bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence or Low Expression of Fas-Associated Protein with Death Domain in Acute Myeloid Leukemia Cells Predicts Resistance to Chemotherapy and Poor Outcome
Authors: Tourneur Léa , Delluc Stéphanie , Lévy Vincent , Valensi Françoise , Radford-Weiss Isabelle , Legrand Ollivier , Vargaftig Jacques , Boix Charlotte , Macintyre Elizabeth A. , Varet Bruno , Chiocchia Gilles , Buzyn Agnès
Published: September 2005

In acute myeloid leukemia (AML), coexpression of death receptors and ligands of the tumor necrosis factor (TNF) receptor/TNF-α superfamily on leukemic cells after chemotherapy is not always accompanied by apoptosis, suggesting that the apoptotic death receptor signaling pathway is disrupted. Because Fas-associated protein with death domain (FADD) is the main adaptor for transmitting the Fas, TNF-related apoptosis-inducing ligand receptors, and TNF receptor 1 death signal, expression of FADD was analyzed by Western blot and immunocytochemistry in leukemic cells of 70 de novo AML patients treated with the European Organization of Research and Treatment of Cancer AML-10 randomized trial before initiation of induction chemotherapy. Thirty seven percent of patients (17 of 46) with FADD negative/low (FADD−/low) leukemic cells had a primary refractory disease compared with 12% of FADD+ patients (3 of 24; P = 0.05). FADD−/low expression was significantly associated with a worse event-free survival [EFS (P = 0.04)] and overall survival (P = 0.04). In multivariate analysis, FADD−/low protein expression was independently associated with a poor EFS and overall survival (P = 0.002 and P = 0.026, respectively). Importantly, FADD−/low protein expression predicted poor EFS even in patients with standard- or good-risk AML (P = 0.009). Thus, we identified low or absent expression of the FADD protein in leukemic cells at diagnosis as a poor independent prognostic factor that can predict worse clinical outcome even for patients with standard- or good-risk AML.
Wnt3a Growth Factor Induces Androgen Receptor-Mediated Transcription and Enhances Cell Growth in Human Prostate Cancer Cells
Authors: Verras Meletios , Brown Jeffrey , Li Xiaomeng , Nusse Roel , Sun Zijie
Published: September 2005

The Wnt signaling pathway plays a critical role in embryogenesis and tumorigenesis. However, biological roles of Wnt growth factors have not been fully characterized in prostate development and the pathogenesis of prostate cancer. In this study, we used Wnt3a-conditioned medium (Wnt3a-CM) and purified Wnt3a proteins to investigate whether there is a direct effect of Wnt3a on androgen receptor (AR)-mediated transcription and to determine its role in the growth of prostate cancer cells. We demonstrated that Wnt3a-CM either induces AR activity in the absence of androgens or enhances AR activity in the presence of low concentrations of androgens, whereas purified Wnt3a showed a pronounced effect in the presence of low concentrations of ligands. We also showed that Wnt3a-CM and the purified Wnt3a enhance the level of cytosolic and nuclear β-catenin, suggesting an involvement of β-catenin in this regulation. Moreover, treatment of LNCaP cells with Wnt3a-CM and purified Wnt3a significantly enhances cell growth in the absence of androgens. Our findings demonstrate that Wnt3a plays an important role in androgen-mediated transcription and cell growth. These results suggest a novel mechanism for the progression of prostate cancer.
Folate Is Associated with the Natural History of High-Risk Human Papillomaviruses
Authors: Piyathilake Chandrika J. , Henao Olga L. , Macaluso Maurizio , Cornwell Phillip E. , Meleth Sreelatha , Heimburger Douglas C. , Partridge Edward E.
Published: September 2005

Several micronutrients have been implicated in cervical carcinogenesis. However, their mode of action is still a matter of speculation. In particular, it is unclear whether certain nutrients reduce the probability of acquiring high-risk human papillomavirus (HPV) or whether they facilitate the clearance of high-risk HPV. We conducted a 24-month prospective follow-up study to test the hypothesis that systemic concentrations of folate are associated with the occurrence and duration of high-risk HPV infections after controlling for other micronutrients (vitamins B12, A, E, and C, total carotene) and known risk factors for high-risk HPV infections and cervical cancer. Circulating concentrations of these micronutrients and risk factors for cervical cancer were determined in a cohort of 345 women who were at risk of developing cervical intraepithelial neoplasia. Using the hybrid capture 2 (HC-2) assay, high-risk HPV status was evaluated at 6-month intervals up to 24 months. All women had at least three consecutive visit high-risk HPV test results. Higher folate status was inversely associated with becoming HC-2 test-positive [odds ratio (OR): 0.27; 95% confidence interval (CI), 0.08–0.91; P = 0.04]. Women with higher folate status were significantly less likely to be repeatedly HC-2 test-positive (OR: 0.33; 95% CI, 0.13–0.86; P = 0.02) and more likely to become test-negative during the study (OR: 2.50; 95% CI, 1.18–5.30; P = 0.02). To our knowledge, this is the first long-term prospective follow-up study reporting an independent protective role of higher folate status on several aspects of the natural history of high-risk HPV after controlling for known risk factors and other micronutrients. Improving folate status in subjects at risk of getting infected or already infected with high-risk HPV may have a beneficial impact in the prevention of cervical cancer.
Vδ1 T Lymphocytes from B-CLL Patients Recognize ULBP3 Expressed on Leukemic B Cells and Up-Regulated by
Trans
-Retinoic Acid
Authors: Poggi Alessandro , Venturino Claudia , Catellani Silvia , Clavio Marino , Miglino Maurizio , Gobbi Marco , Steinle Alexander , Ghia Paolo , Stella Stefania , Caligaris-Cappio Federico , Zocchi Maria Raffaella
Published: September 2005

