bluuebunny/crossref_metadata_2025 huggingface dataset:
A Polymorphism in the
CDKN1B
Gene Is Associated with Increased Risk of Hereditary Prostate Cancer
Authors: Chang Bao-li , Zheng Siqun L , Isaacs Sarah D. , Wiley Kathy E. , Turner Aubrey , Li Ge , Walsh Patrick C. , Meyers Deborah A. , Isaacs William B. , Xu Jianfeng
Published: September 2005

The loss of cell cycle control is believed to be an important mechanism in the promotion of carcinogenesis. CDKN1B (p27) belongs to the Cip/Kip family and functions as an important cell cycle gatekeeper. Several lines of evidence from clinical studies and laboratory experiments demonstrate that CDKN1B is an important tumor suppressor gene in prostate cancer etiology. In addition, a case-control study has shown that the 326T/G (V109G) polymorphism in CDKN1B is associated with advanced prostate cancer. In light of the evidence for linkage between the chromosomal location of the CDKN1B gene (12p13) and prostate cancer susceptibility in several hereditary prostate cancer (HPC) populations, we hypothesized that sequence variants of CDKN1B play a role in HPC. To test this hypothesis, we first resequenced this gene in 96 HPC probands to identify germ-line mutations and sequence variants. We then genotyped the identified sequence variants among all family members of 188 HPC families and tested for their cosegregation with prostate cancer. In total, 10 sequence variants were identified, including three nonsynonymous changes. A family-based test, which is free from the effects of population stratification, revealed a significant association between single nucleotide polymorphism (SNP) -79C/T and prostate cancer (with a nominal P of 0.0005). The C allele of -79C/T was overtransmitted from parents to their affected offspring. Evidence for this association was primarily contributed by affected offspring whose age at diagnosis was <65 years. Together with the previous association study in a sporadic prostate cancer population, our new findings additionally suggest that germ-line variants of this gene play a role in prostate cancer susceptibility.
herg1
Gene and HERG1 Protein Are Overexpressed in Colorectal Cancers and Regulate Cell Invasion of Tumor Cells
Authors: Lastraioli Elena , Guasti Leonardo , Crociani Olivia , Polvani Simone , Hofmann Giovanna , Witchel Harry , Bencini Lapo , Calistri Massimo , Messerini Luca , Scatizzi Marco , Moretti Renato , Wanke Enzo , Olivotto Massimo , Mugnai Gabriele , Arcangeli Annarosa
Published: September 2005

The acquisition of the capacity to invade surrounding tissues confers a more malignant phenotype to tumor cells and is necessary for the establishment of metastases. The understanding of the molecular mechanisms underlying cell invasion in human solid tumors such as colorectal cancers could provide not only more sensitive prognostic analyses but also novel molecular targets for cancer therapy.

We report in this article that K+ ion channels belonging to the HERG family are important determinants for the acquisition of an invasive phenotype in colorectal cancers. The herg1 gene and HERG1 protein are expressed in many colon cancer cell lines, and the activity of HERG channels modulates colon cancer cell invasiveness. Moreover, the amount of HERG1 protein expressed on the plasma membrane is directly related to the invasive phenotype of colon cancer cells.

Finally, both the herg1 gene and HERG1 protein were expressed in a high percentage of primary human colorectal cancers, with the highest incidence occurring in metastatic cancers, whereas no expression could be detected either in normal colonic mucosa or in adenomas.
Hepatocyte Growth Factor Sensitizes Human Ovarian Carcinoma Cell Lines to Paclitaxel and Cisplatin
Authors: Rasola Andrea , Anguissola Sergio , Ferrero Norma , Gramaglia Daniela , Maffe Antonella , Maggiora Piera , Comoglio Paolo M. , Di Renzo M. Flavia
Published: September 2005

The hepatocyte growth factor (HGF) receptor, encoded by the MET oncogene, is expressed in ∼70% of human ovarian carcinomas and overexpressed in 30% of cases. Because HGF is known to protect cells from apoptosis, we investigated whether receptor expression modifies ovarian cancer cell response to chemotherapy. The apoptotic effect of the front-line chemotherapeutic drugs paclitaxel and cisplatin on cells treated with HGF was studied. In ovarian cancer cell lines, pretreatment with HGF surprisingly enhances the apoptotic response to low doses of paclitaxel and cisplatin. HGF empowers specifically the intrinsic apoptotic pathway, whereas it protects cells from extrinsic Fas-induced apoptosis. Chemotherapy sensitization is specific for HGF because another growth factor (e.g., epidermal growth factor) increases ovarian cancer cell survival. In nonovarian cancer cell models, as expected, HGF provides protection from drug-induced apoptosis. These data show that HGF sensitizes ovarian carcinoma cells to low-dose chemotherapeutic agents. This suggests that HGF may be used to improve response to chemotherapy in a set of human ovarian carcinomas molecularly classified based on the MET oncogene expression.
Tumor Suppressor Maspin Is Up-Regulated during Keratinocyte Senescence, Exerting a Paracrine Antiangiogenic Activity
Authors: Nickoloff Brian J. , Lingen Mark W. , Chang Bey-Dih , Shen Mei , Swift Mari , Curry Jonathan , Bacon Patricia , Bodner Barbara , Roninson Igor B.
Published: September 2005

