bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear Hormone Receptor NR4A2 Is Involved in Cell Transformation and Apoptosis
Authors: Ke Ning , Claassen Gisela , Yu De-Hua , Albers Aaron , Fan Wufang , Tan Philip , Grifman Mirta , Hu Xiuyuan , DeFife Kristin , Nguy Vivian , Meyhack Bernd , Brachat Arndt , Wong-Staal Flossie , Li Qi-Xiang
Published: September 2005

HeLaHF cells are transformation revertants of cervical cancer HeLa cells and have lost anchorage-independent growth potential and tumorigenicity. Activation of tumor suppressor(s) was implicated previously in this transformation reversion. In this study, expression profiling analysis was carried out to identify potential oncogenes that are down-regulated in HeLaHF cells. We found that all three members of the NR4A1/Nur77/NGFIB orphan nuclear hormone receptor subfamily (NR4A1, NR4A2, and NR4A3) were down-regulated in the HeLaHF revertant. Small interfering RNA-mediated down-regulation of NR4A2 in HeLa cells, either transiently or stably, resulted in reduced anchorage-independent growth that was largely attributable to increased anoikis. Furthermore, down-regulation of NR4A2 as well as NR4A1 promoted intrinsic apoptosis. These phenotypes were also observed in several other experimental cancer cells, suggesting the observed apoptosis suppression is a more general property of NR4A2 and NR4A1. These phenotypes also suggest that the Nur77/NGFIB subfamily of orphan receptors exhibit certain oncogenic functionalities with regards to cell proliferation and apoptosis and could therefore be evaluated as potential cancer therapeutic targets.
Analysis of Gene Expression Patterns and Chromosomal Changes Associated with Aging
Authors: Geigl Jochen B. , Langer Sabine , Barwisch Simone , Pfleghaar Katrin , Lederer Gaby , Speicher Michael R.
Published: September 2005

Age is the largest single risk factor for the development of cancer in mammals. Age-associated chromosomal changes, such as aneuploidy and telomere erosion, may be vitally involved in the initial steps of tumorigenesis. However, changes in gene expression specific for increased aneuploidy with age have not yet been characterized. Here, we address these questions by using a panel of fibroblast cell lines and lymphocyte cultures from young and old age groups. Oligonucleotide microarrays were used to characterize the expression of 14,500 genes. We measured telomere length and analyzed chromosome copy number changes and structural rearrangements by multicolor interphase fluorescence in situ hybridization and 7-fluorochrome multiplex fluorescence in situ hybridization, and we tried to show a relationship between gene expression patterns and chromosomal changes. These analyses revealed a number of genes involved in both the cell cycle and proliferation that are differently expressed in aged cells. More importantly, our data show an association between age-related aneuploidy and the gene expression level of genes involved in centromere and kinetochore function and in the microtubule and spindle assembly apparatus. To verify that some of these genes may also be involved in tumorigenesis, we compared the expression of these genes in chromosomally stable microsatellite instability and chromosomally unstable chromosomal instability colorectal tumor cell lines. Three genes (Notch2, H2AFY2, and CDC5L) showed similar expression differences between microsatellite instability and chromosomal instability cell lines as observed between the young and old cell cultures suggesting that they may play a role in tumorigenesis.
The Minimal Active Domain of Endostatin Is a Heparin-Binding Motif that Mediates Inhibition of Tumor Vascularization
Authors: Olsson Anna-Karin , Johansson Irja , Åkerud Helena , Einarsson Barbro , Christofferson Rolf , Sasaki Takako , Timpl Rupert , Claesson-Welsh Lena
Published: September 2005

Endostatin constitutes the COOH-terminal 20,000 Da proteolytic fragment of collagen XVIII and has been shown to possess antiangiogenic and antitumorigenic properties. In the present study, we have investigated the role of the heparin-binding sites in the in vivo mechanism of action of endostatin. The majority of the heparin binding is mediated by arginines 155/158/184/270 in endostatin, but there is also a minor site constituted by arginines 193/194. Using endostatin mutants lacking either of these two sites, we show that inhibition of fibroblast growth factor-2–induced angiogenesis in the chicken chorioallantoic membrane requires both heparin-binding sites. In contrast, inhibition of vascular endothelial growth factor-A–induced chorioallantoic membrane angiogenesis by endostatin was only dependent on the minor heparin-binding site (R193/194). These arginines were also required for endostatin to inhibit fibroblast growth factor-2– and vascular endothelial growth factor-A–induced chemotaxis of primary endothelial cells. Moreover, we show that a synthetic peptide corresponding to amino acids 180–199 of human endostatin (which covers the minor heparin-binding site) inhibits endothelial cell chemotaxis and reduces tumor vascularization in vivo. Substitution of arginine residues 193/194 for alanine attenuates the antiangiogenic effects of the peptide. These data show an essential role for heparin binding in the antiangiogenic action of endostatin.
The Anticancer Drug Ellipticine Forms Covalent DNA Adducts, Mediated by Human Cytochromes P450, through Metabolism to 13-Hydroxyellipticine and EllipticineN2-Oxide
Authors: Stiborová Marie , Sejbal Jan , Bořek-Dohalská Lucie , Aimová Dagmar , Poljaková Jitka , Forsterová Kristina , Rupertová Martina , Wiesner Jiří , Hudeček Jiří , Wiessler Manfred , Frei Eva
Published: September 2005

