bluuebunny/crossref_metadata_2025 huggingface dataset:
Capsaicin, a Component of Red Peppers, Inhibits the Growth of Androgen-Independent, p53 Mutant Prostate Cancer Cells
Authors: Mori Akio , Lehmann Sören , O'Kelly James , Kumagai Takashi , Desmond Julian C. , Pervan Milena , McBride William H. , Kizaki Masahiro , Koeffler H. Phillip
Published: March 2006

Capsaicin is the major pungent ingredient in red peppers. Here, we report that it has a profound antiproliferative effect on prostate cancer cells, inducing the apoptosis of both androgen receptor (AR)-positive (LNCaP) and -negative (PC-3, DU-145) prostate cancer cell lines associated with an increase of p53, p21, and Bax. Capsaicin down-regulated the expression of not only prostate-specific antigen (PSA) but also AR. Promoter assays showed that capsaicin inhibited the ability of dihydrotestosterone to activate the PSA promoter/enhancer even in the presence of exogenous AR in LNCaP cells, suggesting that capsaicin inhibited the transcription of PSA not only via down-regulation of expression of AR, but also by a direct inhibitory effect on PSA transcription. Capsaicin inhibited NF-κ activation by preventing its nuclear migration. In further studies, capsaicin inhibited tumor necrosis factor-α–stimulated degradation of IκBα in PC-3 cells, which was associated with the inhibition of proteasome activity. Taken together, capsaicin inhibits proteasome activity which suppressed the degradation of IκBα, preventing the activation of NF-κB. Capsaicin, when given orally, significantly slowed the growth of PC-3 prostate cancer xenografts as measured by size [75 ± 35 versus 336 ± 123 mm3 (±SD); P = 0.017] and weight [203 ± 41 versus 373 ± 52 mg (±SD); P = 0.0006; capsaicin-treated versus vehicle-treated mice, respectively]. In summary, our data suggests that capsaicin, or a related analogue, may have a role in the management of prostate cancer. (Cancer Res 2006; 66(6): 3222-9)
Akt-Mediated Cisplatin Resistance in Ovarian Cancer: Modulation of p53 Action on Caspase-Dependent Mitochondrial Death Pathway
Authors: Yang Xiaokui , Fraser Michael , Moll Ute M. , Basak Ajoy , Tsang Benjamin K.
Published: March 2006

Akt is a determinant of cisplatin [cis-diammine-dichloroplatinum (CDDP)] resistance in ovarian cancer cells, and this may be related to the regulation of p53. Precisely how Akt facilitates CDDP resistance and interacts with p53 is unclear. Apoptotic stimuli induce second mitochondria-derived activator of caspase (Smac) release from mitochondria into the cytosol, where it attenuates inhibitor of apoptosis protein–mediated caspase inhibition. Whereas Smac release is regulated by p53 via the transactivation of proapoptotic Bcl-2 family members, it is unclear whether p53 also facilitates Smac release via its direct mitochondrial activity. Here we show that CDDP induces mitochondrial p53 accumulation, the mitochondrial release of Smac, cytochrome c, and HTR/Omi, and apoptosis in chemosensitive but not in resistant ovarian cancer cells. Smac release was p53 dependent and was required for CDDP-induced apoptosis. Mitochondrial p53 directly induced Smac release. Akt attenuated mitochondrial p53 accumulation and Smac/cytochrome c/Omi release and conferred resistance. Inhibition of Akt facilitated Smac release and sensitized chemoresistant cells to CDDP in a p53-dependent manner. These results suggest that Akt confers resistance, in part, by modulating the direction action of p53 on the caspase-dependent mitochondrial death pathway. Understanding the precise etiology of chemoresistance may improve treatment for ovarian cancer. (Cancer Res 2006; 66(6): 3126-36)
Elongation Factor-2 Kinase Regulates Autophagy in Human Glioblastoma Cells
Authors: Wu Hao , Yang Jin-Ming , Jin Shengkan , Zhang Haiyan , Hait William N.
Published: March 2006

Elongation factor-2 kinase (eEF-2 kinase), also known as Ca2+/calmodulin–dependent kinase III, regulates protein synthesis by controlling the rate of peptide chain elongation. The activity of eEF-2 kinase is increased in glioblastoma and other malignancies, yet its role in neoplasia is uncertain. Recent evidence suggests that autophagy plays an important role in oncogenesis and that this can be regulated by mammalian target of rapamycin (mTOR). Because eEF-2 kinase lies downstream of mTOR, we studied the role of eEF-2 kinase in autophagy using human glioblastoma cell lines. Knockdown of eEF-2 kinase by RNA interference inhibited autophagy in glioblastoma cell lines, as measured by light chain 3 (LC3)-II formation, acidic vesicular organelle staining, and electron microscopy. In contrast, overexpression of eEF-2 kinase increased autophagy. Furthermore, inhibition of autophagy markedly decreased the viability of glioblastoma cells grown under conditions of nutrient depletion. Nutrient deprivation increased eEF-2 kinase activity and decreased the activity of S6 kinase, suggesting an involvement of mTOR pathway in the eEF-2 kinase regulation of autophagy. These results suggest that eEF-2 kinase plays a regulatory role in the autophagic process in tumor cells; and eEF-2 kinase is a downstream member of the mTOR signaling; eEF-2 kinase may promote cancer cell survival under conditions of nutrient deprivation through regulating autophagy. Therefore, eEF-2 kinase may be a part of a survival mechanism in glioblastoma and targeting this kinase may represent a novel approach to cancer treatment. (Cancer Res 2006; 66(6): 3015-23)
Human Leukocyte Antigen-A2–Restricted CTL Responses to Mutated BRAF Peptides in Melanoma Patients
Authors: Somasundaram Rajasekharan , Swoboda Rolf , Caputo Laura , Otvos Laszlo , Weber Barbara , Volpe Patricia , van Belle Patricia , Hotz Susan , Elder David E. , Marincola Francesco M. , Schuchter Lynn , Guerry DuPont , Czerniecki Brian J. , Herlyn Dorothee
Published: March 2006

