bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Papillomavirus Type 16 E6 and E7 Cause Polyploidy in Human Keratinocytes and Up-Regulation of G2-M-Phase Proteins
Authors: Patel Daksha , Incassati Angela , Wang Nancy , McCance Dennis J.
Published: September 2005

Human papillomavirus type 16 proteins E6 and E7 have been shown to cause centrosome amplification and lagging chromosomes during mitosis. These abnormalities during mitosis can result in missegregation of the chromosomes, leading to chromosomal instability. Genomic instability is thought to be an essential part of the conversion of a normal cell to a cancer cell. We now show that E6 and E7 together cause polyploidy in primary human keratinocytes soon after these genes are introduced into the cells. Polyploidy seems to result from a spindle checkpoint failure arising from abrogation of the normal functions of p53 and retinoblastoma family members by E6 and E7, respectively. In addition, E6 and E7 cause deregulation of cellular genes such as Plk1, Aurora-A, cdk1, and Nek2, which are known to control the G2-M-phase transition and the ordered progression through mitosis.
Tumor Necrosis Factor-α-Induced Matrix Proteolytic Enzyme Production and Basement Membrane Remodeling by Human Ovarian Surface Epithelial Cells
Authors: Yang Wan-Lin , Godwin Andrew K. , Xu Xiang-Xi
Published: September 2005

The majority of cancer is of surface/cyst epithelial origin. The ovarian surface epithelial cells are organized by a sheet of basement membrane composed mainly of collagen IV and laminin, and it is believed that the basement membrane greatly influences the physiological properties of ovarian surface epithelial cells. Previous studies in our laboratories indicated that loss of the basement membrane, an obligated step in ovulation, is also a critical step during the morphological transformation and tumor initiation of the ovarian surface epithelium. It is speculated that the loss of basement membrane in ovarian surface epithelial transformation may have similar biological mechanism to the loss of surface epithelial basement membrane in ovulation. However, the mechanisms involved in the ovarian surface epithelial basement membrane removal during ovulation are still not completely understood. In the current study, cultured human ovarian surface epithelial (HOSE) cells were examined for their abilities to produce matrix hydrolyzing enzymes and degrade basement membrane in response to a number of potential local mediators in ovulation. Among the candidate-stimulating factors tested, tumor necrosis factor (TNF)-α and IL-1β (to a lesser extent) were found to drastically increase urokinase type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 activities secreted from HOSE cells. MMP-2, the other major HOSE cell-secreted gelatinase, is constitutively produced but not regulated. As demonstrated by immunofluorenscence staining and Western blot analysis, TNF-α treatment caused the degradation and structural reorganization of collagen IV and laminin secreted and deposited by HOSE cells in culture. Amiloride, an uPA inhibitor, not only inhibited the activity of uPA but was also able to suppress TNF-α-stimulated MMP-9 activity and prevented the TNF-α-stimulated remodeling of the basement membrane extracellular matrix, suggesting the contribution of uPA-mediated proteolytic cascade in this process. This study implicates the potential roles of TNF-α, uPA, and MMP-9 in ovarian surface epithelial basement membrane degradation and remodeling, which are processes during ovulation and may contribute to epithelial transformation. The findings may underscore the importance of TNF-α, uPA, and MMP-9 in ovarian surface epithelial basement membrane remodeling and may provide a molecular mechanism linking ovulation and ovarian cancer risk.
Allelic Imbalance of 12q22–23 Associated with
APAF-1
Locus Correlates with Poor Disease Outcome in Cutaneous Melanoma
Authors: Fujimoto Akihide , Takeuchi Hiroya , Taback Bret , Hsueh Eddy C. , Elashoff David , Morton Donald L. , Hoon Dave S. B.
Published: September 2005

Cutaneous melanoma is a highly aggressive tumor that is relatively resistant to chemotherapy and radiotherapy. This resistance may be in part due to inhibition of apoptosis. Apoptotic protease activating factor-1(APAF-1), a candidate tumor suppressor gene, mediates p53-induced apoptosis, and its loss promotes oncogenic transformation. To determine whether loss of the APAF-1 locus influences tumor progression, we assessed loss of heterozygosity microsatellites on the APAF-1 locus (12q22–23) in 62 primary and 112 metastatic melanomas. We discovered that frequency of allelic imbalance was significantly higher in metastatic tumors (n = 36 of 98; 37%) than in primary melanomas (n = 10 of 54; 19%; P = 0.02). In metastatic melanomas, APAF-1 loss significantly correlated with a worse prognosis (P < 0.05) in the patients, and its loss during melanoma tumor progression suggests that APAF-1 is a tumor suppressor gene. Furthermore, loss of heterozygosity was frequent in the 12q22–23 chromosome region centromeric to the APAF-1 locus suggesting that other tumor-related genes may be present in the 12q22–23 region. In summary, the study demonstrates that allelic imbalance in the 12q22–23 region is a genomic surrogate of poor disease outcome for cutaneous melanoma patients.
SJG-136 (NSC 694501), A Novel Rationally Designed DNA Minor Groove Interstrand Cross-Linking Agent with Potent and Broad Spectrum Antitumor Activity
Authors: Alley Michael C. , Hollingshead Melinda G. , Pacula-Cox Christine M. , Waud William R. , Hartley John A. , Howard Philip W. , Gregson Stephen J. , Thurston David E. , Sausville Edward A.
Published: September 2005

Pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC 694501) selectively cross-links guanine residues located on opposite strands of DNA, and exhibits potent in vitro cytotoxicity. In addition, SJG-136 is highly active in vivo in hollow fiber assays. In the current investigation, SJG-136 was evaluated for in vivo efficacy in 10 tumor models selected on the basis of sensitivity of cells grown in the hollow fiber and in vitro time course assays: LOX IMVI and UACC-62 (melanomas); OVCAR-3 and OVCAR-5 (ovarian carcinomas); MDA-MB-435 (breast carcinoma); SF-295 and C-6 (gliomas); LS-174T (colon carcinoma); HL-60 TB (promyelocytic leukemia); and NCI-H522 (lung carcinoma). SJG-136 was active against small (150 mg) and large (250–400 mg) xenografts with tumor mass reductions in all 10 models. In addition, significant growth delays occurred in nine models, cell kill in six models ranged between 1.9 and 7.2 logs, and there were 1 to 4/6 tumor-free responses in six models. SJG-136 is active following i.v. bolus injections, as well as by 5-day continuous infusions. Of all of the schedules tested, bolus administrations for 5 consecutive days (qd×5) conferred the greatest efficacy. SJG-136 is active over a wide dosage range in athymic mouse xenografts: on a qd×5 schedule, the maximum-tolerated dose was ∼120 μg/kg/dose (total dose: 0.6 mg/kg = 1.8 mg/m2) and the minimum effective dose in the most sensitive model (SF-295) was ∼16 μg/kg/dose (total dose: 0.08 mg/kg = 0.24 mg/m2). Results of this study extend the initial in vivo observations reported in the reference above and confirm the importance of expediting more detailed preclinical evaluations on this novel agent in support of phase I clinical trials in the United Kingdom and the United States, which are planned to commence shortly.
Comprehensive Scanning of Somatic Mitochondrial DNA Alterations in Acute Leukemia Developing from Myelodysplastic Syndromes
Authors: Linnartz Bjoern , Anglmayer Roswitha , Zanssen Stefanie
Published: September 2005

Myelodysplastic syndromes (MDS) are clonal myeloid disorders characterized by ineffective hematopoiesis resulting in refractory cytopenias. Transformation resulting in acute myeloblastic leukemia is the final stage in the multistep process of MDS evolution. Functional relevant mutations of mitochondrial DNA (mtDNA) have been related to sideroblastic anemia and MDS. To investigate the role of mtDNA in malignant transformation to acute leukemia, we used high-resolution techniques such as single-strand conformational polymorphism and fluorescence sequencing for investigation of the whole mitochondrial genome from blood cells of 10 patients with MDS. Functionally relevant point mutations in mitochondrial RNA and polypeptide-encoding genes were detected in 50% of patients with MDS. Their increasing mutation load connects MDS and the developing acute myeloid leukemias. Several point mutations of mtDNA, including secondary point mutations for Leber’s hereditary optic neuropathy, occur in one bone marrow and may synergically affect bone marrow stem cells by an apoptotic pathway.
Expression Pattern of Chemokine Receptor 6 (CCR6) and CCR7 in Squamous Cell Carcinoma of the Head and Neck Identifies a Novel Metastatic Phenotype
Authors: Wang Jun , Xi Liqiang , Hunt Jennifer L. , Gooding William , Whiteside Theresa L. , Chen Zhuo , Godfrey Tony E. , Ferris Robert L.
Published: September 2005

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes predictably to cervical lymph nodes, with low rates of distant metastases. Tumor cells can express various receptors that facilitate such metastatic spread to lymph nodes and other nonlymphoid organs. Chemokine receptors (CCR), normally expressed on lymphocytes, control immune and inflammatory cell migration, providing a link between innate and adaptive immunity. Chemokine receptor expression was evaluated in SCCHN, using paired primary and metastatic tumors cell lines, and paired primary and metastatic biopsies from the same patients. Quantitative reverse transcription-PCR showed a consistent pattern of CCR6 down-regulation and up-regulation of CCR7 in metastatic cells and tissues. Chemotaxis assays, ligand-induced receptor down-regulation, and specific antibody blocking experiments supported the quantitative reverse transcription-PCR results, indicating that these surface receptors were functional on metastatic tumor cells. Cells derived from a highly metastatic mouse model of SCCHN were used to confirm CCR7 up-regulation in tumor cells with higher metastatic potential. CCR6 down-regulation is consistent with its decreased expression in cells emigrating from peripheral mucosal sites, whereas CCR7, important for homing of immune cells to secondary lymphoid organs, was significantly up-regulated. Thus, CCR6, CCR7, and their ligands, normally important in controlling immune cell trafficking in response to inflammatory stimuli, may have an important role in determining the metastasis of SCCHN cells in vivo.
Selective Accumulation of Mature DC-Lamp+ Dendritic Cells in Tumor Sites Is Associated with Efficient T-Cell-Mediated Antitumor Response and Control of Metastatic Dissemination in Melanoma
Authors: Movassagh Mojgan , Spatz Alain , Davoust Jean , Lebecque Serge , Romero Pedro , Pittet Mikaël , Rimoldi Donata , Liénard Danièle , Gugerli Oliver , Ferradini Laurent , Robert Caroline , Avril Marie-Françoise , Zitvogel Laurence , Angevin Eric
Published: September 2005

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.
αCP-4, Encoded by a Putative Tumor Suppressor Gene at 3p21, But Not Its Alternative Splice Variant αCP-4a, Is Underexpressed in Lung Cancer
Authors: Pio Ruben , Zudaire Isabel , Pino Irene , Castaño Zafira , Zabalegui Natalia , Vicent Silvestre , Garcia-Amigot Fermin , Odero Maria D. , Lozano Maria D. , Garcia-Foncillas Jesus , Calasanz Maria J. , Montuenga Luis M.
Published: September 2005

