bluuebunny/crossref_metadata_2025 huggingface dataset:
Degradation of Fibrillar Collagen in a Human Melanoma Xenograft Improves the Efficacy of an Oncolytic Herpes Simplex Virus Vector
Authors: McKee Trevor D. , Grandi Paola , Mok Wilson , Alexandrakis George , Insin Numpon , Zimmer John P. , Bawendi Moungi G. , Boucher Yves , Breakefield Xandra O. , Jain Rakesh K.
Published: March 2006

Oncolytic viral therapy provides a promising approach to treat certain human malignancies. These vectors improve on replication-deficient vectors by increasing the viral load within tumors through preferential viral replication within tumor cells. However, the inability to efficiently propagate throughout the entire tumor and infect cells distant from the injection site has limited the capacity of oncolytic viruses to achieve consistent therapeutic responses. Here we show that the spread of the oncolytic herpes simplex virus (HSV) vector MGH2 within the human melanoma Mu89 is limited by the fibrillar collagen in the extracellular matrix. This limitation seems to be size specific as nanoparticles of equivalent size to the virus distribute within tumors to the same extent whereas smaller particles distribute more widely. Due to limited viral penetration, tumor cells in inaccessible regions continue to grow, remaining out of the range of viral infection, and tumor eradication cannot be achieved. Matrix modification with bacterial collagenase coinjection results in a significant improvement in the initial range of viral distribution within the tumor. This results in an extended range of infected tumor cells and improved virus propagation, ultimately leading to enhanced therapeutic outcome. Thus, fibrillar collagen can be a formidable barrier to viral distribution and matrix-modifying treatments can significantly enhance the therapeutic response. (Cancer Res 2006; 66(5): 2509–13)
Hepatocyte Growth Factor and Inducible Nitric Oxide Synthase Are Involved in Multidrug Resistance–Induced Angiogenesis in Hepatocellular Carcinoma Cell Lines
Authors: Lasagna Nadia , Fantappiè Ornella , Solazzo Michela , Morbidelli Lucia , Marchetti Serena , Cipriani Greta , Ziche Marina , Mazzanti Roberto
Published: March 2006

Based on literature, it is possible to hypothesize that multidrug resistance (MDR) and angiogenic phenotypes are linked to each other in human liver cancer cells. Our goal is to assess whether MDR cells trigger angiogenesis and to study the possible molecular mechanisms involved. Conditioned medium from parental drug-sensitive P5 cells (P5-CM) and MDR-positive P1(0.5) cells [P1(0.5)-CM] stimulated human umbilical vein endothelial cells (HUVEC) survival, proliferation, migration, and microtubular structure formation, but P1(0.5)-CM had a significantly greater effect than P5-CM. Cell implants were done in the rabbit avascular cornea to measure angiogenesis in vivo: P1(0.5) cells induced an important neovascular response in rabbit cornea after 1 week, whereas P5 cells had no effect. P1(0.5) and P5 cells produced vascular endothelial growth factor, but only P1(0.5) secreted hepatocyte growth factor (HGF) into the medium, and small interfering RNA specific for MDR1 clearly reduced HGF production in P1(0.5) cells. The transcription factor Ets-1 and the HGF receptor c-Met were up-regulated in P1(0.5) cells and in HUVEC cultured in P1(0.5)-CM. Inducible nitric oxide synthase (iNOS) seemed to play a major role in the proangiogenic effect of P1(0.5), and its inhibition by 1400W blunted the capacity of P1(0.5) cells to stimulate HUVEC proliferation, migration, and Ets-1 expression. In conclusion, these data show that development of MDR and angiogenic phenotypes are linked to each other in MDR cells. HGF production, Ets-1 and c-Met up-regulation, and iNOS expression can be part of the molecular mechanisms that enhance the angiogenic activity of the MDR-positive hepatocellular carcinoma cell line. (Cancer Res 2006; 66(5): 2673-82)
Normoxic Stabilization of Hypoxia-Inducible Factor-1α by Modulation of the Labile Iron Pool in Differentiating U937 Macrophages: Effect of Natural Resistance–Associated Macrophage Protein 1
Authors: Knowles Helen J. , Mole David R. , Ratcliffe Peter J. , Harris Adrian L.
Published: March 2006

Hypoxia-inducible factor (HIF) is a transcription factor with major roles in many cellular and systemic responses to hypoxia. Activation of HIF pathways under hypoxia is mediated by suppression of the Fe2+- and O2-dependent HIF hydroxylase enzymes that normally inactivate HIFα subunits. Mechanisms underlying induction of HIF in normoxic conditions are less clearly understood. In human cancers, infiltrating macrophages show up-regulation of HIF and it has recently been shown that normoxic expression of HIF-1α is essential for macrophage function. Here, we report studies of HIF-1α induction following phorbol-12-myristate 13-acetate (PMA)–induced differentiation of monocytic U937 and THP1 cells. HIF-1α was markedly up-regulated under normoxia in this setting and this involved failure of HIF-1α prolyl hydroxylation despite the presence of O2. Fluorescence measurements showed that differentiation was associated with marked reduction of the labile iron pool. Both the reduction in labile iron pool and the up-regulation of HIF-1α were suppressed by RNA interference–mediated down-regulation of the iron transporter natural resistance–associated macrophage protein 1. Up-regulation of HIF-1α following PMA-induced differentiation was also abolished by addition of Fe2+ or ascorbate. These results indicate that physiologic changes in macrophage iron metabolism have an important effect on HIF hydroxylase pathways and suggest means by which the system could be manipulated for therapeutic benefit. (Cancer Res 2006; 66(5): 2600-7)
Chromosomal Instability in MYH- and APC-Mutant Adenomatous Polyps
Authors: Cardoso Joana , Molenaar Lia , de Menezes Renee X. , van Leerdam Monique , Rosenberg Carla , Möslein Gabriela , Sampson Julian , Morreau Hans , Boer Judith M. , Fodde Riccardo
Published: March 2006

