bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Efficacy of Tumor Cell Vaccines Transfected with Secretable hsp70
Authors: Massa Chiara , Guiducci Cristiana , Arioli Ivano , Parenza Mariella , Colombo Mario P. , Melani Cecilia
Published: September 2005

Tumor immunotherapy has exploited the ability of heat shock proteins to chaperone precursors of antigenic peptides to antigen-presenting cells and to activate efficiently an immune response against tumor-associated antigens. The most common strategy is based on the purification of heat shock protein-peptide complexes from tumor cell lines or from tumor surgical samples for in vivo administration. In this article, we have modified the murine-inducible hsp70 into a secreted protein and engineered tumor cells to secrete constitutively their antigenic repertoire associated with the hsp70 protein. In vitro studies showed that the relocalization of hsp70 from the cytoplasm to the secretory pathway did not modify the ability of hsp70 to interact with peptides derived either from natural tumor-associated antigens or model antigens, and that antigen-presenting cells specifically took up the secreted hsp70 and presented the chaperoned epitopes to T cells. In vivo studies showed that tumors secreting hsp70 displayed increased immunogenicity, with induction of a strong and specific CTL response. Mice injected with hsp70-secreting tumors showed increased survival and impaired tumor take compared with mice bearing parental tumors. More than 70% of mice rejected tumor cells secreting hsp70 through mechanisms that involve T lymphocytes and natural killer cells, with the induction of a memory response in the case of T lymphocytes. Moreover, hsp70 secretion increased the immunogenic potential of tumor cell vaccines.
SJG-136 (NSC 694501), a Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity
Authors: Hartley John A. , Spanswick Victoria J. , Brooks Natalie , Clingen Peter H. , McHugh Peter J. , Hochhauser Daniel , Pedley R. Barbara , Kelland Lloyd R. , Alley Michael C. , Schultz Robert , Hollingshead Melinda G. , Schweikart Karen M. , Tomaszewski Joseph E. , Sausville Edward A. , Gregson Stephen J. , Howard Philip W. , Thurston David E
Published: September 2005

SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.
An Msh2 Point Mutation Uncouples DNA Mismatch Repair and Apoptosis
Authors: Lin Diana P. , Wang Yuxun , Scherer Stefan J. , Clark Alan B. , Yang Kan , Avdievich Elena , Jin Bo , Werling Uwe , Parris Tchaiko , Kurihara Naoto , Umar Asad , Kucherlapati Raju , Lipkin Martin , Kunkel Thomas A. , Edelmann Winfried
Published: September 2005

Mutations in the human DNA mismatch repair gene MSH2 are associated with hereditary nonpolyposis colorectal cancer as well as a significant proportion of sporadic colorectal cancer. The inactivation of MSH2 results in the accumulation of somatic mutations in the genome of tumor cells and resistance to the genotoxic effects of a variety of chemotherapeutic agents. Here we show that the DNA repair and DNA damage-induced apoptosis functions of Msh2 can be uncoupled using mice that carry the G674A missense mutation in the conserved ATPase domain. As a consequence, although Msh2G674A homozygous mutant mice are highly tumor prone, the onset of tumorigenesis is delayed as compared with Msh2-null mice. In addition, tumors that carry the mutant allele remain responsive to treatment with a chemotherapeutic agent. Our results indicate that Msh2-mediated apoptosis is an important component of tumor suppression and that certain MSH2 missense mutations can cause mismatch repair deficiency while retaining the signaling functions that confer sensitivity to chemotherapeutic agents.
Electroporated DNA Vaccine Clears Away Multifocal Mammary Carcinomas in Her-2/neuTransgenic Mice
Authors: Quaglino Elena , Iezzi Manuela , Mastini Cristina , Amici Augusto , Pericle Federica , Di Carlo Emma , Pupa Serenella M. , De Giovanni Carla , Spadaro Michela , Curcio Claudia , Lollini Pier Luigi , Musiani Piero , Forni Guido , Cavallo Federica
Published: September 2005

The transforming rat Her-2/neu oncogene embedded into the genome of virgin transgenic BALB/c mice (BALB-neuT) provokes the development of an invasive carcinoma in each of their 10 mammary glands. i.m. vaccination with DNA plasmids coding for the extracellular and transmembrane domains of the protein product of the Her-2/neu oncogene started when mice already display multifocal in situ carcinomas temporarily halts neoplastic progression, but all mice develop a tumor by week 43. By contrast, progressive clearance of neoplastic lesions and complete protection of all 1-year-old mice are achieved when the same plasmids are electroporated at 10-week intervals. Pathological findings, in vitro tests, and the results from the immunization of both IFN-γ and immunoglobulin gene knockout BALB-neuT mice, and of adoptive transfer experiments, all suggest that tumor clearance rests on the combination of antibodies and IFN-γ-releasing T cells. These findings show that an appropriate vaccine effectively inhibits the progression of multifocal preneoplastic lesions.
Overexpression of the Colony-Stimulating Factor (CSF-1) and/or Its Receptor
c-fms
in Mammary Glands of Transgenic Mice Results in Hyperplasia and Tumor Formation
Authors: Kirma Nameer , Luthra Roopa , Jones Jeremy , Liu Ya-Guang , Nair Hareesh B. , Mandava Usha , Tekmal Rajeshwar Rao
Published: September 2005