Ellipticine is an antineoplastic agent, the mode of action of which is considered to be based on DNA intercalation and inhibition of topoisomerase II. We found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. We now identified the ellipticine metabolites formed by human CYPs and elucidated the metabolites responsible for DNA binding. The 7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine, and ellipticine N2-oxide are generated by hepatic microsomes from eight human donors. The role of specific CYPs in the oxidation of ellipticine and the role of the ellipticine metabolites in the formation of DNA adducts were investigated by correlating the levels of metabolites formed in each microsomal sample with CYP activities and with the levels of the ellipticine-derived deoxyguanosine adducts in DNA. On the basis of this analysis, formation of 9-hydroxyellipticine and 7-hydroxyellipticine was attributable to CYP1A1/2, whereas production of 13-hydroxyellipticine and ellipticine N2-oxide, the metabolites responsible for formation of two major DNA adducts, was attributable to CYP3A4. Using recombinant human enzymes, oxidation of ellipticine to 9-hydroxyellipticine and 7-hydroxyellipticine by CYP1A1/2 and to 13-hydroxyellipticine and N2-oxide by CYP3A4 was corroborated. Homologue modeling and docking of ellipticine to the CYP3A4 active center was used to explain the predominance of ellipticine oxidation by CYP3A4 to 13-hydroxyellipticine and N2-oxide.
Metallothionein-I/II Double Knockout Mice Are Hypersensitive to Lead-Induced Kidney Carcinogenesis
Authors: Waalkes Michael P. , Liu Jie , Goyer Robert A. , Diwan Bhalchandra A.
Published: September 2005

Lead is an environmental nephrotoxicant and probable human carcinogen. Elucidating factors predisposing populations to lead toxicity is an important public health issue. Recently, we found that metallothionein-I/-II double knockout (metallothionein-null) mice that are unable to produce the major forms of metallothionein do not produce lead inclusion bodies, which are thought to mitigate lead toxicity, and were sensitive to the subchronic toxic effects of lead exposure (10 weeks), showing modestly diminished renal function and nephromegaly compared with wild-type (WT) mice. It is unclear how this knockout might impact lead carcinogenesis. Thus, the effects of lead(II) acetate were tested in groups (n = 25) of male metallothionein-null and WT mice receiving drinking water with 0, 1,000, 2,000, or 4,000 parts per million lead for up to 104 weeks. Renal proliferative lesions (adenoma and cystic tubular atypical hyperplasia) were much more common and more severe in lead-exposed metallothionein-null mice than in WT mice. A metastatic renal cell carcinoma also occurred in a lead-treated metallothionein-null mouse, whereas none occurred in WT mice. Lead-induced renal proliferative lesions showed marked overexpression of cyclin D1, a common feature of human renal tumors. Renal lead-containing nuclear inclusion bodies were frequently observed in WT mice but did not form in metallothionein-null mice. Metallothionein was often found associated with the outer portion of these inclusion bodies. Thus, the metallothionein-null mice cannot form renal inclusion bodies, even after protracted lead exposure, and this increases the carcinogenic potential of lead. Poor production of metallothionein may predispose human populations to lead carcinogenicity.
NIMA-Related Protein Kinase 1 Is Involved Early in the Ionizing Radiation-Induced DNA Damage Response
Authors: Polci Rosaria , Peng Aimin , Chen Phang-Lang , Riley Daniel J. , Chen Yumay
Published: September 2005

Cellular functions of the NimA-related mammalian kinase Nek1 have not been demonstrated to date. Here we show that Nek1 is involved early in the DNA damage response induced by ionizing radiation (IR) and that Nek1 is important for cells to repair and recover from DNA damage. When primary or transformed cells are exposed to IR, Nek1 kinase activity is increased within 4 minutes, and Nek1 expression is up-regulated shortly thereafter and sustained for hours. At the same early time frame after IR that its kinase activity is highest, a portion of Nek1 redistributes in cells from cytoplasm to discrete nuclear foci at sites of DNA double-strand breaks. There it colocalizes with γ-H2AX and NFBD1/MDC1, two key proteins involved very early in the response to IR-induced DNA double-strand breaks. Finally, Nek1-deficient fibroblasts are much more sensitive to the effects of IR-induced DNA damage than otherwise identical fibroblasts expressing Nek1. These results suggest that Nek1 may function as a kinase early in the DNA damage response pathway.
The FLT3 Internal Tandem Duplication Mutation Prevents Apoptosis in Interleukin-3-Deprived BaF3 Cells Due to Protein Kinase A and Ribosomal S6 Kinase 1–Mediated BAD Phosphorylation at Serine 112
Authors: Yang Xinping , Liu Liyun , Sternberg David , Tang Liren , Galinsky Ilene , DeAngelo Daniel , Stone Richard
Published: September 2005