The vast majority of colorectal cancers display genetic instability, either in the chromosomal instability (CIN) or microsatellite instability (MIN) forms. Although CIN tumors are per definition aneuploid, MIN colorectal cancers, caused by loss of mismatch repair function, are usually near diploid. Recently, biallelic germ line mutations in the MYH gene were found to be responsible for MYH-associated polyposis (MAP), an autosomal recessive predisposition to multiple colorectal polyps, often indistinguishable from the dominant familial adenomatous polyposis (FAP) syndrome caused by inherited APC mutations. Here, we analyzed MYH- and APC-mutant polyps by combining laser capture microdissection, isothermal genomic DNA amplification, and array comparative genomic hybridization. Smoothed quantile regression methods were applied to the MAP and FAP genomic profiles to discriminate chromosomes predominantly affected by gains and losses. Up to 80% and 60% of the MAP and FAP polyps showed aneuploid changes, respectively. Both MAP and FAP adenomas were characterized by frequent losses at chromosome 1p, 17, 19, and 22 and gains affecting chromosomes 7 and 13. The aneuploid changes detected at early stages of MYH-driven tumorigenesis may underlie accelerated tumor progression, increased cancer risk, and poor prognosis in MAP. (Cancer Res 2006; 66(5): 2514-9)
Induction of Nevi and Skin Tumors in Ink4a/Arf Xpa Knockout Mice by Neonatal, Intermittent, or Chronic UVB Exposures
Authors: van Schanke Arne , van Venrooij Gemma M.C.A.L. , Jongsma Marjan J. , Banus H. Alexander , Mullenders Leon H.F. , van Kranen Henk J. , de Gruijl Frank R.
Published: March 2006

Nevi and melanomas correlate to childhood and intermittent solar UV exposure, xeroderma pigmentosum patients run increased risk, and p16Ink4a expression is often lost in malignant progression. To ascertain the effect of these risk factors, pigmented hairless Ink4a/Arf−, Xpa− knockout mice were subjected to various combinations of neonatal [7,12-dimethylbenz(a)anthracene (DMBA) or UVB exposure] and adult treatments (12-O-tetradecanoylphorbol-13-acetate or subacute daily UVB exposure or intermittent overexposure). Nevi occurred earliest, grew largest, and were most numerous in mice exposed to DMBA followed by intermittent UVB overexposure [effect of six minimal edemal doses (MED), 1×/2 weeks > 4 MED 1×/wk]. Neonatal UV exposure enhanced nevus induction but lost its effect after 200 days. The Xpa−/− mice proved exquisitely sensitive to UV-driven nevus induction, indicating the involvement of pyrimidine dimer DNA lesions, but Xpa+/+ mice developed many more nevi (>40 per mouse) at high UV dosages not tolerated by Xpa−/− mice. Ink4a/Arf−/− mice developed most skin tumors faster, but surprisingly developed nevi slower than their heterozygous counterparts especially after neonatal UV exposure. Despite raising >1,600 nevi, only six melanomas arose in our experiments with Ink4a/Arf knockout mice (five of which in Xpa+/+ mice at high UV dosages). In contrast to human nevi, these nevi lacked hotspot mutations in Braf or Ras genes, possibly explaining the lack of malignant progression in the Ink4a/Arf−/− mice. Hence, although our experiments did not effectively emulate human melanoma, they provided clear evidence that intermittent UV overexposure strongly stimulates and the Ink4a/Arf−/− genotype may actually impair nevus development. (Cancer Res 2006; 66(5): 2608-15)
FHIT Is Up-Regulated by Inflammatory Stimuli and Inhibits Prostaglandin E2–Mediated Cancer Progression
Authors: Mimori Koshi , Ishii Hideshi , Nagahara Hisashi , Sudo Tomoya , Yamashita Keishi , Inoue Hiroshi , Barnard Graham F. , Mori Masaki
Published: March 2006

The FHIT gene is known to be susceptible to environmental carcinogens. Formation of prostaglandin E2 (PGE2) is catalyzed by cyclooxygenase-2 (COX-2) and may influence malignant phenotype in colorectal cancer. We explored whether FHIT might play a role in progression of colorectal cancer through inflammation-associated PGE2 activity. Immunohistochemical study of COX-2 and FHIT expression was done in 92 colorectal cancer tumors. We also used a FHIT-expressing cancer cell line (H460) induced by ponasterone A and two FHIT small interfering RNA–treated colorectal cancer cell lines (CCK81 and DLD1). After PGE2 stimulation, we compared synthesis of PGE2 (ELISA assay) and cell proliferation [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay]. Immunohistochemistry showed a significant association between COX-2 and FHIT expression in colorectal cancers (P < 0.01). In a subset of 41 COX-2–expressing tumors, 12 FHIT− tumors showed deeper cancer invasion than 29 FHIT+ tumors (P < 0.01). Experimental study, however, showed there was no direct interaction between FHIT and COX-2. Considered with results from another experiment with epidermal growth factor receptor (EGFR), we hypothesize that FHIT and COX-2 might be regulated by a common molecule, such as EGFR. Additionally, there was an inverse and direct correlation between PGE2 synthesis and FHIT in vitro, suggesting that FHIT's postulated antiaggressive effect on tumor goes through PGE2 but not COX-2. Loss of FHIT expression in colorectal cancer suggests higher malignant potential. We conclude that FHIT suppressed cancer cell proliferation in this malignancy by directly inhibiting synthesis of PGE2 but not affecting that of COX-2. (Cancer Res 2006; 66(5): 2683-90)
Genetic Polymorphisms in Antioxidant Enzymes Modulate Hepatic Iron Accumulation and Hepatocellular Carcinoma Development in Patients with Alcohol-Induced Cirrhosis
Authors: Sutton Angela , Nahon Pierre , Pessayre Dominique , Rufat Pierre , Poiré Aurélie , Ziol Marianne , Vidaud Dominique , Barget Nathalie , Ganne-Carrié Nathalie , Charnaux Nathalie , Trinchet Jean-Claude , Gattegno Liliane , Beaugrand Michel
Published: March 2006

