bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA Methylation in Serum and Tumors of Cervical Cancer Patients
Authors: Widschwendter Andreas , Müller Hannes M. , Fiegl Heidi , Ivarsson Lennart , Wiedemair Annemarie , Müller-Holzner Elisabeth , Goebel Georg , Marth Christian , Widschwendter Martin
Published: September 2005

Purpose: Promoter hypermethylation has been recognized to play an important role in carcinogenesis. Numerous studies have demonstrated tumor-specific alterations, such as aberrant promoter hypermethylation, in DNA recovered from plasma or serum of patients with various malignancies. The aim of this study was to investigate the methylation status of various genes in cervical cancer patients and their association with clinicopathological characteristics and outcome of the disease.

Experimental Design: The methylation status of CALCA, hTERT, MYOD1, PGR (progesterone receptor), and TIMP3 was investigated in serum samples from 93 cervical cancer patients and 19 corresponding tissue samples using the MethyLight technique.

Results: Aberrant promoter hypermethylation was detected in any of these genes in 87% (81 of 93) of the serum samples studied. Methylation of MYOD1 was detected more frequently in advanced stage. All of the genes found to be methylated in serum samples were also methylated in the corresponding tissue sample, except in one patient. Patients with unmethylated MYOD1 serum DNA had significantly better disease-free (P = 0.04) and overall survival (P = 0.02) in comparison with patients with methylated MYOD1.

Conclusions: To the best of our knowledge, this is, thus far, the largest study investigating aberrant promoter hypermethylation in serum samples from cancer patients and the first study investigating methylation patterns in sera of cervical cancer patients. Our results suggest that serological detection of MYOD1 promoter hypermethylation may be of potential use as a prognostic marker for discriminating cervical cancer patients at high risk for lymph node metastasis or relapse. Additional studies, including a panel of additional genes, are necessary to elucidate the role of aberrant methylation in serum as a tool for surveillance of cervical cancer.
In Vivo
Assessment of Antiangiogenic Activity of SU6668 in an Experimental Colon Carcinoma Model
Authors: Marzola Pasquina , Degrassi Anna , Calderan Laura , Farace Paolo , Crescimanno Caterina , Nicolato Elena , Giusti Anna , Pesenti Enrico , Terron Andrea , Sbarbati Andrea , Abrams Tinya , Murray Lesley , Osculati Francesco
Published: September 2005

Purpose: The purpose of this research was to assess in vivo by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) the antiangiogenic effect of SU6668, an oral, small molecule inhibitor of the angiogenic receptor tyrosine kinases vascular endothelial growth factor receptor 2 (Flk-1/KDR), platelet-derived growth factor receptor, and fibroblast growth factor receptor 1.

Experimental Design: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. DCE-MRI with a macromolecular contrast agent was used to measure transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. CD31 immunohistochemical staining was used for assessing microvessels density and vessels area. Experiments were performed after 24 h, and 3, 7, and 14 days of treatment.

Results: DCE-MRI clearly detected the early effect (after 24 h of treatment) of SU6668 on tumor vasculature as a 51% and 26% decrease in the average vessel permeability measured in the tumor rim and core (respectively). A substantial decrease was also observed in average fractional plasma volume in the rim (59%) and core (35%) of the tumor. Histological results confirmed magnetic resonance imaging findings. After 3, 7, and 14 days of treatment, postcontrast magnetic resonant images presented a thin strip of strongly enhanced tissue at the tumor periphery; histology examination showed that this hyperenhanced ring corresponded to strongly vascularized tissue adjacent but external to the tumor. Histology also revealed a strong decrease in the thickness of peripheral viable tissue, with a greatly reduced vessel count. SU6668 greatly inhibited tumor growth, with 60% inhibition at 14 days of treatment.

Conclusions: DCE-MRI detected in vivo the antiangiogenic efficacy of SU6668.
VX-710 (Biricodar) Increases Drug Retention and Enhances Chemosensitivity in Resistant Cells Overexpressing P-Glycoprotein, Multidrug Resistance Protein, and Breast Cancer Resistance Protein
Authors: Minderman Hans , O’Loughlin Kieran L. , Pendyala Lakshmi , Baer Maria R.
Published: September 2005

Purpose: The pipecolinate derivative VX-710 (biricodar; Incel) is a clinically applicable modulator of P-glycoprotein (Pgp) and multidrug resistance protein (MRP-1); we studied its activity against the third multidrug resistance (MDR)-associated drug efflux protein, breast cancer resistance protein (BCRP).

Experimental Design: VX-710 modulation of uptake, retention, and cytotoxicity of mitoxantrone, daunorubicin, doxorubicin, topotecan, and SN38 was studied in cell lines overexpressing Pgp, MRP-1 and wild-type (BCRPR482) and mutant (BCRPR482T) BCRP.

Results: In 8226/Dox6 cells (Pgp), VX-710 increased mitoxantrone and daunorubicin uptake by 55 and 100%, respectively, increased their retention by 100 and 60%, respectively, and increased their cytotoxicity 3.1- and 6.9-fold, respectively. In HL60/Adr cells (MRP-1), VX-710 increased mitoxantrone and daunorubicin uptake by 43 and 130%, increased their retention by 90 and 60%, and increased their cytotoxicity 2.4- and 3.3-fold. In 8226/MR20 cells (BCRPR482), VX-710 increased mitoxantrone uptake and retention by 60 and 40%, respectively, and increased cytotoxicity 2.4-fold. VX-710 increased daunorubicin uptake and retention by only 10% in 8226/MR20 cells, consistent with the fact that daunorubicin is not a substrate for BCRPR482, but, nevertheless, it increased daunorubicin cytotoxicity 3.6-fold, and this increase was not associated with intracellular drug redistribution. VX-710 had little effect on uptake, retention, or cytotoxicity of mitoxantrone, daunorubicin, doxorubicin, topotecan, or SN38 in MCF7 AdVP3000 cells (BCRPR482T).