Internal tandem duplication (ITD) mutations in the FLT3 tyrosine kinase have been detected in ∼20% of acute myeloid leukemia (AML) patients. Patients harboring FLT3/ITD mutations have a relatively poor prognosis. FLT3/ITD results in constitutive autophosphorylation of the receptor and factor-independent survival. Previous studies have shown that FLT3/ITD activates the signal transducers and activators of transcription 5 (STAT5), p42/p44 mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase (ERK) 1/2], and phosphatidylinositol 3-kinase/Akt pathways. We herein provide biochemical and biological evidence that ribosomal S6 kinase 1 (RSK1) and protein kinase A (PKA) are the two principal kinases that mediate the antiapoptotic function of FLT3/ITD via phosphorylation of BAD at Ser112. Inhibiting both MAPK kinase (MEK)/ERK and PKA pathways by a combination of U0126 (10 μmol/L) and H-89 (5 μmol/L) reduced most of BAD phosphorylation at Ser112 and induced apoptosis to a level comparable with that induced by FLT3 inhibitor AG1296 (5 μmol/L) in BaF3/FLT3/ITD cells. RNA interference of RSK1 or PKA catalytic subunit reduced BAD phosphorylation and induced apoptosis. The MEK inhibitor U0126 and/or the PKA inhibitor H-89 greatly enhanced the efficacy of the FLT3 inhibitor AG1296, suggesting that combining FLT3/ITD downstream pathway inhibition with FLT3 inhibitors may be a viable therapeutic strategy for AML caused by a FLT3/ITD mutation.
Polycyclic Aromatic Hydrocarbon-DNA Adducts in Prostate Cancer
Authors: Rybicki Benjamin A. , Rundle Andrew , Savera Adnan T. , Sankey Steadman S. , Tang Deliang
Published: September 2005

The formation of DNA adducts can lead to DNA replication errors and the potential for carcinogenesis. DNA adducts have been detected in prostate cells, but the distribution of adducts with respect to prostate cancer risk factors and histology is unknown. In a study of 130 Caucasian (n = 61) and African-American (n = 69) men with prostate cancer who underwent radical prostatectomy, we quantified polycyclic aromatic hydrocarbon (PAH)-DNA adducts in prostate tumor and adjacent nontumor cells by immunohistochemistry. A strong correlation between paired adduct levels in the two cell types was observed (r = 0.56; P < 0.0001); however, nontumor cells had a significantly higher level of adducts compared with tumor (0.30 absorbance units ± 0.05 versus 0.17 absorbance units ± 0.04; P < 0.0001). Variables significantly associated with PAH-DNA adduct levels in tumor cells included primary Gleason grade, tumor volume, and log-transformed prostate-specific antigen (PSA) at time of diagnosis. Tumors with a primary Gleason grade of 5 had significantly lower PAH-DNA adduct levels than tumor cells with a primary Gleason grade of 3 or 4 (P < 0.0001 for both). Tumors that involved 10% or less of the prostate gland had significantly higher PAH-DNA adduct levels than tumors that involved 15 to 20% of the prostate gland (P = 0.004). PSA levels were inversely associated with PAH-DNA adduct levels in tumor cells (P = 0.009). A similar, albeit less significant, inverse association was observed between PSA and PAH-DNA adduct levels in nontumor cells (P = 0.07). Interestingly, increasing primary Gleason grade was associated with increasing PAH-DNA adduct levels in adjacent nontumor cells (P = 0.008). Our results show that PAH-DNA adducts are present in the prostate but vary with regard to cellular histology. In prostate tumor cells, decreased cellular differentiation and increased tumor proliferation may reduce PAH-DNA adduct levels.
Small In-Frame Deletion in the Epidermal Growth Factor Receptor as a Target for ZD6474
Authors: Arao Tokuzo , Fukumoto Hisao , Takeda Masayuki , Tamura Tomohide , Saijo Nagahiro , Nishio Kazuto
Published: September 2005

ZD6474 is an inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) tyrosine kinase, with additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. ZD6474 inhibits angiogenesis and growth of a wide range of tumor models in vivo. Gefitinib (“Iressa”) is a selective EGFR tyrosine kinase inhibitor that blocks signal transduction pathways implicated in cancer cell proliferation. Here, the ability of gefitinib and ZD6474 to inhibit tumor cell proliferation was examined directly in eight cancer cell lines in vitro, and a strong correlation was noted between the IC50 values of gefitinib and ZD6474 (r = 0.79). No correlation was observed between the sensitivity to ZD6474 and the level of EGFR or VEGFR expression. The NSCLC cell line PC-9 was seen to be hypersensitive to gefitinib and ZD6474, and a small (15-bp) in-frame deletion of an ATP-binding site (exon 19) in the EGFR was detected (delE746-A750–type deletion). To clarify the involvement of the deletional mutation of EGFR in the cellular sensitivity to ZD6474, we examined the effect of this agent on HEK293 stable transfectants expressing deletional EGFR that designed as the same deletion site observed in PC-9 cells (293-pΔ15). These cells exhibited a 60-fold higher sensitivity to ZD6474 compared with transfectants expressing wild-type EGFR. ZD6474 inhibited the phosphorylation of the mutant EGFR by 10-fold compared with cells with wild-type EGFR. In conclusion, the findings suggested that a small in-frame deletion in the EGFR increased the cellular sensitivity to ZD6474.
NOD2
3020insC Alone Is Not Sufficient for Colorectal Cancer Predisposition
Authors: Alhopuro Pia , Ahvenainen Taru , Mecklin Jukka-Pekka , Juhola Matti , Järvinen Heikki J. , Karhu Auli , Aaltonen Lauri A.
Published: September 2005