A number of recent studies have suggested that the colony-stimulating factor (CSF-1) and its receptor c-fms may be involved in the development of mammary glands during lactation and breast cancer. To study the role of CSF-1 or its receptor in initiation of mammary tumorigenesis, we have generated two independent lines of transgenic mice that overexpress either CSF-1 or c-fms under the control of the mouse mammary tumor virus promoter. Mammary glands of the virgin CSF-1 transgenic mice show increased ductal branching, hyperplasia, dysplasia, and other preneoplastic changes, which are indicative of increased cellular proliferation. Similar changes were also evident in the mammary glands of the c-fms transgenic mice. These changes became more prominent with age and resulted in mammary tumor formation. Moreover, secondary events like dimethylbenz(a)anthracene treatment accelerated mammary tumor formation in these mice. Although the expression of estrogen receptor α was not significantly changed in either of the transgenic mouse strains, progesterone receptor levels was higher in both transgenic lines as compared with the nontransgenic littermates. Expression of G1 cyclins was prominently increased in the mammary glands of both the CSF-1 and c-fms transgenic lines, suggesting increased cell cycle progression in these strains. In addition, the proliferation marker proliferating cell nuclear antigen (PCNA) and the mitogen-responsive transcription factor c-jun were also increased as compared with the nontransgenic controls. These findings, along with the histological data, support the hypothesis that CSF-1 and its receptor are involved in the etiology of breast cancer.
Molecular Imaging of Drug-Modulated Protein-Protein Interactions in Living Subjects
Authors: Paulmurugan Ramasamy , Massoud Tarik F. , Huang Jing , Gambhir Sanjiv S.
Published: September 2005

Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor κB promoter/enhancer elements using tumor necrosis factor α, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.
Immunoprevention of HER-2/
neu
Transgenic Mammary Carcinoma through an Interleukin 12-Engineered Allogeneic Cell Vaccine
Authors: De Giovanni Carla , Nicoletti Giordano , Landuzzi Lorena , Astolfi Annalisa , Croci Stefania , Comes Alberto , Ferrini Silvano , Meazza Raffaella , Iezzi Manuela , Di Carlo Emma , Musiani Piero , Cavallo Federica , Nanni Patrizia , Lollini Pier-Luigi
Published: September 2005

This study evaluated the ability of cytokine-engineered allogeneic (H-2q) HER-2/neu-positive cells to prevent tumor development in mammary cancer-prone virgin female BALB/c (H-2d) mice transgenic for the transforming rat HER-2/neu oncogene (BALB-neuT mice). Repeated vaccinations with cells engineered to release interleukin (IL)-2, IL-12, IL-15, or IFN-γ showed that IL-12-engineered cell vaccines had the most powerful immunopreventive activity, with >80% of 1-year-old BALB-neuT mice free of tumors. On the contrary all of the untreated mice and all of the mice vaccinated with IL-12-engineered cells lacking either HER-2/neu or allogeneic antigens developed mammary carcinomas within 22 or 33 weeks, respectively. Whole mount, histology, immunohistochemistry, and gene expression profile analysis showed that vaccination with IL-12-engineered cells maintained 26-week mammary glands free of neoplastic growth, with a gene expression profile that clustered with that of untreated preneoplastic glands. The IL-12-engineered cell vaccine elicited a high production of IFN-γ and IL-4 and a strong anti-HER-2/neu antibody response. Immune protection was lost or markedly impaired in BALB-neuT mice lacking IFN-γ or antibody production, respectively. The protection afforded by the IL-12-engineered cell vaccine was equal to that provided by the systemic administration of recombinant IL-12 in combination with HER-2/neu H-2q cell vaccine. However, IL-12-engineered cell vaccine induced much lower circulating IL-12 and IFN-γ, and therefore lower potential side effects and systemic toxicity.
Cysteine Cathepsins Are Central Contributors of Invasion by Cultured Adenosylmethionine Decarboxylase-Transformed Rodent Fibroblasts
Authors: Ravanko Kirsi , Järvinen Kristiina , Helin Jari , Kalkkinen Nisse , Hölttä Erkki
Published: September 2005

Adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of polyamines, is often up-regulated in cancers. We have demonstrated previously that overexpression of AdoMetDC alone is sufficient to transform NIH 3T3 cells and induce highly invasive tumors in nude mice. Here, we studied the transformation-specific alterations in gene expression induced by AdoMetDC by using cDNA microarray and two-dimensional electrophoresis technologies. We specifically tried to identify the secreted proteins contributing to the high invasive activity of the AdoMetDC-transformed cells. We found a significant increase in the expression and secretion of procathepsin L, which was cleaved and activated in the presence of glycosaminoglycans (heparin), and a smaller increase in cathepsin B. Inhibition of the cathepsin L and B activity by specific peptide inhibitors abrogated the invasive capacity of the AdoMetDC transformants in Matrigel. The transformed cells also showed a small increase in the activity of gelatin-degrading matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator activities, neither of which was sensitive to the inhibitors of cathepsin L and B. Furthermore, the invasive potency of the transformed cells remained unaffected by specific inhibitors of MMPs. The results suggest that cysteine cathepsins are the main proteases contributing to the high invasiveness of the AdoMetDC-transformed cells and that the invasion potential is largely independent of activation of the MMPs.
Fifteenth Annual Pezcoller Symposium
Authors: Mihich Enrico , Kaelin William
Published: September 2005

