bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Novel and Widely Expressed Cancer/Testis Gene Isoforms That Elicit Spontaneous Cytotoxic T-Lymphocyte Reactivity to Melanoma
Authors: Hogan Kevin T. , Coppola Michael A. , Gatlin Christine L. , Thompson Lee W. , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H. , Ross Mark M. , Slingluff Craig L.
Published: September 2005

Multiple isoforms (TAG-1, TAG-2a, TAG-2b, and TAG-2c) of a novel cancer/testis antigen gene have been identified and are expressed in 84–88% of melanoma cell lines tested. The tumor antigen (TAG) genes are also expressed in K562, a myelogenous leukemia cell line, and they have homology to two chronic myelogenous leukemia-derived clones and a hepatocellular carcinoma clone in the human expressed sequence tags (EST) database, thus indicating that their expression is not restricted to melanomas. In contrast to the fact that many cancer/testis antigens are poorly immunogenic, the TAG-derived peptide, RLSNRLLLR, is recognized by HLA-A3-restricted, melanoma-specific CTLs that were obtained from a melanoma patient with spontaneous reactivity to the peptide. Unlike most cancer/testis antigen genes which are located on the X chromosome, the TAG genes are located on chromosome 5. The genes have the additional unusual features of being coded for in an open reading frame that is initiated by one of three nonstandard initiation codons, and the sequence coding the RLSNRLLLR peptide crosses an exon-exon boundary. The properties of the TAG antigens indicate that they are excellent vaccine candidates for the treatment of melanoma and perhaps other cancers.
Heat Shock Protein 90 Regulates the Metaphase-Anaphase Transition in a Polo-Like Kinase-Dependent Manner
Authors: de Cárcer Guillermo
Published: September 2005

We have shown previously that the molecular chaperone heat shock protein 90 (Hsp90) is required for a proper centrosome function. Indeed, this Hsp90 function seems to be reflected in Polo-like kinase stability. Inhibition of Hsp90 in HeLa cells results in cell cycle arrest either in G2 stage or at the metaphase-anaphase transition. Here, we show that this inhibition leads to inactivation of the anaphase-promoting complex or cyclosome by both dephosphorylation and induction of the spindle assembly checkpoint. Hsp90 inhibition compromises two of the main mitotic kinases, Polo-like kinase 1 (Plk1) and cdc2. Interestingly, this mitotic arrest does not occur in certain tumor cell lines where Hsp90 and Plk1 are not associated. Those cells are able to process mitosis successfully and have an active Plk1 despite Hsp90 inactivation. Therefore, it seems that Hsp90 regulates completion of mitosis depending on its association with Plk1.
Oncogenic Ras Sensitizes Normal Human Cells to Tumor Necrosis Factor-α-Related Apoptosis-Inducing Ligand-Induced Apoptosis
Authors: Nesterov Alexandre , Nikrad Malti , Johnson Thomas , Kraft Andrew S.
Published: September 2005

Tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces apoptosis in tumor cells but rarely kills normal ones. To determine how normal human cells acquire TRAIL-sensitive phenotype during the process of malignant transformation, we used an experimental system that allows for controlled conversion of human cells from normal to cancerous by introduction of several genes. Human embryonic kidney cells and foreskin fibroblasts were first immortalized by combination of the early region of simian virus 40 and telomerase and then were transformed with oncogenic Ras. Both normal and immortalized cells were resistant to TRAIL-induced apoptosis, whereas Ras-transformed cells were susceptible. Ras transformation enhanced TRAIL-induced activation of caspase 8 by increasing its recruitment to TRAIL receptors. The proapoptotic effects of Ras could be reversed by mutations in its effector loop or by inhibitors of either farnesyl transferase or mitogen-activated protein kinase kinase. The expression of constitutively activated mitogen-activated protein kinase kinase 1 enhanced caspase 8 recruitment and sensitized immortalized human embryonic kidney cells to TRAIL-induced death. These results indicate that in normal human cells the TRAIL-induced apoptotic signal is blocked at the level of caspase 8 recruitment and that this block can be eliminated by Ras transformation, involving activation of the mitogen-activated protein kinase pathway.
Akt and Hypoxia-Inducible Factor-1 Independently Enhance Tumor Growth and Angiogenesis
Authors: Arsham Andrew M. , Plas David R. , Thompson Craig B. , Simon M. Celeste
Published: September 2005

Recent reports have suggested that phosphatidylinositol 3-kinase/Akt signaling can induce angiogenesis and tumor growth by activating the hypoxia-inducible factor-1 (HIF-1). However, the absence of specific biochemical inhibitors of HIF-1 signaling has prevented a direct test of the requirement for HIF-1 activity in Akt-dependent tumorigenesis. To genetically test the relationship between HIF-1 and Akt, activated Akt was expressed in a hepatoma cell line lacking HIF-1. Akt expression was associated with a dramatic increase in tumor size, despite the absence of HIF-1. Tumor size was not further increased in cells with reconstituted HIF-1 activity, indicating that the effects of Akt on tumorigenesis were not limited by the absence of HIF-1. Increased tumor size in Akt-expressing, HIF-deficient cells was associated with vascular endothelial growth factor secretion and tumor vascularization. In addition to vascular endothelial growth factor production, Akt also conferred a cell-autonomous competitive advantage to tumor cells in an in vivo competition experiment. Thus, Akt has potent, HIF-1-independent oncogenic and angiogenic activities.
Molecular Determinants of the Cytotoxicity of Platinum Compounds
Authors: Vekris Antoine , Meynard Delphine , Haaz Marie-Christine , Bayssas Martine , Bonnet Jacques , Robert Jacques
Published: September 2005

