bluuebunny/crossref_metadata_2025 huggingface dataset:
A Mechanism for Cell Size Regulation by the Insulin and Insulin-Like Growth Factor-I Receptors
Authors: Sun Hongzhi , Tu Xiao , Baserga Renato
Published: December 2006

Deletion of the type 1 insulin-like growth factor receptor (IGF-IR) or of the insulin receptor substrate-1 (IRS-1) genes in animals causes a 50% reduction in body size at birth. Decrease in body size is due to both a decreased number of cells and a decreased cell size. Deletion of the insulin receptor (InR) genes results in mice that are normal in size at birth. We have used 32D-derived myeloid cells to study the effect of IGF-IR and InR signaling on cell size. 32D cells expressing the IGF-IR and IRS-1 are almost twice as large as 32D cells expressing the InR and IRS-1. A mechanism for the difference in size is provided by the levels of the upstream binding factor 1 (UBF1), a nucleolar protein that participates in the regulation of RNA polymerase I activity and rRNA synthesis and therefore cell size. When shifted to the respective ligands, UBF1 levels decrease in cells expressing the InR and IRS-1, whereas they remain stable in cells expressing the IGF-IR and IRS-1. The expression of the IGF-IR and IRS-1 is crucial to the stability of UBF1. (Cancer Res 2006; 66(23): 11106-9)
In vivo Color-Coded Imaging of the Interaction of Colon Cancer Cells and Splenocytes in the Formation of Liver Metastases
Authors: Bouvet Michael , Tsuji Kazuhiko , Yang Meng , Jiang Ping , Moossa Abdool R. , Hoffman Robert M.
Published: December 2006

The role of host cells in tumor progression and metastasis is critical. Intrasplenic injection of tumor cells has long been known as an effective method of developing liver metastases in nude mice, whereas portal vein (PV) injection of tumor cells can result in rapid death of the tumor cells. Host cells were thought to play a role in these phenomena. We report here that after splenic injection of tumor cells, splenocytes cotraffic with the tumor cells to the liver and facilitate metastatic colony formation. Human colon cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP) were injected in either the PV or spleen of nude mice and imaged at the subcellular level in vivo. Extensive clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours after PV injection and essentially all the cancer cells died. In contrast, splenic injection of these tumor cells resulted in the aggressive formation of liver and distant metastasis. GFP spleen cells were found in the liver metastases that resulted from intrasplenic injection of the tumor cells in transgenic nude mice ubiquitously expressing GFP. When GFP spleen cells and the RFP cancer cells were coinjected in the PV, liver metastasis resulted that contained GFP spleen cells. These results suggest a novel tumor-host interaction that enables efficient formation of liver metastasis via intrasplenic injection. (Cancer Res 2006; 66(23): 11293-7)
Lithium Treatment Prevents Neurocognitive Deficit Resulting from Cranial Irradiation
Authors: Yazlovitskaya Eugenia M. , Edwards Eric , Thotala Dinesh , Fu Allie , Osusky Kate L. , Whetsell William O. , Boone Braden , Shinohara Eric T. , Hallahan Dennis E.
Published: December 2006

Curative cancer treatment regimens often require cranial irradiation, resulting in lifelong neurocognitive deficiency in cancer survivors. This deficiency is in part related to radiation-induced apoptosis and decreased neurogenesis in the subgranular zone of the hippocampus. We show that lithium treatment protects irradiated hippocampal neurons from apoptosis and improves cognitive performance of irradiated mice. The molecular mechanism of this effect is mediated through multiple pathways, including Akt/glycogen synthase kinase-3β (GSK-3β) and Bcl-2/Bax. Lithium treatment of the cultured mouse hippocampal neurons HT-22 induced activation of Akt (1.5-fold), inhibition of GSK-3β (2.2-fold), and an increase in Bcl-2 protein expression (2-fold). These effects were sustained when cells were treated with lithium in combination with ionizing radiation. In addition, this combined treatment led to decreased expression (40%) of the apoptotic protein Bax. The additional genes regulated by lithium were identified by microarray, such as decorin and Birc1f. In summary, we propose lithium treatment as a novel therapy for prevention of deleterious neurocognitive consequences of cranial irradiation. (Cancer Res 2006; 66(23): 11179-86)
Maintenance of Head and Neck Tumor Gene Expression Profiles upon Lymph Node Metastasis
Authors: Roepman Paul , de Jager Alike , Groot Koerkamp Marian J.A. , Kummer J. Alain , Slootweg Piet J. , Holstege Frank C.P.
Published: December 2006

Spread of cancer and development of solid metastases at distant sites is the main cause of cancer-related deaths. To understand and treat metastases, it is important to determine at which stages the most pivotal steps for development of metastases occur. In head and neck squamous cell carcinoma (HNSCC), metastasis nearly always occurs first in local lymph nodes before development of distant metastasis. Here, we have investigated gene expression patterns in HNSCC lymph node metastases using DNA microarrays. Several types of analyses show that the gene expression patterns in lymph node metastases are most similar to the corresponding primary tumors from which they arose, as long as samples contain sufficient proportions of tumor cells. Strikingly, gene expression patterns of metastatic primary HNSCC are largely maintained upon spread to the lymph node. Only a single gene, metastasis-associated gene 1 (MTA1), was found to show consistently changed expression between a large number of matched primary tumor-lymph node metastasis pairs. The maintained expression pattern includes the predictive signature for HNSCC lymph node metastasis. These results underscore the importance of the primary tumor gene expression profile for development and treatment of metastasis. The findings also agree with the concept that disseminated cancer cells alter the surrounding tissue into a metastatic environment that resembles the primary tumor microenvironment. (Cancer Res 2006; 66(23): 11110-4)
Get Off My Back! Rapid Receptor Internalization through Circular Dorsal Ruffles
Authors: Orth James D. , McNiven Mark A.
Published: December 2006