Manganese superoxide dismutase (MnSOD) converts the superoxide anion into H2O2, which, unless it is detoxified by glutathione peroxidase 1 (GPx1), can increase hepatic iron and can react with iron to form genotoxic compounds. We investigated the role of Ala/Val-MnSOD and Pro/Leu-GPx1 polymorphisms on hepatic iron accumulation and hepatocellular carcinoma development in patients with alcoholic cirrhosis. Genotypes were determined in 162 alcoholic patients with cirrhosis but without hepatocellular carcinoma initially, who were prospectively followed up for hepatocellular carcinoma development. We found that patients with two Val-MnSOD alleles (slow H2O2 production) and two Pro-GPx1 alleles (presumably quick H2O2 detoxification) had a lower risk of hepatocellular carcinoma development than other patients (χ2 trend test, P = 0.001; log-rank, P = 0.0009). Indeed, hepatocellular carcinoma percentage was 0% in subjects with this “2Val-MnSOD/2Pro-GPx1” genotype versus 16%, 27%, and 32% in “2Val-MnSOD/1or2Leu-GPx1,” “1or2Ala-MnSOD/2Pro-GPx1,” and “1or2Ala-MnSOD/1or2Leu-GPx1” patients, respectively. The percentage of patients with stainable hepatic iron increased progressively with these genotypic associations: 22%, 28%, 50%, and 53%, respectively (χ2 trend test, P = 0.005). Stainable iron was a risk factor for hepatocellular carcinoma (log-rank, P = 0.0002; relative risk, 3.40). In conclusion, polymorphisms in antioxidant enzymes modulate hepatic iron accumulation and hepatocellular carcinoma development in French alcoholic patients with cirrhosis. (Cancer Res 2006; 66(5): 2844-52)
Knockdown of Contactin-1 Expression Suppresses Invasion and Metastasis of Lung Adenocarcinoma
Authors: Su Jen-Liang , Yang Ching-Yao , Shih Jin-Yuan , Wei Lin-Hung , Hsieh Chang-Yao , Jeng Yung-Ming , Wang Ming-Yang , Yang Pan-Chyr , Kuo Min-Liang
Published: March 2006

Numerous genetic changes are associated with cancer cell metastasis and invasion. In search for key regulators of invasion and metastasis, a panel of lung cancer cell lines with different invasive ability was screened. The gene for contactin-1 was found to play an essential role in tumor invasion and metastasis. Suppression of contactin-1 expression abolished the ability of lung adenocarcinoma cells to invade Matrigel in vitro as well as the polymerization of filamentous-actin and the formation of focal adhesion structures. Furthermore, knockdown of contactin-1 resulted in extensive inhibition of tumor metastasis and in increased survival in an animal model. RhoA but not Cdc42 or Rac1 was found to serve a critical role in contactin-1–mediated invasion and metastasis. Contactin-1–specific RNA interference resulted in loss of metastatic and invasive capacity in both in vitro and in vivo models. This loss was overturned by constitutive expression of the active form of RhoA. Contactin-1 was differentially expressed in tumor tissues, and its expression correlated with tumor stage, lymph node metastasis, and patient survival. Contactin-1 is proposed to function importantly in the invasion and metastasis of lung adenocarcinoma cells via RhoA-mediated mechanisms. (Cancer Res 2006; 66(5): 2553-61)
Functional Diversity of DNA Methyltransferase Inhibitors in Human Cancer Cell Lines
Authors: Stresemann Carlo , Brueckner Bodo , Musch Tanja , Stopper Helga , Lyko Frank
Published: March 2006

DNA methyltransferase inhibitors represent promising new drugs for cancer therapies. The first of these compounds (5-azacytidine, Vidaza) has recently been approved as an antitumor agent, and others are presently in various stages of their preclinical or clinical development. Most of the archetypal inhibitors have been established and characterized in different experimental systems, which has thus far precluded their direct comparison. We have now established defined experimental conditions that allowed a comparative analysis of the six most widely known DNA methyltransferase inhibitors: 5-azacytidine (5-aza-CR), 5-aza-2′-deoxycytidine (5-aza-CdR), zebularine, procaine, (−)-epigallocatechin-3-gallate (EGCG), and RG108. Of these, 5-aza-CR, 5-aza-CdR, zebularine, and EGCG were found to exhibit significant cytotoxicity in human cancer cell lines. 5-aza-CdR and EGCG were also found to be genotoxic, as evidenced by the induction of micronuclei. In addition, 5-aza-CR, 5-aza-CdR, zebularine, and RG108 caused concentration-dependent demethylation of genomic DNA, whereas procaine and EGCG failed to induce significant effects. Finally, the experiments in cancer cell lines were complemented by a cell-free in vitro assay with purified recombinant DNA methyltransferase, which indicated that RG108 is the only drug capable of direct enzyme inhibition. These results show a substantial diversity in the molecular activities of DNA methyltransferase inhibitors and provide valuable insights into the developmental potential of individual drugs. (Cancer Res 2006; 66(5): 2794-800)
Matrix Metalloproteinases Play an Active Role in Wnt1-Induced Mammary Tumorigenesis
Authors: Blavier Laurence , Lazaryev Alisa , Dorey Frederick , Shackleford Gregory M. , DeClerck Yves A.
Published: March 2006