Mutated BRAF (BRAFV600E) is a potential immunotherapeutic target for melanoma because of its tumor specificity and expression in the majority of these lesions derived from different patients. BRAFV600E is expressed intracellularly and not on the cell surface, therefore providing a target for T cells but not B cells. Demonstration of patients' T cell responses to BRAFV600E would suggest the feasibility of active specific immunotherapy targeting the mutation in these patients. In the present study, BRAFV600E peptides with putative binding sites for human leukocyte antigen (HLA)-A2 were used to stimulate T lymphocytes of HLA-A2–positive melanoma patients. Four of five patients with BRAFV600E-positive lesions showed lymphoproliferative responses to BRAFV600E peptide stimulation. These responses were specific for the mutated epitope and HLA-A2 was restricted in three patients. Lymphocytes from these three patients were cytotoxic against HLA-A2–matched BRAFV600E-positive melanoma cells. None of the four patients with BRAFV600E-negative lesions and none of five healthy donors had lymphoproliferative responses specific for the mutated epitope. The high prevalence (∼50%) of HLA-A2 among melanoma patients renders HLA-A2–restricted BRAFV600E peptides attractive candidate vaccines for these patients. (Cancer Res 2006; 66(6): 3287-93)
Inhibition of Lymphotoxin-β Receptor–Mediated Cell Death by Survivin-ΔEx3
Authors: You Ren-In , Chen Mei-Chieh , Wang Hsei-Wei , Chou Yang-Chieh , Lin Chi-Hung , Hsieh Shie-Liang
Published: March 2006

TNFSF14/LIGHT is a member of the tumor necrosis factor superfamily that binds to lymphotoxin-β receptor (LTβR) to induce cell death via caspase-dependent and caspase-independent pathways. It has been shown that cellular inhibitor of apoptosis protein-1 inhibits cell death by binding to LTβR-TRAF2/TRAF3 complexes and caspases. In this study, we found that both Kaposi's sarcoma–associated herpesvirus K7 (KSHV-K7), a viral inhibitor of apoptosis protein, and the structurally related protein survivin-ΔEx3 could inhibit LTβR-mediated caspase-3 activation. However, only survivin-ΔEx3 could protect cells from LTβR-mediated cell death. The differential protective effects of survivin-ΔEx3 and KSHV-K7 can be attributed to the fact that survivin-ΔEx3, but not KSHV-K7, is able to maintain mitochondrial membrane potential and inhibit second mitochondria-derived activator of caspase/DIABLO release. Moreover, survivin-ΔEx3 is able to inhibit production of reactive oxygen species and can translocate from nucleus to cytosol to associate with apoptosis signal-regulating kinase 1 after activation of LTβR. Furthermore, survivin-ΔEx3 protects LTβR-mediated cell death in caspase-3-deficient MCF-7 cells. Thus, survivin-ΔEx3 is able to regulate both caspase-dependent and caspase-independent pathways, whereas inhibition of caspase-independent pathway is both sufficient and necessary for its protective effect on LTβR-mediated cell death. (Cancer Res 2006; 66(6): 3051-61)
Hypoxic Culture Induces Expression of Sialin, a Sialic Acid Transporter, and Cancer-Associated Gangliosides Containing Non–Human Sialic Acid on Human Cancer Cells
Authors: Yin Jun , Hashimoto Ayako , Izawa Mineko , Miyazaki Keiko , Chen Guo-Yun , Takematsu Hiromu , Kozutsumi Yasunori , Suzuki Akemi , Furuhata Kimio , Cheng Feng-Leng , Lin Chun-Hung , Sato Chihiro , Kitajima Ken , Kannagi Reiji
Published: March 2006

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-GM2, a cancer-associated ganglioside containing non–human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-GM2, whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non–human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-GM2. We propose that the preferential expression of NeuGc-GM2 in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non–human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically GM2, containing non–human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-GM2 is a potential therapeutic target for hypoxic cancer cells. (Cancer Res 2006; 66(6): 2937-45)
HMGA1 Inhibits the Function of p53 Family Members in Thyroid Cancer Cells
Authors: Frasca Francesco , Rustighi Alessandra , Malaguarnera Roberta , Altamura Sandro , Vigneri Paolo , Del Sal Giannino , Giancotti Vincenzo , Pezzino Vincenzo , Vigneri Riccardo , Manfioletti Guidalberto
Published: March 2006

HMGA1 is an architectural transcription factor expressed at high levels in transformed cells and tumors. Several lines of evidence indicate that HMGA1 up-regulation is involved in the malignant transformation of thyroid epithelial cells. However, the mechanisms underlying the effect of HMGA1 on thyroid cancer cell phenotype are not fully understood. We now show that in thyroid cancer cells, HMGA1 down-regulation by small interfering RNA and antisense techniques results in enhanced transcriptional activity of p53, TAp63α, TAp73α, and, consequently, increased apoptosis. Coimmunoprecipitation and pull-down experiments with deletion mutants showed that the COOH-terminal oligomerization domain of p53 family members is required for direct interaction with HMGA1. Moreover, inhibition of HMGA1 expression in thyroid cancer cells resulted in increased p53 oligomerization in response to the DNA-damaging agent doxorubicin. Finally, electrophoretic mobility shift assay experiments showed that the p53-HMGA1 interaction results in reduced DNA-binding activity. These results indicate a new function of HMGA1 in the regulation of p53 family members, thus providing new mechanistic insights in tumor progression. (Cancer Res 2006; 66(6): 2980-89)
Significant Effect of Homologous Recombination DNA Repair Gene Polymorphisms on Pancreatic Cancer Survival
Authors: Li Donghui , Liu Hui , Jiao Li , Chang David Z. , Beinart Garth , Wolff Robert A. , Evans Douglas B. , Hassan Manal M. , Abbruzzese James L.
Published: March 2006