Internalization and subsequent trafficking of receptor tyrosine kinases (RTKs) play an important role in the modulation of growth factor–stimulated signaling events that affect different cellular processes, from cell growth and mitosis to motility and invasion. The intracellular transport of these receptors has traditionally been viewed as being initiated via clathrin-coated pits. However, nonclathrin pathways have been implicated as well, although these remain poorly understood. Most recently, the formation of dynamic, transient endocytic membrane structures termed circular dorsal ruffles or “dorsal waves” have been reported to selectively sequester and internalize a large percentage of a specific RTK from the surface of growth factor–stimulated cells. This process is dependent on dynamin and cortactin, two endocytic proteins that are also associated with the actin cytoskeleton, whereas it is independent of traditional coat proteins, such as clathrin and caveolin. Additionally, dorsal wave formation requires the participation and remodeling of a dynamic actin cytoskeleton. Most importantly, the formation of these structures may be less frequent in tumor cells and thereby have significant effects on receptor signaling and cell growth. (Cancer Res 2006; 66(23): 11094-6)
Polymorphisms in Genes Related to Oxidative Stress (CAT, MnSOD, MPO, andeNOS) and Acute Toxicities from Radiation Therapy following Lumpectomy for Breast Cancer
Authors: Ahn Jiyoung , Ambrosone Christine B. , Kanetsky Peter A. , Tian Chunqiao , Lehman Teresa A. , Kropp Silke , Helmbold Irmgard , von Fournier Dietrich , Haase Wulf , Sautter-Bihl Marie Luise , Wenz Frederik , Chang-Claude Jenny
Published: December 2006

Purpose: Because radiotherapy exerts cytotoxic effects via generation of massive oxidative stress, we hypothesized that catalase, manganese superoxide dismutase, myeloperoxidase (MPO), and endothelial nitric oxide synthase (eNOS) genotypes might result in greater risk of radiotoxicity.

Experimental Design: Cases (n = 446) were Caucasian women with breast cancer who received radiotherapy following lumpectomy. Genotypes were determined by matrix-assisted laser desorption/ionization time-of-flight. The development of acute reactions (moist desquamation) associated with genotypes was modeled using the Cox proportional hazards model, accounting for cumulative biologically effective radiation dose.

Results: Genotypes associated with higher levels of reactive oxygen species (ROS) were not associated with risk of radiotoxicity. However, relationships between overweight/obesity [body mass index (BMI), >25] and radiotoxicity risk seemed to be modified by eNOS and MPO genotypes associated with higher generation of nitric oxide and ROS, respectively. Women with high BMI (>25) and eNOS GG genotypes were at more than a 6-fold increase in risk (hazard ratio, 6.39; 95% confidence interval, 2.53-16.15) compared with those with BMI <25, and for MPO, those with high BMI (>25) and GG genotypes also had greater risk of radiotoxicity (hazard ratio, 3.61; 95% confidence interval, 1.78-7.35) compared with those with BMI <25. Overweight/obesity was not a strong risk factor among women with other eNOS and MPO genotypes. Exploratory analysis using classification and regression trees indicated that total number of risk alleles contributed, in part, to acute toxicity outcomes among a subgroup of women.

Conclusions: Associations between BMI and radiotoxicity risk may be most apparent among women with genotypes related to higher levels of oxidative stress. Regression trees may be useful in future studies to examine the contributions of multiple factors to individual susceptibility to adverse effects of cancer treatment.
Modulation of Cell Cycle Progression in Human Tumors: A Pharmacokinetic and Tumor Molecular Pharmacodynamic Study of Cisplatin Plus the Chk1 Inhibitor UCN-01 (NSC 638850)
Authors: Perez Raymond P. , Lewis Lionel D. , Beelen Andrew P. , Olszanski Anthony J. , Johnston Nicholas , Rhodes C. Harker , Beaulieu Bernard , Ernstoff Marc S. , Eastman Alan
Published: December 2006

Background: UCN-01, a Chk1 inhibitor, abrogates S and G2 arrest and enhances cancer cell killing by DNA-damaging drugs in preclinical models. UCN-01 avidly binds α1-acid glycoprotein in plasma; whether sufficient drug concentrations are achieved in human tumors is unknown. A phase I trial tested the hypothesis that UCN-01 abrogates cisplatin-induced cell cycle arrest (in tumors) at tolerable doses.

Methods: Patients with advanced cancer received i.v. cisplatin, followed 22 hours later by UCN-01 (3-day continuous i.v. infusion of a 28-day cycle). Platinum was measured by atomic absorption, UCN-01 by high-performance liquid chromatography, and cell cycle progression in tumor biopsies by geminin immunostaining (biomarker for S/G2 phases of cell cycle).