Cell senescence is a physiological program of terminal growth arrest, which is believed to play an important role in cancer prevention. Senescent cells secrete multiple growth-regulatory proteins, some of which can affect tumor growth, survival, invasion, or angiogenesis. Changes in expression of different senescence-associated genes were analyzed in cultured human skin keratinocytes (KCs) that underwent replicative senescence or confluence-induced accelerated senescence. Senescent KC cultures showed a strong increase in mRNA and protein expression of maspin, a member of serine protease inhibitor family and an epithelial cell tumor suppressor with anti-invasive and antiangiogenic activities. Immunohistochemical analysis of 14 normal human skin samples (age range from 3 months to 84 years) showed that maspin is expressed by KCs in vivo and that the extent and intensity of maspin expression in the skin is significantly (P = 0.01) correlated with chronological age. Antiangiogenic activity of maspin secreted by senescent KCs was investigated in vitro by testing the effect of conditioned media from different KC cultures on endothelial cell migration in the presence or absence of several angiogenic factors. Media conditioned by senescent cultures (undergoing replicative or accelerated senescence), but not by proliferating KCs, strongly inhibited the stimulation of endothelial cell migration by all of the tested angiogenic factors. Neutralizing antibody against maspin abrogated this effect of conditioned media. These findings indicate that senescent KCs exert a paracrine antiangiogenic activity, and maspin is the principal contributor to this potentially tumor-suppressive effect of cellular senescence.
FGFR3
and
P53
Characterize Alternative Genetic Pathways in the Pathogenesis of Urothelial Cell Carcinoma
Authors: van Rhijn Bas W. G. , van der Kwast Theo H. , Vis André N. , Kirkels Wim J. , Boevé Egbert R. , Jöbsis Adriaan C. , Zwarthoff Ellen C.
Published: September 2005

Fibroblast growth factor receptor 3 (FGFR3) and P53 mutations are frequently observed in bladder cancer. We here describe the distribution of FGFR3 mutations and P53 overexpression in 260 primary urothelial cell carcinomas. FGFR3 mutations were observed in 59% and P53 overexpression in 25%. Interestingly, FGFR3 and P53 alterations were mutually exclusive, because they coincided in only 5.7% of tumors. Consequently, we propose that they characterize two alternative genetic pathways in urothelial cell carcinoma pathogenesis. The genetic alterations were reflected in the pathology and the clinical outcome, i.e., FGFR3 mutations were found in low-stage/-grade tumors and were associated with a favorable disease course, whereas P53 alterations were tied to adverse disease parameters.
Suppression of Prostate Carcinogenesis by Dietary Supplementation of Celecoxib in Transgenic Adenocarcinoma of the Mouse Prostate Model
Authors: Gupta Sanjay , Adhami Vaqar M. , Subbarayan Murugesan , MacLennan Gregory T. , Lewin Jonathan S. , Hafeli Urs O. , Fu Pingfu , Mukhtar Hasan
Published: September 2005

Epidemiological studies and clinical observations suggest that nonsteroidal anti-inflammatory drugs and certain selective cyclooxygenase (COX)-2 inhibitors may reduce the relative risk of clinically evident prostate cancer. This prompted us to investigate the chemopreventive potential of celecoxib, a selective COX-2 inhibitor, against prostate carcinogenesis in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Similar to prostate cancer in humans, prostate malignancies in TRAMP mice progress from precursor intraepithelial lesions, to invasive carcinoma that metastasizes to lymph nodes, liver, lungs, and occasionally to bone. The basal enzyme activity and protein expression of COX-2 is significantly higher (>4-fold) in the dorsolateral prostate of TRAMP mice up to 24 weeks of age compared with their nontransgenic littermates. Eight-week-old TRAMP mice were randomly divided and fed either control diet (AIN 76A) or a custom prepared AIN 76A diet containing 1500-ppm celecoxib ad libitum for 24 weeks, a dosage that would compare with the normal recommended dose for the treatment of human disease. Studies from two independent experiments, each consisting of 10 mice on test, showed that the cumulative incidence of prostate cancer development at 32 weeks of age in animals fed with AIN 76A diet was 100% (20 of 20) as observed by tumor palpation, whereas 65% (13 of 20), 35% (7 of 20), and 20% (4 of 20) of the animals exhibited distant site metastases to lymph nodes, lungs, and liver. Celecoxib supplementation to TRAMP mice from 8–32 weeks of age exhibited significant reduction in tumor development (5 of 20) with no signs of metastasis. Celecoxib feeding resulted in a significant decrease in prostate (56%; P < 0.0003) and genitourinary weight (48%; P < 0.008). Sequential magnetic resonance imaging analysis of celecoxib-fed mice documented lower prostate volume compared with the AIN 76A-fed group. Histopathological examination of celecoxib-fed animals showed reduced proliferation, and down-modulation of COX-2 and prostaglandin E2 levels in the dorsolateral prostate and plasma, respectively. These results correlated with retention of antimetastasis markers, viz E-cadherin, and α- and β-catenin, along with a significant decrease in vascular endothelial growth factor protein expression. Celecoxib supplementation also resulted in enhanced in vivo apoptosis in the prostate as monitored by several techniques including a recently perfected technique of 99mTc-labeled annexin V in live animals followed by phosphor imaging. One striking observation in an additional study was that celecoxib feeding to mice with established tumors (16 weeks of age) significantly improved their overall survival (P = 0.014), compared with AIN 76A-fed group. Our findings suggest that celecoxib may be useful in chemoprevention of prostate cancer.
Comparative Measurements of Hypoxia in Human Brain Tumors Using Needle Electrodes and EF5 Binding
Authors: Evans Sydney M. , Judy Kevin D. , Dunphy Isolde , Jenkins W. Timothy , Nelson Peter T. , Collins Ruth , Wileyto E. Paul , Jenkins Kevin , Hahn Stephen M. , Stevens Craig W. , Judkins Alexander R. , Phillips Peter , Geoerger Birgit , Koch Cameron J.
Published: September 2005