Genetic variation in DNA repair may affect the clinical response to cytotoxic therapies. We investigated the effect of six single nucleotide polymorphisms of the RecQ1, RAD54L, XRCC2, and XRCC3 genes on overall survival of 378 patients with pancreatic adenocarcinoma who were treated at University of Texas M.D. Anderson Cancer Center during February 1999 to October 2004 and were followed up to October 2005. Genotypes were determined using the MassCode method. Survival was determined from pathologic diagnosis to death. Patients who were alive at the last follow-up evaluation were censored at that time. Kaplan-Meier plot, log-rank test, and Cox regression were used to compare overall survival by genotypes. A significant effect on survival of all patients was observed for RecQ1 and RAD54L genes. The median survival time was 19.2, 14.7, and 13.2 months for the RecQ1 159 AA, AC, and CC genotypes, and 16.4, 13.3, and 10.3 months for RAD54L 157 CC, CT, and TT genotypes, respectively. A significantly reduced survival was associated with the variant alleles of XRCC2 R188H and XRCC3 A17893G in subgroup analysis. When the four genes were analyzed in combination, an increasing number of adverse alleles were associated with a significantly decreased survival. Subgroup analyses have shown that the genotype effect on survival was present among patients without metastatic disease or among patients who receive radiotherapy. These observations suggest that polymorphisms of genes involved in the repair of DNA double-strand breaks significantly affect the clinical outcome of patients with pancreatic cancer. (Cancer Res 2006; 66(6): 3323-30)
A Novel Mechanism of Synergistic Cytotoxicity with 5-Fluorocytosine and Ganciclovir in Double Suicide Gene Therapy
Authors: Boucher Paul D. , Im Michael M. , Freytag Svend O. , Shewach Donna S.
Published: March 2006

The combination of cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) suicide gene protocols has resulted in enhanced antitumor activity in cultured tumor cells and animal models. In this study, we show that concurrent addition of prodrugs 5-fluorocytosine (5-FC) and ganciclovir (GCV) was less efficacious than sequential treatment in human DU145 prostate carcinoma cells infected with an adenovirus containing a CD/HSV-TK fusion gene. If cells were incubated for 24 hours with 5-FC followed by a 24-hour GCV treatment, GCV triphosphate levels were 2-fold higher, incorporation of GCV monophosphate into DNA was 2.5-fold higher, and growth inhibition was increased 4-fold compared with simultaneous treatment. As expected, cellular dTTP levels were reduced during the 5-FC preincubation. However, dGTP pools also declined parallel to the dTTP decrease. Similar results were obtained when 5-fluorouracil or 5-fluoro-2′-deoxyuridine was used instead of CD/5-FC. These data allowed us to propose a novel hypothesis for the synergistic interaction between CD/5-FC and HSV-TK/GCV treatments. We suggest that the CD/5-FC–mediated reduction of dTTP results in a concurrent decrease of dGTP due to allosteric regulation of ribonucleotide reductase. Because dGTP is the endogenous competitor of GCV triphosphate, depleted dGTP at the time of GCV addition results in increased GCV in DNA and cell kill. In fact, addition of deoxyguanosine during the 5-FC incubation reverses the dGTP depletion, reduces the amount of GCV monophosphate incorporated into DNA, and prevents the CD/5-FC–mediated enhancement of HSV-TK/GCV cytotoxicity. Understanding this mechanistic interaction may help recognize better strategies for creating more efficacious clinical protocols. (Cancer Res 2006; 66(6): 3230-7)
4-Oxo-Fenretinide, a Recently Identified Fenretinide Metabolite, Induces Marked G2-M Cell Cycle Arrest and Apoptosis in Fenretinide-Sensitive and Fenretinide-Resistant Cell Lines
Authors: Villani Maria Grazia , Appierto Valentina , Cavadini Elena , Bettiga Arianna , Prinetti Alessandro , Clagett-Dame Margaret , Curley Robert W. , Formelli Franca
Published: March 2006