We analyzed 38 untreated patients with chronic lymphocytic leukemia of B-cell type (B-CLL): 24 low-, 8 intermediate-, and 6 high-risk stage. In 15 patients (13 low risk and 2 intermediate risk), circulating Vδ1 T lymphocytes were significantly increased (100 to 300 cells/μL) compared with most intermediate, all high-risk stage, and 15 healthy donors (50 to 100 cells/μL). We studied these Vδ1 T lymphocytes and observed that they proliferated in vitro and produced tumor necrosis factor α or IFN-γ in response to autologous leukemic B cells but not to normal lymphocytes. However, they were unable to kill resting autologous B cells, which lack the MHC-related MIC-A antigen and express low levels of the UL16-binding protein (ULBP) 3 and undetectable levels of ULBP1, ULBP2, and ULBP4. All these molecules are reported ligands for the NKG2D receptor, which is expressed by γδ T cells and activates their cytolytic function. The Vδ1 T lymphocytes studied were able to lyse the ULBP3+ C1R B-cell line upon transfection with MIC-A. More importantly, they also lysed autologous B-CLL cells when transcription and expression of MIC-A or up-regulation of ULBP3 were achieved either by activation or by exposure to trans-retinoic acid. The NKG2D receptor expressed on Vδ1 T cells was involved in the recognition of B-CLL. Finally, in six patients with low numbers of circulating Vδ1 T cells and undetectable ULBP3, the disease progressed over 1 year, whereas no progression occurred in patients with high Vδ1 T lymphocytes and detectable/inducible ULBP3. These data suggest that Vδ1 T lymphocytes may play a role in limiting the progression of B-CLL.
Inhibition of Chk1 by CEP-3891 Accelerates Mitotic Nuclear Fragmentation in Response to Ionizing Radiation
Authors: Syljuåsen Randi G. , Sørensen Claus Storgaard , Nylandsted Jesper , Lukas Claudia , Lukas Jiri , Bartek Jiri
Published: September 2005

The human checkpoint kinase Chk1 has been suggested as a target for cancer treatment. Here, we show that a new inhibitor of Chk1 kinase, CEP-3891, efficiently abrogates both the ionizing radiation (IR)-induced S and G2 checkpoints. When the checkpoints were abrogated by CEP-3891, the majority (64%) of cells showed fragmented nuclei at 24 hours after IR (6 Gy). The formation of nuclear fragmentation in IR-treated human cancer cells was directly visualized by time-lapse video microscopy of U2-OS cells expressing a green fluorescent protein-tagged histone H2B protein. Nuclear fragmentation occurred as a result of defective chromosome segregation when irradiated cells entered their first mitosis, either prematurely without S and G2 checkpoint arrest in the presence of CEP-3891 or after a prolonged S and G2 checkpoint arrest in the absence of CEP-3891. The nuclear fragmentation was clearly distinguishable from apoptosis because caspase activity and nuclear condensation were not induced. Finally, CEP-3891 not only accelerated IR-induced nuclear fragmentation, it also increased the overall cell killing after IR as measured in clonogenic survival assays. These results demonstrate that transient Chk1 inhibition by CEP-3891 allows premature mitotic entry of irradiated cells, thereby leading to accelerated onset of mitotic nuclear fragmentation and increased cell death.
Androgen-Independent Prostate Cancer Is a Heterogeneous Group of Diseases
Authors: Shah Rajal B. , Mehra Rohit , Chinnaiyan Arul M. , Shen Ronglai , Ghosh Debashis , Zhou Ming , MacVicar Gary R. , Varambally Soorynarayana , Harwood Jason , Bismar Tarek A. , Kim Robert , Rubin Mark A. , Pienta Kenneth J.
Published: September 2005

Understanding the biology of prostate cancer metastasis has been limited by the lack of tissue for study. We studied the clinical data, distribution of prostate cancer involvement, morphology, immunophenotypes, and gene expression from 30 rapid autopsies of men who died of hormone-refractory prostate cancer. A tissue microarray was constructed and quantitatively evaluated for expression of prostate-specific antigen, androgen receptor, chromogranin, synaptophysin, MIB-1, and α-methylacylCoA-racemase markers. Hierarchical clustering of 16 rapid autopsy tumor samples was performed to evaluate the cDNA expression pattern associated with the morphology. Comparisons were made between patients as well as within the same patient. Metastatic hormone-refractory prostate cancer has a heterogeneous morphology, immunophenotype, and genotype, demonstrating that “metastatic disease” is a group of diseases even within the same patient. An appreciation of this heterogeneity is critical to evaluating diagnostic and prognostic biomarkers as well as to designing therapeutic targets for advanced disease.
Pten Loss Causes Hypertrophy and Increased Proliferation of Astrocytes
In vivo
Authors: Fraser Melissa M. , Zhu Xiaoyan , Kwon Chang-Hyuk , Uhlmann Erik J. , Gutmann David H. , Baker Suzanne J.
Published: September 2005