αCP-4 is an RNA-binding protein coded by PCBP4, a gene mapped to 3p21, a common deleted region in lung cancer. In this study we characterized the expression of αCP-4 and αCP-4a, an alternatively spliced variant of αCP-4, in lung cancer cell lines and non-small cell lung cancer (NSCLC) samples from early stage lung cancer patients. In NSCLC biopsies, an immunocytochemical analysis showed cytoplasmic expression of αCP-4 and αCP-4a in normal lung bronchiolar epithelium. In contrast, αCP-4 immunoreactivity was not found in 47% adenocarcinomas and 83% squamous cell carcinomas, whereas all of the tumors expressed αCP-4a. Besides, lack of αCP-4 expression was associated with high proliferation of the tumor (determined by Ki67 expression). By fluorescence in situ hybridization, >30% of NSCLC cell lines and tumors showed allelic losses at PCBP4, correlating with the absence of the protein. On the other hand, no mutations in the coding region of the gene were found in any of the 24 cell lines analyzed. By Northern blotting and real-time reverse transcription-PCR, we detected the expression of αCP-4 and αCP-4a messages in NSCLC and small cell lung cancer cell lines. Our data demonstrate an abnormal expression of αCP-4 in lung cancer, possibly associated with an altered processing of the αCP-4 mRNA leading to a predominant expression of αCP-4a. This may be considered as an example of alternative splicing involved in tumor suppressor gene inactivation. Finally, induction of αCP-4 expression reduced cell growth, in agreement with its proposed role as a tumor suppressor, and suggesting an association of this RNA-binding protein with lung carcinogenesis.
The Tumor Suppressor hSNF5/INI1 Modulates Cell Growth and Actin Cytoskeleton Organization
Authors: Medjkane Souhila , Novikov Eugene , Versteege Isabella , Delattre Olivier
Published: September 2005

hSNF5/INI1, which encodes a component of the ATP-dependent chromatin remodeling hSWI-SNF complex, is a tumor suppressor gene mutated in malignant rhabdoid tumors. We have developed a tetracycline-based hSNF5/INI1-inducible system in a hSNF5/INI1-deficient malignant rhabdoid tumor cell line and studied time course variation of 22,000 genes/expressed sequence tags upon hSNF5/INI1 induction. A total of 482 responsive genes were identified and further clustered into 9 groups of coregulated genes. Among genes with early and strong inductions, the use of a fusion protein with the hormone-binding domain of the estrogen receptor enabled the identification of a subset of direct targets regulated independently of de novo protein synthesis. We show that the G1 arrest induced by hSNF5/INI1 is reversible and associated with the down-regulation of components of the DNA replication complex. We also identify an unsuspected role of hSNF5/INI1 in cytoskeleton organization. Indeed, induction of hSNF5/INI1 induces dramatic modifications of the cell shape including complete disruption of the actin stress fiber network and disappearance of focal adhesions associated with up-regulation of genes involved in the organization of the actin cytoskeleton. We document a strong decrease of Rho activity upon hSNF5/INI1 expression, suggesting that the regulation of this activity constitutes a crucial step of the hSNF5/INI1-induced reorganization of the actin network. This study identifies hSNF5/INI1 target genes and provides evidence that hSNF5/INI1 may modulate the cell cycle control and cytoskeleton organization through the regulation of the retinoblastoma protein-E2F and Rho pathways.
Immunotherapeutic Potential of B7-DC (PD-L2) Cross-Linking Antibody In Conferring Antitumor Immunity
Authors: Radhakrishnan Suresh , Nguyen Loc Tan , Ciric Bogoljub , Flies Dallas , Keulen Virginia P. Van , Tamada Koji , Chen Lieping , Rodriguez Moses , Pease Larry R.
Published: September 2005

A naturally occurring human antibody potentiates dendritic cell function on cross-linking B7-DC (PD-L2), supporting robust T-cell responses in vitro. Moreover, treatment of dendritic cells with B7-DC cross-linking antibody resulted in secretion of interleukin-12, suggesting a TH1 polarization of this response. Here we show an in vivo immunotherapeutic effect of this B7-DC cross-linking antibody using a poorly immunogenic B16 melanoma tumor model. Treatment of mice systemically with antibody at the time of tumor cell engraftment prevented tumor growth in a CD4 and CD8 T-cell-dependent manner. The protective effect of B7-DC cross-linking antibody treatment was independent of endogenous antibody responses. Tumor-specific CTL precursors could be isolated from lymph nodes draining the tumor site in animals treated with B7-DC cross-linking antibody, but not from those treated with isotype control antibodies. The elicited antitumor responses in vivo were specific and long-lasting. More strikingly, treatment of mice with B7-DC cross-linking antibody after the tumors were established in the lungs resulted in protection in a CD8-, perforin-, and granzyme B-dependent fashion. Depletion of natural killer cells did not block the effects of treatment with B7-DC cross-linking antibody. Together, these findings demonstrate that cross-linking B7-DC with the human IgM antibody sHIgM12 can induce a protective immune response against a weakly antigenic experimental tumor and therefore has potential as a novel immunotherapeutic approach for treating cancer.
5-Fluoro-2′-Deoxyuridine-Induced cdc25A Accumulation Correlates with Premature Mitotic Entry and Clonogenic Death in Human Colon Cancer Cells
Authors: Parsels Leslie Anne , Parsels Joshua David , Tai Daniel Chung-Ho , Coughlin Daniel James , Maybaum Jonathan
Published: September 2005