Hypoxia is known to be an important prognostic marker in many human cancers. We report the use of two oxygen measurement techniques in human brain tumors and compare these data with semiquantitative histological end points. Oxygenation was measured using the Eppendorf needle electrode and/or EF5 binding in 28 brain tumors. These data were compared with necrosis, mitosis, and endothelial proliferation. In some tumors, absolute EF5 binding was converted to tissue pO2 based on in vitro calibrations. Eppendorf electrode readings could not be used to identify WHO grade 1/2 versus WHO grade 3/4 tumors, they could not differentiate grade 3 versus grade 4 glial-derived neoplasms, nor did they correlate with necrosis or endothelial proliferation scores. EF5 binding increased as the tumor grade increased and was significantly associated with necrosis and endothelial proliferation. There was no statistically significant correlation between the two hypoxia detection techniques, although both methods indicated similar absolute ranges of tissue pO2. There was substantial inter- and intratumoral heterogeneity of EF5 binding in WHO grade 4 glial neoplasms. The majority of cells in glial-derived tumor had levels of hypoxia that were mild to moderate (defined herein as 10% to 0.5% pO2) rather than severe (defined as approximately 0.1% pO2). Immunohistochemical detection of EF5 binding tracks histological parameters in adult brain tumors, with increased binding associated with increasing necrosis and endothelial proliferation. The proportion of moderately to severely hypoxic cells is relatively low, even in the high-grade tumors. Human brain tumors are dominated by oxic to moderately hypoxic cells.
A Molecular Signature of the Nottingham Prognostic Index in Breast Cancer
Authors: Yu Kun , Lee Chee How , Tan Puay Hoon , Hong Ga Sze , Wee Siew Bok , Wong Chow Yin , Tan Patrick
Published: September 2005

The Nottingham prognostic index (NPI) is a widely used clinicopathological staging system for breast cancer prognostication. Using a step-wise classification approach where breast tumor expression profiles were first divided into general “molecular subtypes” [estrogen receptor (ER)+, ER−, ERBB2+], followed by an independent analysis of each subtype, we identified a 62-gene expression signature (NPI-ES) highly correlated to the NPI in ER+ tumors. The NPI-ES classified the majority of ER+ tumors into two distinct groups with high confidence and was significantly correlated to NPI status in two independent sets of ER+ tumors derived from different centers. The NPI-ES is comparable to the classical NPI in identifying patients likely to exhibit a poor clinical prognosis, as well as to a recently described “prognosis signature” for breast cancer. Our findings demonstrate how expression profiling can complement classical staging systems employing histopathological parameters scored over a continuous range of values.
Biomarkers of Esophageal Adenocarcinoma and Barrett’s Esophagus
Authors: McManus Damian T. , Olaru Andreea , Meltzer Stephen J.
Published: September 2005

The rising incidence and poor prognosis of esophageal adenocarcinoma in the Western world have intensified research efforts into earlier methods of detection of this disease and its relationship to Barrett’s esophagus. The progression of Barrett’s esophagus to adenocarcinoma has been the focus of particular scrutiny, and a number of potential tissue and serum-based disease biomarkers have emerged. The epidemiology and pathogenesis of esophageal adenocarcinoma are outlined. Tissue biomarkers allowing risk stratification of Barrett’s are reviewed as well as strategies currently being used to discover novel biomarkers that will facilitate the early detection of esophageal adenocarcinoma. Finally, the uses of biomarkers as predictive tests for targeted treatments and as surrogate endpoints in chemoprevention trials are considered.
Differentiation between Cell Death Modes Using Measurements of Different Soluble Forms of Extracellular Cytokeratin 18
Authors: Kramer Gero , Erdal Hamdiye , Mertens Helena J. M. M. , Nap Marius , Mauermann Julian , Steiner Georg , Marberger Michael , Bivén Kenneth , Shoshan Maria C. , Linder Stig
Published: September 2005

Cytokeratins are released from carcinoma cells by unclear mechanisms and are commonly used serum tumor markers (TPA, TPS, and CYFRA 21–1). We here report that soluble cytokeratin-18 (CK18) is released from human carcinoma cells during cell death. During necrosis, the cytosolic pool of soluble CK18 was released, whereas apoptosis was associated with significant release of caspase-cleaved CK18 fragments. These results suggested that assessments of different forms of CK18 in patient sera could be used to examine cell death modes. Therefore, CK18 was measured in local venous blood collected during operation of patients with endometrial tumors. In most patient sera, caspase-cleaved fragments constituted a minor fraction of total CK18, suggesting that tumor apoptosis is not the main mechanism for generation of circulating CK18. Monitoring of different CK18 forms in peripheral blood during chemotherapy of prostate cancer patients showed individual differences in the patterns of release. Importantly, several examples were observed where the increase of apoptosis-specific caspase-cleaved CK18 fragments constituted only a minor fraction of the total increase. These results suggest that cell death of epithelially derived tumors can be assessed in patient serum and suggest that tumor apoptosis may not necessarily be the dominating death mode in many tumors in vivo.
Development of a Cancer-Targeted Tissue-Specific Promoter System
Authors: Fukazawa Takuya , Maeda Yutaka , Sladek Frances M. , Owen-Schaub Laurie B.
Published: September 2005