Results: The first two patients treated with cisplatin (20 mg/m2 plus UCN-01 45 mg/m2/d) experienced dose-limiting toxicities (subarachnoid hemorrhage, hyperglycemia, hypoxia, cardiac ischemia, and atrial fibrillation). Following 25% UCN-01 dose reduction, no toxicities greater than grade 2 were seen. Median plasma UCN-01 half-life (T1/2) was 405 hours. Salivary UCN-01 concentrations showed a rapid initial decline (median T1/2α, 29.9 hours), followed by a terminal decay parallel to that in plasma. UCN-01 pharmacokinetics, and the timing of clinical toxicities, suggests that UCN-01 is bioavailable despite α1-acid glycoprotein binding. Marked suppression of cells in S/G2 in tumor biopsies was seen by geminin immunohistochemistry, suggesting that UCN-01 is bioavailable at concentrations sufficient to inhibit Chk1.

Conclusions: Cisplatin (30 mg/m2), followed 22 hours later by UCN-01 (34 mg/m2/d for 3 days), is well tolerated clinically and yields UCN-01 concentrations sufficient to affect cell cycle progression in tumors.
Sorting Nexin 1 Down-Regulation Promotes Colon Tumorigenesis
Authors: Nguyen Lananh N. , Holdren Matthew S. , Nguyen Anthony P. , Furuya Momoko H. , Bianchini Michele , Levy Estrella , Mordoh José , Liu Annie , Guncay Gabriela D. , Campbell Jean S. , Parks W. Tony
Published: December 2006

Purpose: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development.

Experimental Design: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done.

Results: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log2 ratio less than −1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis.

Conclusions: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.
Lymphokine-Activated Killer T-Cell-Originated Protein Kinase Phosphorylation of Histone H2AX Prevents Arsenite-Induced Apoptosis in RPMI7951 Melanoma Cells
Authors: Zykova Tatyana A. , Zhu Feng , Lu Chengrong , Higgins LeeAnn , Tatsumi Yasuaki , Abe Yasuhito , Bode Ann M. , Dong Zigang
Published: December 2006

Purpose: Arsenic is a valuable therapeutic tool in cancer treatment. Lymphokine-activated killer T-cell-originated protein kinase (TOPK) is highly expressed in cancer cells, but its specific function is still unknown. We investigated the role of TOPK in arsenic-induced apoptosis in RPMI7951 human melanoma cells.

Experimental Design: Expression of TOPK was evaluated in different melanoma cell lines, and liquid chromatography-tandem mass spectrometry analysis was used to identify proteins binding with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of arsenic on TOPK, histone H2AX, and apoptosis in RPMI7951 cells.

Results: Melanoma cell lines expressing high levels of TOPK were more resistant to arsenite (As3+)-induced apoptosis. As3+ treatment induced phosphorylation of TOPK and histone H2AX in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry results indicated that TOPK could bind with histone H2AX, and in vitro and in vivo assays confirmed that TOPK binds with and phosphorylates histone H2AX. As3+ treatment caused phosphorylation of TOPK, which colocalized with phosphorylated histone H2AX in the nucleus. TOPK small interfering RNA cells exhibited a decreased phosphorylation of histone H2AX with As3+ treatment. As3+-induced apoptosis was decreased in H2AX−/− cells but increased in TOPK small interfering RNA cells.

Conclusions: TOPK binds with histone H2AX and inhibits As3+-induced apoptosis through phosphorylation of histone H2AX. Melanoma cell lines with high levels of TOPK are more resistant to As3+-induced apoptosis. Therefore, inhibition of TOPK activity combined with As3+ treatment may be helpful in the treatment of melanomas.
Dysregulated Expression of Stem Cell Factor Bmi1 in Precancerous Lesions of the Gastrointestinal Tract
Authors: Tateishi Keisuke , Ohta Miki , Kanai Fumihiko , Guleng Bayasi , Tanaka Yasuo , Asaoka Yoshinari , Tada Motohisa , Seto Motoko , Jazag Amarsanaa , Lianjie Lin , Okamoto Makoto , Isayama Hiroyuki , Tada Minoru , Yoshida Haruhiko , Kawabe Takao , Omata Masao
Published: December 2006

Purpose: It is important to identify the definitive molecular switches involved in the malignant transformation of premalignant tissues. Cellular senescence is a specific characteristic of precancerous tissues, but not of cancers, which might reflect tumorigenesis-protecting mechanisms in premalignant lesions. Polycomb protein Bmi1, which is a potent negative regulator of the p16INK4 gene, suppresses senescence in primary cells and is overexpressed in various cancers. We hypothesized that Bmi1 expression would also be dysregulated in precancerous lesions in human digestive precancerous tissues.

Experimental Design: Bmi1 expression was investigated in cancerous and precancerous tissues of the digestive tract. The expression of p16, β-catenin, and Gli1 and the in vivo methylation status of the p16 gene were also analyzed in serial sections of colonic precancerous lesions.

Results: Bmi1 was clearly overexpressed across a broad spectrum of gastrointestinal cancers, and the expression of Bmi1 increased in a manner that reflected the pathologic malignant features of precancerous colonic tissues (low-grade dysplasia, 12.9 ± 2.0%; high-grade dysplasia, 82.9 ± 1.6%; cancer, 87.5 ± 2.4%). p16 was also strongly expressed in high-grade dysplasia, but not in cancers. p16 promoter methylation was detected only in some Bmi1-positive neoplastic cells.

