bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of 5′-AMP-activated protein kinase in stimulation of glucose transport in response to inhibition of oxidative phosphorylation
Authors: Jing Ming , Ismail-Beigi Faramarz
Published: September 2005

Glucose transport is stimulated in a variety of cells and tissues in response to inhibition of oxidative phosphorylation. However, the underlying mechanisms and mediating steps remain largely unknown. In the present study we first tested whether a decrease in the redox state of the cell per se and the resultant increase in generation of reactive oxygen species (ROS) lead to stimulation of glucose transport. Clone 9 cells (expressing the Glut1 isoform of facilitative glucose transporters) were exposed to azide, lactate, and ethanol for 1 h. Although all three agents stimulated glucose transport and increased cell NADH-to-NAD+ ratio and phospho-ERK1/2, signifying increased ROS generation, the response to the stimuli was not blocked by N-acetyl-l-cysteine (an agent that counteracts ROS); moreover, the response to azide was not blocked by diamide (an intracellular sulfhydryl oxidizing agent). We then found that cell AMP-to-ATP and ADP-to-ATP ratios were increased and 5′-AMP-activated protein kinase (AMPK) was stimulated by all three agents, as evidenced by increased phosphorylation of AMPK and acetyl-CoA carboxylase. We conclude that although azide, lactate, and ethanol increase NADH-to-NAD+ ratios and ROS production, their stimulatory effect on glucose transport is not mediated by increased ROS generation. However, all three agents increased cell AMP-to-ATP ratio and stimulated AMPK, making it likely that the latter pathway plays an important role in the glucose transport response.
In vivo inhibition of nitric oxide synthase impairs upregulation of contractile protein mRNA in overloaded plantaris muscle
Authors: Sellman Jeff E. , DeRuisseau Keith C. , Betters Jenna L. , Lira Vitor A. , Soltow Quinlyn A. , Selsby Joshua T. , Criswell David S.
Published: September 2005

Inhibition of nitric oxide synthase (NOS) activity in vivo impedes hypertrophy in the overloaded rat plantaris. We investigated the mechanism for this effect by examining early events leading to muscle growth following 5 or 12 days of functional overload. Male Sprague-Dawley rats (∼350 g) were randomly divided into three treatment groups: control, NG-nitro-l-arginine methyl ester (l-NAME; 90 mg·kg−1·day−1), and 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM; 10 mg·kg−1·day−1). Unilateral removal of synergists induced chronic overload (OL) of the right plantaris. Sham surgery performed on the left hindlimb served as a normally loaded control. l-NAME and TRIM treatments prevented OL-induced skeletal α-actin and type I (slow) myosin heavy chain mRNA expression at 5 days. Conversely, neither l-NAME nor TRIM affected hepatocyte growth factor or VEGF mRNA responses to OL at 5 days. However, OL induction of IGF-I and mechanogrowth factor mRNA was greater ( P < 0.05) in the TRIM group compared with the controls. Furthermore, the phosphorylated-to-total p70 S6 kinase ratio was higher in OL muscle from NOS-inhibited groups, compared with control OL. At 12 days of OL, the cumulative proliferation of plantaris satellite cells was assessed by subcutaneous implantation of time release 5′-bromo-2′-deoxyuridine pellets during the OL-inducing surgeries. Although OL caused a fivefold increase in the number of mitotically active (5′-bromo-2′-deoxyuridine positive) sublaminar nuclei, this was unaffected by concurrent NOS inhibition. Therefore, NOS activity may provide negative feedback control of IGF-I/p70 S6 kinase signaling during muscle growth. Moreover, NOS activity may be involved in transcriptional regulation of skeletal α-actin and type I (slow) myosin heavy chain during functional overload.
PIK3CA Mutations Correlate with Hormone Receptors, Node Metastasis, and ERBB2, and Are Mutually Exclusive with PTEN Loss in Human Breast Carcinoma
Authors: Saal Lao H. , Holm Karolina , Maurer Matthew , Memeo Lorenzo , Su Tao , Wang Xiaomei , Yu Jennifer S. , Malmström Per-Olof , Mansukhani Mahesh , Enoksson Jens , Hibshoosh Hanina , Borg Åke , Parsons Ramon
Published: September 2005

Deregulation of the phosphatidylinositol 3-kinase (PI3K) pathway either through loss of PTEN or mutation of the catalytic subunit α of PI3K (PIK3CA) occurs frequently in human cancer. We identified PIK3CA mutations in 26% of 342 human breast tumor samples and cell lines at about equal frequency in tumor stages I to IV. To investigate the relationship between PTEN and PIK3CA, we generated a cohort of tumors that had lost PTEN expression and compared it with a matched control set that had retained PTEN. A highly significant association between PIK3CA mutations and retention of PTEN protein expression was observed. In addition, PIK3CA mutations were associated with expression of estrogen and progesterone receptors (ER/PR), lymph node metastasis, and ERBB2 overexpression. The fact that PIK3CA mutations and PTEN loss are nearly mutually exclusive implies that deregulated phosphatidylinositol-3,4,5-triphosphate (PIP3) is critical for tumorigenesis in a significant fraction of breast cancers and that loss of PIP3 homeostasis by abrogation of either PIK3CA or PTEN relieves selective pressure for targeting of the other gene. The correlation of PIK3CA mutation to ER/PR-positive tumors and PTEN loss to ER/PR-negative tumors argues for disparate branches of tumor evolution. Furthermore, the association between ERBB2 overexpression and PIK3CA mutation implies that more than one input activating the PI3K/AKT pathway may be required to overcome intact PTEN. Thus, mutation of PIK3CA is frequent, occurs early in carcinoma development, and has prognostic and therapeutic implications.
Heightened Expression of CTCF in Breast Cancer Cells Is Associated with Resistance to Apoptosis
Authors: Docquier France , Farrar Dawn , D'Arcy Vivien , Chernukhin Igor , Robinson Abigail F. , Loukinov Dmitry , Vatolin Sergei , Pack Svetlana , Mackay Alan , Harris Robert A. , Dorricott Heather , O'Hare Michael J. , Lobanenkov Victor , Klenova Elena
Published: September 2005