The ability to inappropriately progress through S phase during drug treatment is a key determinant of tumor cell sensitivity to thymidylate synthase inhibitors such as 5-fluoro-2′-deoxyuridine (FdUrd). Previous studies suggest that SW620 cells, which are relatively resistant to FdUrd, have an intact early S-phase checkpoint that protects against FdUrd-induced DNA damage and cytotoxicity and that this checkpoint is defective in the relatively sensitive HT29 cells, which continue to progress through S phase during drug treatment. To test this hypothesis, we examined the expression and activation of known S-phase checkpoint mediators in FdUrd-treated SW620 and HT29 cells. FdUrd induced degradation of cdc25A in SW620, but not HT29 cells, in a manner that correlated with the previously described drug-induced S-phase arrest. This difference, however, could not be attributed to differences in either chk1 activation, which was similar in both cell lines, or chk2 activation, which only occurred in HT29 cells and correlated with uracil misincorporation/misrepair-induced DNA double-stranded breaks. These observations suggest that although FdUrd-induced S-phase arrest and associated cdc25A degradation are impaired in HT29 cells, signaling by ATM/ATR is intact upstream of chk1 and chk2. Finally, FdUrd induced premature mitotic entry, a phenomenon associated with deregulated cdc25A expression, in HT29 but not SW620 cells. Blocking cdc25A expression in HT29 cells with small interfering RNA attenuated FdUrd-induced premature mitotic entry, suggesting that progression of HT29 cells through S phase during drug treatment results in part from the inability of these cells to degrade cdc25A in response to FdUrd-induced DNA damage.
In Vitro
Characterization of Enzymatic Properties and Inhibition of the p53R2 Subunit of Human Ribonucleotide Reductase
Authors: Shao Jimin , Zhou Bingsen , Zhu Lijun , Qiu Weihua , Yuan Yate-Ching , Xi Bixin , Yen Yun
Published: September 2005

p53R2 is a newly identified subunit of ribonucleotide reductase (RR) and plays a crucial role in supplying precursors for DNA repair in a p53-dependent manner. In our current work, all three human RR subunit proteins (p53R2, hRRM2, and hRRM1) were prokaryotically expressed and highly purified. Using an in vitro [3H]CDP reduction assay, the activity of RR reconstituted with either p53R2 or hRRM2 was found to be time, concentration, and hRRM1 dependent. The kinetic activity of p53R2-containing RR was about 20–50% lower than that of hRRM2-containing RR. Using a synthetic heptapeptide to inhibit RR activity, it was shown that p53R2 bound to hRRM1 through the same COOH-terminal heptapeptide as hRRM2. However, hRRM2 had a 4.76-fold higher binding affinity for hRRM1 than p53R2, which may explain the reduced RR activity of p53R2 relative to hRRM2. Of interest, p53R2 was 158-fold more susceptible to the iron chelator deferoxamine mesylate than hRRM2, although the iron content of the two proteins determined by atomic absorption spectrometer was almost the same. To the contrary, p53R2 was 2.50-fold less sensitive than hRRM2 to the radical scavenger hydroxyurea, whereas EPR showed similar spectra of the tyrosyl radical in two proteins. Triapine, a new RR inhibitor, was equally potent for p53R2 and hRRM2. These inhibition studies showed that the iron center and tyrosyl radical are involved in RR activity for both p53R2 and hRRM2. The susceptibility differences to RR inhibitors between p53R2 and hRRM2 may lead to a new direction in drug design for human cancer treatment.
Effector Function of Human Tumor-Specific CD8 T Cells in Melanoma Lesions: A State of Local Functional Tolerance
Authors: Zippelius Alfred , Batard Pascal , Rubio-Godoy Verena , Bioley Gilles , Liénard Danielle , Lejeune Ferdy , Rimoldi Donata , Guillaume Philippe , Meidenbauer Norbert , Mackensen Andreas , Rufer Nathalie , Lubenow Norbert , Speiser Daniel , Cerottini Jean-Charles , Romero Pedro , Pittet Mikaël J.
Published: September 2005

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.
NY-ESO-1 Expression and Immunogenicity in Malignant and Benign Breast Tumors
Authors: Sugita Yurika , Wada Hisashi , Fujita Shoichiro , Nakata Tetsuya , Sato Shuichiro , Noguchi Yuji , Jungbluth Achim A. , Yamaguchi Masae , Chen Yao-Tseng , Stockert Elisabeth , Gnjatic Sacha , Williamson Barbara , Scanlan Matthew J. , Ono Toshiro , Sakita Isao , Yasui Masayoshi , Miyoshi Yasuo , Tamaki Yasuhiro , Matsuura Nariaki , Noguchi Shinzaburo , Old Lloyd J. , Nakayama Eiichi , Monden Morito
Published: September 2005

NY-ESO-1 is a cancer/testis antigen expressed in normal adult tissues solely in the testicular germ cells of normal adults and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1-expressing cancer. The aim of this study was to determine the frequency of NY-ESO-1 mRNA and protein expression in malignant and benign breast tumors. NY-ESO-1 mRNA expression was detected by conventional reverse transcription-PCR and real-time PCR, and that of the protein expression by immunohistochemistry and Western blot analysis. Expression of NY-ESO-1 mRNA was detected in 37 of 88 (42%) cancer specimens, whereas that of the NY-ESO-1 protein was detected only in 1 mRNA-positive specimen. In the latter case, expression level of NY-ESO-1 mRNA relative to that in the testis was relatively high (75% of testicular expression) and to the other among breast cancer specimens. In benign breast lesions, 21 of 31 (68%) specimens expressed low levels of NY-ESO-1 mRNA. In 1 case of fibroadenoma, NY-ESO-1 mRNA was 8% of the testicular level, and protein was detected by Western blot analysis. Only 1 breast cancer patient had detectable antibody at time of surgery, which disappeared within 2 years. Tumor specimen from this patient was both NY-ESO-1 mRNA and protein positive, and NY-ESO-1-specific CD8 T cells were detected in this patient by IFN-γ enzyme-linked immunospot assay using NY-ESO-1 recombinant adeno and vaccinia virus. A higher rate of NY-ESO-1 expression was noted in breast cancer with high histological grade and negative hormone receptor status, suggesting NY-ESO-1 as a potential tumor antigen for immunotherapy in patients with breast cancer and poor prognosis.
Luteolin Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis; Inhibition of Endothelial Cell Survival and Proliferation by Targeting Phosphatidylinositol 3′-Kinase Activity
Authors: Bagli Eleni , Stefaniotou Maria , Morbidelli Lucia , Ziche Marina , Psillas Konstantinos , Murphy Carol , Fotsis Theodore
Published: September 2005

