ognizant Communication Corporation

ONCOLOGY RESEARCH

ABSTRACTS
VOLUME 12, NUMBER 3, 2000

Oncology Research, Volume 12, pp. 113-119, 2000
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Structure-Activity Studies of Novobiocin Analogs as Modulators of the Cytotoxicity of Etoposide (VP-16)

Germana Rappa,1,2 Krishnamurthy Shyam,2 Aurelio Lorico,1,2 Øystein Fodstad,1 and Alan C. Sartorelli2

1Department of Tumor Biology, Norwegian Radium Hospital, Montebello, Oslo 0310, Norway
2Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, CT 06520

We have previously reported that the antibiotic novobiocin enhanced the toxicity of the anticancer agent etoposide (VP-16) to several drug-sensitive and -resistant tumor cell lines. The increase in VP-16 cytotoxicity produced by novobiocin was not due to the combined effects of these agents on topoisomerase II, but to inhibition by novobiocin of VP-16 efflux, which in turn led to increased accumulation of VP-16 and increased formation of potentially lethal VP-16-stabilized topoisomerase II-DNA covalent complexes. We have now identified novobiocin analogs that are essentially equivalent to novobiocin as inhibitors of the activity of topoisomerase II, but that are more potent than novobiocin (a) as modulators of the cytotoxicity of VP-16 to WEHI-3B leukemia and A549 lung carcinoma cells and (b) in increasing VP-16 accumulation in these cell lines. Thus, removal of the sugar moiety of novobiocin to form novobiocic acid enhanced the potency of the antibiotic as a modulator of VP-16, whereas the substituted coumarin ring alone (U-7587) was devoid of VP-16 modulatory activity. Modifications of the side chain of novobiocin significantly influenced modulatory activity, with cyclonovobiocic acid, which was formed from novobiocic acid by acid-catalyzed cycloaddition, being the most active in enhancing the cytotoxicity of VP-16. The increased potency of novobiocic acid and cyclonovobiocic acid as modulators of VP-16 activity was achieved with no change from novobiocin in the capacity of these analogs to inhibit the catalytic activity of mammalian topoisomerase II, indicating a change in the specificity of these analogs.

Key words: Novobiocin; VP-16; Topoisomerase II; Drug efflux

Address correspondence to Dr. Alan C. Sartorelli, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Tel: (203) 785-4533; Fax: (203) 737-2045.




Oncology Research, Volume 12, pp. 121-126, 2000
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Induction of Hepatocellular Carcinoma With High Metastatic Potential in WS/Shi Rats: Discovery of an Inbred Strain Highly Susceptible to the Liver Carcinogen N-Nitrosomorpholine

Takashi Murai,1,2 Satoru Mori,1 Motoko Hosono,2 Yoshiko Iwakura,2 Akira Takashima,2 Tadao Oohara,2 Susumu Makino,2 Reiji Takeda,2 and Shoji Fukushima1

1First Department of Pathology Osaka City University Medicaol School, 1-4-3 Asahi-machi Abeno-ku, Osaka 545-8585, Japan
2Aburahi Laboratories Shionogi Research Laboratories Shionogi Co., Ltd. 1405 Koka-cho Koka-gun, Shiga 520-3423, Japan

We investigated the susceptibility of three inbred strains of rats to the hepatocarcinogen, N-nitrosomorpholine (NNM), to establish a spontaneous metastatic model of hepatocellular carcinoma (HCC). WS/Shi, SD/gShi, and F344/DuCrj rats were given 0.02% NNM in drinking water for 8 weeks and thereafter left without any treatment. The experiment ceased at week 20, because mortality markedly increased after this time point in WS/Shi rats. Liver weight was highest in WS/Shi rats among the three strains examined. The incidence of HCC was 15/15 (100%) in WS/Shi rats, 1/16 (6%) in SD/gShi rats, and 13/16 (81%) in F344/DuCrj rats surviving after NNM treatment. Metastasis to the lung was observed in HCC-bearing rats at an incidence of 13/15 (87%) in WS/Shi, 1/1 in SD/gShi, and 6/13 (46%) in F344/DuCrj. Four-week administration of NNM resulted in a significantly higher BrdU-labeling index of hepatocytes in WS/Shi rats than in the other strains. These findings indicated that WS/Shi is the most sensitive strain to NNM and may be the most suitable strain for use as a spontaneous metastatic model of HCC among the strains of rats examined in the present study.