CTCF is a candidate tumor suppressor gene encoding a multifunctional transcription factor. Surprisingly for a tumor suppressor, the levels of CTCF in breast cancer cell lines and tumors were found elevated compared with breast cell lines with finite life span and normal breast tissues. In this study, we aimed to investigate the possible cause for this increase in CTCF content and in particular to test the hypothesis that up-regulation of CTCF may be linked to resistance of breast cancer cells to apoptosis. For this purpose, apoptotic cell death was monitored following alterations of CTCF levels induced by transient transfection and conditional knockdown of CTCF in various cell lines. We observed apoptotic cell death in all breast cancer cell lines examined following CTCF down-regulation. In addition, overexpression of CTCF partially protected cells from apoptosis induced by overexpression of Bax or treatment with sodium butyrate. To elucidate possible mechanisms of this phenomenon, we used a proteomics approach and observed that levels of the proapoptotic protein, Bax, were increased following CTCF down-regulation in MCF7 cells. Taken together, these results suggest that in some cellular contexts CTCF shows antiapoptotic characteristics, most likely exerting its functions through regulation of apoptotic genes. We hypothesize that CTCF overexpression may have evolved as a compensatory mechanism to protect breast cancer cells from apoptosis, thus providing selective survival advantages to these cells. The observations reported in this study may lead to development of therapies based on selective reduction of CTCF in breast cancer cells.
Ron Receptor Signaling Augments Mammary Tumor Formation and Metastasis in a Murine Model of Breast Cancer
Authors: Peace Belinda E. , Toney-Earley Kenya , Collins Margaret H. , Waltz Susan E.
Published: September 2005

The tyrosine kinase receptor Ron has been implicated in several types of cancer, including overexpression in human breast cancer. This is the first report describing the effect of Ron signaling on tumorigenesis and metastasis in a mouse model of breast cancer. Mice with a targeted deletion of the Ron tyrosine kinase signaling domain (TK−/−) were crossed to mice expressing the polyoma virus middle T antigen (pMT) under the control of the mouse mammary tumor virus promoter. Both pMT-expressing wild-type control (pMT+/− TK+/+) and pMT+/− TK−/− mice developed mammary tumors and lung metastases. However, a significant decrease in mammary tumor initiation and growth was found in the pMT+/− TK−/− mice compared with controls. An examination of mammary tumors showed that there was a significant decrease in microvessel density, significantly decreased cellular proliferation, and a significant increase in terminal deoxynucleotidyl transferase–mediated nick end labeling–positive staining in mammary tumor cells from the pMT+/− TK−/− mice compared with the pMT+/− TK+/+ mice. Biochemical analyses on mammary tumor lysates showed that whereas both the pMT-expressing TK+/+ and TK−/− tumors have increased Ron expression compared with normal mammary glands, the pMT-expressing TK−/− tumors have deficits in mitogen-activated protein kinase and AKT activation. These results indicate that Ron signaling synergizes with pMT signaling to induce mammary tumor formation, growth, and metastasis. This effect may be mediated in part through the regulation of angiogenesis and through proliferative and cell survival pathways regulated by mitogen-activated protein kinase and AKT.
CCAAT/Enhancer-Binding Protein δ: A Molecular Target of 1,25-Dihydroxyvitamin D3 in Androgen-Responsive Prostate Cancer LNCaP Cells
Authors: Ikezoe Takayuki , Gery Sigal , Yin Dong , O'Kelly James , Binderup Lise , Lemp Nathan , Taguchi Hirokuni , Koeffler H. Phillip
Published: September 2005

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, inhibits the proliferation of prostate cancer cells. However, the molecular mechanisms by which 1,25(OH)2D3 inhibits the proliferation of these cells remain to be fully elucidated. In this study, we used microarray technology to identify target genes of 1,25(OH)2D3 in androgen-responsive prostate cancer LNCaP cells. 1,25(OH)2D3 up-regulated CCAAT/enhancer-binding protein δ (C/EBPδ) by ∼5-fold in these cells. Knockdown of C/EBPδ expression by RNA interference showed that C/EBPδ is essential for the significant growth inhibition of LNCaP cells in response to 1,25(OH)2D3 treatment. Moreover, we found that 1,25(OH)2D3 induced C/EBPδ in other cancer cells, including the estrogen receptor (ER)–expressing MCF-7 and T47D breast cancer cells that are sensitive to the growth inhibitory effects of 1,25(OH)2D3. On the other hand, 1,25(OH)2D3 was not able to induce C/EBPδ in either androgen receptor–negative PC-3 and DU145 or ER-negative breast cancer MDA-MB-231 cells that were relatively resistant to growth inhibition by 1,25(OH)2D3. Furthermore, forced expression of C/EBPδ in prostate cancer LNCaP as well as breast cancer MCF-7 and T47D cells dramatically reduced their clonal growth. Taken together, forced expression of C/EBPδ in cancer cells may be a promising therapeutic strategy.
Human Splicing Factor SPF45 (RBM17) Confers Broad Multidrug Resistance to Anticancer Drugs When Overexpressed— a Phenotype Partially Reversed By Selective Estrogen Receptor Modulators
Authors: Perry William L. , Shepard Robert L. , Sampath Janardhan , Yaden Benjamin , Chin William W. , Iversen Philip W. , Jin Shengfang , Lesoon Andrea , O'Brien Kathryn A. , Peek Victoria L. , Rolfe Mark , Shyjan Andrew , Tighe Michelle , Williamson Mark , Krishnan Venkatesh , Moore Robert E. , Dantzig Anne H.
Published: September 2005