Conclusions: VX-710 modulates Pgp, MRP-1, and BCRPR482, and has potential as a clinical broad-spectrum MDR modulator in malignancies such as the acute leukemias in which these proteins are expressed.
Epigenetic Inactivation of
TMS1/ASC
in Ovarian Cancer
Authors: Terasawa Katsuhiko , Sagae Satoru , Toyota Minoru , Tsukada Kuniko , Ogi Kazuhiro , Satoh Ayumi , Mita Hiroaki , Imai Kohzoh , Tokino Takashi , Kudo Ryuichi
Published: September 2005

Purpose: The purpose of this work was to explore the role of epigenetic inactivation of apoptotic pathways in ovarian cancer by examining the DNA methylation and expression status of four proapoptotic genes in primary ovarian cancers and cancer cell lines and to correlate those findings with the clinicopathological features of ovarian cancer patients.

Experimental Design: Genomic DNA was isolated from 15 ovarian cancer cell lines, 80 primary ovarian cancer specimens, and 4 normal ovary specimens using phenol-chloroform extraction. The methylation status of the DNA was evaluated using combined bisulfite restriction analysis, gene expression was evaluated using reverse transcription-PCR, and histone acetylation was evaluated using chromatin immunoprecipitation.

Results: Of the four proapoptotic genes studied, expression of TMS1/ASC was absent in six ovarian cancer cell lines. Dense methylation of the 5′ region of TMS1/ASC was detected in cells not expressing TMS1/ASC. Treating methylated cells with 5-aza-deoxycytidine restored gene expression, confirming the role of methylation in silencing the gene. Chromatin immunoprecipitation revealed histone to be deacetylated in cells not expressing TMS1/ASC, indicating that histone deacetylation is also involved in silencing TMS1/ASC. Aberrant methylation of TMS1/ASC was detected in 15 of 80 ovarian cancer tissues (19%) but in none of the normal ovary specimens. Aberrant methylation of TMS1/ASC was observed significantly more often in clear cell-type ovarian cancers than in other tumor types (P < 0.0001).

Conclusions: Methylation-mediated silencing of TMS1/ASC confers a survival advantage to tumor cells by enabling them to escape apoptosis. The role for aberrant methylation in human ovarian tumorigenesis may be particularly important for ovarian cancers with the clear cell phenotype.
Phase I Clinical Trial of the Farnesyltransferase Inhibitor BMS-214662 Given as a 1-Hour Intravenous Infusion in Patients with Advanced Solid Tumors
Authors: Ryan David P. , Eder Joseph P. , Puchlaski Thomas , Seiden Michael V. , Lynch Thomas J. , Fuchs Charles S. , Amrein Philip C. , Sonnichsen Darrell , Supko Jeffrey G. , Clark Jeffrey W.
Published: September 2005

Purpose: BMS-214662 is a nonsedating benzodiazepine derivative that exhibits broad spectrum cytotoxicity against human solid tumor cell lines and potently inhibits farnesylation of the H-ras and K-ras oncogenic proteins. This report describes the initial Phase I clinical trial of the compound. The main objective of the study was to determine the dose-limiting toxicities and the maximum tolerated dose of BMS-214662 when administered as a single dose i.v. over 1 h every 21 days to patients with advanced solid tumors.

Experimental Design: Patients with advanced solid tumors and adequate organ function were eligible for the study. The dose was escalated according to a modified Fibonacci schedule after evaluating groups of at least three patients for toxicity during the first cycle of therapy at each dose level. Pharmacokinetic and pharmacodynamic studies were performed after administration of the two initial doses.

Results: The dose of BMS-214662 was escalated from 36 to 225 mg/m2 through 5 intermediate dose levels in a total of 44 patients. Dose-limiting toxicities occurred in 3 of the 13 (23%) patients during the first cycle of treatment with 225 mg/m2, consisting of grade 3 nausea/vomiting in 2 patients and grade 3 diarrhea in another patient. In addition, four of these patients experienced reversible grade 3 transaminitis, which was not considered to be dose-limiting. At the recommended dose for Phase II studies, 200 mg/m2, the most common side effects were reversible transaminitis, nausea, and vomiting. Although there were no objective responses, one patient with pancreatic cancer continues to receive treatment more than 3.5 years after entering the study. BMS-214662 exhibited linear pharmacokinetics and had a mean biological half-life of 1.55 ± 0.27 h and a total body clearance of 21.8 ± 10.8 liters/h/m2, with a low apparent volume of distribution at steady state (31.5 ± 12.9 liters/m2). In patients treated with the recommended Phase II dose, the mean maximum plasma concentration of the drug was 6.57 ± 2.94 μg/ml, and farnesyltransferase activity in peripheral blood mononuclear cells decreased to a nadir of 10.5 ± 6.4% of baseline at the end of the infusion but fully recovered within 24 h.

Conclusions: BMS-214662 can be delivered safely as a single 1-h i.v. infusion at a dose that results in pronounced inhibition of farnesyltransferase activity in peripheral blood mononuclear cells. However, the duration of enzyme inhibition was transient, recovering in parallel with the decline in plasma concentrations of this rapidly eliminated drug. Because indications of anticancer activity were observed in several patients, further optimization of the administration schedule for this promising new compound is warranted.
Phase I and Pharmacokinetic Study of Prinomastat, a Matrix Metalloprotease Inhibitor
Authors: Hande Kenneth R. , Collier Mary , Paradiso Linda , Stuart-Smith Jill , Dixon Mary , Clendeninn Neil , Yeun Geoff , Alberti Donna , Binger Kim , Wilding George
Published: September 2005

Purpose: Prinomastat is a matrix metalloprotease (MMP) inhibitor with selectivity for MMPs 2, 3, 9, 13, and 14. Inhibition of these MMPs has been postulated to block tumor invasion and metastasis. This Phase I, dose-escalation study was designed to evaluate the acute and chronic toxicities of various doses of prinomastat and to determine prinomastat pharmacokinetics.