Somatic mutations of PTEN are found in many types of cancers including glioblastoma, the most malignant astrocytic tumor. PTEN mutation occurs in 25 to 40% of glioblastomas but is rarely observed in low-grade glial neoplasms. To determine the role of Pten in astrocytes and glial tumor formation, we inactivated Pten by a Cre-loxP approach with a GFAP-cre transgenic mouse that induced Cre-mediated recombination in astrocytes. Pten conditional knockout mice showed a striking progressive enlargement of the entire brain. Increased nuclear and soma size was observed in both astrocytes and neurons, which contributed in part to the increase in brain size. Pten-deficient astrocytes showed accelerated proliferation in vitro and aberrant ongoing proliferation in adult brains in vivo. In contrast, neurons lacking Pten did not show alterations in proliferation. This study shows cell-type dependent effects of Pten loss in the adult brain, including increased astrocyte proliferation that may render astroglial cells susceptible to neoplastic transformation or malignant progression.
Loss of Neural Cell Adhesion Molecule Induces Tumor Metastasis by Up-regulating Lymphangiogenesis
Authors: Crnic Ivana , Strittmatter Karin , Cavallaro Ugo , Kopfstein Lucie , Jussila Lotta , Alitalo Kari , Christofori Gerhard
Published: September 2005

Reduced expression of neural cell adhesion molecule (NCAM) has been implicated in the progression to tumor malignancy in cancer patients. Previously, we have shown that the loss of NCAM function causes the formation of lymph node metastasis in a transgenic mouse model of pancreatic β cell carcinogenesis (Rip1Tag2). Here we show that tumors of NCAM-deficient Rip1Tag2 transgenic mice exhibit up-regulated expression of the lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and -D (17% in wild-type versus 60% in NCAM-deficient Rip1Tag2 mice) and, with it, increased lymphangiogenesis (0% in wild-type versus 19% in NCAM-deficient Rip1Tag2 mice). Repression of VEGF-C and -D function by adenoviral expression of a soluble form of their cognate receptor, VEGF receptor-3, results in reduced tumor lymphangiogenesis (56% versus 28% in control versus treated mice) and lymph node metastasis (36% versus 8% in control versus treated mice). The results indicate that the loss of NCAM function causes lymph node metastasis via VEGF-C- and VEGF-D-mediated lymphangiogenesis. These results also establish Rip1Tag2;NCAM-deficient mice as a unique model for stochastic, endogenous tumor lymphangiogenesis and lymph node metastasis in immunocompetent mice.
Cul4A Physically Associates with MDM2 and Participates in the Proteolysis of p53
Authors: Nag Alo , Bagchi Srilata , Raychaudhuri Pradip
Published: September 2005

The cullin 4A (Cul4A) gene is amplified and overexpressed in breast and hepatocellular carcinomas. Cul4A functions as an E3 ligase and participates in the proteolysis of several regulatory proteins through the ubiquitin-proteasome pathway. Here, we show that Cul4A associates with MDM2 and p53. Depletion of Cul4A leads to an accumulation of p53. Moreover, expression of Cul4A increases the decay-rate of p53 and delays the accumulation of p53 in response to DNA damage. Cul4A fails to increase the decay of p53 in mouse embryonic fibroblasts lacking MDM2. In addition, the Cul4A-mediated rapid decay of p53 is blocked by p19ARF. The results provide evidence for a role of Cul4A in the MDM2-mediated proteolysis of p53.
Androgenic Suppression of ATP-binding Cassette Transporter A1 Expression in LNCaP Human Prostate Cancer Cells
Authors: Fukuchi Junichi , Hiipakka Richard A. , Kokontis John M. , Hsu Stephen , Ko Andrew L. , Fitzgerald Michael L. , Liao Shutsung
Published: September 2005

Alteration of lipid metabolism is commonly observed in sex hormone-dependent cancer cells, yet its mechanistic involvement in cancer cell proliferation and progression is unclear. We have found that the expression of the cholesterol transporter, ATP-binding cassette transporter A1 (ABCA1), was 15- to 20-fold higher in androgen-dependent than in androgen-independent LNCaP human prostate cancer cells, indicating a possible relationship between the expression levels of ABCA1 and prostate cancer progression. On the basis of real-time quantitative PCR and Western blot analysis, expression of ABCA1 in androgen-dependent cells was inhibited by androgen. The antiandrogen Casodex blocked the effect of androgen, implicating the androgen receptor in regulation of ABCA1 expression by androgens. Using an ABCA1 promoter-reporter gene assay, androgenic suppression was observed at the transcriptional level in androgen-dependent but not in androgen-independent prostate cancer cells. ABCA1 appears to have a role in modulating cell proliferation because knockdown of ABCA1 expression by RNA interference in androgen-dependent cells increased their rate of proliferation. Therefore, a suppressive effect of androgen on ABCA1 expression may be one of the mechanisms by which androgens regulate proliferation in prostate cancer cells. Attenuated ABCA1 expression in androgen-independent cells thus may contribute, in part, to prostate cancer progression.
Natural Killer Cell-Mediated Killing of Freshly Isolated Neuroblastoma Cells
Authors: Castriconi Roberta , Dondero Alessandra , Corrias Maria Valeria , Lanino Edoardo , Pende Daniela , Moretta Lorenzo , Bottino Cristina , Moretta Alessandro
Published: September 2005

