bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet-Derived Growth Factor Production by B16 Melanoma Cells Leads to Increased Pericyte Abundance in Tumors and an Associated Increase in Tumor Growth Rate
Authors: Furuhashi Masao , Sjöblom Tobias , Abramsson Alexandra , Ellingsen Jens , Micke Patrick , Li Hong , Bergsten-Folestad Erika , Eriksson Ulf , Heuchel Rainer , Betsholtz Christer , Heldin Carl-Henrik , Östman Arne
Published: September 2005

Platelet-derived growth factor (PDGF) receptor signaling participates in different processes in solid tumors, including autocrine stimulation of tumor cell growth, recruitment of tumor stroma fibroblasts, and stimulation of tumor angiogenesis. In the present study, the B16 mouse melanoma tumor model was used to investigate the functional consequences of paracrine PDGF stimulation of host-derived cells. Production of PDGF-BB or PDGF-DD by tumor cells was associated with an increased tumor growth rate. Characterization of tumors revealed an increase in pericyte abundance in tumors derived from B16 cells producing PDGF-BB or PDGF-DD. The increased tumor growth rate associated with PDGF-DD production was not seen in mice expressing an attenuated PDGF β-receptor and was thus dependent on host PDGF β-receptor signaling. The increased pericyte abundance was not associated with an increased tumor vessel density. However, tumor cell apoptosis, but not proliferation, was reduced in tumors displaying PDGF-induced increased pericyte coverage. Our findings thus demonstrate that paracrine PDGF production stimulates pericyte recruitment to tumor vessels and suggest that pericyte abundance influences tumor cell apoptosis and tumor growth.
Adjuvant-Mediated Tumor Regression and Tumor-Specific Cytotoxic Response Are Impaired in MyD88-Deficient Mice
Authors: Akazawa Takashi , Masuda Hisayo , Saeki Yoshiko , Matsumoto Misako , Takeda Kiyoshi , Tsujimura Kunio , Kuzushima Kiyotaka , Takahashi Toshitada , Azuma Ichiro , Akira Shizuo , Toyoshima Kumao , Seya Tsukasa
Published: September 2005

The Mycobacterium bovis bacillus Calmette-Guérin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN. BCG-CWS has been used for adjuvant immunotherapy for patients with cancer. We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/6). We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88−/− mice immunized with tumor debris and/or BCG-CWS. Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor. BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-α) induction. Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity. Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.
Preferential Inhibition of Akt and Killing of Akt-Dependent Cancer Cells by Rationally Designed Phosphatidylinositol Ether Lipid Analogues
Authors: Castillo S. Sianna , Brognard John , Petukhov Pavel A. , Zhang Chunyu , Tsurutani Junji , Granville Courtney A. , Li Min , Jung Michael , West Kip A. , Gills Joell G. , Kozikowski Alan P. , Dennis Phillip A.
Published: September 2005

Activation of the PI3k/Akt pathway controls key cellular processes and contributes to tumorigenesis in vivo, but investigation of the PI3k/Akt pathway has been limited by the lack of specific inhibitors directed against Akt. To develop Akt inhibitors, we used molecular modeling of the pleckstrin homology (PH) domain of Akt to guide synthesis of structurally modified phosphatidylinositol ether lipid analogues (PIAs). Here, we characterize the biochemical and cellular effects of PIAs. Of 24 compounds tested, five PIAs with modifications at two sites on the inositol ring inhibited Akt with IC50s < 5 μm. Molecular modeling identified putative interactions of PIAs with the phosphoinositide-binding site in the PH domain of Akt, and growth factor-induced translocation of Akt to the plasma membrane was inhibited by PIA administration. Inhibition of Akt occurred rapidly and was maintained for hours. PIAs decreased phosphorylation of many downstream targets of Akt without affecting upstream kinases, such as PI3k or phosphoinositide-dependent kinase-1, or members of other kinase pathways such as extracellular signal-regulated kinase. Importantly, PIAs increased apoptosis 20–30-fold in cancer cell lines with high levels of endogenous Akt activity but only 4–5-fold in cancer cell lines with low levels of Akt activity. These studies identify PIAs as effective Akt inhibitors, and provide proof of principle for targeting the PH domain of Akt.
B-Cell Receptor Translocation to Lipid Rafts and Associated Signaling Differ between Prognostically Important Subgroups of Chronic Lymphocytic Leukemia
Authors: Allsup David J. , Kamiguti Aura S. , Lin Ke , Sherrington Paul D. , Matrai Zoltan , Slupsky Joseph R. , Cawley John C. , Zuzel Mirko
Published: September 2005

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which interaction of the malignant cells with antigen is thought to play a key role. Individual CLL-cell clones markedly differ in their ability to respond to B-cell receptor ligation, but the mechanism underlying the frequent hyporesponsiveness is incompletely understood. Our aim was to further clarify the extent and cause of the B-cell receptor signaling abnormality in CLL and to assign pathophysiologic relevance to the presence or absence of B-cell receptor responsiveness. We show that extracellular signal-regulated kinase-2 phosphorylation, intracellular Ca2+ increases, CD79a phosphorylation, and translocation of the B-cell receptor to lipid rafts in response to ligation with anti–immunoglobulin M (as a surrogate for antigen) are features of CLL cells with relatively unmutated VH genes (<5% deviation from germ line) and a poor prognosis. B-cell receptor stimulation in these cases also promoted cell survival. In clones with mutated VH genes (>5% deviation from germ line), surface immunoglobulin M ligation failed to induce receptor translocation to rafts or to prolong cell survival. This failure of receptor translocation observed in mutated CLL cells was associated with the constitutive exclusion of the B-cell receptor from rafts by a mechanism involving src-dependent interactions between the B-cell receptor and the actin cytoskeleton. We conclude that exposure to antigen promotes the survival of unmutated CLL clones, contributing to the poor prognosis of this group. In contrast, hyporesponsive mutated CLL clones may have developed into a stage where continuous exposure to antigen results in relative tolerance to antigenic stimulation mediated by the exclusion of the B-cell receptor from lipid rafts.
Induction of Apoptosis in Leukemic Cells by Homovanillic Acid Derivative, Capsaicin, through Oxidative Stress
Authors: Ito Keisuke , Nakazato Tomonori , Yamato Kenji , Miyakawa Yoshitaka , Yamada Taketo , Hozumi Nobumichi , Segawa Kaoru , Ikeda Yasuo , Kizaki Masahiro
Published: September 2005

Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is a homovanillic acid derivative found in pungent fruits. Several investigators have reported the ability of capsaicin to inhibit events associated with the promotion of cancer. However, the effects of capsaicin on human leukemic cells have never been investigated. We investigated the effects of capsaicin on leukemic cells in vitro and in vivo and further examined the molecular mechanisms of capsaicin-induced apoptosis in myeloid leukemic cells. Capsaicin suppressed the growth of leukemic cells, but not normal bone marrow mononuclear cells, via induction of G0-G1 phase cell cycle arrest and apoptosis. Capsaicin-induced apoptosis was in association with the elevation of intracellular reactive oxygen species production. Interestingly, capsaicin-sensitive leukemic cells were possessed of wild-type p53, resulting in the phosphorylation of p53 at the Ser-15 residue by the treatment of capsaicin. Abrogation of p53 expression by the antisense oligonucleotides significantly attenuated capsaicin-induced cell cycle arrest and apoptosis. Pretreatment with the antioxidant N-acetyl-l-cystein and catalase, but not superoxide dismutase, completely inhibited capsaicin-induced apoptosis by inhibiting phosphorylation of Ser-15 residue of p53. Moreover, capsaicin effectively inhibited tumor growth and induced apoptosis in vivo using NOD/SCID mice with no toxic effects. We conclude that capsaicin has potential as a novel therapeutic agent for the treatment of leukemia.
Insulin-Like Growth Factor-Binding Protein 3 Induces Caspase-Dependent Apoptosis through a Death Receptor-Mediated Pathway in MCF-7 Human Breast Cancer Cells
Authors: Kim Ho-Seong , Ingermann Angela R. , Tsubaki Junko , Twigg Stephen M. , Walker Gillian E. , Oh Youngman
Published: September 2005

Insulin-like growth factor-binding protein (IGFBP)-3 has been shown to potently inhibit cell proliferation in various cell systems. However, the specific mechanisms involved in the antiproliferative action of IGFBP-3 have yet to be elucidated. In the present study, we demonstrate that IGFBP-3 induces apoptosis in an insulin-like growth factor (IGF)-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. Induction of IGFBP-3 using an ecdysone-inducible expression system inhibited DNA synthesis in an IGF-IGF receptor axis-independent fashion and resulted in the subsequent induction of apoptosis and an increase in caspase activity. Similar results were obtained when cells were transfected with GGG-IGFBP-3, an IGFBP-3 mutant unable to bind IGFs, corroborating the IGF-independent action of IGFBP-3. Additional caspase activity studies and immunoblot analyses using specific caspase substrates and/or caspase inhibitors revealed that the growth-inhibitory effect of IGFBP-3 results mainly from its induction of apoptosis (in particular, activation of caspase-8 and -7). Analyses of caspase-9 activity and release of cytochrome c into the cytosol confirmed that the mitochondria-mediated pathway is not involved. Taken together, these results show that IGFBP-3 expression leads to the induction of apoptosis through the activation of caspases involved in a death receptor-mediated pathway and that IGFBP-3 functions as a negative regulator of breast cancer cell growth, independent of the IGF-IGF receptor axis.
CD47 Mediates Killing of Breast Tumor Cells via Gi-Dependent Inhibition of Protein Kinase A
Authors: Manna Partha Pratim , Frazier William A.
Published: September 2005

Thrombospondins (TSPs) have been implicated as antitumor and antimetastasis factors in breast cancer. Although this effect has been attributed to the antiangiogenic activity of TSPs, recent observations suggest other mechanisms may be at work. The TSP receptor CD47 (integrin-associated protein) has recently been reported to mediate a novel form of apoptosis. Here, we have studied the response of breast cancer cells to CD47 ligands TSP-1, the CD47 agonist peptide 4N1K derived from TSP-1, and the anti-CD47 monoclonal antibody 1F7. All of these ligands killed four different breast cancer cell lines. This CD47-mediated cell death did not require active caspases or Bcl-2 degradation and did not cause DNA laddering or cytochrome c release. Pertussis toxin (PTX) prevented CD47-mediated death, indicating the involvement of Giα. 4N1K dramatically reduced intracellular cAMP levels, an effect reversed with PTX. Forskolin, 8-bromo cAMP, and isobutylmethylxanthine (IBMX) all prevented CD47-mediated apoptosis, indicating the involvement of cAMP. H89 and protein kinase A (PKA) inhibitor peptide prevented rescue of breast cancer cells by PTX, 8-Br-cAMP, and forskolin, suggesting that the effects of cAMP are mediated via PKA-dependent phosphorylation events. Epidermal growth factor also inhibited CD47-induced apoptosis via a PKC-dependent but ERK-independent pathway. Thus, CD47-mediated killing of breast cancer cells occurs by a novel pathway involving regulation of cAMP levels by heterotrimeric Gi with subsequent effects mediated by PKA.
Prion Protein Prevents Human Breast Carcinoma Cell Line from Tumor Necrosis Factor α-Induced Cell Death
Authors: Diarra-Mehrpour Maryam , Arrabal Samuel , Jalil Abdelali , Pinson Xavier , Gaudin Catherine , Piétu Geneviève , Pitaval Amandine , Ripoche Hugues , Eloit Marc , Dormont Dominique , Chouaib Salem
Published: September 2005