The splicing factor SPF45 (RBM17) is frequently overexpressed in many solid tumors, and stable expression in HeLa cells confers resistance to doxorubicin and vincristine. In this study, we characterized stable transfectants of A2780 ovarian carcinoma cells. In a 3-day cytotoxicity assay, human SPF45 overexpression conferred 3- to 21-fold resistance to carboplatin, vinorelbine, doxorubicin, etoposide, mitoxantrone, and vincristine. In addition, resistance to gemcitabine and pemetrexed was observed at the highest drug concentrations tested. Knockdown of SPF45 in parental A2780 cells using a hammerhead ribozyme sensitized A2780 cells to etoposide by ∼5-fold relative to a catalytically inactive ribozyme control and untransfected cells, suggesting a role for SPF45 in intrinsic resistance to some drugs. A2780-SPF45 cells accumulated similar levels of doxorubicin as vector-transfected and parental A2780 cells, indicating that drug resistance is not due to differences in drug accumulation. Efforts to identify small molecules that could block SPF45-mediated drug resistance revealed that the selective estrogen receptor (ER) modulators tamoxifen and LY117018 (a raloxifene analogue) partially reversed SPF45-mediated drug resistance to mitoxantrone in A2780-SPF45 cells from 21-fold to 8- and 5-fold, respectively, but did not significantly affect the mitoxantrone sensitivity of vector control cells. Quantitative PCR showed that ERβ but not ERα was expressed in A2780 transfectants. Coimmunoprecipitation experiments suggest that SPF45 and ERβ physically interact in vivo. Thus, SPF45-mediated drug resistance in A2780 cells may result in part from effects of SPF45 on the transcription or alternate splicing of ERβ-regulated genes.
Angiogenesis Inhibition by Vascular Endothelial Growth Factor Receptor-2 Blockade Reduces Stromal Matrix Metalloproteinase Expression, Normalizes Stromal Tissue, and Reverts Epithelial Tumor Phenotype in Surface Heterotransplants
Authors: Vosseler Silvia , Mirancea Nicolae , Bohlen Peter , Mueller Margareta M. , Fusenig Norbert E.
Published: September 2005

Inhibition of vascular endothelial growth factor (VEGF) signaling, a key regulator of tumor angiogenesis, through blockade of VEGF receptor (VEGFR)-2 by the monoclonal antibody DC101 inhibits angiogenesis, tumor growth, and invasion. In a surface xenotransplant assay on nude mice using a high-grade malignant squamous cell carcinoma cell line (A-5RT3), we show that DC101 causes vessel regression and normalization as well as stromal maturation resulting in a reversion to a noninvasive tumor phenotype. Vessel regression is followed by down-regulation of expression of both VEGFR-2 and VEGFR-1 on endothelial cells and increased association of α-smooth muscle actin–positive cells with small vessels indicating their normalization, which was further supported by a regular ultrastructure. The phenotypic regression of an invasive carcinoma to a well-demarcated dysplastic squamous epithelium is accentuated by the establishment of a clearly structured epithelial basement membrane and the accumulation of collagen bundles in the stabilized connective tissue. This normalization of the tumor-stroma border coincided with down-regulated expression of the stromal matrix metalloproteinases 9 and 13, which supposedly resulted in attenuated turnover of extracellular matrix components permitting their structural organization. Thus, in this mouse model of a human squamous cell carcinoma cell line, blockade of VEGF signaling resulted in the reversion of the epithelial tumor phenotype through stromal normalization, further substantiating the crucial role of stromal microenvironment in regulating the tumor phenotype.
Gene Expression Profiling Reveals Cross-talk between Melanoma and Fibroblasts: Implications for Host-Tumor Interactions in Metastasis
Authors: Gallagher Paul G. , Bao Yongde , Prorock Alyson , Zigrino Paola , Nischt Roswitha , Politi Vincenzo , Mauch Cornelia , Dragulev Bojan , Fox Jay William
Published: September 2005

Host-tumor interaction is considered critical in carcinogenesis, tumor invasion, and metastasis. To explore the reciprocal effects of host-tumor interaction, we developed a system to assess the gene expression patterns of A2058 human melanoma cells cocultured in fibrillar collagen with HS-68 primary human fibroblasts. The gene expression pattern of the cocultured A2058 cells was only modestly affected, whereas the HS-68 fibroblast gene expression pattern was significantly altered. Interleukin-11 and inhibitor of DNA-binding domain-1 gene expression in the cocultured A2058 cells was down-regulated, indicative of a proinflammatory response and resistance to apoptosis, respectively. The overall pattern of up-regulated genes indicated triggering of the proinflammatory process. In addition, the melanoma growth and migration stimulatory chemokines CXCL1 and CXCL2 were significantly up-regulated in the cocultured fibroblasts. These results were corroborated by additional coculture experiments with the melanoma cell lines WM-164, BLM, and SK-Mel-28 and immunohistochemistry on invasive human melanoma sections. Taken together, these results indicate that tumor cells cause a proinflammatory and melanoma growth-promoting response in stromal fibroblasts. The role of inflammation in carcinogenesis, tumor promotion, invasion, and metastasis is viewed as being increasingly important and the results of these studies underscore this as well as identify certain key proteins that are expressed as a result of the complex interactive processes in the host-tumor microenvironment.
Protein Kinase C ζ Is Required for Epidermal Growth Factor–Induced Chemotaxis of Human Breast Cancer Cells
Authors: Sun Ronghua , Gao Ping , Chen Lin , Ma Dalong , Wang Jiming , Oppenheim Joost J. , Zhang Ning
Published: September 2005

