|ognizant Communication Corporation|
VOLUME 12, NUMBER 8
Oncology Research, Volume 12, pp. 309-314
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Copyright © 2001 Cognizant Comm. Corp.
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Giuseppe S. A. Longo,1 Julie Izzo,1 Richard Gorlick,2 Debabrata Banerjee,1 Suresh C. Jhanwar,3 and Joseph R. Bertino1
1Laboratory of Molecular Pharmacology and Experimental Therapeutics and Departments of 2Pediatrics and 3Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY
Four new cell lines were established from the primary tumors of patients with untreated colorectal adenocarcinoma. Drug sensitivity and characterization of these cell lines was performed. Three of the four cell lines formed colonies in soft agar and all were tumorigenic in nude mice. The cell lines were morphologically similar but had differences in growth characteristics. Two of the cell lines, C18 (CCCL-4) and C29 (CCCL-6), had a longer doubling time compared with C85 (CCCL-1) and C86 (CCCL-2). The C18 and C29 cell lines had chromosome 17 abnormalities and evidence by immunohistochemistry of a mutant p53 and had decreased levels of thymidylate synthase and dihydrofolate reductase proteins, associated with decreased thymidylate synthase catalytic activity in C18 and no detectable activity in C29. Raltitrexed and GW1843U89 showed potent cytotoxic activity and all four cell lines displayed similar cytotoxicity to these folate thymidylate synthase inhibitors. The C18 and C29 cell lines were in general resistant to the other agents tested (methotrexate, 5-fluorouracil, nolatrexed) when compared with the C85 and C86 cell lines. These new cell lines may be useful for the study of colorectal adenocarcinoma and for evaluating new drugs or treatment schedules.
Key words: Colorectal; Adenocarcinoma; Antifolate; Thymidylate synthase
Address correspondence to Joseph R. Bertino, M.D., Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue Box #78, New York, NY 10021. Tel: (212) 639-8230; Fax: (212) 639-2767; E-mail: firstname.lastname@example.org
Selective Susceptibility of Transformed T Lymphocytes to Induction of Apoptosis by PSC 833, an Inhibitor of P-Glycoprotein
Janeen Azare,1 Irene Pankova-Kholmyansky,2 Konstantin Salnikow,1 Dalia Cohen,3 and Eliezer Flescher2
1Nelson Institute of Environmental Medicine, New York University
Medical Center, Tuxedo, NY 10987
2Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 69978, Israel
3Novartis Institute for Biomedical Research, Summit, NJ 07901
P-glycoprotein is a cellular efflux pump. The P-glycoprotein inhibitor PSC 833 causes apoptosis of cancer cells and induces a rise in the intracellular levels of ceramide. Our aims were to determine whether a cause and effect relationship exists between these two actions of PSC 833, and to assess whether the PSC 833-induced apoptosis is restricted to transformed cells. Apoptosis was determined by flow cytometry and radioactive quantitation of DNA fragmentation. PSC 833 induced apoptosis in the human T leukemia cell lines: Molt-4 and Jurkat. Analysis of the apoptosis in Molt-4 and Jurkat cells revealed that PSC 833 induced a rise in the cellular ceramide levels (as measured by the DG kinase assay). PSC 833-induced apoptosis was significantly reduced by specific inhibitors of ceramide de novo synthesis (i.e., fumonisin B1 and L-cycloserine). On the other hand, PSC 833 did not induce apoptosis in normal peripheral blood T cells regardless of whether these cells were quiescent, activated, or proliferating. Our results suggest that PSC 833 induces apoptotic death in human transformed T lymphocytes through an increase in ceramide de novo synthesis. In addition, normal lymphocytes are not susceptible to induction of apoptosis by PSC 833. This difference between normal lymphocytes and leukemia cells presents a potential target for chemotherapy.