4-Oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a recently identified metabolite of fenretinide (4-HPR). We explored the effectiveness of 4-oxo-4-HPR in inducing cell growth inhibition in ovarian, breast, and neuroblastoma tumor cell lines; moreover, we investigated the molecular events mediating this effect in two ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/HPR) to 4-HPR. 4-Oxo-4-HPR was two to four times more effective than 4-HPR in most cell lines, was effective in both 4-HPR–sensitive and 4-HPR–resistant cells, and, in combination with 4-HPR, caused a synergistic effect. The tumor growth-inhibitory effects of 4-oxo-4-HPR seem to be independent of nuclear retinoid receptors (RAR), as indicated by the failure of RAR antagonists to inhibit its effects and by its poor ability to bind and transactivate RARs. Unlike 4-HPR, which only slightly affected the G1 phase of the cell cycle, 4-oxo-4-HPR caused a marked accumulation of cells in G2-M. This effect was associated with a reduction in the expression of regulatory proteins of G2-M (cyclin-dependent kinase 1 and cdc25c) and S (cyclin A) phases, and with an increase in the expression of apoptosis-related proteins, such as p53 and p21. Apoptosis was induced by 4-oxo-4-HPR in both 4-HPR–sensitive and 4-HPR–resistant cells and involved activation of caspase-3 and caspase-9 but not caspase-8. We also showed that 4-oxo-4-HPR, similarly to 4-HPR, increased reactive oxygen species generation and ceramide levels by de novo synthesis. In conclusion, 4-oxo-4-HPR is an effective 4-HPR metabolite that might act as therapeutic agent per se and, when combined with 4-HPR, might improve 4-HPR activity or overcome 4-HPR resistance. (Cancer Res 2006; 66(6): 3238-47)
Behavioral Profiling of Human Transitional Cell Carcinoma Ex vivo
Authors: Estrada Carlos R. , Salanga Matthew , Bielenberg Diane R. , Harrell W. Bruce , Zurakowski David , Zhu Xuping , Palmer Matthew R. , Freeman Michael R. , Adam Rosalyn M.
Published: March 2006

Outcome studies of many types of cancer have revealed that tumors of indistinguishable histologic appearance may differ significantly in aggressiveness and in their response to therapy. A strategy that would enable early identification of patients at high risk for disease progression and allow screening of multiple therapeutic agents simultaneously for efficacy would improve clinical management. We have developed an orthotopic organ culture model of bladder cancer in which quantum dot–based fluorescent imaging approaches are used to obtain quantitative measurements of tumor cell behavior. Human transitional cell carcinoma (TCC) cells are labeled with quantum dot nanoparticles, and the cells instilled into the rat bladder in vivo, after which the bladder is excised and cultured ex vivo. Cell implantation, proliferation, and invasion into the organ wall are monitored using epifluorescence imaging and two-photon laser scanning confocal microscopy. Using this approach, we were able to assign distinct phenotypes to two metastatic bladder cancer cell lines based on different patterns of invasiveness into the bladder wall. We also showed that established tumor cell masses regressed following intravesical administration of the chemotherapeutic drug thiotepa. Collectively, these findings suggest that this assay system, which we have named EViTAS (for ex vivo tumor assay system), can recapitulate salient aspects of tumor growth in the host and is amenable to behavioral profiling of human cancer. (Cancer Res 2006; 66(6): 3078-86)
Signal Transducers and Activators of Transcription 3 Activation Is Involved in Nuclear Accumulation of β-Catenin in Colorectal Cancer
Authors: Kawada Mayumi , Seno Hiroshi , Uenoyama Yoshito , Sawabu Tateo , Kanda Naoki , Fukui Hirokazu , Shimahara Yasuyuki , Chiba Tsutomu
Published: March 2006

Nuclear accumulation of β-catenin is a key event for the development of colorectal cancer. Little is known, however, about the mechanisms underlying translocation of β-catenin from the cytoplasm or the membrane to the nucleus. The present study examined whether signal transducers and activators of transcription 3 (STAT3) activation is involved in the nuclear accumulation of β-catenin in colorectal cancer cells. Of the 90 primary colorectal cancer tissues, 40 (44.4%) were positive for nuclear staining of p-STAT3 and 63 (70.0%) were positive for nuclear staining of β-catenin. The nuclear staining of both p-STAT3 and β-catenin were observed predominantly in the periphery of the cancer tissues. Importantly, of the 40 tumors with p-STAT3 nuclear staining, 37 (92.5%) were also positive for nuclear β-catenin staining and there was a significant correlation between p-STAT3 and β-catenin nuclear staining (P < 0.01). Coexpression of nuclear p-STAT3 and β-catenin was associated with lower patient survival (P < 0.01). In an in vitro study using a human colon cancer cell line, SW480, inhibition of STAT3 by dominant negative STAT3 or the Janus kinase inhibitor, AG490, induced translocation of β-catenin from the nucleus to the cytoplasm or membrane. Luciferase assays revealed that STAT3 inhibition resulted in significant suppression of β-catenin/T-cell factor transcription in association with significant inhibition of cell proliferation (P < 0.05). These findings suggest that in colorectal cancer, STAT3 activation is involved in the nuclear accumulation of β-catenin, resulting in poor patient survival. (Cancer Res 2006; 66(6): 2913-7)
Enhanced Photocarcinogenesis in Interleukin-12–Deficient Mice
Authors: Maeda Akira , Schneider Stefan W. , Kojima Motohiro , Beissert Stefan , Schwarz Thomas , Schwarz Agatha
Published: March 2006

UV-induced DNA damage is the basis for the development of UV-mediated skin cancer because reduction of DNA damage lowers the risk for photocarcinogenesis. The cytokine interleukin (IL)-12 was shown to exhibit the capacity to reduce UV-induced DNA damage presumably via induction of nucleotide excision repair. Because IL-12 is also produced in the skin, we wondered whether endogenous IL-12 protects from photocarcinogenesis. Therefore, we used knockout mice that lack the IL-12p40 chain and thus do not secrete biologically active IL-12. IL-12p40 knockout (IL-12p40−/−) and wild-type (wt) mice were exposed thrice weekly to UV. Skin biopsies obtained after 6 weeks revealed significantly increased numbers of sunburn cells in IL-12p40−/− mice. Additionally, a higher load of UV-induced pyrimidine dimers could be detected in the skin of UV-exposed IL-12p40−/− mice. Staining of epidermal sheets with an antibody against the tumor suppressor gene p53 revealed a higher number of p53 patches in the skin of IL-12p40−/− mice. After ∼200 days, first skin tumors developed. Kaplan-Meier analysis indicated a significantly increased probability of tumor development in the IL-12p40−/− mice. In addition, the number of tumors developing in the individual mice was significantly higher in IL-12p40−/− mice than in wt mice. Tumors obtained in IL-12p40−/− mice grew faster than those obtained from wt mice on inoculation into nu/nu mice. This was confirmed in an electrophysiologic assay evaluating the intrinsic invasive potency of tumor cells. Together, these data indicate that IL-12 deficiency is associated with an increased risk to develop UV-induced skin cancer, implying that endogenous IL-12 may protect from photocarcinogenesis. (Cancer Res 2006; 66(6): 2962-9)
Transplantable Tumor Lines Generated in Clonal Zebrafish
Authors: Mizgireuv Igor V. , Revskoy Sergei Y.
Published: March 2006