Present cancer gene therapy using proapoptotic genes has had limited success because the therapy is prone to cause side effects as a result of the lack of tissue and cancer specificity. To target cancer cells without damaging normal cells, we have designed a novel dual promoter system in which a tissue-specific transcription system under the control of a cancer-specific promoter drives expression of a therapeutic gene. The applicability of this system was demonstrated by adapting it to target lung cancer. We termed this lung cancer system TTS (TTF1 gene under the control of human telomerase reverse transcriptase promoter and human surfactant protein A1 promoter). The TTS system showed much higher promoter activity in lung cancer cells compared with other kinds of cancer and normal lung cells, including stem cells. Moreover, insertion of negative glucocorticoid responsive elements in the system allows it to be drug controllable. The approaches that we have used could be adapted to target other types of cancer. We report a novel cancer-targeted tissue-specific dual promoter system designed for gene therapy.
Conditional Deletion of
Rb
Causes Early Stage Prostate Cancer
Authors: Maddison Lisette A. , Sutherland Brent W. , Barrios Roberto J. , Greenberg Norman M.
Published: September 2005

Prostate cancer remains the second leading cause of cancer-related death for men in the United States. Mutations in tumor suppressor genes including retinoblastoma (Rb), p53, and PTEN have been linked to the development of prostate cancer in man and mouse models, and loss of heterozygosity of the Rb locus has been observed in up to 60% of clinical cases. In this study we demonstrate that conditional somatic deletion of even a single Rb allele in the epithelial cells of the mouse prostate causes focal hyperplasia, thereby establishing a causal relationship between Rb loss and development of early stage prostate cancer. As a consequence of Rb ablation we observed increased expression of E2F target genes and a concomitant increase in proliferation in the epithelial compartment. However, by 52 weeks of age these lesions had not become malignant and represent an early stage of the disease. Nevertheless, the multifocal nature of the phenotype in the mice closely resembled multifocality of clinical disease. Taken together, our data demonstrated that loss of pRB-mediated cell cycle control directly caused the initiation of proliferative prostate disease but was insufficient to cause malignancy. Establishment of this early initiation model will aid efforts to thoroughly characterize early prostate disease as well as the elucidation of molecular mechanisms that cooperate with Rb loss to facilitate progression and metastasis.
Cyclolignans as Inhibitors of the Insulin-Like Growth Factor-1 Receptor and Malignant Cell Growth
Authors: Girnita Ada , Girnita Leonard , Prete Fabrizio del , Bartolazzi Armando , Larsson Olle , Axelson Magnus
Published: September 2005

The insulin-like growth factor-1 receptor (IGF-1R) plays a pivotal role in transformation, growth, and survival of malignant cells, and has emerged as a general and promising target for cancer treatment. However, no fully selective IGF-1R inhibitors have thus far been found. This is explained by the fact that IGF-1R is highly homologous to the insulin receptor, coinhibition of which may cause diabetic response. The receptors are both tyrosine kinases, and their ATP binding sites are identical, implying that ATP inhibitors cannot discriminate between them. Therefore, the current strategy has been to identify compounds interfering with receptor autophosphorylation at the substrate level. In this study we investigated the effects of cyclolignans and related molecules on IGF-1R activity. We report that certain cyclolignans are potent and selective inhibitors of tyrosine phosphorylation of the IGF-1R. Of particular interest was picropodophyllin (PPP), which is almost nontoxic (LD50 >500 mg/kg in rodents). PPP efficiently blocked IGF-1R activity, reduced pAkt and phosphorylated extracellular signal regulated kinase 1 and 2 (pErk1/2), induced apoptosis in cultured IGF-1R-positive tumor cells, and caused complete tumor regression in xenografted and allografted mice. PPP did not affect the insulin receptor or compete with ATP in an in vitro kinase assay, suggesting that it may inhibit IGF-1R autophosphorylation at the substrate level. This is also in agreement with our molecular model of how the cyclolignans may act on the IGF-1R kinase. Our results open the possibility to use PPP or related compounds with inhibitory effects on IGF-1R as lead compounds in development of anticancer agents.
c-Kit-Targeting Immunotherapy for Hereditary Melanoma in a Mouse Model
Authors: Kato Masashi , Takeda Kozue , Kawamoto Yoshiyuki , Tsuzuki Toyonori , Hossain Khaled , Tamakoshi Akiko , Kunisada Takahiro , Kambayashi Yasuhiro , Ogino Keiki , Suzuki Haruhiko , Takahashi Masahide , Nakashima Izumi
Published: September 2005