Conclusions: Bmi1 overexpression was correlated with the malignant grades of human digestive precancerous tissues, which suggests that advanced Bmi1 dysregulation might predict malignant progression. The abnormal Bmi1 expression might link to malignant transformation via the disturbance of orderly histone modification.
Haplotypes in Matrix Metalloproteinase Gene Cluster on Chromosome 11q22 Contribute to the Risk of Lung Cancer Development and Progression
Authors: Sun Tong , Gao Yang , Tan Wen , Ma Sufang , Zhang Xuemei , Wang Yonggang , Zhang Qingrun , Guo Yongli , Zhao Dan , Zeng Changqing , Lin Dongxin
Published: December 2006

Purpose: Matrix metalloproteinases (MMP) play important roles in cancer development and single nucleotide polymorphisms (SNP) in some MMP genes were shown to confer susceptibility to certain cancers. This study examined the association between genotypes and haplotypes in the MMP1-MMP3-MMP12 gene cluster and risk of lung cancer development and metastasis.

Experimental Design: A two-stage investigation was conducted. First, 35 SNPs covering these genes were selected and validated in 190 patients and 190 controls. Twenty-two validated SNPs were then analyzed in an entire case-control panel consisting of 711 patients and 716 controls. Associations with the risk of lung cancer were estimated by logistic regression.

Results: The investigated MMP gene region could be partitioned into two major haplotype blocks. One common haplotype in the block composed of major part of MMP1 transcription region was significantly associated with increased risk for the development [odds ratio (OR), 1.35; 95% confidence interval (95% CI), 1.11-1.63; P = 0.01; permutated P = 0.134] and distant metastasis of lung cancer (ORs for stage IV versus stages I-III, 1.67; 95% CI, 1.12-2.50; P = 0.009; permutated P = 0.048) and the other showed a protective effect against metastasis (ORs for stage IV versus stages I-III, 0.22; 95% CI, 0.07-0.62; P = 0.001; permutated P = 0.011). Another common haplotype in the block across MMP3 was significantly associated with decreased risk for developing lung cancer (OR, 0.71; 95% CI, 0.59-0.86; P = 0.003; permutated P = 0.027).

Conclusions: The observed multiple cancer-associated genetic variants suggested that the MMP1-MMP3-MMP12 gene cluster plays important roles in lung cancer development and progression.
Regulation of Angiogenesis by Id-1 through Hypoxia-Inducible Factor-1α–Mediated Vascular Endothelial Growth Factor Up-regulation in Hepatocellular Carcinoma
Authors: Lee Terence K. , Poon Ronnie T.P. , Yuen Anthony P. , Ling Ming Tat , Wang Xiang Hong , Wong Yong Chuan , Guan Xin Yuan , Man Kwan , Tang Zao You , Fan Sheung Tat
Published: December 2006

Purpose: Metastasis is commonly associated with poor prognosis of hepatocellular carcinoma (HCC). Being an important angiogenic factor, vascular endothelial growth factor (VEGF) plays a central role in HCC growth and metastasis. Recently, Id-1 (inhibitor of differentiation/DNA synthesis) has been suggested to be a key factor in cancer progression but the molecular mechanism remains unknown.

Experimental Design: We first showed that overexpression of Id-1 was correlated with HCC metastasis (P < 0.001) and its expression was significantly correlated with VEGF expression by tissue microarray. By ectopic transfection of Id-1 into HCC cells, Id-1 was able to induce VEGF secretion through activation of VEGF transcription.

Results: Increased VEGF secretion in Id-1 transfectants induced morphologic change and proliferation of human umbilical vascular endothelial cell resulting in promotion of angiogenesis. Id-1 induced transcriptional activation of VEGF by stabilizing hypoxia-inducible factor-1α protein. Down-regulation of Id-1 by antisense approach led to suppression of hypoxia-inducible factor-1α–mediated VEGF production. In addition, Id-1 suppression resulted in retardation of cell invasion through down-regulation of VEGF.

Conclusions: Id-1 is a novel angiogenic factor for HCC metastasis and down-regulation of Id-1 may be a novel target to inhibit HCC metastasis through suppression of angiogenesis.
ABCC2 (MRP2, cMOAT) Can Be Localized in the Nuclear Membrane of Ovarian Carcinomas and Correlates with Resistance to Cisplatin and Clinical Outcome
Authors: Surowiak Pawel , Materna Verena , Kaplenko Irina , Spaczynski Marek , Dolinska-Krajewska Barbara , Gebarowska Elzbieta , Dietel Manfred , Zabel Maciej , Lage Hermann
Published: December 2006

Purpose: Cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma. ABCC2 is commonly localized in apical cell membranes and could confer cisplatin resistance. Here, we show that ABCC2 can be localized in the cytoplasmic membrane as well as in the nuclear membrane of various human tissues including ovarian carcinoma cells.

Experimental Design: For the subcellular detection of ABCC2, immunohistochemistry was done using 41 Federation Internationale des Gynaecologistes et Obstetristes stage III ovarian carcinoma specimens prepared before treatment with cisplatin-based schemes and 35 specimens from the same group after chemotherapy. Furthermore, 11 ovarian carcinoma cell lines as well as tissue microarrays consisting of various human tissues were analyzed.

Results: Nuclear membranous localization of ABCC2 was associated with response to first-line chemotherapy at primary (P = 0.0013) and secondary surgery (P = 0.0060). Cases with relapse showed higher nuclear membrane expression at primary (P = 0.0003) and secondary surgery (P = 0.0024). Kaplan-Meier analyses showed that weak nuclear membrane ABCC2 expression before treatment was associated with significantly longer overall (P = 0.04) and progression-free survival (P = 0.001); following chemotherapy, it correlated with significantly longer progression-free survival (P = 0.038). Tissue microarrays confirmed nuclear membranous localization of ABCC2, in particular, in poorly differentiated cells. In ovarian carcinoma cells, it correlated with resistance against cisplatin, whereas localization in the cytoplasmic membrane did not.