Transplantable zebrafish tumors are a novel and very promising model in cancer research. However, further progress in this field has been contained by a lack of true inbred lines in zebrafish. To overcome this problem, we generated two lines of homozygous diploid clonal zebrafish lines (i.e., CB1 and CW1), which allowed us to carry out transplantation of any tissue, including tumors, from one fish to another within a line without rejection of the graft. The primary tumors in CB1 fish were induced by N-nitrosodiethylamine (DEN). The histologic analysis of these tumors revealed different types of hepatocellular carcinomas, hepatoblastomas, hepatoma, cholangiocarcinoma, and pancreatic carcinoma. Four spontaneous acinar cell carcinomas of pancreas were also found in 10- to 18-month-old CB1 fish. Small pieces of tissue or cell suspensions of either DEN-induced or spontaneous tumors were serially transplanted into the peritoneal cavity of syngeneic fish at different stages of development from 5-day-old larvae to adult fish. The development of grossly visible tumors occurred from 2 weeks to 3 months after tumor grafting and grew either as solitary smooth nodules or as an amorphous jelly-like mass infiltrating abdominal organs. The majority of tumors were also successfully transplanted to isogeneic (F1 generation from crossing CB1 × CW1) fish. At the present time, 19 transplantable zebrafish tumor lines have been generated and maintained for as long as 3 to 25 passages. This model provides a novel tool for studying experimental tumor biology and therapy and will become a cost effective system for high throughput screening of anticancer drugs. (Cancer Res 2006; 66(6): 3120-5)
Levels of HdmX Expression Dictate the Sensitivity of Normal and Transformed Cells to Nutlin-3
Authors: Patton John T. , Mayo Lindsey D. , Singhi Aatur D. , Gudkov Andrei V. , Stark George R. , Jackson Mark W.
Published: March 2006

Hdm2 and HdmX coordinately regulate the stability and function of p53. Each is overexpressed in subsets of many different types of malignancy, and most of these subsets maintain wild-type p53. Nutlins, newly discovered small-molecule inhibitors of the Hdm2-p53 interaction, offer a novel strategy for therapy of tumors with wild-type p53. We now show that Nutlin-3 efficiently induces apoptosis and diminishes long-term survival of human fibroblasts transformed in vitro by Hdm2 but not HdmX. The resistance of cells overexpressing HdmX to Nutlin-3 is due to its inability to disrupt the p53-HdmX interaction, resulting in continued suppression of p53 activity. Although HdmX overexpression yielded cells resistant to Nutlin-3, ablation of HdmX expression by short hairpin RNA sensitized tumor cells to Nutlin-3–mediated cell death or arrest. Furthermore, deletion of the COOH-terminal RING finger domain of HdmX completely reversed the resistance to Nutlin-3, probably reflecting the requirement of the RING finger for interaction with Hdm2. Thus, the relative abundance of Hdm2 and HdmX and the specificity of Nutlin-3 for Hdm2 influence the sensitivity of cells to p53-dependent apoptosis or arrest in response to Nutlin-3. Our findings establish Hdm2 and HdmX as independent therapeutic targets with respect to reactivating wild-type p53 as a means for cancer therapy. (Cancer Res 2006; 66(6): 3169-76)
A Novel Transcriptional Inhibitor Induces Apoptosis in Tumor Cells and Exhibits Antiangiogenic Activity
Authors: Radhakrishnan Senthil K. , Gartel Andrei L.
Published: March 2006

Using a high-throughput cell-based assay, we identified a nucleoside analogue 4-amino-6-hydrazino-7-β-d-ribofuranosyl-7H-pyrrolo(2,3-d)-pyrimidine-5-carboxamide (ARC), which has the properties of a general transcriptional inhibitor. Specifically, ARC inhibits the phosphorylation of RNA polymerase II by positive transcription elongation factor-b, leading to a block in transcriptional elongation. ARC was able to potently repress p53 targets p21 and hdm2 (human homologue of mdm2) protein levels, but dramatically increased p53 levels similar to other transcriptional inhibitors, including flavopiridol. This increase in p53 corresponded to the down-regulation of short-lived protein hdm2, which is a well-established negative regulator of p53. Remarkably, ARC induced potent apoptosis in human tumor and transformed, but not in normal cells, and possessed strong antiangiogenic activity in vitro. Although ARC promoted the accumulation of p53, ARC-induced apoptosis in tumor cells was p53-independent, suggesting that it may be useful for the treatment of tumors with functionally inactive p53. Furthermore, cell death induced by ARC had a strong correlation with down-regulation of the antiapoptotic gene survivin, which is often overexpressed in human tumors. Taken together, our data suggests that ARC may be an attractive candidate for anticancer drug development. (Cancer Res 2006; 66(6): 3264-70)
Global Gene Expression Profiling of Circulating Endothelial Cells in Patients with Metastatic Carcinomas
Authors: Smirnov Denis A. , Foulk Bradley W. , Doyle Gerald V. , Connelly Mark C. , Terstappen Leon W.M.M. , O'Hara S. Mark
Published: March 2006