The role of c-Kit in the development of melanoma was studied in line 304/B6 of RET-transgenic mice, in which melanoma spontaneously develops. In Wv/Wv-RET (304/B6)-transgenic mice, in which c-Kit function was severely impaired, development of melanoma was strongly suppressed. Although 31 of the 44 original RET-transgenic mice died of rapidly growing melanoma within 12 months after birth, only 8 of the 44 Wv/Wv-RET-transgenic mice developed slowly growing melanocytic tumors with a greatly prolonged mean tumor-free period, 2 of which died of melanoma at a late stage. Even Wv/+-RET-transgenic mice had a clearly prolonged tumor-free period and definitely reduced frequency (6 of 61) of tumor death within 12 months after birth. Melanin production in the skin of these mice was not strongly impaired, suggesting that c-Kit affects the development of melanomas in these mice with only minor effects in melanin production. c-Kit expression in skin soon after birth was promoted in RET-transgenic mice, and c-Kit was expressed at high levels at the benign but not malignant stage of the tumor. A single injection of anti-c-Kit antibody (ACK2) into RET-transgenic mice soon after birth caused a surprisingly long-lasting suppression of development of melanoma, greatly prolonging the tumor-free period, and none of the 28 ACK2-treated RET-transgenic mice died from tumors at 12 months of age. The c-Kit function needed for melanin production was also suppressed for an unusually long time in ACK2-treated, RET-transgenic mice. These results suggest that c-Kit can be a unique target molecule for melanoma treatment.
Microarray Analysis Reveals Glucocorticoid-Regulated Survival Genes That Are Associated With Inhibition of Apoptosis in Breast Epithelial Cells
Authors: Wu Wei , Chaudhuri Shamita , Brickley Deanna R. , Pang Diana , Karrison Theodore , Conzen Suzanne D.
Published: September 2005

Activation of the glucocorticoid receptor (GR) results in diverse physiological effects depending on cell type. For example, glucocorticoids (GC) cause apoptosis in lymphocytes but can rescue mammary epithelial cells from growth factor withdrawal-induced death. However, the molecular mechanisms of GR-mediated survival remain poorly understood. In this study, a large-scale oligonucleotide screen of GR-regulated genes was performed. Several of the genes that were found to be induced 30 min after GR activation encode proteins that function in cell survival signaling pathways. We also demonstrate that dexamethasone pretreatment of breast cancer cell lines inhibits chemotherapy-induced apoptosis in a GR-dependent manner and is associated with the transcriptional induction of at least two genes identified in our screen, serum and GC-inducible protein kinase-1 (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP-1). Furthermore, GC treatment alone or GC treatment followed by chemotherapy increases both SGK-1 and MKP-1 steady-state protein levels. In the absence of GC treatment, ectopic expression of SGK-1 or MKP-1 inhibits chemotherapy-induced apoptosis, suggesting a possible role for these proteins in GR-mediated survival. Moreover, specific inhibition of SGK-1 or MKP-1 induction by the introduction of SGK-1- or MKP-1-small interfering RNA reversed the antiapoptotic effects of GC treatment. Taken together, these data suggest that GR activation in breast cancer cells regulates survival signaling through direct transactivation of genes that encode proteins that decrease susceptibility to apoptosis. Given the widespread clinical administration of dexamethasone before chemotherapy, understanding GR-induced survival mechanisms is essential for achieving optimal therapeutic responses.
Gene Therapy with Secretory Leukoprotease Inhibitor Promoter-Controlled Replication-Competent Adenovirus for Non-Small Cell Lung Cancer
Authors: Maemondo Makoto , Saijo Yasuo , Narumi Ko , Kikuchi Toshiaki , Usui Kazuhiro , Tazawa Ryushi , Matsumoto Kunio , Nakamura Toshikazu , Sasaki Katsunori , Takahashi Minoru , Niitsu Yoshiro , Nukiwa Toshihiro
Published: September 2005

Secretory leukoprotease inhibitor (SLPI) is highly expressed in almost all non-small cell lung cancers (NSCLCs), but not in the majority of other tumor types. In an attempt to create a specific gene therapy for NSCLC, we constructed AdSLPI.E1AdB, an adenovirus vector with a double expression cassette consisting of E1A driven by the SLPI promoter gene followed by E1B-19K under the control of the cytomegalovirus (CMV) promoter that can selectively replicate only in NSCLC cells. Infection with AdSLPI.E1AdB yielded E1A protein expression and adenovirus replication resulting in a >100-fold increase of the virus titers only in SLPI-producing NSCLC cells (A549, H358, and HS24 cells). In contrast, neither E1A protein nor replication was detected in non-SLPI-producing HepG2 cells. Treatment with AdSLPI.E1AdB significantly inhibited the proliferation of NSCLC cells in vitro in a dose-dependent manner, whereas the cell growth of HepG2 or normal human bronchial epithelial cells was not affected by AdSLPI.E1AdB infection. Direct injection of AdSLPI.E1AdB into A549 and H358 tumors in nude mice resulted in a marked reduction in tumor growth compared with controls (A549, 57%, P < 0.02; H358, 67%, P < 0.03). Histological examination revealed the replication of AdSLPI.E1AdB and strong induction of necrosis and apoptosis. In addition, we evaluated the combination of AdSLPI.E1AdB and AdCMV.NK4 encoding NK4 protein, which has strong antiangiogenic activity. E1A expressed by AdSLPI.E1AdB trans-acts on the replication of AdCMV.NK4 and thus increases the expression of NK4. Injection of these two vectors into H358 tumors resulted in a more striking reduction of tumor growth compared with single injection of each vector. These results suggest that AdSLPI.E1AdB could provide a selective therapeutic modality for NSCLC and that the combination of AdSLPI.E1AdB and AdCMV.NK4 may be a more effective gene therapy for NSCLC.
Bax Mediates the Apoptosis-Sensitizing Effect of Maspin
Authors: Liu Jiayou , Yin Shuping , Reddy Neelima , Spencer Craig , Sheng Shijie
Published: September 2005