Conclusions: ABCC2 confers resistance to cisplatin of ovarian carcinoma in cell culture systems and in clinics when expressed in the nuclear membrane. Thus, ABCC2 localization can predict platinum therapy outcome. Furthermore, expression of ABCC2 in nuclear membranes in human tissues is specific for poorly differentiated cells including stem cells.
Joint Effect of Mutagen Sensitivity and Insulin-Like Growth Factors in Predicting the Risk of Developing Secondary Primary Tumors and Tumor Recurrence in Patients with Head and Neck Cancer
Authors: Wu Xifeng , Gu Jian , Dong Qiong , Huang Maosheng , Do Kim-Anh , Hong Waun Ki , Spitz Margaret R.
Published: December 2006

Purpose: Early-stage head and neck cancer patients are at high risks for tumor recurrence and secondary primary tumor (SPT) development. We hypothesized that latent genetic instability and proliferation potential may be associated with elevated risks of SPT and recurrence.

Experimental Design: We conducted a nested case-control study within a randomized, placebo-controlled chemoprevention trial in patients with early-stage head and neck cancer. We compared prediagnostic bleomycin-induced chromatid breaks in peripheral blood lymphocyte cultures (as an indicator of latent genetic instability) between 298 cases (patients with SPT/recurrence) and 693 controls (patients without SPT/recurrence). We also determined the joint effects of latent genetic instability and elevated proliferation potential [indicated by serum insulin-like growth factor (IGF) level] in modulating the risk of SPT and recurrence.

Results: In the Cox proportional hazards model, patients with higher mutagen sensitivity (using a cutoff of ≥0.50 breaks per cell) exhibited a significantly increased risk of developing SPT/recurrence [hazard ratio, 1.38; 95% confidence interval (95% CI), 1.02-1.86]. Cases also exhibited significantly higher levels of IGF-I and IGF-binding protein-3 than controls (P = 0.022 and 0.042, respectively). Moreover, there were joint effects between mutagen sensitivity and IGFs in modulating SPT/recurrence risk. Using patients with low IGF-I level and low mutagen sensitivity profile as the reference group, the odds ratios of developing SPT/recurrence for patients with high IGF-I level alone, high mutagen sensitivity alone, and both high IGF-I level and high mutagen sensitivity were 2.85 (95% CI, 0.92-8.82), 3.92 (95% CI, 1.28-11.97), and 6.16 (95% CI, 2.03-18.71), respectively. A similar joint effect was observed for mutagen sensitivity and IGF-binding protein-3 level.

Conclusions: This is the largest prospective study to evaluate mutagen sensitivity as a prognosis marker in head and neck cancer because mutagen sensitivity data were derived from baseline samples drawn before the development of SPT or tumor recurrence. The results also show for the first time that latent genetic instability and elevated proliferation potential jointly elevate the risk of second tumors in early-stage head and neck cancers.
Monitoring of Singlet Oxygen Is Useful for Predicting the Photodynamic Effects in the Treatment for Experimental Glioma
Authors: Yamamoto Junkoh , Yamamoto Seiji , Hirano Toru , Li Shaoyi , Koide Masayo , Kohno Eiji , Okada Mitsuo , Inenaga Chikanori , Tokuyama Tsutomu , Yokota Naoki , Terakawa Susumu , Namba Hiroki
Published: December 2006

Purpose: Singlet oxygen (1O2) generated in photodynamic therapy (PDT) plays a very important role in killing tumor cells. Using a new near-IR photomultiplier tube system, we monitored the real-time production of 1O2 during PDT and thus investigated the relationship between the 1O2 production and photodynamic effects.

Experimental Design: We did PDT in 9L gliosarcoma cells in vitro and in an experimental tumor model in vivo using 5-aminolevulinic acid and nanosecond-pulsed dye laser. During this time, we monitored 1O2 using this system. Moreover, based on the 1O2 monitoring, we set the different conditions of laser exposure and investigated whether they could affect the tumor cell death.

Results: We could observe the temporal changes of 1O2 production during PDT in detail. At a low fluence rate the 1O2 signal gradually decreased with a low peak, whereas at a high fluence rate it decreased immediately with a high peak. Consequently, the cumulative 1O2 at a low fluence rate was higher, which thus induced a strong photodynamic effect. The proportion of apoptosis to necrosis might therefore be dependent on the peak and duration of the 1O2 signal. A low fluence rate tended to induce apoptotic change, whereas a high fluence rate tended to induce necrotic change.

Conclusions: The results of this study suggested that the monitoring of 1O2 enables us to predict the photodynamic effect, allowing us to select the optimal laser conditions for each patient.
Implication of Polycomb Members Bmi-1, Mel-18, and Hpc-2 in the Regulation of p16INK4a, p14ARF, h-TERT, and c-Myc Expression in Primary Breast Carcinomas
Authors: Silva Javier , García José M. , Peña Cristina , García Vanesa , Domínguez Gemma , Suárez Dolores , Camacho Francisca I. , Espinosa Ruth , Provencio Mariano , España Pilar , Bonilla Félix
Published: December 2006

Purpose: Deregulation of mammalian Polycomb group (PcG) members may contribute to human carcinogenesis. p16INK4a and p14ARF tumor suppressors, human telomerase reverse transcriptase (h-TERT), and oncoprotein c-Myc have been implicated in the regulation of the cell cycle and proliferation mediated by PcG proteins, mainly Bmi-1, in mice and in cell culture experiments. Here, we examine whether these in vitro findings can be extrapolated to the in vivo situation.