Experimental Design: Seventy-five patients with advanced cancer were given 1, 2, 5, 10, 25, 50, or 100 mg prinomastat orally twice daily until tumor progression or development of significant toxicities. Prinomastat pharmacokinetics were measured on day 29 of therapy.

Results: The primary toxicities identified were joint and muscle-related pain, which were generally reversible with treatment rest and/or dose reduction. No dose-limiting toxicities were noted within the first 4 weeks of treatment, but grade 2–3 arthralgias and myalgias were noted 2–3 months after initiation of therapy in >25% of patients at doses >25 mg twice a day. The frequency and severity of symptoms were dose related. Plasma prinomastat concentrations greater than the Ki for MMPs 2 and 9 were achieved at all of the dose levels.

Conclusions: Doses of 5–10 mg bid were recommended for additional trials, because this dose range was well tolerated for a treatment duration of at least 3 months and achieves trough plasma concentrations 10–100-fold greater than the Ki (in vitro inhibition constant) for the targeted MMPs (2 and 9).
A Human Cytotoxic T-Lymphocyte Epitope and Its Agonist Epitope from the Nonvariable Number of Tandem Repeat Sequence of MUC-1
Authors: Tsang Kwong-Yok , Palena Claudia , Gulley James , Arlen Philip , Schlom Jeffrey
Published: September 2005

Purpose: MUC-1/DF-3 remains an attractive target for vaccine therapy. It is overexpressed in the majority of human carcinomas and multiple myeloma. Clinical trials using MUC-1-based vaccines have demonstrated safety, clinical responses, and the induction of T-cell responses directed against MUC-1. Previous studies in experimental models and in clinical trials have demonstrated that altering the amino acid sequence of a “self” epitope can lead to the generation of an enhancer agonist epitope capable of eliciting stronger T-cell responses than the native epitope can.

Experimental Design and Results: We describe here the identification of six novel class I HLA-A2 epitopes of MUC-1 that reside outside of the variable number of tandem repeat region. Each is shown to have the ability to activate human T cells as measured by IFN-γ production. One epitope (ATWGQDVTSV, at amino acid position 92–101 and designated P-92), which demonstrated the highest level of binding to HLA-A2 and which induced the highest level of IFN-γ in human T cells, was further studied for the generation of potential enhancer agonist epitopes. Of four potential agonists identified, one epitope (ALWGQDVTSV, designated P-93L) was identified as an enhancer agonist. Compared with the native P-92 peptide, the P-93L agonist (a) bound HLA-A2 at lower peptide concentrations, (b) demonstrated a higher avidity for HLA-A2 in dissociation assays, (c) when used with antigen-presenting cells, induced the production of more IFN-γ by T cells than with the use of the native peptide, and (d) was capable of more efficiently generating MUC-1-specific human T-cell lines from normal volunteers and pancreatic cancer patients. Most importantly, the T-cell lines generated using the agonist epitope were more efficient than those generated with the native epitope in the lysis of targets pulsed with the native epitope and in the lysis of HLA-A2 human tumor cells expressing MUC-1.

Conclusions: In addition to the identification of novel MUC-1 epitopes outside the variable number of tandem repeat region, the studies reported here describe the first agonist epitope of MUC-1. The employment of this agonist epitope in peptide-, protein-, and vector-based vaccines may well aid in the development of effective vaccines for a range of human cancers.
Cellular Immunity in Breast Cancer Patients Completing Taxane Treatment
Authors: Carson William E. , Shapiro Charles L. , Crespin Timothy R. , Thornton Lisa M. , Andersen Barbara L.
Published: September 2005

Purpose: A field study of postchemotherapy immune functioning relative to the use of taxanes is reported. Immune responses in breast cancer patients were analyzed as a function of whether patients received taxane as part of their adjuvant chemotherapy.

Experimental Design: Immune levels of 227 stage II/III breast cancer patients were measured immediately after surgery prior to chemotherapy and again 12 months later when all chemotherapies had been completed. T-cell blastogenesis and natural killer (NK) cell lysis levels of patients receiving taxanes (n = 55) were compared with levels of patients not receiving taxanes (n = 172).

Results: Regression analyses were conducted. The administration of taxane as part of combination chemotherapy predicted increased T-cell blastogenesis and NK cell cytotoxicity after the conclusion of all chemotherapies. For the Taxane group, average phytohemagglutinin-induced blastogenesis was 37% higher and NK cell cytotoxicity was 39% higher than the values for the No-Taxane group.

Conclusions: Data from group comparisons with appropriate controls in a sizable clinical sample contravene traditional wisdom that taxanes suppress patients’ immune cell functions. Problems in generalizing direct-contact laboratory models to the field of cancer treatment are highlighted.
Identification of Genes with Differential Expression in Acquired Drug-Resistant Gastric Cancer Cells Using High-Density Oligonucleotide Microarrays
Authors: Kang Hio Chung , Kim Il-Jin , Park Jae-Hyun , Shin Yong , Ku Ja-Lok , Jung Mi Sun , Yoo Byong Chul , Kim Hark Kyun , Park Jae-Gahb
Published: September 2005

Purpose: A major obstacle in chemotherapy is treatment failure due to anticancer drug resistance. The emergence of acquired resistance results from host factors and genetic or epigenetic changes in the cancer cells. The purpose of this study was to identify differentially expressed genes associated with acquisition of resistance in human gastric cancer cells.

Experimental Design: We performed global gene expression analysis in the acquired drug-resistant gastric cancer cell lines to the commonly used drugs 5-fluorouracil, doxorubicin, and cisplatin using Affymetrix HG-U133A microarray. The gene expression patterns of 10 chemoresistant gastric cancer cell lines were compared with those of four parent cell lines using fold-change and Wilcoxon’s test for data analysis.