Chemotaxis plays an important role in cancer cell metastasis. In this study, we showed that epidermal growth factor (EGF) was a more potent chemoattractant than chemokine SDF-1α/CXCL12 for human breast cancer cell MDA-MB-231. Different inhibitors were used to evaluate the involvement of 12 protein kinase C (PKC) isotypes in the chemotactic signaling pathway. Chelerythrine chloride, an inhibitor of all PKC isotypes, blocked chemotaxis, whereas inhibitors of classic and novel PKC, such as Gö6976, Gö6850, or calphostin C, only impaired EGF-induced chemotaxis to a minor extent by ≯32% inhibition. These data suggested that atypical PKC were involved. The ligand-induced actin polymerization and cell adhesion were also similarly dependent on atypical PKC. Immunofluorescent staining showed an EGF-induced, LY294002-sensitive translocation of PKCζ from the cytosol to the plasma membrane, indicating that EGF was capable of activating PKCζ, probably via phosphoinositide 3 kinases. A myristoylated PKCζ pseudosubstrate blocked the chemotaxis with an IC50 of 20 μmol/L. To expand our investigation, we further showed that in MCF-7 and T47D, two additional human breast cancer cell lines, EGF-activated PKCζ and the PKCζ pseudosubstrate, inhibited chemotaxis. Taken together, our data suggest that PKCζ is an essential component of the EGF-stimulated chemotactic signaling pathway in human breast cancer cells.
CDK4 and MDM2 Gene Alterations Mainly Occur in Highly Proliferative and Aggressive Mantle Cell Lymphomas with Wild-type INK4a/ARF Locus
Authors: Hernández Luis , Beà Silvia , Pinyol Magda , Ott German , Katzenberger Tiemo , Rosenwald Andreas , Bosch Francesc , López-Guillermo Armando , Delabie Jan , Colomer Dolors , Montserrat Emili , Campo Elías
Published: September 2005

Amplification of 12q13 locus occurs in some mantle cell lymphomas (MCL), potentially involving CDK4 and MDM2 genes. To determine the role of these genes in MCL, we have examined their gene status and expression and their relationship to INK4a/ARF and p53 gene aberrations in 69 tumors. Increased CDK4 gene copy number was detected in 4 of 19 (21%) highly proliferative blastoid variants and was associated with mRNA and protein overexpression. Three additional cases showed mRNA overexpression with no structural alterations of the gene. MDM2 gene overexpression was detected in three blastoid tumors (16%) with no relationship to gene copy gains. INK4a/ARF and p53 aberrations were observed in 13 and 12 tumors, respectively. Four of the seven lymphomas with CDK4 aberrations had concurrent inactivation of p53 gene, whereas only one case had a concomitant homozygous deletion of INK4a/ARF. No other gene alterations were found in the three cases with MDM2 overexpression. Patients with INK4a/ARF deletions or simultaneous aberrations of p53 and CDK4 had a significantly shorter median survival (17 months) than patients with isolated alterations of p53, MDM2, or CDK4 (32 months) and patients with no alterations in any of these genes (77 months). The prognostic impact of the concomitant oncogenic alterations of the p14ARF/p53 and p16INK4a/CDK4 pathways was independent of the proliferation of the tumors. These findings indicate that CDK4 and MDM2 gene alterations mainly occur in MCL with a wild-type INK4a/ARF locus and may contribute to the higher proliferation and more aggressive behavior of the tumors.
Fate of Premalignant Clones during the Asymptomatic Phase Preceding Lymphoid Malignancy
Authors: Moulés Vincent , Pomier Carole , Sibon David , Gabet Anne-Sophie , Reichert Michal , Kerkhofs Pierre , Willems Luc , Mortreux Franck , Wattel Eric
Published: September 2005

Almost all cancers are preceded by a prolonged period of clinical latency during which a combination of cellular events helps move carcinogen-exposed cells towards a malignant phenotype. Hitherto, investigating the fate of premalignant cells in vivo remained strongly hampered by the fact that these cells are usually indistinguishable from their normal counterparts. Here, for the first time, we have designed a strategy able to reconstitute the replicative history of the bona fide premalignant clone in an animal model, the sheep experimentally infected with the lymphotropic bovine leukemia virus. We have shown that premalignant clones are early and clearly distinguished from other virus-exposed cells on the basis of their degree of clonal expansion and genetic instability. Detectable as early as 0.5 month after the beginning of virus exposure, premalignant cells displayed a two-step pattern of extensive clonal expansion together with a mutation load ∼6 times higher than that of other virus-exposed cells that remained untransformed during the life span of investigated animals. There was no fixation of somatic mutations over time, suggesting that they regularly lead to cellular death, partly contributing to maintain a normal lymphocyte count during the prolonged premalignant stage. This equilibrium was finally broken after a period of 18.5 to 60 months of clinical latency, when a dramatic decrease in the genetic instability of premalignant cells coincided with a rapid increase in lymphocyte count and lymphoma onset.
p73-Dependent Apoptosis through Death Receptor: Impairment by Human Cytomegalovirus Infection
Authors: Terrasson Jérôme , Allart Sophie , Martin Hélène , Lulé Jacqueline , Haddada Hedi , Caput Daniel , Davrinche Christian
Published: September 2005