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor α (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrPc), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrPc is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrPc, our data demonstrate that PrPc overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.
Endothelial Cell Surface ATP Synthase-Triggered Caspase-Apoptotic Pathway Is Essential for K1-5-Induced Antiangiogenesis
Authors: Veitonmäki Niina , Cao Renhai , Wu Lin-Hua , Moser Tammy L. , Li Bo , Pizzo Salvatore V. , Zhivotovsky Boris , Cao Yihai
Published: September 2005

We have recently reported the identification of kringle 1-5 (K1-5) of plasminogen as a potent and specific inhibitor of angiogenesis and tumor growth. Here, we show that K1-5 bound to endothelial cell surface ATP synthase and triggered caspase-mediated endothelial cell apoptosis. Induction of endothelial apoptosis involved sequential activation of caspases-8, -9, and -3. Administration of neutralizing antibodies directed against the α- and β-subunits of ATP synthase to endothelial cells attenuated activation of these caspases. Furthermore, inhibitors of caspases-3, -8, and -9 also remarkably blocked K1-5-induced endothelial cell apoptosis and antiangiogenic responses. In a mouse tumor model, we show that caspase-3 inhibitors abolished the antitumor activity of K1-5 by protecting the tumor vasculature undergoing apoptosis. These results suggest that the specificity of the antiendothelial effect of K1-5 is attributable, at least in part, to its interaction with the endothelial cell surface ATP synthase and that the caspase-mediated endothelial apoptosis is essential for the angiostatic activity of K1-5. Thus, our findings provide a mechanistic insight with respect to the angiostatic action and signaling pathway of K1-5 and angiostatin.
Pivotal Role of CXCR3 in Melanoma Cell Metastasis to Lymph Nodes
Authors: Kawada Kenji , Sonoshita Masahiro , Sakashita Hiromi , Takabayashi Arimichi , Yamaoka Yoshio , Manabe Toshiaki , Inaba Kayo , Minato Nagahiro , Oshima Masanobu , Taketo Makoto Mark
Published: September 2005

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis to specific organs. Here we show that mouse B16F10 melanoma cells constitutively express chemokine receptor CXCR3, and that its ligands CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC induce cellular responses in vitro, such as actin polymerization, migration, invasion, and cell survival. To determine whether CXCR3 could play a role in metastasis to lymph nodes (LNs), we constructed B16F10 cells with reduced CXCR3 expression by antisense RNA and investigated their metastatic activities after s.c. inoculations to syngeneic hosts, C57BL/6 mice. The metastatic frequency of these cells to LNs was markedly reduced to ∼15% (P < 0.05) compared with the parental or empty vector-transduced cells. On the other hand, pretreatment of mice with complete Freund’s adjuvant increased the levels of CXCL9 and CXCL10 in the draining LNs, which caused 2.5–3.0-fold increase (P < 0.05) in the metastatic frequency of B16F10 cells to the nodes with much larger foci. Importantly, such a stimulation of metastasis was largely suppressed when CXCR3 expression in B16F10 cells was reduced by antisense RNA or when mice were treated with specific antibodies against CXCL9 and CXCL10. We also demonstrate that CXCR3 is expressed on several human melanoma cell lines as well as primary human melanoma tissues (5 of 9 samples tested). These results suggest that CXCR3 inhibitors may be promising therapeutic agents for treatment of LN metastasis, including that of melanoma.
Enhanced Tumor Formation in Cyclin D1 × Transforming Growth Factor β1 Double Transgenic Mice with Characterization by Magnetic Resonance Imaging
Authors: Deane Natasha G. , Lee Haakil , Hamaamen Jalal , Ruley Anna , Washington M. Kay , LaFleur Bonnie , Thorgeirsson Snorri S. , Price Ronald , Beauchamp R. Daniel
Published: September 2005

Transgenic mice that overexpress cyclin D1 protein in the liver develop liver carcinomas with high penetrance. Transforming growth factor β (TGF-β) serves as either an epithelial cell growth inhibitor or a tumor promoter, depending on the cellular context. We interbred LFABP-cyclin D1 and Alb-TGF-β1 transgenic mice to produce cyclin D1/TGF-β1 double transgenic mice and followed the development of liver tumors over time, characterizing cellular and molecular changes, tumor incidence, tumor burden, and tumor physiology noninvasively by magnetic resonance imaging. Compared with age-matched LFABP-cyclin D1 single transgenic littermates, cyclin D1/TGF-β1 mice exhibited a significant increase in tumor incidence. Tumor multiplicity, tumor burden, and tumor heterogeneity were higher in cyclin D1/TGF-β1 mice compared with single transgenic littermates. Characteristics of cyclin D1/TGF-β1 livers correlated with a marked induction of the peripheral periductal oval cell/stem cell compartment of the liver. A number of cancerous lesions from cyclin D1/TGF-β1 mice exhibited unique features such as ductal plate malformations and hemorrhagic nodules. Some lesions were contiguous with the severely diseased background liver and, in some cases, replaced the normal architecture of the entire organ. Cyclin D1/TGF-β1 lesions, in particular, were associated with malignant features such as areas of vascular invasion by hepatocytes and heterogeneous hyperintensity of signal on T2-weighted magnetic resonance imaging. These findings demonstrate that TGF-β1 promotes stem cell activation and tumor progression in the context of cyclin D1 overexpression in the liver.
Serine/Threonine Kinase AKT Is Frequently Activated in Human Bile Duct Cancer and Is Associated with Increased Radioresistance
Authors: Tanno Satoshi , Yanagawa Nobuyuki , Habiro Atsuya , Koizumi Kazuya , Nakano Yasuhiro , Osanai Manabu , Mizukami Yusuke , Okumura Toshikatsu , Testa Joseph R. , Kohgo Yutaka
Published: September 2005