Recent advances in the understanding of the molecular phenomena underlying neoplastic development are leading to the recognition of metabolic features characteristic of the cancer cell. It has become possible to visualize the presence of these cells in vivo and to follow their progression toward increasing anaplastic behavior through measurements of molecular markers that can be achieved by physical or biochemical means.
Thioredoxin Is Downstream of Smad7 in a Pathway That Promotes Growth and Suppresses Cisplatin-Induced Apoptosis in Pancreatic Cancer
Authors: Arnold Nichole Boyer , Ketterer Knut , Kleeff Jörg , Friess Helmut , Büchler Markus W. , Korc Murray
Published: September 2005

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive human malignancy in which Smad7 is commonly overexpressed. Analysis by differential display identified thioredoxin-1 (TRX) as a gene whose basal expression is increased in COLO-357 pancreatic cancer cells engineered to overexpress Smad7. To delineate the biological consequences of TRX overexpression, we assessed TRX mRNA levels in PDAC and studied the effects of increased TRX levels in Smad7-overexpressing cells. By northern blotting, TRX mRNA levels were increased in PDAC samples by comparison with the normal pancreas. Moreover, analysis of laser-captured pancreatic cancer cells revealed parallel increases in Smad7 and TRX mRNA levels. Retroviral infection of an antisense TRX cDNA suppressed TRX protein levels and blunted the increased capacity of Smad7-overexpressing cells to form colonies in soft agar. 1-Methyl-propyl-2-imidazolozyl disulfide, a TRX inhibitor, markedly suppressed the growth of sham-transfected COLO-357 cells and enhanced the growth inhibitory actions of cis-diamminedichloroplatinum(II) (CDDP). CDDP also induced apoptosis, as evidenced by induction of DNA laddering, PARP cleavage, and caspase-3/9 activities. These pro-apoptotic actions were greatly attenuated in Smad7-overexpressing cells, which exhibited a more prolonged association of TRX with the apoptosis inducer apoptosis signal-regulating kinase-1, and enhanced nuclear factor κB activation in response to CDDP. These findings suggest that TRX is downstream of Smad7 in a pathway that confers a growth advantage to pancreatic cancer cells and that increases their resistance to CDDP-mediated apoptosis, implying novel regulatory functions for Smad7.
Promoter Methylation Inhibits
APC
Gene Expression by Causing Changes in Chromatin Conformation and Interfering with the Binding of Transcription Factor CCAAT-Binding Factor
Authors: Deng Guoren , Song Geun-Am , Pong Erik , Sleisenger Marvin , Kim Young S.
Published: September 2005

As an important regulator in Wnt-signaling pathway, the APC gene is involved in apoptosis and cell cycle arrest. The loss of APC function is observed in most familial adenomatous polyposis-associated and sporadic colorectal cancer. APC gene is frequently inactivated by DNA mutations. However, hypermethylation in APC gene promoter was also observed in different cancers. In this study, by analyzing the methylation status of APC promoter in 22 colorectal cancer cell lines with different APC expression levels, we identified Regions A and B in the promoter, where the methylation of CpG sites was invariably correlated with the loss of gene expression. By nuclease accessibility assay, we also observed a correlation between the closed chromatin conformation in APC promoter and loss of gene expression. When the nonexpressing cell lines were treated with a DNA methyltransferase inhibitor, 5-Aza-2′-Deoxycytidine, the APC expression in these cells was induced, CpG sites were demethylated, and closed chromatin conformation was opened. However, when these cell lines were treated with a histone deacetylase inhibitor, Trichostatin A, no significant changes in APC expression, methylation status, and chromatin conformation were observed. Using transient transfection assay, a CCAAT box located in Region B was identified, which was involved in up-regulation of APC expression. Methylation of CpG sites around the CCAAT box resulted in a significant inhibition in the gene expression. The specific binding of a transcription factor CCAAT-binding factor (CBF) to the CCAAT box was determined by electrophoretic mobility shift analysis. The binding was inhibited after CpG sites close to the CCAAT box were methylated, indicating that DNA methylation can silence gene expression through interfering with the binding of transcription factors to the promoter. The biological function of CBF in APC gene regulation was further indicated by the decrease of luciferase activities in cells cotransfected with a plasmid carrying APC promoter/luciferase gene and a plasmid expressing dominant negative CBF mutant. In summary, methylation of CpG sites around CCAAT box in APC promoter inhibits the gene expression by changing the chromatin conformation and interfering with the binding of transcription factor CBF to CCAAT box.
Structure-Based Assessment of Missense Mutations in Human BRCA1
Authors: Mirkovic Nebojsa , Marti-Renom Marc A. , Weber Barbara L. , Sali Andrej , Monteiro Alvaro N. A.
Published: September 2005