Maspin, a serine protease inhibitor (serpin), can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. This may occur through maspin-mediated inhibition of pericellular proteolysis. In a recent report, we provided evidence that maspin may also suppress tumor progression by enhancing cellular sensitivity to apoptotic stimuli. To our knowledge, maspin is the only proapoptotic serpin among all of the serpins implicated thus far in apoptosis regulation. The goal of the present study is to identify the specific target molecule(s), the modification of which by maspin renders tumor cells sensitive to chemotherapeutic agents. Our cellular, molecular, and biochemical studies demonstrate an essential role of Bax in the proapoptotic effect of maspin. First, Bax was up-regulated in maspin-transfected prostate and breast tumor cells, whereas the levels of other Bcl-2 family members including Bcl-2, Bcl-xl, and Bak remained unchanged. Second, on apoptosis induction, a greater amount of Bax was translocated from cytosol to mitochondria in maspin-transfected cells. After treatment with a Bax-silencing small interfering RNA, maspin-transfected cells became significantly more resistant to drug-induced apoptosis. Consistently, the release of cytochrome c and Smac/DIABLO from mitochondria was more responsive to apoptosis stimuli in maspin-transfected cells than in the mock-transfected cells. Third, the apoptosis induction of maspin-transfected cells was associated with increased activation of both caspase-8 and caspase-9. However, a caspase-9-specific inhibitor blocked the sensitization effect of maspin in a dose-dependent and time-dependent manner, demonstrating a rate-limiting role for caspase-9. In line with the central role of the Bax-mediated mitochondrial apoptotic pathway, maspin sensitized the apoptotic response of breast and prostate carcinoma cells to various drugs, ranging from death ligands to endoplasmic reticulum stress. The link between maspin and Bax up-regulation explains the loss of maspin-expressing tumor cells in invasive breast and prostate carcinomas. Our data reveal a novel mechanism for tumor suppressive maspin and suggest that maspin may be used as a modifier for apoptosis-based cancer therapy.
Identification of Retinoid-Modulated Proteins in Squamous Carcinoma Cells Using High-Throughput Immunoblotting
Authors: Kim Hyun-Jung , Lotan Reuben
Published: September 2005

Retinoids have shown clinical efficacy in cancer chemoprevention and therapy presumably by modulating the growth, differentiation, and apoptosis of normal, premalignant, and malignant cells. To better understand the mechanisms by which retinoids exert their effects, we used a high-throughput Western blotting method (Becton-Dickinson PowerBlot) to evaluate changes in the levels of cellular signaling proteins in head and neck squamous cell carcinoma cells treated with the cytostatic all-trans-retinoic acid or with the proapoptotic retinoids 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid or N-(4-hydroxyphenyl)retinamide. Treatments of the head and neck squamous cell carcinoma cells with these retinoids for 24 h resulted in increased levels of 14, 22, and 22 proteins and decreased levels of 5, 10, and 7 proteins, respectively. The changes in the levels of the following proteins were confirmed by conventional western immunoblotting: all-trans-retinoic acid increased ELF3, topoisomerase II α, RB2/p130, RIG-G, and EMAPII and decreased MEF2D and cathepsin L. N-(4-Hydroxyphenyl)retinamide up-regulated ELF3, c-Jun, Rb2/p130, JAK1, p67phox, Grb2, O6-methylguanine-DNA methyltransferase, and Ercc-1. 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid increased Rb2/p130, c-Jun, Sp1, Sin, and tomosyn and decreased cathepsin L, Mre11, and topoisomerase II α. Some of these proteins were also modulated by these retinoids in other human cancer cell lines. A subset of the proteins were modulated similarly by the different retinoids, whereas changes in other proteins were unique for each retinoid. These results suggest that the mechanisms by which these retinoids modulate proteins are distinct but may overlap. Some of the retinoid-modulated proteins identified in this study may be novel candidates for mediating different responses to retinoids.
Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) Kills Human Glioblastoma Cells in Brain Xenografts by an Apoptosis-Like Mechanism and Prolongs Survival
Authors: Fischer Walter , Gustafsson Lotta , Mossberg Ann-Kristin , Gronli Janne , Mork Sverre , Bjerkvig Rolf , Svanborg Catharina
Published: September 2005

Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human α-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from α-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and α-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all α-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm3). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.
Silencing Expression of the Clusterin/Apolipoprotein J Gene in Human Cancer Cells Using Small Interfering RNA Induces Spontaneous Apoptosis, Reduced Growth Ability, and Cell Sensitization to Genotoxic and Oxidative Stress
Authors: Trougakos Ioannis P. , So Alan , Jansen Burkhard , Gleave Martin E. , Gonos Efstathios S.
Published: September 2005

Clusterin/Apolipoprotein J (CLU) is a heterodimeric ubiquitously expressed secreted glycoprotein that is implicated in several physiological processes and is differentially expressed in many severe physiological disturbances, including tumor formation and in vivo cancer progression. Despite extensive efforts, clarification of CLU’s biological role has been exceptionally difficult and its precise function remains elusive. Short RNA duplexes, referred to as small interfering RNAs (siRNAs), provide a new approach for the elucidation of gene function in human cells. Here, we describe siRNA-mediated CLU gene silencing in osteosarcoma and prostate human cancer cells and illustrate that CLU mRNA is amenable to siRNA-mediated degradation. Our data demonstrate that CLU knockdown in human cancer cells induces significant reduction of cellular growth and higher rates of spontaneous endogenous apoptosis. Moreover, CLU knockdown cancer cells were significantly sensitized to both genotoxic and oxidative stress induced by chemotherapeutic drugs and H2O2, respectively. These effects were more pronounced in cell lines that express high endogenous steady-state levels of the CLU protein and occur through hyperactivation of the cellular apoptotic machinery. Overall, our results reveal that, in the distinct cellular contexts of the osteosarcoma and prostate cancer cells assayed, CLU is a central molecule in cell homeostasis that exerts a cytoprotective function. The described CLU-specific siRNA oligonucleotides that can potently silence CLU gene expression may thus prove valuable agents during antitumor therapy or at other pathological conditions where CLU has been implicated.
Blocking Angiogenesis and Tumorigenesis with GFA-116, a Synthetic Molecule that Inhibits Binding of Vascular Endothelial Growth Factor to its Receptor
Authors: Sun Jiazhi , Blaskovich Michelle A. , Jain Rishi K. , Delarue Frederic , Paris Daniel , Brem Steven , Wotoczek-Obadia Marguerite , Lin Qing , Coppola Domenico , Choi Kihang , Mullan Michael , Hamilton Andrew D. , Sebti Saïd M.
Published: September 2005