In the present study, we assessed the susceptibility of freshly isolated neuroblastoma cells to killing mediated by normal human natural killer (NK) cells and analyzed the receptor–ligand interactions that regulate this event. We show that killing of freshly isolated neuroblasts, similar to neuroblastoma cell lines, involves NKp46 and NKp30 (natural cytotoxicity receptors). However, freshly isolated neuroblasts were generally more resistant to NK-mediated lysis than conventional neuroblastoma cell lines. Moreover, a significant heterogeneity in susceptibility to lysis existed among neuroblastomas derived from different patients. Remarkably, susceptibility to lysis directly correlated with the surface expression, on neuroblasts, of poliovirus receptor [PVR (CD155)], a ligand for the DNAX accessory molecule-1 [DNAM-1 (CD226)] triggering receptor expressed by NK cells. Indeed, PVR-expressing neuroblastomas were efficiently killed by NK cells. Moreover, monoclonal antibody-mediated masking of either DNAM-1 (on NK cells) or PVR (on neuroblasts) resulted in strong inhibition of tumor cell lysis. Thus, assessment of the PVR surface levels may represent a novel useful criterion to predict the susceptibility/resistance of neuroblastomas to NK-mediated killing.
Elevated Flk1 (Vascular Endothelial Growth Factor Receptor 2) Signaling Mediates Enhanced Angiogenesis in β3-Integrin–Deficient Mice
Authors: Reynolds Andrew R. , Reynolds Louise E. , Nagel Tobi E. , Lively Julie C. , Robinson Stephen D. , Hicklin Daniel J. , Bodary Sarah C. , Hodivala-Dilke Kairbaan M.
Published: September 2005

Tumor growth, tumor angiogenesis, and vascular endothelial growth factor (VEGF)–specific angiogenesis are all enhanced in β3-integrin–null mice. Furthermore, endothelial cells isolated from β3-null mice show elevated levels of Flk1 (VEGF receptor 2) expression, suggesting that β3-integrin can control the amplitude of VEGF responses by controlling Flk1 levels or activity. We now show that Flk1 signaling is required for the enhanced tumor growth and angiogenesis seen in β3-null mice. Moreover, β3-null endothelial cells exhibit enhanced migration and proliferation in response to VEGF in vitro, and this phenotype requires Flk1 signaling. Upon VEGF stimulation, β3-null endothelial cells exhibit higher levels of phosphorylated Flk1 and extracellular-related kinases 1 and 2 than wild-type endothelial cells. Furthermore, signaling via ERK1/2 is required to mediate the elevated responses to VEGF observed in β3-null endothelial cells and aortic rings in vitro. These data confirm that VEGF signaling via Flk1 is enhanced in β3-integrin–deficient mice and suggests that this increase may mediate the enhanced angiogenesis and tumor growth observed in these mice in vivo.
A Novel Nuclear Protein, MGC5306 Interacts with DNA Polymerase β and Has a Potential Role in Cellular Phenotype
Authors: Wang Liming , Bhattacharyya Nandan , Chelsea Diane M. , Escobar Pedro F. , Banerjee Sipra
Published: September 2005

A novel protein MGC5306 has been identified in yeast–two-hybrid analysis by screening a HeLa cDNA library with a truncated DNA polymeraseβ (polβΔ) as bait. The polβΔ is expressed in various types of cancers. Co-immunoprecipitation–Western blot analysis confirms not only its interaction with polβΔ but also with wild-type polβ. Binding to polβ indicates potential function of MGC5306 in repair pathway. Transfection of cells with MGC5306-GFP and Western blot analysis with anti-MGC5306 antibody reveal its nuclear localization. MGC5306 is expressed in human carcinomas and tumor cell lines but not in normal tissues, suggesting MGC5306 is most likely involved in carcinogenesis. An antigrowth activity and modulations of cell cycle events are identified in cells expressing siRNAMGC5306.
Oral Consumption of Green Tea Polyphenols Inhibits Insulin-Like Growth Factor-I–Induced Signaling in an Autochthonous Mouse Model of Prostate Cancer
Authors: Adhami Vaqar Mustafa , Siddiqui Imtiaz Ahmad , Ahmad Nihal , Gupta Sanjay , Mukhtar Hasan
Published: September 2005

We earlier demonstrated that oral infusion of green tea polyphenols inhibits development and progression of prostate cancer in transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Evidence indicates that elevated levels of IGF-I with concomitant lowering of IGF binding protein (IGFBP)-3 are associated with increased risk for prostate cancer development and progression. In this study, we examined the role of IGF/IGFBP-3 signaling and its downstream and other associated events during chemoprevention of prostate cancer by green tea polyphenols in TRAMP mice. Our data demonstrated an increase in the levels of IGF-I, phosphatidylinositol 3′-kinase, phosphorylated Akt (Thr-308), and extracellular signal-regulated kinase 1/2 with concomitant decrease in IGFBP-3 in dorso-lateral prostate of TRAMP mice during the course of cancer progression, i.e., as a function of age. Continuous green tea polyphenol infusion for 24 weeks to these mice resulted in substantial reduction in the levels of IGF-I and significant increase in the levels of IGFBP-3 in the dorso-lateral prostate. This modulation of IGF/IGFBP-3 was found to be associated with an inhibition of protein expression of phosphatidylinositol 3′-kinase, phosphorylated forms of Akt (Thr-308) and extracellular signal-regulated kinase 1/2. Furthermore, green tea polyphenol infusion resulted in marked inhibition of markers of angiogenesis and metastasis most notably vascular endothelial growth factor, urokinase plasminogen activator, and matrix metalloproteinases 2 and 9. Based on our data, we suggest that IGF-I/IGFBP-3 signaling pathway is a prime pathway for green tea polyphenol-mediated inhibition of prostate cancer that limits the progression of cancer through inhibition of angiogenesis and metastasis.
Combined Total Genome Loss of Heterozygosity Scan of Breast Cancer Stroma and Epithelium Reveals Multiplicity of Stromal Targets
Authors: Fukino Koichi , Shen Lei , Matsumoto Satoshi , Morrison Carl D. , Mutter George L. , Eng Charis
Published: September 2005