Mutations in NOD2 have been shown to associate with increased susceptibility to Crohn’s disease. A recent Polish study linked the truncating NOD2 3020insC variant with an increased risk of colorectal cancer (CRC) at an older age (>50 years) of disease onset, with an odds ratio of 2.23. We studied the possible contribution of the 3020insC variant to CRC risk in a series of 1,042 Finnish population-based patients from which 926 samples were successfully analyzed and in 348 anonymous cancer-free controls. The frequency of the 3020insC mutation was 3.7% in both CRC patients (34 of 926, including 1 homozygote) and cancer-free controls (13 of 348; odds ratio, 0.98; confidence interval, 0.51–1.88). Contrary to the Polish study, there was no significant difference in the mutation rates between CRC patients > 50 years of age (25 of 576; 4.3%) and controls in the present series. We studied respective tumor tissue DNAs of all patients displaying heterozygous 3020insC changes for loss of heterozygosity. Loss of heterozygosity at NOD2 was observed in only 1 of the 33 CRC samples. Our results suggest that NOD2 3020insC alone does not contribute to CRC risk. If this variant predisposes to CRC, additional factors not present in the Finnish population need to be involved.
Mitogen- and Stress-Activated Protein Kinase 1 Activity and Histone H3 Phosphorylation in Oncogene-Transformed Mouse Fibroblasts
Authors: Drobic Bojan , Espino Paula S. , Davie James R.
Published: September 2005

Activation of the Ras-Raf-mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase-ERK signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinase 1 (MSK1). This activation of MSK1 leads to a rapid phosphorylation of histone H3 at Ser10. Previously, we had demonstrated that Ser10 phosphorylated H3 was elevated in Ciras-3 (c-Ha-ras–transformed 10T1 2) mouse fibroblasts and that H3 phosphatase activity was similar in Ciras-3 and 10T1 2 cells. Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Ciras-3 cells relative to these activities in the parental 10T1 2 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T1 2 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the Ras-mitogen-activated protein kinase signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in these cells.
Adenoviral E1A Targets Mdm4 to Stabilize Tumor Suppressor p53
Authors: Li Zheng , Day Chi-Ping , Yang Jer-Yen , Tsai Wen-Bin , Lozano Guillermina , Shih Hsiu-Ming , Hung Mien-Chie
Published: September 2005

The adenoviral protein E1A associates with multiple anticancer activities, including stabilization of p53 tumor suppressor, and has been tested through gene therapy approaches in clinical trials. To identify potential E1A-binding proteins involved in E1A’s anticancer activities, we screened a yeast two-hybrid library and identified Mdm4, an Mdm2-related p53-binding protein, as a novel E1A-binding protein. The NH2-terminal region of Mdm4 and the CR1 domain of E1A were required for the interaction between E1A and Mdm4. E1A preferentially bound to Mdm4 rather than Mdm2 and formed a complex with p53 in the presence of Mdm4, resulting in the stabilization of p53 in a p14ARF-independent manner. E1A failed to stabilize p53 in the absence of Mdm4, showing that Mdm4 was required for p53 stabilization by E1A. Moreover, E1A-mediated stabilization of p53 occurred in nucleus. Although it had no effect on the p53-Mdm2 interaction, E1A facilitated Mdm4 binding to p53 and inhibited Mdm2 binding to Mdm4, resulting in decreased nuclear exportation of p53. Thus, our findings highlighted a novel mechanism, whereby E1A stabilized the p53 tumor suppressor through Mdm4.
Cyclin B1 Is a Critical Target of RhoB in the Cell Suicide Program Triggered by Farnesyl Transferase Inhibition
Authors: Kamasani Uma , Huang Minzhou , DuHadaway James B. , Prochownik Edward V. , Donover P. Scott , Prendergast George C.
Published: September 2005

Farnesyl transferase inhibitors (FTIs) have displayed limited efficacy in clinical trials, possibly because of their relatively limited cytotoxic effects against most human cancer cells. Therefore, efforts to leverage the clinical utility of FTIs may benefit from learning how these agents elicit p53-independent apoptosis in mouse models of cancer. Knockout mouse studies have established that gain of the geranylgeranylated isoform of the small GTPase RhoB is essential for FTI to trigger apoptosis. Here we demonstrate that Cyclin B1 is a crucial target for suppression by RhoB in this death program. Steady-state levels of Cyclin B1 and its associated kinase Cdk1 were suppressed in a RhoB-dependent manner in cells fated to undergo FTI-induced apoptosis. These events were not derivative of cell cycle arrest, because they did not occur in cells fated to undergo FTI-induced growth inhibition. Mechanistic investigations indicated that RhoB mediated transcriptional suppression but also accumulation of Cyclin B1 in the cytosol at early times after FTI treatment, at a time before the subsequent reduction in steady-state protein levels. Enforcing Cyclin B1 expression attenuated apoptosis but not growth inhibition triggered by FTI. Moreover, enforcing Cyclin B1 abolished FTI antitumor activity in graft assays. These findings suggest that Cyclin B1 suppression is a critical step in the mechanism by which FTI triggers apoptosis and robust antitumor efficacy. Our findings suggest that Cyclin B1 suppression may predict favorable clinical responses to FTI, based on cytotoxic susceptibility, and they suggest a rational strategy to address FTI nonresponders by coinhibition of Cdk1 activity.
Significant Contribution of Germline
BRCA2
Rearrangements in Male Breast Cancer Families
Authors: Tournier Isabelle , Paillerets Brigitte Bressac-de , Sobol Hagay , Stoppa-Lyonnet Dominique , Lidereau Rosette , Barrois Michel , Mazoyer Sylvie , Coulet Florence , Hardouin Agnès , Chompret Agnès , Lortholary Alain , Chappuis Pierre , Bourdon Violaine , Bonadona Valérie , Maugard Christine , Gilbert Brigitte , Nogues Catherine , Frébourg Thierry , Tosi Mario
Published: September 2005