The Wnt signaling transduction pathway plays a critical role in the pathogenesis of several murine and human epithelial cancers. Here, we have used mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice, which develop spontaneous mammary adenocarcinoma, to examine whether matrix metalloproteinases (MMPs)—a family of extracellular proteases implicated in multiple steps of cancer progression—contributed to Wnt1-induced tumorigenesis. An analysis of the expression of several MMPs by RT-PCR and in situ hybridization revealed an increase in the expression of MMP-2, MMP-3, MMP-9, MMP-13, and MT1-MMP (MMP-14) in hyperplastic glands and in mammary tumors of MMTV-Wnt1 transgenic mice. Interestingly, whereas MMP-2, MMP-3, and MMP-9 were exclusively expressed by stromal cells in mammary tumors, MMP-13 and MT1-MMP were expressed by transformed epithelial cells in addition to the tumor stroma. To determine whether these MMPs contributed to tumorigenesis, MMTV-Wnt1 mice were crossed with transgenic mice overexpressing tissue inhibitor of metalloproteinase-2—a natural MMP inhibitor—in the mammary gland. In the double MMTV-Wnt1/tissue inhibitor of metalloproteinases-2 transgenic mice, we observed an increase in tumor latency and a 26.3% reduction in tumor formation. Furthermore, these tumors grew at a slower rate, exhibited an 18% decrease in proliferative rate, and a 12.2% increase in apoptotic rate of the tumor cells in association with a deficit in angiogenesis when compared with tumors from MMTV-Wnt1 mice. Thus, for the first time, the data provides evidence for the active role of MMPs in Wnt1-induced mammary tumorigenesis. (Cancer Res 2006; 66(5): 2691-9)
Lysophosphatidic Acid Is a Major Regulator of Growth-Regulated Oncogene α in Ovarian Cancer
Authors: Lee Zendra , Swaby Ramona F. , Liang Yuewei , Yu Shuangxing , Liu Shuying , Lu Karen H. , Bast Robert C. , Mills Gordon B. , Fang Xianjun
Published: March 2006

Growth-regulated oncogene α (GROα), a member of the chemokine superfamily, is commonly expressed in transformed cells and contributes to angiogenesis and tumorigenesis. Here, we report that increased GROα levels are detected in the plasma and ascites of ovarian cancer patients. Ovarian cancer cell lines in culture express and secrete GROα. However, when they are starved in serum-free medium, ovarian cancer cells ceased producing GROα, suggesting that GROα is not constitutively expressed but rather is produced in response to exogenous growth factors in ovarian cancer cells. The prototype peptide growth factors present in serum such as platelet-derived growth factor, insulin-like growth factor I, and insulin do not stimulate GROα production by ovarian cancer cells. In contrast, lysophosphatidic acid (LPA), a glycerol backbone phospholipid mediator present in serum and ascites of ovarian cancer patients, is a potent inducer of GROα expression in ovarian cancer cell lines. Treatment of ovarian cancer cells with LPA leads to transcriptional activation of the GROα gene promoter and robust accumulation of GROα protein in culture supernatants. The action of LPA on GROα expression is mediated by LPA receptors, particularly the LPA2 receptor in that ectopic expression of these receptors restores the LPA-dependent GROα production in nonresponsive cells. Down-regulation of LPA2 expression by small interfering RNA (siRNA) in ovarian cancer cells desensitizes GROα production in response to LPA. The effect of serum on GROα production is also significantly decreased by siRNA inhibition of LPA2 expression. These studies identify LPA as a primary regulator of GROα expression in ovarian cancer. (Cancer Res 2006; 66(5): 2740-8)
Prognostic Value of an Activation State Marker for Epidermal Growth Factor Receptor in Tissue Microarrays of Head and Neck Cancer
Authors: Kong Anthony , Leboucher Pierre , Leek Russell , Calleja Véronique , Winter Stuart , Harris Adrian , Parker Peter J. , Larijani Banafshé
Published: March 2006

Overexpression and mutation of epidermal growth factor receptors (EGFR) have been shown to be important in the prognosis of several cancers, including head and neck cancers. However, our inability to define the activation status of these and other receptors limits our ability to assess the importance of these pathways and to exploit effectively new molecularly targeted treatments directed at their catalytic activities. Here we describe the use of automated, high-throughput fluorescence lifetime imaging microscopy to measure EGFR autophosphorylation status by fluorescence resonance energy transfer (FRET) in head and neck tumors. We have correlated FRET efficiency with the clinical and survival data. The results from head and neck arrays show that high FRET efficiency is correlated with worsening disease-free survival but not with overall survival. This powerful tool could be exploited as a new independent quantitative prognostic factor in clinical decisions and cancer management. (Cancer Res 2006; 66(5): 2834-43)
7,12-Dimethylbenz(a)Anthracene Treatment of a c-rel Mouse Mammary Tumor Cell Line Induces Epithelial to Mesenchymal Transition via Activation of Nuclear Factor-κB
Authors: Shin Sangmin Ryan , Sánchez-Velar Nuria , Sherr David H. , Sonenshein Gail E.
Published: March 2006