The BRCA1 gene from individuals at risk of breast and ovarian cancers can be screened for the presence of mutations. However, the cancer association of most alleles carrying missense mutations is unknown, thus creating significant problems for genetic counseling. To increase our ability to identify cancer-associated mutations in BRCA1, we set out to use the principles of protein three-dimensional structure as well as the correlation between the cancer-associated mutations and those that abolish transcriptional activation. Thirty-one of 37 missense mutations of known impact on the transcriptional activation function of BRCA1 are readily rationalized in structural terms. Loss-of-function mutations involve nonconservative changes in the core of the BRCA1 C-terminus (BRCT) fold or are localized in a groove that presumably forms a binding site involved in the transcriptional activation by BRCA1; mutations that do not abolish transcriptional activation are either conservative changes in the core or are on the surface outside of the putative binding site. Next, structure-based rules for predicting functional consequences of a given missense mutation were applied to 57 germ-line BRCA1 variants of unknown cancer association. Such a structure-based approach may be helpful in an integrated effort to identify mutations that predispose individuals to cancer.
Oncogenic Action of Secreted Phospholipase A2 in Prostate Cancer
Authors: Sved Paul , Scott Kieran F. , McLeod Duncan , King Nicholas J. C. , Singh Jas , Tsatralis Tania , Nikolov Blagoy , Boulas John , Nallan Laxman , Gelb Michael H. , Sajinovic Mila , Graham Garry G. , Russell Pamela J. , Dong Qihan
Published: September 2005

Mortality from prostate cancer is associated with progression of tumors to androgen-independent growth and metastasis. Eicosanoid products of both the cyclooxygenase (COX) and lipoxygenase (LOX) pathways are important mediators of the proliferation of prostate cancer cells in culture and regulate tumor vascularization and metastasis in animal models. Pharmacologic agents that block either COX or LOX products effectively reduce the size of prostate cancer xenografts. Phospholipase A2 (PLA2) enzymes regulate the provision of arachidonic acid to both COX- and LOX-derived eicosanoids, and a secreted form of the enzyme (sPLA2-IIA) is elevated in prostate cancer tissues. Here, we show by immunohistochemistry, in patients receiving androgen ablation therapy, that sPLA2-IIA remains elevated in remaining cancer cells relative to benign glands after treatment. Furthermore, sPLA2-IIA expression seen in benign glands is substantially decreased after androgen depletion, whereas cytosolic PLA2-α (cPLA2-α) levels are unchanged. sPLA2-IIA mRNA expression is detectable and inducible by androgen (0.01–10 nmol/L) in the androgen-sensitive cell line LNCaP, and exogenous addition of sPLA2-IIA (1–100 nmol/L), but not an inactive sPLA2-IIA mutant (H48Q), results in a dose-dependent increase in cell numbers or the fraction of cells in G2-M phase, which is inhibited by sPLA2-IIA-selective inhibitors. The effect of exogenous sPLA2-IIA can also be blocked by inhibition of cPLA2-α, suggesting a role for cPLA2-α in mediating sPLA2-IIΑ action. sPLA2-IIA inhibitors suppressed basal proliferation in LNCaP cells and in the androgen-independent, sPLA2-positive cell line PC3 but not in the sPLA2-IIA-negative androgen-independent cell line DU145. Established PC3 xenograft tumors grew more slowly in mice treated with sPLA2-IIA inhibitors than those treated with saline only. The PLA2 enzymes, and sPLA2-IIA in particular, thus represent important targets for the treatment of sPLA2-IIA-positive androgen-independent prostate cancer.
Hypoxia Enhances Metastatic Efficiency by Up-Regulating Mdm2 in KHT Cells and Increasing Resistance to Apoptosis
Authors: Zhang Li , Hill Richard P.
Published: September 2005

Tumor hypoxia has been reported to be a negative prognostic factor in a number of tumor sites. Both clinical and experimental studies have suggested a positive correlation between tumor hypoxia and increased metastatic efficiency; however, the mechanisms are not understood. In this study, the mechanisms of hypoxia-enhanced metastasis have been investigated in murine KHT fibrosarcoma and SCC VII cells. We have observed that hypoxia-pretreated KHT-C cells have a higher survival rate than control KHT-C cells after being arrested in mouse lungs. cDNA microarray analysis revealed many hypoxia-regulated genes, most of which have been reported to be involved in cell survival and growth. Among these genes, we have confirmed the up-regulation of Mdm2 by hypoxia and have demonstrated that this up-regulation is p53 independent. The up-regulation of Mdm2 by hypoxia is associated with decreased p53 protein and inhibition of the transactivation of p53 downstream proapoptotic genes. Overexpression of Mdm2 or suppression of p53 by transient transfection increased metastatic efficiency in KHT-C cells. These data suggest that hypoxia can increase tumor cell metastatic efficiency by rendering the tumor cells less sensitive to stress-induced cell death, e.g., through modifying the levels of Mdm2 and p53.
RNA Interference-Mediated Knockdown of DNA Methyltransferase 1 Leads to Promoter Demethylation and Gene Re-Expression in Human Lung and Breast Cancer Cells
Authors: Suzuki Makoto , Sunaga Noriaki , Shames David S. , Toyooka Shinichi , Gazdar Adi F. , Minna John D.
Published: September 2005