Although screening for large deletions or duplications of the BRCA1 gene is becoming a routine component of the molecular diagnosis of familial breast cancer, little is known about the occurrence of such rearrangements in the BRCA2 gene. Because of the high frequency of BRCA2 mutations in breast cancer families with at least one case of male breast cancer, we selected a cohort of 39 such families, tested negative for mutations in the coding regions of BRCA1 and BRCA2, and developed an assay for BRCA2 rearrangements, based on quantitative multiplex PCR of short fluorescent fragments (QMPSF). We found three rearrangements: (1) a deletion of exons 12 and 13; (2) a duplication of exons 1 and 2; and (3) a complete deletion of BRCA2. We determined the boundaries of the deletion of exons 12 and 13, showing that it resulted from an unequal recombination between Alu sequences. We mapped the complete BRCA2 deletion, which extends over at least 298 kb and showed that it does not affect APRIN/AS3, previously characterized as a tumor suppressor gene, but it comprises several loci corresponding to proven or putative transcripts of unknown functional significance. These data suggest that screening for BRCA2 rearrangements should be done, especially in male breast cancer families tested negative for BRCA1 and BRCA2 mutations.
Elimination of Hepatic Metastases of Colon Cancer Cells via p53-Independent Cross-Talk between Irinotecan and Apo2 Ligand/TRAIL
Authors: Ravi Rajani , Jain Ajay J. , Schulick Richard D. , Pham Vui , Prouser Traci S. , Allen Heather , Mayer Elizabeth Garrett , Yu Hua , Pardoll Drew M. , Ashkenazi Avi , Bedi Atul
Published: September 2005

The majority of colorectal cancers have lost/inactivated the p53 tumor suppressor gene. Using isogenic human colon cancer cells that differ only in their p53 status, we demonstrate that loss of p53 renders tumor cells relatively resistant to the topoisomerase I inhibitor, irinotecan. Whereas irinotecan-induced up-regulation of the proapoptotic proteins PUMA and Noxa requires p53, we find that irinotecan inhibits Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 and 5 (STAT3/5) signaling in both p53-proficient and p53-deficient tumor cells. We show that irinotecan inhibits JAK2-STAT3/5-dependent expression of survival proteins (Bcl-xL and XIAP) and cooperates with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) to facilitate p53-independent apoptosis of colon cancer cells. Whereas xenografts of p53-deficient colon cancer cells are relatively resistant to irinotecan compared with their p53-proficient counterparts, combined treatment with irinotecan and Apo2L/TRAIL eliminates hepatic metastases of both p53-proficient and p53-deficient cancer cells in vivo and significantly improves the survival of animals relative to treatment with either agent alone. Although the synergy between chemotherapy and Apo2L/TRAIL has been ascribed to p53, our data demonstrate that irinotecan enhances Apo2L/TRAIL-induced apoptosis of tumor cells via a distinct p53-independent mechanism involving inhibition of JAK2-STAT3/5 signaling. These findings identify a novel p53-independent channel of cross-talk between topoisomerase I inhibitors and Apo2L/TRAIL and suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of irinotecan against both p53-proficient and p53-deficient colorectal cancers, including those that have metastasized to the liver.
Recombinant Human Erythropoietin α Targets Intratumoral Blood Vessels, Improving Chemotherapy in Human Xenograft Models
Authors: Tóvári József , Gilly Réka , Rásó Erzsébet , Paku Sándor , Bereczky Bíborka , Varga Norbert , Vágó Ágnes , Tímár József
Published: September 2005

Recombinant human erythropoietin (rHuEPO) is widely used for correction of hemoglobin level in cancer patients. However, apart from hematopoiesis, rHuEPO reportedly has an effect on endothelial cells. We describe here how rHuEPOα can modulate tumor vasculature in human squamous cell (A431) and colorectal carcinoma (HT25) xenograft models. In vivo rHuEPO treatment of xenografts at human-equivalent dose significantly increased the proliferation index of the tumor-associated endothelial cells and the size of CD31-positive intratumoral blood vessels, whereas the pericyte coverage became fragmented. Moreover, rHuEPO administration resulted in decreased expression of vascular endothelial growth factor both by cancer cells and tumor stroma, measured by quantitative PCR. Due to the morphologic alterations in tumoral microvessels, DNA-binding agents (Hoechst and Doxorubicin) labeled significantly larger areas in the tumor mass. Furthermore, rHuEPO treatment led to a significantly improved efficacy of 5-fluorouracil (5-FU) chemotherapy in the case of both tumor xenografts. Meanwhile, rHuEPO had no effect on the in vitro proliferation of erythropoietin receptor–positive tumor cells, and did not interfere with the effects of 5-FU either. These data reveal a new effect of rHuEPO administration: remodeling tumoral microvessels, suppressing vascular endothelial growth factor expression, thereby augmenting antitumor effects of a cancer drug, 5-FU, even in erythropoietin receptor–positive human cancer xenografts.
JAGGED1 Expression Is Associated with Prostate Cancer Metastasis and Recurrence
Authors: Santagata Sandro , Demichelis Francesca , Riva Alberto , Varambally Sooryanarayana , Hofer Matthias D. , Kutok Jeffery L. , Kim Robert , Tang Jeffery , Montie James E. , Chinnaiyan Arul M. , Rubin Mark A. , Aster Jon C.
Published: September 2005