Key words: Hepatocarcinogenesis; N-Nitrosomorpholine; Spontaneous lung metastasis

Address correspondence to Takashi Murai, D.V.M., Ph.D., Aburahi Laboratories Shionogi Research Laboratories Shionogi Co., Ltd.1405, Koka-cho Koka-gun, Shiga 520-3423, Japan. Tel: (81) 0748-88-6512; Fax: (81) 0748-88-6508; E-mail: takashi murai@shionogi.co.jp




Oncology Research, Volume 12, pp. 127-135, 2000
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Tumor Amplified Protein Expression Therapy: Salmonella as a Tumor-Selective Protein Delivery Vector

Li-mou Zheng, Xiang Luo, Ming Feng, Zujin Li, Trung Le, Martina Ittensohn, Mark Trailsmith, David Bermudes, Stanley L. Lin, and Ivan C. King

Vion Pharmaceuticals, Inc., 4 Science Park, New Haven, CT 06511

Attenuated strains of Salmonella typhimurium, VNP20009 and YS7212, when injected systemically to tumor-bearing mice, accumulated preferentially in tumors at levels at least 200-fold and, more commonly, 1000-fold greater than in other normal tissues. This selectivity occurred in subcutaneously implanted murine tumors, including B16F10 melanoma, M27 lung carcinoma, and colon 38 carcinoma. The preferential accumulation was also manifested in animals bearing human tumor xenografts, including Lox, C8186, DLD1, SW620, HCT116, HTB177, DU145, MDA-MB-231, and Caki. Four to five days after a single IV injection of 1 x 106 colony-forming unit (cfu)/mouse, we routinely detected VNP20009 proliferation and accumulation at levels ranging from 1 x 108 to 2 x 109 cfu/g tumor. The amount of VNP20009 accumulated in the liver ranged from 3 x 104 to 2 x 106 cfu/g. The distribution of Salmonella in tumors was homogenous; YS7212 could be detected from the periphery to the interior portion of the tumors. Using mice with various immunodeficiencies, we also discovered the same preferential accumulation of Salmonella in tumors implanted in these mice. The use of Salmonella as a protein delivery vector was shown by IV administration of the bacteria expressing either green fluorescent protein (GFP) or cytosine deaminase (CD) into tumor-bearing mice. GFP and CD were detected in tumors, but not in livers, taken from mice inoculated with Salmonella carrying these genes. Bacteria accumulation and CD expression persisted in the tumors for up to 14 days after a single bolus IV administration of bacteria to tumor-bearing mice.

Key words: Salmonella; Vector; Targeting; Cancer; Delivery; Prodrug-converting enzyme

Address correspondence to Dr. Ivan King, Vion Pharmaceuticals, Inc., 4 Science Park, New Haven, CT 06511. Tel: (203) 498-4210; Fax: (203) 781-8090; E-mail: iking@vionpharm.com




Oncology Research, Volume 12, pp. 137-148, 2000
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Schedule-Dependent Interaction Between Raltitrexed and 5-Fluorouracil in Human Colon Cancer Cell Lines In Vitro

Yasuhiko Kano,1 Miyuki Akutsu,1 Kenichi Suzuki,2 Yasuo Yazawa,3 Saburo Tsunoda,1 and Yusuke Furukawa4

1Divisions of Medical Oncology, 2Laboratory Medicine, and 3Orthopedic Surgery, Tochigi Cancer Center, Yonan 4-9-13, Utsunomiya, Tochigi, 320-0834, Japan
4Center for Molecular Medicine, Department of Hematology, Jichi Medical School, Tochigi, 329-0431, Japan

Raltitrexed (Tomudex) is a novel thymidylate synthase inhibitor with significant activity against advanced colorectal cancer. We studied the cytotoxic interactions of raltitrexed and 5-fluorouracil (5-FU) in four human colon cancer cell lines on various schedules. The cell growth inhibition after 5 days was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytotoxic interactions at the IC80 level were evaluated by the isobologram method. Simultaneous exposure to raltitrexed and 5-FU for 5 days produced additive to synergistic effects in Colo201 cells, and produced additive effects in Colo321, LoVo, and WiDr cells. Simultaneous exposure to raltitrexed and 5-FU for 24 h produced additive effects in Colo201, LoVo, and WiDr cells, and produced antagonistic effects in Colo320 cells. Sequential exposure to raltitrexed for 24 h followed by 5-FU for 24 h produced additive effects in Colo201, Colo320, and LoVo cells, and produced antagonistic effects in WiDr cells. The reverse sequence produced additive effects in Colo201 cells, and produced antagonistic effects in Colo320, LoVo, and WiDr cells. Simultaneous exposure to raltitrexed and 5-FU for 4 h and sequential exposure to raltitrexed for 4 h followed by 5-FU for 4 h with a 20-h interval produced additive effects, while the reverse sequence produced antagonistic effects in LoVo and WiDr cells. These findings suggest that the simultaneous administration of raltitrexed and 5-FU or the sequential administration of raltitrexed followed by 5-FU may be the optimal sequence, while the reverse sequence may be inappropriate. Preclinical and clinical studies of the simultaneous administration of raltitrexed and 5-FU and the sequential administration of raltitrexed followed by 5-FU are required to better understand the antitumor, toxic, and pharmacokinetic interactions of this combination in order to develop the combination chemotherapy of raltitrexed and 5-FU.