Recent breast cancer studies have highlighted the importance of interactions between cancer epithelium and tumor stroma. Recently, the focus of solid tumor investigations has shifted from mutations in carcinomatous epithelium to disturbances of tissue organization in cancer. The genetic basis of this microenvironment, however, remains to be clarified. To begin to resolve this problem, a total genome loss of heterozygosity (LOH) scan was done on epithelial and stromal DNA from 134 sporadic invasive breast carcinomas. In addition to detecting more frequent LOH at three loci in stroma than in epithelium, we found strong evidence that LOH frequencies were significantly elevated in specific regions of each chromosome. We detected 57 markers, which were preferentially lost either in stroma (n = 38) or epithelium (n = 19), relative to the background LOH frequencies on their respective chromosomes. This multiplicity of stromal cell LOH, and hence loss of genetic material, provides a possible mechanism for interpatient variation in host-stromal response to invading adenocarcinoma cells. This is consistent with a model in which initial, random LOH occurs equally among epithelium and stroma, but subsequent clonal selection is driven by factors, which appear to be distinctly different between malignant epithelial and surrounding stromal cells. Genetic alterations in stroma did not mimic those in epithelium, but they could play a different and parallel role in carcinogenesis and tumor progression, probably by modifying some features specific to breast cancer.
Mutation of the
PIK3CA
Gene in Ovarian and Breast Cancer
Authors: Campbell Ian G. , Russell Sarah E. , Choong David Y. H. , Montgomery Karen G. , Ciavarella Marianne L. , Hooi Christine S. F. , Cristiano Briony E. , Pearson Richard B. , Phillips Wayne A.
Published: September 2005

Phosphatidylinositol 3′-kinases are lipid kinases with important roles in neoplasia. Recently, a very high frequency of somatic mutations in PIK3CA has been reported among a large series of colorectal cancers. However, the relevance of PIK3CA mutation in other cancer types remains unclear because of the limited number of tumors investigated. We have screened a total of 284 primary human tumors for mutations in all coding exons of PIK3CA using a combination of single stranded conformational polymorphism and denaturing high-performance liquid chromatography analysis. Among 70 primary breast cancers, 40% (28 of 70) harbored mutations in PIK3CA, making it the most common mutation described to date in this cancer type. Mutations were not associated with histologic subtype, estrogen receptor status, grade or presence of tumor in lymph nodes. Among the primary epithelial ovarian cancers only 11 of 167 (6.6%) contain somatic mutations, but there was a clear histologic subtype bias in their distribution. Only 2 of 88 (2.3%) of serous carcinomas had PIK3CA mutations compared with 8 of 40 (20.0%) endometrioid and clear cell cancers, which was highly significant (P = 0.001). In contrast, PIK3CA gene amplification (>7-fold) was common among all histologic subtypes (24.5%) and was inversely associated with the presence of mutations. Overall, PIK3CA mutation or gene amplification was detected in 30.5% of all ovarian cancers and 45% of the endometrioid and clear cell subtypes. Our study is the first direct evidence that PIK3CA is an oncogene in ovarian cancer and greatly extends recent findings in breast cancer.
Androgen-Induced Differentiation and Tumorigenicity of Human Prostate Epithelial Cells
Authors: Berger Raanan , Febbo Phillip G. , Majumder Pradip K. , Zhao Jean J. , Mukherjee Shayan , Signoretti Sabina , Campbell K. Thirza , Sellers William R. , Roberts Thomas M. , Loda Massimo , Golub Todd R. , Hahn William C.
Published: September 2005

Androgen ablation is the primary treatment modality for patients with metastatic prostate cancer; however, the role of androgen receptor signaling in prostate cancer development remains enigmatic. Using a series of genetically defined immortalized and tumorigenic human prostate epithelial cells, we found that introduction of the androgen receptor induced differentiation of transformed prostate epithelial cells to a luminal phenotype reminiscent of organ-confined prostate cancer when placed in the prostate microenvironment. Moreover, androgen receptor expression converted previously androgen-independent, tumorigenic prostate epithelial cells into cells dependent on testosterone for tumor formation. These observations indicate that androgen receptor expression is oncogenic and addictive for the human prostate epithelium.
Crucial Role of Phospholipase Cε in Chemical Carcinogen-Induced Skin Tumor Development
Authors: Bai Yunfeng , Edamatsu Hironori , Maeda Sakan , Saito Hiromitsu , Suzuki Noboru , Satoh Takaya , Kataoka Tohru
Published: September 2005