Recent studies suggest that NOTCH signaling can promote epithelial-mesenchymal transitions and augment signaling through AKT, an important growth and survival pathway in epithelial cells and prostate cancer in particular. Here we show that JAGGED1, a NOTCH receptor ligand, is significantly more highly expressed in metastatic prostate cancer as compared with localized prostate cancer or benign prostatic tissues, based on immunohistochemical analysis of JAGGED1 expression in human tumor samples from 154 men. Furthermore, high JAGGED1 expression in a subset of clinically localized tumors was significantly associated with recurrence, independent of other clinical parameters. These findings support a model in which dysregulation of JAGGED1 protein levels plays a role in prostate cancer progression and metastasis and suggest that JAGGED1 may be a useful marker in distinguishing indolent and aggressive prostate cancers.
Targeted Disruption of the 3p12 Gene,
Dutt1/Robo1
, Predisposes Mice to Lung Adenocarcinomas and Lymphomas with Methylation of the Gene Promoter
Authors: Xian Jian , Aitchison Alan , Bobrow Linda , Corbett Gerard , Pannell Richard , Rabbitts Terence , Rabbitts Pamela
Published: September 2005

The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene’s promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.
TRAIL Inhibits Tumor Growth but Is Nontoxic to Human Hepatocytes in Chimeric Mice
Authors: Hao Chunhai , Song Jin H. , Hsi Belinda , Lewis Jamie , Song Doyoun K. , Petruk Kenneth C. , Tyrrell David L. J. , Kneteman Norman M.
Published: September 2005

Tumor necrosis factor (TNF) family ligand TNF-α and Fas ligand (FasL) can trigger apoptosis in solid tumors, but their clinical usage has been limited by hepatotoxicity. TNF-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the TNF family, and its clinical application currently is under a similar debate. Here, we report a recombinant soluble form of human TRAIL (114 to 281 amino acids) that induces apoptosis in tumor cells but not human hepatocytes. We first isolated human hepatocytes from patients and showed that the human hepatocytes expressed Fas but no TRAIL death receptor DR4 and little DR5 on the cell surface. Antibody cross-linked FasL, but not TRAIL, triggered apoptosis of the human hepatocytes through cleavage of caspases. We then examined TRAIL hepatotoxicity in severe combined immunodeficient/Alb-uPA chimeric mice harboring human hepatocytes. Intravenous injection of FasL, but not TRAIL, caused apoptotic death of human hepatocytes within the chimeric liver, thus killing the mice. Finally, we showed that repeated intraperitoneal injections of TRAIL inhibited intraperitoneal and subcutaneous tumor growth without inducing apoptosis in human hepatocytes in these chimeric mice. The results indicate that the recombinant soluble human TRAIL has a profound apoptotic effect on tumor cells but is nontoxic to human hepatocytes in vitro and in vivo.
Reactive Oxygen Species Regulate Caspase Activation in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Human Colon Carcinoma Cell Lines
Authors: Izeradjene Kamel , Douglas Leslie , Tillman David M. , Delaney Addison B. , Houghton Janet A.
Published: September 2005

The effects of reactive oxygen species (ROS) on tumor necrosis factor–related apoptosis–inducing ligand (TRAIL)–induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in ΔΨm and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of ΔΨm and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9–independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9–independent activation of caspase 3.
Analysis of Wnt Gene Expression in Prostate Cancer
Authors: Zhu Hanneng , Mazor Michal , Kawano Yoshiaki , Walker Marjorie M. , Leung Hing Y. , Armstrong Kelly , Waxman Jonathan , Kypta Robert M.
Published: September 2005

The Wnt signaling pathway is aberrantly activated in many tumor types, including those of the prostate, in which β-catenin accumulates in cell nuclei and acts as a transcriptional coregulator for the androgen receptor. Because activating mutations in the β-catenin gene are rare in prostate cancer, we have looked for altered expression of other components of the Wnt signaling pathway in prostate cancer cells. Here we determined the expression levels of Wnt family genes in cultured human prostate cells and prostate cancer cell lines. We found that WNT11 expression is elevated in hormone-independent prostate cancer cell lines. Additional analysis indicated that WNT11 expression is also elevated in high-grade prostatic tumors and in hormone-independent xenografts. Growth of hormone-dependent LNCaP cells in hormone-depleted media led to increased WNT11 expression, which was repressed by the synthetic androgen R1881. This repression was inhibited by the antiandrogen bicalutamide, suggesting that androgens negatively regulate WNT11 expression through the androgen receptor. Expression of WNT11 inhibited androgen receptor transcriptional activity and cell growth in androgen-dependent cells but not in androgen-independent cells. WNT11 inhibited activation of the canonical Wnt pathway by WNT3A in HEK 293 cells and inhibited basal β-catenin/Tcf transcriptional activity in LNCaP cells. However, expression of stabilized β-catenin did not prevent the inhibition of androgen receptor transcriptional activity by WNT11. Our observations are consistent with a model in which androgen depletion activates WNT11-dependent signals that inhibit androgen-dependent but not androgen-independent cell growth.
Host Acid Sphingomyelinase Regulates Microvascular Function Not Tumor Immunity
Authors: Garcia-Barros Monica , Lacorazza Daniel , Petrie Howard , Haimovitz-Friedman Adriana , Cardon-Cardo Carlos , Nimer Stephen , Fuks Zvi , Kolesnick Richard
Published: September 2005