Increased numbers of endothelial cells are observed in peripheral blood of cancer patients. These circulating endothelial cells (CECs) may contribute to the formation of blood vessels in the tumor or reflect vascular damage caused by treatment or tumor growth. Characterization of these cells may aid in the understanding of the angiogenic process and may provide biomarkers for treatment efficacy of angiogenesis inhibitors. To identify markers typical for CECs in cancer patients, we assessed global gene expression profiles of CD146 immunomagnetically enriched CECs from healthy donors and patients with metastatic breast, colorectal, prostate, lung, and renal cancer. From the generated gene profiles, a list of 61 marker genes for CEC detection was generated, and their expression was measured by real-time quantitative PCR in blood samples from 81 metastatic cancer patients and 55 healthy donors that were immunomagnetically enriched for CECs. A set of 34 genes, among which novel CEC-associated genes, such as THBD, BST1, TIE1, POSTN1, SELE, SORT1, and DTR, were identified that were expressed at higher levels in cancer patients compared with healthy donors. Expression of the VWF, DTR, CDH5, TIE, and IGFBP7 genes were found to discriminate between cancer patients and “healthy” donors with a receiver operating characteristic curve accuracy of 0.93. Assessment of the expression of these genes may provide biomarkers to evaluate treatment efficacy. (Cancer Res 2006; 66(6): 2918-22)
Bcl-2 Inhibition of Autophagy: A New Route to Cancer?
Authors: Pattingre Sophie , Levine Beth
Published: March 2006

Bcl-2 was the first identified cellular protein that functions as an oncogene by blocking apoptotic cell death. Beclin 1, the first identified mammalian autophagy gene product, is a haploinsufficient tumor suppressor that was originally isolated as a Bcl-2-interacting protein. We recently showed that Bcl-2 negatively regulates Beclin 1-dependent autophagy and Beclin 1-dependent autophagic cell death. These findings raise the possibility that Bcl-2 family members may function as oncogenes not only by blocking apoptosis but also by blocking autophagy. (Cancer Res 2006; 66(6): 2885-8)
Safety and Efficacy of Oral Slow Release Morphine for Maintenance Treatment in Heroin Addicts: A 6-Month Open Noncomparative Study
Authors: Vasilev Georgi N. , Alexieva Daniela Z. , Pavlova Rositsa Z.
Published: March 2006

This open-label, noncomparative, single-center trial evaluated the safety and efficacy of once-daily treatment with slow release oral morphine (SROM) capsules for the maintenance treatment of 20 outpatients with heroin dependency over 6 months at the National Institute for Addictions in Sofia, Bulgaria. Doses were individually titrated up to a mean daily maintenance dose of 760 mg (range 440–1,200 mg). SROM was effective in significantly reducing the signs and symptoms of opioid withdrawal and craving for heroin, with stabilization generally evident within two weeks. Nineteen patients completed 6 months of treatment and illicit opioid use was virtually eliminated. One patient withdrew voluntarily at 22 weeks. Validated questionnaires and tests indicated improvements in patients’ well-being from baseline assessments. These included significant improvements with regard to suicidal depression (85%), anxiety and dysphoria (66%), general illness (58%), social dysfunction (54%), sense of hopelessness (34%), attention (25%), and self-reported typical depressive (27%) and disease-related (11%) symptoms. No deaths, serious adverse events, or withdrawals due to adverse events occurred. Five episodes of constipation and one episode of sweating (all nonserious and of mild or moderate severity) were reported. Vital signs were unaffected by SROM and no weight change was evident over the study period. The observations made in this study indicate a promising role for once-daily treatment with SROM in the clinical management of heroin dependency.
Influence of Pretreatment Coping Strategies on the Outcome of Outpatient Treatment of Danish Alcohol Abusers
Authors: Bussey Rask Marie , Jørgensen Tina , Pinnerup Jensen Jeanette , Bech Jørgensen Katrine , Madsen Mia , Nielsen Bent , Nybo Andersen Anne-Marie , Søgaard Nielsen Anette , Grønbæk Morten
Published: March 2006

An important issue regarding treatment for alcohol abuse is the high rate of relapse following treatment. In the research on treatment of alcohol abuse, the concept of coping has been proposed as a relevant factor in the relationship between relapse crises and treatment outcome. The present study investigated the role of pretreatment coping strategies in outcome of outpatient treatment for alcohol abuse. The pretreatment coping strategies of 136 clients receiving outpatient treatment for alcohol abuse were examined as a predictor of drinking pattern after treatment. The pretreatment coping strategies were assessed by the COPE questionnaire. Drinking pattern after treatment was assessed at follow-up one year after treatment was entered. Results indicated that some pretreatment coping strategies are identifiable as adaptive and maladaptive coping strategies, respectively, regarding successful treatment for alcohol abuse. Restraint coping was found predictive of a positive drinking pattern at follow-up while the use of alcohol to cope was found predictive of a negative drinking pattern. Furthermore, the results showed tendencies towards the possibility that some coping strategies co-operated differently with types of treatment methods.
Perceived Causal and Treatment Factors Related to Substance Abuse: Gender Differences
Authors: Lev-Wiesel Rachel , Shuval Ronit
Published: March 2006