Gene expression profiling of tumors allows the establishment of relationships between gene expression profiles and sensitivity to anticancer drugs. In an attempt to study the molecular determinants of the activity of platinum compounds, we explored the publicly available databases of the National Cancer Institute (NCI; http://dtp.nci.nih.gov), which allow access to the gene expression profiles of the 60 cell lines for which drug cytotoxicity patterns already existed. Using this database, we have conducted an in silico research to identify the genes the expression of which was positively or negatively correlated to the sensitivity to four platinum compounds (cisplatin, carboplatin, oxaliplatin and tetraplatin). Important similarities were noticed between cisplatin and carboplatin on one hand, and tetraplatin and oxaliplatin on the other hand. In the restricted panel of 1416 genes and molecular markers, we identified 204 markers, among which 120 corresponded to identified genes, that significantly correlated (P < 0.001) with the cytotoxicity of at least one platinum compound. For example, the functionality of the p53-activated pathway appeared positively correlated with the cytotoxicity of all platinum compounds. More specific are the positive correlations between RAS gene mutations and MYC expression and the cellular sensitivity to oxaliplatin. Among the parameters already known as related to the sensitivity to platinum compounds, we identified, in the complete set of 9400 genes, numerous significant relationships, such as the negative correlations between ERB-B2 and BCL-XL expressions and the cytotoxicity of the platinum compounds. Public databases mining, therefore, appears to be a valuable tool for the identification of determinants of anticancer drug activity in tumors.
Role of p12CDK2-AP1 in Transforming Growth Factor-β1-Mediated Growth Suppression
Authors: Hu Miaofen G. , Hu Guo-Fu , Kim Yong , Tsuji Takanori , McBride Jim , Hinds Philip , Wong David T. W.
Published: September 2005

p12CDK2-AP1 (p12) is a growth suppressor isolated from normal keratinocytes. Ectopic expression of p12 in squamous carcinoma cells reversed the malignant phenotype of these cells, in part due an ability of p12 to bind to both DNA polymerase α/primase and to cyclin-dependent kinase 2 (CDK2), thereby inhibiting their activities. We report in this article that in normal epithelial cells, transforming growth factor β1 (TGF-β1) induces p12 expression transcriptionally, which, in turn, mediates the growth inhibitory activity of TGF-β1. We created inducible p12 antisense HaCaT cell lines [ip12 (−) HaCaT] and showed that selective reduction of cellular p12 resulted in an increase in: (a) CDK2-associated kinase activity; (b) protein retinoblastoma (pRB) phosphorylation; and (c) [3H]thymidine incorporation, and partially reversed TGF-β1-mediated inhibition of CDK2 kinase activity, pRB phosphorylation, and cell proliferation. Furthermore, we generated p12-deficient mouse oral keratinocytes (MOKp12−/−) and compared their growth characteristics and response to TGF-β1 with that of wild-type mouse oral keratinocytes (MOKWT). Under normal culture conditions, the number of MOKp12−/− in S phase is 2-fold greater than that of MOKWT. Concomitantly, fewer cells are in G2 phase in MOKp12−/− than that in MOKWT. Moreover, response to TGF-β1-mediated growth suppression is compromised in MOKp12−/− cells. Mechanistic studies showed that MOKp12−/− have increased CDK2 activity and reduced sensitivity to inhibition by TGF-β1. Collectively our data suggest that p12 plays a role in TGF-β1-mediated growth suppression by modulating CDK2 activities and pRB phosphorylation.
A Link between Benzyl Isothiocyanate-Induced Cell Cycle Arrest and Apoptosis: Involvement of Mitogen-Activated Protein Kinases in the Bcl-2 Phosphorylation
Authors: Miyoshi Noriyuki , Uchida Koji , Osawa Toshihiko , Nakamura Yoshimasa
Published: September 2005

In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G2-M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G2-M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G2-M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G2-M-arrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G2-M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.
Expression of Complement Factor H by Lung Cancer Cells
Authors: Ajona Daniel , Castaño Zafira , Garayoa Mercedes , Zudaire Enrique , Pajares Maria J. , Martinez Alfredo , Cuttitta Frank , Montuenga Luis M. , Pio Ruben
Published: September 2005

The complement system is important in immunosurveillance against tumors. However, malignant cells are usually resistant to complement-mediated lysis. In this study, we examine the expression of factor H, an inhibitor of complement activation, and factor H-like protein 1 (FHL-1), its alternatively spliced form, in lung cancer. We also evaluate the potential effect of factor H/FHL-1 in the protection of lung cancer cells against the activation of the complement cascade. By Northern blot analysis we demonstrate a high expression of factor H and FHL-1 in most non-small cell lung cancer cell lines, although neuroendocrine pulmonary tumors (small cell lung carcinoma and carcinoid cell lines) had undetectable levels. Western blot analysis of conditioned medium showed the active secretion of factor H and FHL-1 by cells that were positive by Northern blot. Expression of factor H/FHL-1 mRNA was also shown in a series of non-small cell lung cancer biopsies by in situ hybridization. Interestingly, many cultured lung cancer cells were able to bind fluorescence-labeled factor H to their surfaces. Deposition of C3 fragments from normal human serum on H1264, a lung adenocarcinoma cell line, was more efficient when factor H/FHL-1 activity was blocked by specific antibodies. Blocking factor H/FHL-1 activity also enhanced the release of anaphylatoxin C5a and moderately increased the susceptibility of these cells to complement-mediated cytotoxicity. In summary, we demonstrate the expression of factor H and FHL-1 by some lung cancer cells and analyze the contribution of these proteins to the protection against complement activation.
Endothelin B Receptor Blockade Inhibits Dynamics of Cell Interactions and Communications in Melanoma Cell Progression
Authors: Bagnato Anna , Rosanò Laura , Spinella Francesca , Di Castro Valeriana , Tecce Raffaele , Natali Pier Giorgio
Published: September 2005