Results: We identified over 250 genes differentially expressed in 5-fluorouracil-, cisplatin-, or doxorubicin-resistant gastric cancer cell lines. Our expression analysis also identified eight multidrug resistance candidate genes that were associated with resistance to two or more of the tested chemotherapeutic agents. Among these, midkine (MDK), a heparin-binding growth factor, was overexpressed in all drug-resistant cell lines, strongly suggesting that MDK might contribute to multidrug resistance in gastric cancer cells.

Conclusions: Our investigation provides comprehensive gene information associated with acquired resistance to anticancer drugs in gastric cancer cells and a basis for additional functional studies.
Survival Advantage with Imatinib Mesylate Therapy in Chronic-Phase Chronic Myelogenous Leukemia (CML-CP) after IFN-α Failure and in Late CML-CP, Comparison with Historical Controls
Authors: Kantarjian Hagop , O’Brien Susan , Cortes Jorge , Giles Francis , Shan Jianqin , Rios Mary Beth , Faderl Stefan , Verstovsek Srdan , Garcia-Manero Guillermo , Wierda William , Kornblau Steven , Ferrajoli Alessandra , Keating Michael , Talpaz Moshe
Published: September 2005

Purpose: The purpose of this research was to compare the survival of patients with Philadelphia chromosome (Ph) -positive chronic myelogenous leukemia (CML) post-IFN-α failure treated with imatinib to historical experiences with standards of care or other therapies.

Experimental Design: The outcome of 261 patients with Ph-positive chronic phase CML post-IFN failure treated with imatinib was compared with 204 historical control patients treated for a similar disease status with existing therapies. A subset of 147 patients in late chronic phase CML and 100% Ph-positive status treated with imatinib was compared with 95 patients in a similar disease status treated with IFN. Multivariate analyses were conducted to assess the independent prognostic effect of therapy (imatinib versus other) on survival.

Results: In the first analysis involving 261 patients on imatinib plus 204 historical patients, the complete cytogenetic response rates were 62% and 19%, respectively (P < 0.001). A multivariate analysis identified pretreatment peripheral blasts and thrombocytosis to be independent poor prognostic factors for survival. Imatinib therapy (versus others) was a significant independent favorable prognostic factor for survival (hazard ratio, 0.17; P < 0.0001). In the second analysis involving the subset of 147 patients receiving imatinib plus 95 historical patients treated with IFN regimens, the complete cytogenetic response rates were 41% and 7%, respectively (P < 0.001). A multivariate analysis selected pretreatment anemia and peripheral blasts to be significant independent poor prognostic factors for survival. Imatinib therapy (versus IFN) was an independent favorable prognostic factor for survival (hazard ratio, 0.20; P < 0.0001). Three-month and 6-month landmark analyses showed that patients in all cytogenetic response categories (major, minor, and none) after imatinib therapy had survival outcomes better than the historical control population. Within each cytogenetic response category, survival was also better with imatinib than with other therapies.

Conclusions: This analysis provides evidence for a survival advantage with imatinib versus other therapies in chronic-phase CML post-IFN failure, and for a survival advantage with imatinib versus IFN in late chronic-phase CML.
Radiosensitization by Pan ErbB Inhibitor CI-1033
in Vitro
and
in Vivo
Authors: Nyati Mukesh K. , Maheshwari Divya , Hanasoge Sheela , Sreekumar Arun , Rynkiewicz Susan D. , Chinnaiyan Arul M. , Leopold Wilbur R. , Ethier Stephen P. , Lawrence Theodore S.
Published: September 2005

Purpose: Overexpression of the ErbB family of receptor tyrosine kinases has been associated with uncontrolled growth of many tumor types and, therefore, presents a promising molecular target for cancer therapy. CI-1033 is a small molecule tyrosine kinase inhibitor that differs from other 4-anilinoquinazolines by being a pan ErbB (instead of epidermal growth factor receptor-specific) irreversible (instead of reversible) inhibitor. Therefore, we investigated the antitumor effect of CI-1033 alone and in combination with ionizing radiation in vitro and in vivo.

Experimental Design: We selected three human colon carcinoma cell-lines (LoVo, Caco-2, which express activated epidermal growth factor receptor and ErbB-2 family members, and SW620, which does not), and analyzed the effects of CI-1033 both in vitro and in vivo. For in vivo studies LoVo and Caco-2 cells were implanted s.c. in the flank of nude mice. After the tumor reached ∼100 mm3, treatment was initiated with 20 mg/kg of CI-1033 (orally once daily × 5 for 3 successive weeks), radiation treatment (a total of 30 Gy given in 2 Gy once daily × 5 for 3 successive weeks), or a combination of both CI-1033 and radiation treatment.

Results: We found that exposure of LoVo and Caco-2, but not SW620 cells, to CI-1033 in the range of 1–3 μm could inhibit constitutive signaling by tyrosine kinases, arrest cell growth, inhibit cells in G1, stimulate expression of p53, and induce apoptosis. The inhibition of cell growth by CI-1033 seemed to produce only minimal radiosensitization in LoVo and Caco-2 cells. In contrast, the combination of CI-1033 and radiation produced significant (P < 0.0005 and P = 0.0002, respectively) and prolonged suppression of tumor growth in both the tumor types when compared with either treatment alone.