Experimental Design: We measure the expression of PcG members Bmi-1, Mel-18, and Hpc-2 and their potential targets by reverse transcription-PCR, immunostaining, and Western blotting in a series of 134 breast carcinomas and correlate the data with several clinical-pathologic variables of the tumors.

Results: Expression of PcG genes was variably detected, but overexpression of Bmi-1 was the most frequent PcG alteration observed. In addition, statistical direct correlation in expression level of the three PcG members was detected. A correlation between c-Myc and Bmi-1 expression levels was observed; however, there was no correlation between expression of Bmi-1 and p16INK4a, p14ARF, or h-TERT. However, expression of the other PcG members Mel-18 and Hpc-2 correlated with the cell cycle regulators. Moreover, PcG mRNA–altered expression correlated significantly with certain clinical-pathologic variables associated with poor prognosis.

Conclusions: Our data suggest that the oncogenic role of Bmi-1 in human primary breast carcinomas is not determined by its capacity to inhibit INK4a/ARF proteins or to induce telomerase activity.
Complete Antitumor Protection by Perioperative Immunization with GM3/VSSP Vaccine in a Preclinical Mouse Melanoma Model
Authors: Gabri Mariano R. , Mazorra Zaima , Ripoll Giselle V. , Mesa Circe , Fernandez Luis E. , Gomez Daniel E. , Alonso Daniel F.
Published: December 2006

Purpose: The GM3/VSSP vaccine is composed of very small sized proteoliposomes resulting from the hydrophobic conjugation of GM3 ganglioside with membrane proteins from Neisseria meningitidis. Previously, we showed that preventive vaccination with GM3/VSSP induces a specific antitumor response and elicits the rejection of syngeneic GM3-positive melanoma cells in immunized mice. Our aim was to explore the antitumor properties of perioperative GM3/VSSP vaccination in a preclinical mouse model.

Experimental Design: The highly metastatic B16F10 mouse melanoma was used to investigate perioperative vaccination with GM3/VSSP. The vaccine was administered i.m. in doses of 120 μg emulsified with the adjuvant Montanide ISA 51 at weekly or biweekly intervals, and s.c. tumors were excised 25 to 31 days after tumor cell implantation. The persistence of antitumor protection and dose dependency was also examined in preimmunized animals. To evaluate the immune performance of tumor-bearing and tumor-operated mice, ovoalbumin-specific delayed-type hypersensitivity, cytokine secretion, and cell proliferation responses were studied.

Results: Surgical excision of B16F10 tumors improved survival, and perioperative immunization with four biweekly GM3/VSSP doses yielded survival for all animals (P = 0.04; log-rank test). Mice showed neither local recurrence nor lung metastasis at the end of the experiment. An impairment of CD4+ T-cell responses was observed in tumor-bearing animals measured as neoantigen-specific delayed-type hypersensitivity, with a significant recovery after surgery. A strong interleukin-4 secretion was induced in B16F10-operated mice, whereas IFN-γ remained unaffected.

Conclusion: Preclinical evidence suggests that GM3/VSSP vaccine might have therapeutic potential to induce antitumor immunity in patients with minimal residual disease after surgery, thereby preventing or prolonging the time to recurrence.
In vivo Loss of Expression of Argininosuccinate Synthetase in Malignant Pleural Mesothelioma Is a Biomarker for Susceptibility to Arginine Depletion
Authors: Szlosarek Peter W. , Klabatsa Astero , Pallaska Arben , Sheaff Michael , Smith Paul , Crook Tim , Grimshaw Matthew J. , Steele Jeremy P. , Rudd Robin M. , Balkwill Frances R. , Fennell Dean A.
Published: December 2006

Purpose: Malignant pleural mesothelioma (MPM) is an increasing health burden on many societies worldwide and, being generally resistant to conventional treatment, has a poor prognosis with a median survival of <1 year. Novel therapies based on the biology of this tumor seek to activate a proapoptotic cellular pathway. In this study, we investigated the expression and biological significance of argininosuccinate synthetase (AS), a rate-limiting enzyme in arginine production.

Experimental Design: Initially, we documented down-regulation of AS mRNA in three of seven MPM cell lines and absence of AS protein in four of seven MPM cell lines. We confirmed that the 9q34 locus, the site of the AS gene, was intact using a 1-Mb comparative genomic hybridization array; however, there was aberrant promoter CpG methylation in cell lines lacking AS expression, consistent with epigenetic regulation of transcription. To investigate the use of AS negativity as a therapeutic target, arginine was removed from the culture medium of the MPM cell lines.

Results: In keeping with the cell line data, 63% (52 of 82) of patients had tumors displaying reduced or absent AS protein, as assessed using a tissue microarray. Cell viability declined markedly in the AS-negative cell lines 2591 and MSTO but not in the AS-positive cell line, 28. This response was apparent by day 4 and maintained by day 9 in vitro. Arginine depletion induced BAX conformation change and mitochondrial inner membrane depolarization selectively in AS-negative MPM cells.