Previous studies provided evidence that MCA/129 fibrosarcomas and B16 melanomas grow 2- to 4-fold faster in acid sphingomyelinase (asmase)–deficient mice than in asmase+/+ littermates and are resistant to single-dose irradiation due to inability to mount an apoptotic response in tumor microvascular endothelium. However, others postulated the differences might be associated with a host antitumor immune response in asmase+/+ mice that is not expressed in asmase−/− mice due to phenotypic deficiency in antitumor immunity. The present studies demonstrate that none of the tumor–host combinations displayed the classic criteria of an immunogenic tumor because they lacked endotumoral or peritumoral infiltrates almost entirely. Furthermore, neither MCA/129 fibrosarcoma nor B16 melanoma tumors showed differences in growth or radioresponsiveness when implanted into mutant mouse models (Rag−/− and MEF−/−) lacking functional immune cell [natural killer (NK), NK-T, T, and B cells] populations. Additionally, development and function of B-, T-, and NK-cell populations in asmase−/− mice were normal, indistinguishable from their wild-type littermates. These data provide definitive evidence that MCA/129 fibrosarcomas and B16F1 melanomas do not elicit a host immune response in wild-type mice and that the asmase−/− phenotype is not deficient in antitumor immunity, supporting the notion that the patterns of tumors growth and radiation response are conditionally linked to the ability of the tumor endothelium to undergo ASMase-mediated apoptosis.
Identification and Characterization of a Novel and Specific Inhibitor of the Ataxia-Telangiectasia Mutated Kinase ATM
Authors: Hickson Ian , Zhao Yan , Richardson Caroline J. , Green Sharon J. , Martin Niall M. B. , Orr Alisdair I. , Reaper Philip M. , Jackson Stephen P. , Curtin Nicola J. , Smith Graeme C. M.
Published: September 2005

The serine/threonine protein kinase ATM signals to cell cycle and DNA repair components by phosphorylating downstream targets such as p53, CHK2, NBS1, and BRCA1. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia, which is characterized by hypersensitivity to ionizing radiation and a failure of cells to arrest the cell cycle after the induction of DNA double-strand breaks. It has thus been proposed that ATM inhibition would cause cellular radio- and chemosensitization. Through screening a small molecule compound library developed for the phosphatidylinositol 3′-kinase–like kinase family, we identified an ATP-competitive inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), that inhibits ATM with an IC50 of 13 nmol/L and a Ki of 2.2 nmol/L. KU-55933 shows specificity with respect to inhibition of other phosphatidylinositol 3′-kinase–like kinases. Cellular inhibition of ATM by KU-55933 was demonstrated by the ablation of ionizing radiation-dependent phosphorylation of a range of ATM targets, including p53, γH2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase.
Agents with Selective Estrogen Receptor (ER) Modulator Activity Induce ApoptosisIn vitroandIn vivoin ER-Negative Glioma Cells
Authors: Hui Ai-Min , Zhang Wei , Chen Wei , Xi Dan , Purow Benjamin , Friedman Glenn C. , Fine Howard A.
Published: September 2005

Tamoxifen, a member of the selective estrogen receptor modulator (SERM) family, is widely used in the treatment of estrogen receptor (ER)-expressing breast cancer. It has previously been shown that high-dose tamoxifen has cytotoxic activity against glioma cells, but whether this effect is drug specific or represents a general property of SERMs is unknown. In this study, we demonstrate that tamoxifen and CC-8490, a novel benzopyranone with SERM activity, induce glioma cell apoptosis in a dose- and time-dependent manner. Moreover, administration of tamoxifen and CC-8490 suppresses tumor growth in vivo and extends animal survival in glioma xenograft models. None of the eight glioma cell lines examined express either ER-α or -β, suggesting the mechanism for tamoxifen– and CC-8490–induced glioma cell apoptosis is independent of the ER signaling pathway. Complementary DNA microarray expression profiling allowed us to identify a subset of genes specifically regulated by tamoxifen and CC-8490, and not by other apoptotic stimuli, including nuclear factor (NF)-κB with its target genes IEX-3, SOD2, IL6, and IL8. We demonstrate that suppression of NF-κB activation markedly enhances SERM-induced apoptosis, suggesting a role for NF-κB in protecting glioma cells from SERM-induced cytotoxicity. These findings demonstrate for the first time that a SERM other than tamoxifen can induce glioma cell apoptosis in vitro and in vivo and that the clinical efficacy of SERMs for the treatment of malignant gliomas could potentially be enhanced by simultaneous inhibition of the NF-κB pathway.
Mutations of the
Epidermal Growth Factor Receptor
Gene in Lung Cancer
Authors: Kosaka Takayuki , Yatabe Yasushi , Endoh Hideki , Kuwano Hiroyuki , Takahashi Takashi , Mitsudomi Tetsuya
Published: September 2005