Phenotypic and genotypic analyses of cutaneous melanoma have identified the endothelin B receptor (ETBR) as tumor progression marker, thus representing a potential therapeutic target. Here, we demonstrate that activation of ETBR by endothelin-1 (ET-1) and ET-3 leads to loss of expression of the cell adhesion molecule E-cadherin and associated catenin proteins and gain of N-cadherin expression. Exposure of melanoma cells to ET-1 leads to a 60% inhibition in intercellular communication by inducing phosphorylation of gap junctional protein connexin 43. Additionally, activation of the ETBR pathway increases αvβ3 and α2β1 integrin expression and matrix metalloproteinase (MMP)-2 and MMP-9, membrane type-1-MMP activation, and tissue inhibitor MMP-2 secretion. The ETBR pathway results into the downstream activation of focal adhesion kinase and extracellular signal-regulated kinase 1/2 signaling pathways, which lead to enhanced cell proliferation, adhesion, migration, and MMP-dependent invasion. The small molecule A-192621, an orally bioavailable nonpeptide ETBR antagonist, significantly inhibits melanoma growth in nude mice. These findings demonstrate that ET-1 and ET-3 through ETBR activation trigger signaling pathways involved in events associated with disruption of normal host-tumor interactions and progression of cutaneous melanoma. Pharmacological interruption of ETBR signaling may represent a novel therapeutic strategy in the treatment of this malignancy.
Tissue-Wide Expression Profiling Using cDNA Subtraction and Microarrays to Identify Tumor-Specific Genes
Authors: Amatschek Stefan , Koenig Ulrich , Auer Herbert , Steinlein Peter , Pacher Margit , Gruenfelder Agnes , Dekan Gerhard , Vogl Sonja , Kubista Ernst , Heider Karl-Heinz , Stratowa Christian , Schreiber Martin , Sommergruber Wolfgang
Published: September 2005

With the objective of discovering novel putative intervention sites for anticancer therapy, we compared transcriptional profiles of breast cancer, lung squamous cell cancer (LSCC), lung adenocarcinoma (LAC), and renal cell cancer (RCC). Each of these tumor types still needs improvement in medical treatment. Our intention was to search for genes not only highly expressed in the majority of patient samples but which also exhibit very low or even absence of expression in a comprehensive panel of 16 critical (vital) normal tissues. To achieve this goal, we combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarrays. Seven subtractive libraries consisting of ∼9250 clones were established and enriched for tumor-specific transcripts. These clones, together with ∼1750 additional tumor-relevant genes, were used for cDNA microarray preparation. Hybridizations were performed using a pool of 16 critical normal tissues as a reference in all experiments. In total, we analyzed 20 samples of breast cancer, 11 of LSCC, 11 of LAC, and 8 of RCC. To select for genes with low or even no expression in normal tissues, expression profiles of 22 different normal tissues were additionally analyzed. Importantly, this tissue-wide expression profiling allowed us to eliminate genes, which exhibit also high expression in normal tissues. Similarly, expression signatures of genes, which are derived from infiltrating cells of the immune system, were eliminated as well. Cluster analysis resulted in the identification of 527 expressed sequence tags specifically up-regulated in these tumors. Gene-wise hierarchical clustering of these clones clearly separated the different tumor types with RCC exhibiting the most homogenous and LAC the most diverse expression profile. In addition to already known tumor-associated genes, the majority of identified genes have not yet been brought into context with tumorigenesis such as genes involved in bone matrix mineralization (OSN, OPN, and OSF-2) in lung, breast, and kidney cancer or genes controlling Ca2+ homeostasis (RCN1,CALCA, S100 protein family). EGLN3, which recently has been shown to be involved in regulation of hypoxia-inducible factor, was found to be highly up-regulated in all RCCs and in half of the LSCCs analyzed. Furthermore, 42 genes, the expression level of which correlated with the overall survival of breast cancer patients, were identified. The gene dendogram clearly separates two groups of genes, those up-regulated such as cyclin B1, TGF-β3, B-Myb, Erg2, VCAM-1, and CD44 and those down-regulated such as MIG-6, Esp15, and CAK in patients with short survival time.
A Double-Strand Break Repair Defect in ATM-Deficient Cells Contributes to Radiosensitivity
Authors: Kühne Martin , Riballo Enriqueta , Rief Nicole , Rothkamm Kai , Jeggo Penny A. , Löbrich Markus
Published: September 2005