Conclusions: In summary, we have identified AS negativity as a frequent event in MPM in vivo, leading to susceptibility to cytotoxicity following restriction of arginine. A phase II clinical trial is planned to evaluate the role of arginine depletion in patients with AS-negative MPM.
Dasatinib (BMS-354825) Pharmacokinetics and Pharmacodynamic Biomarkers in Animal Models Predict Optimal Clinical Exposure
Authors: Luo Feng R. , Yang Zheng , Camuso Amy , Smykla Richard , McGlinchey Kelly , Fager Krista , Flefleh Christine , Castaneda Stephen , Inigo Ivan , Kan David , Wen Mei-Li , Kramer Robert , Blackwood-Chirchir Anne , Lee Francis Y.
Published: December 2006

Purpose: Chronic myeloid leukemia (CML) is caused by reciprocal translocation between chromosomes 9 and 22, forming BCR-ABL, a constitutively activated tyrosine kinase. Imatinib mesylate, a selective inhibitor of BCR-ABL, represents current frontline therapy for CML; however, emerging evidence suggests that drug resistance to imatinib may limit its long-term success. To improve treatment options, dasatinib (BMS-354825) was developed as a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC family kinases. To date, dasatinib has shown promising anti-leukemic activity in preclinical models of CML and in phase I/II clinical studies in patients with imatinib-resistant or imatinib-intolerant disease.

Experimental Design: The pharmacokinetic and pharmacodynamic biomarkers of dasatinib were investigated in K562 human CML xenografts grown s.c. in severe combined immunodeficient mice. Tumoral levels of phospho-BCR-ABL/phospho-CrkL were determined by Western blot.

Results: Following a single oral administration of dasatinib at a preclinical efficacious dose of 1.25 or 2.5 mg/kg, tumoral phospho-BCR-ABL/phospho-CrkL were maximally inhibited at ∼3 hours and recovered to basal levels by 24 hours. The time course and extent of the inhibition correlated with the plasma levels of dasatinib in mice. Pharmacokinetic/biomarker modeling predicted that the plasma concentration of dasatinib required to inhibit 90% of phospho-BCR-ABL in vivo was 10.9 ng/mL in mice and 14.6 ng/mL in humans, which is within the range of concentrations achieved in CML patients who responded to dasatinib treatment in the clinic.

Conclusions: Phospho-BCR-ABL/phospho-CrkL are likely to be useful clinical biomarkers for the assessment of BCR-ABL kinase inhibition by dasatinib.
Wnt Antagonist Family Genes as Biomarkers for Diagnosis, Staging, and Prognosis of Renal Cell Carcinoma Using Tumor and Serum DNA
Authors: Urakami Shinji , Shiina Hiroaki , Enokida Hideki , Hirata Hiroshi , Kawamoto Ken , Kawakami Toshifumi , Kikuno Nobuyuki , Tanaka Yuichiro , Majid Shahana , Nakagawa Masayuki , Igawa Mikio , Dahiya Rajvir
Published: December 2006

Purpose: We hypothesized that combined methylation analysis of Wnt antagonist genes could serve as a panel of biomarkers for diagnosis, staging, and prognosis in renal cell carcinoma (RCC).

Experimental Design: Samples (n = 62) of RCC and corresponding normal renal tissue (NRT) were analyzed using methylation-specific PCR for methylation of six Wnt antagonist genes (sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1, and Dkk-3). To increase the sensitivity/specificity of RCC detection, the methylation score (M score) for multigene methylation analysis was developed. Receiver operator characteristic curve analysis was used to determine the optimal sensitivity/specificity of the M score. In addition, the M score was compared with the clinicopathologic outcome. Thirty-three serum DNA samples were also used to investigate the methylation status of Wnt antagonist genes.

Results: The methylation levels of all Wnt antagonists were significantly higher in RCC than in NRT. In multivariate regression analysis, the methylation level of sFRP-1 was a significant independent predictor of RCC, whereas for sFRP-2 and sFRP-4 there was a trend toward significance as independent predictors. The M score of Wnt antagonist genes was significantly higher in RCC than in NRT. Overall, the M score had a sensitivity of 79.0% and a specificity of 75.8% (area under the curve, 0.808) as a diagnostic biomarker. In addition, the M score could significantly distinguish grade, pT category, M category, and overall survival of RCC patients. The M score was independent of age and gender in predicting overall survival by the Cox proportional hazards model. In RCC patients, 72.7% of the methylation-specific PCR results had identical methylation in samples of tumor and serum DNA. No serum DNA in normal controls showed aberrant methylation of the Wnt antagonist genes. In addition, the methylation status of Wnt antagonist genes in serum DNA was significantly correlated with tumor grade and stage.

Conclusions: This is the first report showing that M score analysis of Wnt antagonist genes can serve as an excellent epigenetic biomarker panel for detection, staging, and prognosis of RCC using serum DNA.
Detection of Human Papillomavirus and Relevant Tumor Suppressors and Oncoproteins in Laryngeal Tumors
Authors: Manjarrez María E. , Ocadiz Rodolfo , Valle Leticia , Pacheco Cesar , Marroquin Alberto , De la Torre Carlos , Selman Moisés , Gariglio Patricio
Published: December 2006

Purpose: The mechanism of larynx oncogenesis is complex and controlled by various factors, most of them involved in cell proliferation and apoptosis. In this study, we evaluated the levels of two suppressor proteins (pRb and p53) and two oncogenic proteins (c-Myc and Bcl-2), as well as the apoptotic levels and the presence of human papillomavirus (HPV) in both tumor types.

Experimental Design: Low- or high-risk HPV viral DNA was determined by PCR and in situ PCR; the level of cellular proteins was examined by immunohistochemistry; the presence of apoptotic cells was evaluated by in situ cell death detection.