The prognosis for patients with bile duct cancer (BDC) remains poor. Although BDC cells are essentially radioresistant, recent reports have suggested that radiation therapy, in addition to its palliative role in the management of BDC, may improve patient survival. A better understanding of the mechanisms that lead to cellular radioresistance may assist in the development of more effective BDC therapies based on radiotherapy in combination with radiosensitizing agents. The serine/threonine kinase AKT/protein kinase B, a downstream effector of phosphatidylinositol 3′-kinase, is a well-characterized kinase that is known to play a critical role in antiapoptotic signaling pathways. In this investigation, we sought to clarify the role of AKT signaling in the radioresistance in BDC cells. First, to examine whether activated AKT is expressed in BDCs, tumor specimens were obtained from 19 consecutive BDC cases. Immunohistochemical staining using an anti-phosphorylated-AKT antibody showed that phosphorylated (activated) AKT was expressed in cancer cells but not in neighboring normal mucosa in 16 cases (84.2%). Next, to evaluate the role of AKT activation in the regulation of BDC cell radiosensitivity, clonogenic assays were performed using the phosphatidylinositol 3′-kinase inhibitor LY294002 with and without irradiation. LY294002 inhibited AKT activation in BDC cells and, on irradiation, decreased clonogenic survival in a radiation dose-dependent manner. Only a small decrease in cell viability was observed in cells exposed to LY294002. Expression of constitutively active AKT in BDC cells resulted in decreased radiosensitivity, whereas a dominant-negative AKT increased radiosensitivity. Furthermore, constitutively active AKT also inhibited radiation-induced apoptosis. Collectively, these results indicate that activated AKT in BDC cells is associated with radioresistance and suggest that pharmacological or genetic modulation of AKT activity may have important therapeutic implications in BDC patients treated with radiation.
Predictive Impact of Urokinase-Type Plasminogen Activator
Authors: Manders Peggy , Tjan-Heijnen Vivianne C. G. , Span Paul N. , Grebenchtchikov Nicolai , Foekens John A. , Beex Louk V. A. M. , Sweep C. G. J. (Fred)
Published: September 2005

One of the most thoroughly studied systems in relation to its prognostic relevance in patients with breast cancer, is the plasminogen activation system. This system comprises of, among others, the urokinase-type plasminogen activator (uPA) and its main inhibitor (PAI-1). In this study we investigated whether the uPA:PAI-1 complex is associated with the responsiveness of patients with primary breast cancer to adjuvant systemic therapy.

Quantitative enzyme-linked immunosorbent assays were used to assess the levels of uPA, PAI-1, and uPA:PAI-1 complex in 1119 tumors of patients with primary invasive breast cancer. These patients were followed for a median follow-up time of 59 months (range, 2–267 months) after the primary diagnosis. Correlations with well-known clinicopathological factors, and univariate and multivariate survival analyses were performed.

High uPA:PAI-1 complex levels were correlated with an adverse histological grade, and inversely associated with negative estrogen and progesterone receptor status. High tumor levels of uPA:PAI-1 complex predicted an early relapse in the univariate relapse-free survival analysis (P < 0.001). The multivariate analysis showed that high uPA:PAI-1 complex levels were associated with a decreased relapse-free survival time (P = 0.033), independently of age, tumor size, number of lymph nodes affected, progesterone receptor status, uPA, adjuvant endocrine, and chemotherapy. More important, it was demonstrated that there is a larger benefit from adjuvant chemotherapy for patients with higher versus lower tumor levels of uPA:PAI-1 complex.

The results of this study imply that the expression of uPA:PAI-1 complex independently predicts the efficacy of adjuvant chemotherapy in patients with primary breast cancer.
Expression of Mesenchyme-Specific Gene HMGA2 in Squamous Cell Carcinomas of the Oral Cavity
Authors: Miyazawa Jun , Mitoro Akira , Kawashiri Shuichi , Chada Kiran K. , Imai Kazushi
Published: September 2005

Carcinoma cells of epithelial origin are predisposed to acquire a fibroblastic feature during progression of neoplasm referred to as the epithelial-mesenchymal transition. HMGA2 is an architectural transcriptional factor that is expressed in the undifferentiated mesenchyme and initiates mesenchymal tumor formation. However, the biological consequence of the expression in the pathology of epithelial-type carcinomas is controversial. The present study was conducted to dissect the expression pattern in oral squamous cell carcinomas. HMGA2 was detected exclusively in carcinoma cell lines and tissues, but not in normal keratinocytes and gingival, by conventional reverse transcription-PCR. Quantitative real-time reverse transcription-PCR demonstrated 160-fold more HMGA2 expression in carcinoma tissues than in normal gingiva and 11-fold more HMGA2 expression in carcinoma cell lines than in normal keratinocytes. HMGA2 expression was observed by immunohistochemistry in 73.8% of 42 carcinomas and localized to the invasive front, where the cells exhibit the epithelial-mesenchymal transition. Fourteen patients who had been classified into a group without lymph node metastasis were positive for HMGA2 staining, and the disease recurred. Furthermore, carcinomas from all 23 patients who died of tumor recurrence stained for HMGA2, and HMGA2 staining was correlated to long-term survival of patients (P < 0.01). Multivariate risk factor analysis demonstrated that HMGA2 expression was an independent prognostic value for disease-specific overall survival (P < 0.01). These results suggest that HMGA2 contributes to the aggressiveness of carcinoma and that detection of HMGA2 expression is a useful predictive and prognostic tool in clinical management of oral carcinomas.
Nuclear Accumulation of Globular Actin as a Cellular Senescence Marker
Authors: Kwak In Hae , Kim Hong Seok , Choi Ok Ran , Ryu Min Sook , Lim In Kyoung
Published: September 2005