The aberrant expression of the nuclear factor-κB (NF-κB) c-Rel subunit that occurs in many human breast cancers can play a causal role in tumorigenesis as judged by findings with a mouse mammary tumor virus (MMTV)-c-rel transgenic mouse model, in which 31.6% of mice developed one or more mammary tumors after a long latency. Interestingly, none of the cell lines established from the mammary tumors grew in soft agar. To begin to test the hypothesis that a prototypic carcinogen insult can promote a more invasive, mesenchymal phenotype, a cell line established from a MMTV-c-rel mammary tumor rel-3983 was treated in culture with the polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA; rel-3983D cells) or DMSO vehicle (rel-3983V cells). Rel-3983D cells displayed an increased rate of proliferation, displayed growth to a higher cell density, and acquired the ability to grow in soft agar and in Matrigel compared with the parental rel-3983 or vehicle-treated rel-3983V cells. Consistent with a more mesenchymal phenotype, rel-3983D cells showed loss of E-cadherin expression as judged by immunofluorescence microscopy. Compared with control cells, rel-3983D displayed increased NF-κB binding and higher levels of the NF-κB transactivating subunits c-Rel, RelA, and RelB, which seemed functional as judged by induction of c-Myc and vimentin, products of two NF-κB target genes. Ectopic expression of a super repressor mutant of IκB-α reduced rel-3983D cell growth and invasive morphology in Matrigel, confirming the role of NF-κB in epithelial to mesenchymal transition (EMT). Thus, DMBA treatment of c-Rel–transformed mammary tumor cells in culture is shown here for the first time to result in EMT via activation of NF-κB. The aberrant c-Rel expression present in most human breast cancers suggests that this mechanism may play an important role in carcinogenesis. (Cancer Res 2006; 66(5): 2570-5)
p44 Mitogen-Activated Protein Kinase (Extracellular Signal-Regulated Kinase 1)–Dependent Signaling Contributes to Epithelial Skin Carcinogenesis
Authors: Bourcier Christine , Jacquel Arnaud , Hess Jochen , Peyrottes Isabelle , Angel Peter , Hofman Paul , Auberger Patrick , Pouysségur Jacques , Pagès Gilles
Published: March 2006

Extracellular signal-regulated kinases (ERK) regulate cellular functions in response to a variety of external signals. However, the specific functions of individual ERK isoforms are largely unknown. Hence, we have investigated the specific function of ERK1 in skin homeostasis and tumorigenesis in ERK1 knockout mice. They spontaneously develop cutaneous lesions and hyperkeratosis with epidermis thickness. Skin hyperproliferation and inflammation induced by application of 12-O-tetradecanoylphorbol-13-acetate (TPA) is strongly reduced in mutant mice. ERK1−/− mice are resistant to development of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by TPA. Tumor appearance was delayed, their formation was less frequent, and their number and size were reduced. Keratinocytes obtained from knockout mice showed reduced growth and resistance to apoptotic signals, accompanied by an impaired expression of genes implicated in growth control and invasiveness. These results highlight the importance of ERK1 in skin homeostasis and in the process of skin tumor development. (Cancer Res 2006; 66(5): 2700-7)
Phosphorylation of Ezrin by Cyclin-Dependent Kinase 5 Induces the Release of Rho GDP Dissociation Inhibitor to Inhibit Rac1 Activity in Senescent Cells
Authors: Yang Hi-Su , Hinds Philip W.
Published: March 2006

Normal somatic cells enter a state of irreversible proliferation arrest-designated cellular senescence, which is characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can lead to senescence. Consistent with an antioncogenic role in this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes, including cell cycle exit and cell shape alteration. Here, we show that pRb-induced senescent SAOS-2 cells and senescent human diploid fibroblasts are accompanied by increased phosphorylation of ezrin at T235 by cyclin-dependent kinase 5 and consequent dissociation of Rho GDP dissociation inhibitor (Rho-GDI) from an ezrin/Rho-GDI complex. The release of Rho-GDI results in increased interaction with Rac1 GTPase and inhibition of Rac1 GTPase activity. In addition, reduction of Rho-GDI by small interfering RNA in pRb-transfected cells prevented senescence-associated flat cell formation, suggesting that Rho-GDI plays an important role in contributing to cellular morphology in the process of senescence. (Cancer Res 2006; 66(5): 2708-15)
Oxidative Damage and Aging: Spotlight on Mitochondria
Authors: Linford Nancy J. , Schriner Samuel E. , Rabinovitch Peter S.
Published: March 2006

Whereas free radical damage has been proposed as a key component in the tissue degeneration associated with aging, there has been little evidence that free radical damage limits life span in mammals. The current research shows that overexpression of the antioxidant enzyme catalase in mitochondria can extend mouse life span. These results highlight the importance of mitochondrial damage in aging and suggest that when targeted appropriately, boosting antioxidant defenses can increase mammalian life span. (Cancer Res 2006; 66(5): 2497-9)
Chemopreventive Properties of Black Raspberries in N-Nitrosomethylbenzylamine-Induced Rat Esophageal Tumorigenesis: Down-regulation of Cyclooxygenase-2, Inducible Nitric Oxide Synthase, and c-Jun
Authors: Chen Tong , Hwang Hyejeong , Rose Miranda E. , Nines Ronald G. , Stoner Gary D.
Published: March 2006

Our laboratory has used a rodent model of human esophageal squamous cell carcinoma to identify putative chemopreventive agents for this disease and to determine their mechanisms of action. In the present study, we treated F344 rats with the esophageal carcinogen, N-nitrosomethylbenzylamine (NMBA), thrice per week for 5 weeks. Beginning 1 week later, they were fed a synthetic diet containing 5% black raspberries (BRB) for the duration of the bioassay (25 weeks). Rats were sacrificed at weeks 9, 15, and 25. Esophageal tissues were collected, and tumor data were recorded. The expression and enzymatic activities of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as the expression of c-Jun in the esophagi, were evaluated to investigate the mechanism(s) by which black raspberries modulate tumorigenesis. At week 25, BRB inhibited tumor multiplicity, the standard end point in this tumor model, from 3.78 ± 0.41 tumors per rat in NMBA-treated animals to 2.23 ± 0.21 tumors per rat in animals treated with NMBA plus BRB (P < 0.005). BRB reduced mRNA and protein expression levels of COX-2, iNOS, and c-Jun as well as the level of prostaglandin E2 in preneoplastic lesions of the esophagus at week 25. The berries inhibited mRNA expression of iNOS and c-Jun, but not COX-2, in papillomatous lesions of the esophagus. Prostaglandin E2 and total nitrite levels were also decreased by BRB in papillomas. These results suggest a novel tumor suppressive role of BRB through inhibition of COX-2, iNOS, and c-Jun. (Cancer Res 2006; 66(5): 2853-9)
Large-Scale Molecular Comparison of Human Schwann Cells to Malignant Peripheral Nerve Sheath Tumor Cell Lines and Tissues
Authors: Miller Shyra J. , Rangwala Fatima , Williams Jon , Ackerman Peter , Kong Sue , Jegga Anil G. , Kaiser Sergio , Aronow Bruce J. , Frahm Silke , Kluwe Lan , Mautner Victor , Upadhyaya Meena , Muir David , Wallace Margaret , Hagen Jussara , Quelle Dawn E. , Watson Mark A. , Perry Arie , Gutmann David H. , Ratner Nancy
Published: March 2006

Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14Arf, and p16INK4a proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100β protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool. (Cancer Res 2006; 66(5): 2584-91)
Activation of Human Long Interspersed Nuclear Element 1 Retrotransposition by Benzo(a)pyrene, an Ubiquitous Environmental Carcinogen
Authors: Stribinskis Vilius , Ramos Kenneth S.
Published: March 2006

Long interspersed nuclear elements [LINE-1 (L1)] are abundant retrotransposons in mammalian genomes that remain silent under most conditions. Cellular stress signals activate L1, but the molecular mechanisms controlling L1 activation remain unclear. Evidence is presented here that benzo(a)pyrene (BaP), an environmental hydrocarbon metabolized by mammalian cytochrome P450s to reactive carcinogenic intermediates, increases L1 retrotransposition in HeLa cells. Increased retrotransposition is mediated by up-regulation of L1 RNA levels, increased L1 cDNA synthesis, and stable genomic integration. Activation of L1 is dependent on the ability of BaP to cause DNA damage because it is absent in HeLa cells challenged with nongenotoxic hydrocarbon carcinogens. Thus, the mutations and genomic instability observed in human populations exposed to genotoxic environmental hydrocarbons may involve epigenetic activation of mobile elements dispersed throughout the human genome. (Cancer Res 2006; 66(5): 2616-20)
Novel Nanoliposomal CPT-11 Infused by Convection-Enhanced Delivery in Intracranial Tumors: Pharmacology and Efficacy
Authors: Noble Charles O. , Krauze Michal T. , Drummond Daryl C. , Yamashita Yoji , Saito Ryuta , Berger Mitchel S. , Kirpotin Dmitri B. , Bankiewicz Krystof S. , Park John W.
Published: March 2006

We hypothesized that combining convection-enhanced delivery (CED) with a novel, highly stable nanoparticle/liposome containing CPT-11 (nanoliposomal CPT-11) would provide a dual drug delivery strategy for brain tumor treatment. Following CED in rat brains, tissue retention of nanoliposomal CPT-11 was greatly prolonged, with >20% injected dose remaining at 12 days for all doses. Tissue residence was dose dependent, with doses of 60 μg (3 mg/mL), 0.8 mg (40 mg/mL), and 1.6 mg (80 mg/mL) resulting in tissue half-life (t1/2) of 6.7, 10.7, and 19.7 days, respectively. In contrast, CED of free CPT-11 resulted in rapid drug clearance (tissue t1/2 = 0.3 day). At equivalent CED doses, nanoliposomal CPT-11 increased area under the time-concentration curve by 25-fold and tissue t1/2 by 22-fold over free CPT-11; CED in intracranial U87 glioma xenografts showed even longer tumor retention (tissue t1/2 = 43 days). Plasma levels were undetectable following CED of nanoliposomal CPT-11. Importantly, prolonged exposure to nanoliposomal CPT-11 resulted in no measurable central nervous system (CNS) toxicity at any dose tested (0.06-1.6 mg/rat), whereas CED of free CPT-11 induced severe CNS toxicity at 0.4 mg/rat. In the intracranial U87 glioma xenograft model, a single CED infusion of nanoliposomal CPT-11 at 1.6 mg resulted in significantly improved median survival (>100 days) compared with CED of control liposomes (19.5 days; P = 4.9 × 10−5) or free drug (28.5 days; P = 0.011). We conclude that CED of nanoliposomal CPT-11 greatly prolonged tissue residence while also substantially reducing toxicity, resulting in a highly effective treatment strategy in preclinical brain tumor models. (Cancer Res 2006; 66(5): 2801-6)
Effects of Transferrin Receptor Blockade on Cancer Cell Proliferation and Hypoxia-Inducible Factor Function and Their Differential Regulation by Ascorbate
Authors: Jones Dylan T. , Trowbridge Ian S. , Harris Adrian L.
Published: March 2006

Cellular iron is needed for cell survival and hydroxylation of hypoxia-inducible factor-1α (HIF-α) by prolyl hydroxylases (PHD). One mechanism of iron uptake is mediated by the cell surface transferrin receptor (TfR). Because iron is required for cell growth and suppression of HIF-α levels, we tested the effects of the two anti-TfR monoclonal antibodies (mAb) E2.3 and A27.15 on growth of breast cancer cells and induction of HIF-α and hypoxia-regulated genes. Treatment with both mAbs together synergistically inhibited cell proliferation in a dose-responsive manner by up to 80% following 8 days of exposure, up-regulated HIF-1α and HIF transcription targets, down-regulated TfR expression, and down-regulated cellular labile iron pool by 60%. Because combined treatment with anti-TfR mAbs resulted in the up-regulation of the hypoxia pathway, which may increase tumor angiogenesis, we analyzed the effects of ascorbate on cell viability and HIF-1α levels in cells treated with both anti-TfR mAbs together, as ascorbate has been shown to be required by PHD enzymes for full catalytic activity. Ascorbate at physiologic concentrations (25 μmol/L) suppressed HIF-1α protein levels and HIF transcriptional targets in anti-TfR mAb-treated cells but did not suppress the antiproliferative effect of the mAbs. These results indicate that the addition of ascorbate increased the activity of the PHD enzymes in down-regulating HIF but not the proliferation of iron-starved anti-TfR mAb-treated cells. The use of anti-TfR mAbs and ascorbate in inhibiting both cell proliferation and HIF-1α and angiogenesis under normoxic conditions may be of therapeutic use. (Cancer Res 2006; 66(5): 2749-56)
Physicochemical and Pharmacokinetic Characterization of Highly Potent Recombinant l-Methionine γ-Lyase Conjugated with Polyethylene Glycol as an Antitumor Agent
Authors: Takakura Tomoaki , Takimoto Akio , Notsu Yoshihide , Yoshida Hiroshi , Ito Takaomi , Nagatome Hirofumi , Ohno Masahiro , Kobayashi Yoshinao , Yoshioka Takayuki , Inagaki Kenji , Yagi Shigeo , Hoffman Robert M. , Esaki Nobuyoshi
Published: March 2006