The discovery of p73, a p53-related protein with various isotypes resulting from different promoter usage or splicing events, provided new insights into regulation of neurogenesis and tumorigenesis. Among p73 isoforms described thus far, TA-truncated molecules (ΔN) appeared as key proteins according to their antagonistic activity against transcription factor activity of p53 family members. We previously showed that infection by human cytomegalovirus (HCMV) induced drug resistance and altered p53- and p73-dependent apoptosis of infected cells through accumulation of ΔN-p73α. In accordance with the ability of p53 to induce apoptosis through death receptors, we asked whether p73 activation could compensate for p53 deficiency. We showed that p73 transcriptional activity sensitized cells to apoptosis through death receptors in a caspase-dependent pathway. Expression of the death-inducing signaling complex (DISC) proteins was unchanged, whereas p73 activation through either cisplatin treatment or ectopic overexpression induced up-regulation of Fas transcription and expression at cell surface. According to its ability to flood cells with ΔN-p73α, HCMV inhibited p73-dependent Fas-mediated apoptosis, gaining an additional trick to favor its survival in the host cell. Owing to the involvement of p53- and p73-dependent death receptor signaling in development of the central nervous system, immune surveillance of neural cells, and sensitivity of tumors to drugs, our previous and present data prompt us to consider stabilization of ΔN-p73α by HCMV as a possible mechanism in impairment of embryogenesis and in tumorigenesis.
Differential Expression of Cell Cycle Regulators in Phenotypic Variants of Transgenically Induced Bladder Tumors: Implications for Tumor Behavior
Authors: Garcia-España Antonio , Salazar Edgard , Sun Tung-Tien , Wu Xue-Ru , Pellicer Angel
Published: September 2005

Proteins controlling cell growth, differentiation, apoptosis, and oncogenic stress are often deregulated in tumor cells. However, whether such deregulations affect tumor behavior remains poorly understood in many tumor types. We recently showed that the urothelium-specific expression of activated H-ras and SV40 T antigen in transgenic mice produced two distinctive types of tumors strongly resembling the human superficial papillary tumors and carcinoma in situ of the bladder, respectively. Here we assessed the expression of a key set of cell cycle regulators in these mouse tumors and in a new transgenic line expressing a cyclin D1 oncogene in the urothelium. We found that urothelia of the wild-type and cyclin D1 transgenic mice exhibited a profile of cell cycle regulators found in quiescent (G0) cells, indicating that urothelium overexpressing the cyclin D1 (an 8-fold increase) is reminiscent of normal urothelium and remains slow-cycling. Low-grade superficial papillary tumors induced by activated H-ras had no detectable Rb family proteins (Rb, p107, and p130) and late cell cycle cyclins and kinases (cyclin A, E, and CDK1), but had increased level of p16, p53, and MDM2. These data suggest that the inactivation of the Rb pathway plays an important role in H-ras-induced superficial papillary tumors and that oncogenic H-ras can induce a compensatory activation of alternative tumor suppressor pathways. In contrast, carcinoma in situ of the bladder induced by SV40 T antigen had increased expression of cell cycle regulators mainly active in post-G1 phases. The fact that phenotypically different bladder tumors exhibit different patterns of cell cycle regulators may explain why these tumors have different propensity to progress to invasive tumors. Our results indicate that the transgenic mouse models can be used not only for studying tumorigenesis but also for evaluating therapeutic strategies that target specific cell cycle regulators.
Simultaneous Generation of CD8+ and CD4+ Melanoma-Reactive T Cells by Retroviral-Mediated Transfer of a Single T-Cell Receptor
Authors: Roszkowski Jeffrey J. , Lyons Gretchen E. , Kast W. Martin , Yee Cassian , Van Besien Koen , Nishimura Michael I.
Published: September 2005

Adoptive immunotherapy of cancer requires the generation of large numbers of tumor antigen–reactive T cells for transfer into cancer patients. Genes encoding tumor antigen–specific T-cell receptors can be introduced into primary human T cells by retroviral mediated gene transfer as a potential method of providing any patient with a source of autologous tumor-reactive T cells. A T-cell receptor–specific for a class I MHC (HLA-A2)–restricted epitope of the melanoma antigen tyrosinase was isolated from a CD4+ tumor-infiltrating lymphocyte (TIL 1383I) and introduced into normal human peripheral blood lymphocytes by retroviral transduction. T-cell receptor–transduced T cells secreted various cytokines when cocultured with tyrosinase peptide–loaded antigen-presenting cells as well as melanoma cells in an HLA-A2-restricted manner, and could also lyse target cells. Furthermore, T-cell clones isolated from these cultures showed both CD8+ and CD4+ transduced T cells could recognize HLA-A2+ melanoma cells, giving us the possibility of engineering class I MHC–restricted effector and T helper cells against melanoma. The ability to confer class I MHC–restricted tumor cell recognition to CD4+ T cells makes the TIL 1383I TCR an attractive candidate for T-cell receptor gene transfer–based immunotherapy.
Imatinib Mesylate Inhibits Leydig Cell Tumor Growth: Evidence for In vitro and In vivo Activity
Authors: Basciani Sabrina , Brama Marina , Mariani Stefania , De Luca Gabriele , Arizzi Mario , Vesci Loredana , Pisano Claudio , Dolci Susanna , Spera Giovanni , Gnessi Lucio
Published: September 2005

Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.
Chemopreventive Effect of Peroxisome Proliferator–Activated Receptor γ on Gastric Carcinogenesis in Mice
Authors: Lu Jie , Imamura Kazuhiro , Nomura Sachiyo , Mafune Ken-ichi , Nakajima Atsushi , Kadowaki Takashi , Kubota Naoto , Terauchi Yasuo , Ishii Genichiro , Ochiai Atsushi , Esumi Hiroyasu , Kaminishi Michio
Published: September 2005

Peroxisome proliferator–activated receptor γ (PPARγ) is known to be expressed in several cancers, and the treatment of these cancer cells with PPARγ ligands often induces cell differentiation and apoptosis. Recently, the chemopreventive potential of PPARγ ligands on colon carcinogenesis was reported, although the effect of PPARγ on colon carcinogenesis and the mechanism of the effect remain controversial. In this study, we attempted to elucidate the role of PPARγ in gastric carcinogenesis and explored the possible use of PPARγ ligand as a chemopreventive agent for gastric cancer. N-methyl-N-nitrosourea (MNU, 240 ppm) was given in drinking water for 10 weeks to induce gastric cancer in PPAR wild-type (+/+) and heterozygous-deficient (+/−) mice, followed by treatment with PPARγ ligand [troglitazone, 0.15% (w/w) in powder food] or the vehicle alone for 42 weeks. At the end of the experiment, PPARγ (+/−) mice were more susceptible to MNU-induced gastric cancer than wild-type (+/+) mice (89.5%/55.5%), and troglitazone significantly reduced the incidence of gastric cancer in PPARγ (+/+) mice (treatment 55.5%/vehicle 9%) but not in PPARγ (+/−) mice. The present study showed that (a) PPARγ suppresses gastric carcinogenesis, (b) the PPARγ ligand troglitazone is a potential chemopreventive agent for gastric carcinogenesis, and (c) troglitazone's chemopreventative effect is dependent on PPARγ.
Glucosylceramide Synthase Blockade Down-Regulates P-Glycoprotein and Resensitizes Multidrug-Resistant Breast Cancer Cells to Anticancer Drugs
Authors: Gouazé Valérie , Liu Yong-Yu , Prickett Carlton S. , Yu Jing Y. , Giuliano Armando E. , Cabot Myles C.
Published: September 2005

Overexpression of glucosylceramide synthase (GCS), a pivotal enzyme in glycolipid biosynthesis, contributes to cancer cell resistance to chemotherapy. We previously showed that transfection of doxorubicin-resistant MCF-7-AdrR cells with GCS antisense restored cell sensitivity to doxorubicin and greatly enhanced sensitivity to vinblastine and paclitaxel. In that study, doxorubicin promoted generation of ceramide in MCF-7-AdrR/GCS antisense cells; the present study implicates factors in addition to ceramide that augment sensitivity to chemotherapy. Although GCS antisense cells showed enhanced ceramide formation compared with MCF-7-AdrR when challenged with paclitaxel, GCS antisense cells also showed a 10-fold increase in levels of intracellular drug (paclitaxel and vinblastine). In addition, transfected cells had dramatically decreased expression (80%) of P-glycoprotein and a 4-fold decrease in the level of cellular gangliosides. Chemical inhibition of GCS produced the same effects as antisense transfection: exposure of MCF-7-AdrR cells to the GCS inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 5.0 μmol/L, 4 days) decreased ganglioside levels, restored sensitivity to vinblastine, enhanced vinblastine uptake 3-fold, and diminished expression of MDR1 by 58%, compared with untreated controls. A similar effect was shown in vinblastin-resistant KB-V0.01 cells; after 7 days with PPMP (10 μmol/L), MDR1 expression fell by 84% and P-glycoprotein protein levels decreased by 50%. MCF-7-AdrR cells treated with small interfering RNAs to specifically block GCS also showed a dramatic decrease in MDR1 expression. This work shows that limiting GCS activity down-regulates the expression of MDR1, a phenomenon that may drive the chemosensitization associated with blocking ceramide metabolism. The data suggest that lipids play a role in the expression of multidrug resistance.
Down-regulation of Cx43 by Retroviral Delivery of Small Interfering RNA Promotes an Aggressive Breast Cancer Cell Phenotype
Authors: Shao Qing , Wang Hongling , McLachlan Elizabeth , Veitch Gregory I.L. , Laird Dale W.
Published: September 2005

Connexins are gap junction proteins that assemble into channels that mediate direct intercellular communication. Connexins are well-documented tumor suppressors and are thought to regulate both cell growth and differentiation. As previously reported, most human breast tumors and cell lines down-regulate gap junctions or have defective gap junctional intercellular communication. Furthermore, overexpression of connexins in breast cancer cells inhibits tumor growth in vivo. In this study, we hypothesize that controlled Cx43 down-regulation would induce breast tumor cells to acquire a more aggressive phenotype. Here we report that Cx43 was down-regulated in both normal rat kidney (NRK) cells and human breast cancer cell lines (MDA-MB-231 and Hs578T) by transfection with chemically synthesized small interfering RNA (siRNA) or short hairpin RNA generated from a retroviral infection. Furthermore, we show that retroviral delivery and expression of siRNA directed to different coding regions of Cx43 resulted in differential levels of Cx43 silencing and impaired gap junctional intercellular communication. Cx43-silenced Hs578T cells grew faster and were more migratory. Finally, Western blot analysis revealed that down-regulation of Cx43 resulted in decreased expression of thrombospondin-1, an antiangiogenesis molecule, and increased expression of vascular endothelial growth factor. Taken together, these results suggest that Cx43 is required for maintaining cell differentiation and the regulation of molecules important in angiogenesis.
Farnesyl Transferase Inhibitor (R115777)–Induced Inhibition of STAT3(Tyr705) Phosphorylation in Human Pancreatic Cancer Cell Lines Require Extracellular Signal-Regulated Kinases
Authors: Venkatasubbarao Kolaparthi , Choudary Ahsan , Freeman James W.
Published: September 2005