The study aimed to examine gender differences of causal and treatment factors in substance use as perceived by adults who are addicted to drugs in the southern region of Israel. Eighty-one men and a matched group of 81 women recruited from one methadone center and several welfare agencies, average age was about 31 years, about a third immigrated from the former Soviet Union, were administered self-report questionnaires, which measured the following: demographic variables, perceived causal and treatment factors. Results indicated the following: women drug users perceived familial circumstances such as domestic violence or incest to have higher impact on substance abuse than men; men reported curiosity and the influence of addicted friends to have higher impact on substance abuse than women. In addition, women perceived personality characteristics such as willpower to have greater impact on treatment retention than men; men regarded formal and informal social support to have greater impact on treatment retention than women. Results were discussed in light of planning and developing treatment programs.
Imaging Protein Trafficking
Authors: Brown Dennis
Published: March 2006

The need to understand complex intracellular trafficking mechanisms from both a basic and disease-oriented perspective has stimulated considerable interest in the development of real-time microscopy tools. Recent advances in instrumentation and the development of molecular bioprobes, such as green fluorescent protein and its derivatives, have opened up a new era in our ability to perform close to real-time imaging of cellular events with high spatial and temporal resolution, and with high sensitivity. This review briefly introduces and discusses some of the systems and methodologies that are available from several manufacturers, including laser scanning and spinning disk confocal microscopy, and total internal reflectance microscopy.
Quantitative Microscopic Approaches for Studying Kidney Functions
Authors: Yu Weiming
Published: March 2006

Intravital ratiometric microscopy is a powerful method for quantitative study of kidney functions. As demonstrated in the examples, both the generalized polarity and direct ratio imaging approaches allow investigators to address basic and important questions such as those related to filtration, permeability and reabsorption. Both approaches have similar advantageous for in vivo imaging. However, they have different sensitivities and responses to the component intensity changes. Therefore, investigators must design their experiments based on the characteristics of the generalized polarity function and direct ratio approach.
Imaging Vascular Pathology
Authors: Sutton Timothy A. , Hörbelt Markus , Sandoval Ruben M.
Published: March 2006

Examining the mechanisms of renal microvascular alterations in disease and the ramifications they have for overall organ function has remained a challenge due to the complexity of the renal microvascular system and the inability to examine it with sufficient resolution in vivo. However, advances in intravital microscopy have provided opportunities to meet these challenges. In this review we will examine specific areas where intravital imaging has advanced our knowledge of renal disease processes and present areas where these techniques, especially intravital multiphoton microscopy, have even further potential to integrate our knowledge of renal vascular pathology.
Fluorescent Labeling of Renal Cells in vivo
Authors: Ashworth Sharon L. , Tanner George A.
Published: March 2006

In vivo fluorescence imaging, using confocal or multiphoton microscopes, provides a powerful method to analyze kidney function in experimental animals. In this review, the preparation used for physiological studies in rats is described. A variety of fluorescent probes are available to study glomerular permeability, renal blood flow, peritubular capillary permeability, cell ion concentrations, tubule transport properties, and the functional status of renal cells. We have recently used micropuncture techniques and an adenovirus vector to accomplish gene transfer into kidney tubule and endothelial cells; this new methodology will allow the dynamic study of fluorescently-labeled proteins. Two examples of the use of two-photon fluorescence microscopy to study renal pathophysiology, namely polycystic kidney disease and renal ischemia, are presented. Software is available to quantify data collected from in vivo imaging experiments and to construct 3-dimensional images of renal structures. Two-photon or confocal microscopy offers many opportunities for a better understanding of kidney function in health and disease.
Urocortin and Corticotropin-Releasing Hormone Receptor Type 2 Expression in the Human Gallbladder
Authors: Chatzaki Ekaterini , Euthymiadis Christoforos , Kyriaki Sofia , Lambropoulou Maria , Tsaroucha Alexandra , Laftsidis Prodromos , Simopoulos Konstantinos
Published: March 2006

The corticotropin-releasing hormone (CRH) system, consisting of CRH and the homologue neuropeptide urocortin together with their receptors CRH1 and CRH2 and a specific binding protein (CRH-BP), holds the main role in mediating the response to stressful stimuli. Besides their expression in the brain, CRH peptides and receptors have been found in multiple peripheral sites. Here we investigate the expression of CRH, urocortin, CRH receptors, and CRH-BP in the wall of human normal and inflamed gallbladders, using RT-PCR and immunohistochemistry. Urocortin, but not CRH gene transcripts, was detected in RNA isolated from human gallbladder biopsy specimens. Urocortin immunoreactivity was localized in epithelial cells of the gallbladder mucosa. Gene expression of CRH2 receptor was also detected, and the receptor protein had a localization similar to that of urocortin. Finally, CRH-BP gene expression and low levels of protein immunoreactivity were also shown. There were no differences in the expression profiles of all the above molecules between normal and inflamed tissues. In conclusion, the CRH system is present in the human gallbladder, urocortin being the major ligand expressed, possibly exerting an autocrine/paracrine biological role via activation of the CRH2(α) receptors found locally. Further study is required to enfold the biological role of these effectors in gallbladder physiology and pathogenesis.
Profile Analysis and Immunoglobulin E Reactivity of Wheat Protein Hydrolysates
Authors: Akiyama Hiroshi , Sakata Kozue , Yoshioka Yasuo , Murata Yoshifumi , Ishihara Yoshihiro , Teshima Reiko , Sawada Jun-ichi , Maitani Tamio
Published: March 2006