DNA methyltransferase 1 (DNMT1) is required to maintain DNA methylation patterns in mammalian cells, and is thought to be the predominant maintenance methyltransferase gene. Recent studies indicate that inhibiting DNMT1 protein expression may be a useful approach for understanding the role of DNA methylation in tumorigenesis. To this end, we used RNA interference to specifically down-regulate DNMT1 protein expression in NCI-H1299 lung cancer and HCC1954 breast cancer cells. RNA interference-mediated knockdown of DNMT1 protein expression resulted in >80% reduction of promoter methylation in RASSF1A, p16ink4A, and CDH1 in NCI-H1299; and RASSF1A, p16ink4A, and HPP1 in HCC1954; and re-expression of p16ink4A, CDH1, RASSF1A, and SEMA3B in NCI-H1299; and p16ink4A, RASSF1A, and HPP1 in HCC1954. By contrast, promoter methylation and lack of gene expression was maintained when these cell lines were treated with control small interfering RNAs. The small interfering RNA treatment was stopped and 17 days later, all of the sequences showed promoter methylation and gene expression was again dramatically down-regulated, indicating the tumor cells still were programmed for these epigenetic changes. We saw no effects on soft agar colony formation of H1299 cells 14 days after DNMT1 knockdown indicating that either these genes are not functioning as tumor suppressors under these conditions, or that more prolonged knockdown or other factors are also required to inhibit the malignant phenotype. These results provide direct evidence that loss of DNMT1 expression abrogates tumor-associated promoter methylation and the resultant silencing of multiple genes implicated in the pathogenesis of human lung and breast cancer.
Celecoxib Can Prevent Tumor Growth and Distant Metastasis in Postoperative Setting
Authors: Roh Jong-Lyel , Sung Myung-Whun , Park Seok-Woo , Heo Dae-Seog , Lee Dong Wook , Kim Kwang Hyun
Published: September 2005

Much evidence suggests that an inflammatory condition provides a microenvironment favorable for tumor growth. One of the main components in the healing wound is the induction of cyclooxygenase-2 (COX-2) and prostaglandins, and many solid tumors have been known to overexpress COX-2. The present study investigated the relationship between surgical wounds and tumor growth and the roles of COX-2 and inflammatory reaction in this microenvironment. We created surgical wounds in syngeneic mice for the implantation of SCC VII murine cancer cell line. Accelerated tumor growth and increased angiogenesis by surgical wounds were clearly observed in C3H/HeJ mice with SCC VII tumor. The COX-2 expression of peritumoral tissues and leukocyte infiltration partly explained the accelerated tumor growth, especially in the early phase after surgical wounding. Celecoxib had a significantly suppressive effect on tumor growth, angiogenesis, and metastasis in tumor-implanted mice with surgical wounds. This tumor-suppressive action of celecoxib did not show any noticeable side effects on the late wound healing and on the gastrointestinal tracts. Prophylactic use of the drug can be advocated in many clinical situations, such as residual tumors or contamination of surgical fields by tumor cells.
Bimolecular Interaction of Insulin-Like Growth Factor (IGF) Binding Protein-2 with αvβ3 Negatively Modulates IGF-I-Mediated Migration and Tumor Growth
Authors: Pereira Joseph J. , Meyer Tim , Docherty Susan E. , Reid Hugh H. , Marshall John , Thompson Erik W. , Rossjohn Jamie , Price John T.
Published: September 2005

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the αvβ3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and αvβ3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7β3, which overexpressed the αvβ3 integrin. Collectively, these results indicate that αvβ3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.
Cathepsin B Mediates Caspase-Independent Cell Death Induced by Microtubule Stabilizing Agents in Non-Small Cell Lung Cancer Cells
Authors: Bröker Linda E. , Huisman Cynthia , Span Simone W. , Rodriguez José A. , Kruyt Frank A. E. , Giaccone Giuseppe
Published: September 2005

We have previously reported that the microtubule stabilizing agents (MSAs) paclitaxel, epothilone B and discodermolide induce caspase-independent cell death in non-small cell lung cancer (NSCLC) cells. Here we present two lines of evidence indicating a central role for the lysosomal protease cathepsin B in mediating cell death. First, inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against drug-induced cell death in several NSCLC cells. Second, MSAs trigger disruption of lysosomes and release and activation of cathepsin B. Interestingly, inhibition of cathepsin B prevents the appearance of multinucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in this novel cell death pathway.
Pivotal Role of the Cell Death Factor BNIP3 in Ceramide-Induced Autophagic Cell Death in Malignant Glioma Cells
Authors: Daido Shigeru , Kanzawa Takao , Yamamoto Akitsugu , Takeuchi Hayato , Kondo Yasuko , Kondo Seiji
Published: September 2005