In this study, we report that R115777, a nonpeptidomimetic farnesyl transferase inhibitor, suppresses the growth of human pancreatic adenocarcinoma cell lines and that this growth inhibition is associated with modulation in the phosphorylation levels of signal transducers and activators of transcription 3 (STAT3) and extracellular signal-regulated kinases (ERK). Treatment of cells with R115777 inhibited the tyrosine phosphorylation of STAT3(Tyr705), while increasing the serine phosphorylation of STAT3(Ser727). We found the differential phosphorylation of STAT3 was due to an increased and prolonged activation of ERKs. The biological significance of ERK-mediated inhibition of STAT3(Tyr705) phosphorylation was further assessed by treating the cells with an inhibitor (PD98059) of mitogen-activated protein kinase kinase (MEK) or by transfecting the cells with a vector that expresses constitutively active MEK-1. Expression of constitutively active MEK-1 caused an increase of ERK activity and inhibited STAT3(Tyr705) phosphorylation. Conversely, inhibition of ERK activity by PD98059 reversed the R115777-induced inhibition of STAT3(Tyr705) phosphorylation. R115777 also caused the inhibition of the binding of STAT3 to its consensus binding element. An increase in the activation of ERKs either by overexpressing MEK-1 or treatment of cells with R115777 caused an up-regulation in the levels of a cyclin-dependent kinase (cdk) inhibitor, p21cip1/waf1. These observations suggest that R115777-induced growth inhibition is partly due to the prolonged activation of ERKs that mediates an inhibition of STAT3(Tyr705) phosphorylation and an increase in the levels of p21cip1/waf1 in human pancreatic adenocarcinoma cell lines.
SKI-606, a Src/Abl Inhibitor withIn vivoActivity in Colon Tumor Xenograft Models
Authors: Golas Jennifer M. , Lucas Judy , Etienne Carlo , Golas Jonathan , Discafani Carolyn , Sridharan Latha , Boghaert Erwin , Arndt Kim , Ye Fei , Boschelli Diane H. , Li Fangbiao , Titsch Craig , Huselton Christine , Chaudhary Inder , Boschelli Frank
Published: September 2005

Src up-regulation is a common event in human cancers. In colorectal cancer, increased Src levels are an indicator of poor prognosis, and progression to metastatic disease is associated with substantial increases in Src activity. Therefore, we examined the activity of SKI-606, a potent inhibitor of Src and Abl kinases, against colon tumor lines in vitro and in s.c. tumor xenograft models. SKI-606 inhibited Src autophosphorylation with an IC50 of ∼0.25 μmol/L in HT29 cells. Phosphorylation of Tyr925 of focal adhesion kinase, a Src substrate, was reduced by similar concentrations of inhibitor. Antiproliferative activity on plastic did not correlate with Src inhibition in either HT29 or Colo205 cells (IC50s, 1.5 and 2.5 μmol/L, respectively), although submicromolar concentrations of SKI-606 inhibited HT29 cell colony formation in soft agar. SKI-606 also caused loosely aggregated Colo205 spheroids to condense into compact spheroids. On oral administration to nude mice at the lowest efficacious dose, peak plasma concentrations of ∼3 μmol/L, an oral bioavailability of 18%, and a t1/2 of 8.6 hours were observed. SKI-606 was orally active in s.c. colon tumor xenograft models and caused substantial reductions in Src autophosphorylation on Tyr418 in HT29 and Colo205 tumors. SKI-606 inhibited HT29 tumor growth on once daily administration, whereas twice daily administration was necessary to inhibit Colo205, HCT116, and DLD1 tumor growth. These results support development of SKI-606 as a therapeutic agent for treatment of colorectal cancer.
Modulation of Survival Signaling Pathways and Persistence of the Genotoxic Stress as a Basis for the Synergistic Interaction between the Atypical Retinoid ST1926 and the Epidermal Growth Factor Receptor Inhibitor ZD1839
Authors: Zanchi Chiara , Zuco Valentina , Lanzi Cinzia , Supino Rosanna , Zunino Franco
Published: September 2005

Strategies targeting apoptotic pathways may have relevance to improve the efficacy of antitumor therapy. Because synthetic atypical retinoids are potent inducers of apoptosis, there is an increasing interest in exploiting their potential in novel therapeutic approaches. In the present study, we have investigated the cellular effects of the combination of a novel atypical retinoid, ST1926, and the epidermal growth factor receptor inhibitor ZD1839. The results indicated a synergistic interaction between the two drugs associated with a dramatic enhancement of apoptotic response, up-regulation of the cell death receptor DR5, and caspase 8 activation. Other molecular events induced by the cotreatment included (a) a stabilization of the ST1926-induced genotoxic stress detected by formation of phosphorylated γ-H2AX foci and (b) a complete inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation associated with activation of the proapoptotic protein BAD (i.e., inhibition of phosphorylation on Ser112). In addition, ZD1839 itself inhibited survival pathways by causing a partial dephosphorylation of Akt and a marked down-regulation of survivin. The role of ERK-mediated survival pathways in the cellular response to the drug combination was further supported by the counteracting effect of stimulation of survival pathways by an alternative receptor tyrosine kinase and by the use of a specific inhibitor of the ERK pathway. In conclusion, the results support that the survival pathways activated by epidermal growth factor receptor are determinants of the cell susceptibility to ST1926-induced apoptosis and lowering survival signals may increase the cellular sensitivity to the atypical retinoid. The favorable pharmacologic profiles of both ST1926 and ZD1839 suggest that the combination of these well-tolerated agents may have therapeutic potential.
Increased Expression of Apoptosis Inhibitor Protein XIAP Contributes to Anoikis Resistance of Circulating Human Prostate Cancer Metastasis Precursor Cells
Authors: Berezovskaya Olga , Schimmer Aaron D. , Glinskii Anna B. , Pinilla Clemencia , Hoffman Robert M. , Reed John C. , Glinsky Gennadi V.
Published: September 2005