The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM’s role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of γ-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 h after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells, in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.
Hypermethylation of the 5′ CpG Island of the
FHIT
Gene Is Associated with Hyperdiploid and Translocation-Negative Subtypes of Pediatric Leukemia
Authors: Zheng Shichun , Ma Xiaomei , Zhang Luoping , Gunn Laura , Smith Martyn T. , Wiemels Joseph L. , Leung Kenneth , Buffler Patricia A. , Wiencke John K.
Published: September 2005

The human FHIT (fragile histidine triad) gene is a putative tumor suppressor gene located at chromosome region 3p14.2. Previous studies have shown that loss of heterozygosity, homozygous deletions, and abnormal expression of the FHIT gene are involved in several types of human malignancies. A CpG island is present in the 5′ promoter region of the FHIT gene, and methylation in this region correlates with loss of FHIT expression. To test whether aberrant methylation of the FHIT gene may play a role in pediatric leukemia, we assessed the FHIT methylation status of 10 leukemia cell lines and 190 incident population-based cases of childhood acute lymphocytic and myeloid leukemias using methylation-specific PCR. Conventional and fluorescence in situ hybridization cytogenetic data were also collected to examine aneuploidy, t(12, 21), and other chromosomal rearrangements. Four of 10 leukemia cell lines (40%) and 52 of 190 (27.4%) bone marrows from childhood leukemia patients demonstrated hypermethylation of the promoter region of FHIT. Gene expression analyses and 5-aza-2′-deoxycytidine treatment showed that promoter hypermethylation correlated with FHIT inactivation. Among primary leukemias, hypermethylation of FHIT was strongly correlated with acute lymphoblastic leukemia (ALL) histology (P = 0.008), high hyperdiploid (P < 0.0001), and translocation-negative (P < 0.0001) categories. Hyperdiploid B-cell ALLs were 23-fold more likely to be FHIT methylated compared with B-cell ALL harboring TEL-AML translocations. FHIT methylation was associated with high WBC counts at diagnosis, a known prognostic indicator. These results suggest that hypermethylation of the promoter region CpG island of the FHIT gene is a common event and may play an important role in the etiology and pathophysiology of specific cytogenetic subtypes of childhood ALL.
Human T-Cell Lymphotropic Virus Type I-Infected Cells Extravasate through the Endothelial Barrier by a Local Angiogenesis-Like Mechanism
Authors: Bazarbachi Ali , Merhi Raghida Abou , Gessain Antoine , Talhouk Rabih , El-Khoury Hilda , Nasr Rihab , Gout Olivier , Sulahian Rita , Homaidan Fadia , de Thé Hugues , Hermine Olivier , El-Sabban Marwan E.
Published: September 2005

Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis. Human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATL) is an aggressive disease characterized by visceral invasion. We show that ATL and HTLV-I-associated myelopathy patients exhibit high plasma levels of functional vascular endothelial growth factor and basic fibroblast growth factor. The viral oncoprotein Tax transactivates the promoter of the gap-junction protein connexin-43 and enhances gap-junction-mediated heterocellular communication with endothelial cells. The interaction of HTLV-I-transformed cells with endothelial cells induces the gelatinase activity of matrix metalloproteinase (MMP)-2 and MMP-9 in endothelial cells and down-regulates the tissue inhibitor of MMP. This leads to subendothelial basement membrane degradation followed by endothelial cell retraction, allowing neoplastic lymphocyte extravasation. We propose a model that offers a mechanistic explanation for extravasation of HTLV-I-infected cells: after specific adhesion to endothelia of target organs, tumor cells induce a local and transient angiogenesis-like mechanism through paracrine stimulation and direct cell-cell communication with endothelial cells. This culminates in a breach of the endothelial barrier function, allowing cancer cell invasion. This local and transient angiogenesis-like sequence that may facilitate visceral invasion in ATL represents a potential target for ATL therapy.
Helicobacter pyloriInduces Plasminogen Activator Inhibitor 2 in Gastric Epithelial Cells through Nuclear Factor-κB and RhoA
Authors: Varro Andrea , Noble P-J. M. , Pritchard D. Mark , Kennedy Susan , Hart C. Anthony , Dimaline Rod , Dockray Graham J.
Published: September 2005

The gastric pathogen Helicobacter pylori is associated with a progression to gastric cancer. The specific targets of H. pylori that might influence this progression are still unclear. Previous studies indicated that the gastric hormone gastrin, which may be increased in H. pylori infection, stimulates gastric expression of plasminogen activator inhibitor (PAI)-2, which is an inhibitor of the urokinase plasminogen activator and has previously been shown to be increased in gastric adenocarcinoma. Here, we report that H. pylori also increases PAI-2 expression. In gastric biopsies of H. pylori-positive subjects there was increased PAI-2, including subjects with plasma gastrin concentrations in the normal range. PAI-2 was expressed mainly in chief and mucous cells. In a gastric cancer cell line (AGS), H. pylori increased PAI-2 expression, which was associated with inhibition of H. pylori-stimulated cell invasion and apoptosis. The induction of PAI-2 by H. pylori was mediated by release of interleukin-8 and activation of cyclooxygenase-2, and interestingly, gastrin stimulated PAI-2 expression by similar paracrine pathways. The activation of NFκB was required for interleukin-8 and cyclooxygenase-2 activation but did not occur in cells responding to these paracrine mediators. The data suggest that induction of PAI-2 is a specific target in H. pylori infection, mediated at least partly by paracrine factors; induction of PAI-2 inhibits cell invasion and apoptosis and is a candidate for influencing the progression to gastric cancer.
Resveratrol-induced Autophagocytosis in Ovarian Cancer Cells
Authors: Opipari Anthony W. , Tan Lijun , Boitano Anthony E. , Sorenson Dorothy R. , Aurora Anjili , Liu J. Rebecca
Published: September 2005