Background: Wheat protein hydrolysates have been traditionally used as food additives and are now being used in cooking worldwide. There have been a few studies on the relationship between the molecular mass distribution and the immunoglobulin E (IgE) reactivity of the wheat protein hydrolysates. Method: We analyzed the peptide profile of commercial wheat protein hydrolysate samples from enzymatic or acid hydrolysis of wheat protein using size exclusion chromatography. We further investigated the IgE reactivity of the wheat protein hydrolysates using the inhibition ELISA method and sera of 5 patients sensitive to wheat. Results: The wheat protein enzymatic hydrolysate samples showed high concentrations of peptides with molecular masses greater than 1,050 Da, whereas in contrast, the wheat protein acid hydrolysates showed extremely low concentrations of peptides with molecular masses greater than 1,050 Da. Tested wheat protein acid hydrolysates hardly inhibited the patient IgE binding ability to wheat proteins in the five patient sera. On the contrary, some tested wheat protein enzymatic hydrolysate samples inhibited the IgE binding ability to wheat proteins. Conclusion: These results suggested that the uptake of wheat protein enzymatic hydrolysates might still have the possibility of causing food allergic reactions in patients allergic to wheat and the processed foods containing them.
Functional Intravital Imaging of Leukocytes in Animal Models of Renal Injury
Authors: Atkinson Simon J.
Published: March 2006

An important emerging paradigm in the understanding of renal disease is the recognition of the central role of inflammation in the initiation and progression of acute and chronic kidney injury. These advances have led to an increasing awareness of the importance of leukocytes (white blood cells (WBC)) in the pathogenesis of renal disease, and the necessity for a greater understanding of the specific roles of different WBC lineages. All aspects of WBC function have been implicated in aspects of renal disease. In many cases soluble factors derived from these cells (cytokines, complement, immunoglobulins, etc.) having effects remote from the secreting cells are involved, while in other cases there is apparently more direct involvement of infiltrating cells themselves acting on their immediate surroundings. This highlights the importance of understanding the dynamic behavior of specific WBC cell types and their interactions with the intrinsic cells of the kidney during injury. New insight into this question is promised by recent developments in imaging technology that allow WBC movement and interactions with endothelial or epithelial cells or with the extracellular matrix to be visualized within tissues, even in the relatively unperturbed setting of intact organs in the live animal.
Development of a Novel Method to Detect Helicobacter pyloricagA Genotype from Paraffin-Embedded Materials: Comparison between Patients with Duodenal Ulcer and Gastric Cancer in Young Japanese
Authors: Ueda Hiroyuki , Ito Masanori , Eguchi Hidetaka , Tanaka Shinji , Yoshihara Masaharu , Haruma Ken , Hatakeyama Masanori , Chayama Kazuaki
Published: March 2006

Background/Aim:cagA gene polymorphism of Helicobacter pylori contributes to clinical outcome of patients. We investigated the implication of the cagA polymorphism in young Japanese patients using paraffin-embedded sections. Methods: We studied 71 young patients with gastric cancer or with duodenal ulcer. H. pylori infection was confirmed by sections with Giemsa staining and immunohistochemical staining and the degree of gastritis was evaluated. DNA was extracted from paraffin-embedded sections of 20 patients both from the gastric corpus and the antrum. A portion of cagA gene was amplified with polymerase chain reaction, followed by direct sequencing of the fragment. Results: We established a novel method to determine the cagA subtype using paraffin-embedded sections. We found that all the samples possessed East-Asian type cagA both in the corpus and the antrum, not only in patients with gastric cancer but also with duodenal ulcer. Although the cagA gene sequence was completely identical between the gastric corpus and the antrum in all patients, the corpus gastritis was more prominent in patients with gastric cancer than those with duodenal ulcer. Conclusions:cagA polymorphism can be evaluated with the use of paraffin-embedded sections. The degree of corpus gastritis may not be regulated by cagA diversity only.
Angiotensin II Potentiates the Slow Component of Delayed Rectifier K+Current via the AT1Receptor in Guinea Pig Atrial Myocytes
Authors: Zankov Dimitar P. , Omatsu-Kanbe Mariko , Isono Takahiro , Toyoda Futoshi , Ding Wei-Guang , Matsuura Hiroshi , Horie Minoru
Published: March 2006

Background—Angiotensin II (Ang II) has diverse actions on cardiac electrical activity. Little information is available, however, regarding immediate electrophysiological effects of Ang II on cardiac repolarization.

Methods and Results—The present study investigated the immediate effects of Ang II on the slow component of delayed rectifier K+current (IKs) and action potentials in guinea pig atrial myocytes using the whole-cell patch-clamp technique. Bath application of Ang II increased the amplitude ofIKs(EC50, 6.16 nmol/L) concentration dependently. The stable analogue Sar1–Ang II was also effective at increasingIKs. The voltage dependence ofIKsactivation and the kinetics of deactivation were not significantly affected by these agonists. The enhancement ofIKswas blocked by the Ang II type 1 (AT1) receptor antagonist valsartan (1 μmol/L) and was markedly attenuated by inclusion of GDPβS (2 mmol/L) in the pipette, indicating an involvement of G protein–coupled AT1receptor. The stimulatory effect was also significantly reduced by the phospholipase C inhibitor compound 48/80 (100 μmol/L) and the protein kinase C inhibitors bisindolylmaleimide I (200 nmol/L) and H-7 (10 μmol/L), suggesting that AT1receptor acts through phospholipase C–protein kinase C signaling cascade to potentiateIKs. As expected from its stimulatory action onIKs, Sar1–Ang II markedly shortened the action potential duration, which could be reversed by valsartan.

Conclusions—The potentiation ofIKsvia AT1stimulation in atrial myocytes, accompanied by a shortening of the action potential duration, suggests a potential mechanism by which elevated levels of Ang II may promote atrial fibrillation in heart failure and warrants further investigation.