Key words: P-glycoprotein; Apoptosis; Lymphoma; T lymphocytes; Ceramide; PSC 833
Address correspondence to Eliezer Flescher, Ph.D., Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Tel: +972 3 640-6063; Fax: +972 3 640-9160; E-mail: email@example.com
4-[3-(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline Hydrochloride (NLCQ-1), a Novel Bioreductive Agent as Radiosensitizer In Vitro and In Vivo: Comparison With Tirapazamine
Maria V. Papadopoulou, Ming Ji, Mira K. Rao, and William D. Bloomer
The Radiation Medicine Institute at Evanston Northwestern Healthcare, 2650 Ridge Avenue, Evanston, IL 60201
The novel hypoxia-selective cytotoxin NLCQ-1, which is a weak DNA intercalator, was studied in conjunction with radiation against V79 cultured cells and EMT6 or SCCVII tumors in their syngeneic mice and compared with tirapazamine (TPZ). NLCQ-1 was a very potent and efficient radiosensitizer of hypoxic V79 cells, providing SER values of 2.27-2.56 at 20-80 mM concentration (measured at 10% survival level). Its C1.6 (concentration for an SER of 1.6 to be obtained) was 7.2 ± 0.2 mM. Its in vitro therapeutic index (ThI, defined as CT50(Air)/C1.6) varied by the exposure time from 57 (1-h exposure) to 145 (4.5-h exposure). The corresponding C1.6 value for TPZ was 16.9 mM whereas its in vitro therapeutic index was 49 (3-h exposure). A schedule-dependent synergistic interaction was observed between NLCQ-1 or TPZ and 20 Gy of radiation in both tumor models examined, by using the in vivo-in vitro assay as endpoint. Optimal synergism (>1 log) was observed in EMT6 tumors when each bioreductive drug was given between 45 and 60 min before irradiation. NLCQ-1 alone had no significant antitumor activity at 10 mg/kg (28% of its single LD50), whereas a 0.4 surviving fraction was obtained by TPZ at 30 mg/kg (38% of its single LD50). SER values of 1.52 and 1.25 were obtained with 10 mg/kg NLCQ-1 and 30 mg/kg TPZ, respectively, in EMT6 tumors. An SER value of 1.58 was obtained for both hypoxia-selective cytotoxins, at equitoxic doses, in SCCVII tumors, by using a fractionated regimen. These results suggest a possible use of NLCQ-1 or TPZ as adjuvants to radiotherapy.
Key words: NLCQ-1; Tirapazamine; Radiosensitizers; Bioreductive drugs; Hypoxic cytotoxins
Address correspondence to Maria V. Papadopoulou, Evanston Northwestern Healthcare, Department of Radiation Medicine, 2650 Ridge Avenue, Evanston, IL 60201. Tel: (847) 570-2262; Fax: (847) 570-1878; E-mail: firstname.lastname@example.org
Demethylation by 5-aza-2´-deoxycytidine (5-azadC) of p16INK4A Gene Results in Downregulation of Vascular Endothelial Growth Factor Expression in Human Lung Cancer Cell Lines
Keisuke Miki,1 Eiji Shimizu,2 Seiji Yano,1 Kenji Tani,1 and Saburo Sone1
1Third Department of Internal Medicine, University of Tokushima
School of Medicine, Tokushima, Japan
2Third Department of Internal Medicine, University of Tottori School of Medicine, Tottori, Japan
Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor progression via angiogenesis. Recently, gene transduction of wild-type p16INK4A, tumor suppressor gene, has been shown to result in downregulation of VEGF expression in p16INK4A-deleted glioma cells. Because expression of p16INK4A is regulated by methylation of the p16INK4A gene, we examined whether demethylation of the p16INK4A gene by 5-aza-2´-deoxycytidine (5-azadC) could cause the protein expression of VEGF as well as of p16INK4A in human lung cancer cells. For this, five different lung cancer cell lines with or without loss of p16 activity were used. H841 and Ma-10 cells had the methylated p16INK4A gene without expression of p16INK4A protein, whereas Ma-1 and H209 cells had the unmethylated p16INK4A gene with constitutive expression of p16INK4A protein. Neither the p16INK4A gene nor p16INK4A protein was detected in A549 cells. Treatment with 5-azadC caused demethylation of the p16INK4A gene with reexpression of p16INK4A protein in H841 and Ma-10 (methylated p16INK4A gene dominant) cell, but not in other cell lines such as Ma-1, H209 (unmethylated p16INK4A gene dominant), or A549 (p16INK4A gene deleted). In a parallel experiment, 5-azadC inhibited production of VEGF protein by H841 and Ma-10 cells, especially in the later hypermethylated cells, but not Ma-1, H209, or A549 cells. RT-PCR analysis showed that Ma-10 cells expressed VEGF isoforms 121, 165, and 189, all of which were inhibited by 5-azadC. These findings indicate that the methylation status of the p16INK4A gene plays an important role in the regulation of angiogenesis associated with progression of lung cancer, through regulation of VEGF expression.