Resveratrol (3,5,4-trihydroxystilbene), a natural phytoalexin present in grapes, nuts, and red wine, has antineoplastic activities. Several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo. In the present study, the response of ovarian cancer cells to resveratrol is explored. Resveratrol inhibited growth and induced death in a panel of five human ovarian carcinoma cell lines. The response was associated with mitochondrial release of cytochrome c, formation of the apoptosome complex, and caspase activation. Surprisingly, even with these molecular features of apoptosis, analysis of resveratrol-treated cells by light and electron microscopy revealed morphology and ultrastructural changes indicative of autophagocytic, rather than apoptotic, death. This suggests that resveratrol can induce cell death through two distinct pathways. Consistent with resveratrol’s ability to kill cells via nonapoptotic processes, cells transfected to express high levels of the antiapoptotic proteins Bcl-xL and Bcl-2 are equally sensitive as control cells to resveratrol. Together, these findings show that resveratrol induces cell death in ovarian cancer cells through a mechanism distinct from apoptosis, therefore suggesting that it may provide leverage to treat ovarian cancer that is chemoresistant on the basis of ineffective apoptosis.
Overexpression of a Dominant-Negative Mutant Ubc9 Is Associated with Increased Sensitivity to Anticancer Drugs
Authors: Mo Yin-Yuan , Yu Yanni , Ee P. L. Rachel , Beck William T.
Published: September 2005

Ubc9 is an E2-conjugating enzyme required for sumoylation and has been implicated in regulating several critical cellular pathways. We have shown previously that Ubc9 is important for sumoylation and nucleolar delocalization of topoisomerase (topo) I in response to topo I inhibitors such as topotecan. However, the role for Ubc9 in tumor drug responsiveness is not clear. In this study, we found that although MCF7 cells expressing a Ubc9 dominant-negative mutant (Ubc9-DN) display decreased activity of topo I, these cells are more sensitive to the topo I inhibitor topotecan and other anticancer agents such as VM-26 and cisplatin. In addition, we found that alteration of Ubc9 expression correlates with drug responsiveness in tumor cell lines. To understand possible mechanisms of Ubc9-associated drug responsiveness, we examined several proteins that have been shown to interact with Ubc9 and that may be involved in drug responsiveness. One such protein is Daxx, which is a Fas-associated protein that plays a role in Fas-mediated apoptosis by participating in a caspase-independent pathway through activation of apoptosis signal-regulating kinase 1 and c-Jun NH2-terminal kinase. We found that cells expressing Ubc9-DN accumulate more cytoplasmic Daxx than the control cells. Because cytoplasmic Daxx is believed to participate in cellular apoptosis, we suggest that the interaction of Ubc9 with Daxx and subsequent alteration in the subcellular localization of Daxx may contribute to the increased sensitivity to anticancer drugs in the cells expressing Ubc9-DN. Finally, we found that overexpression of Daxx sensitizes cells to anticancer drugs possibly in part through alterations of the ratio of cytoplasmic and nuclear Daxx. Together, our results suggest a role for Ubc9 in tumor drug responsiveness.
Resveratrol Inhibits Drug-Induced Apoptosis in Human Leukemia Cells by Creating an Intracellular Milieu Nonpermissive for Death Execution
Authors: Ahmad Kashif Adil , Clement Marie-Veronique , Hanif Ismail Muhamad , Pervaiz Shazib
Published: September 2005

Efficient apoptotic signaling is a function of a permissive intracellular milieu created by a decrease in the ratio of superoxide to hydrogen peroxide and cytosolic acidification. Resveratrol (RSV) triggers apoptosis in some systems and inhibits the death signal in others. In this regard, the inhibitory effect on hydrogen peroxide-induced apoptosis is attributed to its antioxidant property. We provide evidence that exposure of human leukemia cells to low concentrations of RSV (4–8 μm) inhibits caspase activation, DNA fragmentation, and translocation of cytochrome c induced by hydrogen peroxide or anticancer drugs C2, vincristine, and daunorubicin. Interestingly, at these concentrations, RSV induces an increase in intracellular superoxide and inhibits drug-induced acidification. Blocking the activation of NADPH oxidase complex neutralized RSV-induced inhibition of apoptosis. Furthermore, our results implicate intracellular hydrogen peroxide as a common effector mechanism in drug-induced apoptosis that is inhibited by preincubation with RSV. Interestingly, decreasing intracellular superoxide with the NADPH oxidase inhibitor diphenyliodonium reversed the inhibitory effect of RSV on drug-induced hydrogen peroxide production. These data show that low concentrations of RSV inhibit death signaling in human leukemia cells via NADPH oxidase-dependent elevation of intracellular superoxide that blocks mitochondrial hydrogen peroxide production, thereby resulting in an intracellular environment nonconducive for death execution.
LynIs a Target Gene for Prostate Cancer
Authors: Goldenberg-Furmanov Mirela , Stein Ilan , Pikarsky Eli , Rubin Hila , Kasem Shafika , Wygoda Marc , Weinstein Irina , Reuveni Hadas , Ben-Sasson Shmuel A.
Published: September 2005