In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have previously shown that certain flavonoids inhibit in vitro angiogenesis. Here, we show that the flavonoid luteolin inhibited tumor growth and angiogenesis in a murine xenograft model. Furthermore, luteolin inhibited vascular endothelial growth factor (VEGF)-induced in vivo angiogenesis in the rabbit corneal assay. In agreement, luteolin inhibited both VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVECs) with an IC50 of about 5 μmol/L. Luteolin inhibited VEGF-induced phosphatidylinositol 3′-kinase (PI3K) activity in HUVECs, and this inhibition was critical for both the antisurvival and antimitotic affects of the compound. Indeed, luteolin abolished VEGF-induced activation of Akt, a downstream target of PI3K conveying both survival and mitotic downstream signals. Because overexpression of a constitutively active form of Akt rescued HUVECs only from the antisurvival effects of luteolin, the result indicated that luteolin targeted mainly the survival signals of the PI3K/Akt pathway. With regard to its antimitotic activity, luteolin inhibited VEGF-induced phosphorylation of p70 S6 kinase (S6K), a downstream effector of PI3K responsible for G1 progression. Indeed, VEGF-induced proliferation of HUVECs was sensitive to rapamycin, an inhibitor of p70 S6K activation. Surprisingly, luteolin did not affect VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, a pathway that is considered important for the mitotic effects of VEGF. Thus, blockade of PI3K by luteolin was responsible for the inhibitory effects of the compound on VEGF-induced survival and proliferation of HUVECs. The antisurvival effects of luteolin were mediated via blockage of PI3K/Akt-dependent pathways, whereas inhibition of the PI3K/p70 S6K pathway mediated the antimitotic effects of the compound.
Slippage of Mitotic Arrest and Enhanced Tumor Development in Mice with
BubR1
Haploinsufficiency
Authors: Dai Wei , Wang Qi , Liu Tongyi , Swamy Malisetty , Fang Yuqiang , Xie Suqing , Mahmood Radma , Yang Yang-Ming , Xu Ming , Rao Chinthalapally V.
Published: September 2005

A compromised spindle checkpoint is thought to play a key role in genetic instability that predisposes cells to malignant transformation. Loss of function mutations of BubR1, an important component of the spindle checkpoint, have been detected in human cancers. Here we show that BubR1+/− mouse embryonic fibroblasts are defective in spindle checkpoint activation, contain a significantly reduced amount of securin and Cdc20, and exhibit a greater level of micronuclei than do wild-type cells. RNA interference-mediated down-regulation of BubR1 also greatly reduced securin level. Moreover, compared with wild-type littermates, BubR1+/− mice rapidly develop lung as well as intestinal adenocarcinomas in response to challenge with carcinogen. BubR1 is thus essential for spindle checkpoint activation and tumor suppression.
A Functional Polymorphism in the
EGF
Gene Is Found with Increased Frequency in Glioblastoma Multiforme Patients and Is Associated with More Aggressive Disease
Authors: Bhowmick Deb A. , Zhuang Zhengping , Wait Scott D. , Weil Robert J.
Published: September 2005

Glioblastoma multiforme, the most aggressive form of primary brain tumor in adults, is nearly universally fatal, with 5-year survivals of <5% (P. Kleihues and W. K. Cavenee, eds., pp. 1–314, Lyon: IARC, 2000). Alterations in the epidermal growth factor receptor (EGFR) are common events in many glioblastoma. We hypothesized that a polymorphism in the 5′-untranslated region of the epidermal growth factor (EGF) gene, a natural ligand of the EGFR, may play a role in the genesis of these malignant gliomas. We find that patients with the GA or GG genotype have higher tumoral levels of EGF, irrespective of EGFR status, that they are more likely to recur after surgery, and that they have a statistically significant shorter overall progression-free survival than patients with the AA genotype. These findings suggest that a single nucleotide polymorphism in EGF may play a role in the formation of glioblastomas, is a useful and powerful prognostic marker for these patients, and may be a target for tumor therapy.
Topoisomerase I-Mediated Inhibition of Hypoxia-Inducible Factor 1
Authors: Rapisarda Annamaria , Uranchimeg Badarch , Sordet Olivier , Pommier Yves , Shoemaker Robert H. , Melillo Giovanni
Published: September 2005