The sphingolipid ceramide has been recognized as an important second messenger implicated in regulating diverse signaling pathways especially for apoptosis. Very little is known, however, about the molecular mechanisms underlying nonapoptotic cell death induced by ceramide. In the present study, we first demonstrate that ceramide induces nonapoptotic cell death in malignant glioma cells. The cell death was accompanied by several specific features characteristic of autophagy: presence of numerous autophagic vacuoles in the cytoplasm, development of the acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3), and a marked increase in expression levels of two forms of LC3 protein (LC3-I and LC3-II). We additionally demonstrate that ceramide decreases mitochondrial membrane potential and activates the transcription of death-inducing mitochondrial protein, BNIP3, resulting in increased expression levels of its mRNA and protein in malignant glioma cells. Moreover, tumor cells transfected with BNIP3 gene undergo autophagy in the absence of ceramide. These results suggest that ceramide induces autophagic cell death in malignant glioma cells via activation of BNIP3. This study adds a new concept to characterize the pathways by which ceramide acts to induce nonapoptotic autophagic cell death in malignant gliomas.
Semaphorin-3F Is an Inhibitor of Tumor Angiogenesis
Authors: Kessler Ofra , Shraga-Heled Niva , Lange Tali , Gutmann-Raviv Noga , Sabo Edmond , Baruch Limor , Machluf Marcelle , Neufeld Gera
Published: September 2005

The neuropilin-1 (np1) and neuropilin-2 (np2) receptors form complexes with type-A plexins. These complexes serve as signaling receptors for specific class-3 semaphorins. Np1 and np2 function in addition as receptors for heparin-binding forms of vascular endothelial growth factor (VEGF), such as VEGF165. Human umbilical vein endothelial cells (HUVEC) express tyrosine-kinase receptors for VEGF and basic fibroblast growth factor (bFGF), as well as np1, np2, and several type-A plexins. We have found that semaphorin-3F (s3f), a semaphorin which signals through the np2 receptor, was able to inhibit VEGF165, as well as bFGF-induced proliferation of HUVECs. Furthermore, s3f inhibited VEGF as well as bFGF-induced phosphorylation of extracellular signal-regulated kinase-1/2. Our experiments indicate that bFGF does not bind to neuropilins, nor does s3f inhibit the binding of bFGF to FGF receptors. It is therefore possible that s3f inhibits the activity of bFGF by a mechanism that requires active s3f signal transduction rather than by inhibition of bFGF binding to FGF receptors. s3f also inhibited VEGF165, as well as bFGF-induced in vivo angiogenesis as determined by the alginate micro-encapsulation and Matrigel plug assays. Overexpression of s3f in tumorigenic human HEK293 cells inhibited their tumor-forming ability but not their proliferation in cell culture. The tumors that did develop from s3f-expressing HEK293 cells developed at a much slower rate and had a significantly lower concentration of tumor-associated blood vessels, indicating that s3f is an inhibitor of tumor angiogenesis.
Cell Surface Expression of Epidermal Growth Factor Receptor and Her-2 with Nuclear Expression of Her-4 in Primary Osteosarcoma
Authors: Hughes Dennis P. M. , Thomas Dafydd G. , Giordano Thomas J. , Baker Laurence H. , McDonagh Kevin T.
Published: September 2005

There is controversy over the role of Her-2 in osteosarcoma, with some investigators reporting association between expression and adverse outcome, whereas others point to the lack of gene amplification and membranous expression by immunohistochemistry (IHC) as inconsistent with biological significance. Her-2 normally requires pairing with epidermal growth factor receptor (EGFR), Her-3, or Her-4, but these have been less well studied in osteosarcoma. We evaluated the expression of each of these receptors in osteosarcoma and their potential to contribute to pathogenesis by examining a panel of low-passage primary osteosarcoma cell lines, comparing these with archival tumor specimens. Her-2 immunoreactivity was seen frequently in the diffuse staining pattern described previously. We observed EGFR in all samples by IHC. Her-3 expression was not observed. Her-4 expression was nuclear in distribution in all tumor samples and many cell line samples, consistent with activation and cleavage of the receptor. Quantified expression of Her-2 and EGFR mRNA by quantitative, real-time PCR in cell lines correlated with IHC for Her-2 but not for EGFR. Western blot identified full-length receptors for EGFR and Her-2 in all expected cell lines and showed Her-4 to be predominantly in the p80 form. Flow cytometry identified cell surface Her-2 and EGFR in all lines with receptor expression by IHC. We conclude that the cell surface expression of Her-2 and EGFR and the nuclear localization of the activated p80 fragment of Her-4 suggest that all three may be contributing to osteosarcoma pathogenesis. Therapy directed against this family of receptors may be beneficial for patients with osteosarcoma.
Blockade of Epidermal Growth Factor- or Heregulin-Dependent ErbB2 Activation with the Anti-ErbB2 Monoclonal Antibody 2C4 Has Divergent Downstream Signaling and Growth Effects
Authors: Jackson James G. , St. Clair Patricia , Sliwkowski Mark X. , Brattain Michael G.
Published: September 2005