The Src-related protein kinase Lyn plays an important role in B-cell activation. However, several lines of evidence suggest that it is also involved in the control of cellular proliferation and the inhibition of apoptosis. We have discovered that Lyn is expressed in normal prostate epithelia, in 95% of primary human prostate cancer (PC) specimens examined, and in all of the PC cell lines that we assayed. Moreover, Lyn knockout mice display abnormal prostate gland morphogenesis, which suggests that Lyn plays an important role in prostate epithelium development and implies that Lyn is a candidate target for specific therapy for PC. Using a drug-design strategy to construct sequence-based peptide inhibitors, a Lyn-specific inhibitor, KRX-123, targeting a unique interaction site within Lyn, was synthesized. KRX-123 was found to inhibit cellular proliferation in three hormone-refractory PC cell lines, DU145, PC3, and TSU-Pr1 with IC50 values of 2–4 μm. In vivo, tumor volume of DU145 explants in nude mice was significantly reduced after once-a-week injections of KRX-123, at a dose of 10 mg/kg, for a period of 5 weeks. Histological analyses of the treated tumors indicated extensive apoptosis. Thus, we suggest that Lyn inhibition may serve as a prime target for the treatment of hormone-refractory PC.
In Vitro
Studies with Methylproamine
Authors: Martin Roger F. , Broadhurst Sam , Reum Monica E. , Squire Christopher J. , Clark George R. , Lobachevsky Pavel N. , White Jonathan M. , Clark Chris , Sy Denise , Spotheim-Maurizot Melanie , Kelly David P.
Published: September 2005

New analogues of the minor groove binding ligand Hoechst 33342 have been investigated in an attempt to improve radioprotective activity. The synthesis, DNA binding, and in vitro radioprotective properties of methylproamine, the most potent derivative, are reported. Experiments with V79 cells have shown that methylproamine is ∼100-fold more potent than the classical aminothiol radioprotector WR1065. The crystal structures of methylproamine and proamine complexes with the dodecamer d(CGCGAATTCGCG)2 confirm that the new analogues also are minor groove binders. It is proposed that the DNA-bound methylproamine ligand acts as a reducing agent by an electron transfer mechanism, repairing transient radiation-induced oxidizing species on DNA.
Antibodies Directed against Lewis-Y Antigen Inhibit Signaling of Lewis-Y Modified ErbB Receptors
Authors: Klinger Markus , Farhan Hesso , Just Herwig , Drobny Helmut , Himmler Gottfried , Loibner Hans , Mudde Geert C. , Freissmuth Michael , Sexl Veronika
Published: September 2005

The majority of cancer cells derived from epithelial tissue express Lewis-Y (LeY) type difucosylated oligosaccharides on their plasma membrane. This results in the modification of cell surface receptors by the LeY antigen. We used the epidermal growth factor (EGF) receptor family members ErbB1 and ErbB2 as model systems to investigate whether the sugar moiety can be exploited to block signaling by growth factor receptors in human tumor cells (i.e., SKBR-3 and A431, derived from a breast cancer and a vulval carcinoma, respectively). The monoclonal anti-LeY antibody ABL364 and its humanized version IGN311 immunoprecipitated ErbB1 and ErbB2 from detergent lysates of A431 and SKBR-3, respectively. ABL364 and IGN311 blocked EGF- and heregulin-stimulated phosphorylation of mitogen-activated protein kinase [MAPK = extracellular signal-regulated kinase 1/2] in SKBR-3 and A431 cells. The effect was comparable in magnitude with that of trastuzumab (Herceptin) and apparently noncompetitive with respect to EGF. Stimulation of MAPK by ErbB was dynamin dependent and contingent on receptor internalization. ABL364 and IGN311 changed the intracellular localization of fluorescent EGF-containing endosomes and accelerated recycling of intracellular [125I]EGF to the plasma membrane. Taken together, these observations show that antibodies directed against carbohydrate side chains of ErbB receptors are capable of inhibiting ErbB-mediated signaling. The ability of these antibodies to reroute receptor trafficking provides a mechanistic explanation for their inhibitory action.
Transcriptional Regulation of the Melanoma Prognostic Marker Melastatin (TRPM1) by MITF in Melanocytes and Melanoma
Authors: Miller Arlo J. , Du Jinyan , Rowan Sheldon , Hershey Christine L. , Widlund Hans R. , Fisher David E.
Published: September 2005

Determining the metastatic potential of intermediate thickness lesions remains a major challenge in the management of melanoma. Clinical studies have demonstrated that expression of melastatin/TRPM1 strongly predicts nonmetastatic propensity and correlates with improved outcome, leading to a national cooperative prospective study, which is ongoing currently. Similarly, the melanocytic markers MLANA/MART1 and MITF also have been shown to lose relative expression during melanoma progression. Recent studies have revealed that MITF, an essential transcription factor for melanocyte development, directly regulates expression of MLANA. This prompted examination of whether MITF also might transcriptionally regulate TRPM1 expression. The TRPM1 promoter contains multiple MITF consensus binding elements that were seen by chromatin immunoprecipitation to be occupied by endogenous MITF within melanoma cells. Endogenous TRPM1 expression responded strongly to MITF up- or down-regulation, as did TRPM1 promoter-driven reporters. In addition, MITF and TRPM1 mRNA levels were correlated tightly across a series of human melanoma cell lines. Mice homozygously mutated in MITF showed a dramatic decrease in TRPM1 expression. Finally, the slope of TRPM1 induction by MITF was particularly steep compared with other MITF target genes, suggesting it is a sensitive indicator of MITF expression and correspondingly of melanocytic differentiation. These studies identify MITF as a major transcriptional regulator of TRPM1 and suggest that its prognostic value may be linked to MITF-mediated regulation of cellular differentiation.
TIMP-1 Alters Susceptibility to Carcinogenesis
Authors: Rhee Jin-Sae , Diaz Robert , Korets Lidiya , Hodgson J. Graeme , Coussens Lisa M.
Published: September 2005