We have shown previously that the camptothecin analogue topotecan (TPT), a topoisomerase I (Top 1) poison, inhibits hypoxia-inducible factor 1 (HIF-1) transcriptional activity and HIF-1α protein accumulation in hypoxia-treated U251 human glioma cells. In this article, we demonstrate that TPT does not affect HIF-1α protein half-life or mRNA accumulation but inhibits its translation. In addition, we demonstrate that Top 1 is required for the inhibition of HIF-1α protein accumulation by TPT as shown by experiments performed using camptothecin-resistant cell lines with known Top 1 alterations. Experiments performed with aphidicolin indicated that TPT inhibited HIF-1 protein accumulation in the absence of DNA replication. DNA-damaging agents, such as ionizing radiation and doxorubicin, did not affect HIF-1α protein accumulation. Ongoing transcription was essential for the inhibition of HIF-1α protein accumulation by TPT. Our results demonstrate the existence of a novel pathway connecting Top 1-dependent signaling events and the regulation of HIF-1α protein expression and function. In addition, our findings dissociate the cytotoxic activity of TPT from the inhibition of the HIF-1 pathway and raise the possibility of novel clinical applications of TPT aimed at targeting HIF-1-dependent responses.
SHP-2-Dependent Mitogen-Activated Protein Kinase Activation Regulates EGFRvIII but not Wild-Type Epidermal Growth Factor Receptor Phosphorylation and Glioblastoma Cell Survival
Authors: Zhan Yi , O’Rourke Donald M.
Published: September 2005

In human glioblastomas, the most common mutation of epidermal growth factor receptor (EGFR) is an in-frame deletion of an 801-bp sequence in the extracellular domain of EGFR termed EGFRvIII. The EGFRvIII does not bind ligand EGF but has constitutive tyrosine phosphorylation (pTyr) content and kinase activity that result in enhanced transformation, reduced apoptosis, and resistance to therapy. Here we report that the protein tyrosine phosphatase SHP-2 modulates a mitogen-activated protein kinase (MAPK) kinase (MEK)-mediated signaling pathway that regulates EGFRvIII pTyr and cell survival in U87MG.EGFRvIII cells. Overexpression of the phosphatase-inactive form of SHP-2 inhibited EGFRvIII pTyr by decreasing MAPK phosphorylation. Consistent with this, we observed that the MEK inhibitor PD98059, but not the phosphatidylinositol 3′-kinase inhibitor LY294002, inhibited EGFRvIII pTyr. Furthermore, constitutive EGFRvIII pTyr content observed in U87MG, LN229, and U373MG glioblastoma cells, but not in NR6.EGFRvIII fibroblasts, correlated with elevated MAPK levels in these cells. Interestingly, LY294002, but not PD98059, inhibited wild-type EGFR pTyr in response to EGF treatment in U87MG parental cells and in wild-type EGFR-overexpressing U87MG cells. Inhibition of EGFRvIII pTyr by PD98059 was not observed to be phosphorylation site specific. However, LY294002 more specifically inhibited wild-type EGFR pTyr at residues Tyr992 and Tyr1068 in the COOH terminus. Treatment of U87MG.EGFRvIII cells with PD98059, but not LY294002, also resulted in increased cell death in response to cisplatin. Collectively, a distinct MEK-mediated pathway in human glioblastoma cells appears to differentially modulate EGFRvIII and wild-type EGFR pTyr, and inhibition of the MAPK pathway sensitizes EGFRvIII-containing human glioblastoma cells to cisplatin-induced cell death.
Epidermal Growth Factor Receptor-Mediated Activation of Stat3 during Multistage Skin Carcinogenesis
Authors: Chan Keith Syson , Carbajal Steve , Kiguchi Kaoru , Clifford John , Sano Shigetoshi , DiGiovanni John
Published: September 2005

In the present study, we have investigated the possible role of signal transducers and activators of transcription (STATs), particularly Stat3, in mouse skin tumor promotion and multistage carcinogenesis. Stat1, Stat3, and Stat5 were activated in mouse epidermis after treatment with different classes of tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, and chrysarobin. In addition, Stat1, Stat3, and Stat5 were constitutively activated in skin tumors generated by the two-stage carcinogenesis regimen using 7,12-dimethylbenz(a)anthracene as initiator and TPA as promoter. Several approaches were used to examine the possible role of epidermal growth factor receptor (EGFR) in modulating Stat3 activity during tumor promotion. In primary cultures of mouse keratinocytes, addition of exogenous EGF led to activation of Stat3 as shown by an elevation in tyrosine phosphorylation and nuclear translocation. In epidermis of transgenic mice expressing transforming growth factor α under control of the keratin 14 promoter, Stat3 was constitutively activated. Abrogation of EGFR function in mouse epidermis using an EGFR kinase inhibitor or by overexpressing a dominant negative form of EGFR led to a reduction in Stat3 activation in response to TPA treatment. Immunoprecipitation analyses using lysates from TPA-treated epidermis and skin papillomas showed enhanced interaction between the EGFR and Stat3. Finally, Stat3 deficiency in mouse epidermis significantly reduced the proliferative response after TPA treatment. Collectively, the current results suggest that Stat3 activation may be a critical event during mouse skin tumor promotion, possibly through regulation of keratinocyte proliferation. In addition, Stat3 activation in tumor promoter-treated epidermis and in skin papillomas may occur, at least in part, via interaction with and phosphorylation by the EGFR. Finally, constitutive activation of Stat3 in both papillomas and squamous cell carcinomas suggest a role in both the development of autonomous growth and the progression of epithelial tumors in mouse skin.
An Unsupervised Approach to Identify Molecular Phenotypic Components Influencing Breast Cancer Features
Authors: Selaru Florin M. , Yin Jing , Olaru Andreea , Mori Yuriko , Xu Yan , Epstein Steven H. , Sato Fumiaki , Deacu Elena , Wang Suna , Sterian Anca , Fulton Amy , Abraham John M. , Shibata David , Baquet Claudia , Stass Sanford A. , Meltzer Stephen J.
Published: September 2005