We evaluated the nuclear actin accumulation as a new marker of cellular senescence, using human diploid fibroblast (HDF), chondrocyte primary cultures, Mv1Lu epithelial cells, and Huh7 cancer cells. Nuclear accumulation of globular actin (G-actin) and dephosphorylated cofilin was highly significant in the senescent HDF cells, accompanied with inhibition of LIM kinase (LIMK) -1 activity. When nuclear export of the actin was induced by 12-O-tetradecanoylphorbol-13-acetate, DNA synthesis of the senescent cells increased significantly, accompanied with changes of morphologic and biochemical profiles, such as increased RB protein phosphorylation and decreased expressions of p21WAF1, cytoplasmic p-extracellular signal-regulated kinase 1/2, and caveolins 1 and 2. Significance of these findings was strengthened additionally by the fact that nuclear actin export of young HDF cells was inhibited by the treatment with leptomycin B and mutant cofilin transfection, whose LIMK-1 phosphorylation site was lost, and the old cell phenotypes were duplicated with nuclear actin accumulation, suggesting that nuclear actin accumulation was accompanied with G1 arrest during cellular senescence. The aforementioned changes were observed not only in the replicative senescence but also in the senescence induced by treatment of HDF cells, Mv1Lu, primary culture of human chondrocytes, or Huh7 cells with H-ras virus infection, hydroxyurea, deferoxamine, or H2O2. Nuclear actin accumulation was much more sensitive and an earlier event than the well-known, senescence-associated β-galactosidase activity.
The Role of p53 in Suppression of KSHV Cyclin-induced Lymphomagenesis
Authors: Verschuren Emmy W. , Hodgson J. Graeme , Gray Joe W. , Kogan Scott , Jones Nic , Evan Gerard I.
Published: September 2005

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a cyclin D homolog, K cyclin, that is thought to promote viral oncogenesis. However, expression of K cyclin in cultured cells not only triggers cell cycle progression but also engages the p53 tumor suppressor pathway, which probably restricts the oncogenic potential of K cyclin. Therefore, to assess the tumorigenic properties of K cyclin in vivo, we transgenically targeted expression of K cyclin to the B and T lymphocyte compartments via the Eμ promoter/enhancer. Around 17% of Eμ-K cyclin animals develop lymphoma by 9 months of age, and all such lymphomas exhibit loss of p53. A critical role of p53 in suppressing K cyclin-induced lymphomagenesis was confirmed by the greatly accelerated onset of B and T lymphomagenesis in all Eμ-K cyclin/p53−/− mice. However, absence of p53 did not appear to accelerate K cyclin-induced lymphomagenesis by averting apoptosis: Eμ-K cyclin/p53−/− end-stage lymphomas contained abundant apoptotic cells, and transgenic Eμ-K cyclin/p53−/− lymphocytes in vitro were not measurably protected from DNA damage-induced apoptosis compared with Eμ-K cyclin/p53wt cells. Notably, whereas aneuploidy was frequently evident in pre-lymphomatous tissues, end-stage Eμ-K cyclin/p53−/− tumors showed a near-diploid DNA content with no aberrant centrosome numbers. Nonetheless, such tumor cells did harbor more restricted genomic alterations, such as single-copy chromosome losses or gains or high-level amplifications. Together, our data support a model in which K cyclin-induced genome instability arises early in the pre-tumorigenic lymphocyte population and that loss of p53 licenses subsequent expansion of tumorigenic clones.
Topical Tazarotene Chemoprevention Reduces Basal Cell Carcinoma Number and Size in
Ptch1
+/− Mice Exposed to Ultraviolet or Ionizing Radiation
Authors: So Po-Lin , Lee Kenneth , Hebert Jennifer , Walker Patricia , Lu Ying , Hwang Jimmy , Kopelovich Levy , Athar Mohammed , Bickers David , Aszterbaum Michelle , Epstein Ervin H.
Published: September 2005

Oral retinoids can reduce basal cell carcinoma (BCC) incidence in genetically susceptible patients, and one topical retinoid, tazarotene, has been reported to cure some sporadic BCCs. Therefore, we have tested whether this agent would affect BCCs in Ptch1+/− mice in a controlled chemoprevention trial. We found that topical tazarotene dramatically inhibits the formation of BCCs induced with either UV or ionizing radiation. The ability of tazarotene to inhibit BCC formation in this mouse model provides encouragement for the use of tazarotene in skin cancer chemoprevention trials in humans.
A Fragment of Histidine-Rich Glycoprotein Is a Potent Inhibitor of Tumor Vascularization
Authors: Olsson Anna-Karin , Larsson Helena , Dixelius Johan , Johansson Irja , Lee Chunsik , Oellig Cornelia , Björk Ingemar , Claesson-Welsh Lena
Published: September 2005