Tissue inhibitors of metalloproteinases (TIMPs) are a family of multifunctional proteins known to possess a broad range of biological activities, including inhibition of metalloproteinase activity, regulation of proliferation and apoptosis of a variety of cell types, and, depending on the context, differential regulation of angiogenic and inflammatory responses. Elevated mRNA expression of TIMP family members correlates with malignancy and clinical outcome in many human cancer types; however, a protective role for TIMPs also has been observed in various mouse models of human cancer. In the current study, we found distinct spatial-temporal expression patterns for the mRNA of TIMP family members in a mouse model of epithelial carcinogenesis [i.e., keratin 14-human papillomavirus 16 (K14-HPV16) transgenic mice]. To test the hypothesis that elevated expression of TIMP-1 functionally regulates epithelial carcinogenesis, we introduced a human TIMP-1 transgene into K14-HPV16 transgenic mice and assessed neoplastic progression. Results from these studies suggest that TIMP-1 enhances tumorgenicity by potentiating keratinocyte hyperproliferation and appearance of chromosomal aberrations in premalignant cells, thereby increasing their risk to undergo malignant conversion. In addition, TIMP-1 inhibits tissue gelatinolytic activity in tumor stroma, affects stabilization of collagen fibrils, but does not inhibit malignant conversion of dysplasias into carcinomas or development of metastases. The combined implications of these studies suggest that TIMP-1 is an important contributor to epithelial neoplastic progression and supports the concept that TIMP-1 exerts differential regulation on tissues in a stage-dependent manner.
Improved Grading of Breast Adenocarcinomas Based on Genomic Instability
Authors: Kronenwett Ulrike , Huwendiek Sören , Östring Carin , Portwood Neil , Roblick Uwe J. , Pawitan Yudi , Alaiya Ayodele , Sennerstam Roland , Zetterberg Anders , Auer Gert
Published: September 2005

Numerous investigations have shown that in primary breast adenocarcinomas DNA aneuploidy in contrast to DNA diploidy indicates high malignancy potential. On the basis of the study of 104 breast carcinomas, we describe a subtype of aneuploidy, which demonstrates a low degree of malignancy. In image cytometric DNA histograms, this subtype possessed a low percentage (≤8.8%) of nonmodal DNA values as measured by the stemline scatter index (SSI), which is defined as sum of the percentage of cells in the S-phase region, the G2 exceeding rate and the coefficient of variation of the tumor stemline. The cut point of SSI = 8.8% (P = 0.03) enabled us to also subdivide diploid and tetraploid tumors into clinically low and high malignant variants. One possible reason for aneuploidy is impaired distribution of chromosomes at mitosis caused by numerical or structural centrosome aberrations. Cyclins A and E seem to be involved in centrosome duplication. Real-time quantitative PCR measurements of cyclin A and E transcript levels and immunohistochemical determination of cyclin A protein expression showed statistically significantly increased values in the tumors with a high SSI (>8.8%), compared with those with a low SSI. A pilot study demonstrated centrosomal aberrations in an average of 9.6% of the measured cells in four aneuploid carcinomas with high SSI values and in an average of 2.5% of the cells in three aneuploid and three diploid tumors with low SSI. Our data indicate that the SSI, most likely reflecting the degree of genomic instability, allows additional classifying of the known aneuploid, diploid, and tetraploid categories of primary breast adenocarcinomas into low and high malignant subtypes.
The Role of Cytidine Deaminase and GATA1 Mutations in the Increased Cytosine Arabinoside Sensitivity of Down Syndrome Myeloblasts and Leukemia Cell Lines
Authors: Ge Yubin , Jensen Tanya L. , Stout Mark L. , Flatley Robin M. , Grohar Patrick J. , Ravindranath Yaddanapudi , Matherly Larry H. , Taub Jeffrey W.
Published: September 2005

