medulloblastoma. In line with the gene expression profiling analysis, a positive staining for STK15 (P = 0.0006), stathmin 1 (P = 0.001), and cyclin D1 (P = 0.03) was associated with an unfavorable OS, whereas cyclin B1, DAXX, Ki-67, MYC, NRAS, and p53 showed no statistical significant effect. In comparison to clinically defined parameters such as gender, age, metastatic stage, extent of tumor resection, application of chemotherapy, and tumor grade, positive staining for STK15 was identified as an independent prognostic factor for OS (P = 0.026). Moreover, additional gene copy numbers of MYC (P = 0.003) and STK15 (P = 0.05) predicted for poor survival. The combination of gene expression profiling with tissue microarray experiments allowed the identification of a series of candidate genes that predicts for survival in medulloblastoma. Of the results highlighted by the various data analysis procedures, genes associated with cell proliferation (cyclin D1), transcription (MYC), and especially mitosis (stathmin 1, STK15) appear particularly intriguing with respect to medulloblastoma pathomechanism.
Microarray Analysis of Epigenetic Silencing of Gene Expression in the KAS-6/1 Multiple Myeloma Cell Line
Authors: Pompeia Celine , Hodge David R. , Plass Christoph , Wu Yue-Zhong , Marquez Victor E. , Kelley James A. , Farrar William L.
Published: September 2005

The epigenetic control of gene transcription in cancer has been the theme of many recent studies and therapeutic approaches. Carcinogenesis is frequently associated with hypermethylation and consequent down-regulation of genes that prevent cancer, e.g., those that control cell proliferation and apoptosis. We used the demethylating drug zebularine to induce changes in DNA methylation, then examined patterns of gene expression using cDNA array analysis and Restriction Landmark Genomic Scanning followed by RNase protection assay and reverse transcription-PCR to confirm the results. Microarray studies revealed that many genes were epigenetically regulated by methylation. We concluded that methylation decreased the expression of, or silenced, several genes, contributing to the growth and survival of multiple myeloma cells. For example, a number of genes (BAD, BAK, BIK, and BAX) involved in apoptosis were found to be suppressed by methylation. Sequenced methylation-regulated DNA fragments identified by Restriction Landmark Genomic Scanning were found to contain CpG islands, and some corresponded to promoters of genes that were regulated by methylation. We also observed that after the removal of the demethylating drug, the addition of interleukin 6 restored CpG methylation and re-established previously silenced gene patterns, thus implicating a novel role of interleukin 6 in processes regulating epigenetic gene repression and carcinogenesis.