In this study, we show that recombinant human histidine-rich glycoprotein (HRGP) has potent antiangiogenic properties as judged from effects on a syngeneic tumor model in C57/bl6 mice. Growth of fibrosarcoma, a very aggressive tumor, was reduced by >60% by HRGP treatment, and tumor angiogenesis was dramatically decreased. Treatment with HRGP led to increased apoptosis and reduced proliferation in the tumors. In contrast, HRGP did not affect apoptosis or DNA synthesis in endothelial cells or tumor cells in vitro. The mechanism of action of HRGP involves rearrangement of focal adhesions and decreased attachment of endothelial cells to vitronectin and, as a consequence, reduced endothelial cell migration. By using truncated versions of HRGP, we demonstrate that the isolated 150 amino acid-residue His/Pro-rich domain, which is also released by spontaneous proteolysis from purified HRGP, mediates the inhibitory effect on chemotaxis. Moreover, the His/Pro-rich domain must be released from HRGP to exert its effect. This study shows for the first time inhibitory effects of HRGP on tumor vascularization in vivo, thus providing proof of concept that HRGP is an angiogenesis inhibitor.
Angiogenic Acceleration of Neu Induced Mammary Tumor Progression and Metastasis
Authors: Oshima Robert G. , Lesperance Jacqueline , Munoz Varinia , Hebbard Lionel , Ranscht Barbara , Sharan Niki , Muller William J. , Hauser Craig A. , Cardiff Robert D.
Published: September 2005

The Neu (ErbB2, HER2) member of the epidermal growth factor receptor family is implicated in many human breast cancers. We have tested the importance of increased angiogenic signaling in the NeuYD [mouse mammary tumor virus (MMTV)-Neundl-YD5] mammary tumor model. Transgenic mice expressing vascular endothelial growth factor (VEGF)164 from the MMTV promoter were generated. These mice expressed VEGF164 RNA and protein at 20- to 40-fold higher levels throughout mammary gland development but exhibited normal mammary gland development and function. However, in combination with the NeuYD oncogene, VEGF164 expression resulted in increased vascularization of hyperplastic mammary epithelium and dramatic acceleration of tumor appearance from 111 to 51 days. Gene expression profiling also indicated that the VEGF-accelerated tumors were substantially more vascularized and less hypoxic. The preferential vascularization of early hyperplastic portions of mammary epithelia in NeuYD;MMTV-VEGF animals was associated with NeuYD RNA expression, disorganization of the tight junctions, and overlapping transgenic VEGF expression. NeuYD;MMTV-VEGF164 bigenic, tumor-bearing animals resulted in an average of 10 tumor cell colonies/lung lodged within vascular spaces. No similar lung colonies were found in control NeuYD mice with similar tumor burdens. Overall, these results demonstrate the angiogenic restriction of early hyperplastic mammary lesions. They also reinforce in vivo the importance of activated Neu in causing disorganization of mammary luminal epithelial cell junctions and provide support for an invasion-independent mechanism of metastasis.
Simultaneous Suppression of Epidermal Growth Factor Receptor and c-erbB-2 Reverses Aneuploidy and Malignant Phenotype of a Human Ovarian Carcinoma Cell Line
Authors: Pack Svetlana D. , Alper Özgül M. , Stromberg Kurt , Augustus Meena , Özdemirli Metin , Miermont Anne M. , Klus Greg , Rusin Marek , Slack Rebecca , Hacker Neville F. , Ried Thomas , Szallasi Zoltan , Alper Özge
Published: September 2005

Coexpression of epidermal growth factor receptor (EGFR) and c-erbB-2 in 47–68% of ovarian cancer cells indicate their strong association with tumor formation. We examined the effects of simultaneous antisense- or immunosuppression of EGFR and c-erbB-2 expression on the invasive phenotype, aneuploidy, and genotype of cultured human ovarian carcinoma cells (NIH:OVCAR-8). We report here that suppression of both EGFR and c-erbB-2 results in regression of aneuploidy and genomic imbalances in NIH:OVCAR-8 cells, restores a more normal phenotype, and results in a more normal gene expression profile. Combined with cytogenetic analysis, our data demonstrate that the regression of aneuploidy is due to the selective apoptosis of double antisense transfected cells with highly abnormal karyotype.
Expression Profiling of Purified Normal Human Luminal and Myoepithelial Breast Cells
Authors: Jones Chris , Mackay Alan , Grigoriadis Anita , Cossu Antonio , Reis-Filho Jorge S. , Fulford Laura , Dexter Tim , Davies Susan , Bulmer Karen , Ford Emily , Parry Suzanne , Budroni Mario , Palmieri Giuseppe , Neville A. Munro , O’Hare Michael J. , Lakhani Sunil R.
Published: September 2005

The normal duct-lobular system of the breast is lined by two epithelial cell types, inner luminal secretory cells and outer contractile myoepithelial cells. We have generated comprehensive expression profiles of the two normal cell types, using immunomagnetic cell separation and gene expression microarray analysis. The cell-type specificity was confirmed at the protein level by immunohistochemistry in normal breast tissue. New prognostic markers for survival were identified when the luminal- and myoepithelial-specific molecules were evaluated on breast tumor tissue microarrays. Nuclear expression of luminal epithelial marker galectin 3 correlated with a shorter overall survival in these patients, and the expression of SPARC (osteonectin), a myoepithelial marker, was an independent marker of poor prognosis in breast cancers as a whole. These data provide a framework for the interpretation of breast cancer molecular profiling experiments, the identification of potential new diagnostic markers, and development of novel indicators of prognosis.
Accelerated Induction of Bladder Cancer in
Patched
Heterozygous Mutant Mice
Authors: Hamed Sahar , LaRue Hélène , Hovington Hélène , Girard Johanne , Jeannotte Lucie , Latulippe Eva , Fradet Yves
Published: September 2005