Conclusions: These findings suggest that CI-1033 can increase the effectiveness of radiation therapy. The extent of suppression of tyrosine kinase activity by CI-1033, rather than the amount of activity in untreated cells, seemed to be more closely associated with the efficacy of combination treatment.
Synergistic Cytotoxicity of Pyrazoloacridine with Doxorubicin, Etoposide, and Topotecan in Drug-Resistant Tumor Cells
Authors: Hu YanPing , Krishan Awtar , Nie WeiJia , Sridhar Kasi S. , Mayer Lawrence D. , Bally Marcel
Published: September 2005

Pyrazoloacridine (NSC 366140, PD115934, PZA) is a new class of acridine anticancer agents under investigation in Phase II clinical trials in patients with advanced cancers. Although poor responses in patients to the treatment with PZA alone have been observed, this class of agents remains of interest because of its distinct mechanism of action from other topoisomerase poisons. Therefore, the combination of PZA with conventional anticancer agents presents an attractive approach to treat drug-resistant human tumors. In the present study, the cytotoxic effects of PZA combined with doxorubicin, topotecan, and etoposide were determined using paired parental and doxorubicin-resistant human colon carcinoma (SW-620 and SW620/AD-300) and breast cancer cell lines (MCF-7 and MCF-7/TH). Cytotoxicity was measured by soft agar clonogenic assays. Dose effect and combination effects were analyzed by the method of Chou and Talalay. The combination of PZA with doxorubicin, topotecan, and etoposide in fixed ratios demonstrated synergistic cytotoxicity on both SW-620 and SW620/AD-300 cell lines. The combination of PZA with doxorubicin also exhibited synergistic cytotoxicity against both MCF-7 and MCF-7/TH cell lines. The mechanism of synergism appeared independent of topoisomerase I and II inhibition, and interference with protein-DNA complexes. Strategies to define optimal drug combinations are proving to be of significant value when considering potential clinical applications of new and established agents.
Imexon-Induced Apoptosis in Multiple Myeloma Tumor Cells Is Caspase-8 Dependent
Authors: Evens Andrew M. , Prachand Sheila , Shi Bo , Paniaqua Mary , Gordon Leo I. , Gartenhaus Ronald B.
Published: September 2005

Purpose: Imexon is a 2-cyanoaziridine agent that has been shown to inhibit growth of chemotherapy-sensitive myeloma cells through apoptosis with decreased cellular stores of glutathione and increased reactive oxygen species (ROS). We examined the mechanism of imexon cytotoxicity in a diverse panel of dexamethasone and chemotherapy-sensitive and -resistant myeloma cell lines.

Experimental Design: We examined cellular cytotoxicity, apoptosis, and changes in redox state in dexamethasone-sensitive (C2E3), dexamethasone-resistant (1-310 and 1-414), chemotherapy-sensitive (RPMI-8226), and chemotherapy-resistant (DOX-1V and DOX-10V) myeloma cell lines.

Results: We found significant cytotoxicity after 48-h incubation with imexon (80–160 μm) in dexamethasone and chemotherapy-sensitive and -resistant myeloma cell lines in a time- and dose-dependent manner. The mechanism of imexon cytotoxicity in all cell lines was related to induction of apoptosis with the presence of cleaved caspase-3. Moreover, after imexon exposure in C2E3 and 1-414 cell lines, we demonstrated caspase-8-dependent apoptosis. Bcl-2:bax was proapoptotic with imexon in C2E3, whereas bcl-2:bax was independent of steroid resistance, chemotherapy sensitivity, and chemotherapy resistance. Depletion of intracellular glutathione was documented in RPMI-8226 at high imexon concentrations (≥225 μm) but not in other cell lines. Furthermore, ROS were found in C2E3, RPMI-8226, and 1-310 only at high imexon concentrations, whereas a sensitive marker of oxidative DNA damage, 8-hydroxydeoxyguanosine, was not increased in any cell line.

Conclusions: Our results demonstrate that imexon has significant broad antimyeloma activity that is mediated through apoptotic mechanisms that is not dependent on production of ROS. Moreover, we have identified a mechanism of cytotoxicity in dexamethasone-sensitive and -resistant myeloma cells induced by imexon that is caspase-8 dependent.
A Prominent Low-pH Methotrexate Transport Activity in Human Solid Tumors
Authors: Zhao Rongbao , Gao Feng , Hanscom Marie , Goldman I. David
Published: September 2005

Whereas the major folate transporter, the reduced folate carrier (RFC), has a physiological pH optimum, transport activities for folates and antifolates have been detected with low pH optima. Because the interstitial pH in solid tumors is generally acidic, the mechanisms by which antifolates are transported at low pH could be an important determinant of drug activity under these conditions. The current study quantitated the low pH methotrexate (MTX) transport activity in human solid tumor cell lines from the National Cancer Institute tumor panel and other sources. MTX influx at pH 5.5 was equal to, or greater than, influx at pH 7.4 in 29 of 32 cell lines. To assess the role of RFC in transport at low pH in one of these cell lines, a HeLa clonal line (R5) was selected for MTX resistance due to a genomic deletion of the carrier gene. MTX influx was depressed by 70% in R5 versus wild-type HeLa cells at pH 7.4. At pH 6.5, influx in these two lines was similar; as the pH was decreased to 5.5 influx increased in both cell lines. Similarly, whereas net MTX uptake over 1 h was markedly decreased in R5 cells at pH 7.4, net uptake in HeLa and R5 cells was comparable at pH 6.5. Also, as compared with MCF7 breast cancer cells, MTX uptake was markedly decreased at pH 7.4, but only minimally at pH 6.5, in the MDA-MB-231 human breast cancer cell line that lacks RFC expression. When grown with folic acid (2 μm) at pH 7.4, the IC50 for MTX was 14-fold higher in R5 as compared with wild-type HeLa cells; the difference was only 4-fold when cells when grown at pH 6.9; the IC50s were identical at this pH when the medium folate was 25 nm 5-formyltetrahydrofolate. These data demonstrate that transport activity at low pH is prevalent in human solid tumors, is RFC-independent in R5 cells and MDA-MB-231 breast cancer cells, and can preserve MTX activity in the absence of RFC at an acidic pH relevant to solid tumors in vivo.
Identification of HLA-A24-Restricted CTL Epitope from Cancer-Testis Antigen, NY-ESO-1, and Induction of a Specific Antitumor Immune Response
Authors: Yamaguchi Hiroshi , Tanaka Fumiaki , Ohta Mitsuhiko , Inoue Hiroshi , Mori Masaki
Published: September 2005

Purpose: For the development of peptide-based, cancer-specific immunotherapy, the identification of CTL epitopes from additional tumor antigens is very important. NY-ESO-1, a cancer–testis antigen, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A24-expressing individuals cover >60% in the population of Japan, we aim at identifying NY-ESO-1-encoded peptide presented by HLA-A24.