Due to heterodimerization and a variety of stimulating ligands, the ErbB receptor system is both diverse and flexible, which proves particularly advantageous to the aberrant signaling of cancer cells. However, specific mechanisms of how a particular receptor contributes to generating the flexibility that leads to aberrant growth regulation have not been well described. We compared the utilization of ErbB2 in response to epidermal growth factor (EGF) and heregulin stimulation in colon carcinoma cells. Anti-ErbB2 monoclonal antibody 2C4 blocked heregulin-stimulated phosphorylation of ErbB2 and ErbB3; activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3′-kinase (PI3K), and Akt; proliferation; and anchorage-independent growth. 2C4 blocked EGF-mediated phosphorylation of ErbB2 and inhibited PI3K/Akt and anchorage-independent growth but did not affect ErbB1 or MAPK. Immunoprecipitations showed that ErbB3 and Grb2-associated binder (Gab) 1 were phosphorylated and associated with PI3K activity after heregulin treatment and that Gab1 and Gab2, but not ErbB3, were phosphorylated and associated with PI3K activity after EGF treatment. These data show that monoclonal antibody 2C4 inhibited all aspects of heregulin signaling as well as anchorage-independent and monolayer growth. Furthermore, we identify ErbB2 as a critical component of EGF signaling to the Gab1/Gab2-PI3K-Akt pathway and anchorage-independent growth, but EGF stimulation of MAPK and monolayer growth can occur efficiently without the contribution of ErbB2.
Identification of the Apoptosis Activation Cascade Induced in Mammary Carcinomas by Energy Restriction
Authors: Thompson Henry J. , Zhu Zongjian , Jiang Weiqin
Published: September 2005

Energy restriction (ER) inhibits mammary carcinogenesis and results in a marked reduction in tumor size, effects likely to be explained by ER-mediated induction of apoptosis. The goal of this study was to investigate the molecular mechanism(s) accounting for apoptosis induction. To do this, chemically induced mammary carcinomas were evaluated from rats that were ad libitum fed (control), 40% ER, or 40% ER but energy repleted for 7 days before study termination (ER-REP); the ER-REP group permitted the determination of the reversibility of ER-mediated effects. Cleaved products of poly(ADP-ribose) polymerase 1 were elevated by ER (P < 0.025) providing biochemical evidence of apoptosis induction. cDNA microarray analysis identified the Bcl-2, CARD, and IAP functional gene groupings as being involved in apoptosis induction. Consistent with the microarray data, the activities of caspases 9 and 3 were observed to be ∼2-fold higher in carcinomas from ER rats (P ≤ 0.01), whereas caspase 8 activity was similar in carcinomas from all three of the groups. This evidence that ER-induced apoptosis mediated by the mitochondrial pathway was additionally supported by the finding that levels of Bcl-2, Bcl-xl, and XIAP protein were significantly lower (P < 0.01), and levels of Bax and Apaf-1 were elevated (P < 0.02) in ER carcinomas versus those carcinomas from control or ER-REP rats. Additional studies revealed that Akt phosphorylation (activation) was reduced in mammary carcinomas from ER rats. Thus, it appears that ER induces apoptosis in mammary carcinomas via a cell survival factor-dependent pathway.
Matrix Metalloproteinase Activity Modulates Tumor Size, Cell Motility, and Cell Invasiveness in Murine Aggressive Fibromatosis
Authors: Kong Yuan , Poon Raymond , Nadesan Puviindran , Di Muccio Tamara , Fodde Riccardo , Khokha Rama , Alman Benjamin A.
Published: September 2005

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) regulate the degradation of extracellular matrix components and play important roles in the progression of select neoplastic processes. The locally invasive soft tissue tumor, aggressive fibromatosis (also called desmoid tumor), is caused by mutations resulting in β-catenin-mediated T-cell factor (tcf)-dependent transcriptional activity. Because β-catenin can regulate MMP expression, we investigated the expression of several MMPs and TIMPs in aggressive fibromatosis tumors that develop in Apc+/Apc1638N mice. Mmp-3 and Timp-1 were differentially regulated (5-fold and 0.5-fold, respectively) in tumors compared with normal fibrous tissue. Conditioned media from tumor cells showed an increased ability to degrade collagen, and inhibition of MMPs using GM6001 decreased the ability of the tumor cells to invade through Matrigel. Both the treatment of Apc/Apc1638N mice with GM6001 or crossing with a transgenic mouse that overexpresses Timp-1 resulted in a significant reduction in tumor volume. Surprisingly, overexpression of Timp-1 also resulted in a 50% increase in tumor number. Although TIMP-1 can induce growth stimulatory effects in some cell types, we found no difference in proliferation or apoptosis rate in cells from tumors that developed in the Timp-1–transgenic mice compared with mice that did not express the Timp-1 transgene, suggesting that TIMP-1 promotes aggressive fibromatosis tumor formation through an alternate mechanism. These data suggest that MMPs play a crucial role in regulating the invasiveness of mesenchymal cells and in modulating aggressive fibromatosis tumor progression. Because this is a locally invasive tumor, MMP inhibition could slow tumor growth and may prove to be an effective adjuvant therapy.
Genome-Wide Identification of Genes Conferring Resistance to the Anticancer Agents Cisplatin, Oxaliplatin, and Mitomycin C
Authors: Wu H. Irene , Brown James A. , Dorie Mary J. , Lazzeroni Laura , Brown J. Martin
Published: September 2005