Key words: Raltitrexed; 5-Fluorouracil; Drug combination; Isobologram

Address correspondence to Y. Kano, Division of Medical Oncology, Tochigi Cancer Center, Yonan 4-9 -13, Utsunomiya, Tochigi. 320-0834, Japan. Tel: 028-658-5151; Fax: 028-658-5488; E-mail: ykano@tcc.pref.tochigi.jp




Oncology Research, Volume 12, pp. 149-155, 2000
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Radiation-Induced Cell Cycle Delays and p53 Status of Early Passage Melanoma Cell Lines

Jayamala Parmar,1,2 Elaine S. Marshall,1 Geoffrey A. Charters1,2 Karen M. Holdaway,1 Andrew N. Shelling,2 and Bruce C. Baguley1

1Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland, New Zealand
2Department of Obstetrics and Gynaecology, University of Auckland, Private Bag 92019, Auckland, New Zealand

Cell cultures exposed to DNA-damaging agents such as gamma radiation respond by arresting at cell cycle checkpoints, and the p53 tumor suppressor protein is strongly implicated in this behavior. We have investigated the TP53 status and cell cycle response to ionizing radiation of a series of early passage cell lines (designated NZM1 to NZM15) previously developed from patients with metastatic melanoma. The TP53 status of each of the cell lines was determined by single-strand conformation polymorphism and DNA sequence analysis. The majority of the lines appeared to have a wild-type TP53 gene sequence, consistent with published studies. Two lines (NZM4 and NZM7.2) were found to have an identical T-C transition mutation in nucleotide 721 (exon 7) of the coding region. NZM7.2 (mutant) and NZM7.4 (wild-type) were clonally derived from the same line (NZM7). The existence of radiation-induced cell cycle arrest in G1 and/or G2M phase was determined 16 h after irradiation (6.3 Gy) by DNA staining and flow cytometric analysis. The mitotic inhibitor paclitaxel was used as a reference compound, with or without irradiation, to assess the efficiency of radiation-induced cell cycle arrest. G1 phase arrest was associated only with the presence of the wild-type TP53 gene, but the efficiency of induced arrest varied among the cell lines and the period of G1 phase arrest appeared to be short. A significant difference (P < 0.002) was also found between the efficiency of induction of G2 phase arrest and the presence of wild-type TP53 gene. The results provide evidence that although the melanoma cell lines generally had an intact TP53 gene, the efficiency of p53-mediated cycle arrest might be deficient and contribute to the resistance of this tumor to treatment.

Key words: Single-strand conformation analysis (SSCP); DNA sequencing; Flow cytometry; Polymorphism; Radiation; Melanoma; p53

Address correspondence to Dr. Bruce C. Baguley, Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel: (64-9) 373 7599, ext. 6142; Fax: (64-9) 373 7502; E-mail: b.baguley@auckland.ac.nz




Oncology Research, Volume 12, pp. 157-167, 2000
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Printed in the USA. All rights reserved.

Smad Protein Expression and Activation in Transforming Growth Factor-b Refractory Human Squamous Cell Carcinoma Cells

Wu Yan, Vincent F. Vellucci, and Michael Reiss

Section of Medical Oncology, Department of Internal Medicine and Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520

In contrast to nonneoplastic keratinocytes, human squamous carcinoma cell lines are able to proliferate in the presence of transforming growth factor-b (TGF-b) in vitro. This has raised the question whether, how frequently, by which mechanism, and at which stage of development squamous carcinomas escape from TGF-b control in vivo. We have developed a method to rapidly identify the most common molecular alterations in the TGF-b signaling pathway by combining measurements of the levels and the activation state of Smad signaling intermediates with DNA-based diagnostic assays. In this report, we demonstrate the validity of this approach using a panel of seven squamous cell carcinoma (SCC) lines known to be refractory to TGF-b-mediated cell cycle arrest. Each of the SCCs expressed the pathway-restricted Smad proteins, Smad2 and -3. Furthermore, treatment with TGF-b induced phosphorylation of Smad2 in each of the SCCs with the exception of the two cell lines that carry inactivating mutations of the TGF-b type II receptor. Three of the remaining SCC lines failed to express the common mediator Smad4, two on the basis of loss of transcription and one by a posttranscriptional mechanism. Thus, a mechanism for TGF-b resistance was identified in five of the seven tumor cell lines. Interestingly, in the two remaining lines, no abnormalities of signaling intermediates were found, and TGF-b was able to activate TGF-b-responsive promoters. This suggests that the ability of these two cell lines to grow in the presence of TGF-b is due to factors extraneous to the TGF-b pathway itself. Application of our protein-based strategy to interrogate the TGF-b signaling pathway should allow us to determine whether or not and, if so, how and at which stage human squamous cell carcinomas become TGF-b resistant in vivo.

Key words: Transforming growth factor-b; Receptor; Smad; Mutation; Head & neck cancer; Squamous cell carcinoma

Address correspondence to Michael Reiss, M.D., Section of Medical Oncology, Department of Internal Medicine and Yale Cancer Center, NS291, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8032. Tel: (203) 785-6221; Fax: (203) 785-7531; E-mail: michael.reiss@yale.edu