The PATCHED (PTC) gene is recognized as a tumor suppressor in basal cell carcinoma. Mapping of a minimal region of deletion at 9q22.3 and observation of a decreased PTC expression in superficial papillary bladder tumors led us to hypothesize that it could also be involved in this cancer. To further investigate this hypothesis, we submitted Ptc+/− heterozygous mutant mice and their wild-type littermates to chemical carcinogenesis by adding N-butyl-N-(4-hydroxybutyl) nitrosamine to their drinking water. Preneoplastic and neoplastic changes were observed significantly earlier in the Ptc+/− than in the wild-type mice. Our data support the hypothesis of Ptc acting as a tumor suppressor gene in bladder cancer.
Pseudopalisades in Glioblastoma Are Hypoxic, Express Extracellular Matrix Proteases, and Are Formed by an Actively Migrating Cell Population
Authors: Brat Daniel J. , Castellano-Sanchez Amilcar A. , Hunter Stephen B. , Pecot Marcia , Cohen Cynthia , Hammond Elizabeth H. , Devi Sarojini N. , Kaur Balveen , Van Meir Erwin G.
Published: September 2005

Necrosis and vascular proliferation are the pathologic features that distinguish the most malignant infiltrative astrocytoma, glioblastoma (GBM), from those of lower grades. In GBM, hypercellular zones called pseudopalisades typically surround necrotic foci. Although these cells are known to secrete high levels of proangiogenic factors that promote tumor growth, their origins are ill defined. We propose that pseudopalisades represent differing stages and histologic samplings of astrocytoma cells migrating away from a hypoxic/anoxic focus, often triggered by a central vaso-occlusive event. This proposition is based on our findings that pseudopalisading cells are 5–50% less proliferative and 6–20 times more apoptotic than adjacent astrocytoma, indicating that cell accumulation does not result from increased proliferation or resistance to apoptosis. Coexisting inflammatory cells account for <2% of pseudopalisading cells and cannot account for hypercellularity. Pseudopalisading cells show nuclear expression of hypoxia-inducible factor 1α, consistent with their hypoxic nature, and hypoxia induces a 20–60% increase in glioma cell migration in vitro. Hypoxic cells in vitro and pseudopalisades in GBM specimens show enhanced gelatinase activity, typical of an invasive phenotype. These results suggest that pseudopalisading cells are migrating at the periphery of a hypoxic center. To uncover a potential source of hypoxia and sequence of structural events leading to pseudopalisade formation, we performed a morphometric analysis of 234 pseudopalisades from 85 pretreatment GBMs. We found distorted, degenerating, or thrombosed blood vessels within the center of more than half the pseudopalisades, suggesting that at least a subset of pseudopalisades are two-dimensional histologic representations of tumor cells migrating away from a vaso-occlusive event.
TC-1 Is a Novel Tumorigenic and Natively Disordered Protein Associated with Thyroid Cancer
Authors: Sunde Margaret , McGrath Kristine C. Y. , Young Lei , Matthews Jacqueline M. , Chua Elizabeth L. , Mackay Joel P. , Death Alison K.
Published: September 2005

A novel gene, thyroid cancer 1 (TC-1), was found recently to be overexpressed in thyroid cancer. TC-1 shows no homology to any of the known thyroid cancer-associated genes. We have produced stable transformants of normal thyroid cells that express the TC-1 gene, and these cells show increased proliferation rates and anchorage-independent growth in soft agar. Apoptosis rates also are decreased in the transformed cells. We also have expressed recombinant TC-1 protein and have undertaken a structural and functional characterization of the protein. The protein is monomeric and predominantly unstructured under conditions of physiologic salt and pH. This places it in the category of natively disordered proteins, a rapidly expanding group of proteins, many members of which play critical roles in cell regulation processes. We show that the protein can be phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, and the activity of both of these kinases is up-regulated when cells are stably transfected with TC-1. These results suggest that overexpression of TC-1 may be important in thyroid carcinogenesis.
A Novel Protein Tyrosine Kinase NOK that Shares Homology with Platelet- Derived Growth Factor/Fibroblast Growth Factor Receptors Induces Tumorigenesis and Metastasis in Nude Mice
Authors: Liu Li , Yu Xin-Zi , Li Tie-Shi , Song Lian-Xia , Chen Pei-La , Suo Ta-Lin , Li Ying-Hua , Wang Shi-Dong , Chen Yue , Ren Yong-Ming , Zhang Shu-Ping , Chang Zhi-Jie , Fu Xin-Yuan
Published: September 2005

Receptor protein tyrosine kinases (RPTKs) play important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Here, we report the identification and characterization of a novel RPTK-like molecule that has a critical role in induction of tumorigenesis and metastasis and is termed Novel Oncogene with Kinase-domain (NOK). NOK contains a putative single transmembrane domain and a conserved intracellular tyrosine kinase domain that shares homology with members of the platelet-derived growth factor/fibroblast growth factor receptor superfamily. NOK was exclusively located in the cytoplasm. NOK mRNAs were detected in limited human organs and expressed with the highest abundance in the prostate. A variety of tumor cells also expressed the NOK mRNAs. We demonstrated that NIH3T3 and BaF3 cells could be strongly transformed by the expression of the NOK gene as examined by colony formation experiment. In addition, BaF3 cells with the stable expression of NOK induced rapid tumorigenesis in nude mice. Interestingly, these NOK-expressing tumor cells could promptly invade and spread into various distinct organs and form metastatic foci, eventually leading to the rapid death of these animals. Moreover, molecular mechanism studies indicated that NOK could concomitantly activate both MAP kinase and phosphatidylinositol 3′-kinases (PI3K) pathways in stable BaF3 cells. Thus, our results both in vitro and in vivo suggest that NOK is a novel oncogene with the capacity of promoting cell transformation, tumorigenesis, and metastasis.
Polymorphisms of DNA Repair Genes and Risk of Glioma
Authors: Wang Li-E , Bondy Melissa L. , Shen Hongbing , El-Zein Randa , Aldape Kenneth , Cao Yumei , Pudavalli Vinay , Levin Victor A. , Yung W. K. Alfred , Wei Qingyi
Published: September 2005