Cisplatin is a crucial agent in the treatment of many solid tumors, yet many tumors have either acquired or intrinsic resistance to the drug. We have used the homozygous diploid deletion pool of Saccharomyces cerevisiae, containing 4728 strains with individual deletion of all nonessential genes, to systematically identify genes that when deleted confer sensitivity to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. We found that deletions of genes involved in nucleotide excision repair, recombinational repair, postreplication repair including translesional synthesis, and DNA interstrand cross-link repair resulted in sensitivity to all three of the agents, although with some differences between the platinum drugs and mitomycin C in the spectrum of required translesional polymerases. Putative defective repair of oxidative damage (imp2′Δ strain) also resulted in sensitivity to platinum and oxaliplatin, but not to mitomycin C. Surprisingly in light of their different profiles of clinical activity, cisplatin and oxaliplatin have very similar sensitivity profiles. Finally, we identified three novel genes (PSY1–3, “platinum sensitivity”) that, when deleted, demonstrate sensitivity to cisplatin and oxaliplatin, but not to mitomycin C. Our results emphasize the importance of multiple DNA repair pathways responsible for normal cellular resistance to all three of the agents. Also, the similarity of the sensitivity profiles of the platinum agents with that of the known DNA interstrand cross-linking agent mitomycin C, and the importance of the gene PSO2 known to be involved in DNA interstrand cross-link repair strongly suggests that interstrand cross-links are important toxic lesions for cisplatin and oxaliplatin, at least in yeast.
Facilitating Role of Preprotachykinin-I Gene in the Integration of Breast Cancer Cells within the Stromal Compartment of the Bone Marrow
Authors: Rao Gayatri , Patel Prem S. , Idler Suji P. , Maloof Paul , Gascon Pedro , Potian Julius A. , Rameshwar Pranela
Published: September 2005

Despite early detection of breast cancer, patients’ survival may be compromised if the breast cancer cells (BCCs) enter the bone marrow (BM). It is highly probable that BCCs enter the BM long before clinical detection. An in vitro coculture model with BM stroma and BCCs (cell lines; primary cells from stage III BC, n = 7, and stage M0, n = 3) mimicked early entry of BCCs into the BM. In coculture, BCCs exhibit contact inhibition and do not require otherwise needed growth supplements. Stromal growth rate was increased 2-fold in coculture. The inclusion of BCCs in stromal support of long-term culture-initiating cell assay frequencies show no difference (38 ± 3 versus 36 ± 6). Nontumorigenic breast cells (patients and cell lines) did not survive in coculture, suggesting that the model could select for malignant population in surgical breast tissues. Cocultures were able to select cells with 73 ± 7% cloning efficiencies and with the ability to form cocultures with BM stroma. Preprotachykinin-I (PPT-I), a gene that is conserved by evolution, facilitates BCC integration as part of the stromal compartment. This was deduced as follows: (a) nontumorigenic breast cells (n = 4) genetically engineered to express PPT-I and led to anchorage-independent growth, foci formation, and formation of cocultures; and (b) suppression of PPT-I in BCCs (n = 5) with pPMSKH1-PPT-I small interfering RNA reverted the cells to nontumorigenic phenotypes and was undetectable in the BM nude mice. The evidence supports that the PPT-I gene facilitates the integration of BCCs in the stromal compartment during a period before clinical detection, without disrupting hematopoietic activity.
Lipoptosis
Authors: Pohle Tina , Brändlein Stephanie , Ruoff Nele , Müller-Hermelink Hans Konrad , Vollmers H. Peter
Published: September 2005

A balanced lipid metabolism is crucial for all cells. Disturbance of this homeostasis by nonphysiological intracellular accumulation of fatty acids can result in apoptosis. This was proven in animal studies and was correlated to some human diseases, like lipotoxic cardiomyopathy. Some metabolic mechanisms of lipo-apoptosis were described, and some causes were discussed, but reagents, which directly induce lipo-apoptosis, have thus far not been identified. The human monoclonal IgM antibody SAM-6 was isolated from a stomach cancer patient by using the conventional human hybridoma technology (trioma technique). The addition of SAM-6 to tumor cells leads to an increase in the intracellular accumulation of neutral lipids, followed by tumor cell apoptosis. The antibody SAM-6 does not react with noncancerous human epithelial and fibroblastic cells, because the Mr 140,000 membrane molecule, recognized by the antibody, is specifically expressed on human malignant cells. The antibody is coded by the germ-line genes IgHV3-30.3*01 and IgLV3-1*01 and is a component of the innate immunity to cancer. In this article, we describe an antibody-induced tumor-specific cell death, named lipoptosis. This is, to our knowledge, the first description of this specific form of lipo-apoptosis as an antibody-mediated mechanism of tumor cell killing.