A highly potent recombinant l-methionine γ-lyase (METase) conjugated with polyethylene glycol (PEG) was characterized physicochemically and pharmacokinetically in vivo and in vitro. Pegylated METase (PEG-METase), which contains pyridoxal 5′-phosphate (PLP) as a cofactor in the molecule, is a potent anticancer agent that can deplete l-methionine from plasma. Although pegylation decreased its specific activity, dithiothreitol (DTT) treatment increased it over three times with the detachment of one PEG moiety modified with a cysteine residue. We can produce DTT-treated PEG-METase on a large scale in sufficient quality for therapeutic use. The superiority of DTT-treated PEG-METase was confirmed by the enhancement of l-methionine depletion and amelioration of pharmacokinetics in mice. The holoenzyme of DTT-treated PEG-METase gave a several times larger area under the plasma concentration curve than that of DTT-untreated PEG-METase, not because of an increase of the half-life but because of high specific activity. Conversely, simultaneous PLP infusion led to a greatly increased half-life of the holoenzyme. DTT-treated PEG-METase administration with PLP infusion was the most useful combination for maximizing the potency of the enzyme. We showed that serum albumin interfered with holoenzyme activity in vitro. The decrease of holoenzyme activity was dependent on the type of serum albumin. We concluded that PLP was released from PEG-METase by serum albumin in vivo and in vitro. The deleterious effect of PLP dissociation from PEG-METase could be improved by supplementing PLP and oleic acid. Their synergistic effect in preventing a decrease of the holoenzyme activity was also observed. (Cancer Res 2006; 66(5): 2807-14)
Cyclooxygenase-1 Is Overexpressed in Multiple Genetically Engineered Mouse Models of Epithelial Ovarian Cancer
Authors: Daikoku Takiko , Tranguch Susanne , Trofimova Irina N. , Dinulescu Daniela M. , Jacks Tyler , Nikitin Alexander Yu. , Connolly Denise C. , Dey Sudhansu K.
Published: March 2006

Cyclooxygenases-1 and -2 (Cox-1 and Cox-2) are two distinct isoforms that catalyze the conversion of arachidonic acid to prostaglandins. The role of Cox-2 in a variety of cancers is well recognized, but the contribution of Cox-1 remains much less explored. We have previously shown that human epithelial ovarian tumors have increased levels of Cox-1, but not Cox-2. We also observed that Cox-1 is highly expressed in a mouse model of epithelial ovarian cancer (EOC), which lacks p53 but overexpresses c-myc and K-ras or c-myc and Akt. More importantly, a Cox-1-selective inhibitor, SC-560, attenuates EOC growth. In the present investigation, we used various genetically engineered mouse models of EOC to determine whether Cox-1 overexpression is unique to specific genetic and oncogenic alterations or is widespread. These models include: (a) deletion of both p53 and Rb, (b) induction of the transforming region of SV40 under the control of Mullerian inhibitory substance type II receptor, or (c) activation of K-Ras in the absence of Pten locally in the ovarian surface epithelium. We found that these three models, which produce spontaneous EOC, also show up-regulated expression of Cox-1, but not Cox-2. The results provide further evidence that Cox-1 overexpression is common in various models of EOC. Thus, Cox-1 serves as a potential marker of EOC and is a possible target for the prevention and/or treatment of this deadly disease. (Cancer Res 2006; 66(5): 2527-31)
Down-regulation of Notch-1 Inhibits Invasion by Inactivation of Nuclear Factor-κB, Vascular Endothelial Growth Factor, and Matrix Metalloproteinase-9 in Pancreatic Cancer Cells
Authors: Wang Zhiwei , Banerjee Sanjeev , Li Yiwei , Rahman K.M. Wahidur , Zhang Yuxiang , Sarkar Fazlul H.
Published: March 2006

Notch signaling plays a critical role in the pathogenesis and progression of human malignancies but the precise role and mechanism of Notch-1 for tumor invasion remains unclear. In our earlier report, we showed that down-regulation of Notch-1 reduced nuclear factor-κB (NF-κB) DNA-binding activity and matrix metalloproteinase-9 (MMP-9) expression. Because NF-κB, VEGF, and MMPs are critically involved in the processes of tumor cell invasion and metastasis, we investigated the role and mechanism(s) by which Notch-1 down-regulation (using molecular approaches) may lead to the down-regulation of NF-κB, vascular endothelial growth factor (VEGF), and MMP-9, thereby inhibiting invasion of pancreatic cancer cells through Matrigel. We found that the down-regulation of Notch-1 by small interfering RNA decreased cell invasion, whereas Notch-1 overexpression by cDNA transfection led to increased tumor cell invasion. Consistent with these results, we found that the down-regulation of Notch-1 reduced NF-κB DNA-binding activity and VEGF expression. Down-regulation of Notch-1 also decreased not only MMP-9 mRNA and its protein expression but also inactivated the pro-MMP-9 protein to its active form. Taken together, we conclude that the down-regulation of Notch-1 could be an effective approach for the down-regulation and inactivation of NF-κB and its target genes, such as MMP-9 and VEGF expression, resulting in the inhibition of invasion and metastasis. (Cancer Res 2006; 66(5): 2778-84)
From Integrated Genomics to Tumor Lineage Dependency
Authors: Garraway Levi A. , Sellers William R.
Published: March 2006