To discover a biological basis for clinical subgroupings within breast cancers, we applied principal components (PCs) analysis to cDNA microarray data from 36 breast cancers. We correlated the resulting PCs with clinical features. The 35 PCs discovered were ranked in order of their impact on gene expression patterns. Interestingly, PC 7 identified a unique subgroup consisting of estrogen receptor (ER); (+) African-American patients. This group exhibited global molecular phenotypes significantly different from both ER (−) African-American women and ER (+) or ER (−) Caucasian women (P < 0.001). Additional significant PCs included PC 4, correlating with lymph node metastasis (P = 0.04), and PC 10, with tumor stage (stage 2 versus stage 3; P = 0.007). These results provide a molecular phenotypic basis for the existence of a biologically unique subgroup comprising ER (+) breast cancers from African-American patients. Moreover, these findings illustrate the potential of PCs analysis to detect molecular phenotypic bases for relevant clinical or biological features of human tumors in general.
Overexpression of ADAM9 in Non-Small Cell Lung Cancer Correlates with Brain Metastasis
Authors: Shintani Yasushi , Higashiyama Shigeki , Ohta Mitsunori , Hirabayashi Hirohisa , Yamamoto Sakae , Yoshimasu Tatsuya , Matsuda Hikaru , Matsuura Nariaki
Published: September 2005

The “a disintegrin and metalloprotease” (ADAM) family contributes to regulation of the cell–cell and cell–matrix interactions that are critical determinants of malignancy. To determine the relationship between metastasis and ADAM proteins, we compared the mRNA levels of ADAM9, -10, -12, -15, and -17 in sublines of an EBC-1 lung cancer cell line that were highly metastatic to either brain or bone. ADAM9 mRNA levels were significantly higher in highly brain-metastatic sublines than in the parent or highly bone-metastatic sublines. To elucidate the role of ADAM9 in brain metastasis, we stably transfected A549 and EBC-1 cells with a full-length ADAM9 expression vector. Compared with mock-transfectants, ADAM9 overexpression resulted in increased invasive capacity in response to nerve growth factor, increased adhesion to brain tissue, and increased expression of integrin α3 and β1 subunits. Administration of the anti-β1 monoclonal antibody attenuated this increase in invasive and adhesive activity. Intravenous administration of ADAM9-overexpressing A549 cells to mice resulted in micrometastatic foci in the brain and multiple metastatic colonies in the lungs. In contrast, administration of parent and mock-transfected A549 cells to mice resulted in lung tumors without brain metastasis. These results suggest that ADAM9 overexpression enhances cell adhesion and invasion of non-small cell lung cancer cells via modulation of other adhesion molecules and changes in sensitivity to growth factors, thereby promoting metastatic capacity to the brain.
Capsaicin Inhibits
in Vitro
and
in Vivo
Angiogenesis
Authors: Min Jeong-Ki , Han Kyu-Yeon , Kim Eok-Cheon , Kim Young-Myeong , Lee Sae-Won , Kim Ok-Hee , Kim Kyu-Won , Gho Yong Song , Kwon Young-Guen
Published: September 2005

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a natural product of Capsicum species, is known to induce excitation of nociceptive terminals involved in pain perception. Recent studies have also shown that capsaicin not only has chemopreventive properties against certain carcinogens and mutagens but also exerts anticancer activity. Here, we demonstrated the antiangiogenic activity of capsaicin using in vitro and in vivo assay systems. In vitro, capsaicin inhibited vascular endothelial growth factor (VEGF) -induced proliferation, DNA synthesis, chemotactic motility, and capillary-like tube formation of primary cultured human endothelial cells. Capsaicin inhibited both VEGF-induced vessel sprouting in rat aortic ring assay and VEGF-induced vessel formation in the mouse Matrigel plug assay. Moreover, capsaicin was able to suppress tumor-induced angiogenesis in chick chorioallantoic membrane assay. Capsaicin caused G1 arrest in endothelial cells. This effect correlated with the down-regulation of the expression of cyclin D1 that led to inhibition of cyclin-dependent kinase 4-mediated phosphorylation of retinoblastoma protein. Signaling experiments show that capsaicin inhibits VEGF-induced p38 mitogen-activated protein kinase, p125FAK, and AKT activation, but its molecular target is distinct from the VEGF receptor KDR/Flk-1. Taken together, these results demonstrate that capsaicin is a novel inhibitor of angiogenesis and suggest that it may be valuable to develop pharmaceutical drugs for treatment of angiogenesis-dependent human diseases such as tumors.
A Susceptibility Locus at Chromosome 3p21 Linked to Familial Nasopharyngeal Carcinoma
Authors: Xiong Wei , Zeng Zhao Yang , Xia Jia Hui , Xia Kun , Shen Shou Rong , Li Xiao Ling , Hu Dong Xu , Tan Chen , Xiang Juan Juan , Zhou Jie , Deng Hao , Fan Song Qing , Li Wei Fang , Wang Rong , Zhou Ming , Zhu Shi Guo , Lü Hong Bin , Qian Jun , Zhang Bi Cheng , Wang Jie Ru , Ma Jian , Xiao Bing Yi , Huang He , Zhang Qiu Hong , Zhou Yan Hong , Luo Xiao Min , De Zhou Hou , Yang Yi Xin , Dai He Ping , Feng Guo Yin , Pan Qian , Wu Ling Qian , He Lin , Li Gui Yuan
Published: September 2005