Experimental Design: In our study, a HLA-A24-restricted CTL epitope was identified by using the following four-step procedure: (a) computer-based epitope prediction from the amino acid sequence of NY-ESO-1 antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A24 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward various carcinoma cells expressing NY-ESO-1 antigen and HLA-A24.

Results: Of the tested peptides, effectors induced by a peptide of NY-ESO-1 at residue position 158–166 lysed three kinds of carcinoma cells expressing both NY-ESO-1 and HLA-A24. Our results indicate that peptide NY-ESO-1 (158–166) (LLMWITQCF) is a new HLA-A24-restricted CTL epitope capable of inducing NY-ESO-1-specific CTLs in vitro mediating HLA class I-restricted manner.

Conclusions: We identified a novel HLA-A24-restricted NY-ESO-1-derived epitope peptide (LLMWITQCF) that could induce specific CTLs from the peripheral blood mononuclear cells of HLA-A24+ healthy donors. This peptide would be useful in further evaluating the clinical utility of peptide-based, cancer-specific immunotherapy against various histological tumors.
Detection of Promoter Hypermethylation of Multiple Genes in the Tumor and Bronchoalveolar Lavage of Patients with Lung Cancer
Authors: Topaloglu Ozlem , Hoque Mohammad Obaidul , Tokumaru Yutaka , Lee Juna , Ratovitski Edward , Sidransky David , Moon Chul-so
Published: September 2005

Purpose: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the bronchoalveolar lavage (BAL) samples of lung cancer patients.

Experimental Design: We examined the tumor and the matched BAL DNA for aberrant methylation of eight gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-β2, and ARF) from 31 patients with primary lung tumors by quantitative fluorogenic real-time PCR. BAL from 10 age-matched noncancer patients was used as a control.

Results: Promoter hypermethylation of at least one of the genes studied was detected in all 31 lung primary tumors; 27 (87%) CDH1, 17 (55%) APC, 14 (45%) RASSF1A, 12 (39%) MGMT, 7 (23%) p16, 3 (10%) GSTP1, 3 (10%) RAR-β2, and 0 (0%) ARF. Methylation was detected in CDH1 (48%), APC (29%), RASSF1A (29%), MGMT (58%), p16 (14%), GSTP1 (33%), RAR-β2 (0%), and ARF (0%) of BAL samples from matched methylation-positive primary tumors, and in every case, aberrant methylation in BAL DNA was accompanied by methylation in the matched tumor samples. BAL samples from 10 controls without evidence of cancer revealed no methylation of the MGMT, GSTP1, p16, ARF, or RAR-β2 genes whereas methylation of RASSF1, CDH1, and APC was detected at low levels. Overall, 21 (68%) of 31 BAL samples from cancer patients were positive for aberrant methylation.

Conclusion: Our findings suggest that promoter hypermethylation in BAL can be detected in the majority of lung cancer patients. This approach needs to be evaluated in large early detection and surveillance studies of lung cancer.
Predictive Models for Breast Cancer Susceptibility from Multiple Single Nucleotide Polymorphisms
Authors: Listgarten Jennifer , Damaraju Sambasivarao , Poulin Brett , Cook Lillian , Dufour Jennifer , Driga Adrian , Mackey John , Wishart David , Greiner Russ , Zanke Brent
Published: September 2005

Hereditary predisposition and causative environmental exposures have long been recognized in human malignancies. In most instances, cancer cases occur sporadically, suggesting that environmental influences are critical in determining cancer risk. To test the influence of genetic polymorphisms on breast cancer risk, we have measured 98 single nucleotide polymorphisms (SNPs) distributed over 45 genes of potential relevance to breast cancer etiology in 174 patients and have compared these with matched normal controls. Using machine learning techniques such as support vector machines (SVMs), decision trees, and naïve Bayes, we identified a subset of three SNPs as key discriminators between breast cancer and controls. The SVMs performed maximally among predictive models, achieving 69% predictive power in distinguishing between the two groups, compared with a 50% baseline predictive power obtained from the data after repeated random permutation of class labels (individuals with cancer or controls). However, the simpler naïve Bayes model as well as the decision tree model performed quite similarly to the SVM. The three SNP sites most useful in this model were (a) the +4536T/C site of the aldosterone synthase gene CYP11B2 at amino acid residue 386 Val/Ala (T/C) (rs4541); (b) the +4328C/G site of the aryl hydrocarbon hydroxylase CYP1B1 at amino acid residue 293 Leu/Val (C/G) (rs5292); and (c) the +4449C/T site of the transcription factor BCL6 at amino acid 387 Asp/Asp (rs1056932). No single SNP site on its own could achieve more than 60% in predictive accuracy. We have shown that multiple SNP sites from different genes over distant parts of the genome are better at identifying breast cancer patients than any one SNP alone. As high-throughput technology for SNPs improves and as more SNPs are identified, it is likely that much higher predictive accuracy will be achieved and a useful clinical tool developed.
Matrix Metalloproteinase 3 Polymorphism
Authors: Blons Hélène , Gad Sophie , Zinzindohoué Franck , Manière Isabelle , Beauregard Janie , Tregouet David , Brasnu Daniel , Beaune Philippe , Laccourreye Ollivier , Laurent-Puig Pierre
Published: September 2005

Purpose: Treatment of head and neck cancer often associates different therapeutic modalities, including surgery, radiotherapy, and chemotherapy. In an attempt to optimize therapeutics, the identification of molecular markers linked to response to chemotherapy remains important. Recently, the involvement of metalloproteinases in resistance to chemotherapy was suggested through their interaction with the Fas/Fas ligand pathway. Indeed metalloproteinases enhance Fas ligand shedding modulating chemotherapy efficiency. On the basis of these findings, we tested the existence of a correlation between response to chemotherapy and four metalloproteinase polymorphisms in a prospective series of 148 head and neck cancer patients.