Mutational activation of the ras proto-oncogenes is frequently found in skin cancers. However, the nature of downstream signaling pathways from Ras involved in skin carcinogenesis remains poorly understood. Recently, we and others identified phospholipase C (PLC) ε as an effector of Ras. Here we have examined the role of PLCε in de novo skin chemical carcinogenesis by using mice whose PLCε is genetically inactivated. PLCε−/− mice exhibit delayed onset and markedly reduced incidence of skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the papillomas formed in PLCε−/− mice fail to undergo malignant progression into carcinomas, in contrast to a malignant conversion rate of approximately 20% observed with papillomas in PLCε+/+ mice. In all of the tumors analyzed, the Ha-ras gene is mutationally activated irrespective of the PLCε background. The skin of PLCε−/− mice fails to exhibit basal layer cell proliferation and epidermal hyperplasia in response to TPA treatment. These results indicate a crucial role of PLCε in ras oncogene-induced de novo carcinogenesis and downstream signaling from TPA, introducing PLCε as a candidate molecular target for the development of anticancer drugs.
Analysis of Diepoxide-Specific Cyclic
N
-Terminal Globin Adducts in Mice and Rats after Inhalation Exposure to 1,3-Butadiene
Authors: Boysen Gunnar , Georgieva Nadia I. , Upton Patricia B. , Jayaraj Karupiah , Li Yutai , Walker Vernon E. , Swenberg James A.
Published: September 2005

1,3-Butadiene is an important industrial chemical used in the production of synthetic rubber and is also found in gasoline and combustion products. It is a multispecies, multisite carcinogen in rodents, with mice being the most sensitive species. 1,3-Butadiene is metabolized to several epoxides that form DNA and protein adducts. Previous analysis of 1,2,3-trihydroxybutyl-valine globin adducts suggested that most adducts resulted from 3-butene-1,2-diol metabolism to 3,4-epoxy-1,2-butanediol, rather than from 1,2;3,4-diepoxybutane. To specifically examine metabolism of 1,3-butadiene to 1,2;3,4-diepoxybutane, the formation of the 1,2;3,4-diepoxybutane–specific adduct N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was evaluated in mice treated with 3, 62.5, or 1250 ppm 1,3-butadiene for 10 days and rats exposed to 3 or 62.5 ppm 1,3-butadiene for 10 days, or to 1000 ppm 1,3-butadiene for 90 days, using a newly developed immunoaffinity liquid chromatography tandem mass spectrometry assay. In addition, 2-hydroxy-3-butenyl-valine and 1,2,3-trihydroxybutyl-valine adducts were determined. The analyses of several adducts derived from 1,3-butadiene metabolites provided new insight into species and exposure differences in 1,3-butadiene metabolism. Mice formed much higher amounts of N,N-(2,3-dihydroxy-1,4-butadiyl)–valine than rats. The formation of 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)–valine was similar in mice exposed to 3 or 62.5 ppm 1,3-butadiene, whereas 2-hydroxy-3-butenyl-valine was 3-fold higher at 1250 ppm. In both species, 1,2,3-trihydroxybutyl-valine adducts were much higher than 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)–valine. Together, these data show that 1,3-butadiene is primarily metabolized via the 3-butene-1,2-diol pathway, but that mice are much more efficient at forming 1,2;3,4-diepoxybutane than rats, particularly at low exposures. This assay should also be readily adaptable to molecular epidemiology studies on 1,3-butadiene-exposed workers
Protein Expression Profiles in Pancreatic Adenocarcinoma Compared with Normal Pancreatic Tissue and Tissue Affected by Pancreatitis as Detected by Two-Dimensional Gel Electrophoresis and Mass Spectrometry
Authors: Shen Jianjun , Person Maria D. , Zhu Jijiang , Abbruzzese James L. , Li Donghui
Published: September 2005

Pancreatic cancer is a rapidly fatal disease, and there is an urgent need for early detection markers and novel therapeutic targets. The current study has used a proteomic approach of two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) to identify differentially expressed proteins in six cases of pancreatic adenocarcinoma, two normal adjacent tissues, seven cases of pancreatitis, and six normal pancreatic tissues. Protein extracts of individual sample and pooled samples of each type of tissues were separated on 2D gels using two different pH ranges. Differentially expressed protein spots were in-gel digested and identified by MS. Forty proteins were identified, of which five [i.e., α-amylase; copper zinc superoxide dismutase; protein disulfide isomerase, pancreatic; tropomyosin 2 (TM2); and galectin-1] had been associated previously with pancreatic disease in gene expression studies. The identified proteins include antioxidant enzymes, chaperones and/or chaperone-like proteins, calcium-binding proteins, proteases, signal transduction proteins, and extracellular matrix proteins. Among these proteins, annexin A4, cyclophilin A, cathepsin D, galectin-1, 14–3-3ζ, α-enolase, peroxiredoxin I, TM2, and S100A8 were specifically overexpressed in tumors compared with normal and pancreatitis tissues. Differential expression of some of the identified proteins was further confirmed by Western blot analyses and/or immunohistochemical analysis. These results show the value of a proteomic approach in identifying potential markers for early diagnosis and therapeutic manipulation. The newly identified proteins in pancreatic tumors may eventually serve as diagnostic markers or therapeutic targets.
Depletion of Mutant p53 and Cytotoxicity of Histone Deacetylase Inhibitors
Authors: Blagosklonny Mikhail V. , Trostel Shana , Kayastha Ganesh , Demidenko Zoya N. , Vassilev Lyubomir T. , Romanova Larisa Y. , Bates Susan , Fojo Tito
Published: September 2005