Nasopharyngeal carcinoma (NPC) poses one of the serious health problems in southern Chinese, with an incidence rate ranging from 15 to 50/100,000. Chromosome translocation t(1;3) and frequent loss of heterogeneity on short arms of chromosome 3 and 9 have been reported to be associated with NPC, and a genome-wide scan identified an NPC susceptibility locus on chromosome 4p15.1-q12 recently. In our study, we collected samples from 18 families at high risk of NPC from the Hunan province in southern China, genotyped with a panel of polymorphic markers on short arms of chromosomes 3, 9, and 4p15.1-q12. A locus on 3p21 was identified to link to NPC with a maximum logarithm of odds for linkage score of 4.18. Fine mapping located the locus to a 13.6-cM region on 3p21.31-21.2, where a tumor suppressor gene cluster resided. Our findings identified a novel locus for NPC and provided a map location for susceptibility genes candidates. In contrast to a recent study, no significant evidence for NPC linkage to chromosomes 4 and 9 was observed.
Detection of Primary Lung Tumors in Rodents by Magnetic Resonance Imaging
Authors: Garbow Joel R. , Zhang Zhongqiu , You Ming
Published: September 2005

This report describes recent efforts to develop and apply small animal magnetic resonance imaging methods to the study of lung tumors in mice. Magnetic resonance (MR) images obtained with respiratory gating, with data collection synchronized with the respiration of the animal, allow visualization of submillimeter tumors in animals treated with a lung carcinogen. Comparison of the MR images with gross pathology of these lungs demonstrates the utility of the imaging methods for measuring tumor burden. As a noninvasive imaging modality that uses nonionizing radiation, MR is well suited to longitudinal studies aimed at understanding the factors that control the onset and development of lung tumors and their response to therapy in a wide variety of animal models.
Cyclooxygenase-2-Dependent Expression of Angiogenic CXC Chemokines ENA-78/CXC Ligand (CXCL) 5 and Interleukin-8/CXCL8 in Human Non-Small Cell Lung Cancer
Authors: Põld Mehis , Zhu Li X. , Sharma Sherven , Burdick Marie D. , Lin Ying , Lee Peter P. N. , Põld Anu , Luo Jie , Krysan Kostyantyn , Dohadwala Mariam , Mao Jenny T. , Batra Raj K. , Strieter Robert M. , Dubinett Steven M.
Published: September 2005

Elevated tumor cyclooxygenase (COX)-2 activity plays a multifaceted role in non-small cell lung cancer (NSCLC). To elucidate the role of COX-2 in the in vitro and in vivo expression of two known NSCLC angiogenic peptides, CXC ligand (CXCL) 8 and CXCL5, we studied two COX-2 gene-modified NSCLC cell lines, A549 and H157. COX-2 overexpression enhanced the in vitro expression of both CXCL8 and CXCL5. In contrast, specific COX-2 inhibition decreased the production of both peptides as well as nuclear translocation of nuclear factor κB. In a severe combined immunodeficient mouse model of human NSCLC, the enhanced tumor growth of COX-2-overexpressing tumors was inhibited by neutralizing anti-CXCL5 and anti-CXCL8 antisera. We conclude that COX-2 contributes to the progression of NSCLC tumorigenesis by enhancing the expression of angiogenic chemokines CXCL8 and CXCL5.
TheEws/Fli-1Fusion Gene Switches the Differentiation Program of Neuroblastomas to Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumors
Authors: Rorie Checo J. , Thomas Venetia D. , Chen Pengchin , Pierce Heather Hanson , O’Bryan John P. , Weissman Bernard E.
Published: September 2005

Neuroblastoma (NB) and the Ewing sarcoma (ES)/peripheral primitive neuroectodermal tumor (PNET) family are pediatric cancers derived from neural crest cells. Although NBs display features of the sympathetic nervous system, ES/PNETs express markers consistent with parasympathetic differentiation. To examine the control of these differentiation markers, we generated NB × ES/PNET somatic cell hybrids. NB-specific markers were suppressed in the hybrids, whereas ES/PNET-specific markers were unaffected. These results suggested that the Ews/Fli-1 fusion gene, resulting from a translocation unique to ES/PNETs, might account for the loss of NB-specific markers. To test this hypothesis, we generated two different NB cell lines that stably expressed the Ews/Fli-1 gene. We observed that heterologous expression of the Ews/Fli-1 protein led to the suppression of NB-specific markers and de novo expression of ES/PNET markers. To determine the extent of changes in differentiation, we used the Affymetrix GeneChip Array system to observe global transcriptional changes of genes. This analysis revealed that the gene expression pattern of the Ews/Fli-1-expressing NB cells resembled that observed in pooled ES/PNET cell lines and differed significantly from the NB parental cells. Therefore, we propose that Ews/Fli-1 contributes to the etiology of ES/PNET by subverting the differentiation program of its neural crest precursor cell to a less differentiated and more proliferative state.
Phosphorylation of Tuberin as a Novel Mechanism for Somatic Inactivation of the Tuberous Sclerosis Complex Proteins in Brain Lesions
Authors: Han Sangyeul , Santos Túlio M. , Puga Ana , Roy Jenn , Thiele Elizabeth A. , McCollin Mia , Stemmer-Rachamimov Anat , Ramesh Vijaya
Published: September 2005

Tuberous sclerosis complex is caused by mutations in tumor suppressor genes TSC1 or TSC2 and is characterized by the presence of hamartomas in many organs. Although tuberous sclerosis complex is a tumor suppressor gene syndrome with classic “second hits” detectable in renal tumors, conventional genetic analysis has not revealed somatic inactivation of the second allele in the majority of human brain lesions. We demonstrate a novel mechanism of post-translational inactivation of the TSC2 protein, tuberin, by physiologically inappropriate phosphorylation, which is specific to tuberous sclerosis complex-associated brain lesions. Additional analysis shows that tissue specificity is due to abnormal activation of the Akt and mitogen-activated protein kinase pathways in brain but not in renal tumors. These results have widespread implications for understanding the tissue specificity of tumor suppressor gene phenotypes.