DNA repair genes play a major role in maintaining genomic stability through different repair pathways that are mediated by cell cycle control genes such as p53. We found previously that glioma patients were susceptible to γ-ray-induced chromosomal breaks, which may be influenced by genetic variation in genes involved in DNA strand breaks, such as XRCC1 in single-strand break repair, XRCC3 and RAD51 in homologous recombination repair, and XRCC7 in nonhomologous end joining double-strand break repair. Therefore, we tested the hypothesis that genetic polymorphisms in XRCC1, XRCC3, RAD51, XRCC7, and p53 were associated with risk of glioma in 309 patients with newly diagnosed glioma and 342 cancer-free control participants frequency matched on age (±5 years), sex, and self-reported ethnicity. We did not find any statistically significant differences in the distributions of XRCC1 Arg399Gln, XRCC3 Thr241Met, RAD51 G135C, and P53 Arg72Pro polymorphisms between the cases and the controls. However, the XRCC7 G6721T variant T allele and TT genotype were more common in the cases (0.668 and 43.4%, respectively) than in the controls (0.613 and 38.9%, respectively), and the differences were statistically significant (P = 0.045 and 0.040, respectively). The adjusted odds ratios were 1.78 (95% confidence interval, 1.08–2.94) and 1.86 (95% confidence interval, 1.12–3.09) for the GT heterozygotes and TT homozygotes, respectively. The combined T variant genotype (GT+TT) was associated with a 1.82-fold increased risk of glioma (95% confidence interval, 1.13–2.93). These results suggest that the T allele may be a risk allele, and this XRCC7 polymorphism may be a marker for the susceptibility to glioma. Larger studies are needed to confirm our findings and unravel the underlying mechanisms.
Human CTLs to Wild-Type and Enhanced Epitopes of a Novel Prostate and Breast Tumor-Associated Protein, TARP, Lyse Human Breast Cancer Cells
Authors: Oh SangKon , Terabe Masaki , Pendleton C. David , Bhattacharyya Anu , Bera Tapan K. , Epel Malka , Reiter Yoram , Phillips John , Linehan W. Marston , Kasten-Sportes Claude , Pastan Ira , Berzofsky Jay A.
Published: September 2005

Vaccine therapy for prostate and breast cancer may have potential for treating these major causes of death in males and females, respectively. Critical to the development of tumor-specific vaccines is finding and characterizing novel antigens to be recognized by CD8+ T cells. To define new CD8+ T-cell tumor antigens, we determined two wild-type HLA-A2 epitopes from a recently found tumor-associated protein, TARP (T-cell receptor γ alternate reading frame protein), expressed in prostate and breast cancer cells. We were also able to engineer epitope-enhanced peptides by sequence modifications. Both wild-type and enhanced epitopes induced peptide-specific CD8+ T-cell responses in A2Kb transgenic mice. In vitro restimulation of human CD8+ T cells from a prostate cancer patient resulted in CD8+ T cells reactive to the peptide epitopes that could lyse HLA-A2+ human breast cancer cells (MCF-7) expressing TARP. Epitope-specific human CD8+ T cells were also enumerated in patients’ peripheral blood by tetramer staining. Our data suggest that HLA-A2-binding TARP epitopes and enhanced epitopes discovered in this study could be incorporated into a potential vaccine for both breast and prostate cancer.
Ovarian Carcinoma Expresses the NKG2D Ligand Letal and Promotes the Survival and Expansion of CD28− Antitumor T Cells
Authors: Conejo-Garcia Jose R. , Benencia Fabian , Courreges Maria C. , Gimotty Phyllis A. , Khang Eugene , Buckanovich Ronald J. , Frauwirth Kenneth A. , Zhang Lin , Katsaros Dionyssios , Thompson Craig B. , Levine Bruce , Coukos George
Published: September 2005

The role of the NKG2D immunoreceptor and its ligands in antitumor immune response is incompletely understood. Here, we report that effector immune cells infiltrating ovarian carcinoma are mostly CD8+ lymphocytes lacking CD28 but expressing the NKG2D costimulatory receptor. Human ovarian carcinoma expresses the novel NKG2D ligand lymphocyte effector cell toxicity-activating ligand (Letal). Letal was found to be an independent prognosticator of improved survival in advanced ovarian cancer. Higher levels of tumor-derived Letal were associated with stronger lymphocyte infiltration. Letal exerted marked costimulatory effects and induced type-1 polarization in CD8+CD28− tumor-infiltrating lymphocytes ex vivo. Letal engagement increased the expression of the glucose transporter Glut-1, enhanced glucose up-take, and protected CD8+ lymphocytes from cisplatin-induced killing. Letal also down-regulated the expression of Fas in CD8+ cells and rendered them resistant to Fas ligand-induced apoptosis. Our results indicate that Letal promotes tumor immune surveillance by promoting the survival and intratumoral expansion of antitumor cytotoxic lymphocytes. We propose that Letal could be used for the ex vivo expansion of apoptosis-resistant tumor-reactive cytotoxic lymphocytes for adoptive transfer.