A small synthetic library of cyclohexapeptidomimetic calixarenes was prepared to identify disrupters of vascular endothelial growth factor (VEGF) binding to its receptor that inhibits angiogenesis. From this library, we discovered GFA-116, which potently inhibits 125I-VEGF binding to Flk-1 in Flk-1-overexpressing NIH 3T3 cells and human prostate tumor cells with an IC50 of 750 nm. This inhibition is highly selective for VEGF in that 125I- platelet-derived growth factor binding to its receptor is not affected. GFA-116 inhibits VEGF-stimulated Flk-1 tyrosine phosphorylation and subsequent activation of Erk1/2 mitogen-activated protein kinases. Furthermore, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor-dependent stimulation of Erk1/2 phosphorylation are not affected at concentrations as high as 10 μm. In vitro, GFA-116 inhibits angiogenesis as measured by inhibition of migration and formation of capillary-like structures by human endothelial cells as well as suppression of microvessel outgrowth in rat aortic rings and rat cornea angiogenesis. In vivo, GFA-116 (50 mpk/day) inhibits tumor growth and angiogenesis as measured by CD31 staining of A-549 human lung tumors in nude mice. Furthermore, GFA-116 is also effective at inhibiting tumor growth and metastasis to the lung of B16-F10 melanoma cells injected into immunocompetent mice. Taken together, these results demonstrate that a synthetic molecule capable of disrupting the binding of VEGF to its receptor selectively inhibits VEGF-dependent signaling and suppresses angiogenesis and tumorigenesis.
Combinatorial Screenings in Patients
Authors: Zurita Amado J. , Troncoso Patricia , Cardó-Vila Marina , Logothetis Christopher J. , Pasqualini Renata , Arap Wadih
Published: September 2005

Direct screening of combinatorial peptide libraries in patients may allow the identification of ligands that target biochemical differences in the endothelium of blood vessels. In a screening performed in a patient, we selected and isolated a mimic motif of interleukin 11 (IL-11) from prostate biopsies after an i.v. administration of a phage display peptide library. We also demonstrated that the IL-11 phage mimic (displaying the cyclic nonapeptide CGRRAGGSC) bound specifically to a corresponding IL-11 receptor (IL-11Rα). Here we show that IL-11Rα is a potential target for intervention in human prostate cancer through morphological and functional analyses. First, a comprehensive serial immunohistochemical analysis of primary and metastatic prostate cancer samples showed increased stage-specific expression of IL-11Rα during disease progression. Second, a proapoptotic peptide was specifically targeted and internalized through this functional IL-11Rα-based ligand-receptor pair: treatment of prostate cancer cells in vitro with a proapoptotic peptide guided by the CGRRAGGSC peptide to the IL-11Rα resulted in dose-dependent apoptosis. Together, these data indicate that the IL-11Rα is a candidate target for translational clinical trials against advanced and metastatic prostate cancer. Moreover, our results illustrate the ability of direct combinatorial screening systems in cancer patients for identification of relevant targets in the context of human disease.
Are
ATM
Mutations 7271T→G and IVS10-6T→G Really High-Risk Breast Cancer-Susceptibility Alleles?
Authors: Szabo Csilla I. , Schutte Mieke , Broeks Annegien , Houwing-Duistermaat Jeanine J. , Thorstenson Yvonne R. , Durocher Francine , Oldenburg Rogier A. , Wasielewski Marijke , Odefrey Fabrice , Thompson Deborah , Floore Arno N. , Kraan Jaennelle , Klijn Jan G. M. , van den Ouweland Ans M. W. ,   the BRCA-X Consortium,   Cooperative Family Registry Breast Cancer Study,   Interdisciplinary Health Research International Team on Breast Cancer Susceptibility, Wagner Teresa M. U. , Devilee Peter , Simard Jacques , van ‘t Veer Laura J. , Goldgar David E. , Meijers-Heijboer Hanne
Published: September 2005

Two mutations of the ATM gene were recently suggested to confer breast cancer risks similar to mutations of BRCA1 or BRCA2. Here, we set out to confirm these findings in 961 families with non-BRCA1/BRCA2 breast cancer from diverse geographical regions. We did not detect the ATM 7271T→G mutation in any family. The ATM IVS10–6T→G mutation was detected in eight families, which was similar to its frequency among population-matched control individuals (pooled Mantel-Haenszel odds ratio = 1.60; 95% confidence interval = 0.48 to 5.35; P = 0.44). Bayesian analysis of linkage in the ATM IVS10–6T→G-positive families showed an overall posterior probability of causality for this mutation of 0.008. We conclude that the ATM IVS10–6T→G mutation does not confer a significantly elevated breast cancer risk and that ATM 7271T→G is a rare event in familial breast cancer.
Hypoxia Targeted Gene Therapy to Increase the Efficacy of Tirapazamine as an Adjuvant to Radiotherapy
Authors: Cowen Rachel L. , Williams Kaye J. , Chinje Edwin C. , Jaffar Mohammed , Sheppard Freda C. D. , Telfer Brian A. , Wind Natasha S. , Stratford Ian J.
Published: September 2005