Myeloblasts from Down syndrome (DS) children with acute myeloid leukemia (AML) are significantly more sensitive in vitro to 1-β-d-arabinofuranosylcytosine (ara-C) and generate higher 1-β-d-arabinofuranosylcytosine 5′-triphosphate (ara-CTP) than non-DS AML myeloblasts. Semiquantitative reverse transcription-PCR analyses demonstrated that transcripts for cytidine deaminase (CDA) were 2.7-fold lower in DS than for non-DS myeloblasts. In contrast, transcripts of cystathionine-β-synthase and deoxycytidine kinase were a median 12.5- and 2.6-fold higher in DS compared with non-DS myeloblasts. The ratio of deoxycytidine kinase/CDA transcripts significantly correlated with ara-C sensitivities and ara-CTP generation. In clinically relevant AML cell line models, high cystathionine-β-synthase transcripts in DS CMK cells were accompanied by 10-fold greater ara-C sensitivity and 2.4-fold higher levels of ara-CTP compared with non-DS CMS cells. Overexpression of CDA in non-DS THP-1 cells was associated with a 100-fold decreased ara-C sensitivity and 40-fold decreased ara-CTP generation. THP-1 cells secreted CDA into the incubation media and converted extracellular ara-C completely to 1-β-d-arabinofuranosyluracil within 30 min. Rapid amplification of 5′-cDNA ends (5′-RACE) and reverse transcription-PCR assays identified short- (sf) and long-form (lf) CDA transcripts in THP-1 cells with different 5′ untranslated regions and translational start sites; however, only the latter resulted in the active CDA. Although 5′ flanking sequences for both CDA transcripts exhibited promoter activity in reporter gene assays, activity for the CDAlf was low. The presence of several GATA1 binding sites in the CDAsf promoter and the uniform detection of GATA1 mutations in DS megakaryocytic leukemia suggested the potential role of GATA1 in regulating CDA transcription and the CDAsf promoter acting as an enhancer. Transfection of GATA1 into Drosophila Mel-2 cells stimulated the CDAlf promoter in a dose-dependent fashion. Additional identification of the mechanisms of differential expression of genes encoding enzymes involved in ara-C metabolism between DS and non-DS myeloblasts may lead to improvements in AML therapy.
Basal Cell Carcinoma and Its Development
Authors: Mancuso Mariateresa , Pazzaglia Simonetta , Tanori Mirella , Hahn Heidi , Merola Paola , Rebessi Simonetta , Atkinson Michael J. , Di Majo Vincenzo , Covelli Vincenzo , Saran Anna
Published: September 2005

Loss-of-function mutations in Patched (Ptch1) are implicated in constitutive activation of the Sonic hedgehog pathway in human basal cell carcinomas (BCCs), and inherited Ptch1 mutations underlie basal cell nevus syndrome in which a typical feature is multiple BCC occurring with greater incidence in portals of radiotherapy. Mice in which one copy of Ptch1 is inactivated show increased susceptibility to spontaneous tumor development and hypersensitivity to radiation-induced tumorigenesis, providing an ideal in vivo model to study the typical pathologies associated with basal cell nevus syndrome. We therefore examined BCC development in control and irradiated Ptch1neo67/+ mice. We show that unirradiated mice develop putative BCC precursor lesions, i.e., basaloid hyperproliferation areas arising from both follicular and interfollicular epithelium, and that these lesions progress to nodular and infiltrative BCCs only in irradiated mice. Data of BCC incidence, multiplicity, and latency support the notion of epidermal hyperproliferations, nodular and infiltrative BCC-like tumors representing different stages of tumor development. This is additionally supported by the pattern of p53 protein expression observed in BCC subtypes and by the finding of retention of the normal remaining Ptch1 allele in all nodular, circumscribed BCCs analyzed compared with its constant loss in infiltrative BCCs. Our data suggest chronological tumor progression from basaloid hyperproliferations to nodular and then infiltrative BCC occurring in a stepwise fashion through the accumulation of sequential genetic alterations.
Selective Potentiation of the Hypoxic Cytotoxicity of Tirapazamine by Its 1-
N
-Oxide Metabolite SR 4317
Authors: Siim Bronwyn G. , Pruijn Frederik B. , Sturman Joanna R. , Hogg Alison , Hay Michael P. , Brown J. Martin , Wilson William R.
Published: September 2005

Tirapazamine (TPZ), a bioreductive drug with selective toxicity for hypoxic cells in tumors, is currently in Phase III clinical trials. It has been suggested to have a dual mechanism of action, both generating DNA radicals and oxidizing these radicals to form DNA breaks; whether the second (radical oxidation) step is rate-limiting in cells is not known. In this study we exploit the DNA radical oxidizing ability of the 1-N-oxide metabolite of TPZ, SR 4317, to address this question. SR 4317 at high, but nontoxic, concentrations potentiated the hypoxic (but not aerobic) cytotoxicity of TPZ in all four of the human tumor cell lines tested (HT29, SiHa, FaDu, and A549), thus providing a 2–3-fold increase in the hypoxic cytotoxicity ratio. In potentiating TPZ, SR 4317 was 20-fold more potent than the hypoxic cell radiosensitizers misonidazole and metronidazole but was less potent than misonidazole as a radiosensitizer, suggesting that the initial DNA radicals from TPZ and radiation are different. SR 4317 had favorable pharmacokinetic properties in CD-1 nude mice; coadministration with TPZ provided a large increase in the SR 4317 plasma concentrations relative to that for endogenous SR 4317 from TPZ. It also showed excellent extravascular transport properties in oxic and anoxic HT29 multicellular layers (diffusion coefficient 3 × 10−6 cm2s−1, with no metabolic consumption). Coadministration of SR 4317 (1 mmol/kg) with TPZ at a subtherapeutic dose (0.133 mmol/kg) significantly enhanced hypoxic cell killing in HT29 tumor xenografts without causing oxic cell killing, and the combination at its maximum tolerated dose was less toxic to hypoxic cells in the retina than was TPZ alone at its maximum tolerated dose. This study demonstrates that benzotriazine mono-N-oxides have potential use for improving the therapeutic utility of TPZ as a hypoxic cytotoxin in cancer treatment.