Mutant p53 is a cancer-specific target for pharmacologic intervention. We show that histone deacetylase inhibitors such as FR901228 and trichostatin A completely depleted mutant p53 in cancer cell lines. This depletion was preceded by induction of p53-regulated transcription. In cells with mutant p53 pretreated with histone deacetylase inhibitors, DNA damage further enhanced the p53 trans-function. Furthermore, histone deacetylase inhibitors were preferentially cytotoxic to cells with mutant p53 rather than to cells lacking wild-type p53. We suggest that, by either restoring or mimicking p53 trans-functions, histone deacetylase inhibitors initiate degradation of mutant p53. Because mutant p53 is highly expressed, a sudden restoration of p53-like functions is highly cytotoxic to cells with mutant p53. In a broader perspective, this shows how selectivity may be achieved by targeting a non-cancer-specific target, such as histone deacetylases, in the presence of a cancer-specific alteration, such as mutant p53.
Spermine Oxidation Induced by
Helicobacter pylori
Results in Apoptosis and DNA Damage
Authors: Xu Hangxiu , Chaturvedi Rupesh , Cheng Yulan , Bussiere Francoise I. , Asim Mohammad , Yao Micheal D. , Potosky Darryn , Meltzer Stephen J. , Rhee Juong G. , Kim Sung S. , Moss Steven F. , Hacker Amy , Wang Yanlin , Casero Robert A. , Wilson Keith T.
Published: September 2005

Oxidative stress is linked to carcinogenesis due to its ability to damage DNA. The human gastric pathogen Helicobacter pylori exerts much of its pathogenicity by inducing apoptosis and DNA damage in host gastric epithelial cells. Polyamines are abundant in epithelial cells, and when oxidized by the inducible spermine oxidase SMO(PAOh1) H2O2 is generated. Here, we report that H. pylori up-regulates mRNA expression, promoter activity, and enzyme activity of SMO(PAOh1) in human gastric epithelial cells, resulting in DNA damage and apoptosis. H. pylori-induced H2O2 generation and apoptosis in these cells was equally attenuated by an inhibitor of SMO(PAOh1), by catalase, and by transient transfection with small interfering RNA targeting SMO(PAOh1). Conversely, SMO(PAOh1) overexpression induced apoptosis to the same levels as caused by H. pylori. Importantly, in H. pylori-infected tissues, there was increased expression of SMO(PAOh1) in both human and mouse gastritis. Laser capture microdissection of human gastric epithelial cells demonstrated expression of SMO(PAOh1) that was significantly attenuated by H. pylori eradication. These results identify a pathway for oxidative stress-induced epithelial cell apoptosis and DNA damage due to SMO(PAOh1) activation by H. pylori that may contribute to the pathogenesis of the infection and development of gastric cancer.
Caspase-1 Is a Direct Target Gene of ETS1 and Plays a Role in ETS1-Induced Apoptosis
Authors: Pei Huiping , Li Chunyang , Adereth Yair , Hsu Tien , Watson Dennis K. , Li Runzhao
Published: September 2005

ETS1, the founding member of Ets transcriptional factor family, plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. Previous work has shown that ETS1 represses tumorigenicity of colon carcinoma cells in vivo, and that the p42-ETS1 protein bypasses a defect in apoptosis in colon carcinoma cells through the up-regulation of caspase-1 expression. In this report, we show that expression of p42-ETS1 inhibits tumorigenicity of colon cancer DLD-1 cells through induction of apoptosis in vivo. In support of the hypothesis that caspase-1 might be a target involved in the sensitization of DLD-1 cells to Fas-induced apoptosis by ETS1, overexpression of caspase-1 bypasses Fas-induced apoptosis in these cells as well. Furthermore, ETS1-mediated apoptosis was observed in MOP8 cells, a transformed mouse NIH3T3 cell line. To determine whether ETS1 activates the transcription of caspase-1, luciferase reporters driven by the wild-type and mutant caspase-1 promoters were generated. Both p51-ETS1 and p42-ETS1 transactivated the caspase-1 transcription and a functional Ets binding site is identified in the caspase-1 promoter. Wild-type caspase-1 promoter (pGL3-ICE) was strongly transactivated by ETS1 and this transactivation was dramatically diminished by the mutation of the potential Ets binding site (−525 bp). In addition, electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed complex formation between this binding site and ETS1 proteins. Taken together, ETS1 transcriptionally induces the expression of caspase-1; as such, the regulatory control of caspase-1 expression by ETS1 may underlie the apoptotic susceptibility modulated by ETS1 in specific tumor cells.
Expression and Activity of Src Regulate Interleukin-8 Expression in Pancreatic Adenocarcinoma Cells: Implications for Angiogenesis
Authors: Trevino Jose G. , Summy Justin M. , Gray Michael J. , Nilsson Monique B. , Lesslie Donald P. , Baker Cheryl H. , Gallick Gary E.
Published: September 2005

Interleukin-8 (IL-8) is an angiogenic factor that promotes growth of pancreatic tumors. The purpose of this study was to determine if c-Src, a protein tyrosine kinase frequently overexpressed in pancreatic cancer, regulated IL-8 expression and to elucidate the Src-mediated signaling pathways that contribute to angiogenesis in pancreatic adenocarcinoma cells. In a panel of pancreatic cancer cell lines, expression of total and activated Src correlated with IL-8 production. Furthermore, ectopic expression of activated Src in PANC-1 cells with low endogenous Src activity significantly increased IL-8 production (P < 0.005). In contrast, pharmacologic inhibition of endogenous c-Src kinase activity or small interfering RNA–mediated “knockdown” of c-Src expression in L3.6pl cells with high Src expression and activity caused significant decreases in IL-8 production (P < 0.005). Inhibition of c-Src activity resulted in decreased phosphorylation of Akt, p38, and extracellular signal-regulated kinase (Erk)-1/2. Significant (P < 0.005) dose-dependent decreases were observed in IL-8 expression by inhibiting Src-dependent signaling molecules Erk-1/2 and p38 but not phosphatidylinositol 3-kinase. To assess the relevance of Src inhibition to angiogenesis, in vivo gelfoam assays were done. Robust infiltration of vessels was observed in gelfoam saturated with conditioned medium from pancreatic carcinoma cells. This angiogenesis was nearly abrogated in gelfoams saturated with conditioned medium from cells treated with the Src family kinase inhibitor, PP2 (P < 0.001). Thus, c-Src regulates critical “downstream” signaling pathways that contribute to expression of IL-8 in human pancreatic tumor cells, suggesting c-Src may be a target for therapeutic intervention in pancreatic adenocarcinoma.
Evaluating Hypoxia-Inducible Factor-1α as a Cancer Therapeutic Target via Inducible RNA Interference In vivo
Authors: Li Leiming , Lin Xiaoyu , Staver Michael , Shoemaker Alexander , Semizarov Dimitri , Fesik Stephen W. , Shen Yu
Published: September 2005