Experimental Design: Patients were genotyped using automated fragment analysis and 5′-nuclease allelic discrimination assay. Response to chemotherapy was clinically assessed without knowledge of the genotype status.

Results: A significant relation between the metalloproteinase type 3 (MMP3) −1612insA polymorphism and response to chemotherapy was identified. Indeed, patients with the 6A/6A genotype responded more frequently (86%) to treatment as compared with patients with the 5A/6A (65%) or 5A/5A (55%) genotypes (P = 0.04). A multivariate analysis, including tumor stage, gender, TP53 mutations, and MMP3 polymorphism, showed that the 6A/6A genotype was an independent factor of response to 5-fluorouracil-cisplatin chemotherapy in head and neck cancer patients with an odds ratio of 6.7 as compared with the 5A/5A genotype.

Conclusions: This work showed that genotyping the MMP3 gene enhancer polymorphism −1612insA could help predict chemosensitivity in head and neck cancer patients.
PC Cell-Derived Growth Factor Expression in Prostatic Intraepithelial Neoplasia and Prostatic Adenocarcinoma
Authors: Pan Chong-Xian , Kinch Michael S. , Kiener Peter A. , Langermann Solomon , Serrero Ginette , Sun Le , Corvera Joseph , Sweeney Christopher J. , Li Lang , Zhang Shaobo , Baldridge Lee Ann , Jones Timothy D. , Koch Michael O. , Ulbright Thomas M. , Eble John N. , Cheng Liang
Published: September 2005

Purpose: PCDGF (PC cell-derived growth factor), also called progranulin, is a Mr 88,000 glycoprotein precursor of granulin. It is a novel growth factor that stimulates cell proliferation, confers epithelial tumorigenesis, and promotes tumor invasion. Here we investigate the potential of PCDGF as a therapeutic target for prostate cancer.

Experimental Design: We studied the expression of PCDGF in invasive prostate cancer, adjacent high-grade prostatic intraepithelial neoplasia (PIN), and benign prostate tissue from 99 human prostate specimens. The level of PCDGF expression was correlated with various clinicopathological characteristics.

Results: Normal prostate tissue did not express (53/99), or expressed low levels (46/99) of PCDGF. In the 46 normal prostate specimens that expressed PCDGF, most of them had less than 10% of cells expressing PCDGF. PCDGF expression could be detected in more than 50% of cells in all specimens of PIN and invasive prostate cancer. The expression of PCDGF in normal prostate tissue was much less intense and in a smaller fraction of cells than in PIN and invasive adenocarcinoma (P < 0.0001). There was no correlation of PCDGF expression with age, Gleason score, pathological stage, status of lymph node metastasis, extraprostatic extension, perineural invasion, surgical margins, and vascular invasion.

Conclusions: Our data suggest that the induction of PCDGF expression occurs during the development of PIN. PCDGF may be a new molecular target for the treatment and prevention of prostate cancer.
Alterations of
p16
and Prognosis in Biliary Tract Cancers from a Population-Based Study in China
Authors: Ueki Takashi , Hsing Ann W. , Gao Yu-Tang , Wang Bing-Sheng , Shen Ming-Chang , Cheng Jiarong , Deng Jie , Fraumeni Joseph F. , Rashid Asif
Published: September 2005

Purpose: Biliary tract cancer is an uncommon malignancy with a poor survival rate. We evaluated p16 gene alteration as a prognostic marker for this disease.

Experimental Design: We studied p16 gene alterations by sequencing, methylation, and loss of heterozygosity of chromosome 9p in 118 biliary tract carcinomas, including 68 gallbladder cancers, 33 extrahepatic bile duct cancers, and 17 ampullary cancers. Survival was evaluated in 57 patients with gallbladder carcinomas, 27 with bile duct carcinomas, and 16 with ampullary carcinomas with and without somatic p16 alterations detected by two different methods.

Results: p16 gene alterations including silent mutations were present in 61.8% gallbladder cancers, 54.5% bile duct cancers, and 70.6% ampullary cancers. p16 gene nonsilent mutations, p16 methylation, and loss of chromosome 9p21-22 that targets p14, p15, and p16 genes were present in 13 of 53 (24.5%), 8 of 54 (14.8%), and 32 of 44 (72.7%) gallbladder tumors; 5 of 25 (20.0%), 5 of 31 (16.1%), and 12 of 21 (57.1%) bile duct tumors; and 3 of 13 (23.1%), 6 of 15 (40.0%), and 8 of 16 (50.0%) ampullary tumors, respectively. The mean survival of patients with gallbladder cancers without p16 alterations was 21.5 ± 14.8 months compared with 12.1 ± 11.4 months for patients with p16 alterations (P = 0.02).

Conclusions: Alteration of p16 gene alone or in combination with alterations of other tumor suppressor genes on chromosome 9p is a prognostic indicator in gallbladder carcinoma, with more favorable survival rates associated with carcinomas lacking p16 gene alterations.
Induction of Apoptosis in Human Myeloid Leukemic Cells by 1′-Acetoxychavicol Acetate through a Mitochondrial- and Fas-Mediated Dual Mechanism
Authors: Ito Keisuke , Nakazato Tomonori , Murakami Akira , Yamato Kenji , Miyakawa Yoshitaka , Yamada Taketo , Hozumi Nobumichi , Ohigashi Hajime , Ikeda Yasuo , Kizaki Masahiro
Published: September 2005

Purpose: The purpose of this investigation was to determine the antileukemic effects of 1′-acetoxychavicol acetate (ACA) obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberaceae).