In principle, genomic information derived from tumors should illuminate critical cellular dependencies that are tractable to therapeutic targeting; however, realizing this ideal remains difficult. Using an integrated analysis of high-resolution single nucleotide polymorphism maps and gene expression databases associated with the NCI60 collection cancer cell lines, we identified the transcription factor MITF as an amplified oncogene in melanoma that is critical for anchoring lineage dependence and malignant character. Similar combined genomic approaches may be useful in other cancer types to learn how critical regulators of tumor lineage are linked to genomic alterations in cancer cells. (Cancer Res 2006; 66(5): 2506-8)
Notch Signaling Is Required to Maintain All Neural Stem Cell Populations – Irrespective of Spatial or Temporal Niche
Authors: Alexson Tania O. , Hitoshi Seiji , Coles Brenda L. , Bernstein Alan , van der Kooy Derek
Published: March 2006

Recently, Notch signaling has been reported to underscore the ability of neural stem cells (NSCs) to self-renew. Utilizing mice deficient in presenilin-1(PS1), we asked whether the function of Notch signaling in NSC maintenance was conserved. At embryonic day 14.5, all NSCs – both similar (cortex-, ganglionic eminence- and hindbrain-derived) and distinct (retinal stem cell) – require Notch signaling in a gene-dosage-sensitive manner to undergo expansionary symmetric divisions, as assessed by the clonal, in vitro neurosphere assay. Within the adult, however, Notch signaling modulates cell cycle time in order to ensure brain-derived NSCs retain their self-renewal property. At face value, the effects in the embryo and adult appear different. We propose potential hypotheses, including the ability of cell cycle to modify the mode of division, in order to resolve this discrepancy. Regardless, these findings demonstrate that PS1, and presumably Notch signaling, is required to maintain all NSCs.
Loss of Notch Activity in the Developing Central Nervous System Leads to Increased Cell Death
Authors: Mason Heather A. , Rakowiecki Staci M. , Gridley Thomas , Fishell Gord
Published: March 2006

Many cells in the mammalian brain undergo apoptosis as a normal and critical part of development but the signals that regulate the survival and death of neural progenitor cells and the neurons they produce are not well understood. The Notch signaling pathway is involved in multiple decision points during development and has been proposed to regulate the survival and apoptosis of neural progenitor cells in the developing brain; however, previous experiments have not resolved whether Notch activity is pro- or anti-apoptotic. To elucidate the function of Notch signaling in the survival and death of cells in the nervous system, we have produced single and compound Notch conditional mutants in which Notch1 and Notch3 are removed at different times during brain development and in different populations of cells. We show here that a large number of neural progenitor cells, as well as differentiating neurons, undergo apoptosis in the absence of Notch1 and Notch3, suggesting that Notch activity promotes the survival of both progenitors and newly differentiating cells in the developing nervous system. Finally, we show that postmitotic neurons do not require Notch activity indefinitely to regulate their survival since elevated levels of cell death are observed only during embryogenesis in the Notch mutants and are not detected in neonates.
Notch3 Signaling Promotes Radial Glial/Progenitor Character in the Mammalian Telencephalon
Authors: Dang Louis , Yoon Keejung , Wang Mike , Gaiano Nicholas
Published: March 2006

The Notch signaling pathway is known to influence cell fate in the developing mammalian nervous system. Previous work in the mouse telencephalon has shown that activated Notch1 promotes radial glial and astrocytic character in vivo, and fibroblast growth factor (FGF)-responsive neural progenitor character in vitro. In light of studies suggesting that Notch3 can antagonize Notch1, we tested the effects of activated Notch3 (NICD3) in the mouse telencephalon. Infection of embryonic day 9.5 telencephalic progenitors in vivo with NICD3 promoted radial glial/progenitor character embryonically and astrocyte fate postnatally. In addition, expression of NICD3 in telencephalic progenitors in vitro increased neurosphere frequency in FGF2, but was incompatible with neurosphere growth in epidermal growth factor (EGF). Thus, in the developing telencephalon, Notch1 and Notch3 function similarly, and may activate similar signaling cascades. Consistent with this notion, expression of an activated form of the Notch effector CBF1 (CBF1-VP16), or of the pathway target Hes5 promoted radial glial/progenitor character in vivo. Interestingly, unlike NICD1 and NICD3, CBF1-VP16 and Hes5 did not inhibit neurosphere growth in EGF, suggesting that this effect may be mediated at least in part by CBF1/Hes-independent signaling.
Hes1 and Hes5 Regulate the Development of the Cranial and Spinal Nerve Systems
Authors: Hatakeyama Jun , Sakamoto Susumu , Kageyama Ryoichiro
Published: March 2006

The basic helix-loop-helix genes Hes1 and Hes5, known Notch effectors, regulate the maintenance of neural stem cells and the development of the central nervous system (CNS). In the absence of Hes1 and Hes5, the size, shape and cytoarchitecture of the CNS are severely disorganized, but the development of the peripheral nervous system remains to be analyzed. Here, we found that in Hes1;Hes5 double-mutant mice, the cranial and spinal nerve systems are also severely disorganized. In these mutant mice, axonal projections from the mesencephalic neurons to the trigeminal (V) ganglion become aberrant and the proximal parts of the glossopharyngeal (IX) and vagus (X) nerves are fused. The hypoglossal (XII) nerve is also formed poorly. Furthermore, the dorsal root ganglia are fused with the spinal cord, and the dorsal and ventral roots of the spinal nerves are lacking in many segments. These results indicate that Hes1 and Hes5 play an important role in the formation of the cranial and spinal nerve systems.