Survival in lymph or blood is an essential prerequisite for metastasis of carcinoma cells to distant organs. Recently, we reported isolation and initial biological characterization of circulating metastatic cells in a fluorescent, orthotopic, metastatic nude-mouse model of human prostate cancer. Here we show that the metastatic human prostate carcinoma cells selected for survival in the circulation have increased resistance to anoikis, which is apoptosis induced by cell detachment. Using gene silencing and gene transfer techniques, we show that increased expression of the apoptosis inhibitory protein XIAP contributes to anoikis resistance of the circulating metastatic human prostate carcinoma cells. We also provide initial preclinical data on the antimetastatic efficacy of recently discovered small-molecule antagonists of XIAP.
HER2 Overexpression Increases Sensitivity to Gefitinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, through Inhibition of HER2/HER3 Heterodimer Formation in Lung Cancer Cells
Authors: Hirata Akira , Hosoi Fumihito , Miyagawa Miho , Ueda Shu-ichi , Naito Seiji , Fujii Teruhiko , Kuwano Michihiko , Ono Mayumi
Published: September 2005

Gefitinib (Iressa), an epidermal growth factor receptor targeting drug, has been clinically useful for the treatment of patients with non–small cell lung cancer (NSCLC). Gefitinib is currently being applied in clinical studies as either a monotherapy, or as part of a combination therapy against prostate, head and neck, gastric, breast, and colorectal tumors. However, success rates vary between different tumor types, and thus it is important to understand which molecular target(s) are responsible for limiting the therapeutic efficacy of the drug. In this study, we ask whether expression of HER2 affects sensitivity to gefitinib in human lung cancer cells. We established two clones, LK2/HER2-32 and LK2/HER2-57, by transfecting HER2 cDNA into LK2, a NSCLC line with a low expression level of HER2. We observed no mutations in exons 18, 19, and 21 of EGFR gene in LK2, LK2/mock- and two HER2-trasfectants when we observed in-frame deletion mutations (E746-A750) adjacent to K745 in a gefitinib-sensitive NSCLC cell line, PC9. These LK2/HER2-32 and LK2/HER2-57 were much more sensitive to the cytotoxic effects of gefitinib than the parental LK2 lines. Treatment with 0.5 to 1 μmol/L gefitinib specifically blocked Akt activation in both HER2-transfectant lines, but not in the parental LK2 cells. Extracellular signal-regulated kinase-1/2 activation, however, was not blocked by gefitinib up to 10 μmol/L in either the parent or transfectant lines. Gefitinib was also shown to induce cell cycle arrest in the G1-S phase, and an accompanying increase of p27Kip1 was observed. LK2/HER2 transfectants showed constitutive formation of HER2/HER3 heterodimer, which were seen to associate with a regulatory subunit of phosphoinositide-3-kinase, p85α, when active. Treatment of LK2/HER2 cells with gefitinib markedly decreased the formation of HER2/HER3 heterodimers, HER3 basal phosphorylation, and the association of p85α with HER3. This study is the first to show that under basal growth conditions, HER2 sensitizes low-EGFR NSCLC cell lines to growth inhibition by gefitinib.
p300 Regulates Androgen Receptor–Independent Expression of Prostate-Specific Antigen in Prostate Cancer Cells Treated Chronically with Interleukin-6
Authors: Debes Jose D. , Comuzzi Barbara , Schmidt Lucy J. , Dehm Scott M. , Culig Zoran , Tindall Donald J.
Published: September 2005

Prostate cancer is the most frequent non–skin cancer in men. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor has been shown to play an important role. Androgen receptor is expressed in both early and late-stage prostate cancer. Also, androgen-regulated pathways are thought to be active as evidenced by elevated levels of prostate-specific antigen (PSA). In addition, several androgen receptor coactivators and cytokines are involved in prostate cancer progression. In this regard, we have shown previously that the coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), a cytokine involved in late-stage prostate cancer. In this study, we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronically with IL-6. We found that p300 but not CREB-binding protein induced activation of PSA in these cells and that the histone acetyltransferase activity of p300 was critical. This effect was independent of the presence of androgens or antiandrogens. Moreover, we found markedly reduced levels of androgen receptor in these cells and p300 transfection did not affect those levels, suggesting that the p300 effect on PSA could be bypassing the androgen receptor. Transfection with exogenous androgen receptor showed minimal response of PSA to androgens but higher response to p300. We found similar effects of p300 on the androgen response element III, which mediates the androgen receptor–dependent activation of PSA. Finally, we showed that p300 alone regulates expression of the endogenous PSA gene in the IL-6–treated cells. These findings reveal a new insight in the progression of prostate cancer, suggesting that coactivators, such as p300, play more important roles in late-stage prostate cancer, and could regulate androgen-dependent genes in the absence or with very low levels of androgen receptor.