Solid tumors are characterized by regions of hypoxia that are inherently resistant to both radiotherapy and some chemotherapy. To target this resistant population, bioreductive drugs that are preferentially toxic to tumor cells in a hypoxic environment are being evaluated in clinical trials; the lead compound, tirapazamine (TPZ), is being used in combination with cisplatin and/or with radiotherapy. Crucially, tumor response to TPZ is also dependent on the cellular complement of reductases. In particular, NADPH:cytochrome P450 reductase (P450R) plays a major role in the metabolic activation of TPZ. In a gene-directed enzyme prodrug therapy (GDEPT) approach using adenoviral delivery, we have overexpressed human P450R specifically within hypoxic cells in tumors, with the aim of harnessing hypoxia as a trigger for both enzyme expression and drug metabolism. The adenovirus used incorporates the hypoxia-responsive element (HRE) from the lactate dehydrogenase gene in a minimal SV40 promoter context upstream of the cDNA for P450R. In a human tumor model in which TPZ alone does not potentiate radiotherapeutic outcome (HT1080 fibrosarcoma), we witnessed complete tumor regression when tumors were virally transduced before treatment.
Short-Term Human Prostate Primary Xenografts
Authors: Gray Danny R. , Huss Wendy J. , Yau Jeffrey M. , Durham Lori E. , Werdin Eric S. , Funkhouser William K. , Smith Gary J.
Published: September 2005

Transgenic spontaneously occurring and transplantable xenograft models of adenocarcinoma of the prostate (CaP) are established tools for the study of CaP progression and metastasis. However, no animal model of CaP has been characterized that recapitulates the response of the human prostate vascular compartment to the evolving tumor microenvironment during CaP progression. We report that primary xenografts of human CaP and of noninvolved areas of the human prostate peripheral zone transplanted to athymic nude mice provide a unique model of human angiogenesis occurring in an intact human prostate tissue microenvironment. Angiogenesis in human kidney primary xenografts established from human renal cell carcinoma and noninvolved kidney tissue, a highly vascular organ and cancer, was compared with angiogenesis in xenografts from the relatively less vascularized prostate. Immunohistochemical identification of the human versus mouse host origin of the endothelial cells and of human endothelial cell proliferation in the human prostate and human kidney xenografts demonstrated that: (a) the majority of the vessels in primary xenografts of benign and malignant tissue of both organs were lined with human endothelial cells through the 30-day study period; (b) the mean vessel density was increased in both the CaP and benign prostate xenografts relative to the initial tissue, whereas there was no significant difference in mean vessel density in the renal cell carcinoma and benign kidney xenografts compared with the initial tissue; and (c) the number of vessels with proliferating endothelial cells in primary xenografts of CaP and benign prostate increased compared with their respective initial tissue specimens, whereas the number of vessels with proliferating endothelial cells decreased in the benign kidney xenografts. Short-term primary human prostate xenografts, therefore, represent a valuable in vivo model for the study of human angiogenesis within a human tissue microenvironment and for comparison of angiogenesis in CaP versus benign prostate.
Involvement of Interferon Regulatory Factor 1 and S100C/A11 in Growth Inhibition by Transforming Growth Factor β1 in Human Hepatocellular Carcinoma Cells
Authors: Miyazaki Masahiro , Sakaguchi Masakiyo , Akiyama Ichiro , Sakaguchi Yoshihiko , Nagamori Seishi , Huh Nam-ho
Published: September 2005

Growth inhibition by transforming growth factor (TGF)-β1 has been attributed to the induction of cyclin-dependent kinase inhibitors, among which p21/Waf1 plays a major role in many biological contexts. In the present study, two new intracellular mediators for the induction of p21/Waf1 by TGF-β1 were identified in a human hepatocellular carcinoma cell line (JHH-5) expressing mutant-type p53. After addition of TGF-β1 to JHH-5 cells, a marked increase of the p21/Waf1 expression preceded the inhibition of DNA synthesis. Expression of IFN regulatory factor (IRF)-1, a known transacting factor for p21/Waf1 promoter, was elevated just before or in parallel with the increase of p21/Waf1. Transduction of antisense IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-β1 and partially released the cells from the growth arrest by TGF-β1. Expression of S100C/A11, a member of the Ca2+-binding S100 protein family, also markedly increased after addition of TGF-β1. S100C/A11 protein was translocated to and accumulated in nuclei of TGF-β1-treated JHH-5 cells, where p21/Waf1 was concomitantly accumulated. When a recombinant S100C/A11 protein was introduced into nuclei of JHH-5 cells, DNA synthesis was markedly inhibited in a dose-dependent manner in the absence of TGF-β1. Prior transfection of p21/Waf1-targeted small interfering RNA efficiently blocked decrease of DNA synthesis in JHH-5 cells caused by TAT-S100C/A11 or TGF-β1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that IRF-1 and S100C/A11 mediate growth inhibition by TGF-β1 via induction of p21/Waf1.