Recently it has been reported that mutations in the tyrosine kinase domain of the epidermal growth factor receptor(EGFR) gene occur in a subset of patients with lung cancer showing a dramatic response to EGFR tyrosine kinase inhibitors. To gain further insights in the role of EGFR in lung carcinogenesis, we sequenced exons 18–21 of the tyrosine kinase domain using total RNA extracted from unselected 277 patients with lung cancer who underwent surgical resection and correlated the results with clinical and pathologic features. EGFR mutations were present in 111 patients (40%). Fifty-two were in-frame deletions around codons 746–750 in exon 19, 54 were point mutations including 49 at codon 858 in exon 21 and 4 at codon 719 in exon 18, and 5 were duplications/insertions mainly in exon 20. They were significantly more frequent in female (P < 0.001), adenocarcinomas (P = 0.0013), and in never-smokers (P < 0.001). Multivariate analysis suggested EGFR mutations were independently associated with adenocarcinoma histology (P = 0.0012) and smoking status (P < 0.001), but not with female gender (P = 0.9917). In adenocarcinomas, EGFR mutations were more frequent in well to moderately differentiated tumors (P < 0.001) but were independent of patient age, disease stages, or patient survival. KRAS and TP53 mutations were present in 13 and 41%, respectively. EGFR mutations never occurred in tumors with KRAS mutations, whereas EGFR mutations were independent of TP53 mutations. EGFR mutations define a distinct subset of pulmonary adenocarcinoma without KRAS mutations, which is not caused by tobacco carcinogens.
Proteasomal Degradation of Topoisomerase I Is Preceded by c-Jun NH2-Terminal Kinase Activation, Fas Up-Regulation, and Poly(ADP-Ribose) Polymerase Cleavage in SN38-Mediated Cytotoxicity against Multiple Myeloma
Authors: Catley Laurence , Tai Yu-Tzu , Shringarpure Reshma , Burger Renate , Son Moni Thi , Podar Klaus , Tassone Pierfrancesco , Chauhan Dharminder , Hideshima Teru , Denis Louis , Richardson Paul , Munshi Nikhil C. , Anderson Kenneth C.
Published: September 2005

Topoisomerase I inhibitors are effective anticancer therapies and have shown activity in hematologic malignancies. Here we show for the first time that SN38, the potent active metabolite of irinotecan, induces c-Jun NH2-terminal kinase activation, Fas up-regulation, and caspase 8-mediated apoptosis in multiple myeloma (MM) cells. Proteasomal degradation of nuclear topoisomerase I has been proposed as a resistance mechanism in solid malignancies. SN38-induced proteasomal degradation of topoisomerase I was observed during SN38-mediated cytotoxicity against MM.1S myeloma cell line but occurred after c-Jun NH2-terminal kinase activation, Fas up-regulation, and poly(ADP-ribose) polymerase cleavage and failed to protect cells from apoptosis. Differential toxicity was observed against MM cells versus bone marrow stromal cells, and SN38 inhibited adhesion-induced up-regulation of MM cell proliferation when MM cells adhere to bone marrow stromal cells. In addition, SN38 directly inhibited constitutive and inducible interleukin 6 and vascular endothelial growth factor secretion by bone marrow stromal cells. Synergy was observed when SN38 was used in combination with doxorubicin, bortezomib, as well as poly(ADP-ribose) polymerase inhibitor NU1025 and Fas-activator CH11. These findings have clinical significance, because identification of downstream apoptotic signaling after topoisomerase I inhibition will both elucidate mechanisms of resistance and optimize future combination chemotherapy against MM.
Myh
Deficiency Enhances Intestinal Tumorigenesis in Multiple Intestinal Neoplasia (
Apc

Min
/+) Mice
Authors: Sieber Oliver M. , Howarth Kimberley M. , Thirlwell Christina , Rowan Andrew , Mandir Nikki , Goodlad Robert A. , Gilkar Ashfaq , Spencer-Dene Bradley , Stamp Gordon , Johnson Victoria , Silver Andrew , Yang Hanjing , Miller Jeffrey H. , Ilyas Mohammad , Tomlinson Ian P. M.
Published: September 2005

Monoallelic APC and biallelic MYH (homolog of Escherichia coli mutY) germ-line mutations are independently associated with a strong predisposition to colorectal adenomas and carcinoma in humans. Whereas mice heterozygous for mutant Apc develop intestinal tumors, mice homozygous for mutant Myh do not show increased tumor susceptibility. We analyzed the phenotype of ApcMin/+/Myh−/− mice and found that they developed significantly more adenomas in the small intestine than did ApcMin/+/Myh+/+ or ApcMin/+/Myh+/− mice (median 231 versus 151 versus 152). In the large bowel, ApcMin/+/Myh−/− mice showed significant increases in the number of aberrant crypt foci. In addition, ApcMin/+/Myh−/− mice developed an increased number of mammary tumors. Molecular analyses suggested that at least 19% of intestinal tumors from ApcMin/+/Myh−/− mice had acquired intragenic Apc mutations rather than allelic loss. Consistent with a defect in base excision repair, three intragenic Apc mutations in polyps without allelic loss from ApcMin/+/Myh−/− mice were shown to be G:C to T:A transversions which resulted in termination codons; no such mutations were found in polyps from ApcMin/+/Myh+/+ or ApcMin/+/Myh+/− mice. Tumors from ApcMin/+/Myh+/− mice harbored neither somatic mutations nor allelic loss at Myh. Thus, homozygous, but not heterozygous, Myh deficiency enhanced intestinal tumorigenesis in ApcMin/+ mice. The excess small-bowel adenomas in ApcMin/+/Myh−/− mice, therefore, appear to be a model of MYH-associated polyposis in humans.