Results: Most laryngeal papillomatosis samples contained low-risk HPV determined by both techniques. However, 25% of laryngeal carcinoma samples were positive for HPV employing PCR or in situ PCR. In papillomatosis, pRb and p53 levels were higher than in normal larynxes, whereas laryngeal cancer presented the lowest levels. c-Myc oncogene expression was very low in normal and cancer tissues but highly increased in papillomatosis. Bcl-2 expression was low and showed no significant difference between laryngeal papillomatosis and normal larynxes. By contrast, Bcl-2 was clearly up-regulated in cancer. Normal larynx samples and those from laryngeal papillomatosis exhibited similar relatively high numbers of apoptotic cells, whereas in malignant tumors, these cells were scarce.

Conclusion: Our results suggest that HPV is an important risk factor in papillomatosis and in some malignant larynx tumors with a strong participation of cellular genes, specifically involved in proliferation and apoptosis. In benign papillomatosis lesions but not in larynx cancer, high p53 activity might preserve the apoptosis process. In larynx cancer, low p53 levels and high bcl-2 expression may be playing an important role to block apoptosis.
Gene Expression Profiling Separates Chromophobe Renal Cell Carcinoma from Oncocytoma and Identifies Vesicular Transport and Cell Junction Proteins as Differentially Expressed Genes
Authors: Rohan Stephen , Tu Jiangling J. , Kao Jean , Mukherjee Piali , Campagne Fabien , Zhou Xi K. , Hyjek Elizabeth , Alonso Miguel A. , Chen Yao-Tseng
Published: December 2006

Purpose: To compare gene expression profiles of chromophobe renal cell carcinoma (RCC) and benign oncocytoma, aiming at identifying differentially expressed genes.

Experimental Design: Nine cases each of chromophobe RCC and oncocytoma were analyzed by oligonucleotide microarray. Candidate genes that showed consistent differential expression were validated by reverse transcription-PCR using 25 fresh-frozen and 15 formalin-fixed, paraffin-embedded tumor samples. Immunohistochemical analysis was also done for two selected gene products, claudin 8 and MAL2.

Results: Unsupervised hierarchical clustering separated the chromophobe RCC and oncocytoma into two distinct groups. By a combination of data analysis approaches, we identified 11 candidate genes showing consistent differential expression between chromophobe RCC and oncocytoma. Five of these genes, AP1M2, MAL2, PROM2, PRSS8, and FLJ20171, were shown to effectively separate these two tumor groups by quantitative reverse transcription-PCR using fresh tissue samples, with similar trends seen on formalin-fixed tissues. Immunohistochemical analysis revealed selective expression of MAL2 and claudin 8 in distal renal tubules, with MAL2 antibody showing differential expression between chromophobe RCC and oncocytoma. Functional analyses suggest that genes encoding tight junction proteins and vesicular membrane trafficking proteins, normally expressed in distal nephrons, are retained in chromophobe RCC and lost or consistently down-regulated in oncocytoma, indicating that these two tumor types, believed to be both derived from distal tubules, are likely distinctive in their histogenesis.

Conclusions: We showed that chromophobe RCC and oncocytoma are distinguishable by mRNA expression profiles and a panel of gene products potentially useful as diagnostic markers were identified.
Heterogeneous CD52 Expression among Hematologic Neoplasms: Implications for the Use of Alemtuzumab (CAMPATH-1H)
Authors: Rodig Scott J. , Abramson Jeremy S. , Pinkus Geraldine S. , Treon Steven P. , Dorfman David M. , Dong Henry Y. , Shipp Margaret A. , Kutok Jeffery L.
Published: December 2006

Purpose: CD52 is a GPI-linked glycoprotein expressed by B cells, T cells, monocytes, and macrophages. The humanized monoclonal antibody alemtuzumab (CAMPATH-1H) is specific for CD52 and is Food and Drug Administration–approved for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL). The utility of CAMPATH in the treatment of other hematologic neoplasms has been explored; however, a comprehensive survey of CD52 expression among a broad spectrum of WHO-defined tumor types has not been completed.

Experimental Design: We evaluated 294 hematologic neoplasms for the presence of CD52 using standard immunohistochemical techniques on paraffin-embedded biopsy specimens fixed with formalin, B-Plus, Zenker's acetic acid, or B5-formalin.

Results: The vast majority of low-grade B cell lymphoproliferative disorders (CLL/small lymphocytic leukemia, follicular lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and mucosa-associated lymphoid tissue lymphomas) express CD52. In addition, we found that the majority of precursor B cell acute lymphoblastic leukemia/lymphomas express this antigen. In contrast, there is surprising heterogeneity in CD52 expression among more aggressive B cell lymphomas, with 25% of cases of diffuse large B cell lymphoma and Burkitt lymphoma demonstrating no detectable CD52. In addition, the majority of neoplasms of the T cell lineage are negative for the antigen, including most cases of precursor T cell acute lymphoblastic leukemia/lymphoma, anaplastic large cell lymphoma, and peripheral T cell lymphoma, not otherwise specified. Finally, the vast majority of cases of acute myeloid leukemia, Hodgkin lymphoma, and multiple myeloma are negative for CD52 expression.

Conclusion: In contrast with CLL, the variable expression of CD52 among other hematologic malignancies suggests that target validation on a case-by-case basis will likely be necessary to guide the rational analysis of CAMPATH therapy.