Key words: 5-aza-2´-deoxycytidine (5-azadC); p16INK4A; VEGF; Lung cancer cell line
Address correspondence to Saburo Sone, Third Department of Internal Medicine, Tokushima University School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. Tel: +81-88-633-7127; Fax: +81-88-633-2134; E-mail: email@example.com
Tatsuaki Ishiguro and Hirokazu Nagawa
Department of Surgical Oncology, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-8655, Japan
We formally reported that ATF3 gene regulated HT29 colon cancer cell metastasis through cell adhesion and invasion. We report here our findings that, on wound filling assay, the ATF3 antisense oligonucleotide changed cell form to a rounder shape and suppressed the cell migration ability of HT29, although FACScan analysis showed that it had no effect on the cell cycle. The growing area of HT29 cells treated with the antisense oligonucleotide decreased, compared with that treated with the sense oligonucleotide. These factors were thought to relate to adhesion and invasion of HT29 cells, hence they influenced metastatic potential.
Key words: ATF3 gene; HT29 colon cancer cells; Cell migration; Cell form
Address correspondence to Dr. Tatsuaki Ishiguro, Department of Surgical Oncology, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-8655, Japan. Tel. and Fax: 81-3-3811-6822.
Human Papilloma Virus 16 E7 Oncogene Does Not Cooperate With RET/PTC 3 Oncogene in the Neoplastic Transformation of Thyroid Cells in Transgenic Mice
Giuseppe Portella,1 Cristina Borselli,1 Massimo Santoro,1 Diego Gerbasio,2 Maria Rosaria D'Armiento,2 Jacques E. Dumont,3 Catherine Ledent,3 Jay L. Rothstein,4 Giancarlo Vecchio,1 and Alfredo Fusco5
1Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio
Nazionale delle Ricerche c/o Dipartimento di Biologia e Patologia Cellulare
e Molecolare and 2Dipartimento di Scienze Biomorfologiche e
Funzionali-Istituto di Anatomia Patologica, Università ``Federico
II,'' 80131 Naples, Italy
3University of Brussels, School of Medicine, Institute of Interdisciplinary Research (IRIBHN), B 1070 Brussels, Belgium
4Department of Otolaryngology-HNS, Thomas Jefferson University, Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, PA
5Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università ``Magna Graecia,'' 88100 Catanzaro, Italy
We have previously reported that the thyroid-targeted expression of the RET/PTC3 oncogene (Tg-RET/PTC3) in transgenic mice induces follicular hyperplasia with papillary architecture, resulting in a modest increase of the thyroid gland volume, followed by the appearance of papillary carcinomas in approximately 1-year-old animals. In order to analyze the genetic alterations that may cooperate with RET/PTC3 in the development or progression of thyroid tumors, we interbred Tg-RET/PTC3 mice with Tg-E7 transgenic mice, which express the E7 oncogene of the human papilloma virus 16 in thyroid cells. Tg-E7 mice develop large colloid goiters with small papillae and well-differentiated thyroid carcinomas in older animals. Here we show that thyroid lesions in Tg-RET/PTC3-Tg-E7 double transgenics were morphologically different from those occurring in Tg-RET/PTC3 mice, while they were virtually indistinguishable from those occurring in Tg-E7 mice. In addition, the coexpression of RET/PTC3 and E7 oncogenes neither enhanced the malignant phenotype nor reduced the latency period of thyroid lesions with respect to parental transgenic lines. We conclude that the coexpression of RET/PTC3 and E7 lacks any cooperative effect in the neoplastic transformation of thyroid cells and that the E7-induced thyroid phenotype is dominant with respect to the RET/PTC3 one.
Key words: Thyroid; RET/PTC3 oncogene; E7 oncogene; Tumorigenesis; Neoplasia; Goiter
Address correspondence to Giuseppe Portella, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia di Napoli, Università ``Federico II,'' via S. Pansini 5, 80131 Napoli, Italy. Tel: 39-081-7463056; Fax: 39-081-7463037; E-mail: Portella@cds.unina.it