Experimental Design: We evaluated the effects of ACA on various myeloid leukemic cells in vitro and in vivo. We further examined the molecular mechanisms of ACA-induced apoptosis in myeloid leukemic cells.

Results: Low-dose ACA dramatically inhibited cellular growth of leukemic cells by inducing apoptosis. Because NB4 promyelocytic leukemic cells were most sensitive to ACA, we used NB4 cells for further analyses. Production of reactive oxygen species triggered ACA-induced apoptosis. ACA-induced apoptosis in NB4 cells was in association with the loss of mitochondrial transmembrane potential (ΔΨm) and activation of caspase-9, suggesting that ACA-induced death signaling is mediated through a mitochondrial oxygen stress pathway. In addition, ACA activated Fas-mediated apoptosis by inducing of casapse-8 activity. Pretreatment with the thiol antioxidant N-acetyl-l-cysteine (NAC) did not inhibit caspase-8 activation, and the antagonistic anti-Fas antibody ZB4 did not block generation of reactive oxygen species, indicating that both pathways were involved independently in ACA-induced apoptosis. Furthermore, ACA had a survival advantage in vivo in a nonobese diabetic/severe combined immunodeficient mice leukemia model without any toxic effects.

Conclusions: We conclude that ACA induces apoptosis in myeloid leukemic cells via independent dual pathways. In addition, ACA has potential as a novel therapeutic agent for the treatment of myeloid leukemia.
The Androgen Axis in Recurrent Prostate Cancer
Authors: Mohler James L. , Gregory Christopher W. , Ford O. Harris , Kim Desok , Weaver Catharina M. , Petrusz Peter , Wilson Elizabeth M. , French Frank S.
Published: September 2005

Purpose. Prostate cancer that recurs during androgen deprivation therapy is referred to as androgen-independent. High levels of expression of androgen receptor and androgen receptor-regulated genes in recurrent prostate cancer suggest a role for androgen receptor and its ligands in prostate cancer recurrence.

Experimental Design. Recurrent prostate cancer specimens from 22 men whose prostate cancer recurred locally during androgen deprivation therapy and benign prostate specimens from 48 men who had received no prior treatment were studied. Androgen receptor expression was measured using monoclonal antibody and automated digital video image analysis. Tissue androgens were measured using radioimmunoassay.

Results. Epithelial nuclei androgen receptor immunostaining in recurrent prostate cancer (mean optical density, 0.284 ± SD 0.115 and percentage positive nuclei, 83.7 ± 11.6) was similar to benign prostate (mean optical density, 0.315 ± 0.044 and percentage positive nuclei, 77.3 ± 13.0). Tissue levels of testosterone were similar in recurrent prostate cancer (2.78 ± 2.34 pmol/g tissue) and benign prostate (3.26 ± 2.66 pmol/g tissue). Tissue levels of dihydrotestosterone, dehydroepiandrosterone, and androstenedione were lower (Wilcoxon, P = 0.0000068, 0.00093, and 0.0089, respectively) in recurrent prostate cancer than in benign prostate, and mean dihydrotestosterone levels, although reduced, remained 1.45 nm. Androgen receptor activation in recurrent prostate cancer was suggested by the androgen-regulated gene product, prostate-specific antigen, at 8.80 ± 10.80 nmol/g tissue.

Conclusions. Testosterone and dihydrotestosterone occur in recurrent prostate cancer tissue at levels sufficient to activate androgen receptor. Novel therapies for recurrent prostate cancer should target androgen receptor directly and prevent the formation of androgens within prostate cancer tissue.
Camptothecin Analogs (Irinotecan or Topotecan) plus High-Dose Cyclophosphamide as Preparative Regimens for Antibody-Based Immunotherapy in Resistant Neuroblastoma
Authors: Kushner Brian H. , Kramer Kim , Modak Shakeel , Cheung Nai-Kong V.
Published: September 2005

Purpose: We used high-dose cyclophosphamide plus topotecan/vincristine (CTV) or irinotecan (C/I) in patients with resistant neuroblastoma. The aim was to use a regimen with little risk to major organs to (a) achieve or consolidate remission in heavily treated patients and to (b) induce an immunological state conducive to passive immunotherapy with the murine 3F8 antibody.

Experimental Design: CTV and C/I included cyclophosphamide 140 mg/kg (∼4200 mg/m2). With CTV, topotecan 2 mg/m2 was infused i.v. (30 min) on days 1–4 (total, 8 mg/m2), and vincristine 0.067 mg/kg was injected on day 1. With C/I, irinotecan, 50 mg/m2 was infused i.v. (1 h) on days 1–5 (total, 250 mg/m2). Mesna and granulocyte colony-stimulating factor were used.

Results: Twenty-nine patients received 38 courses of CTV, and 26 patients received 38 courses of C/I. All patients had previously received topotecan, a hemopoietic stem-cell transplant, and/or high-dose cyclophosphamide. CTV and C/I caused myelosuppression of comparably prolonged duration as follows: absolute neutrophil counts <500/μl lasted 5–12 days in patients who had not previously received transplant and 7–21 days in patients who were post-transplant. Other significant toxicities included typhlitis (two CTV-treated patients, one C/I-treated patient) and hemorrhagic cystitis (one C/I-treated patient). Major responses were seen in 4 (15%) of 26 CTV and 4 (17%) of 24 C/I-treated patients with assessable disease. Bone marrow disease resolved in 5 (28%) of 18 CTV-treated patients and in 4 (27%) of 15 C/I-treated patients. 3F8 after CTV or C/I was not blocked by neutralizing antibodies, consistent with the desired immunosuppressive effect of high-dose cyclophosphamide.

Conclusions: CTV and C/I require transfusional and antibiotic support but otherwise entail tolerable morbidity. They have modest antineuroblastoma activity in heavily treated patients and are good preparative regimens for passive immunotherapy with monoclonal antibodies.