Validating potential targets is an important step in the drug discovery process. In this study, we tested the feasibility of using inducible RNA interference (RNAi) in vivo to obtain an unbiased evaluation on the efficacy of inhibiting hypoxia-inducible factor-1α (HIF-1α) in established tumors. We showed that HIF-1α inhibition resulted in transient tumor stasis or tumor regression, and inhibiting HIF-1α in early-stage tumors was found to be more efficacious than inhibiting HIF-1α in more established tumors. A differential requirement of HIF-1α for tumor growth was also observed among different tumor types. Examination of tumors resistant to HIF-1α inhibition suggested that the resistance might result from a less hypoxic tumor environment and the level of HIF-1α expression in tumors may be a useful marker for predicting tumor response to HIF-1 inhibition. This study shows that inducible RNAi is a versatile tool for evaluating cancer targets in vivo. In addition to broad implications on in vivo validation of cancer targets, results from this study will also be instructive for practical applications of HIF-1–based cancer therapeutics.
Integrative Genomic Analysis Identifies NDRG2 as a Candidate Tumor Suppressor Gene Frequently Inactivated in Clinically Aggressive Meningioma
Authors: Lusis Eriks A. , Watson Mark A. , Chicoine Michael R. , Lyman Meghan , Roerig Peter , Reifenberger Guido , Gutmann David H. , Perry Arie
Published: September 2005

Although meningiomas are common central nervous system tumors, little is known about the genetic events responsible for malignant progression. In this study, we employed gene expression profiling to identify transcripts whose expression was lost in anaplastic (WHO grade III) versus benign (WHO grade I) meningioma. Approximately 40% of genes down-regulated in anaplastic meningioma were localized to chromosomes 1p and 14q. One specific gene located at 14q11.2, NDRG2, was consistently down-regulated in grade III meningioma, a finding which we validated at both the transcript and protein levels in independent sets of clinically and pathologically diverse meningiomas. Loss of NDRG2 expression was also seen in a subset of lower-grade meningiomas, including atypical meningiomas (WHO grade II) with clinically aggressive behavior. Furthermore, we found that the loss of NDRG2 expression was significantly associated with hypermethylation of the NDRG2 promoter. Collectively, these data identify NDRG2 as the first specific candidate tumor suppressor gene on chromosome 14q that is inactivated during meningioma progression. In addition, these findings highlight the utility of combining genomic, epigenetic, and expression data to identify clinically significant tumor biomarkers, and suggest that NDRG2 expression will be a useful and functionally relevant biomarker to predict aggressive behavior in patients with meningioma.
Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals
Authors: Paulmurugan Ramasamy , Gambhir Sanjiv S.
Published: September 2005

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule–mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction–mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 ± 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.
15(S)-Hydroxyeicosatetraenoic Acid Induces Angiogenesis via Activation of PI3K-Akt-mTOR-S6K1 Signaling
Authors: Zhang Baolin , Cao Huiqing , Rao Gadiparthi N.
Published: September 2005

To determine whether the lipoxygenase metabolites of arachidonic acid, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids [5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, respectively] are angiogenic, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All three HETEs stimulated HDMVEC tube formation and migration. Because 15(S)-HETE was found to be more potent than 5(S)-HETE and 12(S)-HETE in HDMVEC tube formation, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 15(S)-HETE stimulated Akt and S6K1 phosphorylation in HDMVEC in a time-dependent manner. Wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3K), blocked both Akt and S6K1 phosphorylation, whereas rapamycin, a specific inhibitor of Akt downstream effector, mammalian target of rapamycin (mTOR), suppressed only S6K1 phosphorylation induced by 15(S)-HETE suggesting that this eicosanoid activates the PI3K-Akt-mTOR-S6K1 signaling in HDMVEC. Wortmannin, LY294002, and rapamycin also inhibited 15(S)-HETE-induced HDMVEC tube formation and migration. In addition, all three HETEs stimulated angiogenesis as measured by in vivo Matrigel plug assay with 15(S)-HETE being more potent. Pharmacologic inhibition of PI3K-Akt-mTOR-S6K1 signaling completely suppressed 15(S)-HETE-induced in vivo angiogenesis. Consistent with these observations, adenoviral-mediated expression of dominant-negative Akt also blocked 15(S)-HETE–induced HDMVEC tube formation and migration and in vivo angiogenesis. Together, these results show for the first time that 15(S)-HETE stimulates angiogenesis via activation of PI3K-Akt-mTOR-S6K1 signaling.